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ANATOMY OF A CORTICAL-STRIATAL-THALAMIC NETWORK MEDIATING
DIRECTED ATTENTION IN THE RAT
JOSEPH LATON CHEATWOOD
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
Joseph Laton Cheatwood
For the great neuroscientists, men and women, who have gone before, and those
who will come. Giants, thank you for lending your shoulders, on which I stand.
I now offer mine.
I thank the innumerable people who have contributed to my making it to this point
in life. I thank my folks and sisters for bringing me up as best they could and giving me a
head start in life. I thank Nancy Smith, my AP Biology teacher at Lake Gibson High
School in Lakeland, Florida. Under her guidance, I saw how little of the natural world
we understand. I thank the great professors at Stetson University, especially Peter May
and Terence Farrell, for encouraging my interest in the sciences and being great friends.
I couldn't have done all of the processing without Maggie Stoll. Tanya McGraw-
Ferguson showed me the wonder of teaching, and the value of strength. Diana Sarko sat
next to me for the last year or two, and I do not know how I would have made it without
her as my cheering section. I thank all of the members of my committee for giving of
their time to oversee my work. Jim Corwin has always been able to make me think and,
above all, laugh aloud despite myself. He also gave me several pieces of sound advice
that changed my life in many ways. I appreciate it, no matter how many ivy-leaguers
laugh in my face. Above all, I thank Roger Reep for being an excellent mentor. His
support has helped me get through the tough times, which unavoidably accompany life.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF FIGURES ............................. ............ .................................... vii
ABSTRACT ................................................... ................. viii
1 IN T R O D U C T IO N .............. .......................................... ......................... .. .. 1..
Striatal Anatom y and Topography ............... .............. ..... ...............1...
Functional Implications of Anatomical Organization ............................................8...
2 CORTICAL AND THALAMIC PROJECTIONS TO THE DORSOCENTRAL
STR IA T U M IN R A T S .... ....................................................................... ............... 14
In tro d u ctio n ............................................................................................................... .. 14
M materials and M ethods .. ..................................................................... ............... 16
R e su lts....................................................................................................... ....... .. 18
Injection Sites ............................................ .. .......... ......... ............... 18
Retrograde Labeling in Cortex and Thalamus................................................19
T opography .......................................................................... ........ ............... 32
D isc u ssio n ................ ............................................................................................ ... 3 2
Corticostriatal Connections ........................................................ 33
Thalam ostriatal Projections ........................................................ 37
C o n c lu sio n s............................................................................................................... .. 3 8
3 OVERLAP AND INTERDIGITATION OF CORTICOSTRIATAL AFFERENTS TO
DORSOCENTRAL STRIATUM IN THE RAT ......................................................40
In tro d u ctio n ................................................................................................................ 4 0
M materials and M ethods .. ..................................................................... ................ 42
R results ....................................................................................................... . 44
Injections in A G m /A G l .................................................................. ................ 45
Injections in A G m /PP C ........................................ ....................... ................ 46
Injections in A G m /V L O ....................................... ....................... ................ 48
Injections in A G m /V L ......................................... ........................ ................ 48
Injection s in A G m /L P .......................................... ........................ ................ 5 1
Injections of BD A in PPC ........................................................ 53
Injections of B D A in O c2M ........................................................... ................ 56
O their B D A Injections......................................... ......................... ................ 56
D discussion ......................................... ............. .................. 56
4 PHOTOTHROMBOTIC LESION OF POSTERIOR PARIETAL CORTEX IN
THE RAT ............................................................................ 62
Introduction ................................................................................... ...................... 62
M materials and M ethods .. ..................................................................... ................ 64
R results and D discussion ................ .............. ............................................ 65
5 CONCLUSIONS AND FUTURE DIRECTIONS....................................................71
L IST O F R E F E R E N C E S ...................................................................................................75
BIO GR APH ICAL SK ETCH .................................................................... ................ 87
LIST OF FIGURES
2-1. Injection sites in D C S .. ...................................................................... ................ 20
2-2. Distribution of labeled cells in case 5, an injection in central DCS, depicted on
selected coronal sections through the cerebral cortex A-C) and thalamus D-F)......23
2-3. Distribution of cortical and thalamic retrograde labeling resulting from injection
of Fast Blue (FB) or Diamidino Yellow (DY) in DCS and adjacent regions of the
d o rsa l striatu m .......................................................................................................... 2 4
2-4. Distribution of labeled cells in case 93, an injection in rostral DCS, depicted on
selected coronal sections through the cerebral cortex A-D) and thalamus E-G) .....26
2-5. Distribution of labeled cells in case 105, an injection in caudal DCS, depicted on
selected coronal sections through the cerebral cortex A-D) and thalamus E-G). ....27
2-6. Distribution of labeled cells in case 45, depicted on selected coronal sections
through the cerebral cortex A-D) and thalamus E-G) .........................................30
2-7. Fluorescent photomicrographs of representative sections from select cases .............31
3-1. Representative fluorescent photomicrographs of cortical and thalamic
inje ctio n site s ........................................................................................................... 4 7
3-2. Striatal labeling following injection of fluorescent anterograde tracers..................49
3-3. Labeling in cases D CS 147 and D CS 173 ............................................. ................ 50
3-4. Striatal labeling pattern in case D C S 191 .............................................. ................ 52
3-5. Labeled axons in the striatum after injection of BDA in cortical area PPC..............54
4-1. Lesion produced via irradiation with the Schott/Omega filter combination ............68
4-2. Lesion produced via irradiation with the YAG laser............................................70
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
ANATOMY OF A CORTICAL-STRIATAL-THALAMIC NETWORK MEDIATING
DIRECTED ATTENTION IN THE RAT
Joseph Laton Cheatwood
Chair: Roger L. Reep
Major Department: Veterinary Medicine
The anatomy of a cortical-subcortical network mediating directed attention in the
rat was investigated via retrograde, single anterograde, and multiple anterograde labeling.
The rat dorsocentral striatum (DCS) is essential for the proper functioning of the directed
attention network. Region DCS is defined as the central terminal field for corticostriatal
axons originating in medial agranular cortex (AGm). Cortical and thalamic afferents to
DCS were first defined by placing a retrograde tracer in DCS, and observing the resultant
pattern of cortical and thalamic labeling. In this way, it was determined that the cortical
areas AGm, PPC, VLO, and Oc2M project most consistently to DCS. Thalamic regions
LP, LD, MD, and VL were labeled selectively, depending on the placement of the
retrograde tracer in DCS. Anterograde tracing experiments were performed to further
explore the pattern of labeling observed in the retrograde study. Multiple injections of
fluorescent anterograde tracers were used to visualize the patterns of overlap and
interdigitation of cortical and thalamic regions with cortical area AGm. Cortical areas
PPC and Oc2M were found to form dense foci of axonal terminals in DCS. These foci
are regions of intense overlap with axons from area AGm, and may represent important
regions of convergence on striatal medium spiny neurons. Lastly, a novel model of focal
photothrombosis was developed in an effort to maximize the physiological similarities of
the rat neglect model and human neglect, which is most frequently caused by a stroke
affecting the middle cerebral artery.
Over the last 20 years, several areas of research have focused on basal ganglia
diseases and disorders with the goal of understanding the pathophysiology, and the
development of therapeutic agents. In humans, diseases of the basal ganglia are most
commonly associated with movement disorders, either hypokinetic (Parkinson's disease),
or hyperkinetic (Huntington's disease) . One broad clinical goal is the successful
development of new, more beneficial treatments and management strategies for disorders
of cortico-basal ganglia-thalamo-cortical loops subtending motion, directed attention, and
other brain functions. To create therapies for complex central nervous system (CNS)
disorders, we need a better understanding of the role of the basal ganglia in functional
CNS loops. This discussion considers what is currently known about the anatomy and
function of these circuits, with a specific focus on striatal projections from cortical and
Striatal Anatomy and Topography
The basal ganglia are a collection of nuclei located in the forebrain and midbrain of
most vertebrate species. These nuclei are known to play a role in movement, attention,
and cognition; but their functions are not fully understood. The basal ganglia are
composed of the caudate nucleus, putamen, globus pallidus, substantial nigra, and the
subthalamic nucleus of the thalamus, which are all interconnected. The neostriatum is a
subset of the basal ganglia, and is composed of the caudate nucleus, the putamen, and the
nucleus accumbens. The nuclei of the neostriatum are discrete in primates, but are fused
in rodents . For this reason, the rat neostriatum is also synonymously referred to as
the caudato-putamen . There are several types of neurons in the striatum, but the
subtype receiving the most attention is the medium spiny neuron.
Efferent projections from cortical areas or thalamic nuclei enter the basal ganglia in
several places, and form synapses on medium spiny neurons in the caudate nucleus and
putamen [40,85,133]. The neostriatum receives convergent inputs via afferent fibers
from sensory, motor, and associational cortical areas; the intralaminar nuclei of the
thalamus; substantial nigra pars compact (SNc); and the dorsal raphe nucleus. Most of
these inputs are glutamatergic, with the exception of those from SNc and the dorsal raphe
nucleus, which are dopaminergic and serotoninergic, respectively [40,85,133]. The
densest projections to the neostriatum come from the cortex and the intralaminar nucleus
of the thalamus. Most synapses (85%) on the primary neurons of the striatum (medium
spiny neurons) are formed by axonal projections from these two contributors [55,66].
This strong interrelation suggests an important integrative role of cortical and thalamic
connections to striatal neurons.
Anatomical evidence suggests that the striatum may play an important role in the
integration of information coming from multiple cortical areas and thalamic nuclei.
Axons from cortical and thalamic neurons arborize in the striatum in a morphology
termed "cruciform axodendritic" by Ram6n y Cajal, in reference to the relatively straight
course the axons follow, forming synapses with dendrites of medium spiny neurons as
they pass . These axons have been shown to enter the striatum and travel long
distances along the rostrocaudal plane, synapsing all along their length with numerous
striatal neurons .
Interestingly, individual striatal neurons receive relatively few synapses from a
given axon as a result of the cruciform axodendritic morphology of cortical afferents
. Considering this fact in concert with the prevalence of cortical synapses on
medium spiny neurons (80%, as previously noted), individual medium spiny neurons
likely receive input from multiple cortical areas . Similarly, the small number of
striatal cells available to receive input relative to the number of cortical cells projecting to
the striatum has led many to hypothesize that the striatum is a site of convergence for
information from multiple brain regions [17,85,133].
Cortical inputs to the striatum have been studied in some detail, but data on the
afferent projections of discrete cortical areas are incomplete, as are the data concerning
the fine topography with which cortical areas project to the striatum. Anatomical and
electrophysiological studies of corticostriatal connections date back to the 1800s. The
first confirmation of the existence of corticostriatal projections came in the mid-1900s.
In 1944, Glees provided the first unequivocal evidence of the existence of corticostriatal
projections. Glees  used a recently developed silver-stain method to visualize the
paths of degenerating axons after the destruction of selected cortical areas, and found that
fibers do indeed project from the cortex to the striatum of the cat, contrary to many prior
Since publication of Glees' paper report in 1944, several generations of axonal
tracing methods (including autoradiography, horseradish-peroxidase, biotinylated
dextran-amines, and a variety of anterograde and retrograde fluorescent tracers) have
been used to expand on Glees' findings . It is now accepted that all functional areas
of the cerebral cortex send projections to some portion of the striatum. This has been
confirmed via retrograde and anterograde tract tracing in cat, rat, and primate models
In addition to documenting which cortical areas send axonal projections to the
striatum, important patterns related to the topography of these projections have been
described. Kemp and Powell  first noted that axonal projections from individual
cortical areas project to discrete and consistent striatal regions that follow the overall
shape of the external capsule, with little overlap between areas. This pattern has been
observed for many cortical areas [10,75,76,92]. In an expansion of this finding, Brown et
al.  and Wright et al.  presented data supporting this pattern, but also noted that
two areas of axon terminals can be seen after closely spaced injections in rat
somatosensory cortex: one area of similar shape to that previously reported, and an
additional area located medially in the striatum. Wright et al.  suggest that this
pattern indicates the presence of at least two systems of corticostriatal projections
terminating in the rat striatum, termed the "discrete" and "diffuse" systems.
The medial area of the striatum discussed by Brown et al. and Wright et al., among
others, is the current focus of a rat model of hemispatial neglect [15,16,18,58,63,64,91-
93,116]. This model focuses on a ~1 mm diameter portion of the rat striatum, located
dorsomedially and extending throughout the rostrocaudal extent of the striatum, as a
potential focal point for convergence of information coming from the medial agranular
cortex (AGm) and the posterior parietal cortex (PPC), among other cortical areas. Focal
injections of a retrograde tracer into this striatal area, referred to as the dorsocentral
striatum (DCS), produce labeled cells in many cortical areas, depending on the precise
location of the injection, but most often in AGm, PPC, and orbital cortex (Chapter 2).
Functional aspects of this model are discussed next.
