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The Florida Manatee Somatosensory System

HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Innervation of follicle-sinus complexes...
 Somatosensory nuclei of the manatee...
 Somatosensory areas of manatee...
 Conclusions and future directi...
 Appendix
 References
 Biographical sketch
 

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1 THE FLORIDA MANATEE SOMATOSENSORY SYSTEM By DIANA KAY SARKO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Diana Kay Sarko

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3 In loving memory of my grandfather, John Sar ko, who always supported my educational pursuits and who I wish could be here on my graduation day, and every day

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4 ACKNOWLEDGMENTS First and foremost, I thank my parents. Thei r support and their ow n pursuit of knowledge, both of them going back to school la ter in life to earn master’s de grees, is an inspiration. I thank my mentor Dr. Roger Reep for showing me ev erything that an adviso r should be—available, good-humored, supportive, and able to balance work with play. I only hope that one day when I have my own lab that I can do him justice. I am grateful to my committee members for their time, wisdom and invaluable contributions to th is project: Dr. Gordon Bauer, Dr. Pete McGuire, and especially Dr. Floyd Thompson, from whom I have learned a great deal about being an exceptional professor and researcher. Many, many th anks are due to Maggie Stoll, who has not only dealt with troubleshooting on ev ery level, but has been with me through personal trials that I could not have undergone alone. I thank my love and my fianc JJ Kennard fo r his patience and devotion, and especially for helping me to turn my brain to the much -needed, often-ignored “off” position now and then. Besides Roger and Maggie, my time in this lab ha s also afforded me the pleasure and privilege of meeting Dr. Joe Cheatwood, who gave me guidance when I first joined the lab and who will be a lifelong friend. Many thanks to Susan Oliver, my best friend, who always knew that I could get through this, even when I had serious doubts. I also thank Dave Schoenberg, who has been a shoulder and one of my deares t friends since college; and Kevin Chadbourne, who has gone above and beyond what it has taken to maintain my sanity towards the end of this journey. Every little thing that he has done has help ed me more than he will ever know. Certain events from my gradua te career have made me realiz e that life is too short to waste pursuing anything but what yo u love to do. These last four and a half years I have had the privilege to do just that. I have learned so much from so man y, and yet I have only just begun.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION ................................................................................................................ .13 The Florida Manatee............................................................................................................ ...13 Sensory Specializations of the Manatee Body................................................................13 Perioral Vibrissae............................................................................................................1 5 The Manatee Brain: General Attributes..................................................................................18 Cytochrome Oxidase: A Metabolic Marker for Primary Sensory Areas................................19 Brainstem Somatosensory Nuclei and Barrelettes..................................................................21 Thalamus and Barreloids........................................................................................................ 23 Rindenkerne.................................................................................................................... ........24 The Cerebral Cortex: Relating Cyto architecture to Electrophysiology..................................25 2 INNERVATION OF FOLLICLE-SINUS COMPLEXES IN THE FLORIDA MANATEE ....................................................................................................................... .....27 Introduction................................................................................................................... ..........27 Materials and Methods.......................................................................................................... .30 Results........................................................................................................................ .............34 Facial Vibrissae............................................................................................................... 34 Postfacial Vibrissae.........................................................................................................37 Discussion..................................................................................................................... ..........38 Manatee Vibrissae: Overall Comparative Structure........................................................38 Facial Musculature Involved in Explorat ory and Prehensile Vibrissal Behaviors..........39 Sensory Innervation of the Rete Ridge Collar and Epidermis........................................39 Sensory Nerve Endings of the I nner Conical Body and Ring Sinus...............................40 Cavernous Sinus Innervation...........................................................................................43 Marine Mammal Vibrissae..............................................................................................45 Comparative Considerations...........................................................................................46 3 SOMATOSENSORY NUCLEI OF THE MANATEE THALAMUS AND BRAINSTEM...................................................................................................................... ...65 Introduction................................................................................................................... ..........65 Materials and Methods.......................................................................................................... .68

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6 Results........................................................................................................................ .............70 Brainstem...................................................................................................................... ...70 Thalamus....................................................................................................................... ..72 Discussion..................................................................................................................... ..........75 Brainstem: Somatotopic Par cellation in Other Species...................................................75 Thalamus: A Comparative Look at Somatosensory Nuclei............................................77 4 SOMATOSENSORY AREAS OF MANATEE CEREBRAL CORTEX: HISTOCHEMICAL CHARACTERIZATION AND FUNCTIONAL IMPLICATIONS...108 Introduction................................................................................................................... ........108 Materials and Methods.........................................................................................................1 10 Results........................................................................................................................ ...........113 Areal Patterning.............................................................................................................11 3 Neonates....................................................................................................................... .114 Juvenile and Adult.........................................................................................................116 Neonate versus Juvenile and Adult Comparison...........................................................117 Discussion..................................................................................................................... ........118 Somatosensory Cortex...................................................................................................118 Auditory and Visual Cortex...........................................................................................121 5 CONCLUSIONS AND FUTURE DIRECTIONS...............................................................138 Summary and Conclusions...................................................................................................138 Future Directions.............................................................................................................. ....141 Additional Considerations....................................................................................................14 3 APPENDIX: LETTER OF PERMISSION TO REPRODUCE COPYRIGHTED MATERIAL (the entirety of chapter 4)................................................................................145 LIST OF REFERENCES............................................................................................................. 146 BIOGRAPHICAL SKETCH.......................................................................................................158

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7 LIST OF TABLES Table page 2-1 Specimen categorization.................................................................................................... 48 3-1 Summary of specimen information....................................................................................82 3-2 Comparative analysis of percentage of thalamus occ upied by the ventroposterior nucleus (VP; averaged from 3 evenly spaced coronal sections to encompass VP)...........82 4-1 Summary of specimen data..............................................................................................124 4-2 Percentage of cortical area repres ented by presumptive sensory cortex..........................124

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8 LIST OF FIGURES Figure page 2-1 Vibrissae sampling regions of the body and face..............................................................48 2-2 Schematic drawing of the structure and innervation of the U2, BLH, and postfacial vibrissal follicle-sinus comp lexes (FSCS) with innervation types and sensory nerve endings illustrated............................................................................................................ ..49 2-3 Characterization of upper perioral field 2 (U2) follicle innervation..................................51 2-4 Innervation of the cavernous sinus and ha ir shaft medulla in facial follicles....................53 2-5 Innervation present in bristle-like hairs (BLHs)................................................................55 2-6 Representative postfacial vibrissae in nervation includes dense networks of MEs along with LLEs and “tangle” endings..............................................................................57 2-7 Immunolabeling attri butes of innervation..........................................................................59 2-8 Confocal surface reconstructions show ing the three-dimensional structure of representative follicle innervation and nove l mechanoreceptors present in the ICB, RS and CS regions.............................................................................................................6 1 2-9 Confocal three-dimensional images of novel endings stained for neurofilament (NF200) and protein gene product 9.5 (PGP)....................................................................63 3-1 A rostrocaudal series of representative coronal brains tem sections with subnuclei labeled illustrates the size and extent of somatosensory nuclei.........................................83 3-2 Brainstem sections cut in the sagittal plane illustrate the ro strocaudal extent of behaviorally relevant nuclei and in part icular the lobulated appearance of the trigeminal nuclei.............................................................................................................. ..88 3-3 Brainstem sections cut in the horizonta l plane show the topo graphy and orientation of nuclei of interest.......................................................................................................... ..89 3-4 Representative coronal brainstem sections illustrating the appearance of each of the trigeminal subnuclei in an adult specimen.........................................................................90 3-5 A rostrocaudal series of re presentative coronal brainstem sections in a neonate shows that somatosensory nuclei are large and have a parcellated appearance as seen in adult specimens................................................................................................................ ..92

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9 3-6 A rostrocaudal series of representative coronal thalamic sections with lowmagnification images of sections staine d with hematoxylin for myelin and highmagnification details of adjacent sections stained with thionin for Nissl bodies with subnuclei labeled.............................................................................................................. ..95 3-7 A rostrocaudal series of closely spaced coronal sections showing the ventroposterior area (VP) of the thalamus in detail..................................................................................100 3-8 Low-magnification and high-magnificat ion images characterizing Nissl body staining of the lateral ventroposterior (VPL) and medial ventroposterior (VPM) subnuclei of the thalamus.................................................................................................102 3-9 Histochemical and histol ogical staining characterizati on in the ventroposterior nucleus of the thalamus....................................................................................................103 3-10 Coronal thalamus sections stained for cytochrome oxidase (CO) from a neonate (specimen TM0410) and a juvenile (specimen TM0339) show that the ventroposterior thalamus (VP) exhibits homogenous CO-dense staining without clearly distinguishable barreloids....................................................................................104 3-11 Fiber laminae (arrows) seen most distin ctly in the juvenile specimen (TM0339) may separate adjacent projections from adjacent body parts into subnuclei of the thalamus as demonstrated in other species......................................................................................106 3-12 Horizontal myelin-stained section s howing unusual placement of the medial (MGN) with respect to the lateral geniculate nucleus (LGN).......................................................106 3-13 Proposed somatotopy of functional re presentations within the brainstem somatosensory nuclei (cuneate-gracile a nd trigeminal) and the ventroposterior nucleus (VP) of the thalamus in the coronal plane of section..........................................107 4-1 Tangential sections stained with cytochro me oxidase and merged to encapsulate the full extent and persistence of areal pattern ing in left hemisphere flattened cortex preparations for A) neonate (TM0310), C) juvenile (TM0339), and D) adult (TM0406) specimens.......................................................................................................125 4-2 Rostrocaudal series of co ronal sections relating cyto chrome oxidase staining to cytoarchitectural bounda ries (determined by Nissl body a nd myelin stains of adjacent sections) in a neonate brain (TM0410)............................................................................127 4-3 Rostrocaudal series of co ronal sections relating cyto chrome oxidase staining to cytoarchitectural bounda ries in a juvenile brain (TM0339)............................................130 4-4 Coronal cytochrome oxidase sections from an adult specimen (TM0406) revealing trends consistent with the juvenile specime n but distinct from the neonate (see text for details)................................................................................................................... .....133

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10 4-5 Adjacent sections stained for myelin, cyto chrome oxidase, and Nissl bodies illustrate consistently dense staining in layer IV in both myelin and cytochrome oxidase preparations of presumptive primary sensory areas (specimen TM0406, area DL1 shown)......................................................................................................................... .....134 4-6 Localization of cytochrome oxidase-dense staining within cort ical layer boundaries for each cytoarchitectural area.........................................................................................135 4-7 Three-dimensional reconstruc tion of neonatal specimen TM0410.................................137

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE FLORIDA MANATEE SOMATOSENSORY SYSTEM By Diana Kay Sarko December 2006 Chair: Roger L. Reep Major Department: Medical Sciences—Neuroscience Florida manatees are thought to be tactile specialists, and in an effort to systematically characterize this system, the res earch presented here first used immunolabeling to functionally characterize sensory innervation in facial fo llicles with behavioral relevance in object recognition and exploration as well as in follicl es from select perioral and postfacial regions. Facial vibrissae exhi bited dense Cand A -fiber innervation of the epidermis and rete ridge collar, novel “tangle” endings at the inner coni cal body level, dense Merk el cell and moderate longitudinal lanceolate ending di stribution at the ring sinus, an d novel endings located along the trabeculae of the cavernous sinus. Postfacial vi brissae contained Merkel endings and dense Cand A -fiber distribution at the rete ridge collar. Dense Merkel ending networks and “tangle” endings were present at the inner conical body and ring sinus levels along with moderate longitudinal lanceolate ending innervation. No novel ending s were present within the trabeculated cavernous sinus of any postfacial vibrissae. We conclude that the facial vibrissae are in fact more densely innervated, with more varied sensory endings, in accordance with their behavioral importance in active tactile explorat ion. Furthermore, it seems that manatees are heavily invested in directionality detection, an adaptation that w ould enhance their perception of underwater hydrodynamic stimuli.

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12 A histochemical and cytoarchitectural analys is was also completed for the brainstem, thalamus, and neocortex of the Florida manatee in order to localize primary sensory areas. Based on the location of cytochrome oxidase (CO)-den se staining, we found that somatosensory nuclei of the brainstem (Bischoff’s, trigeminal, and cu neate-gracile nuclei) and thalamus (VP) appear disproportionately large and, in the case of the trigeminal a nd cuneate-gracile complex, show evidence of parcellation that ma y be somatotopically related to discrete body areas. Flattened cortex preparations stained for CO were assigne d preliminary functional divisions for S1 with the face represented laterally followed by the f lipper, body and tail representations proceeding medially. Coronal cortical se ctions stained for CO, myeli n, or Nissl bodies were also systematically analyzed in order to accurately lo calize the laminar and cytoarchitectural extent of CO staining. Overall, S1 appears to span se ven cytoarchitectural areas for which we have proposed functional assignments.

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13 CHAPTER 1 INTRODUCTION The Florida Manatee Manatees belong to the order Sirenia, of which over 35 spec ies existed during the past 50 million years, with only 4 remaining presently (Domning, 1982). There are three extant manatee species: the West Indian, of which the Florida manatee ( Trichechus manatus latirostris ) and the Antillean manatee ( T. manatus manatus ) are subspecies; the Amazonian, T. inunguis ; and the West African, T. senegalensis As the only obligate herbivores among marine mammals, sirenians possess unique behavior al, physiological, and neuroana tomical adaptations. The US Fish and Wildlife Service currently classifies the Florida manatee as endangered, a status that is supported by their low total population which was estim ated at the last aerial survey in February 2004 to be 2,568 manatees (provided by the Ma natee Technical Advisory Council and the Manatee Population Status Working Group). In the year 2003 alone, th e Florida Fish and Wildlife Conservation Commission Marine Mammal Pathobiology Labora tory reported a total of 380 manatee deaths—a significant percentage of the population, i ndicating that the opportunity to learn from this unique speci es is rapidly disappearing. Sensory Specializations of the Manatee Body Manatees appear to have reasonably welldeveloped hearing (Ger stein and Gerstein, 1999) but reduced vision (e.g., Bauer et al., 2003 ). Though little is known about the extent of their olfactory or gustatory capab ilities, these appear to be sens es of subordinate importance to the manatee as well (Levin and Pfeiffer, 2002; Mackay-Sim et al., 1985). However, recent evidence suggests the pres ence of a sophisticated tactile sense through a sy stem of sinus-type tactile hairs, or follicle sinus complexes (FSCs) covering the entire pos tfacial body (Reep et al., 2002). The postfacial body is covered in approxim ately 3,000 hairs with hair density decreasing

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14 dorsoventrally (Reep et al., 2002) and calves exhibit greater hair density distribution that is attributed to the fixed number of follicles in a mammal at birth. Hair density then decreases with age as the body, and especially the midsection, of the manatee e xpands (Reep et al., 2002). Each body hair has an external lengt h of 2–9 mm, with most ha irs separated by 20–40 mm, giving each hair an independent field of movement (Reep et al., 2002). A single body vibrissa is innervated by 20–50 axons whereas 40–200 axons s upply innervation to each facial vibrissa (Reep et al., 2001; 2002). The distri bution of vibrissae over the entire postf acial body is a unique arrangement among mammals, most of which have tactile hairs restricted only to certain body regions, and is proposed to be analogous to the la teral line system in fish by functioning as a “touch at a distance” sense through passive deflec tion of tactile hairs by hydrodynamic stimuli. Such a system is potentially capable of conveyi ng crucial information about water currents, the approach of other animals, and other features of the underwater environment (Reep et al., 2002). The manatee face possesses further sensory speci alizations that aid in adaptation to the animal’s unique environmental niche. Facial hair is distributed thirty times more densely than on the rest of the body (Reep et al., 1998), an attribute th at should increase sp atial resolution, and can be distinguished from body hair by the greate r stiffness of facial hair due to smaller length/diameter ratios (Reep et al., 1998). Body hair is located on the supradisk portion of the face posterior to the orofacial ridge and on the chin in addition to the entire postfacial extent of the body (Reep et al., 1998). Manatees have an expanded philtrum called the oral disk that contains bristle-like hairs (BLHs) that are used as tactile “feelers” in additi on to perioral bristles that are essentially modified vibrissae (Reep et al., 1998). Vibr issae provide detailed textural information about objects and surfaces in an animal’s immediate environment, and most mammals use vibrissae exclusively for sensory purposes such as findi ng prey and navigating

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15 successfully when vision is compromised, such as in low-light situations (Brecht et al., 1997; Dehnhardt et al., 1998; Dehnhardt et al., 2001; Ling, 1977). Facial ha ir is crucial in manatee feeding and tactile exploration of the environment, accomplishing dual and synergistic motor and sensory roles. The hair and bristles of the mana tee face are composed of 9 distinct regions, 6 of which are perioral bristle—4 upper perioral fiel ds (U1–U4) on each side of the upper lips and oral cavity, and 2 lower perioral fields (L1–L2) on each side of the lower lip pad (Reep et al., 1998). Each of these follicles can be classified as a vibrissa according to the criteria established by Rice et al. (1986): 1) substantial innervation, 2) a dense connective ti ssue capsule, and 3) a prominent, circumferential blood sinus complex. The 9 regions of the manatee face are discernible by location as well as the number, range of length/di ameter ratios, and behavioral role of follicles within each field (Reep et al., 2001). Perioral Vibrissae The BLHs of the oral disk are the vibrissa e primarily involved in object recognition and tactile exploration, whereas U2 and L1 follicle fiel ds are used in a prehensile grasping fashion during feeding and oripulation (a combined sensorimotor function that is unique among mammals) as well as in social behaviors in cluding mouthing, nuzzli ng, and also pinching a conspecific’s back in an attempt to gain acce ss to food (Reep et al., 2001; Marshall et al., 1998b). The right and left U2 bristle fields specifically act in a pr ehensile manner during feeding by reaching out and grasping food while L1 bristles actively push vegetation farther into the oral cavity (Marshall et al., 1998b). The U1 vibrissae may also be invol ved in some level of tactile exploration during feeding while the U3, U4, and L2 fields may assist L1 bristles in the movement of food (Marshall et al., 1998b). Upon en countering a particularly difficult food item, manatees can use each U2 field independently, even reversing directi on in order to expel undesirable food (Marshall et al ., 1998b). Such evidence reveals a high level of dexterity and

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16 perioral tactile discrimination and is supported by the manat ee’s relative tactile difference threshold of 14%—favorably comparable to that of an Asian elephant’s trunk (Bachteler and Dehnhardt, 1999). Notably, the eyes are often cl osed during feeding a nd tactile exploration (Marshall et al., 1998b; Bachte ler and Dehnhardt, 1999), furthe r indicating an emphasis on haptic over visual input The prehensile ability of facial ta ctile hairs is present in dugongs as well, but absent in pinnipeds despite their higher tactile resolvi ng power (Bachteler and Dehnhardt, 1999; Marshall et al., 1998b; Marshall et al., 2003). In an earlier study of manatee follicle innervation U2s were found to contain the largest FSCs composed of the longest hair shafts, the wide st ring sinuses, the thickest capsules, and the highest degree of innervation at over 200 axons pe r follicle (Reep et al., 2001). The L1 bristles are innervated by the second largest number of axons at approximately 200 per FSC, followed by U3, U4, and L2 bristles (approximately 100) and fi nally U1 bristles, whose range overlaps that of the BLH vibrissae at 49–74. The body hair follicles of the chin and supradisk contain the least axonal innervation with a range of 34–48 axons pe r follicle. Most FSC axons terminate in the mesenchymal sheath and the outer root sheath lini ng the hair follicle proper along the level of the ring sinus. Reep et al. (2001) described gene ral morphological features and axonal counts for each follicle type but the silver staining was ofte n inconsistent with inadequately defined nerve endings. This limitation can be solved through a systematic analysis using immunolabeling, and given the co-varying behavioral and sensory tasks for which each follicle field is specialized, concurrently varying attribut es in innervation patterns might be elucidated through immunofluorescence. Upon examination of muscular supply to facial vibrissae, Reep et al. (1998) discovered that the dorsal and ventral buccal branches of the facial nerve supply the li ps and perioral regions

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17 with the dorsal branch supplyi ng the upper lip and nasal area and the ventral branch terminating in the lower jaw and lip muscles to enable vibr issal eversion and feeding behavior. Furthermore, each facial bristle follicle in the U1–U4 fields is supplied by the infraorbital branch of the maxillary nerve (the sensory trigeminal branch ), making these fields homologous to mystacial vibrissae. Sensory innervation of the lower jaw is provided by the in ferior alveolar branch of the mandibular nerve while the lingual branch inne rvates the tongue a nd the mylohoid branch courses ventrally to innervate M. mylohyoidus and the ventral mandible skin. The inferior alveolar branch separates into 2 mental nerves, supplying L1 and L2 and making them homologues of the mental vibr issae present in other taxa. Comparative trends in mammals indicate that vibrissae have evolve d to perform complex functions in order to provide feedback about an animal’s environment, but although sensory detection is often accompanied by vibrissal m ovement, it is not accompanied by prehensile grasping behaviors (Reep et al ., 2001). Harbor seals appear to use vibrissae in touch discrimination as effectively as a monkey is able to utilize its hands (Dehnhardt and Kaminski, 1995), and pinnipeds as a whole have been found to employ thei r long vibrissae in tactile exploration as well as in soci al display behavior (Dehnhardt, 1994; Dehnhardt and Ducker, 1996; Dehnhardt and Kaminski, 1995; Peterson and Bart holomew, 1967; Miller 1975; Kastelein and Van Gaalen, 1988). Rodents utilize a whisking behavi or of their mystacial vibrissae in tactile exploration (Carvell and Sim ons, 1990; Welker, 1964; Wineski, 1985) and freshwater river dolphins ( Platanistidae ), which have poor vision, use vibrissa e on their upper and lower jaws to locate prey (Ling, 1977). Sensory specializations of the skin and hair in mammals are accompanied by expanded cortical representations to accommodate the greater level of neural input (Johnson, 1990; Kaas and Collins, 2001). A clear example of this can be seen in the star-

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18 nosed mole ( Condylura cristata ). The 22 fleshy nasal appendages, or rays, that it uses to explore the environment can be seen in cytochrome oxidase preparations of somatosensory cortex with a distinct band corresponding to each ray (Catania and K aas, 1995, 1997). Catania and Kaas (1997) further discovered that the eleventh a ppendage, used preferentially in environmental exploration, also assumes the la rgest cortical representation. Th erefore, it is reasonable to hypothesize that in the manatee additional ne uroanatomical space would be allotted to complement tactile specializations with a par ticularly expanded faci al representation. We examine this further by identifying and characteri zing the somatosensory ar eas of the brainstem, thalamus and cortex in order to more complete ly understand any specializ ations that might be present and might complement the Florida mana tee’s adaptation to its environmental niche. The Manatee Brain: General Attributes The Florida manatee brain possesses a unique and intriguing set of attributes that combine more primitive traits with those considered to be quite derived. The former include a very smooth (highly lissencephalic) cortex and an extremely small brain size compared to what would be expected for an animal of its body size, a parameter known as the encephalization quotient, or EQ. The EQ of the manatee was found to be 0.27, or about 1/4 the value expected for its body size (O’Shea and Ree p, 1990). The gyration index, a meas ure of cortical folding, of 1.06 for the Florida manatee quantifies the high level of lissencephaly observed (Reep and O’Shea, 1990). Johnson et al. (1994) also reexamined phylogenetic classifications by examining a number of brain traits that were scored as primitive or derived across 152 mammalian species. Manatees were found to be primitive in possessing the following attributes: 1) an optic tract that terminates in closely apposed nuclei of the thal amus, 2) a lack of fasciculus aberrans, 3) no visible separation of the claustrum from corte x, and 4) no clear separation between external cuneate nucleus and cuneate nucleus. Dietary features such as the low quality and abundance of

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19 food along with a low metabolic ra te are also characteristic of many mammals with EQs in the lower mammalian range, including the manatee (McNab, 1978; 1980). Despite the above evidence, most of the life history, ecological and be havioral traits of the Florida manatee are typical of large-brai ned species with higher EQs (O’Shea and Reep, 1990). With a gestation period of approximately 1 y ear, an age range at sexual maturity spanning 5–10 years, an average interbirth interval of 2–5 or more year s, and longevity in the wild estimated at 50–60 years (Hartman, 1979), manatees appe ar to be more typical of altricial species following principles of K-selec tion. Also, while the manatee brai n is small relative to its body size, the telencephalon comprise s 71% of the total brain volume, 90% of which consists of cerebral cortex. The cortex also possesses well-def ined laminae. These qualities are comparable to taxa with large relative brain size, includ ing primates (Reep and O’Shea, 1990). The Johnson et al. (1994) study further indicated that manatees have the followi ng derived brain traits: 1) lack of accessory olfactory formation, involved in phero mone detection, 2) deep position of the optic tract in the collicular tectum, 3) emergence of th e facial nerve ventral to the trigeminal sensory column, 4) olfactory bulb mitral cells gathered into a monolayer, 5) hemispheres connected by a corpus collosum, 6) medial positi on of the ventral nucleus to the pr incipal nucleus of the inferior olive, 7) presence of Rindenkerne, cell clusters in cortical layer VI, and 8) a delaminated dorsal cochlear nucleus. Johnson et al also proposed that the second ary loss of lamination in the auditory dorsal cochlear nucleus along with lo ss of the accessory olfactory formation indicate convergent evolutionary cons equences of departure from a terrestrial habitat. Cytochrome Oxidase: A Metabolic Marker for Primary Sensory Areas Cytochrome oxidase (CO) is an effective endogenous metabolic marker for neurons due to the tight coupling between neuronal activity and oxidative metabolism (Wong-Riley, 1989 for review). This enzyme is an integral transm embrane protein found in the inner mitochondrial

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20 membrane of all eukaryotes and generates AT P through oxidative phosphorylation (Wikstrom et al., 1981). CO accounts for over 90% of oxygen consumption by eukaryotes (Wikstrom et al., 1981) and is vital for organs lik e the brain that rely on oxidativ e metabolism—in the case of neurons, particularly in the maintenance of i onic balance (Lowry, 1975; Sokoloff, 1974). It has been suggested that dendritic metabolism makes th e single largest contribution to this metabolic activity since the level of oxidative enzymes in dendrites reflects the in tensity and type of synaptic input to a neuron (DiFiglia et al ., 1987; Kageyama and Wong-Riley, 1982; Kageyama and Wong-Riley, 1985; Lowry, 1954; Mourdian a nd Scott, 1988; Wong-Riley, 1984). This is supported by the observation that CO activity leve ls are responsive to ex perimentally induced changes in functional activity (Wong-Riley, 1989 for review). Cerebral cortex stained for CO shows a la minar pattern of activ ity with highly active regions representing the thalamic-recipient and other synapse-rich layers (Carroll and WongRiley, 1984; Jones and Friedman, 1982; Matelli et al., 1985; Price, 1985). Neurons with intense CO activity are likely to be tonically active and maintain a high enzyme capacity for energy production to be able to drive the high rate of spontaneous activity (Wong-Riley, 1989). Since primary sensory areas of the cortex are more t onically active, they are easily discernable when stained for CO, and it has been found that CO ca n be used to separate functionally different cortical areas (e.g., Carroll and Wong-Riley, 1984) In fact, a recent study of human cortex was successful in differentiating primary and secondary sensory areas through CO and acetylcholinesterase (AChE) staining. Primary se nsory areas 3a and 3b showed dark CO staining of layer IV and a low level of AChE positive pyramid s, a pattern also seen in primary visual and auditory areas. Secondary associ ation areas 1 and 2 reve aled dark CO staini ng in layer III with an abundance of AChE positive pyramidal cells (Eskenasy and Clarke, 2000). Physiologically

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21 highly active nuclear groups of the basal ganglia, thalam us, brainstem and spinal cord also show strong enzymatic activity (DiFig lia et al., 1987; Jones et al., 1986; Nomura and Mizuno, 1986; Wallace, 1986; Wiener, 1986; Wong-Riley, 1976 ; Wong-Riley and Kageyama, 1986), making cytochrome oxidase useful in identifying th e primary somatosensory components of the brainstem and thalamus in addition to the cortex. Brainstem Somatosensory Nuclei and Barrelettes Commitment to specific sensory modalities in restricted regions of the body creates a commensurate commitment of neurons from the periphery through the brainstem, thalamus, and cerebral cortex. Following this paradigm, if somatic sensation is prevalent for the manatee, then associated nuclei in the thalamus and brainstem ar e expected to be relatively larger and/or more subdivided in order to accommodate the greate r amount of information being taken in and processed. The brainstem nuclei of interest fo r the manatee somatosensory system include trigeminal, cuneate, gracile, and Bischoff’s nuclei. It has already been noted that cranial nerve V (the trigeminal nerve) is large in the manatee (R eep et al., 1989). Further studies have shown that visual thalamic and brainstem nuclei are reduced whereas trigeminal and other somatosensory nuclei are well developed (Johnson et al., 1986; 1 987; Reep et al., 1989; Welker et al., 1986). Assessments of the relative impor tance of these sensory systems in sirenian behavior parallel these results, particularly for the trigeminal nerv e system extensively associated with the use of the facial vibrissae in tactile exploration, a crucial aspect of manatee behavior. However, these findings have never been revis ited, and more remains to be discovered. Bischoff’s nucleus, a distinct group of cells in th e midline of the caudal medulla (Johnson et al., 1968), has not previously been analyzed in the manatee but ha s been shown, along with the cuneate and gracile nuclei, to project heavily to the ventrobasa l thalamus in the raccoon (Ostapoff and Johnson, 1988). In the raccoon, the tail representation occupies the dorsal portion of this nucleus while the

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22 hindlimb representation occupies the ventral portion (Johnson et al., 1968). In the manatee, Bischoff’s nucleus would represent the fluke. CO st udies of the rat revealed that the afferent projection pattern from individual vibrissa follic les was topographically related to CO-dense cell clusters (“barrelettes”) in the trigeminal prin cipal sensory nucleus (PSN ) with a nearly one-toone ratio between follicles and corresponding CO -dense clusters (Florence and Lakshman, 1995). These results supported earl ier findings by Jacquin et al. (1993) that showed that PSN axon collaterals were concentrated within co rresponding CO-dense subdivisions, and terminal branches of individual trigeminal afferents rarely cro ssed over into adjacent regions. In contrast, in three subdivisions of the spinal trigeminal nuc leus—the pars oralis, pars interpolaris, and pars caudalis—a topographical arrangement still ex isted, but with less specificity and more overlapping representations (Florence and Lakshm an, 1995). Goyal et al. ( 1992) showed that the human principal trigeminal nucleus also demonstrated a parcellate d CO-dense pattern. Therefore, size and parcellation data for the trigeminal nucleus would further elucidate the sensory specializations of manatees. The same principle of CO somatotopic parcel lation is also evident in the cuneate and gracile dorsal column nuclei. Cutaneous inputs from the upper limbs and rostral trunk of the body are represented in the cuneate nucleus while lower limbs and lower trunk are represented in the gracile nucleus. Strata et al. (2003) studi ed the Galago monkey to look at the pattern of peripheral nerve input. Through cell clusters that were identified as CO-dense blotches in both nuclei, they discovered a greater segregation of inputs within the cuneate (fingers and hand representation) than in the grac ile (foot representation), which is consistent with the Galago’s extensive and highly differentiated use of its hand s and fingers relative to its feet. In macaques, inputs from specific parts of the hand relate to CO-dense rostroca udal clusters of cells (Florence

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23 et al., 1991). While the manatee lacks the manual dexterity of a primate, CO analysis of the cuneate and gracile nuclei woul d complete the evidence for manatee somatosensory processing and any concurrent specializations. Thalamus and Barreloids In the thalamus, AChE staini ng reveals robust patterns that allow for the discrimination of different nuclei and that are consistent in rodents, cats an d primates (Jones, 1985). Densest staining occurs in the ventral lateral geniculate nucleus (LGN), intralaminar, anteroventral, anterodorsal, rhomboid, paraventricular, habenul ar, and medioventral nuclei. Lighter staining distinguishes the dorsal LGN, me dial geniculate nucleus (MGN), reticular nucleus, anterior of the lateral posterior nucleus, and parts of latera l and ventral complexes. The principal somatic sensory nucleus in the thalamus consists of an area referred to as th e ventrobasal (VB) or ventroposterior (VP) nucleus. A lateral subnucleus, the ventral pos terior lateral (VPL) nucleus, represents the body while a medi al subnucleus, the ventral post erior medial (VPM) subnucleus, represents the face and most of the head (e.g., Jones, 1985). In rodents and marsupials, the medial division of the VB nucleus (VBm) was di scovered to contain “bar reloids”, or neuronal clusters related to individual vibrissae, that are highly reactive for CO (Jones, 1983; Land and Simons, 1985b; Van der Loos, 1976). Chronic tr imming of the vibrissae results in reduced staining for CO in both the somatosensory co rtical barrels (Land and Simons, 1985a; WongRiley and Welt, 1980) and the thalamic barreloids (Land and Akhtar, 1987) associated with the trimmed vibrissae. These findings were similar to those in Macaca fascicularis monkeys where peripheral nerves were cut, resu lting in reduced staining of “rod s” within the VPM (Jones et al., 1986). Using horseradish peroxidase axonal tracing, Jones et al. al so discovered that CO staining was primarily due to terminations of trigeminal afferent fibers that formed somatotopically organized inputs to the rods. They postulated that each rod of the thalamus formed the basis of

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24 columnarity of afferent input to the so matosensory cortex by providing bundles of thalamocortical axons terminating in focal domains of the cortex. Given the manatee’s reliance on haptic input, the VPM would be expected to be relatively large and may possess barreloid parcellation related to input from the facial vibrissae. Rindenkerne Rindenkerne are cortical cell clusters that stai n darkly for cytochrome oxidase and appear to be unique to sirenia, having been found abse nt in over 150 other mammalian species examined (Reep et al., 1989; Johnson et al., 1994). While thes e cell clusters are remi niscent of “barrels” found in the vibrissae subf ield of somatosensory cortex in rats, mice, and other rodents (Johnson, 1980; Kaas and Collins, 2001), as we ll as in shrews (Catania et al., 1999), opossums (Catania et al., 2000; Frost et al., 2000; Huff man et al., 1999), and hedgehogs (Catania et al., 2000), barrels are hollow aggregates of neurons in layer IV, a major afferent zone. In contrast, Rindenkerne are dense aggregates located in layer VI, an effe rent zone, although they do share histochemical attributes with barrels (Reep et al., 1989). Furthermore, Rindenkern e distribution in the cortex is restricted to 5 cytoarchitectural areas termed cluster cortex (C L) 1–5 by Reep et al. (1989) and Marshall and Reep (1995). The limit ed distribution of Rindenkerne and the fact that they are found exclusively in sirenian cortex implies a f unctional significance. Species possessing barrels show a one-to-one correspondence between barrels a nd vibrissae. However, there appear to be many more clusters than facial br istles in the manatee and it may be that only the larger clusters (approximately 1 mm in diameter) found in CL 1 represent individual bristles while smaller Rindenkerne such as those found in CL2 may corr espond to postfacial hairs (Loerzel and Reep, 1991). However, this hypothesis remains unt ested until the somatosensory cortex, and particularly the presumed facial regi on, can be more precisely delineated.

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25 The Cerebral Cortex: Relating Cyt oarchitecture to Electrophysiology Due to the manatee’s status as an endange red species, traditiona l electrophysiological methods of ascertaining the loca tion of primary somatosensory co rtex (SI) are not possible. Fortunately, the literature provi des a wide range of species fo r which both electrophysiology and histochemical processing have been possible. For example, in the marmoset monkey (Huffman and Krubitzer, 2001), and megachiropteran bats (K rubitzer et al., 1993; Krubitzer and Calford, 1992), microelectrode maps of so matosensory fields were found to be highly correlated with cytoarchitectural boundaries (specifically flattened cortex cut tangentially and stained for myelin). A flattened cortex preparation creates a plane of section that includes most of layer IV, the densest zone of CO staining, while also faci litating the comparative interpretation of areal patterns and allowing more direct assessment of the extent and relative position of architectonic fields (Krubitzer et al., 1995) The flying fox, a megachiropteran, was found to have myelindense zones in hand and face representations in area 3b (or SI) that involved non-habituating cutaneous receptors responding c onsistently to repe titive stimulation, whereas sparse zones rapidly habituated. In marmoset monkey s electrophysiology was also related to myeloarchitecture and revealed that the body map re presentation in area 3a is coextensive with a strip of lightly to moderately myelinated cortex rostral to the darkly myelinated area 3b. Overall, non-habituating neurons corresponded with myelin -dense zones considered homologous to area 3b (Krubitzer et al., 1993). Myeloa rchitecture has also been compar ed with CO staining, tracing methods, and microelectrode recording in th e dorsomedial visual area of owl monkeys (Krubitzer and Kaas, 1993) to re veal functional areas and conn ectivity. In monotremes, which share with the manatee the status of being an evolutionary outlier and having a unique environment to which they have had to adapt, Krubitzer et al. (1995) showed CO staining patterns that reveal somatosens ory specializations and suborganization. Mi croelectrode mapping

PAGE 26

26 was combined with CO and myelin staini ng revealed subdivisions and topography of somatosensory cortex. The neocortices of both the platypus and the short-b illed echidna revealed 4 representations of the body su rface with SI occupying a larg e area and containing neurons mainly responsive to cutaneous stimulation of the contralateral body. The platypus bill had a disproportionately large repr esentation with CO-dense re gions corresponding only to mechanosensory stimulation and CO-light re gions responding to bot h electrosensory and mechanosensory stimulation. In a compilation of cortical sensory maps of additional species, including the squirrel, macaque, and quoll, Krubit zer (1995) depicts homologi es that are present in neocortical organization. Ther efore, in lieu of performing electrophysiological studies on the manatee, a thorough histochemical assessment can still reveal a grea t deal about sensory specializations of the brain. Overall this analysis of the manatee somato sensory system, from an immunofluorescence analysis of innervation at the pe riphery to a systematic histoche mical examination of the central nervous system, aims to elucidate in what ways the manatee is a somatosensory specialist and how it has adapted to evolutionary pressures in herent in the environment that it occupies.

