Title: Collateral sprouting of unmyelinated primary afferents lacking receptors for nerve growth factor
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Title: Collateral sprouting of unmyelinated primary afferents lacking receptors for nerve growth factor
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Language: English
Creator: Petruska, Jeffrey Charles
Publisher: State University System of Florida
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Publication Date: 2000
Copyright Date: 2000
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Subject: Neuroscience thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Neuroscience -- UF   ( lcsh )
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Summary: ABSTRACT: Collateral sprouting occurs in both the peripheral and central nervous systems. It is a mechanism for rapid recovery of function after certain injuries, and also underlies such vital processes as the establishment of new anatomical connections during learning and memory formation. Primary afferent collateral sprouting (PACS) is a process whereby uninjured sensory neurons respond to the denervation of adjacent tissue by extending branches into the denervated territory. It was previously determined that this process was limited to small diameter afferents with high thresholds to electrical stimulation. It was also determined that sensory neurons with receptors for nerve growth factor (NGF) were capable of PACS, and that NGF was paramount for the process. This study examined whether or not the population of small diameter neurons lacking NGF receptors (i.e., non-trkA) was capable of PACS.
Abstract: The non-trkA small diameter afferents are those that become dependent on glial cell line-derived neurotrophic factor (GDNF) during early postnatal life. Since NGF has been shown to regulate PACS of trkA expressing neurons, it is possible that GDNF may have a role in PACS of the GDNF-dependent neurons. This would indicate that another neurotrophic system may be involved in plasticity of adult neurons. It was determined, using multi-labelling immunohistochemistry, that axons with markers specific for non-trkA neurons were present in collaterally reinnervated skin. Further, contrary to the common understanding, non-trkA neurons expressed GAP-43 (a marker associated with axonal growth) in both normal ganglia and those undergoing PACS. These results indicate that the small diameter afferents lacking trkA are likely involved in PACS, but had been missed by previous experiments.
Abstract: It was determined, using 1) selective destruction of neurotrophin receptor (i.e., trkA) bearing neurons and 2) transynaptic neuroanatomical tracing, that the small diameter neurons lacking trkA were not involved in the reflex pathway that had been the standard measure for the success and extent of PACS. This implies that the reflex is not suitable for assessing PACS of non-trkA neurons, and provides a partial explanation as to why the non-trkA neurons had been missed in previous PACS investigations.
Summary: KEYWORDS: neural plasticity, neural injury, pain, skin, sensory neuron, neurotrophic factors
Thesis: Thesis (Ph. D.)--University of Florida, 2000.
Bibliography: Includes bibliographical references (p. 93-113).
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Statement of Responsibility: by Jeffrey Charles Petruska.
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COLLATERAL SPROUTING OF UNMYELINATED PRIMARY AFFERENTS
LACKING RECEPTORS FOR NERVE GROWTH FACTOR















By

JEFFREY CHARLES PETRUSKA


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


2000













ACKNOWLEDGMENTS

I extend my deepest thanks to my parents and family for the various roles that

they have played in helping me to reach this place in my life, not just academically, but

also as a person. I would, quite obviously, not be the person I am today were it not for

you. There were specific events, and then there were the less noticed presence. I often

recount the day I found my pet frog dead in his lovely shoe-box house. I was very

young, but my mother felt that a different kind of life lesson was due. She brought me,

the deceased Rana, a wooden board, some pins, and a razor blade, out to the table in the

yard. She then proceeded to dissect the frog, carefully pointing out all the parts she could

find, and explained to me how things worked. That is the first thing that comes to mind

whenever I am asked "What got you into science?", or "Have you always liked

science?" My father and his brothers developed (often demanded) in me the capacity

and desire to stay vigilant in maintaining reason and ration in matters academic or

applied, big or small. I have been blessed with family, friends, and a loving wife, who

have encouraged me and supported me, and have taught me that humanity and

compassion are necessary partners with reason and ration for a good life. I have learned

a lot, and I am still learning.

I have had the good fortune to have found excellent teachers and mentors: Steve

Lyons without his vision, guidance and support, I may not have been at Boston College;

Michael Numan a phenomenal teacher with unending patience who took a chance on an

eager freshman, first exposed me to how the principles of science and the scientific









method are applied, and helped guide me through the vast array of options after Boston

College; and Richard Johnson I can not write or say enough to express my thanks and

appreciation to my graduate school Mentor. He has been patient, not just with how long

this has taken me, but with the small things that make a difference on a daily basis. He

has been available I can not recall ever being turned away when I came with even a

small question. He has been supportive I have a strong tendency to venture beyond

what is available where I am. This is sometimes quite rewarding, other times quite

frustrating. Dr. Johnson supported and guided me through it all. He also supported me

in other ways sending me to special meetings, encouraging me to present my work as

often as possible, taking extra courses that would benefit me more in the future than now,

and encouraging me to pursue my desire to teach. Thank you, Dr. Johnson.

I extend my gratitude to my advisory committee, Drs. Meyer, Ritz, Streit, and

Munson. You have been patient and supportive, and helped keep things focused

(whenever I gave you the chance).

I must also thank William Luttge and the Department of Neuroscience faculty and

staff. I have interacted with too many folks to name here, but the open-door attitude of

the entire department made my time at UF rewarding. I feel I learned quite a bit, not just

about science, but about the folks who do it. I have enjoyed getting to know you all.

I want to extend my thanks to Dr. Ron Wiley whose generosity and enthusiasm

allowed me to perform some of the experiments vital to this work.

And to my loving wife, Sara...I am happier with you than I ever imagined

possible, and it gets better every day. Thank you for all of the small and big ways you

supported this endeavor. We make a great team.









TABLE OF CONTENTS


A CK N O W LED G M EN T S..................................................................................................ii

KEY TO SYMBOLS AND ABBREVIATIONS.............................................................vi

ABSTRA CT............................. ........................................................... ....................... ....vii

CHAPTERS

1 INTRODUCTION AND LITERATURE REVIEW ..............................................1

O v erview ................. ................. . .............................................. ..... ..... ...... ........ ...
Primary afferent neurons......................... .. .... ............................... 3
Characteristics of primary afferent collateral sprouting (PACS)..........................6
M olecular signalling of PA CS..............................................................................8
Methodological limitations in previous PACS testing........................................11
Evan's Blue dye extravasation...................... .... ........................11
Histochemical analysis of peripheral targets...........................................12
Histochemical analysis of DRG.......................................................13
Electrophysiological assessments of PACS............................................16
Assessments of PACS using the CTM reflex.............................................17
Unmyelinated primary afferents lacking NGF receptors........................... ..18
Hypotheses and plan for testing..................................................21

2 M E T H O D S ..................................................................... ................................. 25

General m ethods....... .............. ............ .... ....................... 25
Histochemical analysis of PACS....... ............................ .................... 25
CTM reflex afferents........................................... 25
Specific m ethods........................................ ................................................26
Histochemical analysis of PACS ...........................................................26
Sprouting surgeries.............. ........................ .......... ............ 26
CTM reflex testing................................ ....................... 28
Histochemical procedures for collaterally reinnervated skin........29
Markers used for skin histochemistry..........................................30
Histological procedures for cell counts.........................................31
Cell counting........................... .........................32
CTM reflex afferents................................................ ......................33
Neurotoxin experiments........................ ...................... 33
Tissue processing......................... ...... ....................35
Pseudorabies virus injections...................... ............................35
Pseudorabies virus control experiments.............................. ..37
Tissue processing and histological procedures............................38









3 RESULTS HISTOCHEMICAL ANALYSIS OF PACS....................................40

Histochemical analysis of collaterally reinnervated skin......................................40
Skin histochemistry controls........................ .......................43
Histochemical analysis of DRG sections...................... ...............................44
D election of GA P-43 protein...............................................................................49

4 RESULTS CTM REFLEX AFFERENTS.......................... ...................... 51

192-saporin injections......................... ..................................... ............................ 51
Electrophysiology...................... ..........................................................51
H istochem istry.................................................................... ..... ........................ 55
Pseudorabies virus circuit tracing.................................................. 59
Control experiments........................ ............................. 60

5 D ISCU SSION ...................... ........................ ..............................63

Unmyelinated Afferents Lacking trkA Participate in PACS...............................63
Skin histochemistry.................... .......... ......................63
DRG Histochemistry ............................ .......... ............................63
The Role of Unmyelinated Afferents Lacking trkA in PACS
Has Gone Unrecognized............... .............................................. 70
Previous Histochemical Examinations Could
Have Been Insufficient....................... ... .........................71
NGF Insensitive Afferents Could Become Sensitive................................73
CTM Reflex Testing Could Be Insufficient For
Non-trkA Afferents.......................... .......................... 75
Primary Afferents and the CTM Reflex.............................. ....................77
Selective Destruction of Neurotrophin Receptor-
Expressing Afferents............................... ........................78
Transynaptic Neuronal Tracing Reveals Afferents
Involved in the CTM Reflex............................... ...................... 81
TrkA-Negative Unmyelinated Afferents and the
C T M R eflex........................................................................... ....... 83
PACS of trkA-Positive Versus trkA-Negative Afferents......................................86
A Role for NGF in PACS of Afferents Lacking trkA?
A Likely Role for Other Factors.......................... ...................88
Sum m ary ............................................................... ....................... ...9 1


R E F E R E N C E S ........................................................................ ....................................93

BIOGRAPHICAL SKETCH............................. ....... .... .....................114









SYMBOLS AND ABBREVIATIONS


192-sap saporin-conjugated antibody 192 (against p75)
ABC avidin-biotin-HRP-complex kit
CAP compound action potential
CGRP calcitonin gene-related peptide
CNS central nervous system
CTM cutaneus trunci muscle
CS collateral sprouting
DPH dopamine-beta-hydroxylase
DPH-sap saporin-conjugated antibody against D3H
DRG dorsal root ganglion/ganglia
DCn(n) dorsal cutaneous nerve (nerves)
FRAP fluoride-resistant acid phosphatase
GDNF glial cell line-derived neurotrophic factor
GS-I-B4 isolectin B4 from Griffonia simplicifolia type I
HRP horseradish peroxidase
IR immunoreactive/immunoreactivity
LCn(n) lateral cutaneous nerve (nerves)
LTn(n) lateral thoracic nerve (nerves)
NGF nerve growth factor
trkA high-affinity nerve growth factor receptor
P2X3 P2X family of ATP-gated ion channels subunit 3
p75 low-affinity neurotrophin receptor
PACS primary afferent collateral sprouting
PBS phosphate buffered saline
PFA paraformaldehyde
PGP 9.5 protein G product 9.5 (ubiquitin hydroxylase)
PNS peripheral nervous system
PRV pseudorabies virus
RET GDNF-receptor tyrosine kinase subunit
RF receptive field
SOM somatostatin
TH tyrosine hydroxylase
TSA tyramide signal amplification kit
VR-1 vanilloid receptor 1 (capsaicin-sensitive ion channel)















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

COLLATERAL SPROUTING OF UNMYELINATED PRIMARY AFFERENTS
LACKING RECEPTORS FOR NERVE GROWTH FACTOR

By

Jeffrey Charles Petruska

August, 2000

Chair: Richard D. Johnson, Ph.D.
Major Department Neuroscience



Collateral sprouting occurs in both the peripheral and central nervous systems. It

is a mechanism for rapid recovery of function after certain injuries, and also underlies

such vital processes as the establishment of new anatomical connections during learning

and memory formation.

Primary afferent collateral sprouting (PACS) is a process whereby uninjured

sensory neurons respond to the denervation of adjacent tissue by extending branches into

the denervated territory. It was previously determined that this process was limited to

small diameter afferents with high thresholds to electrical stimulation. It was also

determined that sensory neurons with receptors for nerve growth factor (NGF) were

capable of PACS, and that NGF was paramount for the process. This study examined

whether or not the population of small diameter neurons lacking NGF receptors (i.e.,

non-trkA) was capable of PACS.









The non-trkA small diameter afferents are those that become dependent on glial

cell line-derived neurotrophic factor (GDNF) during early postnatal life. Since NGF has

been shown to regulate PACS of trkA expressing neurons, it is possible that GDNF may

have a role in PACS of the GDNF-dependent neurons. This would indicate that another

neurotrophic system may be involved in plasticity of adult neurons.

It was determined, using multi-labelling immunohistochemistry, that axons with

markers specific for non-trkA neurons were present in collaterally reinnervated skin.

Further, contrary to the common understanding, non-trkA neurons expressed GAP-43 (a

marker associated with axonal growth) in both normal ganglia and those undergoing

PACS. These results indicate that the small diameter afferents lacking trkA are likely

involved in PACS, but had been missed by previous experiments.

It was determined, using 1) selective destruction ofneurotrophin receptor (i.e.,

trkA) bearing neurons and 2) transynaptic neuroanatomical tracing, that the small

diameter neurons lacking trkA were not involved in the reflex pathway that had been the

standard measure for the success and extent of PACS. This implies that the reflex is not

suitable for assessing PACS of non-trkA neurons, and provides a partial explanation as to

why the non-trkA neurons had been missed in previous PACS investigations.


-viii-















CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW


Overview

Collateral sprouting (CS) is a process by which neurons extend newly-formed

appendages out from their original axonal processes. This phenomenon can be part of a

normal process such as the formation of new synaptic contacts during learning193' 201,221-

223. CS can also be induced in response to an injury where it can play a role in the

restoration of numerous functions17, 26,46,51,52,152,240, 242. However, this response to injury

can sometimes lead to the development of pathologic conditions35' 36'124,183. Thus,

experimentation to determine which neurons are capable of CS under both normal and

pathologic conditions is important for the understanding of the mechanisms of collateral

sprouting.

Collateral sprouting is a dynamic growth process that can be observed in certain

neurons in the central nervous system (CNS) and in the peripheral nervous system (PNS).

In the CNS, indicators of CS are routinely observed in neurons that are involved in the

establishment of new anatomical connections in areas such as the hippocampus and

cerebellum. These new connections are believed to provide a portion of the substrate for

learning and memory. Collateral sprouting in the PNS has been observed in response to

injury of neighboring axons or manipulation of the growth factor environment, and is

displayed by motoneurons7, 24 25, 26, 41, 83, 152, 240, 242, sympathetic ganglion neurons1' 70, 7191,96,

100,102, 103,124, 136,189, and primary afferent neurons, though limited to A8- and C-fibers5' 47 49-









53, but see 58, 82, 84, 90, 93, 101/, 108, 216 Studies focussing on primary afferent collateral sprouting

(PACS) deal with the easily accessed and manipulated periphery, and may provide vital

insights that can be applied to all forms of CS. The experiments detailed herein focus on

PACS, which appears to be limited to the small diameter DRG neurons47 84' 93

Most PACS experimentation thus far has primarily employed the cutaneous trunci

muscle (CTM) reflex system as a monitor of its success and progress (see below). It has

been determined that PACS, as it is currently understood, is a nerve growth factor (NGF)

dependent process involving afferents that respond to directly NGF 49, 52. The methods

used to test and monitor PACS, however, have not addressed the possible role of a

subpopulation of unmyelinated primary afferents lacking receptors for NGF. This

dissertation recounts the investigation of the role of this subpopulation of primary

sensory neurons in PACS.

The sensory neurons in question are a subpopulation of the small diameter, dark

type-B dorsal root ganglion neurons. This subpopulation of small diameter DRG neurons

is the group that does not express neurotrophin receptors, but binds the isolectin B4 from

Griffonia (or Bandeireae) simplicifolia type I (GS-I-B4)7 146,157, 158,160. The role of these

neurons in signal transduction and recovery after injury is unknown. The entire small

diameter, dark type-B population has unmyelinated, or thinly myelinated, processes 120,

122,172 and is generally believed to be involved in transducing noxious stimuli, but may

also subserve other functions which are currently unclear203. The anatomy and regulatory

factors of these GS-I-B4-reactive small diameter neurons have only recently begun to be

elucidated12, 64, 159, 176, 180, 181, 186, 187, 205, 207, 211, 212, 235, 236








Primary Afferent Neurons

Non-cranial primary afferents are located in the intervertebral foramina in gross

structures called dorsal root ganglia (DRG). DRG afferents are derived from the neural

crest, and innervate all tissues including skin, muscle, and viscera.

Afferents of a given DRG have segmentally arranged central termination patterns

as well as having a relatively well defined peripheral dermamyotome. The course of the

peripheral axons from a DRG vary greatly from ganglion to ganglion. DRG from the

cervical and lumbar plexus regions supply a great variety of nerves. Many DRG feed

axons to a single nerve, and a single DRG has neurons that form many different nerves.

The thoracic DRG, however, are more regular and segmentally restricted. The "cross-

talk" between the thoracic cutaneous nerves and the DRG of adjacent segments is very

small247. Therefore, the thoracic DRG are the most appropriate for PACS modeling.

Each DRG houses many different functional families of neurons. The particular

groups and proportions vary with the tissues innervated at each segment. There are

dozens of different functional types of primary afferents. General families include low-

threshold mechanoreceptors, high-threshold mechanoreceptors, thermoreceptors, and

chemoreceptors. These general functions are often mixed in single neurons, and the

latter three are generally associated with nociceptor function (afferents transducing

noxious and/or tissue damaging stimuli).

Certain correlations have been made between afferent structure and function.