In addition to the presence of unique mediolaterally and dorsoventrally discrete
projections from cortical areas, these terminal fields are known to extend great distances
in the rostrocaudal dimension [44,91-93,104,114]. In one seminal paper, Selemon and
Goldman-Rakic  demonstrated that some cortical areas project axons that traverse
the rostrocaudal extent of the primate caudate nucleus. In the same paper, Selemon and
Goldman-Rakic demonstrated conclusively that projections arising from cortical areas: 1)
terminate closely to one another in the striatum, 2) do not limit their projections to
proximal portions of the striatum, and 3) partially overlap and interdigitate in the
striatum. Taken together, these findings indicate that axons arising from individual
cortical areas project to portions of the striatum in a consistent, partially overlapping, and
partially interdigitating fashion, without regard to their initial anatomical proximity to
Axons terminating in a given striatal territory can arise from either ipsilateral or
contralateral cortical areas. Studies using several techniques arrived at this conclusion.
Electrophysiological recordings from single striatal neurons responding to stimulation in
both ipsilateral and contralateral cortical areas  and retrograde or anterograde tract
tracing [75,76,92] have conclusively demonstrated the presence contralateral and
ipsilateral axons sending projections to the striatum. These findings indicate that axons
from both adjacent and distant ipsilateral cortical areas converge on single striatal
neurons; and that convergence of information from homotopic contralateral cortical areas
also occurs in the striatum. These contralateral connections may allow for some
interhemispheric synchronization of striatal activity [75,76].
Two broad methods are used to study corticostriatal projections via anterograde or
retrograde tracers: studies of populations of neurons [10,93] or single-cell studies
[89,101,135]. Each type of study is useful for answering different types of questions
about corticostriatal projections, and both have advantages and disadvantages.
Studies of populations of axons allow for comparisons to be made between the
projections of functional cortical areas, like AGm and PPC . Additionally, these
studies are useful in determining which cortical layers are involved in connections
between two territories . These studies can be difficult to interpret, however, if
injections of anterograde tracers cross cytoarchitectural boundaries (i.e., injection on
border of AGm and forelimb cortex). This potential problem with anterograde studies
mandates that any findings from such studies be confirmed via retrograde injections
directed at targets identified by anterograde methods .
Single-cell studies provide the relative advantage of being able to follow a single
axon from its origin to its termination; noting its path, and noting whether any collateral
projections stem from single axons of interest . These studies are performed by
inserting a pipette into individual neurons of interest, or placing a minute quantity of a
tracer near a neuron, hopefully staining only one cell or very few cells . Because of
the impracticality of doing a large number of single-cell injections to determine all of the
targets of a given cell population, studies of single neurons do not easily provide enough
information about relative numbers of neurons projecting among areas of interest. Given
these benefits and pitfalls, single-neuron studies are excellent for examining the results of
broader tracing studies, in much more detail.
By using single-neuron studies in the above fashion, researchers are able to more
clearly define the way in which axonal arborizations of corticostriatal neurons relate to
dendritic arborizations of striatal neurons. The original single-neuron studies were
conducted by Ramon y Cajal, who examined Golgi-stained sections and described the
structure of axons in the striatum, which he correctly surmised to be corticostriatal
In 1987, Wilson  published an important study using several different
methods to look at individual cortical neurons and their efferent projections. In this
study, Wilson used his data in addition to previous findings, to describe three different
subpopulations of neurons projecting from cortical area AGm:
* A population located in layer V which projects to the brainstem. These cells also
send collateral projections to ipsilateral striatum and at least some of their axons
enter the pyramidal tract .
* A population of corticothalamic neurons in layer VI that send collaterals to striatum
* A larger population of corticostriatal neurons in layer Va that do not project to the
thalamus or brainstem. Single cells of this type form projections to contralateral
cortex, ipsilateral striatum, and contralateral striatum .
As a result of his findings, Wilson speculates about the existence of neurons that
are exclusively corticostriatal, stating that perhaps all of the corticostriatal afferents are
collaterals of axons terminating elsewhere in the central nervous system .
Functional Implications of Anatomical Organization
The most basic functional unit of the striatum is the medium spiny neuron. These
neurons make up the majority of cells in the striatum. Medium spiny neurons are the
targets of afferent projections to the striatum, and are also the cells extending efferent
projections out of the striatum . Medium spiny neurons are considered to be tonically
inactive, firing mainly in response to stimulation from cortical and thalamic inputs
[17,132]. Since so many types of afferents contact medium spiny neurons, but so few
synapses are contributed by any one afferent, the likelihood exists that striatal neurons
may only fire in response to convergent stimulation from multiple afferent sources,
indicating a potential role as an integrator of data from multiple cortical areas .
Cowan and Wilson provided preliminary support for this theory by demonstrating that the
synchronization of multiple cells is needed for depolarization of medium spiny neurons
. The previously discussed interdigitation of cortical afferents allows the hypothesis
that axonal inputs from multiple cortical regions firing together could stimulate striatal
neurons. This hypothesis warrants further examination.
In addition to the convergence of cortical inputs, glutamatergic thalamic afferents,
dopaminergic SNc projections, and serotoninergic projections from the dorsal raphe
nuclei also contact single-medium spiny neurons in the striatum [9,69]. Dopamine also
plays a role in the output of the striatum, via the differential expression of excitatory and
inhibitory dopamine receptors (discussed is more detail below). Despite the lack of a
clear model for the interaction of inputs using different neurotransmitters, one commonly
accepted hypothesis is that dopaminergic inputs play a role in gating the output of striatal
medium spiny neurons [41,83,86,106,124].
The striatum can be broken into functional subsystems with different projection
patterns, neurological markers, neurotransmitter receptors, and end functions [40,78,79].
Gerfen refers to these pathways as the "striatonigral" and "striatopallidal" pathways, but
they are most commonly known as the "direct" and "indirect" pathways, respectively
. Both the direct and indirect pathway share common endpoints: GPi and SNr. The
direct pathway projects directly from the striatum to GPi and SNr, as the name suggests.
The indirect pathway, however, projects first to GPe, then to the subthalamic nucleus,
and finally to GPi and SNr.
The direct pathway encourages excitation of thalamic targets (positive feedback),
while the indirect pathway exerts an inhibitory influence on excitatory thalamic
projections, resulting in an overall inhibitory effect (negative feedback). Interestingly,
medium spiny neurons respond differently to dopaminergic inputs from SNc to the
striatum, depending on the pathway (direct or indirect) to which they project. This
difference results from the selective expression of two types of dopamine receptors
(called Dl and D2) on populations of medium spiny neurons. Striatal medium spiny
neurons contributing to the direct pathway have Dl receptors, which are excitatory; and
neurons contributing to the indirect pathway have D2 receptors, which are inhibitory
The direct and indirect pathways were at one time broadly used to explain the
function of striatal outputs to the basal ganglia. With today's better understanding of
striatal circuitry, the concept of the direct and indirect pathways is recognized as an
oversimplified description of the role of the striatum in several currently hypothesized
cortico-basal ganglia-thalamo-cortical loops.
The concept of multiple cortico-basal ganglia-thalamo-cortical loops was first
popularized by Alexander et al. [1,2,85], who proposed the existence of five parallel
loops involved in processing: motor, oculomotor, dorsolateral prefrontal, lateral
orbitofrontal, and anterior cingulate circuits. Each of these loops uses a direct and an
indirect pathway for striatal efferents .
The exact composition of cortico-basal ganglia-thalamo-cortical loops is currently
unclear. Some have hypothesized that each parallel loop may be even further separated
into subloops for more discrete functions, such as movement preparation and movement
execution [1,79]. An alternate hypothesis to this parallel-loop concept is the existence of
a "tripartite" striatum, with three functional and topographic subdivisions receiving
inputs from closely functionally related cortical areas: the associative territory, the
sensorimotor territory, and the limbic territory .
The two hypotheses are not entirely at odds, but the tripartite hypothesis places
more emphasis on the integrative function of the striatum. A rat model of corticostriatal
mechanisms for directed attention more closely resembles the patterns of connectivity
predicted by Parent . The previously mentioned rat model of hemispatial neglect is
an example of a cortico-striatal network mediating an integrative function: directed
attention. Experimental lesions of cortical area AGm, PPC, or striatal region DCS are
sufficient to produce persistent multimodal neglect [15,16,58,63,64,91,97], as are lesions
of axons connecting the areas [ 11].
In some animals with lesions in AGm or PPC, spontaneous recovery is possible
. Delivery of the dopamine agonist apomorphine either systemically or directly into
DCS induces recovery of function after a lesion in either AGm or PPC [14,15,62], while
more lateral injections do not induce recovery [115,116,118]. In contrast to the cortical
lesions, animals receiving excitotoxic axon-sparing lesions of DCS do not recover
spontaneously, nor do they respond to apomorphine . Therefore, behavioral and
anatomical results from the neglect model support the conclusion that the DCS is a
required element in a cortico-striatal circuit for directed attention.
In addition to the functional and topographic subdivisions previously discussed, the
striatum can be further subdivided at the molecular level. Immunohistochemical staining
of the calcium binding protein calbindin reveals an interesting mosaic pattern of staining
in the rat striatum. The striatum contains opiate receptor-rich/calbindin-poor areas, called
"patches," in a calbindin-rich/opiate receptor-poor sea, termed the "matrix" . It has
been hypothesized that this apparent compartmentalization may reflect the differential
staining of functional units in the striatum [29,38].
Some debate exists, however, about the anatomical and functional importance of
the patch and matrix compartments. Some of the literature supports the hypothesis that
cortical areas project to both patch and matrix compartments in their corresponding
striatal territories, but different areas predominantly project to one compartment or the
Gerfen [39,40] concluded that cortical projections to patch and matrix areas arise
from different cortical layers: layer Vb and VI cortical neurons project principally to
patches; while neurons in layers Va, II, and III project largely to the matrix. Gerfen also
notes that neocortical areas project largely to the matrix, while allocortical areas send
their projections to patches. Kincaid and Wilson confirmed these findings, adding that
there are probably multiple subtypes of corticostriatal neurons, each potentially finding
different targets in the striatum .
Gerfen also hypothesizes that, in addition to receiving input from different cortical
layers, patch and matrix compartments also differ in their efferent projections. He
suggests that axons leaving patch compartments project largely to the dopaminergic
substantial nigra pars compact, and that axons from the matrix compartment project to
the GABAergic neurons of the substantial nigra pars reticulata [37,38,40]. Despite
Gerfen's data, no consensus on the functional importance of striatal patch and matrix
compartments exists at this time. Specifically, the differential roles of patch and matrix
compartments in cortico-basal ganglia-thalamo-cortical loops, if any, remain to be
Though striatal research has been conducted in rodents, cats, and primates, the
majority of findings have been consistent. Obviously, some anatomical differences exist,
especially between the rat and primate models. As previously mentioned, key differences
exist in the basic arrangement of the basal ganglia in rats and primates. In primates and
cats, the caudate and putamen are separate, non-overlapping structures and are separated
from one another by the internal capsule. In rats, the two nuclei are blended together in
the dorsal portion of the striatum, and the fibers that make up the internal capsule in cats
and primates perforate the striatum in a much more diffuse arrangement.
In short, the anatomical and functional intricacies of the striatum, specifically
regarding cortico-basal ganglia-thalamo-cortical loops, are not well understood. Future
research on corticostriatal connections and loops should focus on issues such as
* Formation of synapses from functionally related cortical areas on individual
* The role of convergent stimuli from multiple inputs (cortical areas, thalamic inputs,
etc.) in the generation of an action potentials by striatal neurons
* The definition of the functional subunits of the striatum and their relationship to
cortical, thalamic, and brainstem afferents and efferents
* The development of clinical interventions to modify the function of cortico-basal
ganglia-thalamo-cortical loops and generate functional recovery from lesions and
CORTICAL AND THALAMIC PROJECTIONS TO THE DORSOCENTRAL
STRIATUM IN RATS
The dorsocentral striatum (DCS) is defined as the major site of termination of
corticostriatal inputs originating from the medial agranular cortex (AGm or Fr2) in rats
[6,30,93,94]. In the region of the striatum between the level of the genu and that of the
anterior commissure (ac), DCS constitutes a territory ~1 mm in diameter, centered in the
dorsal striatum -0.5 to 1.5 mm below the external capsule, approximately equidistant
from the medial and lateral boundaries of the striatum . Area AGm is a multimodal
association premotor cortex with diverse cortical connections , and DCS has been
implicated as a multimodal convergence region of striatum [76,93].
As noted by Parent and Hazrati , once it became clear that the striatum has
sensorimotor, associational, and limbic regions, it became important to determine the
organizational subdivisions within these regions. Although association areas of the rat
striatum have not been studied in the same detail as sensorimotor and limbic regions,
several investigations have focused on striatal circuitry related to prefrontal cortical areas
[7,8,46]. The study of association areas is of special interest because the influence of the
striatum on motor function includes cognitive components  mediated by cortical
areas like AGm. Thus, the associative striatum constitutes a substrate for the dynamic
modulation of behaviors having a significant multimodal component involving
The relationship between DCS and AGm is of particular interest, because each of
these regions is a major component of the neuronal circuitry mediating directed attention.