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27 CHAPTER 2 INNERVATION OF FOLLIC LE-SINUS COMPLEXES IN THE FLORIDA MANATEE Introduction Follicle-sinus complexes (FSCs, or vibrissae) form highly innervated tactile arrays generally found on a restricted region of th e mammalian body—principally the mystacial region. However, recent evidence suggest s that the Florida manatee ( Trichechus manatus latirostris ) possesses a sophisticated tactile sense through a system of FSCs distributed over the entire body (Reep et al., 2002). Manatees are large-bodied, obligate aquatic he rbivores (a trait unique among marine mammals) that lack predators, do not pur sue active prey, usually reside in a shallow, turbid water environment and have greatly reduc ed visual systems. They appear to have reasonably developed hearing ca pabilities (Gerstein and Gerstein, 1999; Mann et al., 2005) but reduced sight (Bauer et al., 2003) and though little is known about the extent of their olfactory or taste capabilities these senses also appear to be subordinate based on anatomical assessments (Levin and Pfeiffer, 2002; Mackay-Sim et al., 1985). The haptic sense may therefore be crucial in the manatee’s detection of environmental cues, and this hypothesis is supported by the distribution of sinus-type tactil e hairs over the entire body with specialized and more densely packed vibrissae on the face in addition to th e elaboration of somatosensory areas at the neuroanatomical level (Dexler, 1912; Welker et al., 1986; Johnson et al., 1986, 1987, 1994; Reep et al., 1989, 2001, 2002; Marshall and Reep, 1995; Sarko and Reep, 2007). The postfacial body is supplied with approximately 3,000 hairs, each having an independent field of movement, forming an arrangement unique to sirenia that is proposed to be analogous to the lateral line system in fish (Reep et al., 2002). Such a syst em is potentially capable of conveying crucial information about water currents, the approach of other animals, and other features of the

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28 underwater environment through hydrodynamic stim ulation of mechanoreceptors (Reep et al., 2002). Facial vibrissae are packed thirty times mo re densely than on the rest of the body, an attribute that should increase spa tial resolution, and they can be distinguished from postfacial vibrisse by their greater rigidity due to smaller length/diam eter ratios (Reep et al., 1998). The hair and bristles of the manatee face are composed of 9 distinct regions, 6 of which are perioral bristles (Fig. 2-1B): 4 upper pe rioral (U1–U4) fields on each side of the upper lips and oral cavity, and 2 lower perioral (L1–L2) fields on each side of the lower lip pad (Reep et al., 1998). The 9 follicle regions are distinguishable by loca tion, number, range of length/diameter ratios, and behavioral role (Reep et al., 2001). Each of these follicles can be classified as a follicle-sinus complex (FSC) because the follicl e and its affiliated dense inne rvation are surrounded by a blood sinus encased within a thick connective tissue capsule (Ri ce et al., 1986). Manatees have an expanded philtrum called the oral disk that contai ns bristle-like hairs (BLH s) that are the main tactile exploration component involved in object recognition (Reep et al., 1998). Postfacial vibrissae are located on the supradisk portion of the face posterior to the orofacial ridge and on the chin in addition to the en tire postfacial extent of the body (Bachteler and Dehnhardt, 1999; Reep et al., 1998). Perioral fiel ds U2 and L1 are used in a prehensile grasping fashion (“oripulation,” a behavior unique among mammals) during feeding as well as in social behaviors (Reep et al., 2001; Marshall et al., 1998b). The eyes are often closed durin g feeding and tactile exploration (Marshall et al., 1998b; Bachteler and Dehnhardt, 1999) further emphasizing haptic over visual input. Vibrissae are known to provide detailed textural inform ation about objects and surfaces in an animal’s immediate environment, and most mammals use vibrissae exclusively for sensory purposes such as finding prey and naviga ting successfully when vision is compromised,

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29 such as in low light situati ons (Brecht et al., 1997; Dehnhardt et al., 1998; Dehnhardt et al., 2001; Ling, 1977). In the manatee, facial vibrissae serv e dual and synergistic motor and sensory roles in manatee feeding and direct tactile exploration of the envi ronment (Marshall et al., 1998a, 1998b) with a high level of dexterit y and perioral tactile discrimina tion that is also reflected in the manatee’s relative tactile difference thres hold of 14%—comparable to that of an Asian elephant’s trunk (Bachteler and Deh nhardt, 1999). The prehensile func tion of facial vibrissae is present in dugongs as well but is absent in pinnipeds despite thei r higher tactile resolving power (Bachteler and Dehnhardt, 1999; Marshall et al., 1998b; 2003). In an earlier study of manatee follicle innervation the U2 fi elds were found to contain the largest FSCs having the longest hair shafts, the widest ring sinuses, the thickest capsules, and the highest degree of innervation at over 200 axons pe r follicle (Reep et al., 2001). L1 follicles are innervated by the second largest number of a xons at about 200 per FSC, followed by U3, U4 and L2 follicles (about 100) and finally U1 follicles, whose range overlaps that of the BLHs at 49– 74. The chin and supradisk follicles exhibit the le ast innervation, with a range of 34–48 (Reep et al., 2001; 2002). Although the manatee’s status as an endangered species precludes it from more invasive analysis, Reep et al. (2001) provided data de scribing general morphological features and axonal counts for each follicle type. However, si lver staining did not c onsistently reveal the morphology of nerve endings, a limitation solved here through systematic immunolabeling analysis using anti-PGP (protein gene product 9.5) as a standard pan-neuronal marker in combination with several other antigens in order to functionally characterize the innervation of manatee vibrissal FSCs. Given the varying behavi oral and sensory tasks for which each manatee bristle field is specialized, we w ould expect to reveal similarly va rying attributes in patterns of innervation, with facial vibrissae engaged in tactile behavior (the U2 and BLH follicles)

PAGE 30

30 exhibiting more densely distri buted and varied types of ne rve endings. Also, while the anatomical structure of FSCs remains relatively consistent across a wide range of species, patterns of innervation often vary considerably, presumably due to evolutionary pressures and concurrent behavioral demands (D ehnhardt et al., 1999; Ebara et al., 2002). As an evolutionary outlier, the Florida manatee offers a unique opp ortunity to better understand mammalian sensory systems in general by examining a system of vi brissae that has assumed an expanded functional role. A systematic analysis of manatee FSCs may also elucidat e their potential relationship to cortical cellular aggregates called Rindenkerne (Dexler, 1912; Reep et al., 1989; Marshall and Reep, 1995; Johnson et al., 1990; 199 4) that appear to be sim ilar to barrels found in the somatosensory cortex of other spec ies (Woolsey et al., 1975; Rice, 1995). Materials and Methods Manatees in Florida are endangered and protected under federal law. Postmortem manatee follicle samples were acquired through the statewide manatee salvage program under Federal Fish and Wildlife Permit PRT-684532 and IACUC protocol #C233. For each specimen, necropsy sheets summarizing body morphometrics, body weight, gender, likely cause of death, and condition upon recovery were obtained. Spec imens are outlined in Table 2-1 and included TM0406 (adult male, euthanized after watercraft impact; 3 BLH and 3 U2 follicles sampled), TM9728 (adult male, death due to watercraft; 3 rostrodorsal, 1 dorsocentral, and 1 dorsocaudal follicles sampled), TM0506 (male neonate, suffered multisystemic failure due to immune suppression secondary to cold st ress; 2 U2, 2 BLH, 2 L1, and 1 dorsocaudal body sampled), and MNW0614 (subadult female, death due to watercraf t; 3 follicles from each of 10 body regions of interest sampled). Hair follicles samples were acquired as ava ilable from 6 body regions (Fig. 21A; supradisk, dorsocentral midline, rostrodors al midline, caudodorsal midline, ventrocentral midline, dorsal tail, and tail edge) as well as from perioral fields L1, U2, and BLH (Fig. 2-1B) as

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31 described by Reep et al. (1998) using a #11 scalpel blade to ex tract a block of tissue (roughly 5x5x15 mm) surrounding the follicle of interest. Follicles were cut mediolongitudinally to facilitate fixation and placed in 4% paraform aldehyde overnight. After 24 hours of fixation follicles were removed and placed in 0.1M phospha te buffered saline (PBS) and 30% sucrose. Sections were cut using a cryostat. Sections fo r conventional epifluoresce nce evaluation were cut at 14 m parallel to the long axis of the follicles. These sections we re directly thawed onto slides subbed with chrome-alum gelatin, allowed to air dry, and immunolabeled on the slides. Follicles for confocal analysis were cut at 75 m a nd the sections were immunolabeled free-floating before being mounted onto slides. After labeling, the slides were coverslipped using either 90% glycerin in PBS or Vectashi eld (Vector Laboratories). The sections were processed for single and double immunolabeling with the following primary antibodies: 1. Anti-protein gene product 9.5 (PGP, rabbit polyc lonal, 1:800; UltraClone, Isle of Wright, UK; catalog number RA95101). The antigen wa s human PGP9.5 protei n purified from pathogen-free human brain. The antibody show s one band at 26-28 kD on Western blot and is a universal neuronal cytoplasmic prot ein (Thompson et al., 1983; Wilkinson et al., 1989). 2. Anti-neurofilament 200 kD subunit (NF, rabbit polyclonal, 1:800; Chemicon International, Temecula, CA; catalog number AB1982, lot number 24080051). The antigen was a highly purified bovine neurofilament polypeptide. The antibody labels phosphorylated and nonphosphorylated 200kD NF and shows a ba nd at 200kD and bands around 170-180 kD on Western blot. The NF200 antibody identifies myelinated innervation including Merkel endings, A and A fibers (Rice et al., 1997). 3. Anti-calcitonin gene related peptide (CGRP, guinea pig polyclonal, 1:400; Peninsula Laboratories, Inc., San Carlos, CA; catalog number T-5027, lot number 061121). The antigen is human -CGRP with the following sequen ce: H-Ala-Cys-Asp-Thr-Ala-ThrCys-Val-Thr-His-Arg-Leu-AlaGly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-AsnPhe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Al aPhe-NH2. The antibody has 100% reactivity with human and rat -CGRP, human CGRP (8-37); chicken CGRP, human -CGRP. It has 0.04% cross reactivity with human amylin and 0% cross reactivity with rat amylin and with human and rat calcitonin. The CGRP antibody identifies peptidergic C-fiber innervation and Merkel cells (Rice et al., 1997) and is an endogenous sensory neuropeptide and a G-protein coupled receptor.

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32 4. Anti-S-100 (anti-Schwann cell protein S100, rabb it polyclonal, used neat; Biogenesis Inc., Brentwood, NH, catalog number 8200-0184, lot nu mber A2255). The antigen was purified bovine S100 protein. Anti-S100 has been found to be coextensive with axons, terminal arbors, and mechanoreceptor endings (Rice et al., 1997). 5. Anti-BNaC1 (mammalian brain sodium channel BN aC; rabbit polyclonal; 1:500; gift from Dr. Jaime Garca-Aoveros). Th e antigen was N-terminus peptide MDLKESPSEGSLQPSSC (corresponding to resi dues 1-16 of mouse, rat, and human BNaC1 ). The BNaC antibody has been shown to identify low-threshold mechanoreceptors (Garcia-Anoveros et al., 2001). Primary antibodies against MBP (myelin ba sic protein), VR1 or TrpV1 (vanilloid receptor 1; capsacin binder), NPY (neuropeptid e Y, labeling sympathetic innervation), TH (tyrosine hydroxylase), and GAP43 (growth-associated protein 43, marker for neural growth), used successfully in previous rat, monkey and hum an studies (Albrecht et al., 2006; Fundin et al., 1997; Par et al., 2001) did not produce dete ctable labeling on manatee tissue. All 14 m thick sections were first preincubate d with 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in 0.1M PBS for 1 hour, then incubated with a solution of primary antibody (diluted in PBS with 4% calf serum or 1% BSA and 0.3% Trit on X-100) overnight at 4 C at high humidity. Slides were then rinsed in PBS for 30 minut es and subsequently incubated in the dark at room temperature for 2 hours w ith either Cy3 or Alexa488 for red fluorescence (1:500) or Cy2 for green fluorescence (1:250) conjugated secondary an tibodies (Molecular Probes, Inc., Eugene, OR; Jackson Immunoresearch Laboratories, Inc., We st Grove, PA) diluted in PBS or BSA with 0.3% Triton X-100. Slides were then rinsed in PBS and either temporarily coverslipped under PBS (in the case of future doubl e labeling) or permanently coverslipped. Double labeling was usually accomplished by repeating the immunofluorescence procedure described above. In some cases double labe ling was achieved through a single cycle of incubations beginning with a 1:1 mix of the m onoclonal and polyclonal primary antibodies. To control for non-specific labeling, incubation with primary antibody was omitted or the primary

PAGE 33

33 antibody was preincubated with a specific blocking peptide. The 75 m thick sections were processed free floating in the same dilutions of antibodies as the thinne r sections. Incubations were for 4 days in primary antibodies a nd overnight in secondary antibodies at 4 C. Rinses were for at least 4 hours. Sections were analyzed with an Olym pus Provis AX70 microscope equipped with conventional fluorescence: 1) Cy3 filters (528553 nm excitation, 590-650 nm emission) and 2) Cy2 filters (460-500 nm excitation, 510-560 nm emission). Fluorescence images were captured with a high resolution (1280 x 1024 pixels) th ree chip color CCD camera (Sony, DKC-ST5) interfaced with Northern Eclipse software (Emp ix Imaging, Inc., Mississauga, ON). Images were deblurred using a deconvolution program based on a 1 m 2-dimensional nearest neighbor paradigm (Empix Imaging, Inc., Mississauga, ON). Samples were imaged on a Zeiss LSM 510Meta confocal microscope (Carl Zeiss Mi coImaging, Inc., Thornwood, NY) equipped with an Argon (488 nm exc.) and a green HeNe (543 nm exc.) laser. Emissions were collected using a Band Pass 500-530 nm emission filter for Alexa Fluor 488. For CY3 either a Long Pass 560 nm emission filter or a Band Pass 5650-615 nm em ission filter was used, depending on whether the sample was singly or doubly labeled. Images were collected with a Plan-Neofluor 25x/0.8 Imm corr DIC lens with the pinhole set for 1 Airy Un it. Confocal image Z-stacks were collected at 512 x 512 pixel x-y resolution and 1 m steps in Z. The 3-D red-green stereo anaglyph (Fig. 29B) and the 3-D stereo pairs (Fig. 2-9, A, D, E) were generated using th e Zeiss LM510 software. The 3-D surface rendered images (Fig. 2-8, A-H) were produced using the VolumeJ plugin in the ImageJ software software ( http://rsb.info.nih.gov/ij/ ). Figures were assembled using Adobe Photoshop CS, Adobe Illustrator CS, a nd Microsoft PowerPoint software.

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34 Since the intensity of immunolabeling for th e numerous antibodies used in the present study is attributed to many variables that cannot be individually quantif ied, this study does not attempt to quantify the relative amounts of labe led antigens. These variables include: 1) true differences in the presence and quantity of the antigen, 2) whether the antibody is monoclonal or polyclonal, 3) background labeli ng, 4) antibody concentration, 5) efficacy of the antibody, and 6) location of the antigen (i.e. membrane or cyto sol). Because the labeling intensities differed between the various types of an tibodies, the photomicrographs comp iled for illustrative purposes were adjusted using Northern Eclipse, Adobe Photoshop CS (San Jose, CA), and Microsoft Powerpoint (Redmond, WA) software so that the maximum labeling contrast and intensity were similar for each antibody. Results Facial Vibrissae The basic structure of the U2 follicle was ex amined first due to its size, behavioral significance, and substantial innervation (Reep et al., 2001; 2002). The U2 displayed a pronounced epidermal invagination at the mouth of the FSC before the beginning of the capsule and the follicle proper (Fig. 2-2; Fig. 2-3A). Dermal papillae projecting into the epidermal surface were laden with fine-caliber presumptive C fibers (Fig. 2-3B) that co-labeled for antiPGP and anti-CGRP (Fig. 2-7A), as well as small-caliber presumptive A fibers that co-labeled for anti-PGP and anti-NF200 (Fig. 2-7B) at the level of the rete ridge collar (RRC), but no Merkel cells were observed in the RRC or adjacent epidermis. A small distribution of presumptive Pacinian corpuscles was also obser ved just below the epidermis (Fig. 2-7M). A narrow, short outer conical body (OCB) was also present and a circumfere ntial array of fine caliber fibers with presumptive free nerve endi ngs (FNEs) was evident at the inner conical body (ICB) level (Fig. 2-3C) and was PGP-positive an d with minimal NF-positive innervation (Fig. 2-

PAGE 35

35 7C). No transverse lanceolate e ndings were observed. Also present at the lower extent of the ICB region and the upper extent of the ring sinus was a high distribution of “t angle” nerve endings (Fig. 2-3D), novel endings observed in this study that appear mor phologically similar to reticular endings generally seen in othe r taxa along the mesenchymal sheat h at the upper extent of the trabeculated cavernous sinus (CS). These large nerve endings were supplied by large caliber presumptive A or A fibers and were positive for PGP, S100 and NF200 as well as for BNaC (Fig. 2-7H, I) which classifies them as low th reshold mechanoreceptors responsive to mechanical pressure. A subregion of each ending was also CGRP-positive (Fig. 2-7J). Confocal imaging revealed an intricate mesh of NF-positive fibe rs interspersed among DAPI (4',6-diamidino-2phenylindole)-positive nuclei all within a PGPpositive cytoplasmic ending (Fig. 2-8D, F; Fig. 29A, B). Proceeding to the ring sinus (RS) level, a dense distribution of Merkel cells (MCs) was present in the outer root sheath (Fig. 2-3E). The MCs formed a ci rcumferential array with some branching, but individual MCs wit hout visible innervation from bran ches of the deep vibrissal nerve (DVN) predominated. When present, innervation was supplied by large caliber, presumably A or A fibers. Widely spaced longitudinal lanceolate endings (LLEs) were present but did not form a dense pa lisade as in other species. Each LLE appeared to have a single associated terminal glia, although this was not examined in detail. The majority of LLEs appeared unbranched (Fig. 2-8C) and in seve ral morphologies: a studded blade form; a smooth blade; and a curved hook ending (Fig. 2-3E). The lanceolate endings were supplied by the DVN to the mesenchymal sheath of the RS by larger caliber afferents presumably of A or A classification. Clublike endings we re also found at the RS leve l in close proximity to the rudimentary ringwulst along the mesenchymal sheat h. In the region of the cavernous sinus FNEs were observed in addition to another type of novel nerve ending discove red along the trabeculae

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36 (Fig. 2-4A, E; Fig. 2-8A). The latter stained positively for PGP, S100 and NF200 as well as for BNaC, identifying it too as a lo w threshold mechanoreceptor (Fig. 2-7E, F; Fig. 2-9C, E). Low CGRP activity was also detected (Fig. 2-7G). Re presentative endings from each of the two novel ending groups were reconstructe d using confocal imaging to confirm the three dimensional structural morphology and to ensure that the unusu al structure was not simply a result of an aberrant plane of section through the FSC. No Ruffini or reticula r endings were observed. Within the medulla of the hair shaft, an extensive netw ork of fine caliber fibe rs labeled intensely for anti-PGP and with minimal NF-positive innervat ion present (Fig. 2-4B; Fig. 2-7K). The L1 vibrissae were also examined and found to be structurally similar to U2 vibrissae. Deep epidermal papillae filled with fine caliber fi bers were present at the RRC level along with “tangle” endings at the lower ICB/upper RS level. Single-blade termination LLEs and predominantly uninnervated MCs were also pres ent at the RS level. Peptidergic and nonpeptidergic C fibers sparsely innervated the tr abeculae and interior cap sule of the CS. Novel trabecular endings and extensive FNEs were visible within the CS and notably extensive peptidergic C-fiber innervation was present within the hair shaft medulla as seen in U2 vibrissae (Fig. 2-4C). Bristle-like hairs (BLHs; Fig. 22) from the oral disk region were examined next due to their involvement in tactile exploration and objec t recognition. Merkel e ndings and fine caliber fibers were present at the RRC and epidermal level but the prominent epidermal invagination leading to the follicle proper in U2 and L1 vibr issae was absent in BLHs (Fig. 2-5A). A sparse distribution of presumptive Meissner ’s corpuscles was also observed at this level (Fig. 2-7L). A short OCB proceeded to a highly vascularized ICB region (Fig. 25E) with a dense distribution of “tangle” endings at the lower ICB/upper RS level along the mesenchymal sheath (Fig. 2-5C,

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37 D; Fig. 2-8G). Densely distributed Merkel cells, mainly without vi sible innervation, were present at the RS level along with widely spaced single -blade LLEs and both were innervated by large caliber fibers branching from th e DVN (Fig. 2-5A, D; Fig. 2-8E). Clublike endings were present in the ringwulst region (Fig. 25B). Novel trabecular endings were present along the connective tissue of the CS (Fig. 2-4F) along with FNEs but the medulla of the hair shaft lacked the substantial small caliber fiber innervation seen in the U2 and L1 vibrissae (Fig. 2-5F). Supradisk follicles thought to be morphologically similar to postfacial FSCs (Reep et al., 2001) possessed attributes corresponding to those of the BLH vibrissae including th e presence of “tangle” endings within the upper RS, the presence of novel trabec ular endings (Fig. 2-4D), and the absence of extensive innervation of the hair shaft medu lla, but with the exception of having a wellinnervated Merkel network at the RS level (Fig 2-9D) that was not seen in the BLH follicles. Postfacial Vibrissae Postfacial follicle i nnervation was characterized in 6 body regions (Fig. 2-1A; Fig. 2-2): along the dorsal midline (including rostral, central, and caudal samp les), at the ventral midline, and on the tail (dorsocentral and lateral edge). Fi ne caliber presumptive C fibers (PGP+/CGRP+) as well as small caliber presumptive A fibers (PGP+/NF200+) were found to form extensive arrays projecting into the epidermis of the RRC (Fig. 2-6B, C). Merkel endings were present within the RRC at the base of the epidermis (Fig. 2-6B). At the RS level a small distribution of “tangle” endings (Fig. 2-6D-I; Fig. 2-8H) and si ngle-blade termination LLEs (Fig. 2-6F) were observed along with an extensive network of MEs that was particularly pronounced in the dorsocentral FSCs (Fig. 2-6F; Fig. 2-8B). In contrast to the perioral vibrissae examined, the majority of MCs at the RS level of postfacial vi brissae appeared to be innervated (Fig. 2-6A, FI). In accordance with the facial vibrissae, no Ruffini or reticul ar endings were observed in the postfacial vibrissae. The novel endi ngs present in the trab eculae of the facial follicles were also

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38 notably absent in the postfacial vibrissae examined and t hough presumptive FNEs were visible within the CS, no pronounced innervation was presen t within the medulla of the hair papilla. Discussion Manatee Vibrissae: Overall Comparative Structure The facial and postfacial vibr issae of manatees emanate fr om encapsulated blood-filled sinus complexes (Reep et al., 2002) making them true vibrissae (Rice et al., 1986). The FSCs are relatively short compared to the length and caliber of the hairs (Reep et al., 1998). The facial hairs are keratinized and unusually rigid, including the region clos e to the hair papilla. In contrast, rat and cat vibrissae are soft near the hair papilla and the deep half of the CS and gradually become more rigid near the upper end of the lower CS (Ebara et al., 2002). Vibrissae FSCs in smaller mammalian species also genera lly exhibit blood-filled spaces along the upper extent of the ring sinus but lack well-defined trabeculae, whereas the manatee exhibits welldeveloped trabeculae at the upper RS level wher e the mesenchymal sheath expands to form the ICB. The neck of the manatee FSC at the level of the OCB and ICB regions is very long and may contribute to the facial vibrissae being rigidly maintained within the FSC. In smaller species it is likely that the deep end of the vibrissa is more flexible, with the smaller neck of the FSC acting as a fulcrum against which the hair shaft can leve r within the FSC. As suc h, the trabeculae of the CS in smaller species are likely to function more in lateral stabilization than in the manatee, where the hair shaft is rigidly anchored at the base and neck of the FSC. The attenuated ringwulst of manatee vibrissae extends rigidly from th e mesenchymal sheath rather than hanging down from its point of attachment as seen in othe r species. Dense peptidergic and non-peptidergic Cfiber innervation was also intimately wrapped around the outer surface of the FSC capsule, particularly along the upper half of the capsule. This may be presen t in other species as well, but to a lesser extent, and has not been fully investigated.

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39 Facial Musculature Involved in Explorat ory and Prehensile Vibrissal Behaviors Previous experiments have shown that infraorbital branches of the maxillary nerve insert into the upper bristle pad whereas the inferior alveolar branch of the mandibular nerve supplies the vibrissae of the lower pad of the manatee f ace. The dorsal and ventral buccal branches of the facial nerve supply the s uperficial facial musculature and ar e likely to contribute to bristle eversion and feeding behavior movements (Reep et al., 1998). The U2 follicles are specifically associated with the M. levator nasolabialis muscles (Marshall et al., 1998b), making them homologous to mystacial vibrissae, and although i ndividual follicles within a U2 field are not moved independently, the left and right U2 fields can act independently from each other (Marshall et al., 1998a). The L1 fo llicles are supplied by mental bran ches of the inferior alveolar nerve (Reep et al., 1998) and are protruded by me ntalis muscle contract ion (Marshall et al., 1998b), making them homologous to mental vibrissae. At rest the U2 vi brissae are retracted within skin folds and are everted by volu me displacement through contraction of the M. levator nasolabialis and the circular M. buccinatorius muscles during manipula tive behaviors (Marshall et al., 1998a; b). When presented with a novel obj ect, manatees generally touch the object with the oral disk (involving the BLH follicles) first in a side-to-side sweeping motion and then grasp with the U2 follicles, but the BLH follicles are not actively moved (Marshall et at., 1998a). Sensory Innervation of the Rete Ridge Collar and Epidermis A high density of thin, tapering dermal papilla e curve toward the vibrissae at the mouth of the FSC and penetrate throughout an extremel y thick epidermis (Fig. 2-2). Other species generally lack papillae in non-glabrous skin and exhibit a relatively thin epidermis. Numerous peptidergic and non-peptidergic C fibers enter th e papillae and extend in a straight, unbranched manner far into the overlying epidermis and perpe ndicular to its surface. Little innervation was present between the papillae, but the papillae were very closely spaced, resulting in a high

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40 density of innervation to the epidermis. Thin-calib er NF-positive fibers al so penetrate into most papillae and appear to branch, i ndicating that these fibers may serve as mechanoreceptors of the papillae, but NF labeling was rarely seen on endings penetrating the epidermis. Occasional clusters of Merkel cells and inne rvation are located at the base of the epidermis between papillae, and these appear to be widely spaced over the epidermis. Throughout the upper dermis and particularly at the RRC extends a dense vascular network that is well-innervated with dense sympathetic innervation and, to a somewhat le sser extent, CGRP-positive sensory innervation. Thin NF-positive innervation was also present. Thick-walled, especially well-innervated locations appeared to be arteriovenous shunts. While we di d not fully characterize innervation associated with the vascular supply in the manat ee, there appears to be an extensive network for regulating blood flow to the epidermis, potentia lly representing a thermal regulatory mechanism (Fig. 2-7N). Occasional Pacinian-l ike corpuscles were also seen and appeared similar to those present among arterial networks in the glabrous skin of monkey s (Fig. 2-7M; Par et al., 2002). However, these were at a surprisingly superficia l location at the base of the epidermis in the manatee, which may be related to the manat ees’ extensive superficial vascular network. Sensory Nerve Endings of the Inner Conical Body and Ring Sinus Merkel endings are thought to be low th reshold, slowly adapting mechanoreceptors capable of detecting compression stimuli and directionality (Iggo, 1963, 1966; Iggo and Muir, 1969; Johansson et al., 1982a,b; Johansson and Vallbo, 1983; Munger et al ., 1971; Gottschaldt et al., 1973; Rice et al., 1986; Lichtens tein et al., 1990). Given the de nse distribution of MEs in the outer root sheath of both facial and postfacial vibrissae it seem s that manatee FSCs are heavily invested in detecting directiona lity of hair deflection (Burgess and Perl, 1973; Rice et al., 1986), and a commitment of nerve endings to this task would support our propos al that manatees use tactile hairs to detect hydrodynamic stimuli in a manner analogous to th e lateral line system

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41 present in fish (Reep et al., 2002). In microchi ropteran bats, “touch domes” along the wings are heavily invested with Merk el cells and FNEs that appear to de tect air flow and aid in navigation and maneuvering (Zook, in press; Zook, 2005; Z ook and Fowler, 1986) in much the same way that manatee postfacial vibrissae might perceive water flow. Merkel endings in the manatee were found along the RS and ICB regions (facial and po stfacial vibrissae) as well as in the RRC (postfacial vibrissae and BLHs only). The presence of the same receptor at different locations along the follicle axis may indicate that the MEs are involved in ex tracting different features of a stimulus at these positions. At the RS level, MEs are situated in the external root sheath between the inner root sheath and the glassy membrane, a location that makes them susceptible to smallangle deflections of the follicle (Gottschaldt et al., 1973; Rice et al., 1986) whereas MEs of the RRC are in a location that presumab ly lends itself to detection of large-angle deflections of a vibrissa (Rice et al., 1986). By extension, the postfacial vibrissae and BLHs of the Florida manatee appear to be specialized for the comple te range of deflection intensities, due to the presence of MEs at both the RRC and RS levels, wh ereas perioral facial vibrissae may be more receptive to small-angle hair deflections, due to having MEs at the RS level only. While the significance of most MCs at the RS level of facial vibrissae lack ing visible innervation remains uncertain, it is possible that th ese MCs experience a high turnover rate. The presence of clublike endings at the attachment site of the ringwulst indicates that this region is sensitive to mechanical perturbations as well. Lanceolate endings are thought to be low threshold, rapidly adapting stretch receptors that encode dynamic properties of vibrissal de flection such as acceleration and deceleration (Burgess and Perl, 1973; Gottschaldt et al., 1973; Tuckett, 1978; Tu ckett et al., 1978; Rice et al., 1986, 1997; Lichtenstein et al., 1990 ). Whereas the majority of the longitudinal lanceolate

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42 afferents gave rise to a single blad e-like termination at the RS leve l in the U2 facial vibrissae, a subset of LLEs exhibited a forked terminati on or a morphological vari ant including the studded and hook endings observed. A curved “shepard’s crook” morphology was also observed in LLEs along the mesenchymal sheath at the RS level of th e cat and guinea pig (Ri ce et al., 1986). It is possible that the morphological variants of the LLEs have common inherent physiological properties but may transduce slightly different aspects of mechanosensory perception. The relatively wide spacing and low dens ity of distribution of the LLEs in the mesenchymal sheath of all follicles examined suggest that velocity detection is of lesser importance in both the facial and postfacial vibrissae. Circumferentially orient ed peptidergic and non-peptidergic FNEs were found in the ICB and OCB regions. The density of distribution was far less than the wellorganized, dense circumferential bundl es seen in the ICB of rats and mice, and to a lesser degree in cats. These fibers are thought unlikely to conf er linear or spa tial directionali ty given their circumferential orientation, and the absence of tr ansverse lanceolate e ndings (TLEs) supports the hypothesis that TLEs are related to whisking behavior and generally seen only in species such as hamsters, mice, rats and gerbils that utilize this behavior to explore the environment (Rice et al., 1986). Merkel cell-neurite complexes and lanceolate en dings appear to be responsive to a wide frequency range and may be used to detect sounds when a vibrissa is deflected at the proper frequency (Gottschaldt and Va hle-Hinz, 1981; Hyvrinen 1989, 1995; Stephens et al., 1973), a capability that would support the hypothesis of extensive overlap between auditory and somatosensory areas of manatee cerebral cort ex (Sarko and Reep, 2007). In fact, primary auditory cortex appears to be occupied excl usively by cluster cortex areas that feature Rindenkerne, or “cortical nuclei” located in layer VI and thought to be analogous to barrels seen

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43 in a variety of species and potentially repres entative of individual vi brissae (Dexler 1912; Johnson et al., 1994; Marshall and Reep, 1995; Reep et al., 1989; Rice, 1995). Furthermore, a behavioral study that assessed the underwater au diogram of the West Indian manatee found that one manatee adjusted its responses to lowfrequency (<0.4 kHz) s ounds by pivoting its body roughly 45 degrees and lowering it s head (a response not exhibi ted for higher frequencies), which potentially indicates adjustment of percep tual focus from sound to vibrotactile stimuli (Gerstein and Gerstein, 1999). The “tangle” endings observed at the lower inner conical body/upper ri ng sinus level, and present in all manatee vibrissae examined here, appear to be novel because we are unaware of sensory endings of this morphology and immunologi cal characterization observed at this level in the vibrissae of any other species “Tangle” endings consisted of two or more exceptionally large endings abutting the basement me mbrane and supplied by a large A fiber. Each ending consisted of thick tangles of NF+ processes em bedded in a matrix of PGP-positive cytoplasm and S100-positive terminal glia. Th e endings are concentrated in the mesenchymal sheath at the level of the upper ring sinus trabeculae and ma y be involved in dir ectionality detection associated with deflection of th e hair shaft against the upper trab eculae. These endings are also BNaC-positive and therefore are likely low-threshold mechanoreceptors. Cavernous Sinus Innervation The medulla of the hair papilla extends to an extremely superficial location, well into the neck of the FSC, in U2 and L1 facial vibrissa e. Cats also exhibit a superficially extending medulla, but the interface between the medulla a nd the cortex is smooth whereas in manatees it has a jagged appear ance (Ebara et al., 2002). Manatee U2 a nd L1 vibrissae also have extensive peptidergic and non-peptidergic Cfiber innervation within the me dulla. The FNEs present within the hair shaft medulla and spanning the trabeculae of the CS have been implicated in pain and

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44 temperature sensation (Rice et al., 1986). Altern atively, the FNEs found w ithin the medulla of the hair papillae of U2 and L1 vibrissae may be analogous to dentinal tubule innervation. Given the rigidity of manatee facial fo llicles (particularly the perioral fields) compared to the flexible and easily displaced hair follicles of most mammals it is possible that sensory innervation is committed to stress detection and load applicati on in order to assess force transmission without actual material displacement as seen in the de ntal sensory receptors of tooth pulp (Byers, 1984; Byers and Nri, 1999). This innervation may al so be a sensory adaptation to oripulative behaviors. In another marine ma mmal sensory specialist, the narwhal, dentinal tubules within the unusual tusk are thought to func tion as a hydrodynamic sensor detecting fluid flow, salinity gradients, temperature and pr essure (Nweeia et al., 2005). The absence of reticular and Ruffini endings along the basement membrane in manatee vibrissae is unusual, as is the pr esence of novel endings within the tr abeculae of facial vibrissae. Ruffini endings are affiliated w ith collagen bundles and appear to function as tension receptors residing along the mesenchymal sheath (Rice et al., 1986; Zelena, 1994) whereas reticular endings terminate in the upper third of the CS against the glassy membrane and may be directionally sensitive (Ebara et al., 2002). Spiny and encap sulated endings (previously thought to be Ruffini endings; Rice et al., 1997) were also found at the lower CS level of the rat and cat, and Ebara et al. (2002) speculate d that the cumulative CS innerv ation is responsive to tension generated by the trabeculae during follicle deflection. The nerve endings observed within the tr abeculae of the lower cavernous sinus and present only in the facial vibrissae examined he re (U2, L1, BLH and supradisk follicles) appear to be novel in that they are embedded within th e trabecular matrix rather than against the basement membrane. These endings were supplied by a relatively small-caliber A fiber with

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45 one axon innervating single or multiple endings. I ndividual endings consisted of fine-caliber tangles embedded within a terminal glial matrix and were BNaC-positive, indicating that they too are low threshold mechanoreceptors. It is possible that these trabecular endings are responsive to tension induced by deflection of exceptionally rigi d vibrissae (Reep et al., 1998) which might involve modified sensory endings in order to optimally detect deflection. Alternatively, it has been proposed that the tr abeculae of the CS may function in attenuating vascular pulsations in the arteri al supply entering the base of the FSC, thereby creating more uniform blood flow at the RS level (Melara gno and Montagna, 1953; Rice et al., 1986). Novel endings within the trabeculae may have evolved to provide additional sensitivity in monitoring vascular supply at this level. Marine Mammal Vibrissae Vibrissal sensory nerve endings of a limited number of other marine mammals have also been studied. Hyvrinen (1995) examined the ex ceptionally well-innervat ed mystacial vibrissae of the ringed seal ( Phoca hispida ) using histology and electron microscopy. The length of the upper cavernous sinus (UCS) accounts for 60% of the vibrissa, situating the ring sinus at a low level compared to most mammals. At the RS level, LLEs were found abutting the glassy membrane while MEs were found below the glassy membrane and a prominent ringwulst was present (Hyvrinen and Katajist o, 1984; Hyvrinen, 1989). All MCs appeared innervated and formed a well-developed network (Hyvrinen, 1995) Hyvrinen (1995) also described numerous encapsulated end-organs in the lower CS situ ated within the trabec ulae and morphologically similar to “Ruffin’s corpuscles.” These may be si milar to the nerve endings reported here for the manatee, but documentation that would allow fo r a direct comparison was absent. Dykes (1975) classified and compared the affere nt fibers serving cat and harbor seal vibrissae. He found that a the majority of harbor seal vibrissae respond to vibr ations and that the majority of afferent fibers

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46 from the infraorbital nerve (85% versus 66%) serve harbor seal vibrissae. Approximately twothirds of these fibers were rapidly adapting a nd the remainder were slowly adapting, and most fibers (71% in harbor seals and 75% in cats) were directio nally sensitive. Bearded seal ( Erignathus barbatus ) FSCs have also been characterized and are thought to be extensively innervated active-touch systems adapted to benthic foraging (Marsh all et al., 2006). The mystacial vibrissae have an extensive UCS comparab le to that of the ringed seal but unlike the facial follicles of the manatee wh ere the UCS is minimal. A promin ent ringwulst is present at the RS level along with an extens ive ME network and LLEs. Merkel innervation predominates but was not observed at the RRC level as was obs erved for the manatee BLHs and postfacial vibrissae. Comparative Considerations Through studying a range of sp ecies (including opossums, rodents and pinnipeds) for functional variation and unifying prin ciples of vibrissae, Brecht et al. (1997) found that mystacial macrovibrissae tend to form rows in which eff ective whisker length increases exponentially in the caudal direction, with each row operating as a functional unit by sampling highly overlapping spatial information perpendicular to the rostrocauda l axis. At the cortical level there also appears to be preferential connectivity between barr els within a row (Simons, 1978; Simons and Woolsey, 1979). Macrovibrissae were proposed to func tion in spatial orientat ion associated with distance detection and object location while mi crovibrissae appeared optimized for object recognition (Brecht et al., 1997). By extension, the postfacial vibris sae of the manatee appear to have adopted a function analogous to that of m acrovibrissae through optimiz ation for spatial and directional sensitivity while the or al disk and the BLHs in particul ar serve the microvibrissal role of direct tactile obj ect recognition. Instead of the direct surface contact stimulation that macrovibrissae generally receive, manatee postfacial follicles are exposed to perturbations of the

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47 water and consequently undergo passive deflect ion. It has been suggested that mammalian vibrissae may serve a complementary auditory function at low fre quencies (Gerstein and Gerstein, 1999; Griffin, 1958; Mahler and Ham ilton, 1966; Reep et al., 2002; Yohro, 1977). The perioral vibrissae appear to be more adapted to locating and recognizing food, an important task for a strict herbivore that spends 6-8 hours per day looking for food and that must eat the equivalent of approximately 10% of its body wei ght per day to compensate for a low metabolic rate (McNab, 1978; 1980).

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48 Table 2-1. Specimen categorization. Specimen Sex Length (in cm) Weight (in kg) ClassificationCause of Death TM0406 M 290 393 Adult Watercraft TM9728 M 295 500 Adult Watercraft TM0506 M 172 134 Calf Cold stress MNW0614 F 238 326 Subadult Watercraft Figure 2-1. Vibrissae sampling regions of the body and face. A) Postfacial body regions of interest include the tail (lateral edge and dorsomedial areas), ve ntromedial area, and rostral, central, and caudal areas of the dor sal midline. The supradisk region (asterisk) is caudal to the orofacial ri dge and also thought to cons ist of body vibrissae. B) Frontal view of the manatee face with cheek muscles cut to reveal perioral follicle fields. Facial follicles of interest include the bristle-like hairs (BLHs), U2 and L1 follicle fields due to their behavioral significance.