Unmyelinated axons are the smallest in diameter, and axon diameter increases with

increasing thickness ofmyelination. Conduction velocity also increases with increasing

axon diameter. The neurons that give rise to unmyelinated axons consistently have a

small diameter soma (<35um), while the majority of neurons that give rise to myelinated









axons are medium to large diameter, with there being a trend of larger somal diameter

giving rise to larger axon diameter78, 79,122 However, some neurons with very small

somal diameter have large myelinated axons78, 79,22

Certain correlations have also been demonstrated between structure or function

and various histochemical phenotypes. DRG neurons have been broken up into small-

dark and large-light groups based on their somal size and appearance under light

microscopic examination. These groups were shown to generally correspond to the

myelination state of axons, with the small-dark being unmyelinated and the large-light

being myelinated. Recent work has supported this relation between phenotype and

myelination, but has offered a more accurate interpretation. One new development is the

correction of the "large" in large-light There are, in fact, some small neurons that

display the "light" phenotype. These neurons have been shown to have other indicators

of having myelinated axons 78, 79,22. For this reason, Lawson and colleagues have

dropped the term "large-light" and simply distinguish light neurons from small-dark

neurons. Lawson and colleagues have also generated highly convincing data that the

light population (i.e., myelinated) expresses high levels of neurofilament triplet proteins

(NF-H, NF-M, and NF-L), while the small-dark population expresses very low levels.

The two populations can be reliably distinguished based on their immunoreactivity for

neurofilaments122 172. Thus, neurofilament (NF) immunoreactivity can be an indicator of

myelination state.

The small-dark population can be further subdivided into two groups, those that

express one of more of the major known sensory neuropeptides (peptidergic; eg.,

calcitonin gene-related peptide CGRP, substance P SP, somatostatin SOM), and

those that do not (non-peptidergic). CGRP is considered the prototypical sensory









neuropeptide, being expressed either alone, or co-expressed with other neuropeptides69'

125, 126, 209, 239 The majority of the non-peptidergic small-dark neurons are bound by the

isolectin IB4 from Griffonia simplicifolia type one (GS-I-B4) which binds primarily to a-

D-galactose rdu"- -" -" -1'-. Thus, nearly every DRG neuron will demonstrate either

NF-IR, CGRP-IR, or GS-I-B4-binding, and many will display a combination.

It is the combinations of markers that can dramatically improve the utility and

power of histochemical examinations of DRG neurons. Families can be more accurately

defined by the presence or absence of multiple markers, as opposed to the presence or

absence of a single marker. For example, NF-IR defines myelinated neurons, but does

not distinguish between A3 and A8 afferents, and certainly can not indicate whether any

of these may be nociceptors. CGRP-IR encompasses the vast majority of peptidergic

nociceptors, but will not reveal whether any given CGRP-IR neuron may be myelinated.

However, if used together, the markers will delineate nearly all myelinated non-

nociceptive neurons (NF /CGRP-), unmyelinated peptidergic nociceptors (NF-/CGRP'),

and myelinated nociceptors (NF/CGRP ), and non-peptidergic unmyelinated afferents

(NF-/CGRP-)144

A variety of markers that define sensory neuron subgroups have been described.

These include the expression of sensory neuropeptides (CGRP, SP, SOM),

neurofilaments (NF), intermediate filaments (peripherin and a-intemexin), enzymes

(fluoride-resistant acid phosphatase FRAP; carbonic anhydrase; neuron specific

ubiquitin C-terminal hydroxylase PGP 9.5; choline acetyltransferase ChAT), calcium-

binding proteins (calmodulin, calbindin, parvalbumin), growth factor receptors

(neurotrophin receptor tyrosine kinases trk family or p75; glial cell line-derived

neurotrophic factor tyrosine kinases RET and GFR family), chemical receptors (VR-1,







-6-

P2X family, AChR family), ion channels (PN1, SNS, Kv family), and binding of lectins.

Figure 1 is a schematic representation of some of the overlaps of the markers in the DRG.


GS-I-B4
CGRP
SP
SOM
NF-M
P2x3
trkA
p75
? "_
ret

Figure 1. Schematic representation of the general overlaps of certain markers in small
and medium diameter DRG neurons. Not all comparisons have been tested directly and
therefore some overlaps, or lack thereof, may not be entirely correct. However, the
general scheme is highly representative. Arrows indicate a continuation of the marker
into the large diameter population. The distribution of RET outside of the GS-I-B4-
reactive population in medium and larger neurons has been described159, but the overlaps
with other markers are unclear, as indicated with the question marks.




Characteristics of Primary Afferent Collateral Sprouting (PACS)

Peripheral nerve injury results in the induction of two growth processes -

regeneration of all fiber types in the injured nerve and CS of the adjacent uninjured

nerves47. The second form of growth, CS, is unique in that the uninjured neurons

somehow respond to the injury and begin to grow and restore function. The restoration

of function is achieved by an apparent growth of peripheral axons into denervated areas

of tissue. CS is known to occur in humans, but the extent of the process appears to be

somewhat less than what occurs in animal models5' 82, 90. Neurons involved in this form

of plasticity appear to maintain proper modality transduction and somatotopic







-7-

arrangement47, 51, 52, 173, and are thus able to provide a more rapid restoration of function to

denervated regions than would occur from regeneration alone23 47, 82, but see 102, 103

The primary model for PACS investigations in the rodent has been the dorsal

hairy skin of the back and the cutaneus trunci muscle (CTM) reflex system. Though

others have been used47, 108,109,164, 174,191,216, 238, such as denervating the plantar surface of

the hindpaw by tibial nerve ligation and allowing reinnervation by the saphenous and

caudal cutaneous sural nerves, these do not have the anatomical homogeneity present in

the thoracic segments (the spared nerve/nerves is supplied by a single DRG). When an

area of insensitivity is produced in the rat CTM reflex system by transaction of multiple

adjacent dorsal cutaneous nerves (DCnn), it is the high threshold stimulus transducing

primary afferent axons that begin to sprout into the denervated areas of skin. Thus far,

only examinations with pinch or heat have been reported166 173, 217. Expansion of the low

threshold peripheral fields has been tested using natural stimuli and electrical recording

of the isolated nerve. Such work has shown no evidence of large diameter axon (low

threshold / light touch transducing) collateral sprouting84 92,93, though there may be

evidence for this in other systems58. Certain lines of evidence strongly suggest that this

expansion of high threshold sensitivity is due, at least in part, to an actual growth of

axons through the skin into the denervated areas. Silver stains of behaviorally insensitive

skin (skin areas where noxious stimuli are incapable of driving the CTM reflex) reveal no

visible axons in dermal endoneurial tubes51. These same silver stains used on sections of

skin regions to which high threshold (but not low threshold) sensitivity has returned after

collateral sprouting revealed that axons were present in dermal bundles in areas of skin

that had regained high threshold sensation. Such staining was not present after

pharmacological treatments to prevent sprouting51.









The speed of recovery and expansion of the sensitive field into the denervated

area is very similar to what would be expected based on other neural growth systems51' 109,

225. It can first be reliably detected at 10-12 days post DCn transaction and its maximum

extent (regardless of the size of the denervated field) is reached at approximately 4 weeks

post-transection. In models sparing a single DCn inside of a denervated field, the high

threshold receptive field (RF) can approximately double its size over the course of

PACS. Intravenous administration of Evan's Blue dye in conjunction with antidromic

stimulation of spared sensory nerves at different times after denervation of surrounding

areas revealed a gradual expansion of the area of skin into which the dye was

extravasated. These extravasation fields matched extremely well with the behaviorally

responsive areas of skin at all time points51 59. Further, mRNA for the protein GAP-43,

shown to be a marker of neuronal growth and axonal extension3' 16, 31199, increases in

neurons of the DRG supplying the spared DCn11.





Molecular Signalling of PACS

The expansion of the high threshold receptive field into denervated areas of skin

has been shown to be a nerve growth factor (NGF) dependent process49' 52

Administration of anti-NGF antibodies during PACS prevented any expansion of the

receptive field for the duration of the treatment49 52, 58. The expansion resumed once the

treatment had been terminated. Additionally, exogenous NGF could induce PACS de

novo, as revealed by the ratio of high threshold : low threshold receptive field sizes52' 148

Other systems where NGF concentration has been increased have also lead to collateral

innervation by unmyelinated sensory afferents11.









Message (mRNA) for both the high affinity (trkA) and the low affinity (p75)

NGF receptors has been shown to be upregulated in DRGs that housed neurons involved

in PACS149' 151, as well as in other neurons undergoing CS115. These studies did not

directly examine whether the increases were due to a new population of neurons

beginning to express the receptors or whether those already expressing them simply

increased their production, the latter is more likely. It has been shown that the proportion

of neurons expressing NGF-receptor mRNA was similar between normal and sprouting

groups149, 151. It has also been shown that the supply of target-derived NGF can influence

the expression of NGF receptors112. Therefore, it is more likely that the increases in trkA

and p75 in PACS DRG were due to the increases in expression levels by neurons already

expressing the receptors, as opposed to the recruitment of a new group of neurons to

express trkA and/or p75.

It has also been shown that NGF mRNA was increased in denervated skin15.

These findings support the hypothesis that PACS, and perhaps CS in general, is induced

by a buildup of NGF (or other factors) in a denervated area that is detected by intact

neighboring axons. The buildup is likely due to the increased production of NGF by the

target tissue and the reduced uptake and transport by damaged neurons. These axons

may interpret the change in the growth factor environment as a cue to sprout toward the

source of NGF. It has been shown that NGF-sensitive axons will alter their direction of

growth in response to gradients of NGF 66, 86, 106. It appears that this buildup of

endogenous NGF is local and perhaps even compartmentalized48. Endogenous increases

of NGF as a result of focal denervation is unlikely to cause widespread collateral

sprouting of NGF responsive neurons3.







-10-

Further evidence for PACS being an active growth process and involving, at very

least, trkA-bearing DRG neurons, is found with examinations of the expression of

growth-associated protein 43 (GAP-43). GAP-43 has been definitively shown to be

involved in the growth of neural processes in both the normal and regenerating CNS and

PNS 2,3,14,31, 32, 153,170,200, 241 GAP-43 has also been shown to be expressed by neurons

involved in PACS and other forms of CS 17, 98,148,149, 151,152, and found only in neurons also

expressing NGF receptors48 149, 230, though this observation is questioned by work

presented herein (see below Limitations in testing PACS; Specific questions raised by

previous PACS data).

As mentioned above, PACS has been shown to be limited to the medium-small

diameter DRG neurons, to the exclusion of larger low-threshold neurons, and involves

those with NGF receptors. As indicated in Figure 1 (above), trkA is normally found in

about half of DRG neurons, and overlaps almost entirely with the peptidergic (i.e.,

CGRP-IR) medium and small diameter neurons157. The low-affinity receptor, p75, has a

more extensive distribution, but is limited to the population of neurons expressing one or

more of the high affinity neurotrophin receptors (trk family)243. Quite importantly,

neither of these NGF receptors (nor other trk receptors) are expressed by about 30% of

the small diameter neurons157, 243. These neurons instead express the components of the

glial cell line-derived neurotrophic factor (GDNF) complex (RET, GFRc) and are

included in the population that shows GS-I-B4-binding12 157, 159. Therefore, the small

diameter population consists of two general subpopulations those expressing NGF

receptors, and those expressing GDNF receptors. There is clear evidence for the role of

NGF and the trkA-bearing neurons in PACS. However, there is little evidence regarding

the possible role of GDNF and/or the RET-bearing neurons in PACS. Some of the NGF-







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related data could be taken to indicate that there may not be a role in PACS ofnon-trkA

neurons, but such conclusions would be premature as their role was not directly tested

(see below). Most examinations did not address, or even acknowledge, the non-trkA-

bearing small diameter afferent population23'49'50, 52, 149, 151,173,216



Methodological Limitations in Previous PACS Testing

Examinations of collateral sprouting thus far have focused, either by commission

or omission, on the trkA receptor-expressing DRG neurons. TrkA-expressing neurons

are primarily small-medium diameterpeptidergic neurons157' 227(Figure 1). This focus has

ignored another subpopulation of small diameter DRG neurons which may be involved in

PACS. This other population of neurons has unmyelinated axons, includes the non-

peptidergic neurons43 205,207,235 as well as those peptidergic neurons that express

somatostatin-IR 227,228, 230. All of the neurons in this other population lack expression of

any of the known neurotrophin receptors, including trkA and p75 146,160,227, 228, 243

Examination of normal rat hairy and glabrous skin has revealed the presence of this

population in the shallow dermis and certain areas of epidermis64' 176, 186. A detailed

description of this unique population is provided below (see below Unmyelinated

Primary Afferents Lacking NGF Receptors).



Evan's Blue Dye Extravasation

Many of the experimental techniques that have been used thus far in testing

PACS have provided a great deal of important and conclusive data, but are more limited

in their scope of interpretation than was originally believed. Thus, some of the

conclusions that have been made based on these data are perhaps incomplete, or







-12-

overstated. For example, revealing the functional terminations of axons in skin using the

Evan's Blue dye/antidromic stimulation method actually reveals the terminal fields of

only a subset of axons. The axons revealed are those expressing neuropeptides in

particular SP and CGRP as these peptides are released during antidromic stimulation

and cause vasodilation and extravasation of the dye94' 134,147, 195, 244. This method revealed

that the spared fields expanded23' 139174, 191,238, and that the CTM reflex receptive fields

defined by behavioral testing matched extremely well with extravasation-revealed

terminal innervation field 52'174. However, this method could not address the terminal

distribution of non-peptidergic axons, such as much of the GS-I-B4 reactive population64'

176, 186, r those expressing SOM, which also bind GS-I-B4 235 (See Figure 1).




Histochemical Analysis of Peripheral Targets

Silver staining of axons in the collaterally reinnervated skin was also limited.

This method revealed only axons in dermal endoneurial tubes52, and could not address

terminals or axons in the sub-epidermal plexus, a major path for growth of axons in

skin81, even though such structures were shown to exist in collaterally reinnervated

skin107. Also, it could not discriminate between different histochemical subpopulations

of axons, and was likely also to not label a significant portion of the smallest axons. It

has been shown that silver stains specifically and preferentially label neurofilament

proteins42, 67 138, 177, but not intermediate filament proteins 6. It has also been shown that

myelinated neurons are enriched with neurofilament, while unmyelinated neurons are

neurofilament poor20, 122, 172 (Figure 1), but are enriched with intermediate filament

proteins (eg., peripherin, c-intemexin)73' 5 Thus, silver stains likely revealed only the

A 8 axons involved in PACS, and potentially some C-fibers in dermal bundles.









Immunohistochemical analyses have the potential for greater sensitivity than

silver stains and also to provide far better identification of the populations involved in

PACS. However, very few histochemical analyses of the reinnervated skin have been

reported. Those that have examined the innervation have used antibodies against either

the neuropeptides CGRP and/or SP, or pan-neuronal markers such as PGP 9.5 08, 109 see also

40, 216, or anterograde tracers that also could not distinguish between various

populationso1. No analyses were found that used markers specific for the non-trkA small

diameter DRG neurons.

Electron microscopic examination has produced some promising indirect

evidence for a role of non-trkA small diameter afferents in PACS. Unmyelinated axons

lacking CGRP were identified in collaterally reinnervated root dentin in rat molars216. On

the other hand, dermal bundles in denervated skin from animals treated with anti-NGF

lacked any evidence of axons49. This particular finding should be regarded cautiously,

however, when relating to non-trkA collateral sprouting. First, as mentioned above, the

dermal bundles are not the primary route of growth for collaterally sprouting axons.

They instead primarily use the subepidermal plexus81. Further, no study has addressed

the possibility that anti-NGF treatments could, in fact, affect non-trkA afferent collateral

sprouting via some intermediary (discussed below).



Histochemical Analysis of DRG

Other studies on PACS have examined the DRG housing neurons that were

undergoing PACS. These examinations primarily focused on mRNA of the NGF

receptors (trkA and p75) and GAP-43 115,148,149,151. While these studies furthered the

understanding of the role of the NGF system in PACS, they did not address the







-14-

possibility that other growth factor systems might be involved. This is due in part to the

lack of knowledge at that time about the growth factors regulating the non-trkA-bearing

unmyelinated neurons.

Further, the expression of GAP-43 mRNA was not correlated to any other DRG

family markers (i.e., trkA-IR, CGRP-IR, GS-I-B4-binding, etc.) in those studies. It was

assumed that the GAP-43 mRNA was expressed in, and increased in, the trkA-positive

neurons. While it is very likely that the population of neurons that increased their

production of GAP-43 mRNA included the trkA-positive neurons, it was possible that

lower levels of GAP-43 were also produced by non-trkA-bearing neurons. In fact,

assessment of GAP-43 mRNA in non-trkA-bearing neurons has proven difficult. The

initial studies assessing which populations of normal DRG neurons expressed GAP-43

mRNA revealed two families of GAP-43 expressing neurons those expressing high

levels and those expressing very low levels230. The neurons expressing high levels of

GAP-43 mRNA were consistently those that also expressed trkA, the highest GAP-43

levels being found in neurons that also expressed SP-IR The neural population that

expressed GAP-43 mRNA at low levels was consistently the small diameter neurons that

lacked trkA (some of which expressed SOM-IR). An important finding was that the level

of GAP-43 mRNA signal in neurons other than those with trkA was extremely low, very

close to the level of background signal. Thus, assessments of GAP-43 mRNA in normal

non-trkA-bearing DRG neurons has proven consistently difficult.

This difficulty has extended into GAP-43 detection via immunocytochemistry.