After the initial development of a rat model of hemispatial neglect [18,19], behavioral
experiments identified AGm and the posterior parietal cortex (PPC) as being cortical
areas critical for the normal functions of spatial orientation and directed attention
[15,16,58,63,64]. In rats, lesions in either of these reciprocally interconnected [91,97]
areas produce multimodal neglect, as does disconnection of their corticocortical
connections without direct damage to AGm or PPC . Spontaneous recovery from
neglect resulting from cortical lesions occurs in some rats , and is correlated with
reestablishment of symmetrical expression of immediate early genes and glutamate
receptors in the dorsolateral striatum and DCS [120-122]. In addition, DCS is essential
for spontaneous and pharmaceutical-induced recovery of function [115,116], and this
points to the pivotal role of corticostriatal connections in directed attention.
Because of the critical role of DCS in the circuitry mediating directed attention
and recovery from neglect, it is important to know which cortical areas are capable of
influencing the activity of neurons in DCS. Thus, my study aimed to identify all the
cortical inputs to DCS, and to interpret this pattern with regard to the known
corticocortical connections of these areas. Specifically, because of the close anatomical
and functional relationship between AGm and PPC, I wished to determine if cortical-area
PPC projects to DCS. In addition, I wanted to determine if the thalamic nuclei that
project to DCS include those that project to the cortical areas that provide input to DCS.
For these purposes, I made injections of retrograde axonal tracers in DCS or along its
margins. The results provide new information on corticostriatal and thalamostriatal
topography and convergence, support the behaviorally derived hypothesis that DCS
functions as a striatal association area that is critical for directed attention, and provide a
foundation for experiments intended to promote recovery from neglect, by therapeutic
Materials and Methods
Twenty-five male Long-Evans Hooded rats were anesthetized with an
intraperitoneal injection of a ketamine/xylazine cocktail (90 mg/kg: 10 mg/kg) and placed
in a stereotaxic device. Either Fast Blue (Sigma, 3% in H20) or Diamidino Yellow
(Sigma, 3% in H20), was injected into the striatum. Both are frequently used retrograde
axonal tracers that produce reliable labeling with little spreading of the injection site
relative to other tracers like Fluorogold . Injections were made via Picospritzer
(General Valve Corp., Fairfield, NJ) using 2 or 3 pulses of 20 to 30 psi, 5 to 20 msec
duration; or via a 33 g Hamilton syringe using a volume of 0.05 to 0.10 [tl. Some
animals received a second injection of the other tracer in the opposite hemisphere, but
this analysis involves only one tracer per animal. The injections in cases 93 and 94 were
on the right side; all others were on the left. After a 3 to 5 day survival time, rats were
injected IP with 3 mL chloropent (4.25% chloral hydrate, 0.9% pentobarbital) and
perfused intracardially with 300 mL of potassium-buffered saline (PBS) followed by 300
mL of 4% buffered paraformaldehyde. Brains were then removed and stored in dilute
fixative (0.4% paraformaldehyde), and cryoprotected by sinking in a 30% sucrose
solution. Coronal sections were cut at 40 |tm on a freezing microtome, and placed in
dilute fixative until being mounted on slides. Three spaced series of sections were used
for analysis: one for viewing on a fluorescent microscope, a second for cresyl violet
staining for cytoarchitectural analysis, and a third for fluorescent photomicrography.
Three-dimensional reconstructions of tracer-injection sites were produced by
outlining areas of visible fluorescence and the boundaries of the striatum and cortex from
fluorescent sections, transferring these sketches into vector drawings (Adobe Illustrator
10), then making three-dimensional models and rendering final images using an M5
imaging system (Imaging Research, Inc). Cases 93 and 94 are represented on the left for
comparison purposes, even though their injections were located on the right side.
Thalamic and cortical labeling were evaluated using an Olympus BH-2
fluorescent-equipped microscope. Sections were examined at 20x primary magnification
for labeled cells in any cortical areas or thalamic nuclei. The locations of labeled cells
were determined by cytoarchitectural examination of adjacent cresyl-violet-stained
sections, and with reference to the cytoarchitectural criteria of Zilles and Wree  for
cerebral cortical areas. The terms AGm and AGI are synonymous with their areas Fr2
and Frl, respectively. The terms rAGm and cAGm refer to the rostral and caudal
subregions of AGm that are located rostral and caudal to the level of the genu, and have
different patterns of functional representations [30,82] and anatomical connections .
Area PPC is located at approximately AP -3.4 to -4.4 mm relative to bregma, between
ML 1.5 to 4.5mm [16,91]; and was distinguishable from the caudally adjacent area Oc2M
by the appearance of layer V in Nissl stained sections, in most brains. In PPC, layer V
merges with layer IV superficially and with layer VI basally. In area Oc2M, the
boundaries of layer V with layers IV and VI are more distinct, because of reduced cell
density in the superficial and basal portions of layer V. Thalamic nuclei were identified
and delineated according to the criteria and maps of Jones  and Paxinos and Watson
. Analysis of labeled cells was limited to the hemisphere ipsilateral to the tracer
injection. Cases having injection sites that encroached on the white matter or cortical
tracks significant enough to produce contralateral labeling of corresponding cortical areas
were excluded from analysis.
Figure 2-3 was constructed by viewing adjacent sections throughout the brain
with the fluorescent microscope. A score was assigned based on the relative amount of
fluorescent labeling present in cortical and thalamic regions. Scores range from 0 (no
label) to 3 (most densely labeled region) within brains. This scoring system is shown in
Figure 2-3 as shaded boxes ranging from white (0) to black (3).
To describe the location of striatal injection sites and labeling in some cortical
areas, it is useful to refer to landmarks external to the striatum, because of its
homogeneous appearance. For this purpose, the locations of the following features, here
listed in rostrocaudal order along with their shorthand designations, were used as
landmarks: crossing of the genu of the corpus callosum (genu), presence of the septal
nuclei without the fimbria (septum), crossing of the anterior commissure (ac), and the
most rostral appearance of the fimbria distinct from the septal nuclei (fimbria). In the
descriptions below these locations are referred to as the levels at which these features are
The visible fluorescence of the injection sites for 10 key cases is shown in Figure
2-1. Although uptake of Fast Blue and Diamidino Yellow occurs largely, if not entirely,
in the portion of the injection site that was damaged during placement of the tracer , I
have chosen to illustrate a larger area as a conservative estimate of the affected area.
However, it is improbable that this entire visible region is producing label elsewhere in
the brain . These injections were all located in or around the dorsocentral region of
the striatum, with variations in dorsoventral and mediolateral position. Most injection
sites were elongated in the rostrocaudal dimension. Cases 5, 93 and 105 (Fig. 2-1)
represent injections in mid, rostral and caudal DCS, respectively, and are presented in
that order below, together with four other cases involving those regions. Cases 45 (Fig.
2-1 C,D), 52 (Fig. 2-1 F,G), 84 (Fig. 2-1 F) represent injections in dorsal DCS and these
are presented separately below.
Retrograde Labeling in Cortex and Thalamus
Below are detailed descriptions of the injection sites and labeling patterns for four
important cases, with the other cases discussed in reference to these.
Case 5 (central DCS). The injection site for case 5 was centered 1 mm rostral to
the anterior commissure, at the level of the septum (Fig. 2-1). It was centered 700 |tm
ventral to the white matter, 1.5 mm lateral to the lateral ventricle, and 1.5 mm medial to
the lateral boundary of the striatum. This represents an injection in the central portion of
the AGm projection field in DCS. As discussed below, cases 93 and 105 represent rostral
and caudal extensions (respectively) of the territory encompassed by case 5. The
injection site was cylindrical and extended rostrally from its center to the level of the
genu and caudally to the level of the ac.
A slight track of fluorescence lining the pipette trajectory was seen in cortical area
AGI on the centermost section, but this track does not include the white matter.
Figure 2-1. Injection sites in DCS. A) Three dimensional reconstruction of the 10
principal injection sites seen from a lateral oblique perspective, to show their
relative locations within the dorsal striatum. Stick diagram indicates dorsal,
lateral and rostral directions and applies to panels C and F also. B) Dorsal
perspective of the same set of injections as in A, rostral toward the bottom.
Vertical white line indicates rostrocaudal coordinates with respect to Bregma.
Horizontal white lines indicate the plane of sections D,E and G,H. C) Three
dimensional renderings of the injection sites in the four cases discussed in
detail in the Results. White lines indicate the planes represented by the
corresponding two-dimensional depictions of the injection sites at two levels.
D-E) Location of injection sites for the four cases in C, plotted on two
sections between the level of the genu and ac. Scale bar in D also applies to
panels E, G and H. F) Three dimensional reconstructions of the remaining six
cases. White lines indicate the planes represented by the two-dimensional
depictions in G and H. G-H) Sectional views of the injections in F, at the
same rostrocaudal levels as panels D and E. The injection for case 84 (light
green in panel F) is not shown because it begins caudal to the level in G.
Reprinted with permission from: J.L. Cheatwood, R.L. Reep and J.V. Corwin,
The associative striatum: cortical and thalamic projections to the dorsocentral
striatum in rats, Brain Res, 968 (2003) 1-14; Figure 1, pg 4.
Figure 2-1. Continued
There was no label in contralateral AGI at the level of the injection, indicating that the
track did not influence the labeling pattern observed. In the cortex, retrogradely labeled
cell bodies were present in several areas (Fig. 2-2, Fig. 2-3). Area AGm was labeled
most intensely on sections greater than 1 mm rostral to the level of the ac commissure, in
layers II/III and V (Fig. 2-2 A-C). In all sections in which AGm was labeled, layer V
was labeled most strongly. Areas VLO and LO were weakly labeled in layer V only. In
areas PPC and Oc2M, label was only present in layer V and was most robust rostrally. In
the thalamus, nuclei VL, VM, MD, the intralaminar nuclei (CM, PC, CL), and PF all
contained labeled cells (Fig. 2-2 D-F). Nucleus VL appeared more strongly labeled
medially than laterally, but the other nuclei were labeled throughout. The injection in
case 94 overlapped that of case 5 but extended farther rostrally and lay somewhat lateral
to case 5 (Fig. 2-1). Case 94 produced a broader distribution of cortical labeling (Fig. 2-
3) that included all of AGm, somatic sensory and motor areas AGI, FL, HL, Par 1 and Par
2, and visual association areas Oc2M and Oc2L. The pattern of thalamic labeling was
nearly identical to case 5 (Fig. 2-3).
Case 93 (rostral DCS). The injection site in case 93 was centered at the level of
the genu and represents a more rostral injection than case 5, but one that is also centered
in DCS. The center of the injection was located 1.1 mm ventral to the white matter. The
injection was oval shaped and spanned a distance of 1.1 mm in the dorsoventral plane,
700 |tm in the mediolateral plane, and 1 mm in the rostrocaudal plane. There was a very
faint fluorescent track visible along the trajectory the pipette followed through AG1.
Contralateral AGI contained no labeled cells. No fluorescence was evident in the white
matter. Multiple cortical areas were labeled by the striatal injection.
DCS 5 D A
B mL**^ / VPL M 'P
Figure 2-2. Distribution of labeled cells in case 5, an injection in central DCS, depicted
on selected coronal sections through the cerebral cortex A-C) and thalamus D-
F). Reprinted with permission from: J.L. Cheatwood, R.L. Reep and J.V.
Corwin, The associative striatum: cortical and thalamic projections to the
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 1, pg 6.
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 1, pg 6.
Figure 2-3. Distribution of cortical and thalamic retrograde labeling resulting from
injection of Fast Blue (FB) or Diamidino Yellow (DY) in DCS and adjacent
regions of the dorsal striatum. Four levels of relative labeling density are
depicted: no label (white), light label (light gray), medium (dark gray), and
heavy (black). Reprinted with permission from: J.L. Cheatwood, R.L. Reep
and J.V. Corwin, The associative striatum: cortical and thalamic projections to
the dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Table 1, pg 3.
Labeled cells were evident in layers II/III and V of rAGm (Fig. 2-4 A,B). Label in layer
V of AGm became sparser caudally and was limited to layer II/III by the level of the
The labeling in layer II/III of area AGm also became less intense caudal to the
anterior commissure, but remained faintly visible throughout cAGm. Label was present
in layer V of area Cg at and caudal to the level of the ac. Labeled cells in area HL were
limited to layer V and were present only in small numbers (Fig. 2-3B). Labeled cells in
layer V of areas Oc2M, PPC, Par 1, Par 2, RSA and RSG were distributed evenly
throughout their rostrocaudal extent (Fig. 2-4C,D). In the thalamus, label was observed
in VL, VM, MD, the intralaminar group (CM, PC, CL), and PF with no observable
topography (Fig. 2-4E,G). The injection in case 15 was virtually identical to case 93 but
extended slightly more rostrally and was somewhat more focal (Fig. 2-1C,D; Fig. 2-7C).
Cortical labeling was most dense in areas AGm, PPC and orbital cortex. Labeling was
dense in rAGm, sparse in cAGm. Thalamic labeling was similar to case 93, differing
only in relative intensity of cellular labeling (Fig. 2-3).