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49 Figure 2-2. Schematic drawing of the structure a nd innervation of the U2, BLH, and postfacial vibrissal follicle-sinus complexes (FSCs) with innervation types and sensory nerve endings illustrated (RRC=rete ridge co llar, OCB=outer conical body, ICB=inner conical body, BM=basement membrane, RS=r ing sinus, DVN=deep vibrissal nerve, HP=hair papilla). The relative scales of each FSC are accurate, but innervation is presented for illustrative purposes only (see Fig. 3-9 for accurate scale representations). Overall morphology: The th ickness of the capsule and the diameter of the vibrissa decreases progressively from the U2 to the postfacial FSC. The facial vibrissae also exhibited dua l innervation from the DVN at the base of the follicle whereas the DVN entered as a single bundl e of axons in postfacial vibrissae. Presumptive sympathetic fiber innervati on is also depicted based on general characteristics in other mammals. Epider mis and RRC: The epidermis of the U2, BLH and postfacial vibrissae contains superficially projecting dermal papillae within which are fine-caliber A and C fibers. The base of the epidermis of BLH and postfacial FSCs includes Merkel endings. Th e U2 FSC has a particularly pronounced invagination of the RRC. OCB and ICB: Th e U2 FSC exhibits a dense network of circumferential free nerve endings while th e BLH and postfacial FSCs exhibit only fine-caliber and sympathetic innervation. Novel “tangle” endings are located in the facial and postfacial vibrissae. RS: Dense Merkel networks were present in vibrissae from each body region, but Merkel cells lacki ng visible innervation predominated in facial vibrissae. Less densel y distributed longitudinal lan ceolate endings were also observed at this level for each vibrissal t ype and clublike endings were observed in close association with the rudimentary ri ngwulst in facial vibrissae. CS: The trabeculae in FSCs from each body region cont ained fine-caliber innervation with presumptive free nerve endings. The facial vibrissae only included novel endings that spanned the trabeculae. Ruffini and reticu lar endings were notably absent. HP: Dense fine-caliber innervation was present in the rigid U2 follicle along a medulla that extended to an extremely superficial extent. This innervation was very sparse in the BLH and postfacial vibriss ae, and the medulla of each extended less superficially.

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50

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51 Figure 2-3. Characterization of upper perioral field 2 (U2) follicle innervation. A) A longitudinal U2 section stained for PGP is shown just off the medial axis to reveal various innervation characteristics as well as th e deep invagination of the RRC (specimen TM0406). B) Magnified RRC and epidermal region further off the medial axis showing a dense distribution of Cand A fiber projections (arrow) within dermal evaginations (medial is right). C) Magnifi cation of circumferential FNEs at the ICB level (same plane of section as A). D) Cl ose-up of a representative “tangle” ending including 3 branched mechanoreceptors a nd a Schwaan cell (arrows and arrowhead, respectively) within the lower ICB regi on along the mesenchymal sheath (plane of section along the medial axis). E) A dens e distribution of Merk el cells (arrow) and representative types of LLEs (arrowheads) including a bifurcated ending, hooked formation, and studded blade-like termination (same plane of section as A). RRC, rete ridge collar; OCB, outer c onical body; ICB, inner coni cal body; RS, ring sinus; CS, cavernous sinus; HS, hair shaft. Scale bar = 1mm for A-B, 250m for C-E.

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52

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53 Figure 2-4. Innervation of the cav ernous sinus and hair shaft me dulla in facial follicles. A) Extensive innervation of the trabeculated cavernous sinus of a U2 follicle (specimen TM0406) includes novel endings (arrows) and fine-caliber fibers along with the DVN continuing to the ring sinus. B-C) A dens e network of small caliber axons and presumptive FNEs proceeding to a remarkably superficial extent in the medulla of a U2 follicle (B; specimen TM0406) and an L1 follicle (C; specimen MNW0614). Magnified views show details of novel trabecu lar endings seen in a supradisk follicle (D; specimen MNW0614, arrow) a U2 fo llicle (E; specimen TM0406, arrow) and a BLH follicle (F; specimen TM0406, arrow) Scale bar = 600m (A), 1mm (B-C), 300m (D-F).

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54

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55 Figure 2-5. Innervation present in bristle-like hairs (BLHs). A) A longitudinal section of a BLH just off the medial axis stained for pr otein gene product 9.5 re veals characteristic innervation, including “tangle endings” at the lower ICB/upper RS level (arrowhead), in addition to LLEs (arrow) and MCs (magnifi ed in D) at the RS level. B) “Tangle” endings parallel to a clublike ending (arrow) against the basement membrane at the ringwulst level are shown further from the medial axis. C) “Tangle” endings (arrowheads) with associated Schwaan cells at the superficial extent of the RS (plane of section along medial axis). D) Merkel ce lls lacking visible i nnervation (arrow) in addition to “tangle” endings (arrowhead) at the upper RS level (plane of section well past the medial axis). E) Sympathetic innervation of th e vascularized inner conical body (plane of section well past the medial axis), and F) a dermal hair shaft medulla lacking the extensive FNE innervation seen in U2 follicles (plane of section along the medial axis). RRC, rete ridge collar; OCB, outer conical body; ICB, inner conical body; RS, ring sinus; RW, ringwulst; CS, cavernous sinus; DVN, deep vibrissal nerve. Scale bar = 1mm (A), 300m (B-C), 600m (D-F).

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57 Figure 2-6. Representative postf acial vibrissae innervation in cludes dense networks of MEs along with LLEs and “tangle” endings. A) A dorsorostral postfacial hair (TM9728) shows characteristic Merkel innervati on (arrowheads) at the RS level. F) A dorsocentral postfacial hair (TM9728) exhi bits the presence of LLEs (small arrow), “tangle” endings (large arrow) and a particularly extensiv e network of MEs (between arrowheads) at the RS and ICB levels. G-I) Follicles from the tail edge (G; MNW0614), ventral body (H; MNW0614), and dorsocaudal body (I; TM9728) reveal “tangle” endings and Merkel i nnervation (arrows and arrowh eads, respectively) at the RS and ICB levels. B-C) De tails shown for epidermal innervation (MEs shown with arrowhead; superficial is up) and “tangle” endings (D-E; dor socentral postfacial hair, specimen TM9728 for B-E) at the upper RS/lowe r ICB level. All planes of section shown well past the medial axis. NF, 200 kDa neurofilament subunit; PGP, protein gene product 9.5; CGRP, calcitonin gene-rel ated peptide. Scale bar = 1mm (A), 600m (B-C), 150m (D-E), 750m (F, I), 500m (G-H).

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59 Figure 2-7. Immunolabeling attribut es of innervation. A-B) Dermal papillae projecting into the epidermis at the RRC level contain Cand A -fiber innervation (CGRP-positive and NF-positive fibers, respectively). C) Circumfe rential FNEs at the ICB of a U2 follicle reveal mostly fine caliber fibers interspersed among A (NF+) fibers. D) Largely uninnervated MCs (CGRP+) interspersed among MEs (NF+) at the RS level. E-G) Novel endings along the trabeculae of the CS stain positively for BNaC, PGP, S100, NF, and lightly for CGRP (arrows). H-J) “Tangle” endings also stain positively for BNaC, PGP, S100, NF, and lightly for CG RP. K) Presumptive FNEs within the medulla of a U2 follicle hair shaft include mostly fine caliber fibers (PGP+/NF-) interspersed among A (NF+) fibers. L) Meissner’s corpuscles were sparsely distributed at the level of the epidermis. M) Pacinian ending found at the base of the epidermis. N) An example of vascular supply associated with NF-positive innervation. Scale bars = 300m (A -D), 150m (E-K, N), 75m (L-M).

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61 Figure 2-8. Confocal surface reconstructions sh owing the three-dimensional structure of representative follicle innervation and novel mechanoreceptors present in the ICB, RS and CS regions. A) A trabecular ending with in the CS (same ending shown in Fig. 24E). B) Extensive Merkel ending network in a dorsocentral postf acial vibrissa shows the completeness of innervation (shown in Fig. 2-6F). C) The morphology of a lanceolate ending near Merkel cells at the RS level can be compared to the larger, more intricate “tangle” ending in the lower ICB/upper RS of a U2 follicle (D; also seen in Fig. 2-3D). E) Reconstruction of a DVN penetrating the cavernous sinus illustrating size and associated axons. F-H) Examples of “tangle” endings found in the upper RS/lower ICB level of U2 (F), BLH (G), and dorsocentral postfacial vibrissae (H; also shown in Fig. 2-6E). Scale bars=250m (A-H).

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62

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63 Figure 2-9. Confocal three-dime nsional images of novel endings stained for neurofilament (NF200) and protein gene product 9.5 (PGP). A) Stereo pair depicting a group of “tangle” endings. B) A three-dimensional im age (red/green anaglyph) of two “tangle” endings with shared innervation. C) A single-optical secti on shows a trabecular ending in detail. Red depicts NF-positive endings within a green PGP-positive cytoplasmic meshwork. D) A st ereo pair showing Merkel innervation (left-hand pair) and closely associated clublike endings and “tangle” endings (right-hand pair).

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65 CHAPTER 3 SOMATOSENSORY NUCLEI OF THE MANATEE THALAMUS AND BRAINSTEM Introduction Florida manatees are large-bodi ed herbivorous marine mammal s of the Order Sirenia that appear to be tactile specialists due to the presen ce of tactile hairs (vibri ssae) distributed over the entire body with an especially dense distribution on the face (Reep et al., 1998; 2002). This arrangement is unique among mammals and may allow manatees to compensate for their reduced visual system by using vibrissae to aid wi th navigation in the water. A reliance on haptic input is reflected in the organi zation of the neocortex as well. Recent evidence suggests that primary somatosensory cortex (SI) occupies r oughly 25% of the neocortex (Sarko and Reep, 2007), which is favorably comparable to other soma tosensory specialists such as the naked molerat (Catania and Remple, 2002). Additionally, manatees exhibit co rtical specializ ations known as Rindenkerne, or “cortical nuclei” that appear as ce ll clusters in layer VI and are unique to sirenia (Dexler, 1912; Johnson et al., 1994). Rindenkerne sh are histochemical attri butes with “barrels,” the functional representations of mystacial vibri ssae found in layer IV of SI in rats, mice, and other rodents (Johnson, 1980; Rice, 1995; Kaas and Co llins, 2001), as well as in shrews (Catania et al., 1999), opossums (Huffman et al., 1999; Catania et al., 2000; Fr ost et al., 2000), and hedgehogs (Catania et al., 2000). Rindenkerne may pr ocess information related to vibrissae that are behaviorally relevant in activ e tactile exploration and object rec ognition, as well as in passive detection of hydrodynamic stimuli. Howeve r, this hypothesis remains untested by electrophysiology or axonal tracing methods due to the manatee’s status as an endangered species. Dedication of innervation to a particular sensory modality in the periphery creates a commensurate neural commitment in the brainste m, thalamus, and cerebral cortex. Although the

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66 neocortex of the Florida manatee has been char acterized histochemically (Sarko and Reep, 2007) and the sensory innervation of manatee vibrissae is curr ently being examined using immunofluorescence, no systematic analysis of behaviorally releva nt areas of the thalamus and brainstem has been undertaken. It has previous ly been noted that manatees have a large trigeminal nerve (Reep et al., 1989) and well-developed trigeminal and somatosensory nuclei but reduced visual thalamic and brainstem nuclei (Johnson et al., 1986; 1987; Welker et al., 1986; Reep et al., 1989). Assessments of the relative importance of the visual and somatosensory systems in sirenian behavior parall el these findings, particularly fo r the trigeminal system that is associated with the use of the facial vibrissae in tactile exploration. The somatosensory brainstem nuclei of inte rest for the manatee include Bischoff’s nucleus, the cuneate-gracile complex, and the trig eminal nucleus (in particular, the principal sensory component). Bischoff’s nucleus is a distin ct group of cells in the midline of the caudal medulla that projects heavily to the ventroba sal thalamus in the raccoon (Johnson et al., 1968; Ostapoff and Johnson, 1988) and constitutes the tail representation in most mammals with a well-developed tail (Kappers and Ubbo, 1960). By analogy, Bischoff’s nucleus would represent the fluke in the manatee and might occupy a dispr oportionately large area due to the tactile hairs present on the fluke. Behavioral observations also indicate that manatees carefully manipulate their flukes when navigating thr ough the water, which presumably involves significant sensory feedback via Bischoff’s nucleus (Welker, person al observations). The manatee cuneate-gracile complex would be expected to represent so matosensory input from the upper and lower body trunk and the flippers as it does in other species. The commitment of sensory endings to postfacial vibrissae of the fli ppers and trunk of the body may also create somatotopic parcellation within these nuclei. Finally, it seems reasonable to expect the trigeminal nucleus of the manatee

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67 to be disproportionately large a nd parcellated into “barreloids” (the functional representation of vibrissae in the brainstem) in order to maintain and process se gregated inputs from the facial vibrissae used in direct tactile explorati on of objects in the manatee’s environment. The principal somatosensory nucleus in the th alamus is referred to as the ventrobasal (VB) or more commonly the ventroposterior (VP) nucleus which contains a lateral subdivision (VPL) that represents the body and a medial subdivision (VPM) that represents the face and most of the head (e.g., Jones, 1985a). Th e relative sizes of th ese nuclei vary in other species according to the relative innervation of the body versus th e face, respectively (R ose and Mountcastle, 1952; Cabral and Johnson, 1971; Welker, 1974; Bombar dieri et al., 1975). In rodents and some marsupials, VPM has been discovered to contain “b arreloids,” or neuronal clusters related to individual vibrissae, that ar e highly reactive for cytochrome oxidase (Jones, 1983; Land and Simons, 1985b; Van der Loos, 1976). Given the manatee’s reliance on ha ptic input, the VPM would be expected to be relatively la rge and may possess barreloid parcellation. In the present study we investigate the manat ee brainstem and thalamus using stains for Nissl bodies, myelin, acetylcholin esterase, and cytochrome oxida se in order to localize and determine the size and extent of the principal somatosensory nuclei in each region. Because of the manatee’s reliance on haptic input, we hypothesize that somatosensory nuclei in the brainstem and thalamus would be relatively la rge and potentially subdivided in order to accommodate the large amount of information be ing processed by discre te vibrissae in the periphery—approximately 2,000 on the face and 3,000 on the body (Reep et al., 2001; 2002). Additionally, if Rindenkerne are in fact analogous to cortical ba rrels, it seems reasonable to expect similar functional representa tions of vibrissae to be present in the form of discrete cellular aggregates within the brainstem (as barrelettes in the trigeminal nucleus) and the thalamus (as

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68 barreloids). This analysis adds to our co mprehensive characterization of the manatee somatosensory system and our overall efforts to understand manatees’ specialized adaptations and perceptual capabilities in their unique e nvironmental niche. Examining an evolutionary outlier such as the manatee also contributes significantly to our understanding of general organizing principles of sensory systems. Materials and Methods Four postmortem brains of the Florida manatee, Trichechus manatus latirostris were obtained fresh (the head perfused within 24 hours of death) through the statewide manatee salvage program administered by the Florida De partment of Natural Resources and collected under U.S. Federal Fish and Wildlife Perm it PRT-684532 with IACUC protocol #C233. The heads were perfused in situ by gravity-fed bila teral cannulation of the carotid arteries, with 8–15 L of 0.9% phosphate-buffered saline followed by 8–15 L of 4% phosphate-buffered paraformaldehyde fixative (amounts varied accordi ng to specimen size). The dorsal cap of each skull was removed, the brain extracted, and the meninges removed. Each brain was then placed in 4% paraformaldehyde. A summary of relevant specimen information is provided in Table 3-1 (classifications are in accorda nce with size/age class defi nitions for the manatee photoidentification system, Sirenia Project, National Bi ological Survey, 1994). In each case the animal was considered fresh with minimal degradation of the tissues collected and without potentially confounding factors such as ch ronic pathology or emaciation. The thalamus was removed from the right hemisphere of specimens TM0339 and TM0406 and remained intact (with the cort ex) for specimen TM0410. The brainstem was removed from specimens TM0406, TM0410, and TM 0614b. Serial frozen microtome sections were cut coronally at 60m for these brains and ad jacent series of sections were then stained for cytochrome oxidase (CO), for Nissl substance w ith cresyl violet (CV), for myelin with gold

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69 chloride (GC), and for acetylcholinesterase (AChE) as available (see Table 3-1). Five additional specimens were analyzed from brains in our collection. These specimens included TM84-49, TM85-8, TM84-58, TM86-124, and TM85-32, all of which were celloidin-embedded and sectioned at 40m. Specimen TM85-8 was sectione d in the horizontal plane, specimen TM84-58 was sectioned in the sagittal plane, and specimens TM84-49, TM86-124, and TM85-32 were sectioned in the coronal plane. Adjacent series of sections for these specimens were stained for Nissl bodies with thionin and for myelin w ith hematoxylin. The CO procedure (Wong-Riley, 1979) was modified for manatee tissue by staining overnight, a nd the GC procedure (Schmued, 1990) was used with adjustment of pH to 6.3. The AChE recipe was provided by Dr. Robert Switzer (Neuroscience Associates, Inc., Knoxville, TN). Briefly, sections were cut directly into incubation solution consisting of 0.226 g of acety lthiocholine iodine in 100 ml of deionized water, 25 ml stock glycine, 25 ml stock CuSO4, and 50 ml of 0.2M acetate buffer (pH 5.0). Sections were then placed into incubation solu tion in a 40C hot water bath for 90 minutes, rinsed in distilled water, transferred to 1% silv er nitrate for 4 minutes, ri nsed again, treated with 1% sodium thiosulfate for 6 minutes, and given a final rinse before being mounted onto slides from 0.02M acetate buffer. Once each staining pr ocedure was complete and sections were mounted onto gelatinized slides, the s lides were coverslipped using Eukitt. All thalamic and brainstem sections were viewed under an Olympus BH-2 microscope, a Bausch and Lomb microprojector, a Zeiss Axiophot microscope, and on a light table in order to examine sequential sections for persistence of th e visible patterns and identification of nuclei. Brain atlases of the rat (Paxinos and Wa tson, 1986; 1998), cat (Berman and Jones, 1982; http://www.brainmaps.org), and monkey (Gergan and MacLean, 1962) were used to assist in identification of boundaries of the thalamic nuclei. Representative sections were imaged using a

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70 Zeiss Axioplan 2 microscope or scanned with an HP ScanJet 5370C and contrast and brightness were optimized using Adobe Photoshop CS. In an attempt to quantify th e percentage of thalamus o ccupied by VP in the manatee compared to other species, we analyzed three co ronal sections spaced across the full extent of VP (the first close to the rostral-most extent of VP, with both VPM and VPL distinguishable; the second a middle section; and the third close to the ca udal extent of VP and still retaining distinguishable VPM and VPL). Sections from th ree manatee brains and two rat brains stained for CO were scanned and outlined using AIS (Ana lytical Imaging System) software. The extent of the entire thalamus within each coronal section was measured followed by the extent of VP (based on CO-dense staining). These measurem ents were then completed using Nissl body stained sections from atlases for the rat, cat, and squirrel monkey (Table 3-2). Results Brainstem Since manatee brainstem nuclei have never been fully characterized, we first compiled an atlas illustrating all clearly identifiable nuclei in a representative adult specimen (Fig. 3-1) with a particular focus on the somatosensory component s. Nomenclature is based primarily on the Paxinos and Watson (1986; 1998) rat brain atlas and supplemented where appropriate by the cat brain atlas (http://www.brainmaps.org). The mesen cephalic nucleus of cranial nerve 5 (Me5) is the rostral-most component of the trigeminal subnuclei (Fig. 3-1, A-D; Fig. 3-2, A-C). As in other species, it is visible along the lateral exte nt of the periaqueducta l gray (PAG; Fig. 3-1A) and locus coereleus (LC; Fig. 3-1D). Dispropor tionately large compone nts of the auditory system—the inferior colliculus (IC; Fig. 3-1, AD) and nucleus of the lateral lemniscus (NLL; Fig. 3-1, C-E)—are also visible at this level and are commensurate with the manatee’s welldeveloped auditory system (Gerstein and Gerstein, 1999; Mann et al., 2005).

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71 The motor (Mo5) and principal sensory (Pr5) components of the trigeminal system are seen caudal to Me5 (Fig. 3-1E; Fig. 3-2; Fig. 3-3). Nucleus Mo5 exhibits large motorneuron somata in Nissl body stains (Fig. 1, E-F, left pa nels) that also charac terize the facial motor nucleus (FMN; Fig.3-1, I-J) and lateral vestibular nucleus (LVe; Fig. 3-1, H-K). The Pr5 nucleus appears large and lobulated in bo th coronal (Fig. 3-1, E-G; Fig. 3-4A) and sagittal (Fig. 3-2, AC) preparations. It appears as a distinct nucleus caudolateral to the NLL in horizontal sections (Fig. 3-3, B-F), and Pr5 stains moderate ly for CO (Fig. 3-4A, right panel). Just caudal to the initial appear ance of the facial nerve (7n), Pr5 begins to transition into the presumptive oral division of the spinal trig eminal nucleus (Sp5; Fig. 3-1H; Fig. 3-4B). This shift is also characterized by the appearance of the spinal trigeminal tract (sp5) which assumes a crescent shape surrounding the nucleus. As the facial motor nucleus (FMN) becomes distinct (Fig. 1, I-J) the spinal trigeminal nucleus assumes a flattened and less distinct morphology characteristic of the interpol ar subnucleus of Sp5 (e.g., Paxinos and Watson, 1998; Fig. 3-1, J-K; Fig. 3-4C). The extensive caudal subnucleus of Sp5 continues from the interpolar nucleus (Fig. 3-1, L-P; Fig. 3-4D) and appears to be lobulated. The extensiven ess of the spinal trigeminal nucleus is also clearly evident in sagittal (Fig. 3-2D) and horiz ontal sections (Fig. 3-3, C-F). Each of the spinal trigeminal subnuclei stains moderately for CO (Fig. 3-4, right panels). Although Pr5 and the caudal nucleus of Sp5 appear lobulated, and both the oral and interpolar nuclei of Sp5 appear densely penetrated with fiber bundles (Fig. 3-4, B-C), no evidence of barreloids was present. The cuneate nucleus first becomes evident at the caudal extent of the FMN (Fig. 3-1J). Contrary to previous reports (Johns on et al., 1994) an external cuneate nucleus (ECu) is present, although it is greatly reduced (Fig. 3-1, K-L). As a whole, the cuneate-gracile complex is large,

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72 extensively lobulated, and stains densely for CO (Fig. 3-1L-N; Fig. 3-4D). A large Bischoff’s nucleus is also present at the most caudle exte nt of the gracile nucleus (Fig. 1K) and stains densely for CO (shown in the neonate only but pr esent in all specimens examined, Fig. 3-5E). The proposed somatotopic arrangement of cutane ous inputs from the manatee body is presented later along with the proposed somatotopy for VP within the thalamus (Figure 3-13) based on Welker (1973). A spaced series of representative sections fr om a neonatal specimen demonstrates that the location and disproportionately larg e size of somatosensory nuclei, as well as the organization of brainstem nuclei in general, is consistent between adults and neonates. Figure 3-5A shows a section equivalent to that of Fig. 3-1E with a la rge Pr5 that stains moderately for CO. The plane of section for Fig. 3-5B is equivalent to Fi g. 3-1I, showing Sp5 surr ounded by the crescent of spinal trigeminal tract fibers. The adjacent sect ions shown in Fig. 3-5C are equivalent to Fig. 31K with a small external cuneate nucleus present at the lateral aspect an d large Sp5 and cuneategracile nuclei. Figure 3-5D shows lobulation present in Sp5 and mo re extensively in the cuneategracile complex (CuG) equivalent to Fig. 3-1M. Finally, a large Bischoff’s nucleus encompassing the presumptive tail representation is present at the caudal aspect of the medulla (Fig. 3-5E) along with caudal Sp5. Moderate CO st aining was present in the trigeminal nucleus and dense staining characterized CuG as seen in adults (Fig. 3-4). Thalamus The principal somatosensory nucleus in the th alamus is the ventroposterior (VP) nucleus, one of the most clearly defined thalamic nuclei due to its large size, de nsely staining cells, and lobulated appearance resulting from penetrati ng myelin fiber bundles (Jones, 1985b). In Nissl body preparations, subnucleus VPM contains smalle r, relatively closely packed cells compared to VPL. In addition, AChE staining reveals robust patterns that allow for the discrimination of

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73 different nuclei and these patter ns are generally consistent for rats, cats and primates (Jones, 1985a). Densest staining generally characterizes the ventral lateral geniculate nucleus as well as the intralaminar, anteroventral, anterodorsa l, rhomboid, paraventri cular, habenular and medioventral nuclei while lighter staining distingu ishes the dorsal lateral geniculate nucleus (LGN), medial geniculate nucleus (MGN), reticular nucleus, anteri or of the lateral posterior nucleus, and parts of lateral and ventral complexes. An atlas of the manatee thalamus is provide d for the first time (Fig. 3-6) with a more closely spaced series of sections to show the de tailed extent of the soma tosensory thalamus (Fig. 3-7). Nomenclature is based on Jones, (1985a ). The VPL and VPM nuclei first appear in approximately the same plane of section (Fig. 3-6F). Whereas VPL term inates in Fig. 3-6G, VPM extends more caudally to Fig. 3-6H. The me dial subnucleus was id entifiable in Nissl body preparations as having relatively small, closely pa cked cells in contrast to the lateral subnucleus which displayed large, darkly staining cells th at were more widely spaced (Fig. 3-8). The subnuclei of VP were also distinguishable in AChE staining preparations with VPM exhibiting lighter staining than VPL (Fig. 3-9, left column). As seen in other species, VP was characterized by penetrating fiber bundles visible in myelin pr eparations (Fig. 3-6, FH; Fig. 3-7; Fig. 3-9, right column). The entirety of VP was CO-dense as seen in other species and was consistently CO-dense in adults (Fig. 3-9, middle column), neonates and juveniles (Fig. 3-10) although the medial subnucleus did not appear more densely stained as is the case in some other species. No barreloids were clearly distinguishable, although possible functional divisi ons might be indicated by penetrating fiber laminae that were particul arly pronounced in the juvenile specimen (Fig. 311). The posterior nucleus (Po) al so receives cutaneous input from the periphery and is visible in Fig. 3-6, E-I and in Fig. 3-7.

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74 Overall, the ventroposterior a nd posterior nuclei appear di sproportionately large in the manatee in accordance with their functional re levance to somatic sensation. To quantify the percentage of thalamus occupied by VP in the manatee compared to other species, we analyzed three coronal sections spaced acro ss the full extent of VP (see Materials and Methods) from three manatee brains and two rat brains stained for CO as well as from Nissl body stained sections from atlases for the rat, cat, and squirrel monkey (Table 3-2). Although this yields limited information given that the volume of total thal amus versus VP (and more particularly, VPL versus VPM) could not be calculated, the data in dicate that measures were very similar between the rat sections stained for CO and the outlined se ctions from the rat brain atlas and by extension should be comparable for the cat and squirrel monkey as well. Also, although the measure was across only three coronal sections spaced across VP for each species, it does appear that the percentage of thalamus occupied by VP is higher in manatees, pa rticularly in the adult specimen (28%). Indeed, based on a qualitative assessment of CO-stained coronal sections in the adult (Fig. 3-9), VP appears to occupy approximately one-third of the thalamus. The only other study found to quantify thalamic subnuc lei was done by Kruger (1959). Kruger’s data quantified VP as a percentage of dorsal thalamus, and dorsal as a percentage of total thal amus. By extrapolation, his measurements indicate that VP occupies 6.6 % of total thalamus in rabbits, 2.9% in sheep, 5.0% in cats, 7.0% in monkeys, and 2.6% in dolphins. Other behaviorally relevant thalamic nuclei in clude the lateroposterior subnucleus (LP) and the lateral geniculate nuleus (LGN), both of wh ich are relatively small and are overtaken by the large medial geniculate nucleus (MGN; Fig. 3-6, G-K). This anatomical organization reflects the relative degree of development a nd behavioral importance of the visual and auditory systems, respectively. Our proposed location for MGN diff ers from other species in that MGN appears

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75 rostral to LGN as seen in coronal (Fig. 3-6, G-K) and horizontal (Fig. 3-12) sections. The medial geniculate is also situated dorsal to LGN with Po visible as a wedge between MGN and LGN (Fig. 3-6I) as seen in the horizon tal section. This orientation is conceivable if one considers the overall rostroventral rotation th at the manatee brain appears to have undergone, such that the equivalent of the Sylvian (lateral ) fissure is oriented vertically. Additionally, if th e auditory sense truly dominates visual in the manatee, it is pl ausible that MGN became greatly expanded at the expense of visual thalamic nuclei ther eby forcing them caudal and ventral. Discussion Brainstem: Somatotopic Parcellation in Other Species The brainstem nuclei of interest for the manatee somatosensory system include the trigeminal, cuneate, gracile, and Bischoff’s nuclei. Bischoff’s nucleus, a distinct group of cells in the midline of the caudal medulla (Johnson et al., 1968), has not been identif ied previously in the manatee but has been shown, along with the cuneate and gracile nuclei, to project heavily to the ventroposterior thalamus (VP) in the racc oon (Ostapoff and Johnson, 1988). In the raccoon, the tail representation occupies the dorsal porti on of VP whereas the hindlimb representation occupies the ventral portion (Johnson et al., 1968) The presence of Bischoff’s nucleus has also been noted in rats, shrewmice, kangaroos, great an teaters, some monkeys, and to some extent in cetaceans (Kappers and Ubbo, 1960). In the mana tee, Bischoff’s nucleus would presumably represent the fluke, and does in f act appear to be disproportionate ly large as might be expected given the presence of vibrissae on the fluke. The fluke might also be cr itical during navigation, as manatees have been observed to constantly adjust the angle of their fluke while swimming (Welker, personal observations). Species like the manatee that rely on tactil e exploratory behaviors involving the face (e.g., the star-nosed mole (Crish et al., 2003) and the platypus (Ashwell et al., 2006)) have well

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76 developed trigeminal systems often exhibiting extensive somatotopic organization within the sensory trigeminal nuclei. Studies in the rat rev ealed that the afferent projection pattern from individual facial vibrissa fo llicles was topographically related to CO-dense cell clusters (“barrelettes”) in the trigeminal principal sens ory nucleus (Pr5) with a nearly one-to-one ratio between follicles and corresponding CO-dense cl usters (Florence and Lakshman, 1995). These results supported earlier findings by Jacquin et al (1993) that showed th at Pr5 axon collaterals were concentrated within corre sponding CO-dense subdivisions, and terminal branches of individual trigeminal afferents rarely crossed over into adjacent regions. In contrast, in three subdivisions of the spinal trigeminal nucleus—the pars oralis (Sp5o), pars interpolaris (Sp5i), and pars caudalis (Sp5c)—a topographical arrangem ent still existed, but with less specificity and more overlapping representations (Florence a nd Lakshman, 1995). Whisker-related barrelette patterns are present in Pr5, Sp5i, and Sp5c, but not in Sp5o (e.g., Nomura and Mizuno, 1986). Goyal et al. (1992) showed that the human principal trigeminal nucleus also demonstrated a parcellated CO-dense pattern which was interpre ted as a reflection of high-density peripheral innervation of the face de spite the lack of punctate structures like vibrissa e. In the manatee, the principal sensory and caudal spinal trigeminal nuclei appeared partic ularly lobulated with possible somatotopic parcellati on present. All trigeminal components, and especially the principal sensory nucleus, were el aborated and exceptionally large. In contrast to other studies that have shown CO-dense staining in Pr5 a nd Sp5c with only light staining in Sp5o and moderate staining in Sp5i (Florence and Laks hman, 1995), CO staining appeared consistently moderate throughout Pr5 and Sp5 in the manatee. The somatotopy proposed for the manatee brainstem (cuneate-gracile complex and Sp5c repr esented; Fig. 3-13A) is based on the general arrangement seen in mammals (e.g., Woudenberg, 1970).

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77 Somatotopic parcellation is also evident in the cuneate and gracile dorsal column nuclei in other species where somatosensation is the dominant sensory modality. Cutaneous inputs from the upper limbs and rostral trunk of the body are re presented in the cuneat e nucleus while lower limbs and lower trunk are represented in the gr acile nucleus. Strata et al. (2003) studied a prosimian, the Galago, to look at the pattern of peripheral nerve input. Through cell clusters that were identified as CO-dense blotches in both nuc lei, they discovered a greater segregation of inputs within the cuneate (fingers and hand representation) than in the gracile (foot representation), which corresponds with the Galago’ s extensive and highly differentiated use of its hands and fingers relative to it s feet. In macaques, inputs from sp ecific parts of the hand relate to CO-dense rostrocaudal clusters of cells (Florence et al., 1991) Although the manatee lacks the manual dexterity of a primate, CO analysis of the manatee cuneate-gracile complex revealed dense staining and extensive suborganization th at may be related to discrete functional representations of vibris sae on the postfacial body. Thalamus: A Comparative Look at Somatosensory Nuclei The relative sizes of VPM and VPL vary accord ing to the relative i nnervation of the face versus the body, respectively (Rose and Mountcastle, 1952; Ca bral and Johnson, 1971; Welker, 1974; Bombardieri et al., 1975). For rodents w hose nose, mouth and lips dominate tactile perception, the VPM is larger, and in monotremes VPM dominates the entirety of VP. In cats, VPM and VPL are approximately equal in si ze, but in monkeys VPL predominates to accommodate extensive input from the hands and feet. In some species, for example the cat, VPM extends caudal to VPL, whereas in others (such as monkeys) VPL extends more caudally (Jones et al., 1985b). In rodents and some mars upials, VPM has been discovered to contain “barreloids,” or neuronal clusters related to i ndividual vibrissae, that are highly reactive for CO (Jones, 1983; Land and Simons, 1985b; Van der Loos, 1976). The VPL of the raccoon and slow

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78 loris contains lobulated subreg ions representing palmar and digital skin pads (Welker and Johnson, 1965; Krishnamurti et al., 1972). Chronic vibrissae trimming results in reduced staining for CO in both the somatosensory cortical barrels (Land and Simons, 1985a; Wong-Riley and Welt, 1980) and the thalamic barreloids (Land an d Akhtar, 1987) associat ed with the trimmed vibrissae. These findings were similar to those in Macaca fascicularis monkeys where peripheral nerves were cut, resulting in reduced staining of “rods” with in the VPM (Jones et al., 1986). Using horseradish peroxidase axonal tracing, Jones et al. (1986) also disc overed that CO staining was primarily due to terminations of trigeminal afferent fibers that formed somatotopically organized inputs to the rods. They postulated that each rod of the thalamus formed the basis of columnarity of afferent input to the so matosensory cortex by providing bundles of thalamocortical axons terminating in focal domains of the cortex. No barreloids were found in VPM of the manatee thalamus, but the ventroposte rior nucleus as a whol e was large, reflecting the manatee’s reliance on haptic (somatosenso ry) input. Based on AChE staining, VPM and VPL also appear to divide the vent roposterior nucleus in an approximately equal manner (Fig. 3-9). This seems reasonable given the distribution of vibrissae on the manatee body (2,000 on the face and 3,000 postfacially) balanced by th e fact that facial vibrissae are more densely innervated (Reep et al., 2001; 2002). While our proposed “man ateeunculus” within the thalamus follows Welker (1973; Fig. 3-13B), it should be noted that such artistic ab stractions are limited since the body representation extends in th ree dimensions throughout VP. Thalamic nuclei associated with vision (LGN and LP) appear to be overtaken by those associated primarily with somatosensation (Po) and audition (MGN). While this may simply be due to the fact that somatosensation and auditi on both appear to be dominant sensory modalities in the Florida manatee, it is also possible that there exists extensive multisensory integration. A

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79 previous study from our laborator y found that the manatee cortex exhibits what appears to be extensive—and possibly complete—overlap be tween primary auditory (AI) and primary somatosensory cortex (SI; Sarko and Reep, 2007). Responses to somatosensory and visual stimuli have also been reporte d in AI of the macaque (Werner-Reiss et al., 2003; Ghazanfar et al., 2005; Brosch and Scheich, 2005; Brosch et al., 2005; Zhou and Fuster, 2004). While it is possible that this multisensory in tegration occurs entirely within AI, with nonauditory information relayed to AI from unimodal sensory areas in subcortical or cortical areas, it is also conceivable that AI receives inputs from multisensory cortex. But perhaps the most intriguing possibility, and one that might be rele vant to the present study, involves the integration of auditory and non-auditory inputs at subcortical levels, along the traditional auditory pathways or within multisensory subcortical structures, befo re projecting to AI (Budinger et al., in press). Possible candidates for these subcortical multimodal areas include Po; the dorsal and medial division of MG; the brachium, dorsal and external nuclei of the inferior colliculus; and the superior colliculus, all of which were found to involve connections with AI in the Mongolian gerbil, a species used frequently for auditory re search (Budinger et al., in press). The brachium of the inferior colliculus (bic), Po, and MG and particularly strong candidates as these are reciprocally connected with AI. Indeed the inferi or colliculus appears to be extensively involved in acousticomotor and somatosensory system s (e.g., Huffman and Henson, 1990) and neurons within the bic code for audito ry space (Schnupp and King, 1997) a nd project to IC, SC, and AI (Kudo et al., 1984; Mitani et al., 1987; Rouiller et al., 1989 ) The external subnuc leus of the IC has been found to integrate trigeminal and audito ry stimuli with projections from both CN and Sp5 (Jain and Shore, 2006), and multisensory inte gration has also been demonstrated in DCN (Shore, 2005). The inferior and superior collic uli together coordinate aspects of spatial

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80 orientation (e.g., Oliver and Huerta, 1992; Cohen and Knudsen, 1999), and indeed somatosensation has been found to dominate the SC in the star-nosed mole that relies extensively on its haptic sense (Crish et al., 2003). Subdi visions of MG proce ss visual, vestibular, nociceptive and somatosensory stimuli in addition to the auditory stimuli classically associated with MG (e.g., Linke and Shwegler, 2000). In addition, the trigeminal system appears to influence the superior olivary complex (Shore et al., 2000). Overall, cortical and subcortical areas once rigidly defined within unimodal f unctional boundaries appear to have a broader functional scope, and this may be especially tr ue in the manatee’s case where low-frequency sounds in the water might stimulate mechanor eceptors—Merkel and lanceolate endings in particular—as the vibrissae of the face and postf acial body are perturbed. Such integration has been hypothesized to be benefici al in perception and localizat ion of stimuli, potentially by linking object features or adjusting coordinate frames to a different sensory modality (Budinger et al., in press). Projections from AI to subcortical multimodal areas may also modulate subcortical activity by influencing a selected repr esentation of behaviorally relevant frequencies (e.g., Suga and Ma, 2003), improving temporal and spectral resolution of sound representations (Yan et al., 2005), and attenti on-related gating of auditory information (Yu et al., 2004). Absence of Barrelettes and Barreloids If Rindenkerne are in fact analogous to cort ical “barrels” and co rrespond to functional representations of vibrissae, it seems reasonable to expect the brainstem and thalamic counterparts of barrels (“barrelett es” and “barreloids,” re spectively) to be present. However, the absence of the latter two features in the Florid a manatee could be explai ned by several factors. First, the plane of section may not have been optim al for detection, particul arly in the case of the thalamus. We chose to section the majority of our specimens (and all of the specimens stained

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81 for CO) in the coronal plane as this allowed for clearest identification of subnuclei. This was a priority of the current study si nce nuclei of the manatee thalamus and brainstem have not been characterized previously in a systematic and comprehensive ma nner. Because of their complex three-dimensional morphology, identification of barreloids in the coro nal plane has proven difficult in rodents (Ivy and Killackey, 1982; Land et al., 1995 ). Instead, the oblique horizontal plane has proven optimal for detection of barre loids (Land et al., 1995) Although this hypothesis could be tested by simply alteri ng the plane of section, it is more probable that barreloids are not present in the manatee, because we also did not detect barrelettes in the brainstem, which should be clearly apparent in the coronal plane. Another possible explanation fo r the absence of barrelettes and barreloids is that the facial vibrissae of the Florida manatee are not orga nized in discrete rows as seen in other species including mice, rats and cats whos e organization of facial vibri ssae into rows and columns is maintained within VPM and the trigeminal sensory nuclei (e.g.,Nomura and Mizuno, 1986; Ma, 1991; Florence and Lakshman, 1995; Land et al., 1995). But perhaps the most likely explanation for the absence of barreloids a nd barrelettes lies in the di fference between Rindenkerne and cortical barrels. While barrels are located in layer IV, an afferent zone, Rindenkerne are located in efferent layer VI. This undoubtedly indicates a fundamental difference in the columnarity of organization between barrels and Rindenkerne. Cort icocortical feedback connections originate in infragranular layers, implicating Ri ndenkerne in descending pathways.