Studies examining GAP-43-IR in DRG consistently report numbers of GAP-43-IR

neurons that are far lower than would be expected based on mRNA studies15' 199, 200, 210, 241

While it is possible that some neurons simply produce GAP-43 mRNA but do not







-15-

translate detectible levels of protein, as may be the case with motor neurons21'133'152,231,

versus 240, it is also possible that the immunocytochemical detection of GAP-43 in DRG

neurons has been insufficient. Detection of GAP-43 in axons rarely suffers from such

problems, as the protein is rapidly concentrated in axons, especially at terminals in

muscle or skin. Evidence for insufficient detection of GAP-43-IR in DRG neurons in

previous studies is presented as part of the results of this study.

Since GAP-43 is widely accepted, based on excellent demonstrations, to be

intrinsically involved in neuronal anatomical plasticity, it may have been some of the

seminal studies demonstrating which populations of DRG neurons expressed GAP-43

that prevented earlier examinations of the possibility that non-trkA small diameter

primary afferent may be involved in PACS. For example, it was demonstrated that high

levels of GAP-43 mRNA were selectively expressed by DRG neurons expressing trkA

230. Other DRG populations expressed levels of GAP-43 mRNA that were very close to

background. The combination of this demonstration with the lack of knowledge about

the non-trkA small diameter population is likely to have played a role in the generation of

the general focus on the trkA-positive population in sprouting studies. At the same time,

however, Verge et al. (1990) also recognized that while the level of GAP43 mRNA may

be correlated with the capacity for anatomical plasticity in some neural populations, it

was not necessarily indicative for all populations, such as motor neurons. They realized

that "the possible contribution of GAP43 to sprouting might be clarified by better

histochemical definition of sensory axons that are capable of collateral sprouting" (Verge

et al., 1990, p.933). While the current study does not focus on the role of GAP-43 per se,

it does mirror the sentiment that a better definition of the populations that are capable of

PACS is needed.









Electrophysiological Assessments of PACS

There have also been a limited number of electrophysiological assessments of the

neurons involved in PACS. The most consistent finding was that the sprouted afferents

were limited to high-threshold mechanoreceptors and thermoreceptors5' 47 59, to the

exclusion of low-threshold A-fibers but see 58, 84, 92, 93. The electrophysiological data indicate

quite strongly that PACS is indeed primarily, if not exclusively, limited to small diameter

afferents (including at least a portion of the A8 group). This fits well with what is

known about which histochemical types of afferents participate. As stated above,

afferents responsive to NGF (thus trkA-positive and peptidergic157) have a known role in

PACS. Further, it has been shown that the vast majority of afferents expressing SP

and/or CGRP displayed clear nociceptive capacities, or nociceptor-associated

properties54' 119, 121,142-144, 171. Therefore, the demonstrations of trkA expression and

nociceptive roles for peptidergic neurons go hand-in-hand with the demonstration that

afferents capable of PACS (an NGF-dependent phenomenon) are primarily nociceptors.

While these data fit together nicely and provide compelling evidence for the role

of trkA-positive, peptidergic neurons in PACS, they have at no point actually addressed

whether or not the trkA-negative population could participate in PACS. The same

studies that provided direct evidence for the nociceptive capabilities of SP- and/or

CGRP-expressing afferents gave evidence for nociceptive capabilities ofnon-peptidergic

C-fibers 121, 142-144, 234. This means that the trkA-negative small diameter afferents are

included in the functional types of afferents capable of PACS. Therefore, unless a

previous PACS study had been designed to specifically test for a role ofnon-trkA

(mostly non-peptidergic) afferents in PACS, then any role they played could have been

missed. No such design was found in any of the PACS studies to date. Further, none of







-17-

the PACS studies done to date provided evidence that could eliminate a possible role of

the non-trkA afferents in PACS.



Assessments of PACS Using the CTM Reflex

Assessments of PACS using the CTM reflex are limited because it is unclear

which particular types of afferents are involved in the reflex, and also what stimuli are

adequate for the reflex. As previously stated, anti-NGF treatments have been shown to

halt the expansion of an isolated CTM reflex-inducing sensory receptive field49, 52

Accordingly, if the trkA-negative, GS-I-B4 reactive axons do participate in PACS, then it

is likely that they do not contribute to the CTM reflex, unless they do so by transducing a

sensory modality that has not yet been tested in relation to the CTM reflex. The function

of the GS-I-B4 reactive population is still unclear, and the only modalities tested to date

and shown to be adequate for induction of the CTM reflex were noxious heat and pinch52'

59, 166, 217. It is possible that some types of A-delta and/or C-fibers may still participate in

PACS, but may not be involved in the CTM reflex, and would therefore not have been

observed with the behavioral tests. Given that the trkA-negative C-fiber terminations are

anatomically distinct from those of the trkA-positive neurons, and that the two

populations contain different neurotransmitters and/or neuromodulators, express different

receptors, and also rely on different neurotrophic factors (see below), it is not at all

unreasonable to believe that the two populations may be differentially involved in certain

reflexes and/or afferent processes.









Unmyelinated Primary Afferents Lacking NGF Receptors

The focus of this dissertation was on the subset of unmyelinated primary afferents

which are not directly responsive to NGF, lack trkA, and therefore were not examined in

the previous work on PACS (described above). These neurons are almost entirely

encompassed in the population that binds GS-I-B4 205,207,211,212, 235, 236 and expresses the

enzymes fluoride-resistant acid phosphatase (FRAP) and/or thiamine monophosphatase

(TM P)12, 40, 113, 145, 182, 204,205,207,235

Understanding of this population has lagged behind that regarding the trkA-

bearing group, however, primarily due to the lack of suitable anatomic and functional

markers for the population, as well as a lack of understanding of which factors regulate

their function. FRAP histochemistry has been used to visualize the neuronal somata in

the DRG, and the terminals in the dorsal horn of the spinal cord, but was not suitable for

visualizing most peripheral processes45' 62, but see 77, 113,204, 207. However, Streit and co-

workers described the binding of GS-I-B4 in a subpopulation of small diameter primary

afferent somata and processes211'212 that was subsequently shown to include the FRAP

expressing population of primary aill'renl'-" '. The GS-I-B4-binding population

includes the non-peptidergic unmyelinated DRG neurons, as well as the entirety of the

population expressing the neuropeptide SOM 235. Both the non-peptidergic and the

SOM-expressing GS-I-B4-binding neurons have been shown to lack NGF receptors10'12,

157, 202,227. The distribution of GS-I-B4 reactive processes in peripheral tissues has since

been described64 65' 175, 176, 186, 187, 205-207, 236, as well as their central terminations in the spinal

cord110,111,157,181,213

Although the exact functional subclasses encompassed in this population remains

unclear, certain functions are strongly suggested. Terminal distribution patterns of GS-I-









B4 axons in the spinal cord and skin suggest a possible role as thermoreceptors157' 176

Recent evidence has demonstrated that a subtype ofpurine receptor (P2X3) is almost

exclusively expressed in GS-I-B4 reactive DRG neurons and likely in their cutaneous

axons233. This lends credibility to the possibility that this population may demonstrate

chemosensitivity, or at least have a sensitivity to tissue damage and/or inflammatory

processes. Other recent work offers further evidence that at least a portion of the GS-I-

B4-positive, trkA-negative population is likely to contain nociceptors. The capsaicin-

sensitive ligand-gated ion channel (VR-1) was shown to be primarily localized to this

subpopulation of DRG neurons"7, and pain is the primary sensation elicited by

administration of capsaicin' 117, 20, 215,218

In vitro classification of acutely dissociated DRG neurons by current signature

has provided some more direct evidence that the GS-I-B4-binding neuron population that

lacks peptides (except SOM) contain nociceptors. Classification of neurons based on

their repertoire of voltage activated currents (current signature) has generated a large

number of different subclassifications that maintain a high degree of internal consistency

in regard to histochemistry, action potential shape, and pharmacological sensitivity to

numerous agcnl 30, 44. This classification scheme is very powerful in that it allows the

tracking and compilation of characteristics across experiments. Type 1 and type 2

neurons that had been recorded and then examined for their histochemical characteristics

both consistently displayed GS-I-B4-binding. Type 2 neurons consistently lacked CGRP

and SP, while type 1 neurons expressed CGRP and SOM, but lacked SP, indicating that

both types were part of the non-trkA small diameter afferent population (Petruska, J.C., J.

Napaporn, R.D. Johnson, J.G. Gu, B.Y. Cooper; unpublished observations). Both types

displayed numerous characteristics of nociceptive primary afferents (Petruska et al.,









unpublished observations)29'30'44. These included sensitivity to acidic solutions and

capsaicin, as well as a wide action potential and a long-duration after-hyperpolarization

(AHP), characteristics that correlate extremely well with a nociceptive function in normal

animals54'144,192. Type 2 neurons also displayed ATP-induced currents with rapid kinetics

that were likely mediated by homomeric P2X1 and P2X3 receptors (Petruska et al.,

unpublished observations), a characteristic associated with nociceptors38' 39

It has recently been shown that trkA-negative, GS-I-B4-binding DRG neurons

expressed the components of the glial cell line-derived neurotrophic factor (GDNF)

receptor complex12'159. They bound and transported GDNF and were supported during

development and after injury by GDNF 123, 141. Further, while both NGF and GDNF both

regulated the expression of various proteins including TTX-insensitive Na+ channels,

ATP-sensitive P2X3 receptors, and potentially the VR-1 receptor they did so in

mutually exclusive groups of neurons12' 22, 63, 75, 99, 123, 141, 154. The group of trkA-negative

small diameter neurons regulated by GDNF also completely encompassed the SOM-

expressing DRG neurons12' 235. SOM expression has been shown to be regulated by

GDNF, and SOM-IR neurons have been shown to be insensitive to NGF, as they did not

express either trkA or p75 10,12,157, 202, 227. Based on its expression of GS-I-B4-binding, its

regulation by GDNF, and its lack ofNGF receptors, the SOM-IR population was

included in the group under investigation in these experiments.

These recent discoveries should greatly enhance efforts to understand the role of

these neurons in sensory systems. The findings described above are in line with the

current proposal that these sensory neurons are capable of collateral sprouting since it is

likely that the GDNF system is involved in at least one other form of collateral sprouting,

namely that of motoneurons132 165









Hypotheses and Plan for Testing

It was the basic hypothesis that the non-trkA small diameter DRG afferents were

involved in PACS, but have been missed in previous studies because of methodological

limitations. In order to address this hypothesis initially, collaterally reinnervated skin

was histochemically analyzed for the presence of axons that displayed markers indicative

of the non-trkA small diameter afferent population. As detailed in the results below,

such axons were, in fact, observed. This was taken as a direct indication that the non-

trkA C-fiber population was capable of collateral sprouting. While this finding supported

the basic hypothesis, there were questions that arose from the previous work that needed

to be addressed.

Since non-trkA neurons are likely capable of PACS, then 1) why did anti-NGF

treatments block PACS as observed by microscopy of the skin?, and 2) why did anti-

NGF treatments block PACS as observed by the CTM reflex? These questions can be

addressed individually and are summarized below.

In addressing the first question, namely, the effect of anti-NGF treatments on

PACS as assessed by microscopic examination of skin, two possibilities are raised. First,

it is possible that silver staining of the skin may have missed some of the axons that were

present, or was simply an insufficient stain to reveal the trkA primary afferent

population. There are certain lines of evidence that support this possibility. First, it is

known that silver stains primarily reveal neurofilament content6' 67,177 In regards to DRG

neurons, silver stains would reveal myelinated axons due to their high content of

neurofilament proteins12, 122,172. However, the majority of trkA+ axons, and all of the

trkA- axons (of interest) lack myelin. Second, the only myelinated axons that are

involved in PACS are a subgroup of AS axons. Following that, the only A8 subgroup







-22-

involved in PACS is NGF-sensitive, and therefore likely trkA 28, 190. This means that

any myelinated axons that did participate in PACS as revealed by silver stain were those

that were sensitive to NGF. Lastly, the only axons (of any type) revealed by the silver

stain of skin were axons in dermal bundles52' 166. The highest density and occurrence of

unmyelinated afferent axons is in the subepidermal plexus and in the epidermis. The

silver stain did not reveal any such axons. This speaks directly to the likely insufficiency

of the silver staining technique to reveal unmyelinated axons, the group of primary

interest for these studies. The possibility that silver staining was an insufficient

technique to reveal the full array of axons in collaterally reinnervated skin is addressed

by experiments detailed in Chapter 3.

Second, it is possible that the anti-NGF treatment blocked PACS of both the

trkA+ and trkA- groups because NGF may play some role in PACS of the trkA-

population. It is possible that trkA- DRG neurons may begin to express trkA (and/or

p75) in response to the surgical isolation of their terminal fields. This possibility was

indirectly addressed by experiments detailed in Chapter 3.

In addressing the second question, namely why anti-NGF appeared to block

PACS as assessed by the CTM reflex, two possibilities are raised. First, it is possible that

the anti-NGF treatment blocked only the PACS of the trkA neurons and the PACS of the

trkA- group was not observed. This would occur if the trkA- neurons do not participate

in the CTM reflex, and therefore any collateral sprouting would have been missed since

the CTM reflex was the primary method of assessment. This possibility was directly

tested by experiments detailed in Chapter 4. The second possibility is the same as for the

first question above, namely, thatNGF could somehow affect PACS of this group. These

arguments are summarized in Outline 1.










Since non-trkA neurons are capable ofPACS, then why did anti-NGF block PACS:

1) as assessed by skin histochemistry?
A) silver stains were insufficient (insensitive to the axons of interest):
i) silver stains show myelinated, but the axons of interest are unmyelinated
ii) the only involved myelinated are A8, and all are trkA
iii) primary innervation fields of axons of interest not revealed
B) NGF plays a role in trkA- PACS

2) as assessed by CTM reflex?
A) trkA- PACS not observed because:
i) trkA- not part of CTM reflex
B) NGF plays a role in trkA- PACS

Outline 1. Summary of arguments addressing previous PACS data.



The experiments described herein were designed to address the possibility that

non-trkA small diameter DRG afferents were capable of PACS, and to address some

possible explanations as to why they had not been observed in previous examinations. In

order to demonstrate the presence ofnon-trkA DRG axons in collaterally reinnervated

skin, multi-labelling histochemistry for highly specific markers was performed on

sections of normal and collaterally reinnervated skin. These experiments would also

address the possibility that stains other than silver stains might be better suited to

revealing the true array of innervation in collaterally reinnervated skin (Question 1A in

Outline 1). In order to demonstrate that non-trkA DRG neurons expressed markers

indicative of PACS, multi-labelling histochemistry for GAP-43 and markers highly

specific for the non-trkA small diameter population was carried out on sections from the

DRG involved in PACS. In order to address the possibility that NGF may play some role

in trkA- PACS because the trkA- neurons begin to express trkA during PACS (Question

1B in Outline 1), multi-labelling histochemistry for trkA and markers highly specific for

the non-trkA small diameter population was carried out on sections of skin. The guiding

hypothesis of this set of experiments was that the non-trkA small diameter DRG









afferents were involved in PACS, but have been missed thus far. The specific

hypotheses were: 1) axons displaying markers specific for the trkA-negative small

diameter afferents would be observed in collaterally reinnervated skin, 2) neurons in the

DRG involved in PACS that displayed markers specific for the trkA-negative small

diameter afferents would begin to express GAP-43.

In order to address one of the possible reasons that non-trkA PACS was missed,

namely, that the trkA- small diameter DRG neurons are not involved in the CTM reflex

(Question 2A in Outline 1), experiments were undertaken to demonstrate which afferent

populations were involved in the CTM reflex. The guiding hypothesis of this set of

experiments was that the non-trkA small diameter DRG afferents were not involved

in the CTM reflex. The primary means of examining this possibility was the selective

destruction of p75-expressing DRG neurons (which included the trkA+ neurons) with a

directed neurotoxin. The toxin (saporin) gains access to the interior of only targeted cells

based on the internalization of a transmembrane protein with an external antigen to which

the antibody-neurotoxin complex has bound. The saporin is cleaved from the antibody

and then inactivates ribosomes, which eventually kills the cells178,179. The ability of the

spared neurons to drive the CTM reflex was then examined, and the selective destruction

of the targeted population was confirmed histologically. In addition, transneuronal

tracing with PRV was employed to anatomically retrogradely trace the reflex circuit from

the motoneurons to the primary afferents.















CHAPTER 2
METHODS

General Methods

Histochemical Analysis of PACS

The intent of these experiments was to determine whether or not GS-I-B4-binding

neurons lacking NGF receptors were involved in PACS. This was assessed by

examining which primary afferent subpopulations were present in collaterally

reinnervated skin, and also by examining which primary afferent subpopulations in the

spared DRG expressed the growth associated protein-43 (GAP-43) a marker for axonal

growth. Collateral sprouting was induced by surgical isolation of the T 13 dermatomal

cutaneous nerves. Animals were allowed to survive for either 14 days (for DRG cell

counts and cutaneous innervation examinations; n=6) or 28 days (for cutaneous

innervation examinations; n=4). They were then euthanized, perfused, and the tissue

prepared for histochemical processing.



CTM Reflex Afferents

The intent of these experiments was to determine which subgroup or subgroups of

primary afferents were involved in the induction of the nociceptive specific cutaneus

trunci muscle (CTM) reflex. The first set of experiments involved the injection of a

directed neurotoxin into the left T 13 DRG. The neurotoxin was the ribosomal

inactivating protein saporin conjugated to a monoclonal antibody against p75 (192-sap).