Case 105 (caudal DCS). The injection site for case 105 was centered at a level
0.5 mm rostral to the ac, and represents a location caudally adjacent to case 5 (Fig. 2-1,
Fig. 2-7A). The center of the injection was located 1 mm ventral to the white matter and
1.1 mm lateral to the lateral wall of the lateral ventricle. The injection measured 1.4 mm
in the dorsoventral plane, 600 |tm in the mediolateral plane, and 1.5 mm in the
rostrocaudal plane (it extended from 500 |tm caudal to the level of the genu to the level of
the ac). There was no evidence of fluorescent tracer lining the path through which the
pipette was inserted, nor was there any fluorescent material in the white matter. In the
cortex, several areas contained labeled cells. Areas VLO, LO, and the rostral portions of
AGm and AGI contained labeled cells in layers II/III and V (Fig. 2-5A,B). Area AGm
remained labeled throughout its rostrocaudal extent and densely in cAGm, though layer
V was no longer labeled caudal to the level of the ac. Labeled cells in area AGI were seen
only until the level of the ac. Label was observed in area Cg at the level of the ac and
continued to its caudal boundary. Areas HL, PPC, Oc2M, Par 1, Par 2, and PRh all
contained labeled cells in layer V throughout their rostrocaudal extents (Fig. 2-5C,D). In
the thalamus, nuclei AV, VL, VM, the intralaminar nuclei (CM, PC, CL), MD, PF, and
rostral LP all contained labeled cells (Fig. 2-5E,G).
The injection in case 6 partially overlaps that of case 105 throughout most of its
extent, but is centered more ventrally (Fig. 2-1; compare panels D and G).
DCS 93 E
AGIPVA PT AV
*.* e ** *
V.m *. '/L
B AGmv AGI F
RSG MD CL
RSG ,**** Par I
Figure 2-4. Distribution of labeled cells in case 93, an injection in rostral DCS, depicted
on selected coronal sections through the cerebral cortex A-D) and thalamus E-
G). Reprinted with permission from: J.L. Cheatwood, R.L. Reep and J.V.
Corwin, The associative striatum: cortical and thalamic projections to the
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 3, pg 7.
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 3, pg 7.
A AGI "** t *
*.. *IVLO S
*B FL '4V AGm FoA
-. .,^s. .* VL O
Par 1".* ,... ".. /I
Figure 2-5. Distribution of labeled cells in case 105, an injection in caudal DCS, depicted
on selected coronal sections through the cerebral cortex A-D) and thalamus E-
G). Reprinted with permission from: J.L. Cheatwood, R.L. Reep and J.V.
Corwin, The associative striatum: cortical and thalamic projections to the
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 4, pg 8.
dorsocentral striatum in rats, Brain Res, 968 (2003) 1-14; Figure 4, pg 8.
Cortical labeling included many of the same areas as in case 105 but AGm was labeled
more intensely in rostral portions and somatic sensorimotor labeling did not include area
HL. Thalamic labeling was very similar to case 105 with the addition of labeling in LD
Case 104 had an injection site located just medial to that of case 105 (Fig. 2-1). It
also extended more rostrally than case 105. These two injections overlap partially in the
middle of their a-p ranges. The pattern of cortical labeling for case 104 is similar to the
pattern seen in case 105, with the addition of labeling in areas FL, Par2, and Oc2L, and a
lack of labeled cells in RSA. Thalamic labeling differed from case 105 in the addition of
LD, VPL, and VPM (Fig. 2-3).
Case 45 (dorsal DCS). The injection site in case 45 was centered at the level of
the anterior commissure and extended caudally to the level of the fimbria (Fig. 2-1). The
injection was spherical in shape and measured 500 |tm in diameter. The dorsal border of
the injection lay against the ventral border of the white matter. Therefore, this case
represents a dorsal injection with respect to the boundaries of DCS. A moderate
fluorescent track outlining the path of the pipette could be seen in all layers of area FL
immediately above the center of the injection. Contralateral FL was not labeled,
indicating that the track did not cause a change in the labeling pattern of the striatal
injection. Because of the location of the injection site, a slight amount of fluorescence
was visible in the white matter. In the cortex label could be seen in areas AGm, AG1,
PPC, Oc2M, Oc2L, Par 1, Par2, MO, VLO, LO, Cg, FL, HL, and PRh. Throughout its
extent area AGm was labeled most densely in layers II/III, with infrequent labeling in
layer V. Fluorescence was strongest within 1 mm of the ac (Fig. 2-6A,B). Labeled cells
in layer V of area AGI could be seen from the level of the anterior commisure through the
caudal border of AGl. Area PPC contained numerous labeled cells in layer V throughout
its extent (Fig. 2-6C). Label in layer V of areas Oc2M and Oc2L began at their rostral
pole and continued caudally through the remaining sections (Fig. 2-6D). Area Par 1
contained lightly labeled cells in layer V only (Fig. 2-6C,D). Perirhinal cortex contained
labeled cells in layer V only. In the thalamus, nuclei AV, AD, VL, LD, medial LP, the
intralaminar nuclei (CM, PC, CL), and PF contained labeled cells throughout their extent.
Thalamic nucleus MD was labeled more strongly rostrally (Fig. 2-6E,G).
Case 52 had an injection site that overlapped that of case 45 from the level of the
ac to the level of the fimbria, but extended more caudally (Fig. 2-1). Cortical labeling
differed from case 45 by being sparse in rAGm, absent in MO, and present in areas RSA
and RSG. In the thalamus the distribution was identical to case 45, with the addition of
robust labeling in nuclei VPM and VPL (Fig. 2-3).
Case 84 had an injection site located lateral to both cases 45 and 52, and mostly
caudal to case 45 (Fig. 2-1). Cortical labeling was similar to case 45 with the addition of
labeled cells in RSA and RSG, and an absence of label in HL. The thalamic pattern was
the same as case 45 (Fig. 2-3, Fig. 2-7D).
Case 76 (Fig. 2-7B) involved a large injection that was dorsally located but did
not overlap with the more caudal dorsal injections represented by cases 45, 52 and 84. It
did overlap significantly with several rostral injections including cases 5, 15, 93, 94 and
104. Cortical labeling was extensive but excluded Par 1 and Par 2 (Fig. 2-3). The
thalamic labeling pattern was similar to the other dorsal cases presented above (Fig. 2-3).
Rt CL LM PV
Figure 2-6. Distribution of labeled cells in case 45, depicted on selected coronal sections
through the cerebral cortex A-D) and thalamus E-G). Reprinted with
permission from: J.L. Cheatwood, R.L. Reep and J.V. Corwin, The associative
striatum: cortical and thalamic projections to the dorsocentral striatum in rats,
Brain Res, 968 (2003) 1-14; Figure 5, pg 10.
D Oc2L Oc2M RSA
Par I RSG
Figure 2-7. Fluorescent photomicrographs of representative sections from select cases.
A) The Diamidino Yellow (DY) injection site in case 105 at the level of the
septum. B) Retrograde labeled cells in cortical area AGm, case 76. C) The
Fast Blue (FB) injection site in case 15 at the level of the genu. D) Retrograde
labeled cells in the thalamus, case 45. Reprinted with permission from: J.L.
Cheatwood, R.L. Reep and J.V. Corwin, The associative striatum: cortical and
thalamic projections to the dorsocentral striatum in rats, Brain Res, 968 (2003)
1-14; Figure 6, pg 11.
From the cases presented above it was possible to discern some topographic
patterns of connectivity. With regard to the cortex it is notable that all injections
produced labeled cells in areas AGm, PPC, orbital cortex, and Oc2M. No other cortical
areas were labeled with this degree of consistency. The pattern of labeling in AGm was
correlated with the location of the injection in DCS. Most of the injections had a longer
rostrocaudal than dorsoventral extent and produced at least some labeling across the full
rostrocaudal extent of areas AGm and PPC. However, injections centered rostrally in
DCS (cases 5, 15, 93, 94) tended to produce their most robust labeling in rAGm whereas
injections located in caudal DCS (cases 70104, 105) exhibited sparser labeling in rAGm.
One important exception is case 6, which had an injection located more ventrally in
caudal DCS than cases 104 and 105, and exhibited only slight labeling in cAGm. Area
PPC and visual association cortical areas Oc2M and Oc2L were labeled most densely in
cases with injections on the dorsal border of DCS (cases 45, 52, 76, 84) and those placed
caudally in DCS (104, 105). A rostrocaudal pattern of topography was also noticeable to
a lesser degree in area AG1.
In the thalamus, the nuclei with the most evident topographical relationship with
striatum were LD and LP. Labeled cells were found in LD and LP only in cases with
injections in dorsal portions of DCS.
Two important findings of the present study are the identification of cortical areas
that project to DCS, and the discovery of a high degree of overlap in the projections from
AGm and PPC. Also of significance is the finding that the thalamic nuclei projecting to
DCS are also those that project to the cortical areas that provide input to DCS. Finally,
there is evidence of some topography within DCS.
Many cortical areas have some projections to DCS but the most frequent
contributors are areas AGm, AGI, PPC, VLO, LO, and Oc2M. The projections from
AGm and other frontal cortical areas to the striatum are arranged along rostrocaudal and
mediolateral gradients [5,6,30,93], and the rostrocaudal topography observed in the
present study with regard to the projections from AGm is consistent with these previous
reports. There is also evidence of dorsoventral topography with respect to the labeling
pattern in AGm. The injection in case 6 was the most ventral in the sample, and
produced very little labeling in cAGm. This is consistent with the fact that cAGm
projects to a more dorsal region within caudal DCS .
Evidence for spatial overlap in the terminal fields of axons projecting from AGm
and PPC to the striatum was noted previously [93,97]. In the present cases, all injections
produced some degree of labeling in both PPC and AGm, confirming the previous
anterograde findings based on single injections in different brains. This finding is in
itself suggestive of a potential relationship between information coming from AGm and
PPC into DCS, and relates to functional studies demonstrating that AGm and PPC are
parts of an interconnected network mediating spatial processing and directed attention
. This network also includes orbital cortex and Oc2M, the final two regions labeled
in every brain.
The present retrograde findings demonstrating convergence in the region of DCS
of inputs from AGm, PPC and orbital cortex at the light microscopic level are suggestive
of a possible interaction among these inputs. In primates, Yeterian and Van Hoesen
 first demonstrated convergent striatal projections from distant cortical areas and
related this to the corticocortical connectivity between these areas. A later study by
Selemon and Goldman-Rakic  showed that most convergence of this kind actually
consist of zones of interdigitating projections rather than true overlap, and that
convergence occurs even among projections from cortical areas that are not themselves
interconnected. In rodent somatic sensory striatum there is significant true overlap in
addition to interdigitation in the case of projections from cortical barrels representing the
same whisker row . It is possible that AGm, PPC, and VLO each project to a discrete
territory within DCS, with little or no overlap. Alternatively, there may be convergence
onto single striatal neuron dendritic trees . These issues are discussed in more detail
in a companion study , which found that dense patches of segregated labeling from
AGm and PPC are interspersed with less dense patches of overlapping label in DCS.
The convergence in DCS of input from AGm and PPC is also interesting because
of behavioral and pharmacological data implicating DCS as a critical site necessary for
normal directed attention and spatial processing, and recovery from deficits induced by
cortical lesions [115,116]. The cannula placements in these studies tended to involve the
dorsal portion of DCS between the levels of the septum and ac, exactly that territory
implicated by the present findings as the AGm-PPC overlap zone.
In the present study visual association areas Oc2m and Oc2L were consistently
labeled most densely with dorsal and caudal injections, and this is consistent with the
topography defined by others [72,76]. No labeling was seen in area Ocl, which projects
to a circumscribed region of dorsomedial striatum bordering the lateral ventricle ,
outside the injected territory. These authors noted that areas Oc2M and Oc2L project not
only to this dorsomedial sector but also to the more deeply located territory encompassed
by the current injections, which is consistent with the current findings.
Several other cortical areas labeled by injections in DCS have their densest
corticostriatal terminations outside of DCS. Physiologically identified motor areas for
the forelimb and hindlimb project in a banded pattern to the dorsolateral striatum but the
less dense medial portions of their projection fields appear to be located on the lateral
margin of DCS . The physiologically identified vibrissal-eyefield area located in
AGm projects in a topographic manner to DCS and a dorsolateral shell . Somatic
sensory areas also project most densely to the dorsolateral striatum in banded patterns
[10,50], but in each case the medial-most band is located within DCS between the levels
of the genu and ac . The focal size of these medial bands together with individual
variation may explain the variability observed in labeling of somatic sensory areas.
Orbital and cingulate cortical areas project to territories along the ventral and medial
borders of DCS and appear to have their densest terminations outside of DCS [5,6].
Some discrepancies observed in the present study may relate to the focal,
distributed nature of corticostriatal axon arborizations [4,17,60,61,71] and their potential
for variability across individuals. Cases 15 and 93 appeared to occupy virtually
overlapping regions but there was more widespread cortical labeling in case 93.
Likewise, the dorsal cases 45, 84, and 76 exhibited labeled cells in orbital area MO; cases
52 also a dorsal injection, but did not produce labeling in MO.
The laminar distribution of labeled cortical neurons varied across cortical areas
and within AGm. Labeled neurons were present in layers II/III and V in AGm and AGI
but only in layer V of the other cortical areas. Within AGm it was common to find only
layers II/III labeled caudal to the level of the ac, as described for cases 93, 105 and 45.