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82 Table 3-1. Summary of specimen information. Specimen Areas analyzed Stains SexLength (in cm) Weight (in kg) TM84-49 Thalamus & brainstem intact Thionin, hematoxylin TM85-8 Thalamus & brainstem intact Thionin, hematoxylin TM84-58 Thalamus & brainstem intact Thionin, hematoxylin TM86-124 Thalamus & brainstem intact Thionin, hematoxylin TM85-32 Brainstem Thionin TM0614b Brainstem CO, CV, GC F 326 TM0339 Thalamus CO, CV, GC (bad) F 200 167.8 TM0406 Thalamus& brainstem CO, CV, AChE, GC M 290 393.2 TM0410 Thalamus (intact) & brainstemCO, CV, AChE, GC M 94 14.5 Table 3-2. Comparative analysis of percentage of thalamus occupied by the ventroposterior nucleus (VP; averaged from 3 evenly spaced coronal sections to encompass VP). Species Source of analysis VP Area* (in mm2) Total Thalamus Area* (in mm2) Percentage of Thalamus Occupied by VP Manatee Neonate (TM0410) 39.0 196.4 20% Juvenile (TM0339) 66.9 286.2 23% Adult (TM0406) 93.6 333.5 28% Rat Adult (340) 1.5 7.1 22% Adult (341) 1.6 8.1 20% Brain atlas 4.6 24.7 19% (VPL=4.4%, VPM=14.6%) Cat Brain atlas 18.4 115.5 16% Squirrel Monkey Brain atlas 20.3 101.5 20% 1 Paxinos and Watson, 1986 2 Berman and Jones, 1982 3 Gergan and MacLean, 1962

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83 Figure 3-1. A rostrocaudal series of representative co ronal brainstem sec tions with subnuclei labeled illustrates the size and extent of somatosensory nuclei. A-P) Sections stained with thionin for Nissl bodies (left) sh own with adjacent sections stained with hematoxylin for myelin (right). Section nu mbers are listed at the bottom of each section, and sections were cut at 40m, specimen 84-49. Scale bars=5mm. 4=trochlear nucleus; 7n=faci al nerve; 8n=auditory-ves tibular nerve; 10=vagus nucleus; 12=hypoglossal nucleus; Amb=nucle us ambiguous; bic: brachium of the inferior colliculus; BN=Bischoff’s nucleus; bp=brachium pontis; CuG=cuneategracile; CI=inferior central nucleus; CN=cochlear nucleus; cp=cerebral peduncle; CS=superior central nucleus; Cu=cuneate nucleus; DCN=dorsal cochlear nucleus; DTN=dorsal tegmental nucleus; ECu=external cuneate nucleus; FMN=facial motor nucleus; FTG=gigantocellular te gmental field; g7=genu of facial nerve; IC=inferior colliculus; ICN=interposed cerebellar nuc leus; icp=inferior cerebellar peduncle; INC=nucleus incertus; IO=inferior olive; LC=locus coereleus; LCN=lateral (dentate) cerebellar nucleus; ll=lateral lemniscu s; LPB=lateral parabrachial nucleus; LVe=lateral vestibular nucleus; Me5=me sencephalic nucleus of 5; ML=medial lemniscus; MLF=medial longitudinal fasciculus; MnR=median raphe nuclei; Mo5=motor nucleus of 5; MRt=medullary re ticular nucleus; MVe=medial vestibular nucleus; NLL=nucleus of the lateral lemniscus; PAG=periaqueductal gray; Pn=pontine nuclei; Pr5=principal sensor y nucleus of 5; py=pyramidal tract; Rb=rubrospinal tract; Rt=reticular nucleus; s5=sensory root of 5; SC=superior colliculus; scp=superior cerebellar peduncle; SO=superior olivary nucleus; Sol=nucleus of the solitary tract; Sp5=spinal trigeminal nucleus; sp5=spinal trigeminal tract; TRN=tegmental reticular nucleus; Tz=trapezoid nucleus; VCN=ventral cochlear nucleus; Ve=vestibular nucleus; xpy=pyramidal decussation; xscp=decussation of the supe rior cerebellar peduncle

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84

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85 Figure 3-1. Continued

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86 Figure 3-1. Continued

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87 Figure 3-1. Continued

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88 Figure 3-2. Brainstem sections cut in the sagittal plane illustrate the rostrocaudal extent of behaviorally relevant nuclei and in part icular the lobulated appearance of the trigeminal nuclei. Sections proceed lateral to medial and were stai ned with thionin for Nissl bodies Section numbers are listed at the bottom of each section, and sections were cut at 40m, specimen 84-58. Scale bar=5mm. FMN=facial motor nucleus, IC=inferior colliculus, Me5=mesencephali c nucleus of 5, Mo5=motor nucleus of 5, NLL=nucleus of the lateral lemniscus, Pr5=principal sensory nucleus of 5, SC=superior colliculus, Sp5=spinal trigem inal nucleus, Ve=vestibular nucleus.

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89 Figure 3-3. Brainstem sections cut in the horizonta l plane show the topogr aphy and orientation of nuclei of interest. Sections proceed ventral to dorsal and were stained with thionin for Nissl bodies Section numbers are listed at the bottom of each section, and sections were cut at 40m, specimen 85-8. Scale bar= 5mm. 5=trigeminal nerve, CN=cochlear nucleus, CuG=cuneate-gracile complex, FM N=facial motor nucleus, IO=inferior olivary nucleus, Mo5=motor nucleus of 5, NLL=nucleus of the lateral lemniscus, Pr5=principal sensory nucleus of 5, py=pyram idal tract, SO=superior olivary nucleus, Sp5=spinal trigeminal nucleus, Ve=vestibular nucleus.

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90 Figure 3-4. Representative coronal brainstem secti ons illustrating the app earance of each of the trigeminal subnuclei in an adult specime n. Adjacent sections are shown stained for myelin (with gold chloride, GC) and cyto chrome oxidase (CO). A) The principal sensory nucleus (Pr5) is large and exhibits possible somatotopic parcellation. It also stains moderately for CO, as do all of th e trigeminal subnuclei. Section numbers are listed at the bottom of each section, a nd sections were cut at 60m, specimen TM0614b. Scale bar=5mm. 7=facial nerve, BN=Bischoff’s nucleus, CN=cochlear nucleus, CuG=cuneate-gracile complex, DC N=dorsal cochlear nucleus, FMN=facial motor nucleus, LVe=lateral vestibular nucleus, Mo5=motor nucleus of 5, Pr5=principal sensory nucleus of 5, SO=supe rior olivary nucleus, sp=spinal nerve of 5, sp5=spinal trigeminal tract, Sp5c=spina l trigeminal nucleus caudalis, Sp5i=spinal trigeminal nucleus interpolar is, Sp5o=spinal trigeminal nucleus oralis, VCN=ventral cochlear nucleus.

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91

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92 Figure 3-5. A rostrocaudal series of representative cor onal brainstem sections in a neonate shows that somatosensory nuclei are large and have a parcellated appearance as seen in adult specimens. Sections were stained with cyto chrome oxidase (CO), gold chloride (GC) for myelin, and cresyl violet (CV) for Nissl bodies. Secti on numbers are listed at the bottom of each section, and sections we re cut at 60m, specimen TM0410. Scale bar=5mm. BN=Bischoff’s nucleus, CN=c ochlear nucleus, CuG=cuneate-gracile complex, FMN=facial motor nucleus, M o5=motor nucleus of 5, Pr5=principal sensory nucleus of 5, Sp5=spinal trig eminal nucleus, Ve=vestibular nucleus.

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93

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94 Figure 3-5. Continued

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95 Figure 3-6. A rostrocaudal series of representative coronal thalamic sections with lowmagnification images of sections staine d with hematoxylin for myelin and highmagnification details of adjacent sections stained with thionin for Nissl bodies with subnuclei labeled. Section numbers are list ed at the bottom of each section, and sections were cut at 40m, specimen 84-49. Scale bar=5mm. AM=anteromedial nucleus, AV=anteroventral nucleus, CeM=centr al medial nucleus, CL=central lateral nucleus, CM=centre median nucleus, FF=fields of Forel, fr=fasciculus retroflexus, H=habenular nuclei, IC=inferior collicul us, ic=internal capsule, iml=internal medullary lamina, LD=lateral dorsal nuc leus, LG=lateral geniculate nucleus, LP=lateral posterior nucleus, MD=medi odorsal nucleus, MG=medial geniculate nucleus, ml=medial lemniscus, MV=medioventral nucleus, PARA=anterior paraventricular nucleus, Pc=paracentral nucleus, Pf=parafascicular nucleus, Po=posterior nucleus, PT=parataenial nuc leus, Rh=rhomboid nucleus, SC=superior colliculus, SM=submedial nucleus, st=stria terminalis, SubI=subincertal nucleus, VA=ventral anterior nucleus, VL=ventral lateral nucleus, VM=ventral medial nucleus, VPL=ventral posterior lateral nuc leus, VPM= ventral posterior medial nucleus, ZI=zona incerta.

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96

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97 Figure 3-6. Continued

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98 Figure 3-6. Continued

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99 Figure 3-6. Continued

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100 Figure 3-7. A rostrocaudal series of closely spaced coronal sectio ns showing the ventroposterior area (VP) of the thalamus in detail. Lowmagnification images of sections stained with hematoxylin for myelin and high-ma gnification details of adjacent sections stained with thionin for Ni ssl bodies are labeled for s ubnuclei. Section numbers are listed at the bottom of each section, and sections were cut at 40m, specimen 84-49. Scale bar=5mm. CL=central lateral nucleus, CM=centre median nucleus, fr=fasciculus retroflexus, H=habenular nuclei, LD=lateral dorsal nucleus, MD=mediodorsal nucleus, ml=medial lemn iscus, Pf=parafascicular nucleus, Po=posterior nucleus, st=stria medullaris, VL=ventral lateral nucleus, VPL=ventral posterior lateral nucleus, VPM= ventral posterior medial nucleus.

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101 Figure 3-7. Continued

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102 Figure 3-8. Low-magnification and high-magnification images charact erizing Nissl body staining of the lateral ventroposteri or (VPL) and medial ventr oposterior (VPM) subnuclei of the thalamus. A) Low-magnification view s hows coronal plane of section and areas imaged at high magnification. B) Cells in VP L are visibly larger, stain more densely, and are less densely packed than those in VP M (C). Sections were stained for thionin and cut at 40m, specimen 84-49. S cale bars=3mm (A), 1mm (B-C).

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103 Figure 3-9. Histochemical and hi stological staining characterizat ion in the ventroposterior nucleus of the thalamus. Coronal sections stained for acetylcholinesterase (AChE) allow the lateral extent of the ventroposter ior nucleus (VPL) to be distinguished from the medial subdivision (VPM) by the denser st aining in VPL. The entirety of VP also stains densely for cytochrome oxidase (CO) and exhibits dense penetration by fiber bundles in myelin staining (stained with gold chloride, GC). Section number on bottom left for AChE, adjacent sections s hown in CO and GC preparations. Up is dorsal, right is medial. Adult specimen TM0406. Scale bar=5mm.

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104 Figure 3-10. Coronal thalamus sections stained for cytochrome oxidase (CO) from a neonate (specimen TM0410) and a juvenile (specime n TM0339) show that the ventroposterior thalamus (VP) exhibits homogenous CO-dense staining without clearly distinguishable barreloids. Up is dorsal, right is medial. Adult specimen TM0406. Scale bar=5mm.

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105

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106 Figure 3-11. Fiber laminae (arrows) seen most di stinctly in the juveni le specimen (TM0339) may separate adjacent projections from adjacent body parts into subnuclei of the thalamus as demonstrated in other species. Asteri sks denote dorsal and ve ntral boundaries of the ventroposterior nucleus. Co ronal sections stained for cytochrome oxidase. Up is dorsal, right is medial. Scale bar=5mm. Figure 3-12. Horizontal myelin-stained secti on showing unusual placement of the medial (MGN) with respect to the lateral geniculate nucl eus (LGN). The posterior nucleus (Po) is also visible separating MGN from LGN rost rally. Up is rostral, right is medial. Section number 437, specimen 85-8. Scale bar=5mm.

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107 Figure 3-13. Proposed somatot opy of functional representati ons within the brainstem somatosensory nuclei (cuneate-gracile a nd trigeminal) and the ventroposterior nucleus (VP) of the thalamus in the corona l plane of section. A) As seen in other mammals, the presumptive somatotopical arrangement within the manatee brainstem would include the head at the most lateral aspect within the trigeminal nucleus with the dorsal aspect of the face medial and the ventral face la teral. The rest of the body arrangement would proceed dorsomedially w ithin the cuneate-gracile complex with the flippers, trunk of the body, a nd the fluke. The plane of sect ion is equivalent to Fig. 3-1M. B) In the thalamus, the somatotopy is presumably arranged such that the face is represented at the ventromedial extent of VP with the dorsal aspect of the face positioned dorsally and the ventral aspect positioned ventrally. The somatotopy continues along the lateroventral aspect of the thalamus proceeding laterally and dorsally with the flipper representati on followed by the trunk of the body and the fluke. The plane of section is equivalent to Fig. 3-6F. Up is dorsal, right is medial.

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108 CHAPTER 4 SOMATOSENSORY AREAS OF MANATEE CEREBRAL CORTEX: HISTOCHEMICAL CHARACTERIZATION AND FU NCTIONAL IMPLICATIONS Introduction The Florida manatee appears to have developed a unique combination of neuroanatomical, physiological, an d behavioral traits to accom modate its life as a tactile specialist and the only mammalian obligate aquatic herbivore. For example, whereas most mammals exclusively exhibit tactile hairs on the f ace, particularly the mystacial region, manatee tactile hairs are distributed ove r the entire body. Furthermore, tactile hairs (or “vibrissae,” distinguished by a prominent circumferential bl ood sinus complex, connective tissue capsule and dense innervation) are the only t ype of hair seen in manatees (Reep et al., 2001). This system might function as a distributed array for detecti ng water movements associated with the presence of other animals, river currents, and tidal flows (Reep et al., 2002). Manatees could also use this system to orient and navigate in the murky waters of their habita t with an enhanced tactile sense compensating for poor visual acuity (Bauer et al ., 2003). Tactile hairs are 30 times more densely distributed on the face than on the postcranial bo dy and are crucial in manatee feeding and tactile exploration of the environment, accomplishing du al and synergistic motor and sensory roles (Marshall et al., 1998a, b). The hair and bristles of the manatee face span 9 distinct regions (Reep et al., 1998), 2 of which are used in a pr ehensile grasping fashion during feeding and “oripulation” that is unique among mammals as we ll as in social behavi ors including mouthing, nuzzling, and pinching a conspecific’ s back in an attempt to gain access to food (Marshall et al., 1998b; Reep et al., 2001). Such obser vations suggest a high level of perioral dexterity and tactile discrimination. This is supported by evidence that the relative tactile di fference threshold (or Weber fraction) is 14% for manatees using their peri oral hairs, which is comp arable to that of an Asian elephant’s trunk (Bachteler and Dehnhardt, 1999). Notably, the eyes are often closed

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109 during feeding and tactile expl oration (Marshall et al., 1998b; Bachteler and Dehnhardt, 1999), further suggesting an emphasis on haptic over visual input. Ther efore we hypothesized that the Florida manatee has a large area of cortex devoted to somatosens ory processing, with the facial region assuming a disproportionately large amount of this area. Somatosensory maps have been delineated fo r a wide variety of species, traditionally through the use of electrophysiology but more r ecently in combination with histochemical and axonal tracing studies. Although the endangered status of the Flor ida manatee precludes it from becoming a subject for electrophysiological st udy, studies relating electrophysiology to histochemistry—specifically cytochrome oxidase and myelin staining (Krubitzer, 1995; Kaas and Collins, 2001)—have identified anatomical ch aracteristics that hold predictive value in determining the functional parcella tion of primary sensory areas, a nd can therefore be applied to the manatee through comparative analysis. Given th e variety of behavioral and sensory uses for which the facial vibrissae of the manatee are speci alized, we would expect to find a large cortical primary somatosensory area (SI) with a particul arly large and potential ly specialized facial representation to accommodate a high level of cuta neous input, as seen in a diverse range of animals including the llama (Welker et al., 1976 ), platypus (Bohringer and Rowe, 1977; Johnson, 1990), star-nosed mole (Catania, 2000), and na ked mole-rat (Catania and Remple, 2002; Henry et al., 2006). Presumptive SI of the Florida manatee was first id entified on the basis of location and cytoarchitectural characteristics (Reep et al., 1989; Marshall and Reep, 1995) and was hypothesized to consist of areas cluster cortex areas 1 and 2 (CL1 and CL2) as well as dorsolateral areas 1 and 2 (DL1 and DL2). In add ition, cortical cell cluste rs of layer VI called Rindenkerne (“cortical nuclei”), first identified by Dexler (1912) and re cently investigated in more detail (Reep et al., 1989; Marshall and Reep 1995), are unique to sire nians (Johnson et al.,

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110 1994) and stain darkly for cytochrome oxidase. Rindenkerne are reminis cent of “barrels” found in layer IV of the vibrissae s ubfield of somatosensory cortex in rats, mice, and other rodents (Johnson, 1980; Rice, 1995; Kaas and Collins, 2001), as well as in shrews (Catania et al., 1999), opossums (Huffman et al., 1999; Catania et al., 2000; Frost et al., 2000), a nd hedgehogs (Catania et al., 2000). In our efforts to delineate SI and to assi gn Rindenkerne within a pr esumptive functional scheme for the Florida manatee, preliminary findi ngs revealed four distinct cytochrome oxidasedense patches in the neonate, corresponding to the presumptive forelimb flipper, face, body, and tail representations of SI but only one distinct patch in presumptive SI of juvenile and adult specimens (Sarko and Reep, 2005). This might indi cate modification and refinement of sensory inputs as manatees develop. In order to pursue th ese observations in more detail, we performed a systematic analysis of flattened cortex prepara tions and coronal sections stained for Nissl bodies, myelin and cytochrome oxidase to accurately lo calize primary sensory areas, laminar restriction of staining, and differential pa tterning observed between younger versus older animals. Materials and Methods Four postmortem brains of the Florida manatee, Trichechus manatus latirostris were obtained fresh (the head perfused within 24 hours of death) through the statewide manatee salvage program administered by the Florida De partment of Natural Resources and collected under U.S. Federal Fish and Wildlife Pe rmit PRT-684532 and IACUC protocol #C233. The heads were removed at the necropsy facility of Sea World, FL, and then transported to the University of Florida to be perfused in situ by gravity-fed bilateral ca nnulation of the carotid arteries, with 8–15 L of 0.9% phosphate-buffere d saline followed by 8–15 L of 4% phosphatebuffered paraformaldehyde fixative (amounts varied according to specimen size). The dorsal cap of each skull was removed, the br ain extracted, and the meninges removed. Each brain was then

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111 placed in 4% paraformaldehyde. A summary of re levant specimen information is provided in Table 4-1 (classifications are in accordance with size/age class definitions for the manatee photoidentification system, Sirenia Project, Na tional Biological Survey, 1994). Specimen TM0310 was classified as a neonatal mortality with nonspecific pulmonary and renal congestion, no underlying infectious or degene rative process, and the cause of death was attributed to hypothermia. The death of the second neonate (TM 0410) was classified as perinatal and natural including the involvement of salmonellosis a nd hypoglycemia. The juvenile TM0339 died due to gastric rupture. The death of the adult TM0406 was attributed to watercraft impact. In each case the animal was considered fresh with minimal de gradation of the tissues collected and without potentially confounding factors such as chroni c pathology or emaciation. In an additional specimen, TM2, sections spanning the caudal extent of the basal ganglia to the rostral extent of the thalamus had been previously stained fo r Nissl bodies and cytochrome oxidase in the laboratory of Dr. Robert Switzer (Neuroscience Associates, Inc., Knoxville, TN). These sections were analyzed and found to corroborate findings for the j uvenile and adult. The left hemisphere cortex of each brain wa s removed and flattened in fixative overnight under a uniformly weighted glass plate. Once flat tened, 60 m serial frozen microtome sections were cut tangentially to the pial surface. Adjacent series of s ections were then stained for cytochrome oxidase (all specimens), for Nissl subs tance with cresyl violet (all specimens), and for myelin with gold chloride (TM0339, TM 0406, and TM0410 only). The cytochrome oxidase procedure (Wong-Riley, 1979) was modified for ma natee tissue by staining overnight, and the gold chloride procedure (Schmue d, 1990) was used with adjustment of pH to 6.3. Once staining was complete and sections were mounted onto ge latinized slides, the slides were coverslipped using Eukitt. The right hemisphere of each br ain remained unflattened and was processed to

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112 allow for detailed analysis of cytochrome oxida se staining within cyto architectural boundaries. Sections were cut coronally, but otherwise follo wed the above protocol (frozen-sectioned at 60 m and with identical staining procedures). All cortical sections were viewed under an Olympus BH-2 microscope, a Bausch and Lomb microprojector, and a Zei ss Axiophot microscope. Tangential sections from the flattened left hemisphere stained for cytochrome oxidase were placed on a light Table and sequential sections were examined for persistence of the visible patterns. Representative sections were scanned with a HP ScanJet 5370C and merg ed using Adobe Photoshop. Composites were completed for each brain (Fig. 4-1) with the exception of TM0410, where inadequate perfusion of the left hemisphere only permitted analysis of the right hemisphere coronal sections. Images of flattened composites were then analyzed us ing image analysis software (MCID, Imaging Research, Inc.) to determine the percentage of area that presented cytochrome oxidase-dense staining when using threshold and areal morpho metry analysis and also when areas were outlined by hand (Fig. 4-1B; Table 4-2). Coronal sections were examined for cytochro me oxidase-dense patches and compared to adjacent Nissl body sections in order to loca lize patches according to cytoarchitectural boundaries previously noted (Reep et al., 1989 ; Marshall and Reep, 1995). We subsequently related the above results on corona l sections of the right hemisphe re to tangential patterning seen in the left hemisphere. This allowed for accura te localization of patte rns seen on tangential sections, and compensated for distortion inherent in the flattening process. The laminar locations of cytochrome oxidase-dense bands were also assessed through direct comparison of adjacent coronal cytochrome oxidase and Nissl body sections using the Ba usch and Lomb microprojector and the Zeiss Axiophot microsc ope. Rindenkerne were observed, localized cytoarchitecturally,

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113 and then related to the functional areal de lineations proposed in the above analyses. Representative sections used in figures were imaged with a Zeiss Axioplan2 morphometric microscrope. A spaced series of coronal sections from the right hemisphere of specimen TM0410 was imaged using MCID software in order to cr eate a true three-dimensional reconstruction of cytoarchitectural area overlap with cytochrome oxidase staining (Fig. 4-7). Each section was aligned and imaged using a CCD black and white camera model 72S (Dage MTI, Inc.) attached to a lightbox. The lateral surface of each section was then outli ned by hand with user-specified colors to delineate the extent of cytoarchitectur al areas determined from Nissl-stained sections. The outlining was then repeated for cytochrome oxidase-dense staining and the reconstructions were then overlapped. Results Areal Patterning Tangential sections taken from flattened co rtex preparations for a neonate (TM0310, Fig. 4-1A), a juvenile (TM0339, Fig. 4-1C), and an adult (TM0406, Fig. 4-1D) were stained for cytochrome oxidase and analyzed for genera l identification of primary sensory areas. Presumptive functional areas of somatosensory cortex were assigned based on the location of cytochrome oxidase-dense patches with “F” repr esenting the face, “FL” the flipper, “B” the body, and “T” the tail (Fig. 4-1A, C-D). Although the frontoparietal region of the neonate had 4 distinct patches (Fig. 4-1A), th e juvenile and adult specimens sh ared a similar pattern of one large patch blending the presumptive functional areas into one continuou s domain (Fig. 4-1C, D). Primary auditory (Fig. 4-1A, C-D patch “A1”) and visual (Fig. 4-1A, C-D, patch “V1”) areas were also assigned based on cytochrome oxidase-dense staining and location. The percentage of flattened cortical surface that stained darkly for cytochrome oxidase, and therefore presumably represented primary se nsory areas, was determined using a computer-

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114 based morphometry program (Fig. 4-1B; Table 42). Percentages were calculated for primary sensory areas versus total cortic al surface area, SI versus tota l cortical surface area, primary somatosensory area (SI) versus cortical surface ar ea of the frontal hemisphere (rostral to the lateral fissure), and primary sens ory areas (presumably encompassi ng A1 and V1) versus cortical surface area of the caudal hemisphe re (caudal to the lateral fissu re). A density threshold was chosen across all specimens such so as to minimize false positives and generate a conservative estimate of percentage of cortical area stained. A measurement for SI versus frontal hemisphere area was also drawn by hand and recalculated as the computer-generated outline appeared to underestimate surface area devoted to SI as can be visually assessed for the frontoparietal hemisphere density threshold generated for sp ecimen TM0310 (Fig. 4-1B) versus staining (Fig. 4-1A). Roughly one third of cortical area was de voted to primary sensor y areas overall. Although the MCID software did appear to underestimate the percentage of SI area in the frontal hemisphere compared to manually delineated outlines of SI, in both cases the neonatal specimen produced smaller percentages of SI area than in th e juvenile and adult. If the self-drawn outlines were indeed more accurate, then the adult specimen showed the most area devoted to SI at 75% of the frontal hemisphere, but taking the com puter-generated percentages as a conservative estimate still assigned over 50% of the frontal cortex to SI in the adult and juvenile specimens. In contrast, only 12% of the caudal hemisphere was devoted to primary auditory and visual areas combined, and this percentage was consistent across age groups. Neonates Coronal sections from the right hemisphere of 2 neonate brains (Table 4-1) were analyzed to match cytochrome oxidase-dense staining with cytoar chitectural boundaries. Although ventral aspects of TM0310 were damaged, and tangential sections of TM0410 were not useful for analysis due to shredding, the neonate brains exhibited consistent trends. Cytochrome

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115 oxidase-dense staining in layer IV corresponded to myelin-rich ar eas overall, with presumptive primary sensory areas also staining for myelin in la yers V and VI (Fig. 4-5) consistent with other species (Hassiotis et al., 2004). Staining for cytochrome oxidase activity is characterized as dense (or intense; e.g., area DL1 layer IV staini ng, Fig. 4-2A), moderate (e.g., layer III staining in rhinal cortex (RH), Fig. 4-2A ), or absent (e.g., area dorsomed ial (DM2), Fig. 4-2D). Within the frontal cortex (FR), cytochrome oxidase st aining rostrally was broa d and dense spanning the dorsal to dorsomedial extent of FR (Fig. 4-2A). Intense staining in area FR spanned layer V and moderate staining persisted in layer VI. In area DL1 intense cytochrome oxidase staining became restricted to layer IV with m oderate staining in layer III (Fig. 4-6B). The DL1 band extended throughout DL1 rostrally and then began to termin ate more dorsally within caudal DL1 (Fig. 4-2, A-E). A band of moderate cytochrome oxidase st aining emerged caudally in layer VI of DL1 in neonatal specimens (Figs. 4-2D, E; 4-6B) but not in juvenile (Fig. 4-3, B-D) or adult (Figs. 4-4A, B; 4-5) specimens. Within area DL2, dense st aining was located in layer IV only caudally along with moderate staining in layer II I and additional moderate layer VI seen only in neonates (Figs. 4-2, A-D; 4-6C). As dorsomedial FR transitioned to area DM3 the dense staining of layers V and VI that characterized FR became laminar and re stricted to layer IV w ith moderate layer III staining in DM3 (Figs. 4-2, A-C; 4-6E), and the extent of DM3 was cytochrome oxidase-dense throughout layer IV of each brain (Fig. 4-2, C-I). DM2 consistently lacked staining (Fig. 4-2, CI). Dorsal cortex (DD) area staining was irregu lar, showing no staining rostrally (Fig. 4-2, B-F) but exhibiting moderate laminar staining in laye r IV for a limited caudal extent (Fig. 4-2G, H). Areas RH and olfactory cortex (OLF) were mode rately cytochrome oxidase-dense in layer III (Fig. 4-2, A-C). Rindenkerne in CL1 stained dens ely for cytochrome oxidase in layer VI but were clearest in myelin and Nissl body stai ns for areas CL2 through CL5 in all specimens

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116 examined. Intense layer IV, modera te layer III and additional laye r VI staining in neonates only also appeared throughout the caudal extent of CL2 rostral to the lateral fissure (Figs. 4-2E; 46A). Caudal to the lateral fissure moderate CL2 staining appeared in laye r IV (Fig. 4-2, F-G) whereas CL1 stained only lightly for cytochrome oxidase (Fig. 4-2G). Area DD2 displayed moderate staining in layer III (Figs. 4-2, I-L; 4-6F ). All of CL3 was cytochrome oxidase-dense in layer IV whereas the dorsal extent of areas CL 4 and CL5 showed moderate staining in their combined layer III/IV (Fig. 4-2, H-L). Splenial su lcal cortex (SS) was consistently cytochrome oxidase-dense in layer IV, as was all of DL3 and to a moderate extent th e ventral portion of DL4, both in layer IV (Fig. 4-2, J-L). Juvenile and Adult Adult (TM0406) and juvenile (TM0339) sp ecimens displayed similar cytochrome oxidase-dense staining patterns, a nd therefore a representative seri es from only the juvenile is shown for reference (Fig. 4-3) with selected exam ples from the adult used to illustrate similar trends (Fig. 4-4). Results for areas CL1, CL2, CL3, DD, DD2, DM2 and DM3 were also confirmed in specimen TM2. Rostral area FR exhi bited diffuse, dense stai ning (Fig. 4-3A). Area DL1 demonstrated intense laminar staining restricted to layer IV with moderate staining in layer III (Fig. 4-3, B-D). Only the caudal portion of DL2 stained densely for cytochrome oxidase and staining was restricted to layer IV with moderate layer III stai ning (Fig. 4-3B, C). Area DM3 was consistently cytochrome oxidase-dense in layer IV with moderate staining in layer III, and DM2 displayed diffuse cytochrome oxidase activity rost rally (Fig. 4-3, B-C) but not caudally (Fig. 4-3, D-K), whereas DM1 exhibited no cytochrome oxidase activity. Area DD displayed dense cytochrome oxidase activity in layer IV (Figs. 4-3E, F; 46D) whereas DD2 demonstrated moderate layer III activit y (Fig. 4-3, G-N). In area CL2, dens e staining was present in layer IV both rostral and caudal to the lateral fissure, except at the most rostral extent of CL2, along with

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117 moderate layer III staining (Figs. 4-3, C-J; 4-6A). Area CL1 stained moderately for cytochrome oxidase in the dorsocaudal region bor dering the lateral fissure (Fig. 4-3, G-J). The entire span of CL3 exhibited intense cytochrome oxidase st aining in layer IV (Fig. 4-3J, K). Area CL4 exhibited moderate layer III/IV staining dorsally (Fig. 4-3K) a nd CL5 also expressed moderate staining in layer III/IV (Fig. 43, L-O). Area DL3 had intense staini ng in layer IV (Fig. 4-3L, M) whereas DL4 showed moderate layer IV staini ng (Fig. 4-3N, O). Caudal pole cortex (CP) exhibited moderate to light stai ning, SS stained intensely in laye r IV (Fig. 4-3, L-O), and medial wall cortex area two (MW2) stained mode rately in layer III (Fig. 4-3, K-M). The new histochemical information provided above might indicate a reassessment of previously assigned cytoarchit ectural regions, as the caudal portion of areas CL2 and DL2 stained for cytochrome oxidase whereas the rost ral portion (and lateral po rtion in the case of older specimens) of each region did not (Fig. 4-6A, C). Given this ad ditional histochemical information, we suggest that the rostral and caudal portions of areas CL2 and DL2 be referred to as alpha and beta subdivisions, respectively. Neonate versus Juvenile and Adult Comparison As noted previously (Sarko and Reep, 2005), cytochrome oxidase staining patterns in neonate versus juvenile and a dult specimens differed. Area DL1 staining occurred more dorsally in the more developed animals (Figs. 4-2D; 4-3, B-D; 4-4B) as did CL2 staining (Figs. 4-2E; 43D; 4-4C). Area DD staining was also prominent in the older animals (Figs. 4-3E, F; 4-4A, B) but not in neonatal specimens (Fig. 4-2, B-F) Finally, neonatal specimens exhibited a moderately staining cytochrome oxidase band in layer VI of areas DL1, DL2 and CL2 that was absent in juvenile and adult specimens. Vari ations in cytoarchitecture between neonates and older animals also became apparent. Area DM2 neve r extended to the dorsal aspect of the cortex

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118 in neonates (Fig. 4-2, A-C) but was present in th e juvenile (Fig. 4-3B) and the adult. Also, the medial aspect of area FR extended much further caudally in the neonatal specimens. Discussion Utilizing the ability of cytochrome oxidase to preferentially stain primary sensory areas, we have determined which cytoarchitectural re gions are likely to have specific functional significance for the Florida manatee (Fig. 4-7) Though sample size was necessarily limited due to the manatee’s endangered status, these resu lts expand on, and are ofte n in agreement with, those areas proposed by Mars hall and Reep (1995) based on cytoarchitecture alone. Somatosensory Cortex Primary somatosensory cortex appears to correspond to areas DM3, DL1, CL2, CL1, and portions of DD, DL2, and DM2. Areas DM3, DL1, a nd CL2 each have a well organized layer IV and were previously identified as potentia l candidates for SI (M arshall and Reep, 1995). Although the presence of areas with and without Rindenkerne in presumptive SI remains puzzling, it seems likely that CL areas represen t vibrissal input whereas areas DL, DM, and DD mainly accommodate cutaneous information, with roughly half of presumptive SI devoted to each type of input. Area DM3 likely encompasse s the tail representation of SI, due to its dorsomedial location, pronounced layer IV, and cyto chrome oxidase-dense staining in layer IV. However, a caveat to the proposal that non-cluster cortex repres ents cutaneous input would be that, by extension, the tail repres entation of the cortex lacks clear vibrissal representation because area DM3 lacks Rindenkerne. The manat ee tail contains a hair density distribution comparable to that of the trunk of the body, making the absence of any cort ical representation of tail vibrissae unlikely (Reep et al., 2002). Manatees also manipulate their ta ils as they navigate through the water, implicating th e tail in sensory feedback thr ough a turbid water environment. Therefore, we hypothesize that the ta il vibrissae are in fact represente d in the cortex but that their

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119 small size and innervation (Reep et al., 2001) potentially correspond to relatively small Rindenkerne just as smaller, less innervated mystacial vibrissae pr oject to relatively small barrels in the rodent cortex (Woolsey and Van der Loos, 1970; Lee and Woolsey, 1975; Woolsey et al., 1975; Welker and Van der Loos, 1986; Rice, 1995), and that such Rindenkerne might be detectable by more sensitive methods. Welker and Van der Loos (1986) also discovered that histological cortex preparations only produced visible barrels if the corresponding whiskers had a threshold number of afferents. Therefore it is pos sible that the tail vibr issae of the manatee are below this innervation threshold as has been pr oposed for buccal pad sensory hairs in the naked mole-rat (Henry et al., 2006). Ar ea DL1 is located more laterall y and has a well-developed layer IV that consistently stains for cytochrome oxidase, making it a likely candidate for the body representation. The adjacen t cytoarchitectural area CL2 also ha s a well-developed layer IV that stains for cytochrome oxidase and might lend itsel f to the body representation in its more medial portion and possibly the flipper and face repres entations in its lateral portion. Area DL2 exhibited cytochrome oxidase-dense staining excl usively at its caudal extent, making it possible that this area represents facial information. Due to differential staining patterns, rost ral DL2 is reclassified here as DL2 with the caudal, cytochrome oxidase-dense portion denoted DL2 (Figs. 4-1E; 4-6C). Similarly, the lateral extent of CL2 was found to lack cytochrome oxidase reactiv ity in layer IV and is thus renamed CL2, with CL2 signifying the larger, cytochrome oxidase-dense region (Figs. 4-1E; 46A). Area CL1, though lacking layer IV, maintains cytochrome oxidase staining of the largest Rindenkerne present in the cortex and therefor e remains a likely candidate for the facial representation as well, possibly specialized for the extensive se nsorimotor integration involved with oripulation during use of th e largest vibrissae. Prominent cytochrome oxidase staining in

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120 DD and moderate dorsal DM2 staining that was s een only in the juvenile and adult specimens likely contribute to the body representation as we ll, given the dense staining in a well-developed layer IV in each area. The differences seen in areal patterning between neonate versus juvenile and adult specimens (Fig. 4-1A, C-D) are also reflected in the analysis of coronal sections (Figs. 4-2, A-E; 4-3, A-G; 4-4) and app ear to be due to more dorsal st aining of areas CL2 and DL1 as well as more extensive staining of DD and dorsolate ral DM2 seen in the older animals. Riddle et al. (1992) compared the growth of functional representations within SI in juvenile and mature rats and found that the representation of the head enlarged to a greater ex tent than that of the paw. They also found that SI grew to a greater extent than the neocorte x overall, demonstrating that the cortical expansion is not uniform but rather exhibits re gional growth differences. Due to the close relationship between the periphery and th e cortical map, and the instructional role that the periphery plays in organizing the layout of somatosensory cortex (Killackey et al., 1990; Catalano et al., 1995), the di fferences seen in SI of neonate ve rsus juvenile and adult manatees may reflect modifications of the sensory periphery reflected in reorganization of SI in the more developed animals, although the limitation of small sample size precludes assignment of a definitive causal mechanism. Area DD2 presented moderate staining restricted to layer III. As shown by Eskenasy and Clarke (2000), area SII in huma ns demonstrates cytochrome oxida se activity in layer III whereas cytochrome oxidase-dense activit y in SI was restricted to la yer IV. Due to its location and cytochrome oxidase reactivity in layer III, area DD2 of the manatee brain might be a candidate for SII. Supragranular cytochrome oxidase staini ng was also found in secondary sensory areas in other species such as the tammar wallaby (A shwell et al., 2005), whereas the motor cortex exhibited broad, dense staining as shown here for the manatee. SI appears to occupy roughly

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121 25% of total cortical area (Table 4-2), which is comparable to other somatosensory specialists such as the naked mole-rat in which SI comprise s approximately 31% of neocortex (Catania and Remple, 2002). As hypothesized, SI also appears to occupy proportio nately greater neocortical area than A1 and V1 (Table 4-2) in the manatee. Th is is characteristic of other tactile specialists such as the echidna and the platypus whose 4 so matosensory areas occupy approximately 75% of total sensory cortex (Hassi otis et al., 2004; Krubitzer et al., 1995b; Ulinski, 1984). Auditory and Visual Cortex As hypothesized by Marshall and Reep (1995), pr imary auditory cortex appears to span area CL3, but appears to include area CL2 caudal to the lateral fissure as well. Both CL2 and CL3 presented dense cytochrome oxidase staining of layer IV and of Rindenkerne in layer VI. Layer IV staining characterized th e entirety of each area. Caudal to the lateral fissure, only the Rindenkerne of area CL1 stained for cytochrome oxidase, making this an unlikely primary auditory area but a potential audi tory association area. The presen ce of only cluster cortex areas in what is hypothesized to be primary auditory cortex is puzz ling if Rindenkerne are in fact cortical representations of vibriss ae. However, it is plausible that this could represent an overlap of auditory and somatosensory information an alogous to the parietal ventral (PV), ventral somatosensory (VS), or caudomedial (CM) area s found in other taxa (Cusick et al., 1989; Krubitzer et al., 1995a, b; Beck et al., 1996; Kaas and Collins, 2001; Schroeder et al., 2001). Such multisensory integration could conceiva bly involve low-frequency sounds or other hydrodynamic stimuli detected through movement of vibrissae on the manatee body (Reep et al., 2002). Indeed, in determining the underwater audi ogram of the West Indian manatee, it was discovered that behavioral res ponses to low-frequency (<0.4 kHz) sounds changed such that one manatee pivoted its body roughly 45 degrees and lowered its head (a response not exhibited for frequencies >0.4 kHz), which the authors attributed to switchi ng detection of sound stimuli to

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122 vibrotactile sensation (Gerstein and Gerstein, 1999). However, if Rindenkerne are in fact functional representations of t actile hairs, then the entirety of A1 would overlap with presumptive somatosensory function caudal to the lateral fissure in cytoarchitectural areas CL3 and CL2, each of which has a well-developed laye r IV that stains densely for cytochrome oxidase. Although the bimodal areas such as PV, VS or CM could offer a partial explanation, it seems unusual that a separate A1 would not exist in the manatee cortex unless somatosensory information is so crucial to perception of th e environment that a complete overlap became adaptive. In the opossum Monodelphis domestica Catania et al. (2000) showed that A1 overlaps with the trunk, body, and ear repr esentations in the opossum’s unus ually large S2, but this case appears to be an exception. Primary visual cortex appears to span cyto architectural areas DL3 and DL4, as postulated by Marshall and Reep (1995). Each area has a well-organized layer IV that stains densely for cytochrome oxidase. The dorsal aspects of area s CL4 and CL5 are also cytochrome oxidasedense, but the ventral, larger proportion of each stains only moderately, making these areas likely candidates as visual association areas. Area SS ha s prominent staining in layer IV as well, but layer IV is only moderately organized with cel ls that appear more pyramidal than granular (Marshall and Reep, 1995), making it unlikely to be part of the pr imary visual cortex. The broad, moderate staining of area CP, in addition to it s well-developed cortical layers, mark it as a potential multimodal association area that, based on its location, is mostly dedicated to visual information. In order to completely characterize the mana tee somatosensory system and to elucidate specializations that have presumab ly arisen as adaptations to th e unique ecological niche of this species, it will be necessary to thoroughly examin e this system at each level of processing.