-26-


Animals (n=8) were allowed to survive for 7-23 days, and then underwent a terminal

electrophysiological assessment of the ability of the DCn from the injected DRG to

generate a CTM reflex. Animals were euthanized at the end of the experiment, perfused,

and the tissue prepared for processing to examine the expression of NGF-related markers

(trkA, p75, CGRP, SP) and markers associated with the trkA-negative population of

small diameter afferents (GS-I-B4-binding, SOM, P2X3).

A second set of experiments designed to address the same question involved

injections of the transneuronal tracer pseudorabies virus into the CTM muscle or LTn (or

control tissues) in order to characterize the afferents involved in the CTM reflex.

Animals (n=24) were allowed to survive for 24-72 hours, and were then euthanized,

perfused, and the tissue prepared for processing to localize the virus. The virus was

localized with antibodies directed against the virus. This was combined with other

markers specific for various subpopulations of primary afferents in order to determine if

the virus was localized to particular subpopulations.



Specific Methods

Histochemical Analysis of PACS

Sprouting surgeries

Adult female Wistar rats were anesthetized with ketamine/xylazine. Fluids were

usually administered at this time (1.5 3cc lactated Ringer's i.p.). They were maintained

at 360C (+10C, monitored via a rectally placed thermistor) with an electric heating pad,

and the heart rate was monitored with a stethoscope attached to an audio amplifier. For

the surgical induction of collateral sprouting, an incision was made approximately 1cm to







-27-

the right ofmidline along the low thoracic and upper lumbar dorsal skin. This incision

was designed to leave intact any cutaneous innervation from the left dorsal cutaneous

nerves that may have crossed midline. The skin was pulled away from the body and the

subcutaneous fascia freed from the underlying body wall musculature to reveal the DCnn

emerging from the body wall musculature. The T11, T12, L1, and L2 DCnn were

isolated, ligated with 7-0 monofilament nylon as they emerged from muscle and

transected. For animals that were part of the DRG cell count studies, the corresponding

lateral cutaneous nerves (LTnn) were also identified by approximate landmarks and

ligated and transected. The LTnn were transected in these experiments in order to

provide a greater area of neighboring denervation for the T13 dermatome. This would

increase the probability of quantifying any changes in the PACS DRG, since a greater

proportion of the T13 afferents would be undergoing PACS than if only the DCnn were

transected. The incision was sutured in layers and closed with Michel clips. As the

animals regained mobility during their recovery from anesthesia, the CTM reflex fields

were mapped with a fine pinch stimulus. Fields were marked with a permanent marker

on the skin. At the end of the survival period (28 or 14 days for skin studies, 14 days for

DRG cell count studies), animals were anesthetized with 50-60 mg/kg sodium

pentobarbital and the CTM reflex field mapped again. A survival time of 14 days was

used for cell counting studies, where GAP-43-IR was a major focus, because a previous

study had indicated that GAP-43 mRNA in PACS DRG peaked at 12 days151. Therefore,

14 days should offer an excellent indication of the GAP-43 protein signal in the PACS

DRG. A survival time of 28 days was included for the skin histochemical analyses

because a previous study had indicated that PACS reached its spatial extent at 28 days







-28-

post-denervation52. In all cases the formerly unresponsive fields had regained the ability

to drive the CTM reflex in response to pinch. Animals were then overdosed with

urethane and perfused (see below).

Control experiments were done in order to ensure that any IR or GS-I-B4-binding

observed in the collaterally reinnervated regions were actually axons that had grown into

the area and were not simply residual profiles from axons that had originally innervated

the tissue. These consisted of carrying out the surgical procedures as described above,

but only allowing the animal to survive for 3 days before euthanizing and perfusing as

described above (n=l).

This animal also served as a control for the accuracy of the innervation map

produced by using pinch testing and the CTM reflex. The skin was tested prior to

euthanizing the animal to ensure that the delineations had not changed. The skin sections

were examined microscopically to determine how well the behavioral and the

histochemical innervation patterns matched.

CTM reflex testing

The areas of skin that were capable of driving the CTM reflex were assessed as

the animal recovered from the T13 isolation surgery as well as just prior to sacrifice. The

reflex was sensitive to the ketamine/xylazine anesthesia used during surgery. Therefore,

the testing was not done until the animal was able to move all four limbs and raise its

head. This level of recovery was suitable to give an indication of the CTM reflex

receptive field that did not differ from that revealed under pentobarbital anesthesia, to

which the reflex was highly resistant. Assessment was done by lightly pinching the skin

with fine forceps. The reflex could be induced with light pinch of very small areas of







-29-


skin, and was often induced by the prick of the forceps onto the skin surface. The

forceps were closed perpendicularly to the body axis, so as to provide a very narrow

application of force parallel to the borders of the receptive fields. The borders were

drawn with permanent ink, which was re-applied every 2-3 days. The reflex was readily

visible as a rapidly appearing and disappearing puckering of the skin just rostral to the

pinch site.

Histochemical procedures for collaterally reinnervated skin

Samples of skin that included both the normal T13 dermatome and the

collaterally reinnervated region were sectioned at 20-35!pm and retrieved onto slides.

Procedures for the detection of markers are summarized below. Antisera used for this

and other procedures are listed in Table 1. Tissue underwent an initial blocking

incubation in a solution of 1:30 goat serum in PBS with 0.4% Triton X-100 (GS-PBS-T)

to prevent non-specific protein-protein binding of the subsequent antisera. The sections

were then incubated overnight in a mbbit primary antisera This step, and all others, was

followed by repeated rinses with 1% GS-PBS-T. The primary antisera were then

detected with a 1:75 solution of Texas Red-conjugated, or a 1:100 solution of

AlexaFluor 594-conjugated goat a-rabbit IgG. Detection of the trkA or P2X3 antisera

was done with amplification, described below. This was followed by incubation with

mouse primary antisera. The primary antisera were then detected with a 1:100 solution

of FITC- (or AlexaFluor 488-) conjugated goat a-mouse IgG. The a-mouse antisera

were preadsorbed against rat serum that was prepared in our lab to prevent non-specific

binding of the secondary antisera to rat proteins. Controls for this uniformly showed that

the preadsorbtion procedure eliminated non-specific binding. Following the rinse of the







-30-

secondary antisem, the tissue was incubated overnight in a solution of HRP-conjugated

GS-I-B4, or in some cases, a-PGP 9.5. PGP 9.5 was detected with Pacific Blue-

conjugated a-rabbit antiserum, and the lectin was detected with the coumarin (blue)

conjugate of the TSA amplification system (New England Nuclear, Inc.), similar to that

used for the trkA or P2X3 antisera. This system utilizes the HRP molecule to catalyze

the deposition of conjugated tyramide onto the tissue8, 19, 20, 224. Detection of the trkA or

P2X3 antisera was done by incubation with biotinylated secondary antisera, followed by

the avidin-biotin-HRP complex (ABC kit, Vector Labs). The ABC complex then

catalyzed the deposition of FITC-conjugated TSA. If the TSA-detected lectin was to

follow this, then the HRP present from detection of the trkA or P2X3 was quenched prior

to application of the lectin with a 20 minute incubation in a solution of H202 and

methanol diluted in PBS.

Markers used for skin histochemistry

The classification of a cutaneous axon into a particular histochemical family (and

thus often a functional family) is more difficult than the histochemical classification of

neurons in the DRG. This is primarily because the skin is not only innervated by sensory

axons, but also by sympathetic axons. Sympathetic neurons share many markers found

on families of DRG neurons, including trkA-IR. Morphological features of axons in

normal skin can be a good indicator of whether an axon is sympathetic or sensory.

However, since sympathetic axons also undergo collateral sprouting, and the only

evidence that collaterally sprouted axons retain some morphological features is indirect

and merely suggestive1"', morphology could not be used as the primary indicator of a

sensory or sympathetic identity. Therefore, multi-labelling histochemistry was







-31-


employed. By labelling the skin with markers whose combinations would clearly

identify sympathetic and sensory axons, as well as the various subfamilies of sensory

axons, the types of axons innervating the reinnervated skin could be identified. TrkA

provided an excellent indicator of both sympathetic and trkA-bearing sensory neurons.

Therefore, any axons in collaterally reinnervated skin that bound GS-I-B4, expressed

GAP-43-IR, but lacked trkA-IR could be considered to be axons derived from the

population of interest that had successfully sprouted into the denervated skin. P2X3,

which is not present in sympathetic neurons of normal rats131 or PACS rats (Petruska,

unpublished observations), also provided an excellent marker for axons of interest since

it is primarily expressed in GS-I-B4-binding DRG neurons that lack CGRP 22, and are

therefore unlikely to express trkA 57. Axons binding GS-I-B4 but lacking CGRP are

indicative of successful non-trkA collateral sprouting by the same reasoning.

Multiple samples of skin were taken from the collaterally reinnervated regions

and represented a span of 1.5 2.0 cm of skin. Generally, 2-3 samples from the

reinnervated skin were sectioned, and 3-4 sections with 150-200!tm separation were

placed on slides for staining. This procedure generated a good sampling of the

reinnervated skin regions.

Histological procedures for cell counts

Euthanized animals were transcardially perfused with exsanguination solution

(heparinized phosphate buffered saline) followed by 4% paraformaldehyde in phosphate

buffered saline (PBS). Tissue was removed and placed in a solution of 30% sucrose in

PBS until they were sectioned. Tissue sections were made with a cryostat. Sections

were cut at 10-12alm and collected in a serial series with 10-12 slides in the series. This







-32-


collection procedure generated slides holding sections with 90-132mm between each

section. This separation guaranteed that no single neuron would be present in more than

one section per slide and that one slide would contain a representative sample of the

entire ganglion.

Immunohistochemical detection of markers for cell counts generally followed the

procedures described above, with some modifications. Following the initial blocking

step, the sections were incubated with a solution of mouse-a-GAP-43 overnight. This

was followed by detection similar to that described for P2X3 or trkA in skin

(bitotinylated secondary antiserum and ABC kit). However, instead of using TSA-FITC,

TSA-biotin was used. This was then followed by an incubation with avidin-AlexaFluor

488 (green). The appropriate rabbit antisera were then applied (P2X3, trkA, SOM) and

then detected with AlexaFluor 594-conjugated a-rabbit antiserum. Prior to application of

the lectin, the HRP on the tissue sections from the amplification of the GAP-43 signal

was quenched. The tissue was thoroughly rinsed prior to application of the lectin

conjugate. The sections were incubated overnight in the lectin solution. The following

day the GS-I-B4-binding was detected with TSA-coumarin.

Cell counting

Black-and-white digital images of each section on the stained slides were taken in

montage fashion and saved on computer disk. Images were captured either with a Zeiss

Axiophot equipped with a Dage 72-S 10-bit integrating single-chip CCD camera, or a

Zeiss Axiophot II equipped with a Spot II three-chip CCD camera. Images were opened

in Adobe Photoshop 5.0 and positively-stained neurons with nuclei were circled. The

circled cells and the marking layers were all compared and counted to generate the







-33-


percentages of neurons with various combinations of markers. A minimum of three

sections per slide were used to generate the proportions.

The various staining combinations were assessed and the percentages generated

for each individual animal. These percentages were then subjected to a two-tailed t-test

to determine whether there was any significant difference between the normal control

group and the PACS group.



CTM Reflex Afferents

Neurotoxin experiments

Adult female Wistar rats were anesthetized with ketamine/xylazine. Fluids were

usually administered at this time (1.5 3cc lactated Ringer's ip). They were maintained

at 360C (+1C, monitored via a rectally placed thermistor) with an electric heating pad,

and the heart rate was monitored with a stethoscope attached to an audio amplifier.

Under aseptic conditions, a midline incision was made in the dorsal back skin. The L6

spinal process was identified by subcutaneous palpation, and the T13 vertebra identified

based on counting spinous processes. The left T13 DRG was then exposed. The capsule

was pierced with a 28g needle, and 75-90ng (in 1.0-1.241) of the monoclonal antibody

192 (directed against the low-affinity NGF receptor p75) conjugated to saporin was then

injected into the DRG with 10l Hamilton syringe coupled to a glass micropipette with a

tip outer diameter of 30-50|tm. The injection was done in 0.2pl increments with a few

minutes between each. The injected fluid was visualized to cause brief and minor

swelling inside the DRG capsule and root compartment. There was no visible leakage of

the fluid out of the injection hole either during the injection or during withdrawal of the







-34-

pipette. The muscle layers that had been retracted were sutured together again and the

fascia and skin closed in layers. The wound was finally sealed with Michel clips.

Animals (n=8) were housed separately and allowed to survive for 7-23 days.

At the end of the survival period they were prepared for a terminal physiological

examination. They were anesthetized with sodium pentobarbital (50-60mg/kg) and

placed on a circulating water heating pad to maintain their core tempemture (monitored

with a gastric thermistor). The trachea was intubated to monitor end-expired pCO2.

They were given 0.1cc of atropine (0. mg/ml, s.c.) every 2 hours to counteract the fluid

build-up in the lungs that accompanies pentobarbital anesthesia. A midline incision was

made in the dorsal back skin and the T11, T12, T13, and L1 DCnn isolated bilaterally. In

some cases, the middle branch of the LTn was dissected free from where it entered the

CTM and was placed on recording electrodes. Each of the DCnn were placed

sequentially on bipolar silver-silver chloride stimulating electrodes. The threshold to

elicit a visible contraction of the CTM, or a recordable LTn response, was then

established. The strength of the contraction at various stimulus intensities was also

noted. In cases where the LTn recordings were done, the LTn response was recorded

with a DCn stimulus of 1.5mA (A6 strength) or 3-5mA (C-fiber strength). Finally, a

laminectomy was performed in order to expose the dorsal roots. The dorsal root from the

injected DRG was placed on bipolar recording electrodes and the compound action

potential (CAP) elicited by C-fiber strength stimulation of the appropriate DCn was

averaged and recorded. At the end of the physiological experiment the animals were

overdosed with anesthetic and perfused as described above.







-35-


Tissue processing

Procedures for the detection of histochemical markers in sections of the injected

and control DRG from toxin-injected animals were similar to those described above.

Various combinations of markers were run in order to assess the efficacy and specificity

of the toxin. The following markers (in various combinations) were assessed: mse-a-p75

(clone 192 same as used for toxin conjugation), rbt-a-p75, trkA, SOM, P2X3, GS-I-B4.

Pseudorabies virus injections

Adult female Wistar rats were anesthetized with ketamine/xylazine. Fluids were

usually administered at this time (1.5 3cc lactated Ringer s ip). They were maintained

at 360C (+1C, monitored via a rectally placed thermistor) with an electric heating pad,

and the heart rate was monitored with a stethoscope attached to an audio amplifier.

Under aseptic conditions, a midline incision was made in the dorsal back skin and the

skin/CTM layer was separated from the underlying muscle in order to introduce the

transneuronal tracer pseudorabies virus (PRV) into the cutaneus trunci muscle (CTM)

reflex circuit (n=18). Care was taken to avoid damaging the dorsal cutaneous nerves

(DCn). The skin/CTM layer was held away from the body with hemostats, and the thin

fascia covering a small portion of the CTM was cleared away to facilitate injection of the

muscle. The caudal-most portion of the cleared region was consistently at least 15mm

rostral to the point where the T13 (the segment of focus in this study) DCn pierced the

CTM to innervate the overlying skin. This ensured that the PRV injection was not placed

into the region of CTM directly underlying the T13 dermatome to avoid any direct

uptake of the virus by the DCn axons. Further, previous research determined by

electromyography (EMG) that the zone of shortest latency response (SLR) of the CTM

reflex elicited by electrical stimulation of a single DCn was 1cm rostral to the dermatome







-36-

of the stimulated nerve21. Focussing the injection within the T13 SLR would optimize

the transport of the virus through the CTM reflex circuit to the T13 DRG. In another 6

cases, PRV was injected directly into the middle branch of the left LTn near the origin of

the CTM.

The virus (1-5x 10 plaque-forming units (PFU)/ml) was pulled into a 10ll

Hamilton microsyringe equipped with a 33 gauge needle. The virus was injected directly

into multiple sites within the CTM in 0.5-1.0tl increments. Care was taken to avoid

injections near any penetrating DCn, but injections were often very close to the many

branches of the lateral thoracic nerve (LTn) innervating the CTM. Any residual fluid

was removed with a cotton-tipped applicator. The incision was then sutured closed with

7-0 nylon and secured with Michel clips.

Animals were allowed to recover to the point of regaining mobility of their trunk

while still being monitored for rectal temperature and heart rate. Once they had regained

this level of mobility, the stethoscope and thermistor were removed and the animal

placed in a bedding-free cage which had one half over a heating pad. They were

monitored until regaining mobility of their hindlimbs for weight-support and were then

placed in a normal cage (full bedding and ad libitum water and food) which also was

placed partially on a heating pad. Once they had fully recovered they were removed to a

designated cubicle in the University of Florida Health Center Animal Resources

Department (HCARD) infectious disease suite. The animals were observed at least three

(3) times per day (usually at 8 hour intervals), with more frequent monitoring on the final

day of the survival period. Animals were generally allowed to survive from 24-72 hours,

depending on the particular experiment. Animals were allowed to survive for the entire







-37-


designated survival period unless they began to display signs of a more widespread

neural infection. These signs included significant lethargy, head-bobbing, sporadic bouts

of rapid motion, or ballistic movements. If these behaviors were observed, the animal

was euthanized as soon as possible and perfused. Delays prior to euthanization were

generally less than 30 minutes, as the supplies for this procedure were kept at the ready

from 48 hours post-injection. No signs were ever observed prior to 60 hours post-

injection.