Wilson  also reported labeling in layers II/III and V within ipsilateral AGm, and
labeling restricted to layer V in somatic sensory cortex following injection of a retrograde
tracer into the striatum.
Due to the long rostrocaudal extent of the current injections, the ability to make
statements about highly discrete topography of cortical projections to DCS is limited.
The observed rostrocaudal expansion of the injections could be due to diffusion along
corticostriatal axons, which are known to traverse long distances in the striatum,
synapsing on striatal neurons at varying rostrocaudal levels . Selemon and Goldman-
Rakic  demonstrated that some cortical areas project axons that traverse the entire
rostrocaudal extent of the primate caudate nucleus. Diffusion of this type presents a
unique problem for discrete retrograde analyses in DCS and should be addressed in future
Similarly, the placement of a tracer in the rat striatum raises the possibility of
uptake not only by the axons of interest but also those fibers that are only passing through
the striatum to form terminals elsewhere in the CNS. This is known to occur with
retrograde fluorescent tracers . However, the coordinated pattern of thalamic and
cortical labeling suggests uptake by axon terminals, because it seems unlikely that
interconnected cortical and thalamic regions would be consistently labeled via uptake by
fibers of passage. Rather, it would appear more parsimonious to suggest that the axons
from such linked cortical and thalamic regions terminate in the same striatal territory.
Ultimate resolution of this question will depend on double anterograde tracing.
The pattern of thalamic projections to DCS bears a striking resemblance to the
thalamic projections to those cortical areas that provide input to DCS. The rostral portion
of area AGm receives thalamic input from lateral MD, VL, VM and the intralaminar
nuclei, whereas inputs to caudal AGm emphasize VL, LD, LP and Po [49,93,94]. In the
present study these same thalamic nuclei were found to project to DCS, with the
exception of Po and the addition of the parafascicular nucleus. It is known that single
neurons of the central lateral nucleus project to both the striatum and cortex , and if
this is also an attribute of the projections of the other thalamic nuclei projecting to DCS it
could underlie the registration observed. Anterograde tracing has demonstrated that the
intralaminar nuclei (CM, PC, CL) have dense projections to the dorsal striatum [7,8].
Although their terminal fields are generally located medially (CM), centrally (PC) and
laterally (CL), they overlap to some extent in the dorsocentral striatum, especially rostral
to the level of the ac. This may explain why all three nuclei were labeled in all of the
There is evidence of topography in the thalamic projections to DCS from LD and
LP, and the observed pattern is consistent with the known organization of thalamocortical
projections involving these nuclei and the topography of corticostriatal projections from
their cortical targets. Injections in dorsal DCS consistently labeled thalamic nuclei LD
and LP. Together with the fact that LD and LP project to cAGm , this topography
suggests that dorsal DCS is preferentially related to cAGm. Consistent with this claim,
corticostriatal projections from cAGm project most densely to dorsal DCS .
Thalamic nuclei LD and LP also project to cortical areas PPC and Oc2M , which are
densely connected with cAGm rather than rAGm [91,97]. Finally, cortical input to dorsal
DCS is densest from areas PPC and Oc2M (present study and ; see Chapter 3). Thus,
dorsal DCS appears to represent a subregion of DCS that is distinguished by its network
of thalamic and cortical connections. As an aside, the projections from LP to DCS and
AGm [93,109] originate predominately from its medial portion, whereas the projection
from LP to area PPC originates from all of LP .
The current results are in agreement with the topography of labeling produced
following injections of retrograde tracers (Fluorogold and the cholera toxin B-subunit)
into the dorsomedial caudate-putamen, with the addition of AV and AD labeling .
The description of the rostrocaudal and mediolateral topography of labeled cells in the
lateral and ventral thalamic nuclei following injections in the Erro et al. paper is
consistent with the pattern of labeling produced in the current cases. They reported
labeled cells in the ipsilateral intralaminar nuclei and medial thalamic nuclei in all of their
cases . Additionally, Erro et al. note that they found labeled cells in VPM and VPL
following injections in the caudal striatum, a feature that is not widely accepted. The
current injections also produced robust labeling in VPM and VPL in some, but not all,
caudal (postcommisural) injections .
The present results together with previous findings demonstrate that DCS receives
input from cortical and thalamic regions that are themselves interconnected.
Furthermore, DCS and connected cortical areas are involved in directed attention and its
dysfunctional counterpart, multimodal hemispatial neglect. The data presented herein
suggest that DCS is a key element of a cortico-striatal-thalamic network specialized for
multimodal associations and spatial functions including directed attention. This network
includes cortical areas AGm and PPC, the striatal area DCS, and the thalamic nuclei VL,
MD, LD, LP, and the intralaminar group. This network is similar to the large scale
networks proposed to be involved in spatial functions in primates [48,77,105]. Similar
interconnected cortico-striatal-thalamic networks have been identified for sensory, motor
and limbic systems [1,46,85].
OVERLAP AND INTERDIGITATION OF CORTICOSTRIATAL AFFERENTS TO
DORSOCENTRAL STRIATUM IN THE RAT
Using a rodent model of hemispatial neglect [62-64,92,115-118], our group has
determined that the medial agranular cortex (AGm), a multimodal association premotor
cortex with diverse cortical connections , and posterior parietal cortex (PPC) are
components of a cortico-striato-thalamo-cortical network mediating directed attention in
the rat . The dorsocentral striatum (DCS), defined anatomically as the central striatal
terminal field of projections originating from cortical area AGm, is critical for normal
directed attention. Destruction of DCS results in chronic neglect which does not recover
or respond to dopamine agonists [93,115-117]. For these reasons, region DCS has
become the focus of work on the cortical-striatal-thalamic network mediating directed
attention in the rat.
In previous anterograde and retrograde studies, we demonstrated that the cortical
regions AGm, PPC, ventrolateral orbital cortex (VLO), and secondary visual cortex
(Oc2M) all send projections to each other and to DCS [16,93,92]. Projections from each
of these areas form a dense primary projection field in the dorsal striatum and an
additional diffuse pattern of labeling in or near DCS. This pattern of labeling in the rat
striatum has been described by many authors [3,10,51,74,92,134], and suggests the
presence of at least two systems of corticostriatal projections terminating in the rat
striatum, termed "discrete" and "diffuse" . The same diffuse region of
corticostriatal labeling was also described in the monkey [34,35], and may therefore be
an innate property of corticostriatal organization. A thorough examination of the
band/diffuse pattern of labeling in the rat network for directed attention was recently
published by Reep and colleagues .
Previously, the thalamic nuclei with which the cortical components of the rat
network for directed attention are each reciprocally interconnected were identified .
Because of the known functional and anatomical relationships between cortical areas and
thalamic nuclei in the network [65,91,93,96,97], it is important to understand the
topography of their striatal projections and the spatial relationship of these projections
with respect to DCS. More detailed examination of the relationship between the
convergent terminal fields of these key regions will provide a better basis to understand
how their inputs interact to influence the activity of neurons in the DCS. Cortico-striato-
thalamo-cortical networks have also been proposed as mediators of directed attention in
primates. Similar to the rat network, the primate networks involve regions of prefrontal
cortex and posterior parietal cortex, as well as associated regions of the basal ganglia and
thalamic nuclei [73,77,105].
Interest in these specific cortical areas extends to the more general question of
whether multiple cortical areas terminate on or near the dendrites of individual striatal
medium spiny neurons. This hypothesis has been proposed previously  and is
supported by both electrophysiological and anatomical data [17,33,80], although it
remains untested in a multi-labeling experiment at the light microscopic level. If it exists,
this pattern of connectivity would support the hypothesis that the rat striatum, and
potentially individual medium spiny neurons, plays a key role in integrating multimodal
input from diverse cortical areas, and that the rat DCS is a center for convergence of
diverse corticostriatal inputs related to directed attention. Previous reports support these
hypotheses. In Chapter 2, I found that many cortical areas project to region DCS, but
none more consistently than AGm, PPC, and Oc2M. Subsequently, we demonstrated that
axons from cortical areas AGm and PPC exhibit both overlap and interdigitation in the rat
DCS, using an anterograde tracer placed in individual cortical areas, as well as in two
double-labeled cases .
In the current multiple-labeling study we directly tested the hypothesis that
projections from cortical areas PPC and VLO, as well as thalamic nuclei VL and LD,
overlap and/or interdigitate with projections from cortical area AGm in the striatal region
DCS. Further, I sought to demonstrate whether neurons from anatomically non-adjacent
cortical areas form terminals on or near the dendrites of the same individual medium
spiny neurons in the rat striatum.
Materials and Methods
All animal procedures were conducted according to institutional protocols that
meet or exceed NIH and Society for Neuroscience guidelines. Animals were
anesthetized with ketamine/xylazine (90mg/kg: 10mg/kg). Upon cessation of tail pinch
and eyeblink responses, animals were placed in a small animal stereotaxic device and
holes were drilled in the skull at the selected location. The dura was then incised,
exposing the brain surface. All rats received one injection of a 10,000mw dextran
conjugated to a green-fluorescing molecule (100nl of a 10% AlexaFluor 488 solution in
phosphate buffer; Molecular Probes, Inc.) in area AGm using procedures derived from
previous work by the Reep group [91,92,97]. Additionally, each rat received an
injection of a red fluorescing molecule conjugated to a 10,000mw dextran (100nl of a
10% FluoroRuby or MicroRuby solution in phosphate buffer; Molecular Probes, Inc.) in
one of the following regions: cortical area AGI (case DCS 193), PPC (case DCS 178),
VLO (case DCS 147), or thalamic nucleus VL (case DCS 173), or LP (case DCS 191).
One rat (case DCS 193) also received a third injection: a 3,000mw dextran conjugated to
a biotin molecule (BDA3K; Molecular Probes) was placed in globus pallidus and
retrogradely filled the dendritic trees of medium spiny neurons in DCS.
Injections for all cases were placed in the left hemisphere. Intracerebral injections
were made through a 27-gauge Hamilton syringe except for injections in orbital cortex,
which necessitated the use of iontophoresis. For iontophoretic injections, glass
micropipettes having tip diameters of 20-30 [im were lowered to the proper coordinates
and constant current pulses of 4-5 [tA were delivered from an iontophoresis unit (Kation
Scientific, model BAB-350) in a 5 sec on/5 sec off pattern for 20-25 minutes. All
thalamic injections were made iontophoretically using the same parameters as cortical
iontophoretic injections. After surgical procedures were completed, all skull wounds
were filled with gelfoam and antibiotic ointment was placed between the scalp and skull
to prevent post-surgical infection. The scalp wound was closed using wound clips. After
10-14 days rats received an overdose of a barbiturate solution (Beuthanasia-D; 100mg/kg
IP) and were perfused intracardially with 340C phosphate-buffered saline followed by
4% phosphate-buffered paraformaldehyde. The brain was extracted and post-fixed in 4%
paraformaldehyde fixative for 24hr, then stored in cold 0.4% paraformaldehyde
containing 30% sucrose for 2-3 days prior to cutting.
Coronal frozen brain sections were cut at 40 [m on a sliding microtome and
collected serially in dilute fixative (0.4% paraformaldehyde). Two series of sections
were mounted, dehydrated and coverslipped using Fluoromount (BDH Chemicals).
Fluorescent sections were viewed and photographed on a Biorad MRC-1024 confocal
laser scanning microscope using separate red and green filter channels. Final images
represent combined red/green image stacks of approximately 20 .im total thickness.
A third series of sections stored in dilute fixative was stained for Nissl substance
using cresyl violet and used for cytoarchitectural orientation. Additionally, brains
containing microruby or BDA3K injections were processed as previously described .
The locations of cortical injection sites were determined in each brain according to
the cytoarchitectural criteria of Zilles and Wree . The terms AGm and AGI are
synonymous with their areas Fr2 and Frl, respectively. Thalamic nuclei were identified
and demarcated according to the criteria and maps of Jones  and Paxinos and Watson
Data from single anterograde biotinylated-dextran-amine (BDA) injections are
previously unreported findings resulting from a reanalysis of 13 rats for which methods
were previously reported, as were the methods for calbindin staining on those cases .
For the purpose of this report, only rats with injections which affected only one cortical
area were analyzed.
Injections of anterograde fluorescent tracers in AGm (green) and a second brain
region (red) resulted in the simultaneous red and green labeling of terminal fields in the
striatum. I attempted to determine whether the striatal terminal fields labeled by each
tracer injection overlapped or interdigitated. As noted previously, I viewed the sections
on a confocal microscope and imaged ~20km of each ~40km-thick section in 1 Lm
intervals. The images from each stack were combined via software into one two-
dimensional image. Therefore, axons seen to cross on final images are somewhere
between actually touching and passing within 20tm of one another. Since the two
conditions are indistinguishable on the final images, I considered any image in which
fibers from different injections were observed crossing as an image depicting overlap.
"Interdigitation" was used to describe sections on which very little or no overlap was
seen, yet axons from different injection sites (typically large fields of them) were seen
adjacent to one another.