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123 Whether there exist somatosensory specializations in the manatee thalamus and brainstem, particularly those that might be associated with the cortic al Rindenkerne, remains an open question that we are currently investigating. Characterization of nerv e endings present in vibrissae, especially interregi onal differences and specializa tions, would also further our understanding of the manatee’s perc eptual capabilities and adaptations.

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124 Table 4-1. Summary of specimen data. Animal Number Gender Length (in cm) Weight (in kg) Classification TM0310 F 127 13.6 Neonate TM0339 F 200 167.8 Juvenile TM0406 M 290 393.2 Adult TM0410 M 94 14.5 Neonate Table 4-2. Percentage of cortical area represented by presumptive sensory cortex. 1 areas/total cortex SI/totalSI/frontal area SI (manual delineation)/ frontal area 1 areas/ caudal area TM0310 35 18 35 49 12 TM0339 36 28 55 66 12 TM0406 31 27 53 75 12

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125 Figure 4-1. Tangential sections stained with cyto chrome oxidase and merged to encapsulate the full extent and persistence of areal pattern ing in left hemisphere flattened cortex preparations for A) neonate (TM0310), C) juvenile (TM0339), and D) adult (TM0406) specimens. Presumptive functional areas within SI have been assigned such that “F” represents the face, “FL” the flipper, “B” the body, and “T” the tail. Primary auditory (A1) and visual (V1) areas have also been assigned based on cytochrome oxidase-dense staining pattern s (A, C-D). B) A computer-generated density threshold generated for cytochro me oxidase-dense staining in specimen TM0310. E) A cytoarchitectural schematic of the manatee brain modified from Marshall and Reep (1995) to include and subdivisions of CL2 and DL2. This schematic illustrates differences in lami nar organization as revealed by Nissl body staining and was used as a template for loca lization of cytochrome oxidase staining in the present study. A-E: left is rostral, up is dorsal.

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126

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127 Figure 4-2. Rostrocaudal series of coronal sections relating cytochrome oxidase staining to cytoarchitectural boundari es (determined by Nissl body and myelin stains of adjacent sections) in a neonate brain (TM0410). Black arrows indicate examples of Rindenkerne seen in areas CL1 and CL2 best illustrated in B but evident in A-E. Right is medial, up is dorsal. The insets of a manatee brain schematic reveal planes of section for A-L. Dotted lines within the schematic approximate locations of the lateral and horizontal fissures separating th e frontal and caudal hemispheres.

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128

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129 Figure 4-2. Continued

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130 Figure 4-3. Rostrocaudal series of coronal sections relating cytochrome oxidase staining to cytoarchitectural bounda ries in a juvenile brain (T M0339). Right is medial, up is dorsal. The inset of a manatee brain reveals planes of section for A-O.

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131 Figure 4-3. Continued

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132 Figure 4-3. Continued

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133 Figure 4-4. Coronal cytochrome oxidase sections from an adult specimen (TM0406) revealing trends consistent with the juvenile specimen but distinct from the neonate (see text for details). Right is medial, up is dorsal. The inset of a manatee brain reveals planes of section for A-C.

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134 Figure 4-5. Adjacent sections stained for myelin, cytochrome oxidase, and Nissl bodies illustrate consistently dense staining in layer IV in both myelin and cytochrome oxidase preparations of presumptive primary sensory areas (specimen TM0406, area DL1 shown). Myelin preparations also exhib it moderate layer V and dense layer VI staining.

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135 Figure 4-6. Localization of cytoch rome oxidase-dense staining with in cortical layer boundaries for each cytoarchitectural area. A) Area CL 2 is shown with absence of cytochrome oxidase staining rostrally (l eft; specimen TM0339, Fig. 4-43F equivalent section), presence of cytochrome oxidase staini ng caudally (right; spec imen TM0410, Fig. 4-42E equivalent), and stained for Nissl bodies (center; TM0410, Fig. 4-4-2E equivalent) to show laminar restriction of cytochrome oxidase-dense staining to layer IV, with a moderately staining band also present in la yer VI in neonates. Scale bar = 1mm for all sections. White arrowheads show staining ar tifact in A and B. B) Area DL1 exhibits cytochrome oxidase-dense staining restrict ed to layer IV (specimen TM0410, Fig. 44-2D equivalent) in addition to a moderate ly staining band also present in layer VI (for neonatal specimens). C) Caudal area DL2 is shown with little cytochrome oxidase staining rostrally (l eft) versus cytochrome oxidase-dense staining caudally restricted to layer IV and a moderately dense cytochrome oxidase band in layer VI (right; specimen TM0410, Fig. 4-4-2D; laye r VI band present only in neonatal specimens). D) Area DD exhibits cytochrome oxidase-dense staining restricted to layer IV (specimen TM0339, fig 3F equiva lent sections). E) Area DM3 has cytochrome oxidase-dense staining restrict ed to layer IV (specimen TM0410, Fig. 44-2D). F) Area DD2 (specimen TM0310) demonstrates moderate, supragranular staining.

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136

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137 Figure 4-7. Three-dimensional reconstruction of neonatal specimen TM0410. The color-coded cytoarchitectural map was reconstructed from a series of spaced coronal sections from the right hemisphere, stained for cres yl violet. Superimposed upon this (in gray shading) is a map of cytochrome oxidase staining reconstructed from an adjacent coronal series stained for cytochrome oxidase. The composite was then smoothed to compensate for discrepancies between cy toarchitectural and cytochrome oxidase boundaries drawn by hand. Right is rostral, up is dorsomedial.

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138 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS Summary and Conclusions This work has contributed extensively to our understanding of the Florida manatee somatosensory system. For the first time, imm unofluorescent labeling was used to characterize sensory innervation of tactile hairs (vibrissae) of the manatee body and face. From this we discovered that facial vibri ssae exhibited dense Cand A -fiber innervation of the epidermis and rete ridge collar, dense Merkel cell and moderate longitudinal la nceolate ending distribution at the ring sinus, and fine-caliber innervation located along the tr abeculae of the cavernous sinus. Postfacial vibrissae contained Me rkel endings and dense Cand A -fiber distribution at the rete ridge collar. Dense Merkel endi ng networks were present at th e inner conical body and ring sinus levels along with moderate longitudinal lanceo late ending innervation. The cavernous sinus contained fine-caliber innervation. By knowing th e response properties of each type of sensory ending in other species, this gene ral characterization of innervati on in manatee vibrissae gives us an indication of what stimuli manatees are cap able of detecting in their environment. Two types of novel, presumptive low-threshol d mechanoreceptors (BNaC+), were also discovered. Novel “tangle” endings were found along the mesenchymal sheath at the lower inner conical body/upper ring sinus leve l of both facial and postfacial vibrissae, and novel trabecular endings were found at the level of the cavernou s sinus along the connect ive tissue in facial vibrissae only. Endings with these characteristic s (size, location along the axis of the folliclesinus complex, and immunolabeling characterizati on) have not been previously documented in any species. “Tangle” endings may c onfer additional directionality detection to the follicle-sinus complex (FSC) through association with deflection of the hair sh aft against the upper trabeculae while the trabecular endings may be responsive to tension induced by defl ection of exceptionally

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139 rigid vibrissae which might invol ve modified sensory endings in order to optimally detect deflection. Since only the facial vibrissae exhibited the trabecular endings, these mechanoreceptors may have evolved to facilitate or ipulative behavior that is unique to sirenia and combines sensorimotor functions. Facial vibrissae are in fact more densely inne rvated, with more varied sensory endings, in accordance with their behavioral importance in active tactile e xploration. Furthermore it seems that manatees are heavily invest ed in directionality detection, an adaptation that would enhance their perception of underwat er hydrodynamic stimuli. Histochemical and cytoarchitectural analysis of the brainstem, including stains for cytochrome oxidase (CO), myelin, and Nissl bodies, showed that somatosensory nuclei of the brainstem (Bischoff’s, trigeminal, and cuneate-gr acile nuclei) appear di sproportionately large. The trigeminal and cuneate-gracile complex show evidence of parcellation that may be somatotopically related to disc rete body areas, tho ugh no clear evidence of “barrelettes” was discovered. Similar histochemical and cytoarchitectural an alysis of the thalamus, including stains for CO, myelin, acetylcholinesterase, and Nissl bodies showed that the somatosensory nucleus, the ventroposterior (VP) nucleus w ith its medial (VPM) and latera l (VPL) subdivisions, appears disproportionately large. No evidence of “barreloids” was f ound. Reduced areas related to vision (e.g., LP and LGN) appear to be overtaken by ar eas related to somatosensation (VP and Po) and audition (MGN). We further postu late that many of these subnuclei may be multimodal and function in subcortical inte gration of auditory and somatosensory information. Histochemical and cytoarchitectural analysis was also completed for the neocortex of the Florida manatee in order to localize primar y sensory areas and particularly primary

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140 somatosensory cortex (SI). Based on the locati on of CO-dense staining in flattened cortex preparations, and in coronal sections systematical ly analyzed in order to accurately localize the laminar and cytoarchitectural ex tent of CO staining, prelimin ary functional divisions were assigned for SI with the face represented la terally followed by the flipper, body and tail representations proceeding medially Overall, SI appears to occupy roughly 25% of total cortical area, which is comparable to other somatosensor y specialists such as the naked mole-rat, and spans seven cytoarchitec tural areas. We hypothesize that ar ea dorsomedial cortex (DM3) is dedicated to the tail representation; areas dorsola teral cortex (DL1), dorsal cortex (DD) (in the juvenile and adult specimens) and the dorsal por tion of cluster cortex (CL2) serve as the body representation; the lateral extent of CL2 represents the flipper; and DL2, ventrolateral CL2, and CL1 represent the face. Area DD2 may represent a secondary somatosensory area (SII). The neonate cortex exhibited four distin ct patches in the fr ontoparietal region (presumptive SI) of flattened cortex prepara tions, whereas juvenile and adult specimens demonstrated a distinct pattern in which CO-dense staining appeared to be blended into one large patch extending dorsomedially. This differentia l staining between younger versus older, more developed animals was also seen on coronal sect ions stained for CO, mye lin, or Nissl bodies and may indicate modifications of the sensory periphery reflected in reorganization of SI in the more developed animals. Primary auditory cortex appears to span area CL3 and area CL2 caudal to the lateral fissure. The presence of only cluste r cortex areas in what is hypot hesized to be primary auditory cortex indicates extensive overlap of auditory and somatosensor y information analogous to the parietal ventral (PV), ventral somatosensory (VS), or caudomedial (CM) areas found in other taxa (Cusick et al., 1989; Krubitzer et al., 1995a b; Beck et al., 1996; Kaas and Collins, 2001;

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141 Schroeder et al., 2001). This proposed multisenso ry integration may involve low-frequency sounds or other hydrodynamic stimuli detected through deflection of vibrissae on the manatee body. Primary visual cortex appears to span cy toarchitectural areas DL3 and DL4. Areas CL4 and CL5 are likely candidates as visual associa tion areas while area caudal pole cortex (CP) may function as a multimodal association area primarily dedication to visual processing. This study is the first 1) to characterize sensory endings present in manatee vibrissae using immunofluorescence, and 2) to localize an d characterize primary somatosensory areas of the brainstem, thalamus and cortex in the Flor ida manatee using a metabolic marker (cytochrome oxidase) that distinguishes prim ary sensory areas. This information substantially enhances our understanding of the manatee’s somatosensor y specializations, and adaptations to its environmental niche. Such knowledge is becoming increasingly critical as the numbers of this endangered species dwindle. It is our hope that elucidating the percep tual capabilities of manatees will aid in conservation efforts to preserve them as a species. Furthermore, as an evolutionary outlier, manatees offer the unique opportunity to add significant information to comparative neurobiology and allow a better under standing of organizing pr inciples of sensory systems in general. Future Directions Although innervation of manatee vibrissae wa s characterized for the types of nerve endings present at each level of the follicle sinus complex and compared across 3 facial regions and 7 postfacial regions of the manatee body, exact quantification of the distribution density of nerve endings was not completed. This quantif ication would provide a direct method for comparison with other mammalian somatosensory sp ecialists and allow us to further elucidate

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142 the extent of somatosensory speci alization in the Florida manatee. Furthermore, the information presented here is only a microl evel assessment of the anatomical substrates involved in manatee perceptual abilities; behavioral analyses are needed to confirm what behaviorally relevant stimuli manatees actually detect. The locations of primary somatosensory areas defined here for the brainstem, thalamus and cortex would be greatly enhanced by electrophysiological or axonal tracing studies examining the exact functional boundaries of both the overall somatosensory region and the functional representation of each body region within it. Such invasive techniques are not feasible at present due to the manatee’s status as an e ndangered species, but true functional delineations can only be hypothesized based on comparative neurobiological pr inciples until more direct assessments are possible. Examination of the c onnectivity of individual Rindenkerne, especially in areas containing the largest Rindenkerne (CL1 and CL2) that may be related to facial vibrissae, is also critical in understanding this uniquely sireni an neuroanatomical specialization. Although a “fastDiI” experiment was completed by injecting DiI into non-perfused manatee brain sections in order to exam ine the connectivity of CL1 Rindenke rne, these data have not yet been analyzed. Further characterization of be haviorally relevant subnuclei of the brainstem would also benefit our understandin g of the Florida manatee’s physiology and perceptual abilities. The present study shows that such areas as the vestibular nuclei and the vagus nucleus are exceptionally large in the manatee, but these were not examined in detail. While manatees and dugongs represent evolu tionary outliers as the only remaining families under the order Sirenia, their closest (yet distant) relatives, elephants and hyraxes, have been largely neglected as well in terms of phys iological and neurobiolog ical analysis. This

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143 neglect of an entire supraordinal group, the Af rotheria (Kemp, 2005), represents a major gap in our knowledge of comparative evolution. Gro ss morphological and functional analyses are sparse (see Sale, 1970; Shoshani et al., 2006 for exceptions) and must be expanded upon. Current investigations in our laboratory involve analyz ing the somatosensory system of the rock hyrax for comparison with the Florida manatee. Additional Considerations The present study suggests broader questions about the development and evolutionary divergence involved in creating the unique attributes seen in the Florida manatee. For instance, it is a uniquely sirenian attribute to possess a distribution of tactile hairs on the entire body. The question becomes: how was the developmental pl an modified such that tactile hairs are distributed over the entire manatee body, and not just in restricted re gions? Zelena (1994) describes the development of vibrissal follicles in four stages. First, sensory axons grow from the nerve plexus towards the epidermis and bran ch into the mesenchyme. Mesenchyme then aggregates around the nerve branches, followed by thickening of the epidermis above the mesenchymal condensation. Finall y, the budding follicle grows down toward the nerve branches and into the mesenchymal condensation. Howeve r, whether sensory axons induce mesenchymal condensation or the mesenchyme itself induces fo llicle development remains an open question. It does appear that neural cell adhesion molecule (NCAM) is critical in formation of the coordinated pattern of nerves and whiskers in rat maxillary vibrissae (Scarisbrick and Jones, 1993). Other signals such as WNT, DKK, BMP, FG F, and Lef1 appear to be involved in hair follicle spacing (Sick et al., 2006). However, w hy certain animals retain vibrissae in only restricted regions (e.g., maxillary, carpal and supr aorbital regions), while manatee vibrissae are distributed over the entire body, remains unclear. It is possible, though it s eems unlikely, that this

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144 is the basic mammalian plan and has only been retain ed in its complete form in sirenia, but has been lost (except in restricted, specialized regions of the body) in other mammals. One must assume that because innervation to tactile hairs on the entire body is a metabolically costly commitment, vibrissae have only been retained wh ere they confer information critical to the animal’s successful adapta tion to its environment. Regarding the issue of Rindenkerne developm ent an additional que stion becomes: how did manatees evolve to form barrel-like stru ctures in layer VI? If Rindenkerne are truly analogous to barrels, it seems r easonable to hypothesize that similar developmental principles govern the formation of both neuroanatomical structures. Little is known about the exact mechanisms involved in barrel field formation, although it does appear th at growth-associated protein (GAP-43) is critical to the formation of an ordered vi brissal representation in the rat (Maier et al., 1999). Recent studies using monoa mine oxidase-A (MAO-A), 5-HT transporter, vesicular monoamine transporter-2 (VMAT2) a nd 5-HT1B receptor single, double and triple knockout mice also indicate that th e serotonergic system plays an important role in barrel field formation in the developing somatosensory cort ex (Luo et al., 2003), but the possible genetic basis for barrel field formation remains elusive.

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145 APPENDIX LETTER OF PERMISSION TO REPRODUC E COPYRIGHTED MATERIAL (THE ENTIRETY OF CHAPTER 4) Dear Dr. Sarko, Thank you for your e-mail below. As for your request, I am pleased to inform you that permission is granted herew ith to use the article Sarko DK, Reep RL. 2007. Somatosensory areas of manatee cerebral cort ex: Histochemical characterization and functional im plications. Brain Behav Evol, 69:20–36. in your doctoral dissertation provided that complete credit is given to the original source and S. Karger AG, Basel is mentioned. Best wishes, Isabelle Flckiger Rights and Permissions S. Karger AG Medical and Scientific Publishers Allschwilerstrasse 10 CH 4009 Basel Tel +41 61 306 14 75 Fax +41 61 306 12 34 E-Mail permission@karger.ch

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155 Schmued LC. 1990. A rapid, sensitive histochemical st ain for myelin in frozen brain sections. J Histochem Cytochem 38:717-720. Schnupp JW, King AJ. 1997. Coding for auditory space in the nucleus of the brachium of the inferior colliculus in the ferret. J Neurophysiol 78:2717-2731. Schroeder CE, Lindsley RW, Specht C, Ma rcovici A, Smiley JF, Javitt DC. 2001. Somatosensory input to auditory associ ation cortex in the macaque monkey. J Neurophysiol 85:1322-1327. Shore SE. 2005. Multisensory integration in th e dorsal cochlear nucle us: Unit responses to acoustic and trigeminal ganglion s timulation. Eur J Neurosci, 21:3334-3348. Sick S, Reinker S, Timmer J, Schlake T. 2006. WNT and DKK determine hair follicle spacing through a reaction-diffusion mechanism. Science. Simons DJ. 1978. Response properties of vibrissa units in the rat SI somatosensory neocortex. J Neurophysiol 41:798-820. Simons DJ, Woolsey TA. 1979. Func tional organization in the m ouse barrel cortex. Brain Res 165:327-332. Stephens RJ, Beebe IJ, Poulter TC. 1973. Innervati on of the vibrissae of the California sea lion, Zalophus californianus Anat Rec 176:421-441. Strata F, Coq JO, Kaas JH. 2003. The chemoand somatotopic architecture of the Galago cuneate and gracile nucle i. Neurosci 116:831-850. Suga N, Ma X. 2003. Multiparametric corticofuga l modulation and plasticity in the auditory system. Nat Rev Neurosci, 4:783-794. Tuckett RP. 1978. Response of cutaneous hair an d field mechanoreceptors in rat to paired mechanical stimuli. J Neurophysiol 41:150-156. Tuckett RP, Horsch KW, Burgess PR. 1978. Response of cutaneous fair and field mechanoreceptors in cat to threshold stimuli. J Neurophysiol 41:138-149. Ulinski PS. 1984. Thalamic projections to th e somatosensory cortex of the echidna ( Tachyglossus aculeatus ). J Comp Neurol 229:153-170. Van der Loos H. 1976. Barreloids in the mouse somatosensory thalamus. Neurosci Lett 2:1-6. Welker E, Van der Loos H. 1986. Quantitative correlation between barrel-field size and the sensory innervation of the whiskerpad: A comp arative study in six strains of mice bred for different patterns of mystacial vibrissae. J Neurosci 6:3355-3373. Welker WI. 1973. Principles of organization of the ventrobasa l complex in mammals. Brain Behav Evol 7:253-336.

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156 Welker WI, Johnson J. 1965. Correlation be tween nuclear morphology and somatotopic organization in ventrobasal complex of the raccoon’s thalamus. J Anat 99:761-790. Welker WI. 1974. Principles of organization of the ventrobasa l complex in mammals. Brain Behav Evol 7:253-336. Welker WI, Adrian HO, Lifschitz W, Kaulen R, Caviedes E, Gutman W. 1976. Somatic sensory cortex of llama ( Lama glama ). Brain Behav Evol 13:284-293. Welker WI., Johnson JI, Reep RL. 1986. Morphol ogy and cytoarchitecture of the brain of Florida Manatees ( Trichechus manatus ). Soc Neurosci Abstr 12:110. Werner-Reiss U, Kelly KA, Trause AS, Underhill AM, Groh JM. 2003. Eye position affects activity in primary aud itory cortex of primat es. Curr Biol, 13:554-562. Wong-Riley M. 1979. Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxida se histochemistry. Brain Res 171:11-28. Wong-Riley M.T.T, Welt C. 1980. Histochemical ch anges in cytochrome-oxidase of cortical barrels after vibris sal removal in neonatal and adult mice. PNAS 77:2333-2337. Woolsey TA, Van der Loos H. 1970. The struct ural organization of layer IV in the somatosensory region, SI, of mouse ce rebral cortex. Brain Res 17:205-242. Woolsey TA, Welker C, Schwartz RH. 1975. Compar ative anatomical studies of the SmI face cortex with special references to the occurr ence of “barrels” in la yer IV. J Comp Neurol 164:79-94. Woolsey TA, Welker C, Schwartz RH. 1975. Comp arative anatomical studies of the SmI face cortex with special reference to the occurren ce of “barrels” in layer IV. J Comp Neurol 184:363-380. Woudenberg RA. 1970. Projections of mechanreceptive fields to cuneate-gracile and spinal trigeminal nuclear regions in sheep. Brain Res, 17:417-437. Yan J, Zhang Y, Ehret G. 2005. Corticofugal shapi ng of frequency tuning cu rves in the central nucleus of the inferior colliculus of mice. J Neurophysiol, 93: 71-83. Yohro T. 1977. Structure of the sinus hair follicle in the big-clawed shrew, Sorex unguiculatus J Morph 153:333-354. Yu YQ, Xiong Y, Chan YS, He J. 2004. Corticof ugal gating of auditory information in the thalamus: an in vivo intracellular recording study. J Neurosci, 24:3060-3069. Zelena J. 1994. Mechanoreceptor complexes associated with hair. In: Nerves and Mechanoreceptors: the role of innervati on in the development and maintenance of mammalian mechanoreceptors, London: Chapman & Hall. p 243-260.

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157 Zhou J, Shore SE. (in press). Convergence of spinal trigem inal and cochlear nucleus projections in the inferior colliculus of the guinea pig. J Comp Neurol. Zhou YD, Fuster JM. 2004. Somatosensory cell respons e to an auditory cue in a haptic memory task. Behav Brain Res, 153:573-578. Zook JM. (in press). Somatosensory adaptations of flying mammals. In: Evolution of Nervous Systems (Kaas JH, Krubitzer L, ed s), NY: Elsevier Press. chpt. 8.03. Zook JM. 2005. The neuroethology of touch in ba ts: cutaneous receptors of the wing. Soc Neurosci Abstr 78.21. Zook JM, Fowler BC. 1986. A specialized mechanor eceptor array of the bat wing. Myotis 23-24.

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158 BIOGRAPHICAL SKETCH Diana Kay Sarko was born April 28, 1981 in We st Palm Beach, Florida. She graduated from the Math, Science and Engineering (MSE) program at Suncoast Community High School in 1999. Earning enough advanced placement credits in high school to satisfy a year of college, she graduated magna cum laude from Emory University three years later with a B.S. in neuroscience and behavioral biology. Her honor s thesis involved creating an ethogram for bottlenose dolphin mirror self-recognition behavior. Diana’s enrollment in the interdisciplinary (IDP) graduate program of the College of Medicine at the University of Florida began in the fall of 2002. From there, she entered the Department of Neuroscience to earn her Ph.D. a nd joined the lab of Dr. Roger Reep analyzing the somatosensory system of the Florida mana tee. Through this lab, she became co-affiliated with the College of Veterinary Medicine a nd the Marine Mammal Program, which allowed her to participate in necropsies at Sea World, FL, and the Department of Environmental Protection Florida Marine Research Institute (DEP-FMRI) in St. Petersburg, FL. She also served as a student board member of the Save the Manatee Club in order to f acilitate education outreach and manatee awareness. Diana has accepted a postdoctora l position in the la bs of Dr. Kenneth Catania and Dr. Jon Kaas at Vanderbilt University in Nashville, Te nnessee. She will move there with her fianc, Jeremy (JJ) Kennard, who works at a hospi tal as a nuclear medicine technologist.


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Title: The Florida Manatee Somatosensory System
Physical Description: Mixed Material
Copyright Date: 2008

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Table of Contents
    Title Page
        Page 1
        Page 2
    Dedication
        Page 3
    Acknowledgement
        Page 4
    Table of Contents
        Page 5
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    List of Tables
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    List of Figures
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    Abstract
        Page 11
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    Introduction
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    Innervation of follicle-sinus complexes in the Florida Manatee
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    Somatosensory nuclei of the manatee thalamus and brainstem
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    Somatosensory areas of manatee cerebral cortex etc.
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    Conclusions and future directions
        Page 138
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    Appendix
        Page 145
    References
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    Biographical sketch
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Full Text





THE FLORIDA MANATEE SOMATOSENSORY SYSTEM


By

DIANA KAY SARKO



















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2006

































Copyright 2006

by

Diana Kay Sarko

































In loving memory of my grandfather, John Sarko, who always supported my educational pursuits
and who I wish could be here on my graduation day, and every day









ACKNOWLEDGMENTS

First and foremost, I thank my parents. Their support and their own pursuit of knowledge,

both of them going back to school later in life to earn master's degrees, is an inspiration. I thank

my mentor Dr. Roger Reep for showing me everything that an advisor should be-available,

good-humored, supportive, and able to balance work with play. I only hope that one day when I

have my own lab that I can do him justice. I am grateful to my committee members for their

time, wisdom and invaluable contributions to this project: Dr. Gordon Bauer, Dr. Pete McGuire,

and especially Dr. Floyd Thompson, from whom I have learned a great deal about being an

exceptional professor and researcher. Many, many thanks are due to Maggie Stoll, who has not

only dealt with troubleshooting on every level, but has been with me through personal trials that

I could not have undergone alone.

I thank my love and my fiance JJ Kennard for his patience and devotion, and especially

for helping me to turn my brain to the much-needed, often-ignored "off' position now and then.

Besides Roger and Maggie, my time in this lab has also afforded me the pleasure and privilege of

meeting Dr. Joe Cheatwood, who gave me guidance when I first joined the lab and who will be a

lifelong friend. Many thanks to Susan Oliver, my best friend, who always knew that I could get

through this, even when I had serious doubts. I also thank Dave Schoenberg, who has been a

shoulder and one of my dearest friends since college; and Kevin Chadbourne, who has gone

above and beyond what it has taken to maintain my sanity towards the end of this journey. Every

little thing that he has done has helped me more than he will ever know.

Certain events from my graduate career have made me realize that life is too short to

waste pursuing anything but what you love to do. These last four and a half years I have had the

privilege to do just that. I have learned so much from so many, and yet I have only just begun.









TABLE OF CONTENTS



A C K N O W L E D G M E N T S ..............................................................................................................4

L IST O F T A B L E S ......................................................................................................... ........ .. 7

LIST OF FIGURES ......................................................... ...........................8

A B S T R A C T .......................................................................................................... ..................... 1 1

CHAPTER

1 INTRODUCTION .... .................................... .. ......... ............ ............... 13

T he F lorida M anatee .......................................................................................... .. .... .. ...... 13
Sensory Specializations of the M anatee Body ........................................... ................ 13
Perioral V ibrissae .. ..................................................................................................... 15
The M anatee B rain: G general A ttributes................................................. ......... ................ 18
Cytochrome Oxidase: A Metabolic Marker for Primary Sensory Areas.............................. 19
Brainstem Som atosensory Nuclei and Barrelettes............................................. ................ 21
T halam us and B arreloids ............... .. .................. .................. .......................... .................23
Rindenkerne ............................. ........... .. .............. ........ .................... 24
The Cerebral Cortex: Relating Cytoarchitecture to Electrophysiology...............................25

2 INNERVATION OF FOLLICLE-SINUS COMPLEXES IN THE FLORIDA
M A N A T E E .......................................................................................................... ........ .. 2 7

In tro du ctio n ............................................................................................................. ........ .. 2 7
M materials and M methods .............. .............................................................................. 30
R esu lts ........................................................................................................... 34
F racial V ib rissae ............................................................................................................... 3 4
P o stfacial V ib rissae ......................................................................................................... 3 7
D iscu ssio n ........................... .................................................................................... ......... 3 8
M anatee Vibrissae: Overall Comparative Structure................................... ................ 38
Facial Musculature Involved in Exploratory and Prehensile Vibrissal Behaviors..........39
Sensory Innervation of the Rete Ridge Collar and Epidermis ..................................39
Sensory Nerve Endings of the Inner Conical Body and Ring Sinus ..............................40
C avernous Sinus Innervation .......................................... ......................... ................ 43
M arine M am m al V ibrissae ..................................................................... ................ 45
C om parative C considerations .......................................... ......................... ................ 46

3 SOMATOSENSORY NUCLEI OF THE MANATEE THALAMUS AND
B R A IN ST E M ...................................................................................................... ....... .. 65

In tro du ctio n ............................................................................................................. ........ .. 6 5
M materials and M methods .............. .............................................................................. 68









Results .................................................... .............................. 70
Brainstem .............................................. .............................. 70
T h alam u s .............................................................................................. ..................... 7 2
D iscussion.................... .... ................. ............... ................................75
Brainstem: Somatotopic Parcellation in Other Species ................................................75
Thalamus: A Comparative Look at Somatosensory Nuclei .........................................77

4 SOMATOSENSORY AREAS OF MANATEE CEREBRAL CORTEX:
HISTOCHEMICAL CHARACTERIZATION AND FUNCTIONAL IMPLICATIONS... 108

Introduction .............................................. .............. .................. 108
M materials an d M eth o d s .........................................................................................................1 10
Results .......................................................... .................. 113
A re a l P a tte rn in g .............................................................................................................1 1 3
N e o n a te s ........................................................................................................................1 1 4
Ju v en ile an d A d u lt ........................ ... ............................ ..............................................1 16
Neonate versus Juvenile and Adult Comparison.............................................117
D iscu ssio n ..................... ....................................................... ......................................1 1 8
Som atosensory C ortex .............................................................................................118
A uditory and V isual Cortex .....................................................................................121

5 CONCLUSIONS AND FUTURE DIRECTIONS .......... .....................................138

Su m m ary an d C on clu sion s ...................................................................................................13 8
F u tu re D ire ctio n s ..................................................................................................................14 1
A d edition al C on sid eration s .................................................................................................... 14 3

APPENDIX: LETTER OF PERMISSION TO REPRODUCE COPYRIGHTED
MATERIAL (the entirety of chapter 4)............ .............................145

L IS T O F R E F E R E N C E S .............................................................................................................14 6

B IO G R A P H IC A L SK E T C H .......................................................................................................158



















6









LIST OF TABLES


Table page

2-1 Specim en categorization ................................................. ............................................ 48

3-1 Sum m ary of specim en inform ation...................................... ...................... ............... 82

3-2 Comparative analysis of percentage of thalamus occupied by the ventroposterior
nucleus (VP; averaged from 3 evenly spaced coronal sections to encompass VP). ..........82

4-1 Sum m ary of specim en data ......................................................................... ............... 124

4-2 Percentage of cortical area represented by presumptive sensory cortex....................... 124









LIST OF FIGURES


Figure page

2-1 Vibrissae sam pling regions of the body and face.. ....................................... ................ 48

2-2 Schematic drawing of the structure and innervation of the U2, BLH, and postfacial
vibrissal follicle-sinus complexes (FSCS) with innervation types and sensory nerve
en d in g s illu stated ............................................................................................................. 4 9

2-3 Characterization of upper perioral field 2 (U2) follicle innervation.............................. 51

2-4 Innervation of the cavernous sinus and hair shaft medulla in facial follicles.................53

2-5 Innervation present in bristle-like hairs (BLH s)........................................... ................ 55

2-6 Representative postfacial vibrissae innervation includes dense networks of MEs
along w ith LLE s and "tangle" endings.. ....................................................... ................ 57

2-7 Im m unolabeling attributes of innervation..................................................... ............... 59

2-8 Confocal surface reconstructions showing the three-dimensional structure of
representative follicle innervation and novel mechanoreceptors present in the ICB,
R S an d C S reg io n s............................................................................................................. 6 1

2-9 Confocal three-dimensional images of novel endings stained for neurofilament
(N F200) and protein gene product 9.5 (PGP)............................................... ................ 63

3-1 A rostrocaudal series of representative coronal brainstem sections with subnuclei
labeled illustrates the size and extent of somatosensory nuclei....................................83

3-2 Brainstem sections cut in the sagittal plane illustrate the rostrocaudal extent of
behaviorally relevant nuclei and in particular the lobulated appearance of the
trig em in al n u clei.............................................................................................................. .. 8 8

3-3 Brainstem sections cut in the horizontal plane show the topography and orientation
of nu clei of interest............................................................................................................ 89

3-4 Representative coronal brainstem sections illustrating the appearance of each of the
trigem inal subnuclei in an adult specim en.................................................... ................ 90

3-5 A rostrocaudal series of representative coronal brainstem sections in a neonate shows
that somatosensory nuclei are large and have a parcellated appearance as seen in
a d u lt sp e cim en s................................................................................................................ .. 9 2









3-6 A rostrocaudal series of representative coronal thalamic sections with low-
magnification images of sections stained with hematoxylin for myelin and high-
magnification details of adjacent sections stained with thionin for Nissl bodies with
su b n u clei lab eled ...................................................................................................... 9 5

3-7 A rostrocaudal series of closely spaced coronal sections showing the ventroposterior
area (V P) of the thalam us in detail.. ..................... ................................................ 100

3-8 Low-magnification and high-magnification images characterizing Nissl body
staining of the lateral ventroposterior (VPL) and medial ventroposterior (VPM)
subnuclei of the thalam us.. .................................................................... ............... 102

3-9 Histochemical and histological staining characterization in the ventroposterior
nucleus of the thalamus ................... ............ ............................. 103

3-10 Coronal thalamus sections stained for cytochrome oxidase (CO) from a neonate
(specimen TM0410) and a juvenile (specimen TM0339) show that the
ventroposterior thalamus (VP) exhibits homogenous CO-dense staining without
clearly distinguishable barreloids.. ...................... ................................................. 104

3-11 Fiber laminae (arrows) seen most distinctly in the juvenile specimen (TM0339) may
separate adjacent projections from adjacent body parts into subnuclei of the thalamus
as dem onstrated in other species.................................... ....................... ............... 106

3-12 Horizontal myelin-stained section showing unusual placement of the medial (MGN)
with respect to the lateral geniculate nucleus (LGN)....... .................... ................... 106

3-13 Proposed somatotopy of functional representations within the brainstem
somatosensory nuclei (cuneate-gracile and trigeminal) and the ventroposterior
nucleus (VP) of the thalamus in the coronal plane of section................ ...................107

4-1 Tangential sections stained with cytochrome oxidase and merged to encapsulate the
full extent and persistence of areal patterning in left hemisphere flattened cortex
preparations for A) neonate (TM0310), C) juvenile (TM0339), and D) adult
(T M 0406) specim ens.. ......................................................................................... 125

4-2 Rostrocaudal series of coronal sections relating cytochrome oxidase staining to
cytoarchitectural boundaries (determined by Nissl body and myelin stains of adjacent
sections) in a neonate brain (TM 0410).. ........................................................................ 127

4-3 Rostrocaudal series of coronal sections relating cytochrome oxidase staining to
cytoarchitectural boundaries in a juvenile brain (TM0339).. ....................... 130

4-4 Coronal cytochrome oxidase sections from an adult specimen (TM0406) revealing
trends consistent with the juvenile specimen but distinct from the neonate (see text
for d details) .. .... ........ ............................................................................. .......... 13 3









4-5 Adjacent sections stained for myelin, cytochrome oxidase, and Nissl bodies illustrate
consistently dense staining in layer IV in both myelin and cytochrome oxidase
preparations of presumptive primary sensory areas (specimen TM0406, area DL 1
show n) ................................................................................................... 134

4-6 Localization of cytochrome oxidase-dense staining within cortical layer boundaries
for each cytoarchitectural area...................................... ........................ ............... 135

4-7 Three-dimensional reconstruction of neonatal specimen TM0410 ............................ 137









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

THE FLORIDA MANATEE SOMATOSENSORY SYSTEM

By

Diana Kay Sarko

December 2006

Chair: Roger L. Reep
Major Department: Medical Sciences-Neuroscience

Florida manatees are thought to be tactile specialists, and in an effort to systematically

characterize this system, the research presented here first used immunolabeling to functionally

characterize sensory innervation in facial follicles with behavioral relevance in object

recognition and exploration as well as in follicles from select perioral and postfacial regions.