Pseudorabies virus control experiments

In order to ensure that the virus localized to the lower thoracic and upper lumbar

DRG neurons was indeed derived from retrograde infection of the various neurons of the

CTM reflex, control experiments were performed. The injections of PRV made directly

into the LTn branches near the origin of the CTM also served as a control to ensure that

lower thoracic DRG infection could arise from LTn-derived primary infection (n=6).

Injections of PRV were also made into the CTM as described above but were coupled

with specific nerve transactions (n= 1 each): 1) transaction of the ipsilateral LTn

branches, 2) transaction of the ipsilateral T10-L2 DCnn, 3) transaction of the ipsilateral

LTn and T10-L2 DCnn. These transactions were designed to dissect the routes of entry

of the virus into the nervous system from the CTM. Animals were allowed to survive for

up to 72 hours before being euthanized, perfused, and their tissue processed for the

localization of PRV.

Previous studies had showed that the virus displayed differential tropism for

certain subgroups of neurons27'28. In order to assess whether or not there was any

differential tropism of the virus for the central terminals of subtypes of DRG neurons,







-38-

injections of virus were made directly into the dorsal horn of the T12 or T13 spinal cord

(n=3). Animals were allowed to survive for approximately 24 hours before being

euthanized, perfused, and their tissue processed for the localization of PRV (see below).

Tissue processing and histological procedures

At the end of the designated survival period, or with the onset of symptoms of

serious central viral infection, animals were euthanized with an overdose of urethane

(0.5g/ml). Perfusion and cryostat sectioning of tissue was as described above.

All tissue was obtained from perfusion-fixed animals. Paraformaldehyde (4%) in

PBS was used for all tissue with the exception of the first few PRV animals. These were

perfusion-fixed with a lysine-periodate-paraformaldehyde (2%) fixative as previously

used by other Inab. .

Procedures for the detection of PRV and its overlap with other markers is

summarized below. The sections were then incubated overnight in a 1:1200 solution of

rabbit anti-trkA 37. This step, and all others, was followed by repeated rinses with 1%

GS-PBS-T. The primary antisera were then detected with a 1:75 solution of Texas Red-

conjugated, or a 1:100 solution ofAlexaFluor 594-conjugated goat anti-rabbit IgG. In

order to avoid cross-reactivity with the subsequent antisera also raised in rabbit and

detected with anti-rabbit antisera, the sections were incubated briefly in a 1:50 solution of

unconjugated goat anti-rabbit IgG. This was rinsed only briefly, and the sections were

incubated for 5 minutes with 4% PFA, which was then thoroughly washed off. The

sections were then incubated overnight in a 1:2000 solution of rabbit-anti-PRV 28. The

primary antiserum was then detected with a 1:100 solution of FITC- (or AlexaFluor 488-)

conjugated goat anti-rabbit IgG. Control experiments were performed to establish the

dilutions necessary to completely avoid the second set of rabbit and anti-rabbit antisera







-39-


from cross-reacting with the first. The dilutions used were highly reliable for clean,

strong, and specific signals. Controls were run with each procedure, and all were

negative.

Immunohistochemical localization of PRV in DRG was also compared to the

pattern of GS-I-B4-binding. This was accomplished by processing the tissue as described

above for the localization of PRV. This was followed by an overnight incubation of the

tissue in 1:200 GS-I-B4-biotin. The tissue was rinsed the following morning and the

lectin detected with avidin-FITC.



Table 1. List of antisera used and their sources and dilutions.


Antigen Host Source Dilution

PRV Rbt J.P. Card 28 1:2000

trkA Rbt L.F. Reichardt or Chemicon, Inc.37 1:1200

p75 Rbt M.V. Chao87 88 1:5000

p75 Mse Oncogene, Inc. 5pjg/ml

CGRP Rbt Peninsula Labs, Inc. 1:15k

CGRP Mse RBI, Inc. 1:2000

PGP 9.5 Rbt Biogenesis, Inc. 1:1500

TH Mse Sigma, Inc. 1:1000

P2X3 Rbt E.J. Kidd, Glaxo-Wellcome104 1:1500

P2X3 GP Neuromics, Inc.232 1:3000

SP Rbt Peninsula Labs, Inc. 1:3000

SOM Rbt Peninsula Labs, Inc. 1:1000

SOM Mse Biomeda, Inc. 1:10

GAP-43 Mse Boerhinger-Mannheim, Inc. 5pjg/ml

GAP-43 Rbt P. Caroni2 1:1000

GS-I-B4 Sigma, Inc. 5-0Ipg/ml















CHAPTER 3
RESULTS HISTOCHEMICAL ANALYSIS OF PACS


Histochemical Analysis of Collaterally Reinnervated Skin

Skin regions that had been denervated and subsequently reinnervated by collateral

sprouting of the spared T13 cutaneous axons were examined in order to determine the

characteristics of the axons present in the reinnervated skin.

























Figure 2. Examples of multi-labelling histochemistry of collaterally reinnervated skin.
Skin was stained for GAP-43 (A) and GS-I-B4 (B), CGRP (C) and GS-I-B4 (D), or
GAP-43 (E), trkA (F), and GS-I-B4 (G). The yellow arrow in panels A and B reveals a
GAP-43-IR axon(s) that also has lectin-binding approaching epidermis (white arrow).
The red arrows in C and D indicate a CGRP-IR axon that lacks GS-I-B4-binding, and
the green arrows indicate subepidermal GS-I-B4 axons that lack CGRP-IR. The yellow
arrows in E-G reveal a GAP-43-IR axon (E) that also has GS-I-B4-binding (G), but
completely lacks trkA-IR (F). Epidermis is at the top of all images.







-41-

Skin samples from animals undergoing PACS for 2 weeks (n=6) or 4 weeks (n=4)

were examined with markers specific for axons undergoing active growth (GAP-43), as

well as markers associated with tikA-IR neurons (irkA, CGRP, SP). GAP-43-IR axonal

profiles that were GS-I-B4+ but lacked either CGRP or trkA were consistently observed in

the collaterally reinnervated regions of skin (Figure 2). These regions had been

unresponsive to noxious stimulation (as assessed by attempting to elicit the CTM reflex)


Figure 3. Examples of multi-labelling histochemistry of collaterally reinnervated skin.
Skin was stained for GAP-43 (A, C) and P2X3 (B, D), or GAP-43 (E), and SOM (F).
White arrows (A-D) indicate GAP-43-positive axons that also displayed P2X3-IR.
Arrowheads (A, B) indicate GAP-43-positive axons that lack P2X3-IR. White arrows (E,
F) indicate GAP-43-positive axons that also displayed SOM-IR.







-42-

after the acute transactions, but had regained responsiveness by the end of the survival

period. Further, these regions also displayed axonal profiles that were GAP-43 /GS-I-

B4 /TH indicating that they were likely not sympathetic in origin. This conclusion was

further reinforced by the demonstration of GAP-43-IR and GS-I-B4-binding in axons that

lacked trkA since trkA is also present in sympathetic axons. These profiles were not

observed as frequently as GAP-43-IR axons that displayed trkA-IR or associated

markers. However, their presence was consistent. No difference was observed in the

reinnervation patterns of skin from the two week or four week survival groups.

Skin samples were also examined for markers associated with the non-trkA small

diameter DRG neurons (SOM, P2X3). SOM-IR was found in a few axonal profiles as

revealed by co-labelling with PGP 9.5 or GAP-43 (Figure 3). Further, many P2X3-IR

axon profiles were consistently observed in the collaterally reinnervated regions of skin

(Figure 3). These profiles could sometimes be observed entering the epidermis.

Interestingly, certain staining patterns were observed on axonal profiles in the

underlying CTM in samples from experimental animals that were not observed in those

from control animals. In particular, many CTM motor axons, morphologically identified

based on axon caliber and the direct observation of motor end-plates, were GAP-43-IR

and CGRP-IR (Figure 4). Motor axons with GAP-43-IR and CGRP-IR were found in the

CTM underlying the normal spared T13 dermatome as well as the denervated field.







-43-


Figure 4. Example of CTM from below collaterally reinnervated skin stained for GAP-
43 (A) and CGRP (B). The arrow indicates labelled axons, and the arrowhead indicates
a labelled end-plate.




Skin Histochemistry Controls

Control experiments were run in order to ensure that any IR or GS-I-B4-binding

observed in the collaterally reinnervated regions was actually due to axons that had

grown into the area and was not simply residual signal from axons that had originally

innervated the tissue. One animal underwent the same surgical procedures as the others

used for examination of skin reinnervation, but was allowed to survive for only 3 days.

The regions that had been defined by pinch-induced CTM reflex activity to be denervated

showed a dramatically decreased innervation density that was clear upon histological

examination. The rare profiles that could be observed displayed extremely weak IR for

PGP 9.5, CGRP, SP, trkA, or GAP-43. GS-I-B4-binding could still be observed, but it

was very weak. GS-I-B4+ profiles also lacked the normally accompanying PGP 9.5-IR.







-44-


The same skin samples were examined in order to determine how well the

behaviorally defined innervation fields matched with the histochemically revealed

innervation fields. The pinch-induced CTM reflex provided an excellent behavioral

indicator of the extent of innervation as revealed by immunohistochemistry for axons.

Only the extremely rare axon profile (approximately one axon profile per ten sections

examined) was found more than 500itm beyond the behaviorally-defined border.

Other control tissue was also examined to assess the rate of denervation-induced

marker loss. This tissue included the distal segment of a transected T12 DCn and sciatic

nerve that had been crushed either 7 or 14 days prior. All nerve segments revealed rapid

and nearly complete loss of all markers.



Histochemical Analysis of DRG Sections

Cell counts were done to assess the proportion of trkA-negative neurons

expressing GAP-43-IR in both control (n=7) and PACS (n=4) DRGs. Quantitation of the

proportion of trkA-negative/GAP-43-positive neurons that displayed GS-I-B4-binding

was also done in order to directly assess the GAP-43-IR of the population in question.

The guiding hypothesis was that PACS DRGs would contain more trkA-negative/GS-I-

B4-binding neurons with GAP-43-IR than would controls. This was based on the ideas

that there was a general lack of GAP-43 in non-trkA neurons in normal animals5' 230, but

that GAP-43-IR axons displaying markers of non-trkA afferents were found in

collaterally reinnervated skin (present data).

Qualitatively, in control DRG, there were three clear levels of GAP-43 staining,

namely negative, low-intensity, and high-intensity (Figure 5B). These three levels were







-45-


Figure 5. Histochemistry of both normal (A-C) and PACS (D-F) DRG. Sections were
stained for trkA (red; A, D), GAP-43 (green; B, E), and GS-I-B4-binding (blue; C, F).
Arrows indicate GAP-43-positive neurons that also displayed trkA-IR, while arrowheads
indicate GAP-43-positive neurons that lacked trkA-IR, but expressed GS-I-B4-binding.







-46-


also visible in the experimental DRG. Interestingly, there appeared to be a general

reduction in signal intensity, though certain neurons had even higher intensity GAP-43-

IR than was present in the controls.

As expected from previous reports230, many GAP-43-IR neurons in the control

DRGs also expressed trkA-IR (55.39.8%). Qualitatively, it was clear that those neurons

with high levels of GAP-43-IR were most often trkA-IR (Figure 5). Somewhat

surprisingly in light of previous reports15' 230, there was a large proportion of the GAP-43-

IR population that lacked trkA-IR (44.79.8%). These neurons generally displayed low

levels of GAP-43-IR, but were clearly positive. Most of these trkA-negative/GAP-43-IR

neurons also expressed GS-I-B4-binding (94.22.9%), indicating that they were part of

the population of interest.

The distribution of GAP-43-IR in DRG neurons was not changed in the DRGs

involved in PACS (Table 2). Contrary to the hypothesis, the percentage of neurons

expressing GAP-43-IR but lacking trkA-IR was not found to be significantly different

between control and experimental DRGs by two-tailed t-test. Qualitatively, though the

general GAP-43-IR signal intensity was reduced, GAP-43-IR could still be observed in

trkA-negative DRG neurons (Figure 5).

Table 2. Percentages of neurons in control and PACS ganglia.
trkA- of GAP-43' GS-I-B4' of GAP-43'/trkA-

Control 46.0+7.5 94.22.9

PACS 41.5+5.1 77.3+7.5**
Values represent the mean of the group SD. ** indicates significance at p<0.01


The proportions of the GAP-43-positive/trkA-negative group that expressed GS-

I-B4-binding was also examined. Most of the GAP-43-positive/trkA-negative neurons







-47-


did bind GS-I-B4 (Figure 5). There was, however, a significant difference between

groups for GAP-43 /trkA-/GS-I-B4+ neurons by two-tailed t-test (p=0.01)(Table 2).

In order to focus on the trkA-negative small diameter population, the distribution

of GAP-43-IR was also assessed in relation to SOM-IR or P2X3-IR. Quantitatively,

nearly all SOM-IR neurons in control DRG (n=7) expressed GAP-43-IR (Table 3).

Table 3. Percentages of neurons expressing GAP-43-IR.
SOM P2X3

Control 95.3+7.9 94.47.3

PACS 90.54.7 88.36.2

Qualitatively, the GAP-43-IR signal intensity was mixed. Rarely did SOM-IR

neurons display the highest levels of GAP-43-IR, but they did often display moderate to

strong GAP-43-IR, and rarely displayed the lowest levels of GAP-43-IR (Figure 6).






















Figure 6. Examples of multi-labelling histochemistry of both normal (A-C) and PACS
(D-F) DRG sections. Sections were stained for SOM (red; A, D), GAP-43 (green; B, E),
and GS-I-B4-binding (blue; C, F). Arrows indicate SOM-IR neurons that also displayed
GAP-43-IR. All SOM-IR neurons were GS-I-B4-reactive.







-48-

The P2X3-IR population also consistently displayed GAP-43-IR. In control DRG

(n=5), 94% of P2X3-IR neurons displayed GAP-43-IR. Qualitatively, the majority of

these neurons displayed moderate to weak GAP-43-IR (Figure 7).

These distributions were not different in the PACS group. The proportion of

SOM-IR neurons displaying GAP-43-IR in the PACS DRG was not significantly

different from controls. The same was true for the P2X3-IR population. The proportion

of P2X3-IR neurons displaying GAP-43-IR in the PACS DRG was not significantly

different from controls. Qualitatively, GAP-43-IR was appeared to have a very similar

distribution pattern to control DRG (Figures 6, 7).


Figure 7. Examples of multi-labelling histochemistry of both normal (A, B) and PACS
(C, D) DRG sections. Sections were stained for P2X3 (red; A, C) and GAP-43 (green;
B, D). Arrows indicate P2X3-IR neurons that also displayed GAP-43-IR.







-49-


Detection of GAP-43 Protein

It was clear during the development of the GAP-43 immunohistochemical

protocols that the method of detection employed could significantly affect the

quantitative outcome. This was confirmed with the unexpected discovery of large

proportions ofnon-trkA DRG neurons that displayed GAP-43-IR. A brief assessment

was made of a variety detection methods. This provided an illustration of the likely

cause of the discrepancies between previous reports of GAP-43 mRNA and protein, and

also between the current report and previous ones.

The direct comparison of the detection methods made it clear that strong signal

amplification was a requirement for the accurate assessment of GAP-43-IR in the DRG

soma. Indirect fluorescence revealed very few trkA-negative neurons with GAP-43-IR

(Figure 8A, B), and this was not significantly improved with the use of an avidin-biotin

amplification step (Figure 8C, D). Reliable and reproducible detection of the trkA-

negative GAP-43-IR population required the use of enzymatic amplification. The current

results were obtained using the avidin-biotin-HRP complex (ABC kit Vector Labs, Inc.)

to catalyze the deposition of the tyramide signal amplification system (TSA NEN, Inc.;

Figure 8E, F).







-50-


Figure 8. Examples of multi-labelling histochemistry of normal DRG sections with
different methods of visualizing GAP-43-IR. Sections were stained for GAP-43 (green;
A, C, E) and trkA (B, D, F). Arrows indicate GAP-43-IR neurons that lacked trkA-IR.
GAP-43-IR was visualized with fluorophore-conjugated secondary antiserum (A),
biotinylated secondary with fluorophore-conjugated avidin (C), or with enzymatic
amplification via a biotinylated secondary followed by the avidin-biotin-HRP complex
(ABC kit) which deposited FITC-conjugated tyramide (E). Note the dramatic increase
in the number of clearly GAP-43-IR neurons in E. Note also that there were still many
clearly GAP-43-negative neurons.















CHAPTER 4
RESULTS CTM REFLEX AFFERENTS


192-Saporin Injections

In order to directly examine the contribution of the trkA-IR and non-trkA-IR

DRG neurons to the CTM reflex, a neurotoxin conjugate directed against the p75

neurotrophin receptor was injected into the left T13 DRG. It was previously

demonstrated that p75 mRNA was expressed in nearly all trkA-positive DRG neurons,

but was not expressed in neurons lacking mRNA for any of the trk receptors243. Brief

examinations ofp75-IR, trkA-IR, SOM-IR, and GS-I-B4-binding performed as part of

this work demonstrated that the conclusions made by Wright and Snider (1995) at the

mRNA level were likely to be true at the protein level as well. Nearly all trkA-IR

neurons also expressed p75-IR. Further, even though many non-trkA-IR neurons

displayed p75-IR, none of the SOM-IR neurons expressed trkA-IR or p75-IR, and only

those GS-I-B4-binding neurons that also had trkA-IR showed p75-IR. Thus, since the

desired end was the destruction of trkA-expressing neurons, a toxin directed at p75-

bearing neurons should have been sufficient.