Placement of injection sites is described below in anteroposterior (a-p),
mediolateral (m-1), and dorsoventral (d-v) dimensions.
Injections in AGm/AGI
In case DCS 193, the AGm injection was centered at a-p +1.0 and affected an area
similar to the injection in case 178, extending to all cortical layers without damaging the
white matter. The AGI injection was centered at a-p -3.6 and m-1 +2.0, and affected all
cortical layers, as well as a small amount of the white matter. The AGm labeling in case
193 produced a dorsolateral band and DCS labeling consistent with the previous accounts
. A dorsolateral band of labeling was produced in the striatum following the AGI
injection, and sparse axonal labeling was observed in DCS, as previously described .
The dorsolateral band is distinct from the AGm band, but labeling in DCS overlapped
with AGm labeling.
Additionally, case DCS 193 also received an injection of BDA3K in globus
pallidus (a-p -0.9), resulting in a robust retrograde labeling of the DCS efferent neurons
(medium spiny neurons). With confocal microscopy, both green (AGm) and red (PPC)
fine-caliber axons with varicosities were observed to overlap with individual dark-stained
medium spiny neuron dendrites in this triple-labeled brain (Figure 2C,2D).
Injections in AGm/PPC
Case DCS 178 received fluorescent tracer injections in cortical areas AGm and
PPC and both produced a pattern of labeling in the striatum similar to those seen
previously after single injections of the anterograde tracer BDA . The AGm injection
site was centered at a-p +0.7 and affected all cortical layers but did not affect the white
matter (Fig 3-1A). Terminal fields from area AGm included a dorsolateral band
immediately internal to the white matter, as well as a more diffuse area of labeling in
DCS, as reported previously [92,93]. The PPC injection site for case 178 was centered at
a-p -3.6 (Fig 3-1B), and affected all layers of the cortex without extending into the white
matter. The injection is centered in PPC and has a narrow mediolateral extent (-0.2mm).
The injection is visible over a rostrocaudal distance of approximately 0.3mm (from
spaced serial sections). The primary terminal field of projections from area PPC forms a
dense band located dorsal to the AGm projection field in DCS, and it spans the distance
between DCS and the dorsolateral AGm band but remains distinct from both fields of
AGm axon terminals. The same pattern of termination can be observed along much of
the rostrocaudal extent of the striatum in this case.
In case 178, the PPC injection also produced a dense focus of labeling in DCS
(Fig 3-2A,B). The focal PPC projection in DCS was somewhat distinct from AGm
labeling in its center but overlapped extensively with AGm axons at the edges of the
focus. Numerous axons from each injection were observed in the focus periphery and
crossed each other extensively.
Figure 3-1. Representative fluorescent photomicrographs of cortical and thalamic
injection sites. 25x. Scale bar = 1 mm; applies to A-F. A) Injection of
AlexaFluor 488 in cortical area AGm (case DCS 178). B) Injection of
Fluororuby in cortical area VLO (case DCS 147). C) Injection of Fluororuby
in cortical area PPC (case DCS 178). D) Injection of Fluororuby in the caudal
portion of cortical area AGI (case DCS 193). E) Injection of Microruby in
thalamic nucleus VL (case DCS 173). F) Injection of Microruby in the medial
portion of thalamic nucleus LP (case DCS 191).
Injections in AGm/VLO
Case DCS 147 had an AGm injection site centered at a-p +0.7, m-1 +1.0, which
affected all layers of the cortex without impinging on the white matter. The injection
spans a rostrocaudal distance of ~0.3mm. Striatal labeling from this injection was typical
of that observed following other AGm injections, and was composed of a densely labeled
dorsolateral band and a more diffuse area of labeling in DCS.
The VLO injection site for case DCS 147 was centered at a-p +3.2, m-1 +2.2, and
d-v -4.2. Terminal fields of axons originating in cortical area VLO were located close to
and along the d-v extent of the medial wall in this case (Fig 3-3A). Some labeling
produced by the injection extends to border the AGm labeling in DCS from the medial
side and interdigitates at the light microscopic level with some of the axons from area
AGm, forming a region similar to the focal projection described following PPC
injections. In contrast to the PPC focal DCS projection, the discrete region of VLO label
remained continuous with the primary field of VLO label along the medial wall. Despite
the presence of this seemingly similar focal projection, there was little if any overlap of
axons from AGm and VLO evident in this case.
Injections in AGm/VL
Case DCS 173 had an injection centered in rostral AGm (a-p +1.4). The VL
injection was centered at a-p -2.5, m-1 +1.4, and d-v -6.0, and was limited to nucleus VL
(Fig 3-1E). The terminals of axons originating in thalamic region VL were located
primarily lateral to DCS. Some terminals were observed dorsolateral to DCS,
overlapping with the dorsolateral AGm band, but these were less dense.
Figure 3-2. Striatal labeling following injection of fluorescent anterograde tracers. A)
Labeling following injection in AGm (green) and PPC (red) in case DCS 178.
AGm labeling is visible in a green dorsolateral band. Larger-caliber green
axons are visible in the white matter fascicles passing through the dorsocentral
striatum (DCS). Labeling from the PPC injection also forms a dorsolateral
band and terminals are diffusely present in all of DCS. Additionally, a dense
focus of labeling is visible in DCS (arrow; B). 50x. Scale bar = Imm. B)
Higher-magnification photomicrograph of the dense focus of labeled PPC
axons in case DCS 191. Several AGm axons (green) are visible and cross the
PPC axons. The two either touch or pass within 20[tm of one another.
Larger-caliber green axons are also visible in the white matter fascicles. 100x.
Scale bar = 100tm. C) Photomicrograph of axons from cortical areas AGI
(green) and PPC (red) in close association with dendritic processes of
retrogradely-labeled striatal medium spiny neurons (black) in case DCS 193.
200x. Scale bar = 100tm. D) Line drawing of axons and cell bodies from C.
Figure 3-3. Labeling in cases DCS 147 and DCS 173. A) Striatal labeling following
injection of fluorescent anterograde tracers in AGm (green) and VLO (red) in
case DCS 147. AGm labeling forms a dense green dorsolateral band and a
more diffuse region of labeling in the dorsocentral striatum (DCS). Labeling
from the VLO injection is most dense along the medial wall, and terminals are
diffusely present in all of DCS. Additionally, a dense focus of labeling is
visible in or near DCS (arrow), although no overlap was evident between
axons from AGm and VLO in the dense focus. 50x. Scale bar = 1 mm. B)
Striatal labeling following injection of fluorescent anterograde tracers in
cortical area AGm (green) and thalamic nucleus VL (red) in case DCS 173.
Labeling from the VL injection is most dense along the lateral edge of the
striatum. No terminals were observed in DCS in this case. Large-caliber
fibers can be seen passing through the white matter bundles and probably
account for VL labeling observed in a previous retrograde study of DCS.
l00x. Scale bar = 1 mm.
Heavy fascicular labeling was present throughout much of the rostrocaudal extent of the
striatum and was especially dense in and around DCS. Despite the rostral AGm
injection, the VL injection in this animal produced striatal labeling that was located
rostral to the majority of the observed AGm labeling. There was no overlap between
AGm and VL terminal field labeling observed in this case (Fig 3-3B).
Injections in AGm/LP
Case DCS 191 had an AGm injection centered at a-p +1.2 which affected all
cortical layers but did not damage the white matter. It is visible on only one section in
each spaced series, and therefore had a narrow a-p extent. The MicroRuby injection site
in LP was centered at a-p -3.8, m-1 +1.3, and d-v -4.5 (Fig 3-1F). The injection site was
very focal within medial LP, which resulted in the labeling of a relatively small number
of terminal axons in the striatum. However, those that were labeled were very densely
filled and easily visualized. The primary terminal field ofLP thalamostriatal axons in
this case formed a dorsolateral band visually very similar in placement to the one
typically seen following injections in cortical area PPC (Fig 3-4A,B,C). Thalamostriatal
axons also overlapped diffusely with the dorsolateral AGm band. Within DCS, LP axons
extended in long, relatively straight arborizations, which were not observed to this degree
or in this conformation following injection in any other cortical area or thalamic nucleus
VL (Fig 3-4C,D). These varicosity-dotted long axons overlapped many AGm axons in
both the dorsolateral AGm band and within DCS. Axons from LP were evident in the
dorsolateral band on both the fluorescent sections and those processed for BDA labeling,
but were only visualized effectively in DCS on BDA sections (Fig 3-4).
Figure 3-4. Striatal labeling pattern in case DCS 191 following injection of anterograde
tracers. A) Fluorescent labeling following injection in cortical area AGm
(green) and thalamic nucleus LP (red). Axonal labeling from AGm forms a
dense green dorsolateral bandand a more diffuse region of labeling in the
dorsocentral striatum (DCS). Labeling from the LP injection also forms a
dorsolateral band (arrow; panel B), in which overlap was observed between
AGm and LP axons. 50x. Scale bar = 1 mm. B) Axons from thalamic
nucleus LP (red) form a band in the dorsolateral striatum and both overlap and
interdigitate with axons from cortical area AGm (green). 100x. Scale bar =
100 [tm. C) Adjacent section to the one depicted in A and B, processed for
BDA (Microruby contains both tetramethylrhodamine and biotin molecules).
The dorsolateral band of LP labeling is still evident, and labeling in DCS is
more easily visualized. Arrows point to an especially dense focus of axonal
labeling in DCS (depicted in D). 62.5x. Scale bar = 1 mm. D) An especially
dense focus of labeled axons following injection of Microruby in thalamic
nucleus LP. Many axonal varicosities are evident on the labeled axons. 125x.
Scale bar= 100 [pm.
Injections of BDA in PPC
The brains examined having single injections of BDA in PPC (DCS 60, 77, 88, 91,
and 114) each exhibited a pattern of striatal labeling which included a dense focus of
axonal terminals located at approximately the level of the anterior commisure (AC), with
the exception of DCS 114. Photomicrographs of each PPC focus are presented in Figure
3-5. In all brains with foci, the focus was always located ventromedial to the densest
portion of the primary labeling field (band) within a given section. Foci were typically
ellipsoid in the a-p dimension, having the largest diameter and greatest density in the
approximate center of their anterioposterior extent.
As previously noted, the primary terminal bands of PPC shift in position
commensurate with a medial or lateral placement of the BDA injection site . Case
DCS 91 had an injection placed in medial PPC (PPCm) and had the most visibly dense
focus of axonal labeling (Fig 5A, B). Cases DCS 60, 77, and 114, also had injection sites
confined to PPCm. The primary terminal fields and foci in cases 60 and 77 (Fig 5C-5F)
resembled those observed in DCS 91, although foci in both brains appeared less dense
overall than those in case 91. Case DCS 114 possessed a primary band of label very
similar to those seen in 60 and 77, but lacked an observable focus.
Case DCS 88 received a BDA injection in lateral PPC and the primary terminal
band was located more dorsolaterally than in the other cases. The focus was also shifted
laterally, into the central portion of DCS (Fig 3-5G,H).
In all PPC cases, labeled foci were in the calbindin-positive matrix with few or no
axons visible in nearby patch compartments.
Figure 3-5. Labeled axons in the striatum after injection of BDA in cortical area PPC. In
each case there was a dense dorsolateral band of labeling, a diffuse projection
to DCS, and a dense focus of labeled axons in DCS. A) Photomicrograph of
striatal labeling in case DCS 91. The dense dorsolateral band and dense focus
in DCS (arrows) are clearly visible. 62.5x. Scale bar = 1 mm and applies to
A, C, E, and G. B) Higher-magnification view of the dense focus of labeled
axons in case DCS 91. 125x. Scale bar = 100m and applies to B, D, F, and
H. C) Photomicrograph of striatal labeling in case DCS 60. The dense
dorsolateral band and dense focus in DCS (arrows) are clearly visible. D)
Higher-magnification view of the dense focus of labeled axons in case DCS
60. E) Photomicrograph of striatal labeling in case DCS 77. The dense
dorsolateral band and dense focus in DCS (arrows) are clearly visible. F)
Higher-magnification view of the dense focus of labeled axons in case DCS
77. G) Photomicrograph of striatal labeling in case DCS 88. The dense
dorsolateral band and dense focus in DCS (arrows) are clearly visible. H)
Higher-magnification view of the dense focus of labeled axons in case DCS
Figure 3-5. Continued
Injections of BDA in Oc2M
Both Oc2M cases examined (DCS 95 and 101) exhibited a focus of labeled axons
ventromedial to the primary terminal field. The observed ellipsoid foci qualitatively
resembled those seen in PPC brains and were located at or near the level of the AC, as in
PPC cases. All observed foci were in the calbindin-positive matrix with few to no axons
visible in nearby patch compartments, as in PPC brains.
Other BDA Injections
One brain with a BDA injection in VLO (DCS 110), and another with an AGI
injection (DCS 100), had robust labeling in the primary terminal fields (bands), but no
foci similar to those seen in PPC or Oc2M brains were observed in either case.
One significant finding in my study is that the cortical areas PPC and Oc2M
terminate in DCS diffusely and in a focal manner, overlapping and interdigitating with
AGm axons, while other cortical areas and thalamic nuclei do not form dense foci of
axonal terminals in DCS (cortical areas VLO and AGI and thalamic nuclei LP and VL).