Facial vibrissae exhibited dense C- and A6-fiber innervation of the epidermis and rete ridge

collar, novel "tangle" endings at the inner conical body level, dense Merkel cell and moderate

longitudinal lanceolate ending distribution at the ring sinus, and novel endings located along the

trabeculae of the cavernous sinus. Postfacial vibrissae contained Merkel endings and dense C-

and A6-fiber distribution at the rete ridge collar. Dense Merkel ending networks and "tangle"

endings were present at the inner conical body and ring sinus levels along with moderate

longitudinal lanceolate ending innervation. No novel endings were present within the

trabeculated cavernous sinus of any postfacial vibrissae. We conclude that the facial vibrissae are

in fact more densely innervated, with more varied sensory endings, in accordance with their

behavioral importance in active tactile exploration. Furthermore, it seems that manatees are

heavily invested in directionality detection, an adaptation that would enhance their perception of

underwater hydrodynamic stimuli.









A histochemical and cytoarchitectural analysis was also completed for the brainstem,

thalamus, and neocortex of the Florida manatee in order to localize primary sensory areas. Based

on the location of cytochrome oxidase (CO)-dense staining, we found that somatosensory nuclei

of the brainstem (Bischoff's, trigeminal, and cuneate-gracile nuclei) and thalamus (VP) appear

disproportionately large and, in the case of the trigeminal and cuneate-gracile complex, show

evidence of parcellation that may be somatotopically related to discrete body areas. Flattened

cortex preparations stained for CO were assigned preliminary functional divisions for S with

the face represented laterally followed by the flipper, body and tail representations proceeding

medially. Coronal cortical sections stained for CO, myelin, or Nissl bodies were also

systematically analyzed in order to accurately localize the laminar and cytoarchitectural extent of

CO staining. Overall, S appears to span seven cytoarchitectural areas for which we have

proposed functional assignments.









CHAPTER 1
INTRODUCTION

The Florida Manatee

Manatees belong to the order Sirenia, of which over 35 species existed during the past 50

million years, with only 4 remaining presently (Domning, 1982). There are three extant manatee

species: the West Indian, of which the Florida manatee (Trichechus manatus latirostris) and the

Antillean manatee (T manatus manatus) are subspecies; the Amazonian, T inunguis; and the

West African, T senegalensis. As the only obligate herbivores among marine mammals,

sirenians possess unique behavioral, physiological, and neuroanatomical adaptations. The US

Fish and Wildlife Service currently classifies the Florida manatee as endangered, a status that is

supported by their low total population which was estimated at the last aerial survey in February

2004 to be 2,568 manatees (provided by the Manatee Technical Advisory Council and the

Manatee Population Status Working Group). In the year 2003 alone, the Florida Fish and

Wildlife Conservation Commission Marine Mammal Pathobiology Laboratory reported a total of

380 manatee deaths-a significant percentage of the population, indicating that the opportunity

to learn from this unique species is rapidly disappearing.

Sensory Specializations of the Manatee Body

Manatees appear to have reasonably well-developed hearing (Gerstein and Gerstein,

1999) but reduced vision (e.g., Bauer et al., 2003). Though little is known about the extent of

their olfactory or gustatory capabilities, these appear to be senses of subordinate importance to

the manatee as well (Levin and Pfeiffer, 2002; Mackay-Sim et al., 1985). However, recent

evidence suggests the presence of a sophisticated tactile sense through a system of sinus-type

tactile hairs, or follicle sinus complexes (FSCs), covering the entire postfacial body (Reep et al.,

2002). The postfacial body is covered in approximately 3,000 hairs with hair density decreasing









dorsoventrally (Reep et al., 2002) and calves exhibit greater hair density distribution that is

attributed to the fixed number of follicles in a mammal at birth. Hair density then decreases with

age as the body, and especially the midsection, of the manatee expands (Reep et al., 2002). Each

body hair has an external length of 2-9 mm, with most hairs separated by 20-40 mm, giving

each hair an independent field of movement (Reep et al., 2002). A single body vibrissa is

innervated by 20-50 axons whereas 40-200 axons supply innervation to each facial vibrissa

(Reep et al., 2001; 2002). The distribution of vibrissae over the entire postfacial body is a unique

arrangement among mammals, most of which have tactile hairs restricted only to certain body

regions, and is proposed to be analogous to the lateral line system in fish by functioning as a

"touch at a distance" sense through passive deflection of tactile hairs by hydrodynamic stimuli.

Such a system is potentially capable of conveying crucial information about water currents, the

approach of other animals, and other features of the underwater environment (Reep et al., 2002).

The manatee face possesses further sensory specializations that aid in adaptation to the

animal's unique environmental niche. Facial hair is distributed thirty times more densely than on

the rest of the body (Reep et al., 1998), an attribute that should increase spatial resolution, and

can be distinguished from body hair by the greater stiffness of facial hair due to smaller

length/diameter ratios (Reep et al., 1998). Body hair is located on the supradisk portion of the

face posterior to the orofacial ridge and on the chin in addition to the entire postfacial extent of

the body (Reep et al., 1998). Manatees have an expanded philtrum called the oral disk that

contains bristle-like hairs (BLHs) that are used as tactile "feelers" in addition to perioral bristles

that are essentially modified vibrissae (Reep et al., 1998). Vibrissae provide detailed textural

information about objects and surfaces in an animal's immediate environment, and most

mammals use vibrissae exclusively for sensory purposes such as finding prey and navigating









successfully when vision is compromised, such as in low-light situations (Brecht et al., 1997;

Dehnhardt et al., 1998; Dehnhardt et al., 2001; Ling, 1977). Facial hair is crucial in manatee

feeding and tactile exploration of the environment, accomplishing dual and synergistic motor and

sensory roles. The hair and bristles of the manatee face are composed of 9 distinct regions, 6 of

which are perioral bristle-4 upper perioral fields (U1-U4) on each side of the upper lips and

oral cavity, and 2 lower perioral fields (L1-L2) on each side of the lower lip pad (Reep et al.,

1998). Each of these follicles can be classified as a vibrissa according to the criteria established

by Rice et al. (1986): 1) substantial innervation, 2) a dense connective tissue capsule, and 3) a

prominent, circumferential blood sinus complex. The 9 regions of the manatee face are

discernible by location as well as the number, range of length/diameter ratios, and behavioral

role of follicles within each field (Reep et al., 2001).

Perioral Vibrissae

The BLHs of the oral disk are the vibrissae primarily involved in object recognition and

tactile exploration, whereas U2 and LI follicle fields are used in a prehensile grasping fashion

during feeding and oripulation (a combined sensorimotor function that is unique among

mammals) as well as in social behaviors including mouthing, nuzzling, and also pinching a

conspecific's back in an attempt to gain access to food (Reep et al., 2001; Marshall et al., 1998b).

The right and left U2 bristle fields specifically act in a prehensile manner during feeding by

reaching out and grasping food while L bristles actively push vegetation farther into the oral

cavity (Marshall et al., 1998b). The Ul vibrissae may also be involved in some level of tactile

exploration during feeding while the U3, U4, and L2 fields may assist LI bristles in the

movement of food (Marshall et al., 1998b). Upon encountering a particularly difficult food item,

manatees can use each U2 field independently, even reversing direction in order to expel

undesirable food (Marshall et al., 1998b). Such evidence reveals a high level of dexterity and









perioral tactile discrimination and is supported by the manatee's relative tactile difference

threshold of 14%-favorably comparable to that of an Asian elephant's trunk (Bachteler and

Dehnhardt, 1999). Notably, the eyes are often closed during feeding and tactile exploration

(Marshall et al., 1998b; Bachteler and Dehnhardt, 1999), further indicating an emphasis on

haptic over visual input. The prehensile ability of facial tactile hairs is present in dugongs as

well, but absent in pinnipeds despite their higher tactile resolving power (Bachteler and

Dehnhardt, 1999; Marshall et al., 1998b; Marshall et al., 2003).

In an earlier study of manatee follicle innervation U2s were found to contain the largest

FSCs composed of the longest hair shafts, the widest ring sinuses, the thickest capsules, and the

highest degree of innervation at over 200 axons per follicle (Reep et al., 2001). The LI bristles

are innervated by the second largest number of axons at approximately 200 per FSC, followed by

U3, U4, and L2 bristles (approximately 100) and finally Ul bristles, whose range overlaps that of

the BLH vibrissae at 49-74. The body hair follicles of the chin and supradisk contain the least

axonal innervation with a range of 34-48 axons per follicle. Most FSC axons terminate in the

mesenchymal sheath and the outer root sheath lining the hair follicle proper along the level of the

ring sinus. Reep et al. (2001) described general morphological features and axonal counts for

each follicle type but the silver staining was often inconsistent with inadequately defined nerve

endings. This limitation can be solved through a systematic analysis using immunolabeling, and

given the co-varying behavioral and sensory tasks for which each follicle field is specialized,

concurrently varying attributes in innervation patterns might be elucidated through

immunofluorescence.

Upon examination of muscular supply to facial vibrissae, Reep et al. (1998) discovered

that the dorsal and ventral buccal branches of the facial nerve supply the lips and perioral regions









with the dorsal branch supplying the upper lip and nasal area and the ventral branch terminating

in the lower jaw and lip muscles to enable vibrissal version and feeding behavior. Furthermore,

each facial bristle follicle in the U1-U4 fields is supplied by the infraorbital branch of the

maxillary nerve (the sensory trigeminal branch), making these fields homologous to mystacial

vibrissae. Sensory innervation of the lower jaw is provided by the inferior alveolar branch of the

mandibular nerve while the lingual branch innervates the tongue and the mylohoid branch

courses ventrally to innervate M. mylohyoidus and the ventral mandible skin. The inferior

alveolar branch separates into 2 mental nerves, supplying LI and L2 and making them

homologues of the mental vibrissae present in other taxa.

Comparative trends in mammals indicate that vibrissae have evolved to perform complex

functions in order to provide feedback about an animal's environment, but although sensory

detection is often accompanied by vibrissal movement, it is not accompanied by prehensile

grasping behaviors (Reep et al., 2001). Harbor seals appear to use vibrissae in touch

discrimination as effectively as a monkey is able to utilize its hands (Dehnhardt and Kaminski,

1995), and pinnipeds as a whole have been found to employ their long vibrissae in tactile

exploration as well as in social display behavior (Dehnhardt, 1994; Dehnhardt and Ducker, 1996;

Dehnhardt and Kaminski, 1995; Peterson and Bartholomew, 1967; Miller, 1975; Kastelein and

Van Gaalen, 1988). Rodents utilize a whisking behavior of their mystacial vibrissae in tactile

exploration (Carvell and Simons, 1990; Welker, 1964; Wineski, 1985) and freshwater river

dolphins (Platanistidae), which have poor vision, use vibrissae on their upper and lower jaws to

locate prey (Ling, 1977). Sensory specializations of the skin and hair in mammals are

accompanied by expanded cortical representations to accommodate the greater level of neural

input (Johnson, 1990; Kaas and Collins, 2001). A clear example of this can be seen in the star-









nosed mole (Condylura cristata). The 22 fleshy nasal appendages, or rays, that it uses to explore

the environment can be seen in cytochrome oxidase preparations of somatosensory cortex with a

distinct band corresponding to each ray (Catania and Kaas, 1995, 1997). Catania and Kaas

(1997) further discovered that the eleventh appendage, used preferentially in environmental

exploration, also assumes the largest cortical representation. Therefore, it is reasonable to

hypothesize that in the manatee additional neuroanatomical space would be allotted to

complement tactile specializations with a particularly expanded facial representation. We

examine this further by identifying and characterizing the somatosensory areas of the brainstem,

thalamus and cortex in order to more completely understand any specializations that might be

present and might complement the Florida manatee's adaptation to its environmental niche.

The Manatee Brain: General Attributes

The Florida manatee brain possesses a unique and intriguing set of attributes that

combine more primitive traits with those considered to be quite derived. The former include a

very smooth (highly lissencephalic) cortex and an extremely small brain size compared to what

would be expected for an animal of its body size, a parameter known as the encephalization

quotient, or EQ. The EQ of the manatee was found to be 0.27, or about 1/4 the value expected

for its body size (O'Shea and Reep, 1990). The gyration index, a measure of cortical folding, of

1.06 for the Florida manatee quantifies the high level of lissencephaly observed (Reep and

O'Shea, 1990). Johnson et al. (1994) also reexamined phylogenetic classifications by examining

a number of brain traits that were scored as primitive or derived across 152 mammalian species.

Manatees were found to be primitive in possessing the following attributes: 1) an optic tract that

terminates in closely apposed nuclei of the thalamus, 2) a lack of fasciculus aberrans, 3) no

visible separation of the claustrum from cortex, and 4) no clear separation between external

cuneate nucleus and cuneate nucleus. Dietary features such as the low quality and abundance of









food along with a low metabolic rate are also characteristic of many mammals with EQs in the

lower mammalian range, including the manatee (McNab, 1978; 1980).

Despite the above evidence, most of the life history, ecological and behavioral traits of

the Florida manatee are typical of large-brained species with higher EQs (O'Shea and Reep,

1990). With a gestation period of approximately 1 year, an age range at sexual maturity spanning

5-10 years, an average interbirth interval of 2-5 or more years, and longevity in the wild

estimated at 50-60 years (Hartman, 1979), manatees appear to be more typical of altricial species

following principles of K-selection. Also, while the manatee brain is small relative to its body

size, the telencephalon comprises 71% of the total brain volume, 90% of which consists of

cerebral cortex. The cortex also possesses well-defined laminae. These qualities are comparable

to taxa with large relative brain size, including primates (Reep and O'Shea, 1990). The Johnson

et al. (1994) study further indicated that manatees have the following derived brain traits: 1) lack

of accessory olfactory formation, involved in pheromone detection, 2) deep position of the optic

tract in the collicular tectum, 3) emergence of the facial nerve ventral to the trigeminal sensory

column, 4) olfactory bulb mitral cells gathered into a monolayer, 5) hemispheres connected by a

corpus collosum, 6) medial position of the ventral nucleus to the principal nucleus of the inferior

olive, 7) presence of Rindenkerne, cell clusters in cortical layer VI, and 8) a delaminated dorsal

cochlear nucleus. Johnson et al. also proposed that the secondary loss of lamination in the

auditory dorsal cochlear nucleus along with loss of the accessory olfactory formation indicate

convergent evolutionary consequences of departure from a terrestrial habitat.

Cytochrome Oxidase: A Metabolic Marker for Primary Sensory Areas

Cytochrome oxidase (CO) is an effective endogenous metabolic marker for neurons due

to the tight coupling between neuronal activity and oxidative metabolism (Wong-Riley, 1989 for

review). This enzyme is an integral transmembrane protein found in the inner mitochondrial









membrane of all eukaryotes and generates ATP through oxidative phosphorylation (Wikstrom et

al., 1981). CO accounts for over 90% of oxygen consumption by eukaryotes (Wikstrom et al.,

1981) and is vital for organs like the brain that rely on oxidative metabolism-in the case of

neurons, particularly in the maintenance of ionic balance (Lowry, 1975; Sokoloff, 1974). It has

been suggested that dendritic metabolism makes the single largest contribution to this metabolic

activity since the level of oxidative enzymes in dendrites reflects the intensity and type of

synaptic input to a neuron (DiFiglia et al., 1987; Kageyama and Wong-Riley, 1982; Kageyama

and Wong-Riley, 1985; Lowry, 1954; Mourdian and Scott, 1988; Wong-Riley, 1984). This is

supported by the observation that CO activity levels are responsive to experimentally induced

changes in functional activity (Wong-Riley, 1989 for review).

Cerebral cortex stained for CO shows a laminar pattern of activity with highly active

regions representing the thalamic-recipient and other synapse-rich layers (Carroll and Wong-

Riley, 1984; Jones and Friedman, 1982; Matelli et al., 1985; Price, 1985). Neurons with intense

CO activity are likely to be tonically active and maintain a high enzyme capacity for energy

production to be able to drive the high rate of spontaneous activity (Wong-Riley, 1989). Since

primary sensory areas of the cortex are more tonically active, they are easily discernable when

stained for CO, and it has been found that CO can be used to separate functionally different

cortical areas (e.g., Carroll and Wong-Riley, 1984). In fact, a recent study of human cortex was

successful in differentiating primary and secondary sensory areas through CO and

acetylcholinesterase (AChE) staining. Primary sensory areas 3a and 3b showed dark CO staining

of layer IV and a low level of AChE positive pyramids, a pattern also seen in primary visual and

auditory areas. Secondary association areas 1 and 2 revealed dark CO staining in layer III with

an abundance of AChE positive pyramidal cells (Eskenasy and Clarke, 2000). Physiologically









highly active nuclear groups of the basal ganglia, thalamus, brainstem and spinal cord also show

strong enzymatic activity (DiFiglia et al., 1987; Jones et al., 1986; Nomura and Mizuno, 1986;

Wallace, 1986; Wiener, 1986; Wong-Riley, 1976; Wong-Riley and Kageyama, 1986), making

cytochrome oxidase useful in identifying the primary somatosensory components of the

brainstem and thalamus in addition to the cortex.

Brainstem Somatosensory Nuclei and Barrelettes

Commitment to specific sensory modalities in restricted regions of the body creates a

commensurate commitment of neurons from the periphery through the brainstem, thalamus, and

cerebral cortex. Following this paradigm, if somatic sensation is prevalent for the manatee, then

associated nuclei in the thalamus and brainstem are expected to be relatively larger and/or more

subdivided in order to accommodate the greater amount of information being taken in and

processed. The brainstem nuclei of interest for the manatee somatosensory system include

trigeminal, cuneate, gracile, and Bischoff s nuclei. It has already been noted that cranial nerve V

(the trigeminal nerve) is large in the manatee (Reep et al., 1989). Further studies have shown that

visual thalamic and brainstem nuclei are reduced, whereas trigeminal and other somatosensory

nuclei are well developed (Johnson et al., 1986; 1987; Reep et al., 1989; Welker et al., 1986).

Assessments of the relative importance of these sensory systems in sirenian behavior parallel

these results, particularly for the trigeminal nerve system extensively associated with the use of

the facial vibrissae in tactile exploration, a crucial aspect of manatee behavior. However, these

findings have never been revisited, and more remains to be discovered. Bischoff s nucleus, a

distinct group of cells in the midline of the caudal medulla (Johnson et al., 1968), has not

previously been analyzed in the manatee but has been shown, along with the cuneate and gracile

nuclei, to project heavily to the ventrobasal thalamus in the raccoon (Ostapoff and Johnson,

1988). In the raccoon, the tail representation occupies the dorsal portion of this nucleus while the









hindlimb representation occupies the ventral portion (Johnson et al., 1968). In the manatee,

Bischoff's nucleus would represent the fluke. CO studies of the rat revealed that the afferent

projection pattern from individual vibrissa follicles was topographically related to CO-dense cell

clusters ("barrelettes") in the trigeminal principal sensory nucleus (PSN) with a nearly one-to-

one ratio between follicles and corresponding CO-dense clusters (Florence and Lakshman,

1995). These results supported earlier findings by Jacquin et al. (1993) that showed that PSN

axon collaterals were concentrated within corresponding CO-dense subdivisions, and terminal

branches of individual trigeminal afferents rarely crossed over into adjacent regions. In contrast,

in three subdivisions of the spinal trigeminal nucleus-the pars oralis, pars interpolaris, and pars

caudalis-a topographical arrangement still existed, but with less specificity and more

overlapping representations (Florence and Lakshman, 1995). Goyal et al. (1992) showed that the

human principal trigeminal nucleus also demonstrated a parcellated CO-dense pattern. Therefore,

size and parcellation data for the trigeminal nucleus would further elucidate the sensory

specializations of manatees.

The same principle of CO somatotopic parcellation is also evident in the cuneate and

gracile dorsal column nuclei. Cutaneous inputs from the upper limbs and rostral trunk of the

body are represented in the cuneate nucleus while lower limbs and lower trunk are represented in

the gracile nucleus. Strata et al. (2003) studied the Galago monkey to look at the pattern of

peripheral nerve input. Through cell clusters that were identified as CO-dense blotches in both

nuclei, they discovered a greater segregation of inputs within the cuneate (fingers and hand

representation) than in the gracile (foot representation), which is consistent with the Galago's

extensive and highly differentiated use of its hands and fingers relative to its feet. In macaques,

inputs from specific parts of the hand relate to CO-dense rostrocaudal clusters of cells (Florence









et al., 1991). While the manatee lacks the manual dexterity of a primate, CO analysis of the

cuneate and gracile nuclei would complete the evidence for manatee somatosensory processing

and any concurrent specializations.

Thalamus and Barreloids

In the thalamus, AChE staining reveals robust patterns that allow for the discrimination

of different nuclei and that are consistent in rodents, cats and primates (Jones, 1985). Densest

staining occurs in the ventral lateral geniculate nucleus (LGN), intralaminar, anteroventral,

anterodorsal, rhomboid, paraventricular, habenular, and medioventral nuclei. Lighter staining

distinguishes the dorsal LGN, medial geniculate nucleus (MGN), reticular nucleus, anterior of

the lateral posterior nucleus, and parts of lateral and ventral complexes. The principal somatic

sensory nucleus in the thalamus consists of an area referred to as the ventrobasal (VB) or

ventroposterior (VP) nucleus. A lateral subnucleus, the ventral posterior lateral (VPL) nucleus,

represents the body while a medial subnucleus, the ventral posterior medial (VPM) subnucleus,

represents the face and most of the head (e.g., Jones, 1985). In rodents and marsupials, the

medial division of the VB nucleus (VBm) was discovered to contain "barreloids", or neuronal

clusters related to individual vibrissae, that are highly reactive for CO (Jones, 1983; Land and

Simons, 1985b; Van der Loos, 1976). Chronic trimming of the vibrissae results in reduced

staining for CO in both the somatosensory cortical barrels (Land and Simons, 1985a; Wong-

Riley and Welt, 1980) and the thalamic barreloids (Land and Akhtar, 1987) associated with the

trimmed vibrissae. These findings were similar to those in Macacafascicularis monkeys where

peripheral nerves were cut, resulting in reduced staining of "rods" within the VPM (Jones et al.,

1986). Using horseradish peroxidase axonal tracing, Jones et al. also discovered that CO staining

was primarily due to terminations of trigeminal afferent fibers that formed somatotopically

organized inputs to the rods. They postulated that each rod of the thalamus formed the basis of









columnarity of afferent input to the somatosensory cortex by providing bundles of

thalamocortical axons terminating in focal domains of the cortex. Given the manatee's reliance

on haptic input, the VPM would be expected to be relatively large and may possess barreloid

parcellation related to input from the facial vibrissae.

Rindenkerne

Rindenkerne are cortical cell clusters that stain darkly for cytochrome oxidase and appear

to be unique to sirenia, having been found absent in over 150 other mammalian species examined

(Reep et al., 1989; Johnson et al., 1994). While these cell clusters are reminiscent of "barrels"

found in the vibrissae subfield of somatosensory cortex in rats, mice, and other rodents (Johnson,

1980; Kaas and Collins, 2001), as well as in shrews (Catania et al., 1999), opossums (Catania et

al., 2000; Frost et al., 2000; Huffman et al., 1999), and hedgehogs (Catania et al., 2000), barrels

are hollow aggregates of neurons in layer IV, a major afferent zone. In contrast, Rindenkerne are

dense aggregates located in layer VI, an efferent zone, although they do share histochemical

attributes with barrels (Reep et al., 1989). Furthermore, Rindenkerne distribution in the cortex is

restricted to 5 cytoarchitectural areas termed cluster cortex (CL) 1-5 by Reep et al. (1989) and

Marshall and Reep (1995). The limited distribution of Rindenkerne and the fact that they are

found exclusively in sirenian cortex implies a functional significance. Species possessing barrels

show a one-to-one correspondence between barrels and vibrissae. However, there appear to be

many more clusters than facial bristles in the manatee and it may be that only the larger clusters

(approximately 1 mm in diameter) found in CL1 represent individual bristles while smaller

Rindenkerne such as those found in CL2 may correspond to postfacial hairs (Loerzel and Reep,

1991). However, this hypothesis remains untested until the somatosensory cortex, and

particularly the presumed facial region, can be more precisely delineated.









The Cerebral Cortex: Relating Cytoarchitecture to Electrophysiology

Due to the manatee's status as an endangered species, traditional electrophysiological

methods of ascertaining the location of primary somatosensory cortex (SI) are not possible.

Fortunately, the literature provides a wide range of species for which both electrophysiology and

histochemical processing have been possible. For example, in the marmoset monkey (Huffman

and Krubitzer, 2001), and megachiropteran bats (Krubitzer et al., 1993; Krubitzer and Calford,

1992), microelectrode maps of somatosensory fields were found to be highly correlated with

cytoarchitectural boundaries (specifically flattened cortex cut tangentially and stained for

myelin). A flattened cortex preparation creates a plane of section that includes most of layer IV,

the densest zone of CO staining, while also facilitating the comparative interpretation of areal

patterns and allowing more direct assessment of the extent and relative position of architectonic

fields (Krubitzer et al., 1995). The flying fox, a megachiropteran, was found to have myelin-

dense zones in hand and face representations in area 3b (or SI) that involved non-habituating

cutaneous receptors responding consistently to repetitive stimulation, whereas sparse zones

rapidly habituated. In marmoset monkeys electrophysiology was also related to

myeloarchitecture and revealed that the body map representation in area 3a is coextensive with a

strip of lightly to moderately myelinated cortex rostral to the darkly myelinated area 3b. Overall,

non-habituating neurons corresponded with myelin-dense zones considered homologous to area

3b (Krubitzer et al., 1993). Myeloarchitecture has also been compared with CO staining, tracing

methods, and microelectrode recording in the dorsomedial visual area of owl monkeys

(Krubitzer and Kaas, 1993) to reveal functional areas and connectivity. In monotremes, which

share with the manatee the status of being an evolutionary outlier and having a unique

environment to which they have had to adapt, Krubitzer et al. (1995) showed CO staining

patterns that reveal somatosensory specializations and suborganization. Microelectrode mapping









was combined with CO and myelin staining revealed subdivisions and topography of

somatosensory cortex. The neocortices of both the platypus and the short-billed echidna revealed

4 representations of the body surface with SI occupying a large area and containing neurons

mainly responsive to cutaneous stimulation of the contralateral body. The platypus bill had a

disproportionately large representation with CO-dense regions corresponding only to

mechanosensory stimulation and CO-light regions responding to both electrosensory and

mechanosensory stimulation. In a compilation of cortical sensory maps of additional species,

including the squirrel, macaque, and quoll, Krubitzer (1995) depicts homologies that are present

in neocortical organization. Therefore, in lieu of performing electrophysiological studies on the

manatee, a thorough histochemical assessment can still reveal a great deal about sensory

specializations of the brain.

Overall this analysis of the manatee somatosensory system, from an immunofluorescence

analysis of innervation at the periphery to a systematic histochemical examination of the central

nervous system, aims to elucidate in what ways the manatee is a somatosensory specialist and

how it has adapted to evolutionary pressures inherent in the environment that it occupies.









CHAPTER 2
INNERVATION OF FOLLICLE-SINUS COMPLEXES IN THE FLORIDA MANATEE

Introduction

Follicle-sinus complexes (FSCs, or vibrissae) form highly innervated tactile arrays

generally found on a restricted region of the mammalian body-principally the mystacial region.

However, recent evidence suggests that the Florida manatee (Trichechus manatus latirostris)

possesses a sophisticated tactile sense through a system of FSCs distributed over the entire body

(Reep et al., 2002). Manatees are large-bodied, obligate aquatic herbivores (a trait unique among

marine mammals) that lack predators, do not pursue active prey, usually reside in a shallow,

turbid water environment and have greatly reduced visual systems. They appear to have

reasonably developed hearing capabilities (Gerstein and Gerstein, 1999; Mann et al., 2005) but

reduced sight (Bauer et al., 2003), and though little is known about the extent of their olfactory

or taste capabilities these senses also appear to be subordinate based on anatomical assessments

(Levin and Pfeiffer, 2002; Mackay-Sim et al., 1985). The haptic sense may therefore be crucial

in the manatee's detection of environmental cues, and this hypothesis is supported by the

distribution of sinus-type tactile hairs over the entire body with specialized and more densely

packed vibrissae on the face in addition to the elaboration of somatosensory areas at the

neuroanatomical level (Dexler, 1912; Welker et al., 1986; Johnson et al., 1986, 1987, 1994; Reep

et al., 1989, 2001, 2002; Marshall and Reep, 1995; Sarko and Reep, 2007). The postfacial body

is supplied with approximately 3,000 hairs, each having an independent field of movement,

forming an arrangement unique to sirenia that is proposed to be analogous to the lateral line

system in fish (Reep et al., 2002). Such a system is potentially capable of conveying crucial

information about water currents, the approach of other animals, and other features of the









underwater environment through hydrodynamic stimulation of mechanoreceptors (Reep et al.,

2002).

Facial vibrissae are packed thirty times more densely than on the rest of the body, an

attribute that should increase spatial resolution, and they can be distinguished from postfacial

vibrisse by their greater rigidity due to smaller length/diameter ratios (Reep et al., 1998). The

hair and bristles of the manatee face are composed of 9 distinct regions, 6 of which are perioral

bristles (Fig. 2-1B): 4 upper perioral (U1-U4) fields on each side of the upper lips and oral

cavity, and 2 lower perioral (L1-L2) fields on each side of the lower lip pad (Reep et al., 1998).

The 9 follicle regions are distinguishable by location, number, range of length/diameter ratios,

and behavioral role (Reep et al., 2001). Each of these follicles can be classified as a follicle-sinus

complex (FSC) because the follicle and its affiliated dense innervation are surrounded by a blood

sinus encased within a thick connective tissue capsule (Rice et al., 1986). Manatees have an

expanded philtrum called the oral disk that contains bristle-like hairs (BLHs) that are the main

tactile exploration component involved in object recognition (Reep et al., 1998). Postfacial

vibrissae are located on the supradisk portion of the face posterior to the orofacial ridge and on

the chin in addition to the entire postfacial extent of the body (Bachteler and Dehnhardt, 1999;

Reep et al., 1998). Perioral fields U2 and LI are used in a prehensile grasping fashion

("oripulation," a behavior unique among mammals) during feeding as well as in social behaviors

(Reep et al., 2001; Marshall et al., 1998b). The eyes are often closed during feeding and tactile

exploration (Marshall et al., 1998b; Bachteler and Dehnhardt, 1999), further emphasizing haptic

over visual input. Vibrissae are known to provide detailed textural information about objects and

surfaces in an animal's immediate environment, and most mammals use vibrissae exclusively for

sensory purposes such as finding prey and navigating successfully when vision is compromised,









such as in low light situations (Brecht et al., 1997; Dehnhardt et al., 1998; Dehnhardt et al., 2001;

Ling, 1977). In the manatee, facial vibrissae serve dual and synergistic motor and sensory roles

in manatee feeding and direct tactile exploration of the environment (Marshall et al., 1998a,

1998b) with a high level of dexterity and perioral tactile discrimination that is also reflected in

the manatee's relative tactile difference threshold of 14%-comparable to that of an Asian

elephant's trunk (Bachteler and Dehnhardt, 1999). The prehensile function of facial vibrissae is

present in dugongs as well but is absent in pinnipeds despite their higher tactile resolving power

(Bachteler and Dehnhardt, 1999; Marshall et al., 1998b; 2003).

In an earlier study of manatee follicle innervation the U2 fields were found to contain the

largest FSCs having the longest hair shafts, the widest ring sinuses, the thickest capsules, and the

highest degree of innervation at over 200 axons per follicle (Reep et al., 2001). L follicles are

innervated by the second largest number of axons at about 200 per FSC, followed by U3, U4 and

L2 follicles (about 100) and finally Ul follicles, whose range overlaps that of the BLHs at 49-

74. The chin and supradisk follicles exhibit the least innervation, with a range of 34-48 (Reep et

al., 2001; 2002). Although the manatee's status as an endangered species precludes it from more

invasive analysis, Reep et al. (2001) provided data describing general morphological features and

axonal counts for each follicle type. However, silver staining did not consistently reveal the

morphology of nerve endings, a limitation solved here through systematic immunolabeling

analysis using anti-PGP (protein gene product 9.5) as a standard pan-neuronal marker in

combination with several other antigens in order to functionally characterize the innervation of

manatee vibrissal FSCs. Given the varying behavioral and sensory tasks for which each manatee

bristle field is specialized, we would expect to reveal similarly varying attributes in patterns of

innervation, with facial vibrissae engaged in tactile behavior (the U2 and BLH follicles)









exhibiting more densely distributed and varied types of nerve endings. Also, while the

anatomical structure of FSCs remains relatively consistent across a wide range of species,

patterns of innervation often vary considerably, presumably due to evolutionary pressures and

concurrent behavioral demands (Dehnhardt et al., 1999; Ebara et al., 2002). As an evolutionary

outlier, the Florida manatee offers a unique opportunity to better understand mammalian sensory

systems in general by examining a system of vibrissae that has assumed an expanded functional

role. A systematic analysis of manatee FSCs may also elucidate their potential relationship to

cortical cellular aggregates called Rindenkerne (Dexler, 1912; Reep et al., 1989; Marshall and

Reep, 1995; Johnson et al., 1990; 1994) that appear to be similar to barrels found in the

somatosensory cortex of other species (Woolsey et al., 1975; Rice, 1995).

Materials and Methods

Manatees in Florida are endangered and protected under federal law. Postmortem

manatee follicle samples were acquired through the statewide manatee salvage program under

Federal Fish and Wildlife Permit PRT-684532 and IACUC protocol #C233. For each specimen,

necropsy sheets summarizing body morphometrics, body weight, gender, likely cause of death,

and condition upon recovery were obtained. Specimens are outlined in Table 2-1 and included

TM0406 (adult male, euthanized after watercraft impact; 3 BLH and 3 U2 follicles sampled),

TM9728 (adult male, death due to watercraft; 3 rostrodorsal, 1 dorsocentral, and 1 dorsocaudal

follicles sampled), TM0506 (male neonate, suffered multisystemic failure due to immune

suppression secondary to cold stress; 2 U2, 2 BLH, 2 LI, and 1 dorsocaudal body sampled), and

MNW0614 subadultt female, death due to watercraft; 3 follicles from each of 10 body regions of

interest sampled). Hair follicles samples were acquired as available from 6 body regions (Fig. 2-

lA; supradisk, dorsocentral midline, rostrodorsal midline, caudodorsal midline, ventrocentral

midline, dorsal tail, and tail edge) as well as from perioral fields LI, U2, and BLH (Fig. 2-1B) as









described by Reep et al. (1998) using a #11 scalpel blade to extract a block of tissue (roughly

5x5x15 mm) surrounding the follicle of interest. Follicles were cut mediolongitudinally to

facilitate fixation and placed in 4% paraformaldehyde overnight. After 24 hours of fixation

follicles were removed and placed in 0.1M phosphate buffered saline (PBS) and 30% sucrose.

Sections were cut using a cryostat. Sections for conventional epifluorescence evaluation were cut

at 14 rm parallel to the long axis of the follicles. These sections were directly thawed onto slides

subbed with chrome-alum gelatin, allowed to air dry, and immunolabeled on the slides. Follicles

for confocal analysis were cut at 75 [im and the sections were immunolabeled free-floating

before being mounted onto slides. After labeling, the slides were coverslipped using either 90%

glycerin in PBS or Vectashield (Vector Laboratories).

The sections were processed for single and double immunolabeling with the following

primary antibodies:

1. Anti-protein gene product 9.5 (PGP, rabbit polyclonal, 1:800; UltraClone, Isle of Wright,
UK; catalog number RA95101). The antigen was human PGP9.5 protein purified from
pathogen-free human brain. The antibody shows one band at 26-28 kD on Western blot
and is a universal neuronal cytoplasmic protein (Thompson et al., 1983; Wilkinson et al.,
1989).

2. Anti-neurofilament 200 kD subunit (NF, rabbit polyclonal, 1:800; Chemicon International,
Temecula, CA; catalog number AB 1982, lot number 24080051). The antigen was a highly
purified bovine neurofilament polypeptide. The antibody labels phosphorylated and
nonphosphorylated 200kD NF and shows a band at 200kD and bands around 170-180 kD
on Western blot. The NF200 antibody identifies myelinated innervation including Merkel
endings, AP and A6 fibers (Rice et al., 1997).

3. Anti-calcitonin gene related peptide (CGRP, guinea pig polyclonal, 1:400; Peninsula
Laboratories, Inc., San Carlos, CA; catalog number T-5027, lot number 061121). The
antigen is human a-CGRP with the following sequence: H-Ala-Cys-Asp-Thr-Ala-Thr-
Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-
Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Al a-Phe-NH2. The antibody has 100% reactivity
with human and rat a-CGRP, human CGRP (8-37); chicken CGRP, human P-CGRP. It has
0.04% cross reactivity with human amylin and 0% cross reactivity with rat amylin and
with human and rat calcitonin. The CGRP antibody identifies peptidergic C-fiber
innervation and Merkel cells (Rice et al., 1997) and is an endogenous sensory neuropeptide
and a G-protein coupled receptor.









4. Anti-S-100 (anti-Schwann cell protein S100, rabbit polyclonal, used neat; Biogenesis Inc.,
Brentwood, NH, catalog number 8200-0184, lot number A2255). The antigen was purified
bovine S 100 protein. Anti-S 100 has been found to be coextensive with axons, terminal
arbors, and mechanoreceptor endings (Rice et al., 1997).

5. Anti-BNaClua (mammalian brain sodium channel BNaC; rabbit polyclonal; 1:500; gift
from Dr. Jaime Garcia-Afioveros). The antigen was N-terminus peptide
MDLKESPSEGSLQPSSC (corresponding to residues 1-16 of mouse, rat, and human
BNaC lca). The BNaC antibody has been shown to identify low-threshold
mechanoreceptors (Garcia-Anoveros et al., 2001).

Primary antibodies against MBP (myelin basic protein), VR1 or TrpVl (vanilloid

receptor 1; capsacin binder), NPY (neuropeptide Y, labeling sympathetic innervation), TH

(tyrosine hydroxylase), and GAP43 (growth-associated protein 43, marker for neural growth),

used successfully in previous rat, monkey and human studies (Albrecht et al., 2006; Fundin et al.,

1997; Pare et al., 2001) did not produce detectable labeling on manatee tissue.

All 14tm thick sections were first preincubated with 1% bovine serum albumin (BSA)

and 0.3% Triton X-100 in 0.1M PBS for 1 hour, then incubated with a solution of primary

antibody (diluted in PBS with 4% calf serum or 1% BSA and 0.3% Triton X-100) overnight at

4C at high humidity. Slides were then rinsed in PBS for 30 minutes and subsequently incubated

in the dark at room temperature for 2 hours with either Cy3 or Alexa488 for red fluorescence

(1:500) or Cy2 for green fluorescence (1:250) conjugated secondary antibodies (Molecular

Probes, Inc., Eugene, OR; Jackson Immunoresearch Laboratories, Inc., West Grove, PA) diluted

in PBS or BSA with 0.3% Triton X-100. Slides were then rinsed in PBS and either temporarily

coverslipped under PBS (in the case of future double labeling) or permanently coverslipped.

Double labeling was usually accomplished by repeating the immunofluorescence procedure

described above. In some cases double labeling was achieved through a single cycle of

incubations beginning with a 1:1 mix of the monoclonal and polyclonal primary antibodies. To

control for non-specific labeling, incubation with primary antibody was omitted or the primary









antibody was preincubated with a specific blocking peptide. The 75[tm thick sections were

processed free floating in the same dilutions of antibodies as the thinner sections. Incubations

were for 4 days in primary antibodies and overnight in secondary antibodies at 40C. Rinses were

for at least 4 hours.