Electrophysiology

Terminal electrophysiological experiments were performed 7 to 23 days

following injection of 192-sap (75ng in 1.0-1.2pl) into the T13 DRG (n=7) or T12 DRG

(n= 1; animal had only 5 lumbar vertebrae which caused a mis-identification of T13







-52-


during the injection surgery). When the CTM reflex was elicited by bipolar electrical

stimulation of DCnn from uninjected DRG, it appeared normal in threshold, latency,

duration, and magnitude. In contrast, the CTM elicited from stimulation of the DCn from

the injected DRG was absent, or extremely weak, in comparison with the reflex elicited

from other nerves in the same animal and uninjected animals in 5 of 8 cases (Figure 9).


T-11 T-12 T-13 L-1











ra)






Figure 9. Recorded responses of the Lateral Thoracic nerve (LTn) ipsilateral to the
injected DRG to stimulation of ipsilateral Dorsal Cutaneous nerves (DCnn). The DCn
stimulated is indicated above the columns of traces, and the stimulus intensity is
indicated to the left of the rows of traces. The T13 DRG was injected in this case. The
large waves present in some of the traces is motion artifact due to contraction of the
CTM lying under the recorded LTn.


Further, the threshold for generating a visible response (if possible at all) was greater for

the injected nerve than for the other nerves. Averaged recordings from filaments of the

dorsal root from the injected DRG in response to stimulation of the DCn revealed the

presence of functional A- and C-fibers in the DCn (Figure 10). The A-fiber compound

wave was dramatically reduced compared with both ipsilateral and contralateral control

nerves, as was the C-fiber wave, though to a lesser degree (Figures 10 11).







-53-


T-12


T-13


L-1


0 1 I




Figure 10. Recorded responses of dorsal roots (level indicated above columns of traces),
in response to stimulation of the appropriate DCn. Stimulus intensity is indicated to the
left of the rows of traces. The T13 DRG was injected in this case. Note that the T13
response indicates that there are still functional A- and C-fibers present. Also note that
the A-fiber responses from the uninjected segments have saturated the amplifier,
capping the trace, whereas the responses from the T13 segment have not since they are
much smaller.







-54-


CO3










4-1-










Figure 11. Recorded responses of the dorsal roots of both the injected (Lt T13) and
uninjected (Rt T13) DRG in response to stimulation of their DCn. The higher level of
noise from the Rt T13 recording is a result of the stimulated DCn being intact, as
opposed to the Lt T13 DCn, which had been transected. The delay in the Lt T13 C-fiber
wave could be accounted for by differences in conduction distance.







-55-


Histochemistry

Qualitative histological examinations of both the injected and contralateral

uninjected DRG for trkA and related markers revealed that the neurotoxin had severely

disrupted, or destroyed, nearly all trkA-IR neurons. TrkA-IR was clearly diminished in

terms of the number of IR neurons, and nearly completely abolished in terms of IR

intensity in the injected DRG. The same was true for p75-IR (detected with two separate

antisera), although the extracellular p75-IR remained nearly unchanged between the

DRG (Figure 12). Substance P-IR (SP-IR) neurons were clearly fewer in number and

weaker in intensity in the injected DRG than the contralateral DRG, although there were

many SP-IR axons that appeared normal. CGRP-IR appeared in many neurons in the

injected DRG. However, the pattern of CGRP-IR in many of the neurons was clearly

different from normal DRG. The CGRP-IR was diffuse throughout the cytoplasm, as

opposed to the usual strong and granular morphology of vesicular neuropeptides. In such

neurons, GS-I-B4-binding was either absent or extremely weak.

Qualitative histological examinations of the DRG with markers specific for non-

trkA-IR small diameter neurons revealed that while the trkA-IR population had been

severely reduced, the non-trkA-IR, GS-I-B4-binding neurons appeared unaffected. Large

numbers of GS-I-B4-binding neurons remained in the injected DRG and had a completely

normal appearance. SOM-IR was intact and appeared normal in morphology and number

in the injected DRG (Figure 13). P2X3-IR also appeared completely normal in the

injected DRG (Figure 13). A summary of the data is presented in Table 4.












Normal


Injected


I





Figure 12. Histochemical stains of both injected and contralateral uninjected DRG. For
each group, the trkA and GS-I-B4 stains are from the same sections, and the p75 stains
are from a different section. Note the dramatic loss of both p75 and trkA from neurons
in the injected DRG, while GS-I-B4-binding appears unaffected.


-56-















Normal


Injected


cr














Figure 13. Histochemical stains of both injected and contralateral uninjected DRG. For
each group, the SOM and P2X3 stains are from the same section. Note that the staining
for both markers in the injected DRG appears unaffected.


-57-















a-n,



la


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ir


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V- B 0 O P" 0 V-4 W-4
O










F" 1 P41 I I 1 F 1P


-58-


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*
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8
:?







-59-


Pseudorabies Virus Circuit Tracing

Forty-eight to 76 hours following injections of PRV into either the left CTM or

the left LTn, the C7, C8, T12, and T13 spinal cord segments and the bilateral T12, T13,

and L1 DRG were retrieved and processed to detect the presence of PRV. Many of the

injections did not result in successful infections. An injection was considered to result in

a successful infection if PRV was detected in the CTM motoneuron pool. Of the 17

injections designed to trace the CTM circuit, 4 resulted in clear infection. PRV was

reliably detected in the DRG in experiments where the injections lead to successful

infections. The PRV was present almost exclusively in a subpopulation of small

diameter neurons (Figure 14). Multi-labelling fluorescence experiments were carried out

to determine the relationship of PRV-IR to trkA-IR and GS-I-B4-binding. PRV-IR was

consistently demonstrated to co-exist primarily with trkA-IR (Figure 14). PRV-IR was

observed in GS-I-B4-binding neurons, but the majority of these also expressed trkA-IR.

Therefore, most neurons infected with PRV up to 76 hours post-injection were those that

expressed the NGF receptor trkA.







-60-


Figure 14. Examples of DRG from PRV injected animals. Panel A demonstrates that
PRV primarily infected small diameter DRG neurons (scale bar indicates 25pLm). PRV
(green; B, D, F, G) was also localized primarily to neurons that expressed trkA (red; C,
E), and those that lacked GS-I-B4-binding (blue; F, G). Arrows indicate neurons that are
infected with PRV and co-expressed the other marker. Arrowheads (B-E) indicate
trkA-positive neurons that were not infected with PRV.


Control Experiments

Control experiments to examine whether or not there was any differential tropism

of dorsal root processes for the Becker PRV were performed by injecting PRV directly







-61-


into the dorsal horn of the T12/T13 spinal cord. PRV-IR was detectable in the vast

majority of T12 and T13 DRG neurons 24 hours after injection, indicating that nearly all

neurons were capable of taking up and transporting the virus from their central terminals

(Figure 15).




., ."" .








ALI. WS W




Figure 15. Example of a T12 DRG section stained for PRV 24 hours after injection of
PRV into the T12-T13 dorsal horn. Nearly every neuron showed evidence of infection,
regardless of diameter. Arrows indicate neurons that appear to lack any sign of
infection.


Control experiments were also done to assess whether PRV injected into the

CTM muscle might be gaining access to the CNS from some route other than the CTM

motor axons. Injections of PRV were made into the CTM in the same fashion as circuit

tracing animals. In addition, certain nerve transactions were also made. In order to

investigate the possibility that the PRV was infecting afferents innervating the overlying

skin, a PRV injection was coupled with transaction of all ipsilateral LTn branches near

the origin of the CTM (n=1). This resulted in no sign of infection. In order to investigate

if the PRV was gaining access via some route other than axons innervating the injected







-62-


tissue, a PRV injection was coupled with transaction of the ipsilateral LTn (as above),

and also with transaction of nearby DCnn (n=l). This resulted in no sign of infection. In

order to investigate whether the central infection was due to infection of the CTM motor

axons, a PRV injection was coupled with transaction of the ipsilateral neighboring DCnn

(n=l). This animal showed signs of infection in both the CTM motor neuron pool and

the low thoracic DRG. While the controls that showed no infection certainly provide

evidence in support of the route of infection being limited to the CTM motor axons,

negative results must be interpreted cautiously due to the low rate of infection intrinsic in

this method. The latter control, however, provided positive evidence that the CTM motor

axons did provide a route of infection that could give rise to subsequent infection of the

low thoracic DRG.















CHAPTER 5
DISCUSSION


Unmyelinated Afferents Lacking trkA Participate in PACS

Skin Histochemistry

Histochemical analysis of the collaterally reinnervated skin regions revealed the

presence of axons that appeared to have been derived from the small diameter DRG

neurons lacking trkA and expressing GS-I-B4-binding. While these axons were regularly

and consistently observed, they were not present as frequently as those expressing NGF-

related markers (sensory or sympathetic). This could indicate that while the population

in question was likely capable of PACS, it may not have exhibited quite as robust a

response as the trkA-expressing afferents and sympathetic neurons. This finding

supported one of the guiding hypotheses of this work, namely, that the non-trkA small

diameter DRG afferents were involved in PACS, but have been missed thus far.

In order to be certain that the hypothesis has truly been supported, the

identification of the axons in collaterally reinnervated skin must be highly reliable. The

areas of greatest concern were 1) the possible mis-identification oftrkA-positive axons as

trkA-negative axons; 2) the possible mis-identification of sympathetic axons as sensory

axons; and 3) the possible mis-identification of axon remnants as collaterally sprouted

axons. This latter possibility was of least concern. Previous studies have shown that

axons in target tissues deteriorate rapidly following transaction61 16, 168, 169, 185, 135, 155, 85

No remnants of transected axons should have remained in the denervated skin into the







-64-

time that the collaterally sprouting axons reinnervated the tissue, let alone into the time

when the animal was euthanized and the skin examined for innervation. Nonetheless,

controls were also examined. The skin examined after a 3 day survival following T13

isolation surgery (the same as used for induction of PACS) revealed that the skin was

essentially completely devoid of axons as revealed by stains for PGP 9.5, SP, CGRP, and

GAP-43. Some GS-I-B4-binding did remain on structures that appeared to be axons, but

the intensity was very weak, and was not accompanied by PGP 9.5 as it was in normal or

collaterally reinnervated skin. These same stains were used to examine the distal

segments of transected or crushed nerve (with survival times of 7 to 14 days). None of

the stains, including the lectin, revealed any signal. Therefore, it was concluded that the

axons revealed in the collaterally reinnervated skin were in fact derived from collaterally

sprouted axons from the spared segment, and were not remnants of transected axons.

In order to address the other possible mis-identifications, a number of

combinations of stains and detection methods were used to identify axons in collaterally

reinnervated skin. Since it had been shown previously (by electrophysiological

recording, not CTM reflex) that the only sensory axons present in collaterally

reinnervated skin were small diameter high threshold axons (to the exclusion of larger

myelinated axons)84 92, 93, identifications did not have to be concerned with eliminating

the larger trkA-negative sensory afferent population. The task was to clearly identify

trkA-negative unmyelinated sensory afferents. To do this, GAP-43-IR or PGP 9.5-IR

distinguished axonal structures in the collaterally reinnervated skin, and GAP-43-IR also

identified growing axons. In combination with these, stains for trkA or CGRP were used

(see overlap of trkA and CGRP Figure 1). GS-I-B4-binding, P2X3-IR, and SOM-IR

were also used to identify the trkA-negative unmyelinated sensory afferents.







-65-

GAP-43/trkA/GS-I-B4: The combination of trkA-IR and lectin-binding revealed

trkA-positive axons (sensory or all sympathetic) as trkA /GS-I-B4'/-, or trkA-negative

unmyelinated sensory axons as trkA-/GS-I-B4+ (Figure 2). Since trkA is expressed by all

sympathetic axons, and the detection method employed very strong enzymatic signal

amplification, axons that lacked trkA-IR could definitively be considered trkA-negative

unmyelinated sensory afferents.

GAP-43/CGRP/GS-I-B4: The combination of CGRP-IR and lectin-binding

revealed trkA-positive sensory axons (CGRP is not found in sympathetic axons but has a

strong degree of overlap with trkA in sensory neurons157) as CGRP+/GS-I-B4+/-. TrkA-

negative unmyelinated axons (sensory or sympathetic) were revealed as CGRP-/GS-I-B4+

(Figure 2). Since both trkA-negative unmyelinated sensory axons and nearly all

sympathetic axons display GS-I-B4-binding, morphology of the innervation was used to

determine whether the CGRP-negative axon was sensory or sympathetic. Such axons on

vasculature were considered sympathetic, although some axons on vasculature were

trkA-negative. Such axons that were located in the subepidermal area or extended into

the epidermis were considered trkA-negative sensory axons.

GAP-43/P2X3: P2X3-IR was taken to indicate a tlkA-negative small diameter

unmyelinated sensory axon (Figure 3). The overlap of P2X3 with CGRP and/or trkA is

very small, but P2X3-expression in small diameter sensory neurons is almost completely

encompassed inside of the GS-I-B4-binding population22'232, 233 Further, P2X3 is not

expressed by sympathetic neurons34

GAP-43/SOM: SOM-IR, when not found on/near vasculature, was taken to

indicate trkA-negative unmyelinated sensory afferents (Figure 3). Sensory neurons

expressing SOM are entirely encompassed inside of the trkA-negative, GS-I-B4-binding







-66-


population23. However, since SOM-IR is found in some sympathetic neurons,

morphology was considered when assessing a sensory versus sympathetic identity for

SOM-IR axons.

Numerous combinations of stains conclusively revealed the presence of trkA-

negative unmyelinated sensory axons in collaterally reinnervated skin. The mis-

identification of sympathetic axons as sensory axons, as well as the mis-identification of

trkA-positive axons as trkA-negative was eliminated by staining the skin with antibodies

against trkA and detecting trkA-IR with strong signal amplification. Further, stains

specific for the trkA-negative population were used, and also revealed many axons in

collaterally reinnervated skin. The demonstration of trkA-negative axons in reinnervated

skin also indicated that at least some portion of the unmyelinated sensory axons that

previously lacked trkA-IR remained trkA-negative.



DRG Histochemistry

Histochemical analysis of DRG housing neurons undergoing PACS (PACS DRG)

was undertaken to determine whether or not trkA-negative unmyelinated afferents

expressed GAP-43, an indicator of ongoing plasticity. It had been previously shown that

GAP-43 was preferentially expressed in small diameter trkA-positive neurons of the

DRG 15, 230. It was hypothesized, based on the distribution of GAP-43 in normal DRG

and the novel findings of trkA-negative axons in collaterally reinnervated skin, that the

trkA-negative unmyelinated neurons of the PACS DRG would begin to express

GAP-43 in response to the surgical isolation of the dermatome. It was expected that

there would be an increase in the proportion of GAP-43 /trkA- neurons in PACS DRG.







-67-

Contrary to the hypothesis, it was found that there was no significant difference

in the proportion of GAP-43 /trkA- neurons between the normal and PACS DRG. There

was, however, a unique finding. It was clear that the reason that there was no difference

between the control and PACS DRG was not because there were very few trkA-negative

neurons that expressed GAP-43 in PACS DRG. Instead, the normal DRG housed a large

group of trkA-negative unmyelinated neurons that expressed GAP-43, which was

unexpected based on the literature15'230. The difference between the present results and

what was expected based on the literature is likely due to a much improved means for

visualizing the GAP-43-IR (Figure 8). The current results were obtained using a degree

of signal amplification much greater than what has been previously reported for a

fluorescence immunohistochemical study of GAP-43 15, 200. The novel GAP-43-IR

distribution is demonstrated in the context of other markers in Figure 16.


GS-I-B4
CGRP
SP
SOM
NF-M
P2x3
trkA
? p75
-- ret
SGAP43
Figure 16. Schematic representation of the general overlaps of certain markers in small
and medium diameter DRG neurons. The gray portions of the GAP-43 distribution
represent the new findings reported in this dissertation. The question mark indicates a
portion of the GAP-43 distribution that lacks trkA-IR and GS-I-B4-binding. It is
possible that it may overlap with the larger RET-positive neurons, but this is not yet
known.







-68-


The vast majority of the GAP-43 /trkA- neurons also displayed GS-I-B4-binding.

There was also no significant difference between normal and PACS DRG in the

proportion of neurons displaying SOM-IR and GAP-43-IR. A significant difference was

found, however, between normal and PACS DRG for the proportion of GAP-43+/trkA-

neurons that displayed GS-I-B4-binding. In normal DRG, the vast majority displayed

GS-I-B4-binding. In PACS DRG however, this proportion was significantly lower (94%

vs. 77%). There are multiple possible interpretations for these results.

Since there was not a significant decrease in the proportion of GAP-43 /trkA-

neurons between normal and PACS DRG, it is possible that there was a loss of GS-I-B4-

binding neurons, or more likely, a loss of GS-I-B4-reactivity by some neurons. It has

been proposed that lectin-binding elements (in the case of GS-I-B4 it is a-D-galactose)

play a role in axonal outgrowth, pathfinding, and fasciculation4 55-57, 89, 95, 184. As such, it

seems unlikely that GS-I-B4-binding should be lost during PACS. On the other hand,

GS-I-B4-binding is lost after transaction of peripheral axons156 237. It is possible that the

difference between normal and PACS DRG is due to the transaction of axons from the

PACS DRG that were traveling in one of the neighboring DCnn. While the number of

axons that project via a DCn other than their own is very low247, they do exist. If this

were the case, then transected axons would lose GS-I-B4-binding, but would increase

their levels of irkA-IR. The increase in trkA-IR intensity could change neurons that

would been deemed negative during cell counts of normal DRG to being considered

positive. These two changes together could create a significant difference in the

proportion of GAP-43 /trkA- neurons that displayed GS-I-B4-binding between control

and PACS DRG.