Previously, I found that cortical areas AGm, AGI, PPC, VLO, and Oc2M were
consistently labeled following injections of a retrograde tracer in and around region DCS
(see Chapter 2), and all were observed to form terminal fields near or in DCS in the
current anterograde study, and in an earlier report . These areas have all been shown
previously to be reciprocally interconnected with one another and with several thalamic
Fluorescent or BDA injections in AGI did produce the typical dorsolateral band of
primary axonal labeling as well as sparse labeling in DCS, but those brains did not
exhibit the focal groups of axons in DCS observed following PPC or Oc2M injections.
This is consistent with previous descriptions of AGI labeling . It is demonstrated
herein that corticostriatal axons from areas AGm and AGI cross both one another and the
dendritic processes of individual retrogradely-filled striatal medium spiny neurons at the
light microscopic level. This finding is consistent with the hypothesis that individual
medium spiny neurons integrate inputs from multiple cortical and thalamic regions,
requiring multiple simultaneous input stimuli to depolarize [17,33,80].
Fluorescent tracer injections in AGm and PPC produced a dorsolateral band of
labeled PPC axons between DCS and the dorsolateral AGm band, as well as a discrete
focus of labeled PPC axons within AGm. These foci were first described in another
AGm/PPC double-fluorescent case  and appear to be sites of overlap and
interdigitation between axons from AGm and PPC. Herein, the same type of isolated
focus of labeled axons was found in an additional double-fluorescent AGm/PPC case
(178). Additionally, brains which received single PPC injections of the anterograde
tracer BDA were also examined for foci. Four of the five PPC BDA cases exhibit clear
foci which are detached from both the primary terminal field (band) and from terminals
scattered diffusely throughout DCS. Therefore, it is probable that these foci are a feature
common to the PPC/AGm projection and represent a consistent zone of overlap between
axons from the two cortical areas.
In a previous report, we described the results of a AGm/Oc2M double-labeling
experiment . We demonstrated that Oc2M projects to the striatum in a pattern
consistent with the cortical areas examined herein, forming a dense primary area (band)
of terminals and a more diffuse secondary area which overlaps partially with labeling
from area AGm in DCS. In the current report, BDA-labeled corticostriatal projections
from Oc2M form a focal arborization in DCS similar to those seen in PPC brains.
Small groups of labeled axons similar to those seen following PPC and Oc2M
injections were reported by Brown et. al following injections of an anterograde tracer into
FL and HL . Their presence following injections in both primary somatosensory
cortices (FL and HL) and areas involved in secondary and associative tasks (AGm and
PPC) suggests that these foci represent a larger-scale pattern of corticostriatal projection.
A visually similar focus of labeling was observed following double labeling of
AGm and VLO axons. Although this focus is smaller than the one formed by PPC axons
and no overlap was observed, it could also be a zone of overlap on medium spiny neurons
located near the interface between AGm and VLO terminal fields. As noted by Wilson
, the possibility of individual medium spiny neurons integrating information from
adjacent yet non-overlapping corticostriatal terminals is a distinct and often overlooked
one, due to the size of their dendritic trees. Further, the convergence of axons from non-
overlapping corticostriatal projections has been demonstrated electrophysiologically on
single medium spiny neurons in the macaque . This may also be true in the rat
striatum but it remains to be demonstrated.
It is not surprising that axons from thalamic region VL do not terminate in region
DCS, since VL is primarily associated with motor functions, despite the fact that VL was
heavily labeled following injections of retrograde tracers in and around DCS. Uptake of
retrograde tracers by the large number of labeled axons in the fascicles traveling through
DCS is the likely explanation for the VL labeling seen in the previous retrograde study
(see Chapter 2). The observed pattern of labeling is consistent with previous anatomical
and electrophysiological data localizing the effects of VL stimulation to lateral striatum
. Also, VL is a large nucleus, and the injection did not completely fill it in this case.
It is possible that other portions of VL do project to DCS, although the current data
suggest that previously reported retrograde labeling of VL was the result of tracer uptake
by fibers of passage, as discussed in Chapter 2.
The medial portion of thalamic nucleus LP was found to project densely to a
dorsolateral band which overlapped and interdigitated with the primary dorsolateral AGm
band, and diffusely to DCS. In Chapter 2, a retrograde study, I reported that LP was
labeled only when injections of Fast Blue were placed in DCS itself. Additionally, Erro
et al.  found LP labeling following injection of a retrograde tracer in the dorsal
striatum but not in the ventral striatum.
The overlap of corticostriatal terminal fields was noted by Yeterian and Van
Hoesen . Many authors have since addressed the topic of overlapping and/or
interdigitating corticostriatal terminal fields because of the potent implications. Of
particular interest is the overlap in the striatum of efferents from reciprocally
interconnected cortical and thalamic areas. In one notable report, a network of
reciprocally interconnected cortical and thalamic areas was described in the macaque
, where interconnected motor-related areas project both to distinct, non-overlapping
territories in the basal ganglia, and also project more diffusely to overlapping areas in
both the caudate and putamen. Similarly, several groups have demonstrated that forelimb
representations from different cortical areas overlap in the macaque putamen [52,111].
This pattern of overlap has also been described in thalamostriatal and corticostriatal
projections from rodent somatosensory cortex as well, where there is more convergence
in the striatum from SI areas representing subcomponents of the rodent forelimb than
from cortical areas representing noncontiguous body parts [50,51].
In addition to the reports which demonstrated overlap, there are substantial data
which suggest that the projections from reciprocally interconnected cortical areas project
exclusively to non-overlapping portions of the striatum . One possible explanation
for the disparity between the current findings and those of Selemon and Goldman-Rakic
is that some cortical areas project in an overlapping way and others in a non-overlapping
way, despite their reciprocal interconnection. It is possible that functionally-associated
cortical areas (like AGm and PPC) involved in a cortical-subcortical network that relies
on the striatum for some integrating function project to the striatum with some degree of
overlap depending on the requirements of the specific network, while other cortical areas
are entirely segregated. This pattern of selective overlap and segregation was previously
reported in the macaque .
In my study, as in previous reports, I found that both AGm and PPC form their
primary terminal fields as densely labeled bands in dorsolateral striatum which do not
overlap [92,93], as reported by Brown et al.  for rat somatosensory cortex. Also, it is
known that areas AGm and PPC project diffusely to region DCS . Herein, I report
the existence of a focus of overlap in DCS of projections from AGm and cortical areas
PPC and Oc2M. These foci could represent key regions in DCS for multimodal
integration of information from AGm with that from PPC and Oc2m. Whether PPC and
Oc2M foci overlap with each other in DCS remains an open question and warrants
further study. Areas AGm, PPC, and Oc2M all play a critical role in directed attention in
the rat. Destruction of DCS, but not dorsolateral striatum, produces robust neglect of
contralesional stimuli which does not spontaneously recover and cannot be relieved by
the administration of dopamine agonists [115,116]. The location of these foci within
DCS is therefore a significant finding. Axons in the dense foci observed in BDA brains
were located in the calbindin-rich matrix compartment. This is consistent with the
descriptions of matriosomes in the primate basal ganglia  and may indicate an
anatomical and functional similarity between the rat foci and primate matriosomes.
It is possible that integration in the diffusely-terminating portion of the rat network
mediating directed attention is taking place only on specific subtypes of striatal projection
cells, which are typically segregated into patch and matrix groups . It is unlikely,
however, that all of the diffuse overlap seen in the striatum is occurring in exclusively
patch or matrix compartments. Diffuse overlap is seen at the edge of the AGm/VLO
striatal terminal fields and LP neurons extend relatively long distances in the dorsolateral
AGm band as well as in DCS, forming many distinct en passant type axonal varicosities.
In a previous paper, we reported that the majority of labeled axons in DCS were in the
matrix compartment following cortical placement of BDA . It is likely, therefore,
that the majority of the diffuse overlap observed in multiple-labeled cases is occurring in
the matrix compartment.
PHOTOTHROMBOTIC LESION OF POSTERIOR PARIETAL CORTEX IN
The rat model of hemispatial neglect has yielded key information regarding the role
of the medial agranular cortex (AGm) and the posterior parietal cortex (PPC) in a cortico-
striato-thalamic network mediating directed attention [16,18,92]. An aspiration lesion of
either area AGm or area PPC results in a lasting neglect of contralesional hemispace,
similar to that seen following unilateral PPC damage in humans [11,16,115,117]. In rats,
symptoms of neglect do not spontaneously recover over time. This is also consistent with
the human syndrome, in which 25% of patients still show reduced spatial capacity after
12 years, often after attempting physical therapy .
In previous experiments, lesions were produced by removing either AGm or PPC
via aspiration [14,116]. While aspiration lesions effectively produce neglect, the ability
to induce a more physiologically-relevant lesion would represent an improvement for the
rat neglect model in several ways. First, a craniotomy is required to reach the surgical
site for an aspiration lesion. During this procedure, the bone and meninges immediately
external to the cortical area of interest are removed by the surgeon. Tampering with these
tissues is undesirable because of the increased risk of secondary complications related to
bone or meningial trauma. The dorsal saggital sinus is also placed at risk during this
procedure, presenting the chance that a subject could experience uncontrollable bleeding
during the aspiration lesion.
Second, an aspiration lesion risks damaging the white matter forming the cingulum
bundle, which is located internal to AGm. Damage to the cingulum bundle could result
in inappropriate behavior changes in addition to those expected from the intended lesion
and may interfere with sprouting from contralesional cortex, if occurring. Third, acute
removal of brain tissue most likely does not result in the replication of the cell signal
cascades produced in response to a focal ischemic attack, since the cells are not
undergoing necrosis or apoptosis in situ. Also, spreading depression-like effects may be
artificially minimized by an aspiration lesion. Due to the disruption in cell signaling,
cortical remodeling (as measured by changes in GAP-43 expression), and functional
recovery from behavioral or motor deficits are likely adversely affected following
For these reasons, I explored the practicality of making a photothrombotic lesion
limited to the cortical area of interest, without performing a craniotomy, leaving the
tissues in place to more closely replicate the physiological state of the brain post-infarct.
This procedure relies on the unique qualities of an injectable dye, Erythrosin B, which is
placed in the venous system. When light of a known wavelength and intensity strikes the
molecules of the dye in the blood, a series of events is initiated which results in the
formation of a thrombus localized to the area struck by the light (PPC in this case).
Photothrombosis relies on the natural process of thrombogenesis following a vascular
insult. In the case of Erythrosin-B mediated photothrombosis, the insult is in the form of
photochemically-mediated peroxidation of the lipids in the membranes of cells forming
the vascular endothelium. This vascular damage is caused by the donation of valence
electrons from the dye molecules to the endothelial cells and results in platelet adhesion
and aggregation, which result in the formation of a vascular occlusion thrombuss) .
The highest level of platelet aggregation occurs at the pial surface since it is exposed to
the most direct light. It is possible to monitor the progression of the lesion via nuclear
magnetic resonance imaging, and multiple MRI studies have plotted the time course of
photothrombotic lesions under different parameters [68,70,88,113,123].
Materials and Methods
A total of 24 male Long-Evans Hooded rats were used in my study. Rats were
anesthetized with a Ketamine/xylazine cocktail (87:13 mixture; 80 mg/Kg, ip). The head
was shaved and ointment put in the eyes to prevent drying during surgery. Upon
exhibiting no eyeblink response or withdrawal to ear bar placement, anesthetized rats
were fixed in a stereotaxic apparatus and the skull exposed. A drop of mineral oil was
placed on the skull to render the surface optically smooth. Either rose bengal or
erythrosin-B (20mg/mL in PBS; 20mg/kg; RB:Sigma 3300000; EB:Sigma 200964) were
injected into the medial saphenous vein or the tail vein. The maximum absorbance for
rose bengal and erythrosin-b are 549nm and 533nm, respectively, but both are effective
when excited at 532nm . Immediately following the dye injection, the brain was
transcranially illuminated via either a Schott light source (KL2500-LCD) with a custom
filter limiting the transmitted light to a 50nm band centered on 532nm (Omega Optical)
or a frequency-doubled YAG-laser with output at 532 nm and power -100 mW (250
mW/cm2; LAGR100; Information Unlimited) for a maximum of 8 minutes, after which
time the dye concentration in the blood stream is below that necessary to cause a
thrombus. Following the illumination period, an antibiotic ointment was placed between
the skin and the skull, and the incision was closed with wound clips. After a survival
period of five to ten days, rats were injected IP with a barbiturate solution (Beuthanasia-
D; 100mg/kg IP) and perfused intracardially with 300 mL of phosphate buffered saline
(PBS) followed by 300 mL of 4% buffered paraformaldehyde. Brains were subsequently
removed and cryoprotected by incubation in a 30% sucrose solution. Coronal sections
were cut on a freezing microtome at 40[m and placed in dilute fixative until being
mounted on slides. Cresyl violet staining was performed for cytoarchitectural analysis
and description of the lesion site.