Sections were analyzed with an Olympus Provis AX70 microscope equipped with

conventional fluorescence: 1) Cy3 filters (528-553 nm excitation, 590-650 nm emission) and 2)

Cy2 filters (460-500 nm excitation, 510-560 nm emission). Fluorescence images were captured

with a high resolution (1280 x 1024 pixels) three chip color CCD camera (Sony, DKC-ST5)

interfaced with Northern Eclipse software (Empix Imaging, Inc., Mississauga, ON). Images were

deblurred using a deconvolution program based on a ltm 2-dimensional nearest neighbor

paradigm (Empix Imaging, Inc., Mississauga, ON). Samples were imaged on a Zeiss LSM

510Meta confocal microscope (Carl Zeiss Micolmaging, Inc., Thornwood, NY) equipped with

an Argon (488 nm exc.) and a green HeNe (543 nm exc.) laser. Emissions were collected using a

Band Pass 500-530 nm emission filter for Alexa Fluor 488. For CY3 either a Long Pass 560 nm

emission filter or a Band Pass 5650-615 nm emission filter was used, depending on whether the

sample was singly or doubly labeled. Images were collected with a Plan-Neofluor 25x/0.8 Imm

corr DIC lens with the pinhole set for 1 Airy Unit. Confocal image Z-stacks were collected at

512 x 512 pixel x-y resolution and 1[tm steps in Z. The 3-D red-green stereo anaglyph (Fig. 2-

9B) and the 3-D stereo pairs (Fig. 2-9, A, D, E) were generated using the Zeiss LM510 software.

The 3-D surface rendered images (Fig. 2-8, A-H) were produced using the VolumeJ plugin in the

ImageJ software software (http://rsb.info.nih.gov/ij/). Figures were assembled using Adobe

Photoshop CS, Adobe Illustrator CS, and Microsoft PowerPoint software.









Since the intensity of immunolabeling for the numerous antibodies used in the present

study is attributed to many variables that cannot be individually quantified, this study does not

attempt to quantify the relative amounts of labeled antigens. These variables include: 1) true

differences in the presence and quantity of the antigen, 2) whether the antibody is monoclonal or

polyclonal, 3) background labeling, 4) antibody concentration, 5) efficacy of the antibody, and 6)

location of the antigen (i.e. membrane or cytosol). Because the labeling intensities differed

between the various types of antibodies, the photomicrographs compiled for illustrative purposes

were adjusted using Northern Eclipse, Adobe Photoshop CS (San Jose, CA), and Microsoft

Powerpoint (Redmond, WA) software so that the maximum labeling contrast and intensity were

similar for each antibody.

Results

Facial Vibrissae

The basic structure of the U2 follicle was examined first due to its size, behavioral

significance, and substantial innervation (Reep et al., 2001; 2002). The U2 displayed a

pronounced epidermal invagination at the mouth of the FSC before the beginning of the capsule

and the follicle proper (Fig. 2-2; Fig. 2-3A). Dermal papillae projecting into the epidermal

surface were laden with fine-caliber presumptive C fibers (Fig. 2-3B) that co-labeled for anti-

PGP and anti-CGRP (Fig. 2-7A), as well as small-caliber presumptive A6 fibers that co-labeled

for anti-PGP and anti-NF200 (Fig. 2-7B) at the level of the rete ridge collar (RRC), but no

Merkel cells were observed in the RRC or adjacent epidermis. A small distribution of

presumptive Pacinian corpuscles was also observed just below the epidermis (Fig. 2-7M). A

narrow, short outer conical body (OCB) was also present and a circumferential array of fine

caliber fibers with presumptive free nerve endings (FNEs) was evident at the inner conical body

(ICB) level (Fig. 2-3C) and was PGP-positive and with minimal NF-positive innervation (Fig. 2-









7C). No transverse lanceolate endings were observed. Also present at the lower extent of the ICB

region and the upper extent of the ring sinus was a high distribution of "tangle" nerve endings

(Fig. 2-3D), novel endings observed in this study that appear morphologically similar to reticular

endings generally seen in other taxa along the mesenchymal sheath at the upper extent of the

trabeculated cavernous sinus (CS). These large nerve endings were supplied by large caliber

presumptive Aap or A3 fibers and were positive for PGP, S100 and NF200 as well as for BNaC

(Fig. 2-7H, I) which classifies them as low threshold mechanoreceptors responsive to mechanical

pressure. A subregion of each ending was also CGRP-positive (Fig. 2-7J). Confocal imaging

revealed an intricate mesh of NF-positive fibers interspersed among DAPI (4',6-diamidino-2-

phenylindole)-positive nuclei all within a PGP-positive cytoplasmic ending (Fig. 2-8D, F; Fig. 2-

9A, B). Proceeding to the ring sinus (RS) level, a dense distribution of Merkel cells (MCs) was

present in the outer root sheath (Fig. 2-3E). The MCs formed a circumferential array with some

branching, but individual MCs without visible innervation from branches of the deep vibrissal

nerve (DVN) predominated. When present, innervation was supplied by large caliber,

presumably Aap or A3 fibers. Widely spaced longitudinal lanceolate endings (LLEs) were

present but did not form a dense palisade as in other species. Each LLE appeared to have a single

associated terminal glia, although this was not examined in detail. The majority of LLEs

appeared unbranched (Fig. 2-8C) and in several morphologies: a studded blade form; a smooth

blade; and a curved hook ending (Fig. 2-3E). The lanceolate endings were supplied by the DVN

to the mesenchymal sheath of the RS by larger caliber afferents presumably of Aap or AP

classification. Clublike endings were also found at the RS level in close proximity to the

rudimentary ringwulst along the mesenchymal sheath. In the region of the cavernous sinus FNEs

were observed in addition to another type of novel nerve ending discovered along the trabeculae









(Fig. 2-4A, E; Fig. 2-8A). The latter stained positively for PGP, S100 and NF200 as well as for

BNaC, identifying it too as a low threshold mechanoreceptor (Fig. 2-7E, F; Fig. 2-9C, E). Low

CGRP activity was also detected (Fig. 2-7G). Representative endings from each of the two novel

ending groups were reconstructed using confocal imaging to confirm the three dimensional

structural morphology and to ensure that the unusual structure was not simply a result of an

aberrant plane of section through the FSC. No Ruffini or reticular endings were observed. Within

the medulla of the hair shaft, an extensive network of fine caliber fibers labeled intensely for

anti-PGP and with minimal NF-positive innervation present (Fig. 2-4B; Fig. 2-7K). The LI

vibrissae were also examined and found to be structurally similar to U2 vibrissae. Deep

epidermal papillae filled with fine caliber fibers were present at the RRC level along with

"tangle" endings at the lower ICB/upper RS level. Single-blade termination LLEs and

predominantly uninnervated MCs were also present at the RS level. Peptidergic and non-

peptidergic C fibers sparsely innervated the trabeculae and interior capsule of the CS. Novel

trabecular endings and extensive FNEs were visible within the CS and notably extensive

peptidergic C-fiber innervation was present within the hair shaft medulla as seen in U2 vibrissae

(Fig. 2-4C).

Bristle-like hairs (BLHs; Fig. 2-2) from the oral disk region were examined next due to

their involvement in tactile exploration and object recognition. Merkel endings and fine caliber

fibers were present at the RRC and epidermal level but the prominent epidermal invagination

leading to the follicle proper in U2 and LI vibrissae was absent in BLHs (Fig. 2-5A). A sparse

distribution of presumptive Meissner's corpuscles was also observed at this level (Fig. 2-7L). A

short OCB proceeded to a highly vascularized ICB region (Fig. 2-5E) with a dense distribution

of "tangle" endings at the lower ICB/upper RS level along the mesenchymal sheath (Fig. 2-5C,









D; Fig. 2-8G). Densely distributed Merkel cells, mainly without visible innervation, were present

at the RS level along with widely spaced single-blade LLEs and both were innervated by large

caliber fibers branching from the DVN (Fig. 2-5A, D; Fig. 2-8E). Clublike endings were present

in the ringwulst region (Fig. 2-5B). Novel trabecular endings were present along the connective

tissue of the CS (Fig. 2-4F) along with FNEs but the medulla of the hair shaft lacked the

substantial small caliber fiber innervation seen in the U2 and LI vibrissae (Fig. 2-5F). Supradisk

follicles thought to be morphologically similar to postfacial FSCs (Reep et al., 2001) possessed

attributes corresponding to those of the BLH vibrissae including the presence of "tangle" endings

within the upper RS, the presence of novel trabecular endings (Fig. 2-4D), and the absence of

extensive innervation of the hair shaft medulla, but with the exception of having a well-

innervated Merkel network at the RS level (Fig. 2-9D) that was not seen in the BLH follicles.

Postfacial Vibrissae

Postfacial follicle innervation was characterized in 6 body regions (Fig. 2-1A; Fig. 2-2):

along the dorsal midline (including rostral, central, and caudal samples), at the ventral midline,

and on the tail (dorsocentral and lateral edge). Fine caliber presumptive C fibers (PGP+/CGRP+)

as well as small caliber presumptive A6 fibers (PGP+/NF200+) were found to form extensive

arrays projecting into the epidermis of the RRC (Fig. 2-6B, C). Merkel endings were present

within the RRC at the base of the epidermis (Fig. 2-6B). At the RS level a small distribution of

"tangle" endings (Fig. 2-6D-I; Fig. 2-8H) and single-blade termination LLEs (Fig. 2-6F) were

observed along with an extensive network of MEs that was particularly pronounced in the

dorsocentral FSCs (Fig. 2-6F; Fig. 2-8B). In contrast to the perioral vibrissae examined, the

majority of MCs at the RS level of postfacial vibrissae appeared to be innervated (Fig. 2-6A, F-

I). In accordance with the facial vibrissae, no Ruffini or reticular endings were observed in the

postfacial vibrissae. The novel endings present in the trabeculae of the facial follicles were also









notably absent in the postfacial vibrissae examined and though presumptive FNEs were visible

within the CS, no pronounced innervation was present within the medulla of the hair papilla.

Discussion

Manatee Vibrissae: Overall Comparative Structure

The facial and postfacial vibrissae of manatees emanate from encapsulated blood-filled

sinus complexes (Reep et al., 2002) making them true vibrissae (Rice et al., 1986). The FSCs are

relatively short compared to the length and caliber of the hairs (Reep et al., 1998). The facial

hairs are keratinized and unusually rigid, including the region close to the hair papilla. In

contrast, rat and cat vibrissae are soft near the hair papilla and the deep half of the CS and

gradually become more rigid near the upper end of the lower CS (Ebara et al., 2002). Vibrissae

FSCs in smaller mammalian species also generally exhibit blood-filled spaces along the upper

extent of the ring sinus but lack well-defined trabeculae, whereas the manatee exhibits well-

developed trabeculae at the upper RS level where the mesenchymal sheath expands to form the

ICB. The neck of the manatee FSC at the level of the OCB and ICB regions is very long and may

contribute to the facial vibrissae being rigidly maintained within the FSC. In smaller species it is

likely that the deep end of the vibrissa is more flexible, with the smaller neck of the FSC acting

as a fulcrum against which the hair shaft can lever within the FSC. As such, the trabeculae of the

CS in smaller species are likely to function more in lateral stabilization than in the manatee,

where the hair shaft is rigidly anchored at the base and neck of the FSC. The attenuated ringwulst

of manatee vibrissae extends rigidly from the mesenchymal sheath rather than hanging down

from its point of attachment as seen in other species. Dense peptidergic and non-peptidergic C-

fiber innervation was also intimately wrapped around the outer surface of the FSC capsule,

particularly along the upper half of the capsule. This may be present in other species as well, but

to a lesser extent, and has not been fully investigated.









Facial Musculature Involved in Exploratory and Prehensile Vibrissal Behaviors

Previous experiments have shown that infraorbital branches of the maxillary nerve insert

into the upper bristle pad whereas the inferior alveolar branch of the mandibular nerve supplies

the vibrissae of the lower pad of the manatee face. The dorsal and ventral buccal branches of the

facial nerve supply the superficial facial musculature and are likely to contribute to bristle

version and feeding behavior movements (Reep et al., 1998). The U2 follicles are specifically

associated with the M elevator nasolabialis muscles (Marshall et al., 1998b), making them

homologous to mystacial vibrissae, and although individual follicles within a U2 field are not

moved independently, the left and right U2 fields can act independently from each other

(Marshall et al., 1998a). The LI follicles are supplied by mental branches of the inferior alveolar

nerve (Reep et al., 1998) and are protruded by mentalis muscle contraction (Marshall et al.,

1998b), making them homologous to mental vibrissae. At rest the U2 vibrissae are retracted

within skin folds and are everted by volume displacement through contraction of the M elevator

nasolabialis and the circular M. buccinatorius muscles during manipulative behaviors (Marshall

et al., 1998a; b). When presented with a novel object, manatees generally touch the object with

the oral disk (involving the BLH follicles) first in a side-to-side sweeping motion and then grasp

with the U2 follicles, but the BLH follicles are not actively moved (Marshall et at., 1998a).

Sensory Innervation of the Rete Ridge Collar and Epidermis

A high density of thin, tapering dermal papillae curve toward the vibrissae at the mouth

of the FSC and penetrate throughout an extremely thick epidermis (Fig. 2-2). Other species

generally lack papillae in non-glabrous skin and exhibit a relatively thin epidermis. Numerous

peptidergic and non-peptidergic C fibers enter the papillae and extend in a straight, unbranched

manner far into the overlying epidermis and perpendicular to its surface. Little innervation was

present between the papillae, but the papillae were very closely spaced, resulting in a high









density of innervation to the epidermis. Thin-caliber NF-positive fibers also penetrate into most

papillae and appear to branch, indicating that these fibers may serve as mechanoreceptors of the

papillae, but NF labeling was rarely seen on endings penetrating the epidermis. Occasional

clusters of Merkel cells and innervation are located at the base of the epidermis between papillae,

and these appear to be widely spaced over the epidermis. Throughout the upper dermis and

particularly at the RRC extends a dense vascular network that is well-innervated with dense

sympathetic innervation and, to a somewhat lesser extent, CGRP-positive sensory innervation.

Thin NF-positive innervation was also present. Thick-walled, especially well-innervated

locations appeared to be arteriovenous shunts. While we did not fully characterize innervation

associated with the vascular supply in the manatee, there appears to be an extensive network for

regulating blood flow to the epidermis, potentially representing a thermal regulatory mechanism

(Fig. 2-7N). Occasional Pacinian-like corpuscles were also seen and appeared similar to those

present among arterial networks in the glabrous skin of monkeys (Fig. 2-7M; Pare et al., 2002).

However, these were at a surprisingly superficial location at the base of the epidermis in the

manatee, which may be related to the manatees' extensive superficial vascular network.

Sensory Nerve Endings of the Inner Conical Body and Ring Sinus

Merkel endings are thought to be low threshold, slowly adapting mechanoreceptors

capable of detecting compression stimuli and directionality (Iggo, 1963, 1966; Iggo and Muir,

1969; Johansson et al., 1982a,b; Johansson and Vallbo, 1983; Munger et al., 1971; Gottschaldt et

al., 1973; Rice et al., 1986; Lichtenstein et al., 1990). Given the dense distribution of MEs in the

outer root sheath of both facial and postfacial vibrissae it seems that manatee FSCs are heavily

invested in detecting directionality of hair deflection (Burgess and Perl, 1973; Rice et al., 1986),

and a commitment of nerve endings to this task would support our proposal that manatees use

tactile hairs to detect hydrodynamic stimuli in a manner analogous to the lateral line system









present in fish (Reep et al., 2002). In microchiropteran bats, "touch domes" along the wings are

heavily invested with Merkel cells and FNEs that appear to detect air flow and aid in navigation

and maneuvering (Zook, in press; Zook, 2005; Zook and Fowler, 1986) in much the same way

that manatee postfacial vibrissae might perceive water flow. Merkel endings in the manatee were

found along the RS and ICB regions (facial and postfacial vibrissae) as well as in the RRC

(postfacial vibrissae and BLHs only). The presence of the same receptor at different locations

along the follicle axis may indicate that the MEs are involved in extracting different features of a

stimulus at these positions. At the RS level, MEs are situated in the external root sheath between

the inner root sheath and the glassy membrane, a location that makes them susceptible to small-

angle deflections of the follicle (Gottschaldt et al., 1973; Rice et al., 1986) whereas MEs of the

RRC are in a location that presumably lends itself to detection of large-angle deflections of a

vibrissa (Rice et al., 1986). By extension, the postfacial vibrissae and BLHs of the Florida

manatee appear to be specialized for the complete range of deflection intensities, due to the

presence of MEs at both the RRC and RS levels, whereas perioral facial vibrissae may be more

receptive to small-angle hair deflections, due to having MEs at the RS level only. While the

significance of most MCs at the RS level of facial vibrissae lacking visible innervation remains

uncertain, it is possible that these MCs experience a high turnover rate. The presence of clublike

endings at the attachment site of the ringwulst indicates that this region is sensitive to mechanical

perturbations as well.

Lanceolate endings are thought to be low threshold, rapidly adapting stretch receptors

that encode dynamic properties of vibrissal deflection such as acceleration and deceleration

(Burgess and Perl, 1973; Gottschaldt et al., 1973; Tuckett, 1978; Tuckett et al., 1978; Rice et al.,

1986, 1997; Lichtenstein et al., 1990). Whereas the majority of the longitudinal lanceolate









afferents gave rise to a single blade-like termination at the RS level in the U2 facial vibrissae, a

subset of LLEs exhibited a forked termination or a morphological variant including the studded

and hook endings observed. A curved "shepard's crook" morphology was also observed in LLEs

along the mesenchymal sheath at the RS level of the cat and guinea pig (Rice et al., 1986). It is

possible that the morphological variants of the LLEs have common inherent physiological

properties but may transduce slightly different aspects of mechanosensory perception. The

relatively wide spacing and low density of distribution of the LLEs in the mesenchymal sheath of

all follicles examined suggest that velocity detection is of lesser importance in both the facial and

postfacial vibrissae. Circumferentially oriented peptidergic and non-peptidergic FNEs were

found in the ICB and OCB regions. The density of distribution was far less than the well-

organized, dense circumferential bundles seen in the ICB of rats and mice, and to a lesser degree

in cats. These fibers are thought unlikely to confer linear or spatial directionality given their

circumferential orientation, and the absence of transverse lanceolate endings (TLEs) supports the

hypothesis that TLEs are related to whisking behavior and generally seen only in species such as

hamsters, mice, rats and gerbils that utilize this behavior to explore the environment (Rice et al.,

1986).

Merkel cell-neurite complexes and lanceolate endings appear to be responsive to a wide

frequency range and may be used to detect sounds when a vibrissa is deflected at the proper

frequency (Gottschaldt and Vahle-Hinz, 1981; Hyvarinen 1989, 1995; Stephens et al., 1973), a

capability that would support the hypothesis of extensive overlap between auditory and

somatosensory areas of manatee cerebral cortex (Sarko and Reep, 2007). In fact, primary

auditory cortex appears to be occupied exclusively by cluster cortex areas that feature

Rindenkerne, or "cortical nuclei" located in layer VI and thought to be analogous to barrels seen









in a variety of species and potentially representative of individual vibrissae (Dexler 1912;

Johnson et al., 1994; Marshall and Reep, 1995; Reep et al., 1989; Rice, 1995). Furthermore, a

behavioral study that assessed the underwater audiogram of the West Indian manatee found that

one manatee adjusted its responses to low-frequency (<0.4 kHz) sounds by pivoting its body

roughly 45 degrees and lowering its head (a response not exhibited for higher frequencies),

which potentially indicates adjustment of perceptual focus from sound to vibrotactile stimuli

(Gerstein and Gerstein, 1999).

The "tangle" endings observed at the lower inner conical body/upper ring sinus level, and

present in all manatee vibrissae examined here, appear to be novel because we are unaware of

sensory endings of this morphology and immunological characterization observed at this level in

the vibrissae of any other species. "Tangle" endings consisted of two or more exceptionally large

endings abutting the basement membrane and supplied by a large A3 fiber. Each ending

consisted of thick tangles of NF+ processes embedded in a matrix of PGP-positive cytoplasm

and S 100-positive terminal glia. The endings are concentrated in the mesenchymal sheath at the

level of the upper ring sinus trabeculae and may be involved in directionality detection

associated with deflection of the hair shaft against the upper trabeculae. These endings are also

BNaC-positive and therefore are likely low-threshold mechanoreceptors.

Cavernous Sinus Innervation

The medulla of the hair papilla extends to an extremely superficial location, well into the

neck of the FSC, in U2 and LI facial vibrissae. Cats also exhibit a superficially extending

medulla, but the interface between the medulla and the cortex is smooth whereas in manatees it

has a jagged appearance (Ebara et al., 2002). Manatee U2 and L vibrissae also have extensive

peptidergic and non-peptidergic C-fiber innervation within the medulla. The FNEs present within

the hair shaft medulla and spanning the trabeculae of the CS have been implicated in pain and









temperature sensation (Rice et al., 1986). Alternatively, the FNEs found within the medulla of

the hair papillae of U2 and LI vibrissae may be analogous to dentinal tubule innervation. Given

the rigidity of manatee facial follicles (particularly the perioral fields) compared to the flexible

and easily displaced hair follicles of most mammals, it is possible that sensory innervation is

committed to stress detection and load application in order to assess force transmission without

actual material displacement as seen in the dental sensory receptors of tooth pulp (Byers, 1984;

Byers and Nari, 1999). This innervation may also be a sensory adaptation to oripulative

behaviors. In another marine mammal sensory specialist, the narwhal, dentinal tubules within the

unusual tusk are thought to function as a hydrodynamic sensor detecting fluid flow, salinity

gradients, temperature and pressure (Nweeia et al., 2005).

The absence of reticular and Ruffini endings along the basement membrane in manatee

vibrissae is unusual, as is the presence of novel endings within the trabeculae of facial vibrissae.

Ruffini endings are affiliated with collagen bundles and appear to function as tension receptors

residing along the mesenchymal sheath (Rice et al., 1986; Zelena, 1994) whereas reticular

endings terminate in the upper third of the CS against the glassy membrane and may be

directionally sensitive (Ebara et al., 2002). Spiny and encapsulated endings (previously thought

to be Ruffini endings; Rice et al., 1997) were also found at the lower CS level of the rat and cat,

and Ebara et al. (2002) speculated that the cumulative CS innervation is responsive to tension

generated by the trabeculae during follicle deflection.

The nerve endings observed within the trabeculae of the lower cavernous sinus and

present only in the facial vibrissae examined here (U2, LI, BLH and supradisk follicles) appear

to be novel in that they are embedded within the trabecular matrix rather than against the

basement membrane. These endings were supplied by a relatively small-caliber AB fiber with









one axon innervating single or multiple endings. Individual endings consisted of fine-caliber

tangles embedded within a terminal glial matrix and were BNaC-positive, indicating that they

too are low threshold mechanoreceptors. It is possible that these trabecular endings are

responsive to tension induced by deflection of exceptionally rigid vibrissae (Reep et al., 1998)

which might involve modified sensory endings in order to optimally detect deflection.

Alternatively, it has been proposed that the trabeculae of the CS may function in attenuating

vascular pulsations in the arterial supply entering the base of the FSC, thereby creating more

uniform blood flow at the RS level (Melaragno and Montagna, 1953; Rice et al., 1986). Novel

endings within the trabeculae may have evolved to provide additional sensitivity in monitoring

vascular supply at this level.

Marine Mammal Vibrissae

Vibrissal sensory nerve endings of a limited number of other marine mammals have also

been studied. Hyvarinen (1995) examined the exceptionally well-innervated mystacial vibrissae

of the ringed seal (Phoca hispida) using histology and electron microscopy. The length of the

upper cavernous sinus (UCS) accounts for 60% of the vibrissa, situating the ring sinus at a low

level compared to most mammals. At the RS level, LLEs were found abutting the glassy

membrane while MEs were found below the glassy membrane and a prominent ringwulst was

present (Hyvarinen and Katajisto, 1984; Hyvarinen, 1989). All MCs appeared innervated and

formed a well-developed network (Hyvarinen, 1995). Hyvarinen (1995) also described numerous

encapsulated end-organs in the lower CS situated within the trabeculae and morphologically

similar to "Ruffin's corpuscles." These may be similar to the nerve endings reported here for the

manatee, but documentation that would allow for a direct comparison was absent. Dykes (1975)

classified and compared the afferent fibers serving cat and harbor seal vibrissae. He found that a

the majority of harbor seal vibrissae respond to vibrations and that the majority of afferent fibers









from the infraorbital nerve (85% versus 66%) serve harbor seal vibrissae. Approximately two-

thirds of these fibers were rapidly adapting and the remainder were slowly adapting, and most

fibers (71% in harbor seals and 75% in cats) were directionally sensitive. Bearded seal

(Erignathus barbatus) FSCs have also been characterized and are thought to be extensively

innervated active-touch systems adapted to benthic foraging (Marshall et al., 2006). The

mystacial vibrissae have an extensive UCS comparable to that of the ringed seal but unlike the

facial follicles of the manatee where the UCS is minimal. A prominent ringwulst is present at the

RS level along with an extensive ME network and LLEs. Merkel innervation predominates but

was not observed at the RRC level as was observed for the manatee BLHs and postfacial

vibrissae.

Comparative Considerations

Through studying a range of species (including opossums, rodents and pinnipeds) for

functional variation and unifying principles of vibrissae, Brecht et al. (1997) found that mystacial

macrovibrissae tend to form rows in which effective whisker length increases exponentially in

the caudal direction, with each row operating as a functional unit by sampling highly overlapping

spatial information perpendicular to the rostrocaudal axis. At the cortical level there also appears

to be preferential connectivity between barrels within a row (Simons, 1978; Simons and

Woolsey, 1979). Macrovibrissae were proposed to function in spatial orientation associated with

distance detection and object location while microvibrissae appeared optimized for object

recognition (Brecht et al., 1997). By extension, the postfacial vibrissae of the manatee appear to

have adopted a function analogous to that of macrovibrissae through optimization for spatial and

directional sensitivity while the oral disk and the BLHs in particular serve the microvibrissal role

of direct tactile object recognition. Instead of the direct surface contact stimulation that

macrovibrissae generally receive, manatee postfacial follicles are exposed to perturbations of the









water and consequently undergo passive deflection. It has been suggested that mammalian

vibrissae may serve a complementary auditory function at low frequencies (Gerstein and

Gerstein, 1999; Griffin, 1958; Mahler and Hamilton, 1966; Reep et al., 2002; Yohro, 1977). The

perioral vibrissae appear to be more adapted to locating and recognizing food, an important task

for a strict herbivore that spends 6-8 hours per day looking for food and that must eat the

equivalent of approximately 10% of its body weight per day to compensate for a low metabolic

rate (McNab, 1978; 1980).









Table 2-1. Specimen categorization.
Specimen Sex Length Weight Classification Cause of Death
(in cm) (in kg)
TM0406 M 290 393 Adult Watercraft
TM9728 M 295 500 Adult Watercraft
TM0506 M 172 134 Calf Cold stress
MNW0614 F 238 326 Subadult Watercraft


30 cm


Figure 2-1. Vibrissae sampling regions of the body and face. A) Postfacial body regions of
interest include the tail (lateral edge and dorsomedial areas), ventromedial area, and
rostral, central, and caudal areas of the dorsal midline. The supradisk region (asterisk)
is caudal to the orofacial ridge and also thought to consist of body vibrissae. B)
Frontal view of the manatee face with cheek muscles cut to reveal perioral follicle
fields. Facial follicles of interest include the bristle-like hairs (BLHs), U2 and L
follicle fields due to their behavioral significance.









Figure 2-2. Schematic drawing of the structure and innervation of the U2, BLH, and postfacial
vibrissal follicle-sinus complexes (FSCs) with innervation types and sensory nerve
endings illustrated (RRC=rete ridge collar, OCB=outer conical body, ICB=inner
conical body, BM=basement membrane, RS=ring sinus, DVN=deep vibrissal nerve,
HP=hair papilla). The relative scales of each FSC are accurate, but innervation is
presented for illustrative purposes only (see Fig. 3-9 for accurate scale
representations). Overall morphology: The thickness of the capsule and the diameter
of the vibrissa decreases progressively from the U2 to the postfacial FSC. The facial
vibrissae also exhibited dual innervation from the DVN at the base of the follicle
whereas the DVN entered as a single bundle of axons in postfacial vibrissae.
Presumptive sympathetic fiber innervation is also depicted based on general
characteristics in other mammals. Epidermis and RRC: The epidermis of the U2,
BLH and postfacial vibrissae contains superficially projecting dermal papillae within
which are fine-caliber A6 and C fibers. The base of the epidermis of BLH and
postfacial FSCs includes Merkel endings. The U2 FSC has a particularly pronounced
invagination of the RRC. OCB and ICB: The U2 FSC exhibits a dense network of
circumferential free nerve endings while the BLH and postfacial FSCs exhibit only
fine-caliber and sympathetic innervation. Novel "tangle" endings are located in the
facial and postfacial vibrissae. RS: Dense Merkel networks were present in vibrissae
from each body region, but Merkel cells lacking visible innervation predominated in
facial vibrissae. Less densely distributed longitudinal lanceolate endings were also
observed at this level for each vibrissal type and clublike endings were observed in
close association with the rudimentary ringwulst in facial vibrissae. CS: The
trabeculae in FSCs from each body region contained fine-caliber innervation with
presumptive free nerve endings. The facial vibrissae only included novel endings that
spanned the trabeculae. Ruffini and reticular endings were notably absent. HP: Dense
fine-caliber innervation was present in the rigid U2 follicle along a medulla that
extended to an extremely superficial extent. This innervation was very sparse in the
BLH and postfacial vibrissae, and the medulla of each extended less superficially.












0.
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c~ C





tie







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LL. a









Figure 2-3. Characterization of upper perioral field 2 (U2) follicle innervation. A) A longitudinal
U2 section stained for PGP is shown just off the medial axis to reveal various
innervation characteristics as well as the deep invagination of the RRC (specimen
TM0406). B) Magnified RRC and epidermal region further off the medial axis
showing a dense distribution of C- and AS- fiber projections (arrow) within dermal
evaginations (medial is right). C) Magnification of circumferential FNEs at the ICB
level (same plane of section as A). D) Close-up of a representative "tangle" ending
including 3 branched mechanoreceptors and a Schwaan cell (arrows and arrowhead,
respectively) within the lower ICB region along the mesenchymal sheath (plane of
section along the medial axis). E) A dense distribution of Merkel cells (arrow) and
representative types of LLEs (arrowheads) including a bifurcated ending, hooked
formation, and studded blade-like termination (same plane of section as A). RRC, rete
ridge collar; OCB, outer conical body; ICB, inner conical body; RS, ring sinus; CS,
cavernous sinus; HS, hair shaft. Scale bar = Imm for A-B, 250im for C-E.






























c.0
)CH


















Ni




















IC52









Figure 2-4. Innervation of the cavernous sinus and hair shaft medulla in facial follicles. A)
Extensive innervation of the trabeculated cavernous sinus of a U2 follicle (specimen
TM0406) includes novel endings (arrows) and fine-caliber fibers along with the DVN
continuing to the ring sinus. B-C) A dense network of small caliber axons and
presumptive FNEs proceeding to a remarkably superficial extent in the medulla of a
U2 follicle (B; specimen TM0406) and an LI follicle (C; specimen MNW0614).
Magnified views show details of novel trabecular endings seen in a supradisk follicle
(D; specimen MNW0614, arrow) a U2 follicle (E; specimen TM0406, arrow) and a
BLH follicle (F; specimen TM0406, arrow). Scale bar = 600m (A), Imm (B-C),
300m (D-F).

















msI














54









Figure 2-5. Innervation present in bristle-like hairs (BLHs). A) A longitudinal section of a BLH
just off the medial axis stained for protein gene product 9.5 reveals characteristic
innervation, including "tangle endings" at the lower ICB/upper RS level (arrowhead),
in addition to LLEs (arrow) and MCs (magnified in D) at the RS level. B) "Tangle"
endings parallel to a clublike ending (arrow) against the basement membrane at the
ringwulst level are shown further from the medial axis. C) "Tangle" endings
(arrowheads) with associated Schwaan cells at the superficial extent of the RS (plane
of section along medial axis). D) Merkel cells lacking visible innervation (arrow) in
addition to "tangle" endings (arrowhead) at the upper RS level (plane of section well
past the medial axis). E) Sympathetic innervation of the vascularized inner conical
body (plane of section well past the medial axis), and F) a dermal hair shaft medulla
lacking the extensive FNE innervation seen in U2 follicles (plane of section along the
medial axis). RRC, rete ridge collar; OCB, outer conical body; ICB, inner conical
body; RS, ring sinus; RW, ringwulst; CS, cavernous sinus; DVN, deep vibrissal
nerve. Scale bar = Imm (A), 300tm (B-C), 600tm (D-F).


































































56









Figure 2-6. Representative postfacial vibrissae innervation includes dense networks of MEs
along with LLEs and "tangle" endings. A) A dorsorostral postfacial hair (TM9728)
shows characteristic Merkel innervation (arrowheads) at the RS level. F) A
dorsocentral postfacial hair (TM9728) exhibits the presence of LLEs (small arrow),
"tangle" endings (large arrow), and a particularly extensive network of MEs (between
arrowheads) at the RS and ICB levels. G-I) Follicles from the tail edge (G;
MNW0614), ventral body (H; MNW0614), and dorsocaudal body (I; TM9728) reveal
"tangle" endings and Merkel innervation (arrows and arrowheads, respectively) at the
RS and ICB levels. B-C) Details shown for epidermal innervation (MEs shown with
arrowhead; superficial is up) and "tangle" endings (D-E; dorsocentral postfacial hair,
specimen TM9728 for B-E) at the upper RS/lower ICB level. All planes of section
shown well past the medial axis. NF, 200 kDa neurofilament subunit; PGP, protein
gene product 9.5; CGRP, calcitonin gene-related peptide. Scale bar = Imm (A),
600m (B-C), 150tm (D-E), 750tm (F, I), 500m (G-H).


































































58









Figure 2-7. Immunolabeling attributes of innervation. A-B) Dermal papillae projecting into the
epidermis at the RRC level contain C- and A6-fiber innervation (CGRP-positive and
NF-positive fibers, respectively). C) Circumferential FNEs at the ICB of a U2 follicle
reveal mostly fine caliber fibers interspersed among A6 (NF+) fibers. D) Largely
uninnervated MCs (CGRP+) interspersed among MEs (NF+) at the RS level. E-G)
Novel endings along the trabeculae of the CS stain positively for BNaC, PGP, S 100,
NF, and lightly for CGRP (arrows). H-J) "Tangle" endings also stain positively for
BNaC, PGP, S100, NF, and lightly for CGRP. K) Presumptive FNEs within the
medulla of a U2 follicle hair shaft include mostly fine caliber fibers (PGP+/NF-)
interspersed among A6 (NF+) fibers. L) Meissner's corpuscles were sparsely
distributed at the level of the epidermis. M) Pacinian ending found at the base of the
epidermis. N) An example of vascular supply associated with NF-positive
innervation. Scale bars = 300tm (A-D), 150tm (E-K, N), 75[tm (L-M).










aIM )(f .1MNVO1
SG











MN 01 BII l\(64 -

FK






061431 1 MMN614Dorscental 4 W06


fil, I




-1-M0406 U'-'









Figure 2-8. Confocal surface reconstructions showing the three-dimensional structure of
representative follicle innervation and novel mechanoreceptors present in the ICB, RS
and CS regions. A) A trabecular ending within the CS (same ending shown in Fig. 2-
4E). B) Extensive Merkel ending network in a dorsocentral postfacial vibrissa shows
the completeness of innervation (shown in Fig. 2-6F). C) The morphology of a
lanceolate ending near Merkel cells at the RS level can be compared to the larger,
more intricate "tangle" ending in the lower ICB/upper RS of a U2 follicle (D; also
seen in Fig. 2-3D). E) Reconstruction of a DVN penetrating the cavernous sinus
illustrating size and associated axons. F-H) Examples of "tangle" endings found in
the upper RS/lower ICB level of U2 (F), BLH (G), and dorsocentral postfacial
vibrissae (H; also shown in Fig. 2-6E). Scale bars=250m (A-H).

































































62









Figure 2-9. Confocal three-dimensional images of novel endings stained for neurofilament
(NF200) and protein gene product 9.5 (PGP). A) Stereo pair depicting a group of
"tangle" endings. B) A three-dimensional image (red/green anaglyph) of two "tangle"
endings with shared innervation. C) A single-optical section shows a trabecular
ending in detail. Red depicts NF-positive endings within a green PGP-positive
cytoplasmic meshwork. D) A stereo pair showing Merkel innervation (left-hand pair)
and closely associated clublike endings and "tangle" endings (right-hand pair).


































































64









CHAPTER 3
SOMATOSENSORY NUCLEI OF THE MANATEE THALAMUS AND BRAINSTEM

Introduction

Florida manatees are large-bodied herbivorous marine mammals of the Order Sirenia that

appear to be tactile specialists due to the presence of tactile hairs vibrissaee) distributed over the

entire body with an especially dense distribution on the face (Reep et al., 1998; 2002). This

arrangement is unique among mammals and may allow manatees to compensate for their

reduced visual system by using vibrissae to aid with navigation in the water. A reliance on haptic

input is reflected in the organization of the neocortex as well. Recent evidence suggests that

primary somatosensory cortex (SI) occupies roughly 25% of the neocortex (Sarko and Reep,

2007), which is favorably comparable to other somatosensory specialists such as the naked mole-

rat (Catania and Remple, 2002). Additionally, manatees exhibit cortical specializations known as

Rindenkerne, or "cortical nuclei" that appear as cell clusters in layer VI and are unique to sirenia

(Dexler, 1912; Johnson et al., 1994). Rindenkerne share histochemical attributes with "barrels,"

the functional representations of mystacial vibrissae found in layer IV of SI in rats, mice, and

other rodents (Johnson, 1980; Rice, 1995; Kaas and Collins, 2001), as well as in shrews (Catania

et al., 1999), opossums (Huffman et al., 1999; Catania et al., 2000; Frost et al., 2000), and

hedgehogs (Catania et al., 2000). Rindenkerne may process information related to vibrissae that

are behaviorally relevant in active tactile exploration and object recognition, as well as in passive

detection of hydrodynamic stimuli. However, this hypothesis remains untested by

electrophysiology or axonal tracing methods due to the manatee's status as an endangered

species.

Dedication of innervation to a particular sensory modality in the periphery creates a

commensurate neural commitment in the brainstem, thalamus, and cerebral cortex. Although the









neocortex of the Florida manatee has been characterized histochemically (Sarko and Reep, 2007)

and the sensory innervation of manatee vibrissae is currently being examined using

immunofluorescence, no systematic analysis of behaviorally relevant areas of the thalamus and

brainstem has been undertaken. It has previously been noted that manatees have a large

trigeminal nerve (Reep et al., 1989) and well-developed trigeminal and somatosensory nuclei but

reduced visual thalamic and brainstem nuclei (Johnson et al., 1986; 1987; Welker et al., 1986;

Reep et al., 1989). Assessments of the relative importance of the visual and somatosensory

systems in sirenian behavior parallel these findings, particularly for the trigeminal system that is

associated with the use of the facial vibrissae in tactile exploration.