-69-

It is also possible that there was an increase in the number of neurons expressing

trkA in the PACS DRG. If this occurred primarily in GS-I-B4-binding neurons, then the

proportion of GAP-43 /trkA- neurons that displayed GS-I-B4-binding could have

changed significantly. It has been shown that NGF levels can regulate the expression

levels of its receptors49 220, 226. It has also been shown that trkA and p75 levels were

increased in PACS DRG 149,151, though it was not clear if any neurons began to express

trkA and/or p75 in PACS DRG that did not express them previously. Preliminary

evidence was produced as part of this work that suggested that there was little, if any,

increase in the number of SOM-IR (thus also GS-I-B4-binding) neurons that expressed

trkA. That did not, however, address the remainder of the normally trkA-/GS-I-B4+

population. Both this possibility and the previous could be addressed by quantitatively

measuring the proportion of trkA-expressing and GS-I-B4-binding neurons in normal and

PACS DRG in relation to the entire DRG population. It must also be considered,

however, that although there was not a significant difference between normal and PACS

DRG in the proportion of GAP-43-IR neurons that displayed trkA-IR, the raw number of

neurons being considered in the GAP-43+/trkA- group is much smaller than the GAP-43-

IR group as a whole. Neurons lacking trkA made up only 46% and 41% of the GAP-43-

IR population in normal and PACS DRG, respectively. Therefore, while differences may

not have reached significance when considered in relation to the entire GAP-43-IR

population, they may have become significant when compared to the much smaller group

of GAP-43-IR neurons that lacked trkA.







-70-


As a summary for the possible explanations for the finding of a significant

difference:

1) Not all GS-I-B4-binding neurons expressed SOM. Therefore, there is still a

group of trkA-/GS-I without SOM-IR that could lose GAP-43-IR without there

being a change to the SOM+/GAP+ population;

2) There could have been a decrease in the number of GS-I-B4-binding neurons or

a downregulation of the GS-I-B4-binding elements in some neurons;

3) There could have been an increase in the number of neurons expressing trkA,

which could have occurred together with, or independently from, possibility #2;

4) There could have been an increase in the number of trkA-/GS-I-B4-negative

very small, or larger, DRG neurons, that expressed GAP-43. This would have to

be a small enough group that it would not be significant as part of the full GAP-

43 population, but it could become significant when viewed inside of the smaller

GAP-43 /trkA- population.



The Role of Unmyelinated Afferents Lacking trkA

in PACS Has Gone Unrecognized

PACS of the unmyelinated afferents lacking trkA has been clearly demonstrated.

However, their role in PACS has gone unrecognized until this work. Some of the

possible reasons for this have been presented in Chapter 1, and addressed as part of this

dissertation.







-71-


Previous Histochemical Examinations Could Have Been Insufficient

It was possible that the histochemical stains (silver stains) employed to visualize

axons that had reinnervated skin by PACS were not sufficient to reveal all axons that

may have actually been present. If that were the case, then studies where PACS was

prevented by administration of anti-NGF antiserum52'166 and subsequently found no

axons (by silver stain) in the denervated skin may have simply missed axons that were

actually present, but were not revealed by the silver stains.

The insufficiency of stains also extends to previous work which used

immunohistochemistry. Previous studies used stains for markers (e.g., PGP 9.5) which

were not specific for the various subpopulations of afferents (or sympathetic efferents,

for that matter)108' 109, see also 140, 216. Moreover, most studies focused on the trkA-positive,

peptidergic population, and did not take the trkA-negative population into account at all.

As a result, such studies could not address whether the trkA-negative neurons were

indeed involved in PACS.

Other studies attempted to reveal the terminal distribution pattems of axons that

had undergone PACS, or were in the midst of PACS, by inducing the extravasation of

Evan's Blue dye into the skin23' 52,139,174,191,238. This method was excellent for revealing

the distribution of axons expressing SP and/or CGRP. However, it was completely

insufficient to address the distribution of any non-peptidergic axons (therefore most trkA-

negative unmyelinated afferents), since SP and/or CGRP are required for the

extravasation of the dye94' 134, 147,195, 244

Studies examining mRNA may have also been insufficient to reveal any role of

the non-trkA afferents in PACS. Studies of the distribution of GAP43 mRNA in normal







-72-


DRG revealed that neurons with high levels of GAP-43 mRNA almost invariably co-

expressed trkA 230. Neurons lacking trkA, however, expressed levels of GAP-43 mRNA

that were very low, and very close to the background signal levels. Examination of

GAP-43 mRNA in PACS DRG did not examine which neurons expressed GAP-43

mRNA 51. The low levels of GAP-43 mRNA in non-trkA DRG neurons, and the lack of

correlation of GAP-43 mRNA in PACS DRG with trkA, have made examination of the

distribution of GAP-43 in normal and PACS DRG incomplete.

The histochemical examinations presented here have been designed to address the

insufficiencies present in previous studies. Examinations of axons in skin were

performed with markers specific for the trkA-bearing axons (trkA, CGRP) as well as the

non-trkA bearing unmyelinated afferents (GS-I-B4, P2X3, SOM). Further, the

examinations of the distribution of GAP-43 in normal and PACS DRG presented here

had two significant differences from previous relevant examinations. First, the present

examinations looked at GAP-43 protein, not mRNA. This had the advantage of

examining the actual functional unit of GAP-43, but the disadvantage that if the low

levels of GAP-43 mRNA in non-trkA afferents actually translated to very low levels of

GAP-43 protein, then the protein would be difficult to detect. This was a problem with

previous studies as well15. Second, this very disadvantage was addressed. The current

results were produced with a very strong and clean signal amplification system. This

allowed the clear identification of even very low levels of GAP-43 protein. Thus, the

current examinations have overcome the insufficiencies of the previous studies in order

to directly examine the role of the non-trkA unmyelinated afferents in PACS.







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NGF Insensitive Afferents Could Become Sensitive

Previous studies determined that NGF was paramount for PACS 49, 52, 70, 71,173

These studies detailed excellent data to support this conclusion, but could have suffered

from insufficiencies that could have caused the overstating of conclusions. Some of

these insufficiencies were addressed above. Of particular importance was the data

demonstrating that anti-NGF treatments could halt PACS. This conclusion was reached

after histochemical examinations of skin and behavioral examinations of the CTM reflex

both indicated that there were no axons that had undergone PACS during anti-NGF

treatments49, 52, 70, 71,173. Possible insufficiencies in the histochemical examinations were

discussed above, and possible insufficiencies in the CTM reflex examinations are

presented below. If these examinations truly suffered from such insufficiencies, then the

conclusion reached in those studies that NGF is paramount for PACS could be an

overstatement. It is possible that NGF is paramount only for PA CS of trkA-positive

afferents (and CSofsympathetic Eff-rcat i, and that the PACS of trkA-negative afferents

was missed in those studies for any number of the reasons discussed above.

However, it is also possible that NGF is in fact paramount for all PACS. If this is

the case, then the possible insufficiencies of the previous studies may have been

irrelevant. This possibility could be brought about in a number of ways. First, it is

possible that the afferents that lack trkA and/or p75 in normal DRG began to express

these receptors in response to isolation of their RF. It has been shown that the population

ofunmyelinated afferents that lack trkA in the adult did express trkA during

development, but then ceased expressing the receptor58' 159',202. If this expression

occurred, then their participation in PACS may have been be entirely dependent on their







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newly acquired responsiveness to NGF via the trkA and/or p75 receptors. If this were

the case, then anti-NGF treatments would have prevent PACS of this population as well.

Second, it is possible that the neurons that lacked NGF receptors in normal DRG still did

not express them in PACS DRG, but their PACS mechanism was still somehow

dependent on NGF in another way. This possibility is addressed in detail below.

The current experiments did not test these possibilities directly, though some data

was generated that offers some indication that the possible de novo expression of NGF

receptors was unlikely to have occurred. First, if the neurons that lacked trkA expression

in normal DRG began to express trkA in PACS DRG and were involved in PACS, then it

would be expected that there would be a significant decrease in the proportion of GAP-

43 /trkA- neurons in PACS DRG. This was not the case (Table 2). It is still possible,

however, that a group of neurons did express trkA de novo, but was too small to become

significant. It is also possible that the neurons expressed p75 de novo, since this was not

examined. There is preliminary evidence that p75 knockout mice have a reduced

capacity for PACS. However, they were still capable of PACS with administration of

exogenous NGF, and all examinations were again focused on only the trkA-bearing

populations. Preliminary evidence was produced as part of this work that suggested that

there was little, if any, increase in the number of SOM-IR (thus also GS-I-B4-binding)

neurons that expressed trkA. That did not, however, address the remainder of the

normally trkA-/GS-I-B4+ population. These possibilities could be addressed by

quantitatively measuring the proportion of trkA-expressing and p75-expressing neurons

in normal and PACS DRG in relation to the entire DRG population.







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Further evidence against the possibility of de novo synthesis of trkA in formerly

non-trkA afferents is found in the present histochemical examinations of collaterally

reinnervated skin. Clear evidence of the presence of numerous GAP-43+/trkA- axons

was found in reinnervated skin regions (Figure 2). This provides evidence that at least a

portion of the axons involved in PACS do not express trkA.



CTM Reflex Testing Could Be Insufficient For Non-trkA Afferents

A number of previous studies used induction of the CTM reflex as a test for the

progress of PACS and to reveal its spatial extent49 50, 52, 59151,166,173. It was also shown that

anti-NGF could halt the expansion of the isolated RF capable of inducing the CTM

reflex49, 52 70, 71,173. If non-trkA afferents did participate in PACS, then why did anti-NGF

treatments prevent the expansion of the RF capable of inducing the CTM? This was

possible if 1) the anti-NGF actually prevented PACS of the non-trkA afferents (discussed

above and further below), 2) the non-trkA afferents did sprout and could have evoked the

CTM reflex, but the adequate stimuli were not applied, or 3) the non-trkA afferents were

not part of the CTM reflex circuit.

Certain possibilities regarding a role of NGF in any PACS mechanisms of the

non-trkA afferents do exist. Some of these have been discussed, and others are discussed

below. The possibility that the non-trkA afferents did sprout and could have evoked the

CTM reflex had the adequate stimuli been applied is unlikely. It has been shown that the

CTM reflex in rats can be evoked by noxious pinch and heat217. It has also been shown

that the non-trkA unmyelinated afferent population has a number of properties that

indicate that it encompasses families of neurons that are not only nociceptive, but







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specifically have the capability to transduce noxious heat and/or mechanical stimuli. The

capsaicin receptor VR-1 has been shown to be primarily localized to the non-peptidergic

(thus non-trkA), GS-I-B4-binding afferent population75. Further, the VR-1 receptor has

been shown to be regulated by GDNF154, which also regulates the non-trkA unmyelinated

population12159. Capsaicin application has been shown to induce the sensation of noxious

heat117,196,215,218, and in vitro experiments have shown that DRG neurons that respond to

capsaicin also respond to noxious levels of heat9' 33',60161, 162, 246. Type 2 neurons29'30,44 also

display strong capsaicin sensitivity and have been shown directly to lack SP-IR, CGRP-

IR (and thus are non-trkA), but to express GS-I-B4-binding (Petruska et al, unpublished

observations). It has also been shown that the ATP receptor P2X3 was primarily

localized to the non-peptidergic (thus non-trkA), GS-I-B4-binding afferent population.

This receptor confers rapid ATP-induced currents which have been associated with

mechano-nociceptors39. P2X3 has also been shown to be present on type 2 neurons,

which display rapid ATP-induced currents (Petruska et al, unpublished observations).

Type 2 neurons have also been shown to be sensitized by PGE2 29. This array of

characteristics is consistent with non-trkA GS-I-B4-binding afferents containing families

with the capability to transduce noxious heat and/or mechanical stimuli. Therefore, the

non-peptidergic, non-trkA unmyelinated afferents contain at least some neurons that have

the capacity to transduce stimuli which are also capable of inducing the CTM reflex in

rats, making it unlikely that non-trkA PACS was missed because the proper stimulus for

these afferents to evoke the CTM reflex was not applied.

As an aside, there is another possible mechanism that could account for the

applied heat stimuli in previous reports59'166,173,217 not activating non-trkA afferents that







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may have sprouted into denervated skin during anti-NGF treatments. There is good

evidence that different groups of thermo-nociceptors are responsible for transducing

different cutaneous heating rates. Specifically, it appears that noxious heat applied at a

low rate is transduced by capsaicin-sensitive C-fiber afferents, while that applied at a

high rate is transduced by other nociceptors, likely AS-fiber all lnt'.i -'46. The previous

reports do not provide sufficient detail to determine whether the heating rates fell into

one or the other category. However, the heating rates could very well have been rapid,

as could be expected with the application of heat being through the advancement of a

strong heat source toward the skin. If this were truly the case, then this stimulus may not

have activated any non-trkA afferents that did sprout into the denervated territory, and

thus the CTM reflex would not have been activated, leading to the possible mistaken

conclusion that no sprouting had occurred.

A final possibility was that the non-trkA afferents were simply not involved in the

CTM reflex circuit, and therefore would have been consistently missed in examinations

of PACS that relied on the CTM reflex. This possibility was directly examined in the

present experiments.



Primary Afferents and the CTM Reflex

Experiments were undertaken in order to investigate whether or not non-trkA

small diameter DRG neurons played any role in the CTM reflex. This investigation was

of vital importance in determining whether or not the CTM reflex was a suitable test to

reveal the spatial extent of cutaneous innervation by C-fibers lacking NGF receptors.

Induction of the CTM reflex by application of noxious stimuli to the skin has been the

standard behavioral test for measuring the success and/or extent of PACS in rats49' 52,59,93,







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166,173. Previous work had indicated that PACS, as measured by the CTM reflex, was

entirely dependent on NGF 49, 52, 59,150,151,173. This being the case, the NGF-insensitive C-

fibers have essentially been regarded either as incapable of PACS, or disregarded

altogether in the context of PACS. The previous work left open the possibility that non-

trkA C-fibers may in fact be capable of PACS, but only if these neurons were either not

involved in the CTM reflex, or if they transduced some stimulus that was never applied

as part of previous investigations. Therefore, experiments were undertaken to determine

whether or not non-trkA C-fibers were involved in the CTM reflex.



Selective Destruction of Neurotrophin Receptor-Expressing Afferents

One approach taken to address the question of whether or not non-trkA C-fibers

were involved in the CTM reflex was to acutely destroy the trkA-IR population and test

the CTM reflex. This was accomplished through the use of a ribosome-inactivating toxin

(saporin) that had been conjugated to a monoclonal antibody raised against an

extracellular epitope of the rat low-affinity neurotrophin receptor (p75). No such toxin-

conjugate directed against the trkA receptor itself was available at the time. However,

detailed studies from other lnab-' as well as our own observations, had indicated that the

p75 receptor was restricted in its DRG distribution to neurons expressing at least one of

the high affinity neurotrophin receptors (trkA, trkB, trkC). Further, nearly all trkA-IR

neurons co-expressed p75, and none of the non-trkA, GS-I-B4-binding neurons expressed

p75. As a result, while many more neurons than just the desired target neurons (trkA-IR)

would be destroyed, the neurons in question (non-trkA, GS-I-B4-binding) should have

been immune to the toxin.







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The results of these experiments demonstrated that the toxin did, in fact, severely

affect the target neurons within the survival period. This was indicated histochemically

by the clear loss ofp75-IR (examined with two different antisera) and trkA-IR, as well as

the morphological changes found in CGRP-IR and SP-IR from the injected DRG as

compared to intact control and contralateral control DRGs (Figure 12).

The effects of the toxin also appeared to have been selective based on

histochemical evidence. GS-I-B4-binding was morphologically no different from

controls. There were somewhat fewer neurons displaying GS-I-B4-binding, but this was

expected since many trkA-IR/CGRP-IR neurons also have GS-I-B4-binding157-159 235

More specifically, cell markers specific to the non-trkA, GS-I-B4-binding neurons (P2X3

and SOM) showed no differences between controls and injected DRGs (Figure 13). This

suggested that the synthesis and trafficking of neurotransmitters (SOM), membrane

bound receptors/channels (P2X3), and Golgi apparatus and membrane markers (GS-I-B4-

binding) were intact. This in turn indicated that this subpopulation of neurons was likely

functioning as normal.

Physiological examination of the primary afferent population in the DCn of the

injected DRG indicated that a large complement of C-fibers remained (Figures 10, 11).

This established that any differences between control and injected animals was not due to

a general loss of C-fibers as a result of the injection. These results indicated that the 192-

sap had altered the function of the target population, but had spared the non-trkA, GS-I-

B4-binding population. Further, this population had axons that were still functional.