Results and Discussion
The lesion was much more difficult to make than expected. Many papers
misrepresent the difficulty of the procedure, often as the result of the use of flawed
methodology [59,88,103,129]. In one report, researchers injected Rose Bengal into the
femoral vein in an uncontrolled fashion, sometimes used immense doses (as much as
80mg/kg), illuminated for a much longer amount of time than their dye molecule of
choice could actually be present in the bloodstream, and irradiated via an unfiltered "cold
light source" . In one murine model, authors injected the dye intraperitoneally at a
low dose (10mg/kg), waited five minutes, and irradiated with an unfiltered light source
for 15 minutes, which is much longer than the time in which rose bengal is at an active
concentration following injection . Similarly, researchers designing a rat model of
photothrombotic infarct in epileptic rats expose the brain to unfiltered light (400 nm to
1200 nm) for 20 minutes . Another group irradiated for 30 minutes , which is
similarly well past the eight minutes it takes for the rat liver to remove rose bengal from
the blood stream [126,127].
In all of the above cases, the investigators exposed the brain to unnecessary
wavelengths of light, which are not absorbed by the dye molecules, used an unnecessary
period of illumination to induce infarcts, and did not perform controls to ensure there
were no burns produced by the light source. There is no such thing as a "cold light
source" . Excess light energy does not simply disappear, in accordance with the law
of conservation of energy. In this case, unabsorbed light is converted to heat energy,
which does an unknown amount of tissue damage separate from that caused by the photo-
excited dye molecules .
With the aim of controlling these errors, I began my trials with a Schott light source
and a filter from Omega Optical, which limited the wavelength of the transmitted light to
a 30 nm band centered on 530 nm. Only light capable of exciting the RB in the brain was
emitted while using this apparatus, which greatly limited the chance of causing heat-
related damage. Conversely, a thermometer placed against the surface of the fiber optic
gooseneck on the Schott quickly rose 10 to 150C without the filter in place to restrict the
spectra emitted by the "cool light source." When the Omega filter (green) was placed in
the light path, this temperature rise was eliminated. Control animals which received
filtered irradiation alone (no dye) did not have heat lesions.
I was able to make lesions reasonably consistently using the green Omega filter and
the Schott light source, but the lesions were very large (Fig 4-1). It is possible to limit the
size of the lesions by using an optically opaque substance (i.e. a brass shim) to protect the
non-target portions of the skull and the underlying brain regions from the light source.
Unfortunately, lesions produced with the Schott/Omega are somewhat variable depending
on the distance of the light source from the skull, since the light emitted from the fiber
optic gooseneck spreads rapidly. This spread results in reduced power with distance.
For roughly the same price as the Schott/Omega setup, I purchased a frequency-
doubled YAG-laser with output at 532 nm and power -100 mW (250 mW/cm2;
LAGR100; Information Unlimited). The laser provides several immediate benefits over
the Schott/Omega setup. First, the laser does not produce energy capable of causing heat
damage to the brain. Second, the beam of laser energy does not spread rapidly and
therefore produces lesions which are much more reproducible between animals.
Several factors affected the decision regarding an appropriate injection site for the
dye molecule in the rat model of hemispatial neglect. Many researchers inject their dye
of choice into the femoral vein, including those who developed the initial protocols .
However, gaining access to the femoral vein requires cutting a muscle and is painful for
the animal post-surgery. Since any post-surgical pain could interfere with locomotion,
which is frequently measured in behavioral testing, a deficit resulting from the surgery
would represent a significant confound in nearly any behavioral test and is highly
Therefore, I wanted to design a methodology that would interfere minimally with
behavioral testing. First, I tried using the lateral tail vein. The lateral tail vein is
frequently chosen as a venous access in rats due to its relative availability . Trials
with the lateral tail vein were largely unsuccessful. In albino rat strains, the lateral tail
vein is relatively easy to visualize and inject, since their tails are a pale pink color and the
vein is red. Long-Evans Hooded rats, the strain used in the Corwin/Reep model as well
as in many behavioral models, have a heavily pigmented tail, and visualization of the
lateral tail vein is very difficult. Since the vein is so small, placing a needle in it is
extremely difficult without visualizing the vein.
Figure 4-1. Lesion produced via irradiation with the Schott/Omega filter combination.
A) Gross lesion at seven days post-photothrombosis. B) Cresyl violet section
through the center of the lesion. The lesion is deep and affects the white
matter. Note that the medial cortical areas are intact due to the blood supply
from the anterior cerebral artery (ACA), which is not affected by the
photothrombotic lesion of cortical areas supplied by the branches of the
middle cerebral artery (MCA).
If either Rose Bengal or Erythrosin-B is placed outside the vein, a thrombus will not
develop in the brain after irradiation, and there is a good chance of necrosis of the tail
distal to the injection site. This complication is obviously undesirable. For this reason, I
examined other venous routes.
After consulting an anatomy text , I decided on the medial saphenous vein, a
branch of the femoral vein. The medial saphenous vein is located on the medial side of
each hindlimb and is best visualized transdermally after shaving the medial hindlimb.
Since the injected volume is usually -0.5 mL, I found it easiest to use a 1 mL insulin
syringe with an attached 27-gauge needle.
One benefit of photothrombosis is that the size of the lesion can be easily
controlled. It is simple, for example, to make a large lesion with the filtered Schott light
source, since the aperture of the Schott's fiber optic gooseneck is 4 mm. The laser beam
is only 2 mm diameter, which makes it useful for more precise lesions (Figure 4-2). It is
possible to reduce the size of the lesion resulting from the Schott light source if an
opaque substance is placed over the portions of the skull directly over brain regions
which are to be spared [22,24-27,126,127].
I began this project with rose bengal, the photoreactive dye most commonly used in
the literature [22-28,126-128]. The use of rose bengal is complicated by one major
physiological complication: infusion of the dye over any period shorter than 90 seconds
produces dangerous fluctuations in the animal's blood pressure . This fluctuation
does not occur after infusion of the photoreactive dye erythrosin-b  and it was
chosen because of this property. This lesion works well in PPC and reliably produces a
circular lesion with a ~2mm diameter.
Figure 4-2. Lesion produced via irradiation with the YAG laser. A) Cresyl violet section
through the center of the lesion at 21 days post-photothrombosis (saggital
view). Scale bar = 2 mm. B) Higher magnification view of the lesion site.
The lesion is deep but does not appear to affect the white matter. Scale bar =
The lesion affects all layers of the cortex but appears to stop external to the white matter.
Conversely, I have not successfully created a lesion of AGm via photothrombosis.
This failure is probably due to the protection of AGm by the dorsal sagittal sinus
(which may prevent transmitted laser energy from reaching the cortex), the inability to
effectively occlude branches of the anterior cerebral artery supplying AGm, or some
combination of the two. If photothrombosis is to be used to study the Corwin/Reep
model of directed attention, it will most likely be necessary to study the attentional
effects of a PPC lesion instead of an AGm lesion. Despite this change, the directed
attention model could be improved overall by the use of a carefully controlled
photothrombotic lesion instead of an aspiration lesion, because of the lesion's similarities
to occlusive stroke.
CONCLUSIONS AND FUTURE DIRECTIONS
The work described herein began as a retrograde study of afferents to DCS. That
study, presented in Chapter 2, used the retrograde tracers Fast Blue and Diamidino
Yellow to determine which cortical areas and thalamic nuclei project to the dorsocentral
striatum (DCS) in the rat. Retrograde tracers were placed in DCS where they were
subsequently absorbed by axon terminals and transported retrogradely to the cell bodies
of origin. I then observed which cortical areas and thalamic regions possessed
fluorescent-labeled cell bodies and therefore sent axonal projections to DCS.
The retrograde study revealed that most cortical areas project to some portion of
DCS or an adjacent striatal region. Most cortical areas were only labeled in some cases,
but AGm, PPC, and Oc2M, the cortical areas known to be part of the network mediating
directed attention in the rat, possessed retrogradely-labeled cell bodies in every case.
One inherent problem with studies employing retrograde tracers is the potential for
tracer-uptake by axons which are not actually making synapses in the area of interest, but
are rather simply passing through the area. This potential necessitates that anterograde
studies, where tracers are transported from the cell body toward axon terminals, be
carried out. We published data from a study in which we injected rats with biotinylated
dextran amine (BDA) and examined the terminal fields of individual cortical areas ,
which allowed the production of a general schematic of potential overlap and
interdigitation of cortical inputs to DCS. Additionally, we published double-labelling
observations from one AGm/PPC and one AGm/Oc2M brain in the same paper . The
larger double-anterograde study presented in Chapter 3 goes much further, detailing the
overlap and interdigitation of AGm with cortical areas PPC, Oc2M, and VLO, as well as
with thalamic regions LP and VL. Additionally, Chapter 3 adds descriptions of
previously undescribed dense foci of axonal labeling in DCS following placement of an
anterograde tracer in either PPC or Oc2M, but not VLO. These dense matriosome-like
foci are sites of increased overlap between axons from cortical areas PPC and Oc2M with
axons from AGm. The observed foci may therefore represent key sites of integration of
multimodal spatial information from these anatomically non-adjacent cortical areas.
Importantly, these foci were observed in every brain with an anterograde tracer injection
placed in Oc2M, and every brain except one following PPC injection. Therefore, the
presence of dense foci within DCS appears to be an anatomical pattern and not just an
anomaly. It would be interesting to double-label PPC and Oc2M to determine if their
individual dense DCS foci overlap and/or interdigitate with one another. In addition,
electron-microscopic studies could be performed to further examine the degree to which
axonal projections from cortical areas AGm, PPC, and Oc2M, and thalamic nucleus LP,
form synapses with the same individual medium spiny neurons in DCS.
The anatomical studies presented in Chapters 2 and 3 enhanced the previous
knowledge of the connections of the cortical and subcortical portions of the network
mediating direct attention in the rat. When taken together with the past studies discussed
in Chapter 1 [12,16,92,93,95,96], one can build a more detailed picture of the anatomical
connections of this network, which subsequently allows the formation of hypotheses
regarding the functional aspects of the anatomical network.
The basic anatomy of the system is not extremely complicated. First, it is
commonly accepted that sensory information from all modalities enters the brain at the
level of the sensory thalamus, which sends projections to primary sensory cortices. The
primary sensory areas project to secondary and associative cortices, all of which in turn
project to motor cortices, to the sensory thalamic nuclei, and to the striatum. Cortical
motor areas project to the motor thalamus, to the striatum, to brainstem targets, and
directly into the corticospinal tract. The striatum receives glutamatergic inputs from all
of these cortical regions and thalamic nuclei, limbic cortical regions, and also receives
dopaminergic inputs from substantial nigra. The only efferent targets of DCS are the
substantial nigra and globus pallidus, both of which project to the motor thalamus. The
findings presented herein are consistent with these general patterns and extend our
knowledge regarding how cortical and thalamic striatal afferents relate to one another and
to medium spiny neurons in DCS. DCS appears to be the place where signals from all of
these diverse brain structures are integrated, leading to the successful functioning of a
mechanism for directing attention toward salient objects in space. The myriad
interactions between neurotransmitters, receptors, and the implications for combining
information from these diverse regions are far from being understood and add several
levels of further complexity to understand when forming hypotheses about the precise
functioning of this anatomical network mediating a behavioral process. It is known that
striatal medium spiny neurons require synchronous, convergent input from multiple
neurons in order to depolarize [131,133], and it is very possible that the corticostriatal
projections outlined herein are interacting at some level, but this remains to be
demonstrated. To move beyond the current limited understanding of the functioning of
this network, electrophysiological studies are necessary. It is important to define the
mechanism by which corticostriatal inputs are synchronized. This synchrony could be
the result of interhemispheric corticocortical coupling of contralateral homotopic cortical
areas and this has been hypothesized, but remains unknown . The authors of one
recent paper which studied the function of neurons in DCS found that striatal neurons
which fire in response to spatial information and those which fire in response to rewards
are different populations .
Now is the time to ask how far to go toward an understanding of the function of the
network itself, on a small scale. The main goal of this model's development is to develop
potential therapeutics for human patients, which is something that a deeper understanding
of the actual computational functioning of the network itself may or may not actually aid.
The Corwin/Reep model may well have reached the point now where future studies
should be aimed at increasing the degree of functional recovery following a neglect-
inducing event (stroke, trauma, etc.). The current lesion model is undesirable for
pursuing functional recovery of neglect following stroke, which is the most common
cause of neglect in humans, as discussed in Chapter 4.
Photothrombotic lesions are a good alternative for studies of functional recovery
from neglect because they replicate a stroke much more closely than does an aspiration
lesion , while still allowing the production of a lesion which is limited to one
anatomical region, which is vital to interpreting experimental outcomes.
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Joseph Laton Cheatwood was born and raised in rural Polk County, Florida. He
graduated from Lake Gibson High School in 1994. He received a BS in biology from
Stetson University (DeLand, Florida) on May 10, 1998. On May 11, 1998, he began
graduate school at the University of Florida, completing his MS in Veterinary Medical
Sciences in December, 2000. Immediately thereafter, Joseph joined the lab of Roger
Reep, PhD, and began his studies in the neuroscience. After completion of his PhD in
August 2004, Joseph will begin a postdoctoral fellowship at Loyola University, Chicago.