The somatosensory brainstem nuclei of interest for the manatee include Bischoff s

nucleus, the cuneate-gracile complex, and the trigeminal nucleus (in particular, the principal

sensory component). Bischoff s nucleus is a distinct group of cells in the midline of the caudal

medulla that projects heavily to the ventrobasal thalamus in the raccoon (Johnson et al., 1968;

Ostapoff and Johnson, 1988) and constitutes the tail representation in most mammals with a

well-developed tail (Kappers and Ubbo, 1960). By analogy, Bischoff's nucleus would represent

the fluke in the manatee and might occupy a disproportionately large area due to the tactile hairs

present on the fluke. Behavioral observations also indicate that manatees carefully manipulate

their flukes when navigating through the water, which presumably involves significant sensory

feedback via Bischoff s nucleus (Welker, personal observations). The manatee cuneate-gracile

complex would be expected to represent somatosensory input from the upper and lower body

trunk and the flippers as it does in other species. The commitment of sensory endings to

postfacial vibrissae of the flippers and trunk of the body may also create somatotopic parcellation

within these nuclei. Finally, it seems reasonable to expect the trigeminal nucleus of the manatee









to be disproportionately large and parcellated into "barreloids" (the functional representation of

vibrissae in the brainstem) in order to maintain and process segregated inputs from the facial

vibrissae used in direct tactile exploration of objects in the manatee's environment.

The principal somatosensory nucleus in the thalamus is referred to as the ventrobasal

(VB) or more commonly the ventroposterior (VP) nucleus which contains a lateral subdivision

(VPL) that represents the body and a medial subdivision (VPM) that represents the face and most

of the head (e.g., Jones, 1985a). The relative sizes of these nuclei vary in other species according

to the relative innervation of the body versus the face, respectively (Rose and Mountcastle, 1952;

Cabral and Johnson, 1971; Welker, 1974; Bombardieri et al., 1975). In rodents and some

marsupials, VPM has been discovered to contain "barreloids," or neuronal clusters related to

individual vibrissae, that are highly reactive for cytochrome oxidase (Jones, 1983; Land and

Simons, 1985b; Van der Loos, 1976). Given the manatee's reliance on haptic input, the VPM

would be expected to be relatively large and may possess barreloid parcellation.

In the present study we investigate the manatee brainstem and thalamus using stains for

Nissl bodies, myelin, acetylcholinesterase, and cytochrome oxidase in order to localize and

determine the size and extent of the principal somatosensory nuclei in each region. Because of

the manatee's reliance on haptic input, we hypothesize that somatosensory nuclei in the

brainstem and thalamus would be relatively large and potentially subdivided in order to

accommodate the large amount of information being processed by discrete vibrissae in the

periphery-approximately 2,000 on the face and 3,000 on the body (Reep et al., 2001; 2002).

Additionally, if Rindenkerne are in fact analogous to cortical barrels, it seems reasonable to

expect similar functional representations of vibrissae to be present in the form of discrete cellular

aggregates within the brainstem (as barrelettes in the trigeminal nucleus) and the thalamus (as









barreloids). This analysis adds to our comprehensive characterization of the manatee

somatosensory system and our overall efforts to understand manatees' specialized adaptations

and perceptual capabilities in their unique environmental niche. Examining an evolutionary

outlier such as the manatee also contributes significantly to our understanding of general

organizing principles of sensory systems.

Materials and Methods

Four postmortem brains of the Florida manatee, Trichechus manatus latirostris, were

obtained fresh (the head perfused within 24 hours of death) through the statewide manatee

salvage program administered by the Florida Department of Natural Resources and collected

under U.S. Federal Fish and Wildlife Permit PRT-684532 with IACUC protocol #C233. The

heads were perfused in situ by gravity-fed bilateral cannulation of the carotid arteries, with 8-15

L of 0.9% phosphate-buffered saline followed by 8-15 L of 4% phosphate-buffered

paraformaldehyde fixative (amounts varied according to specimen size). The dorsal cap of each

skull was removed, the brain extracted, and the meninges removed. Each brain was then placed

in 4% paraformaldehyde. A summary of relevant specimen information is provided in Table 3-1

(classifications are in accordance with size/age class definitions for the manatee photo-

identification system, Sirenia Project, National Biological Survey, 1994). In each case the animal

was considered fresh with minimal degradation of the tissues collected and without potentially

confounding factors such as chronic pathology or emaciation.

The thalamus was removed from the right hemisphere of specimens TM0339 and

TM0406 and remained intact (with the cortex) for specimen TM0410. The brainstem was

removed from specimens TM0406, TM0410, and TM0614b. Serial frozen microtome sections

were cut coronally at 60[tm for these brains and adjacent series of sections were then stained for

cytochrome oxidase (CO), for Nissl substance with cresyl violet (CV), for myelin with gold









chloride (GC), and for acetylcholinesterase (AChE) as available (see Table 3-1). Five additional

specimens were analyzed from brains in our collection. These specimens included TM84-49,

TM85-8, TM84-58, TM86-124, and TM85-32, all of which were celloidin-embedded and

sectioned at 40m. Specimen TM85-8 was sectioned in the horizontal plane, specimen TM84-58

was sectioned in the sagittal plane, and specimens TM84-49, TM86-124, and TM85-32 were

sectioned in the coronal plane. Adjacent series of sections for these specimens were stained for

Nissl bodies with thionin and for myelin with hematoxylin. The CO procedure (Wong-Riley,

1979) was modified for manatee tissue by staining overnight, and the GC procedure (Schmued,

1990) was used with adjustment of pH to 6.3. The AChE recipe was provided by Dr. Robert

Switzer (Neuroscience Associates, Inc., Knoxville, TN). Briefly, sections were cut directly into

incubation solution consisting of 0.226 g of acetylthiocholine iodine in 100 ml of deionized

water, 25 ml stock glycine, 25 ml stock CuSO4, and 50 ml of 0.2M acetate buffer (pH 5.0).

Sections were then placed into incubation solution in a 40C hot water bath for 90 minutes,

rinsed in distilled water, transferred to 1% silver nitrate for 4 minutes, rinsed again, treated with

1% sodium thiosulfate for 6 minutes, and given a final rinse before being mounted onto slides

from 0.02M acetate buffer. Once each staining procedure was complete and sections were

mounted onto gelatinized slides, the slides were coverslipped using Eukitt.

All thalamic and brainstem sections were viewed under an Olympus BH-2 microscope, a

Bausch and Lomb microprojector, a Zeiss Axiophot microscope, and on a light table in order to

examine sequential sections for persistence of the visible patterns and identification of nuclei.

Brain atlases of the rat (Paxinos and Watson, 1986; 1998), cat (Berman and Jones, 1982;

http://www.brainmaps.org), and monkey (Gergan and MacLean, 1962) were used to assist in

identification of boundaries of the thalamic nuclei. Representative sections were imaged using a









Zeiss Axioplan 2 microscope or scanned with an HP ScanJet 5370C and contrast and brightness

were optimized using Adobe Photoshop CS.

In an attempt to quantify the percentage of thalamus occupied by VP in the manatee

compared to other species, we analyzed three coronal sections spaced across the full extent of VP

(the first close to the rostral-most extent of VP, with both VPM and VPL distinguishable; the

second a middle section; and the third close to the caudal extent of VP and still retaining

distinguishable VPM and VPL). Sections from three manatee brains and two rat brains stained

for CO were scanned and outlined using AIS (Analytical Imaging System) software. The extent

of the entire thalamus within each coronal section was measured followed by the extent of VP

(based on CO-dense staining). These measurements were then completed using Nissl body

stained sections from atlases for the rat, cat, and squirrel monkey (Table 3-2).

Results

Brainstem

Since manatee brainstem nuclei have never been fully characterized, we first compiled an

atlas illustrating all clearly identifiable nuclei in a representative adult specimen (Fig. 3-1) with a

particular focus on the somatosensory components. Nomenclature is based primarily on the

Paxinos and Watson (1986; 1998) rat brain atlas and supplemented where appropriate by the cat

brain atlas (http://www.brainmaps.org). The mesencephalic nucleus of cranial nerve 5 (Me5) is

the rostral-most component of the trigeminal subnuclei (Fig. 3-1, A-D; Fig. 3-2, A-C). As in

other species, it is visible along the lateral extent of the periaqueductal gray (PAG; Fig. 3-1A)

and locus coereleus (LC; Fig. 3-1D). Disproportionately large components of the auditory

system-the inferior colliculus (IC; Fig. 3-1, A-D) and nucleus of the lateral lemniscus (NLL;

Fig. 3-1, C-E)-are also visible at this level and are commensurate with the manatee's well-

developed auditory system (Gerstein and Gerstein, 1999; Mann et al., 2005).









The motor (Mo5) and principal sensory (Pr5) components of the trigeminal system are

seen caudal to Me5 (Fig. 3-1E; Fig. 3-2; Fig. 3-3). Nucleus Mo5 exhibits large motorneuron

somata in Nissl body stains (Fig. 1, E-F, left panels) that also characterize the facial motor

nucleus (FMN; Fig.3-1, I-J) and lateral vestibular nucleus (LVe; Fig. 3-1, H-K). The Pr5 nucleus

appears large and lobulated in both coronal (Fig. 3-1, E-G; Fig. 3-4A) and sagittal (Fig. 3-2, A-

C) preparations. It appears as a distinct nucleus caudolateral to the NLL in horizontal sections

(Fig. 3-3, B-F), and Pr5 stains moderately for CO (Fig. 3-4A, right panel).

Just caudal to the initial appearance of the facial nerve (7n), Pr5 begins to transition into

the presumptive oral division of the spinal trigeminal nucleus (Sp5; Fig. 3-1H; Fig. 3-4B). This

shift is also characterized by the appearance of the spinal trigeminal tract (sp5) which assumes a

crescent shape surrounding the nucleus. As the facial motor nucleus (FMN) becomes distinct

(Fig. 1, I-J) the spinal trigeminal nucleus assumes a flattened and less distinct morphology

characteristic of the interpolar subnucleus of Sp5 (e.g., Paxinos and Watson, 1998; Fig. 3-1, J-K;

Fig. 3-4C). The extensive caudal subnucleus of Sp5 continues from the interpolar nucleus (Fig.

3-1, L-P; Fig. 3-4D) and appears to be lobulated. The extensiveness of the spinal trigeminal

nucleus is also clearly evident in sagittal (Fig. 3-2D) and horizontal sections (Fig. 3-3, C-F).

Each of the spinal trigeminal subnuclei stains moderately for CO (Fig. 3-4, right panels).

Although Pr5 and the caudal nucleus of Sp5 appear lobulated, and both the oral and interpolar

nuclei of Sp5 appear densely penetrated with fiber bundles (Fig. 3-4, B-C), no evidence of

barreloids was present.

The cuneate nucleus first becomes evident at the caudal extent of the FMN (Fig. 3-1J).

Contrary to previous reports (Johnson et al., 1994) an external cuneate nucleus (ECu) is present,

although it is greatly reduced (Fig. 3-1, K-L). As a whole, the cuneate-gracile complex is large,









extensively lobulated, and stains densely for CO (Fig. 3-1L-N; Fig. 3-4D). A large Bischoffs

nucleus is also present at the most caudle extent of the gracile nucleus (Fig. 1K) and stains

densely for CO (shown in the neonate only but present in all specimens examined, Fig. 3-5E).

The proposed somatotopic arrangement of cutaneous inputs from the manatee body is presented

later along with the proposed somatotopy for VP within the thalamus (Figure 3-13) based on

Welker (1973).

A spaced series of representative sections from a neonatal specimen demonstrates that the

location and disproportionately large size of somatosensory nuclei, as well as the organization of

brainstem nuclei in general, is consistent between adults and neonates. Figure 3-5A shows a

section equivalent to that of Fig. 3-1E with a large Pr5 that stains moderately for CO. The plane

of section for Fig. 3-5B is equivalent to Fig. 3-11, showing Sp5 surrounded by the crescent of

spinal trigeminal tract fibers. The adjacent sections shown in Fig. 3-5C are equivalent to Fig. 3-

1K with a small external cuneate nucleus present at the lateral aspect and large Sp5 and cuneate-

gracile nuclei. Figure 3-5D shows lobulation present in Sp5 and more extensively in the cuneate-

gracile complex (CuG) equivalent to Fig. 3-1M. Finally, a large Bischoff's nucleus

encompassing the presumptive tail representation is present at the caudal aspect of the medulla

(Fig. 3-5E) along with caudal Sp5. Moderate CO staining was present in the trigeminal nucleus

and dense staining characterized CuG as seen in adults (Fig. 3-4).

Thalamus

The principal somatosensory nucleus in the thalamus is the ventroposterior (VP) nucleus,

one of the most clearly defined thalamic nuclei due to its large size, densely staining cells, and

lobulated appearance resulting from penetrating myelin fiber bundles (Jones, 1985b). In Nissl

body preparations, subnucleus VPM contains smaller, relatively closely packed cells compared

to VPL. In addition, AChE staining reveals robust patterns that allow for the discrimination of









different nuclei and these patterns are generally consistent for rats, cats and primates (Jones,

1985a). Densest staining generally characterizes the ventral lateral geniculate nucleus as well as

the intralaminar, anteroventral, anterodorsal, rhomboid, paraventricular, habenular and

medioventral nuclei while lighter staining distinguishes the dorsal lateral geniculate nucleus

(LGN), medial geniculate nucleus (MGN), reticular nucleus, anterior of the lateral posterior

nucleus, and parts of lateral and ventral complexes.

An atlas of the manatee thalamus is provided for the first time (Fig. 3-6) with a more

closely spaced series of sections to show the detailed extent of the somatosensory thalamus (Fig.

3-7). Nomenclature is based on Jones, (1985a). The VPL and VPM nuclei first appear in

approximately the same plane of section (Fig. 3-6F). Whereas VPL terminates in Fig. 3-6G,

VPM extends more caudally to Fig. 3-6H. The medial subnucleus was identifiable in Nissl body

preparations as having relatively small, closely packed cells in contrast to the lateral subnucleus

which displayed large, darkly staining cells that were more widely spaced (Fig. 3-8). The

subnuclei of VP were also distinguishable in AChE staining preparations with VPM exhibiting

lighter staining than VPL (Fig. 3-9, left column). As seen in other species, VP was characterized

by penetrating fiber bundles visible in myelin preparations (Fig. 3-6, F-H; Fig. 3-7; Fig. 3-9,

right column). The entirety of VP was CO-dense as seen in other species and was consistently

CO-dense in adults (Fig. 3-9, middle column), neonates and juveniles (Fig. 3-10) although the

medial subnucleus did not appear more densely stained as is the case in some other species. No

barreloids were clearly distinguishable, although possible functional divisions might be indicated

by penetrating fiber laminae that were particularly pronounced in the juvenile specimen (Fig. 3-

11). The posterior nucleus (Po) also receives cutaneous input from the periphery and is visible in

Fig. 3-6, E-I and in Fig. 3-7.









Overall, the ventroposterior and posterior nuclei appear disproportionately large in the

manatee in accordance with their functional relevance to somatic sensation. To quantify the

percentage of thalamus occupied by VP in the manatee compared to other species, we analyzed

three coronal sections spaced across the full extent of VP (see Materials and Methods) from three

manatee brains and two rat brains stained for CO as well as from Nissl body stained sections

from atlases for the rat, cat, and squirrel monkey (Table 3-2). Although this yields limited

information given that the volume of total thalamus versus VP (and more particularly, VPL

versus VPM) could not be calculated, the data indicate that measures were very similar between

the rat sections stained for CO and the outlined sections from the rat brain atlas and by extension

should be comparable for the cat and squirrel monkey as well. Also, although the measure was

across only three coronal sections spaced across VP for each species, it does appear that the

percentage of thalamus occupied by VP is higher in manatees, particularly in the adult specimen

(28%). Indeed, based on a qualitative assessment of CO-stained coronal sections in the adult

(Fig. 3-9), VP appears to occupy approximately one-third of the thalamus. The only other study

found to quantify thalamic subnuclei was done by Kruger (1959). Kruger's data quantified VP as

a percentage of dorsal thalamus, and dorsal as a percentage of total thalamus. By extrapolation,

his measurements indicate that VP occupies 6.6% of total thalamus in rabbits, 2.9% in sheep,

5.0% in cats, 7.0% in monkeys, and 2.6% in dolphins.

Other behaviorally relevant thalamic nuclei include the lateroposterior subnucleus (LP) and

the lateral geniculate nuleus (LGN), both of which are relatively small and are overtaken by the

large medial geniculate nucleus (MGN; Fig. 3-6, G-K). This anatomical organization reflects the

relative degree of development and behavioral importance of the visual and auditory systems,

respectively. Our proposed location for MGN differs from other species in that MGN appears









rostral to LGN as seen in coronal (Fig. 3-6, G-K) and horizontal (Fig. 3-12) sections. The medial

geniculate is also situated dorsal to LGN with Po visible as a wedge between MGN and LGN

(Fig. 3-61) as seen in the horizontal section. This orientation is conceivable if one considers the

overall rostroventral rotation that the manatee brain appears to have undergone, such that the

equivalent of the Sylvian (lateral) fissure is oriented vertically. Additionally, if the auditory sense

truly dominates visual in the manatee, it is plausible that MGN became greatly expanded at the

expense of visual thalamic nuclei thereby forcing them caudal and ventral.

Discussion

Brainstem: Somatotopic Parcellation in Other Species

The brainstem nuclei of interest for the manatee somatosensory system include the

trigeminal, cuneate, gracile, and Bischoff's nuclei. Bischoff s nucleus, a distinct group of cells in

the midline of the caudal medulla (Johnson et al., 1968), has not been identified previously in the

manatee but has been shown, along with the cuneate and gracile nuclei, to project heavily to the

ventroposterior thalamus (VP) in the raccoon (Ostapoff and Johnson, 1988). In the raccoon, the

tail representation occupies the dorsal portion of VP whereas the hindlimb representation

occupies the ventral portion (Johnson et al., 1968). The presence of Bischoff s nucleus has also

been noted in rats, shrewmice, kangaroos, great anteaters, some monkeys, and to some extent in

cetaceans (Kappers and Ubbo, 1960). In the manatee, Bischoff s nucleus would presumably

represent the fluke, and does in fact appear to be disproportionately large as might be expected

given the presence of vibrissae on the fluke. The fluke might also be critical during navigation,

as manatees have been observed to constantly adjust the angle of their fluke while swimming

(Welker, personal observations).

Species like the manatee that rely on tactile exploratory behaviors involving the face

(e.g., the star-nosed mole (Crish et al., 2003) and the platypus (Ashwell et al., 2006)) have well









developed trigeminal systems often exhibiting extensive somatotopic organization within the

sensory trigeminal nuclei. Studies in the rat revealed that the afferent projection pattern from

individual facial vibrissa follicles was topographically related to CO-dense cell clusters

("barrelettes") in the trigeminal principal sensory nucleus (Pr5) with a nearly one-to-one ratio

between follicles and corresponding CO-dense clusters (Florence and Lakshman, 1995). These

results supported earlier findings by Jacquin et al. (1993) that showed that Pr5 axon collaterals

were concentrated within corresponding CO-dense subdivisions, and terminal branches of

individual trigeminal afferents rarely crossed over into adjacent regions. In contrast, in three

subdivisions of the spinal trigeminal nucleus-the pars oralis (Sp5o), pars interpolaris (Sp5i),

and pars caudalis (Sp5c)-a topographical arrangement still existed, but with less specificity and

more overlapping representations (Florence and Lakshman, 1995). Whisker-related barrelette

patterns are present in Pr5, Sp5i, and Sp5c, but not in Sp5o (e.g., Nomura and Mizuno, 1986).

Goyal et al. (1992) showed that the human principal trigeminal nucleus also demonstrated a

parcellated CO-dense pattern which was interpreted as a reflection of high-density peripheral

innervation of the face despite the lack of punctate structures like vibrissae. In the manatee, the

principal sensory and caudal spinal trigeminal nuclei appeared particularly lobulated with

possible somatotopic parcellation present. All trigeminal components, and especially the

principal sensory nucleus, were elaborated and exceptionally large. In contrast to other studies

that have shown CO-dense staining in Pr5 and Sp5c with only light staining in Sp5o and

moderate staining in Sp5i (Florence and Lakshman, 1995), CO staining appeared consistently

moderate throughout Pr5 and Sp5 in the manatee. The somatotopy proposed for the manatee

brainstem (cuneate-gracile complex and Sp5c represented; Fig. 3-13A) is based on the general

arrangement seen in mammals (e.g., Woudenberg, 1970).









Somatotopic parcellation is also evident in the cuneate and gracile dorsal column nuclei

in other species where somatosensation is the dominant sensory modality. Cutaneous inputs from

the upper limbs and rostral trunk of the body are represented in the cuneate nucleus while lower

limbs and lower trunk are represented in the gracile nucleus. Strata et al. (2003) studied a

prosimian, the Galago, to look at the pattern of peripheral nerve input. Through cell clusters that

were identified as CO-dense blotches in both nuclei, they discovered a greater segregation of

inputs within the cuneate (fingers and hand representation) than in the gracile (foot

representation), which corresponds with the Galago's extensive and highly differentiated use of

its hands and fingers relative to its feet. In macaques, inputs from specific parts of the hand relate

to CO-dense rostrocaudal clusters of cells (Florence et al., 1991). Although the manatee lacks the

manual dexterity of a primate, CO analysis of the manatee cuneate-gracile complex revealed

dense staining and extensive suborganization that may be related to discrete functional

representations of vibrissae on the postfacial body.

Thalamus: A Comparative Look at Somatosensory Nuclei

The relative sizes of VPM and VPL vary according to the relative innervation of the face

versus the body, respectively (Rose and Mountcastle, 1952; Cabral and Johnson, 1971; Welker,

1974; Bombardieri et al., 1975). For rodents whose nose, mouth and lips dominate tactile

perception, the VPM is larger, and in monotremes VPM dominates the entirety of VP. In cats,

VPM and VPL are approximately equal in size, but in monkeys VPL predominates to

accommodate extensive input from the hands and feet. In some species, for example the cat,

VPM extends caudal to VPL, whereas in others (such as monkeys) VPL extends more caudally

(Jones et al., 1985b). In rodents and some marsupials, VPM has been discovered to contain

"barreloids," or neuronal clusters related to individual vibrissae, that are highly reactive for CO

(Jones, 1983; Land and Simons, 1985b; Van der Loos, 1976). The VPL of the raccoon and slow









loris contains lobulated subregions representing palmar and digital skin pads (Welker and

Johnson, 1965; Krishnamurti et al., 1972). Chronic vibrissae trimming results in reduced staining

for CO in both the somatosensory cortical barrels (Land and Simons, 1985a; Wong-Riley and

Welt, 1980) and the thalamic barreloids (Land and Akhtar, 1987) associated with the trimmed

vibrissae. These findings were similar to those in Macacafascicularis monkeys where peripheral

nerves were cut, resulting in reduced staining of "rods" within the VPM (Jones et al., 1986).

Using horseradish peroxidase axonal tracing, Jones et al. (1986) also discovered that CO staining

was primarily due to terminations of trigeminal afferent fibers that formed somatotopically

organized inputs to the rods. They postulated that each rod of the thalamus formed the basis of

columnarity of afferent input to the somatosensory cortex by providing bundles of

thalamocortical axons terminating in focal domains of the cortex. No barreloids were found in

VPM of the manatee thalamus, but the ventroposterior nucleus as a whole was large, reflecting

the manatee's reliance on haptic somatosensoryy) input. Based on AChE staining, VPM and VPL

also appear to divide the ventroposterior nucleus in an approximately equal manner (Fig. 3-9).

This seems reasonable given the distribution of vibrissae on the manatee body (2,000 on the face

and 3,000 postfacially) balanced by the fact that facial vibrissae are more densely innervated

(Reep et al., 2001; 2002). While our proposed "manateeunculus" within the thalamus follows

Welker (1973; Fig. 3-13B), it should be noted that such artistic abstractions are limited since the

body representation extends in three dimensions throughout VP.

Thalamic nuclei associated with vision (LGN and LP) appear to be overtaken by those

associated primarily with somatosensation (Po) and audition (MGN). While this may simply be

due to the fact that somatosensation and audition both appear to be dominant sensory modalities

in the Florida manatee, it is also possible that there exists extensive multisensory integration. A









previous study from our laboratory found that the manatee cortex exhibits what appears to be

extensive-and possibly complete-overlap between primary auditory (AI) and primary

somatosensory cortex (SI; Sarko and Reep, 2007). Responses to somatosensory and visual

stimuli have also been reported in AI of the macaque (Werner-Reiss et al., 2003; Ghazanfar et

al., 2005; Brosch and Scheich, 2005; Brosch et al., 2005; Zhou and Fuster, 2004).

While it is possible that this multisensory integration occurs entirely within AI, with non-

auditory information relayed to AI from unimodal sensory areas in subcortical or cortical areas,

it is also conceivable that AI receives inputs from multisensory cortex. But perhaps the most

intriguing possibility, and one that might be relevant to the present study, involves the integration

of auditory and non-auditory inputs at subcortical levels, along the traditional auditory pathways

or within multisensory subcortical structures, before projecting to AI (Budinger et al., in press).

Possible candidates for these subcortical multimodal areas include Po; the dorsal and medial

division of MG; the brachium, dorsal and external nuclei of the inferior colliculus; and the

superior colliculus, all of which were found to involve connections with AI in the Mongolian

gerbil, a species used frequently for auditory research (Budinger et al., in press). The brachium

of the inferior colliculus (bic), Po, and MG and particularly strong candidates as these are

reciprocally connected with AI. Indeed the inferior colliculus appears to be extensively involved

in acousticomotor and somatosensory systems (e.g., Huffman and Henson, 1990) and neurons

within the bic code for auditory space (Schnupp and King, 1997) and project to IC, SC, and AI

(Kudo et al., 1984; Mitani et al., 1987; Rouiller et al., 1989). The external subnucleus of the IC

has been found to integrate trigeminal and auditory stimuli with projections from both CN and

Sp5 (Jain and Shore, 2006), and multisensory integration has also been demonstrated in DCN

(Shore, 2005). The inferior and superior colliculi together coordinate aspects of spatial









orientation (e.g., Oliver and Huerta, 1992; Cohen and Knudsen, 1999), and indeed

somatosensation has been found to dominate the SC in the star-nosed mole that relies extensively

on its haptic sense (Crish et al., 2003). Subdivisions of MG process visual, vestibular,

nociceptive and somatosensory stimuli in addition to the auditory stimuli classically associated

with MG (e.g., Linke and Shwegler, 2000). In addition, the trigeminal system appears to

influence the superior olivary complex (Shore et al., 2000). Overall, cortical and subcortical

areas once rigidly defined within unimodal functional boundaries appear to have a broader

functional scope, and this may be especially true in the manatee's case where low-frequency

sounds in the water might stimulate mechanoreceptors-Merkel and lanceolate endings in

particular-as the vibrissae of the face and postfacial body are perturbed. Such integration has

been hypothesized to be beneficial in perception and localization of stimuli, potentially by

linking object features or adjusting coordinate frames to a different sensory modality (Budinger

et al., in press). Projections from Al to subcortical multimodal areas may also modulate

subcortical activity by influencing a selected representation of behaviorally relevant frequencies

(e.g., Suga and Ma, 2003), improving temporal and spectral resolution of sound representations

(Yan et al., 2005), and attention-related gating of auditory information (Yu et al., 2004).

Absence of Barrelettes and Barreloids

If Rindenkerne are in fact analogous to cortical "barrels" and correspond to functional

representations of vibrissae, it seems reasonable to expect the brainstem and thalamic

counterparts of barrels ("barrelettes" and "barreloids," respectively) to be present. However, the

absence of the latter two features in the Florida manatee could be explained by several factors.

First, the plane of section may not have been optimal for detection, particularly in the case of the

thalamus. We chose to section the majority of our specimens (and all of the specimens stained









for CO) in the coronal plane as this allowed for clearest identification of subnuclei. This was a

priority of the current study since nuclei of the manatee thalamus and brainstem have not been

characterized previously in a systematic and comprehensive manner. Because of their complex

three-dimensional morphology, identification of barreloids in the coronal plane has proven

difficult in rodents (Ivy and Killackey, 1982; Land et al., 1995). Instead, the oblique horizontal

plane has proven optimal for detection of barreloids (Land et al., 1995). Although this hypothesis

could be tested by simply altering the plane of section, it is more probable that barreloids are not

present in the manatee, because we also did not detect barrelettes in the brainstem, which should

be clearly apparent in the coronal plane.

Another possible explanation for the absence of barrelettes and barreloids is that the

facial vibrissae of the Florida manatee are not organized in discrete rows as seen in other species

including mice, rats and cats whose organization of facial vibrissae into rows and columns is

maintained within VPM and the trigeminal sensory nuclei (e.g.,Nomura and Mizuno, 1986; Ma,

1991; Florence and Lakshman, 1995; Land et al., 1995). But perhaps the most likely explanation

for the absence of barreloids and barrelettes lies in the difference between Rindenkerne and

cortical barrels. While barrels are located in layer IV, an afferent zone, Rindenkerne are located

in efferent layer VI. This undoubtedly indicates a fundamental difference in the columnarity of

organization between barrels and Rindenkerne. Corticocortical feedback connections originate in

infragranular layers, implicating Rindenkerne in descending pathways.









Table 3-1. Summary of specimen information.
Specimen Areas analyzed Stains Sex Length Weight
(in cm) (in kg)
TM84-49 Thalamus & brainstem intact Thionin, hematoxylin
TM85-8 Thalamus & brainstem intact Thionin, hematoxylin
TM84-58 Thalamus & brainstem intact Thionin, hematoxylin
TM86-124 Thalamus & brainstem intact Thionin, hematoxylin
TM85-32 Brainstem Thionin
TM0614b Brainstem CO, CV, GC F 326
TM0339 Thalamus CO, CV, GC (bad) F 200 167.8
TM0406 Thalamus& brainstem CO, CV, AChE, GC M 290 393.2
TM0410 Thalamus (intact) & brainstem CO, CV, AChE, GC M 94 14.5


Table 3-2. Comparative analysis of percentage of thalamus occupied by the ventroposterior
nucleus (VP; averaged from 3 evenly spaced coronal sections to encompass VP).
Species Source of analysis VP Total Percentage of
Area* Thalamus Thalamus Occupied
(in mm2) Area* by VP
(in mm2)
Manatee Neonate (TM0410) 39.0 196.4 20%
Juvenile (TM0339) 66.9 286.2 23%
Adult (TM0406) 93.6 333.5 28%
Rat Adult (340) 1.5 7.1 22%
Adult (341) 1.6 8.1 20%
Brain atlas 4.6 24.7 19% (VPL=4.4%,
VPM=14.6%)
Cat Brain atlas 18.4 115.5 16%
Squirrel Monkey Brain atlas 20.3 101.5 20%
1Paxinos and Watson, 1986
2 Berman and Jones, 1982
3 Gergan and MacLean, 1962









Figure 3-1. A rostrocaudal series of representative coronal brainstem sections with subnuclei
labeled illustrates the size and extent of somatosensory nuclei. A-P) Sections stained
with thionin for Nissl bodies (left) shown with adjacent sections stained with
hematoxylin for myelin (right). Section numbers are listed at the bottom of each
section, and sections were cut at 40tm, specimen 84-49. Scale bars=5mm.
4=trochlear nucleus; 7n=facial nerve; 8n=auditory-vestibular nerve; 10=vagus
nucleus; 12=hypoglossal nucleus; Amb=nucleus ambiguous; bic: brachium of the
inferior colliculus; BN=Bischoff s nucleus; bp=brachium pontis; CuG=cuneate-
gracile; CI=inferior central nucleus; CN=cochlear nucleus; cp=cerebral peduncle;
CS=superior central nucleus; Cu=cuneate nucleus; DCN=dorsal cochlear nucleus;
DTN=dorsal tegmental nucleus; ECu=external cuneate nucleus; FMN=facial motor
nucleus; FTG=gigantocellular tegmental field; g7=genu of facial nerve; IC=inferior
colliculus; ICN=interposed cerebellar nucleus; icp=inferior cerebellar peduncle;
INC=nucleus incertus; IO=inferior olive; LC=locus coereleus; LCN=lateral dentatee)
cerebellar nucleus; ll=lateral lemniscus; LPB=lateral parabrachial nucleus;
LVe=lateral vestibular nucleus; Me5=mesencephalic nucleus of 5; ML=medial
lemniscus; MLF=medial longitudinal fasciculus; MnR=median raphe nuclei;
Mo5=motor nucleus of 5; MRt=medullary reticular nucleus; MVe=medial vestibular
nucleus; NLL=nucleus of the lateral lemniscus; PAG=periaqueductal gray;
Pn=pontine nuclei; Pr5=principal sensory nucleus of 5; py=pyramidal tract;
Rb=rubrospinal tract; Rt=reticular nucleus; s5=sensory root of 5; SC=superior
colliculus; scp=superior cerebellar peduncle; SO=superior olivary nucleus;
Sol=nucleus of the solitary tract; Sp5=spinal trigeminal nucleus; sp5=spinal
trigeminal tract; TRN=tegmental reticular nucleus; Tz=trapezoid nucleus;
VCN=ventral cochlear nucleus; Ve=vestibular nucleus; xpy=pyramidal decussation;
xscp=decussation of the superior cerebellar peduncle








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Figure 3-2. Brainstem sections cut in the sagittal plane illustrate the rostrocaudal extent of
behaviorally relevant nuclei and in particular the lobulated appearance of the
trigeminal nuclei. Sections proceed lateral to medial and were stained with thionin for
Nissl bodies Section numbers are listed at the bottom of each section, and sections
were cut at 40. m, specimen 84-58. Scale bar-5mm. FMN=facial motor nucleus,
IC=inferior colliculus, Me5=mesencephalic nucleus of 5, Mo5=motor nucleus of 5,
NLL=nucleus of the lateral lemniscus, Pr5=principal sensory nucleus of 5,
SC=superior colliculus, Sp5=spinal trigeminal nucleus, Ve=vestibular nucleus.

















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Figure 3-3. Brainstem sections cut in the horizontal plane show the topography and orientation of
nuclei of interest. Sections proceed ventral to dorsal and were stained with thionin for
Nissl bodies Section numbers are listed at the bottom of each section, and sections
were cut at 40gm, specimen 85-8. Scale bar=5mm. 5=trigeminal nerve, CN=cochlear
nucleus, CuG=cuneate-gracile complex, FMN=facial motor nucleus, IO=inferior
olivary nucleus, Mo5=motor nucleus of 5, NLL=nucleus of the lateral lemniscus,
Pr5=principal sensory nucleus of 5, py=pyramidal tract, SO=superior olivary nucleus,
Sp5=spinal trigeminal nucleus, Ve=vestibular nucleus.









Figure 3-4. Representative coronal brainstem sections illustrating the appearance of each of the
trigeminal subnuclei in an adult specimen. Adjacent sections are shown stained for
myelin (with gold chloride, GC) and cytochrome oxidase (CO). A) The principal
sensory nucleus (Pr5) is large and exhibits possible somatotopic parcellation. It also
stains moderately for CO, as do all of the trigeminal subnuclei. Section numbers are
listed at the bottom of each section, and sections were cut at 60im, specimen
TM0614b. Scale bar=5mm. 7=facial nerve, BN=Bischoff's nucleus, CN=cochlear
nucleus, CuG=cuneate-gracile complex, DCN=dorsal cochlear nucleus, FMN=facial
motor nucleus, LVe=lateral vestibular nucleus, Mo5=motor nucleus of 5,
Pr5=principal sensory nucleus of 5, SO=superior olivary nucleus, sp=spinal nerve of
5, sp5=spinal trigeminal tract, Sp5c=spinal trigeminal nucleus caudalis, Sp5i=spinal
trigeminal nucleus interpolaris, Sp5o=spinal trigeminal nucleus oralis, VCN=ventral
cochlear nucleus.












Figure 3-5. A rostrocaudal series of representative coronal brainstem sections in a neonate shows
that somatosensory nuclei are large and have a parcellated appearance as seen in adult
specimens. Sections were stained with cytochrome oxidase (CO), gold chloride (GC)
for myelin, and cresyl violet (CV) for Nissl bodies. Section numbers are listed at the
bottom of each section, and sections were cut at 60m, specimen TM0410. Scale
bar=5mm. BN=Bischoff's nucleus, CN=cochlear nucleus, CuG=cuneate-gracile
complex, FMN=facial motor nucleus, Mo5=motor nucleus of 5, Pr5=principal
sensory nucleus of 5, Sp5=spinal trigeminal nucleus, Ve=vestibular nucleus.



















%go C'?









AW
-4A






JOS

it A.;









CO GC CV
















Figure 3-5. Continued









Figure 3-6. A rostrocaudal series of representative coronal thalamic sections with low-
magnification images of sections stained with hematoxylin for myelin and high-
magnification details of adjacent sections stained with thionin for Nissl bodies with
subnuclei labeled. Section numbers are listed at the bottom of each section, and
sections were cut at 40tm, specimen 84-49. Scale bar=5mm. AM=anteromedial
nucleus, AV=anteroventral nucleus, CeM=central medial nucleus, CL=central lateral
nucleus, CM=centre median nucleus, FF=fields of Forel, fr=fasciculus retroflexus,
H=habenular nuclei, IC=inferior colliculus, ic=internal capsule, iml=internal
medullary lamina, LD=lateral dorsal nucleus, LG=lateral geniculate nucleus,
LP=lateral posterior nucleus, MD=mediodorsal nucleus, MG=medial geniculate
nucleus, ml=medial lemniscus, MV=medioventral nucleus, PARA=anterior
paraventricular nucleus, Pc=paracentral nucleus, Pf=parafascicular nucleus,
Po=posterior nucleus, PT=parataenial nucleus, Rh=rhomboid nucleus, SC=superior
colliculus, SM=submedial nucleus, st=stria terminalis, Subl=subincertal nucleus,
VA=ventral anterior nucleus, VL=ventral lateral nucleus, VM=ventral medial
nucleus, VPL=ventral posterior lateral nucleus, VPM= ventral posterior medial
nucleus, ZI=zona incerta.










A


AV


VA PARA

AM
VL

/' 4 ',,
w V
Ic

1017 1018

B .2>

.LD
'AV
AM
VA PARA


~MD'
VL











..CL-:.
VM.V


ic.











1097 1098












LD


CL H
& PARA

VL MD
Pc


VM
ZI ml



1138


LD


H
Po
CL. PARA

MD
VL I

VM -
SM '
Z. r:
FF .







st
Po .,


VL
MD
VPM CM


ml

Subi


1218


Figure 3-6. Continued


1137


1178


1177


L


























I






ii
F1g t
Figure 3-6. Continued


fr


*LD


VPL

F"'


1258


VPM


1298


Po -'


LG..


1338













r -
~MG

4



LO -.


42


1378


.... -. 4*;M


4
7 [*1. **'
St
tiF


* j7,5


1414


1466


Figure 3-6. Continued


-Sc
I,


1377














St -.
PO
H

VPL VL

CM t'
VPM .


ml


1234

.' LD


O-'.


;4 VPL

' IN
"* ',"


MG
Po ,



CM ", "
VPM


ml .


1246


Figure 3-7. A rostrocaudal series of closely spaced coronal sections showing the ventroposterior
area (VP) of the thalamus in detail. Low-magnification images of sections stained
with hematoxylin for myelin and high-magnification details of adjacent sections
stained with thionin for Nissl bodies are labeled for subnuclei. Section numbers are
listed at the bottom of each section, and sections were cut at 40gm, specimen 84-49.
Scale bar=5mm. CL=central lateral nucleus, CM=centre median nucleus,
fr=fasciculus retroflexus, H=habenular nuclei, LD=lateral dorsal nucleus,
MD=mediodorsal nucleus, ml=medial lemniscus, Pf=parafascicular nucleus,
Po=posterior nucleus, st=stria medullaris, VL=ventral lateral nucleus, VPL=ventral
posterior lateral nucleus, VPM= ventral posterior medial nucleus.










100


1233


b,