The ability of electrical stimulation of the DCn from the injected DRG to induce

the CTM reflex was also examined. This was a direct test of the involvement of the non-







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trkA, GS-I-B4-binding DRG neurons in the CTM reflex. Stimulation of the adjacent and

contralateral DCnn revealed similar stimulus-intensity thresholds to CTM induction, as

well as generating a normal-appearing CTM reflex. Electrical stimulation of the DCn

from the injected DRG, however, revealed a higher threshold than the other nerves (if the

reflex could be induced at all), as well as a severely weakened CTM reflex, even at

stimulus intensities greater than those eliciting maximal responses when delivered to

other nerves. This was true only in animals where the histological examination of the

injected DRG showed that the p 75-bearing population had been affected. Those that had

a normal CTM response elicited from the injected dermatome were also histochemically

revealed to have had normal appearing NGF-related markers. As an example,

histochemical examination of the injected DRG from Case 2 (Table 4) revealed that a

very small group of neurons with normal trkA-IR neurons was present in the injected

DRG, as well as a group of axons with normal CGRP-IR and SP-IR morphology. This

may indicate that the CTM-induction capacities that remained, weakened though they

were, could have been due to those neurons and/or axons. Even if the remaining CTM-

induction ability of the DCn from the injected DRG was due to the non-trkA, GS-I-B4-

binding neurons, it is clear that these neurons must play a very small role in the CTM

reflex. It is most likely, however, that the weakened remaining capacities were due to

residual functionality of the axons of moribund neurons, since the CTM reflex induction

was completely absent, not simply weakened, in injected DRG from other animals.

Eight animals were involved in the toxin experiments. All underwent the same

surgical procedures, and all received similar amounts of toxins in similar volumes of

fluid. In spite of this, 3 of 8 cases showed no signs of destruction of the p75-bearing







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neurons. They also showed normal CTM reflex induction from the injected dermatome.

These animals therefore served as excellent controls for any possible effects of surgery or

injection on the CTM reflex. It was likely that the Case 1 failed because of a short

survival time (7 days). The other 2 cases, however, had 17 and 21 day survival periods,

and received different doses of toxin. There appeared to be no correlation between

failure of the toxin and dosage (within the range used here).



Transynaptic Neuronal Tracing Reveals Afferents Involved in the CTM Reflex

A neuroanatomical tracing approach was also taken to determine if the non-trkA

C-fibers were synaptically connected to the CTM reflex circuitry. This technique was

designed to retrogradely trace the elements of the CTM reflex circuit While there may

have been numerous other elements labelled, the only ones examined in this investigation

were the CTM motoneurons and the low thoracic primary afferents. The CTM

motoneuron pool was examined in all cases to ensure that there was infection of the

motoneurons. In all cases where infection was successful, the extent of PRV infection of

primary afferents from the level of injection was limited to a subgroup of small diameter

afferents. Multiple-labelling histochemistry revealed that the vast majority of these

neurons expressed trkA. This result indicated that the majority of the afferents that

supplied input to the CTM reflex were limited to those expressing trkA. This conclusion

supported the hypothesis that non-trkA C-fibers are not involved in the CTM reflex,

making the CTM reflex an unsuitable measure to indicate the spatial extent ofnon-trkA

C-fiber cutaneous innervation.







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Control experiments were performed to examine the possibility that some route

other than the CTM motoneurons had lead to the infection of the primary afferents.

There were a number of possible routes of entry of the PRV tracer into the nervous

system from the site of injection (CTM or LTn). These were the motoneurons,

sympathetic neurons, and sensory neurons in the muscle and/or overlying skin. It was

highly improbable that the specific infections that were observed were due to viral spread

through the vasculature to some remote site28. Further, direct infection of the DCn

cutaneous afferents from the overlying skin did not occur, which suggests that infection

of the LTn afferents was also unlikely. Infection of the sympathetic axons in the injected

CTM or overlying skin was another possible route of entry. This route of entry proved

extremely difficult to control. Since there was no elimination of this route of infection in

this study, the results of the PRV tracing studies must be interpreted with caution. As

stated, the majority of DRG neurons that became infected by the PRV were trkA-

positive, many were non-trkA, and often expressed GS-I-B4-binding. One possible

interpretation of this is that non-trkA afferents were synaptically connected to the CTM

reflex circuit (this is discussed below). Another interpretation is that the populations of

afferents infected with PRV represent both CTM reflex afferents and those that influence

local sympathetic outflow.

Another set of control experiments were performed to assess whether or not all

types, particularly the non-trkA C-fibers, were capable of being infected from their

central terminals. It was unlikely that all neurons would be infected, since the

terminations of neurons in a single DRG spread over a larger rostro-caudal area than was

actually injected, and the injections also likely did not reach all lamina where the







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afferents terminate. Since the PRV was rapidly taken up and transported, using the

presence of the virus as an indicator of the injection sites was not possible. The results

showed that the majority of DRG neurons were infected after PRV injections into the

dorsal horn. More importantly, PRV was found in many non-trkA small diameter

neurons, and in far more GS-I-B4-binding neurons than when PRV was injected into the

CTM or LTn. These results indicated that the both trkA-IR and non-trkA primary

afferents were capable of being infected by exposure of their central terminals to PRV.

This indicated that the reduced infection in non-trkA DRG neurons compared to trkA-

positive neurons was not due to an inability of their central terminals to become infected

by the virus.

The initial indication of the lack of non-trkA involvement as assessed by PRV-

tracing must be accompanied by the possibility that the non-trkA, GS-I-B4-binding

primary afferents could have been involved in the CTM reflex, but were synaptically

connected via a more complex path than the trkA-IR neurons. If this were the case, then

the 72 hour PRV transport time may not have been sufficient for the virus to have

reached them.



TrkA-Negative Unmyelinated Afferents and the CTM Reflex

The results of the two sets of experiments described above, when taken together,

strongly support the hypothesis that the non-trkA, GS-I-B4-binding DRG neurons do not

significantly participate in the induction of the CTM reflex. Further, any ability that they

may have to induce the CTM reflex is likely to be transmitted via a pathway that is

distinct from that of the trkA-IR DRG neurons. Therefore, the CTM reflex is an

insufficient test/monitor for PACS of the non-trkA afferent population.







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It is certainly interesting to find that two different groups ofunmyelinated

afferents, each of which contains mechano-nociceptors and thermo-nociceptors, are

differentially connected to a reflex circuit that is activated by noxious heat or mechanical

stimui. There are a number of possible reasons for this, but it must still be considered

that the non-trkA unmyelinated afferent may be capable of inducing the CTM reflex.

The present data and discussion make it clear that the non-trkA afferents are

unlikely to drive the CTM reflex under the conditions studied, but this may not hold true

for other conditions. It was clear from behavioral assessments that a strong CTM reflex

could be induced in normal animals with very brief natural stimuli that was spatially

constrained (and thus would not activate a large number of afferent fibers). Further,

electrophysiological experiments showed that a full CTM reflex could be evoked with

just a single electrical pulse. In light of the revelation that destruction of the trkA-

bearing neurons eliminated these capacities, one must believe that these capacities were

conferred by the trkA-bearing neurons.

It is unlikely that the elimination of the ability of a nerve to induce the CTM

reflex was due to the destruction of a large population of C-fibers in a simple population

type of effect. As mentioned above, a strong CTM could easily be evoked with a

spatially constrained natural stimulus. Further, the dorsal root recordings from the toxin-

injected animals revealed that a large group of C-fibers still existed and functioned in the

injected segment, though these axons clearly were not capable of evoking a CTM reflex.

Instead a general population type effect, the elimination of the ability to evoke a CTM

reflex is most likely a specific type of effect. It indicates that the non-trkA afferents do

not have the same capacity to rapidly and reliably induce a CTM reflex as do the trkA

afferents.







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It must be considered, however, that the non-trkA afferents might be capable of

driving the CTM reflex under other circumstances or conditions. The PRV tracing

studies did reveal that some non-trkA afferents became infected. This could have been a

result of the possible sympathetic route of entry, or the result of some circuit path that

connected the CTM motoneurons and non-trkA afferents. Further, a wide variety of

electrical stimuli were not tested in the toxin experiments. Perhaps trains or bursts of

stimuli would have driven the reflex. Perhaps the non-trkA afferents gain access to the

CTM reflex when the target tissue is injured or inflamed. It is also possible that the non-

trkA afferents might be capable of driving the CTM reflex as a result of PACS, but this

was not investigated as part of the present experiments. If this were the case, the effects

of anti-NGF treatments on expansion of the isolated CTM RF would have to be

considered.

Regardless of whether the non-trkA afferents can drive the CTM reflex under

some condition, it is clear that the trkA and non-trkA afferents are differentially

connected to the reflex circuitry. This differential connectivity could be related to the

development of the two populations. It has been shown that all unmyelinated afferents

initially express trkA, and that a subgroup downregulates their expression of trkA by

postnatal day 21 58, 202. This subgroup of small diameter afferents that ceases to express

trkA begins to express the components of the GDNF receptor complex and becomes

responsive to GDNF in late embryonic and early postnatal life12' 59. These two

populations also have different central termination patterns in the spinal cord.

Unmyelinated afferents that express trkA terminate primarily in lamina IIo, whereas

those that lack trkA primarily terminate in lamina IIi 157. The differences in development

and central termination could directly underlie the differences in input to the CTM reflex.







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PACS of trkA-Positive Versus trkA-Negative Afferents

Previous work has clearly demonstrated that trkA-positive afferents are capable

of robust PACS, and that this PACS is a robust and rapid growth response to an acute

denervation that is dependent on NGF 49, 52, 59,150,151,173. The current work demonstrates

that the non-trkA unmyelinated afferents are also capable of PACS. There may be many,

or few, differences between the two processes. It was noted that there were fewer trkA-

negative axons present in collaterally reinnervated skin than trkA-positive axons. This

could be due to a number of reasons. First, trkA-positive axons would represent not only

trkA positive sensory afferents that had sprouted, but also sympathetic efferents that had

sprouted70, 71, 103, 163, 164. Second, there are more trkA-positive than trkA-negative

unmyelinated afferents in DRG 157. In response to that argument, however, it should be

noted that a greater percentage of cutaneous afferents express GS-I-B4-binding and lack

trkA than express trkA or CGRP ". Finally, it is possible that fewer trkA-negative

afferents successfully sprouted into the denervated territory.

A recent study that examined PACS in great detail provided strong indications

that NGF is paramount for PACS 173. This study included examinations of the intraspinal

changes that may occur in conjunction with the peripheral innervation changes during

PACS. They used a retrograde tracer to show that the termination area of an isolated

DCn that had undergone PACS was significantly larger than in normal animals. Further,

they demonstrated that the number of second-order neurons that displayed IR for the

inducible protein c-Fos increased significantly in response to noxious pinch of the RF of

the sprouted DCn. All of these changes were shown to be due to PACS, as opposed to

the sensitization that is known to occur in response to acute administration of exogenous

NGF 127,129,130, and also to be prevented by administration of anti-NGF antibodies. While







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the data would appear to strongly suggest that NGF is the primary, if not the sole,

controlling factor in PACS, some other possibilities need to be addressed.

First, the tracer used to visualize the central terminals of the isolated DCn was

HRP-conjugated wheat germ agglutinin (WGA-HRP). While this tracer has been widely

used, it has intrinsic technical limitations. It was applied to the DCn by transecting the

nerve and suturing it into a sealed tube containing the WGA-HRP. It has been shown

that transaction of the peripheral axons of a nerve prevent the transganglionic transport of

GS-I-B4-HRP by GS-I-B4-binding afferents156. The lectin is still transported to the DRG

cell bodies, but will not go centrally as it would if the axons had not been transected. It is

possible that this same phenomenon could occur for WGA-HRP, since WGA is also a

lectin and was applied to a transected nerve. If this were the case, then trkA-negative

PACS would have been missed, and the effects of anti-NGF treatments overstated.

Further, it is currently unclear whether WGA-HRP may have a selective affinity for

particular afferent types when applied to a transected nerve74, but this could have obvious

implications for interpretation of the data. The data presented by Pertens et al. (1999) do

not demonstrate whether or not the WGA-HRP in normal cord or on the contralateral

control side of PACS animals is present in lamina IIi.







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The fact that the increase in the number of c-Fos-expressing dorsal horn neurons

could also be prevented by anti-NGF treatments brings us back to the possibility that

NGF is the sole controlling factor in PACS. The arguments in favor of this data

supporting the idea that NGF insensitive afferents do not sprout is clear, the changes are

prevented by anti-NGF. However, this suggestion must be reconciled with the current

data demonstrating that trkA-negative afferents are involved in PACS. One approach is

to address the induction of c-Fos. It was not detailed by Pertens et al. (1999), nor was it

found elsewhere, which specific lamina (in particular IIo or IIi) housed second order

neurons that could be induced to express c-Fos in normal animals. The data presented by

Pertens et al. (1999) do not demonstrate whether or not lamina Ii has any c-Fos-IR

neurons in these control groups. If stimulation of the afferents that terminate in lamina

IIi (non-trkA unmyelinated) do not induce significant c-Fos expression in lamina IIi

neurons in normal animals, then it is reasonable to expect that they may not induce c-Fos

expression after PACS, either, regardless of the presence of NGF. If this were the case,

then it would be possible that the non-trkA afferents did in fact undergo PACS, with or

without anti-NGF treatments, but would have been missed by the c-Fos studies.



A Role for NGF in PACS of Afferents Lacking trkA? A Likely Role for Other

Factors.

While there may be many reasons that non-trkA PACS was missed by previous

studies, it must be considered that the major line of evidence against a role for non-trkA

afferent PACS may have been correct in spite of technical challenges in demonstrating

the participation of these afferents. This evidence is the consistent and dramatic effects

that anti-NGF treatments had on PACS-induced changes. How can the clear effects of







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anti-NGF on numerous measures of PACS, and on PACS-related/induced changes, be

reconciled with the clear demonstration of PACS by the non-trkA unmyelinated afferents

provided by the current data? Possibly the non-trkA afferents express some unknown

receptor for NGF. This is highly unlikely since it has been shown that trkA and p75

account very well for all NGF binding in DRG neurons226. The possibility that non-trkA

unmyelinated afferents become sensitive to NGF during PACS has been discussed above.

In short, this is also unlikely, at least for the majority of non-tikA unmyelinated afferents,

based on 1) the demonstration of non-trkA axons in collaterally reinnervated skin, 2) the

lack of any difference in the GAP-43+/trkA- population between normal and PACS

DRG, and 3) preliminary evidence from direct examination of trkA-IR with markers

specific for the non-trkA population.

Contrary to possibilities based on the direct sensitivity/sensitization of the non-

trkA unmyelinated afferents to NGF, if there is a role for NGF in PACS of these neurons,

then it is far more likely to be via an intermediary. Such an intermediary would have to

itself be sensitive to NGF, and in turn produce some factor that would influence the non-

trkA unmyelinated afferent population. The most likely candidate for a controlling factor

at this point must be considered to be GDNF. The non-tikA unmyelinated afferents seem

to display requirements of, and sensitivities to, GDNF that are very similar to those of the

trkA-expressing afferents with NGF. These include survival dependency during

development and regulation of cellular constituents in adulthood12' 22'63, 72,141,157,159, 188, 219,

228,229







-90-


Directly related to PACS, both GDNF and NGF are capable of affecting the

neurite outgrowth of adult DRG neurons, but each affects mutually exclusive populations

(those with the appropriate receptor)68' 123. In addition, the current data has revealed that

the GDNF-sensitive population of DRG neurons (non-trkA, GS-I-B4-binding neurons)

expresses GAP-43 protein in the normal adult, contrary to previous understanding. This

expression is clearly present, but much weaker than that exhibited by trkA-IR neurons.

Coincidentally, the current data also demonstrated that PACS of the non-trkA

unmyelinated afferents was not as robust as that of the trkA-bearing neurons (sensory or

sympathetic), and it was recently shown that although GDNF significantly enhanced

neurite outgrowth of adult DRG neurons in vitro, its effect on total neurite length was far

less than that of NGF 68,123

GDNF therefore appears to be a suitable and logical candidate for one possible

factor that may influence PACS of the non-trkA unmyelinated afferents. The logical

cellular intermediary is likely to be (at least) Schwann cells. It is known that denervated

Schwann cells begin to express p75 137, 248. Further, it has been shown that denervated

Schwann cells begin to express significant levels of GDNF 8,76

It is likely that the GDNF signal transduction system participates in at least one

other form of peripheral plasticity, namely CS of motoneurons, and also some central CS

193. It is known that GDNF levels play a role in the innervation of muscle65' 167, 214. It has

recently been shown that GDNF is upregulated in denervated muscle132. It has also been

shown that GAP-43 is upregulated in motoneurons and their axons innervating partially

paralyzed or denervated muscle17 80, 152, 231. These pieces of information are quite similar

to those demonstrating the process ofNGF-dependent PACS and sympathetic efferent

CS.







-91-


The current data have demonstrated that non-trkA unmyelinated afferents are

indeed capable of PACS. Their capacity for PACS, however, was clearly less than that

for trkA-positive neurons. This could be related to their intrinsic low level of GAP-43

expression, or, if the role of GDNF is accepted, the differences between the NGF and

GDNF systems on neurite outgrowth mechanisms68. It is possible that non-trkA PACS

could be more robust under different conditions. Perhaps the PACS of this population

would be stronger if the trkA-positive axons were not present. Perhaps PACS of this

population would be more robust into inflamed or injured skin where the production of

TGF-P, a necessary co-factor for full GDNF signalling114' 198, would be increased97, 105, 194,

197




Summary

The current data demonstrate that the trkA-negative unmyelinated DRG afferents

are capable of PACS. The factors) controlling this process are still unclear. If NGF

plays no role, then it is most likely that PACS of this population has been overlooked in

previous studies for numerous reasons. One major reason could be that, as demonstrated

by the current data, this population of neurons does not participate in the CTM reflex, a

primary monitor for PACS. It is quite possible, however, that NGF does play a role in

PACS of this population, in spite of their lack of NGF receptors. If this is the case, then

the influence of NGF on non-trkA PACS is likely via an intermediary. As a corollary to

that, PACS of this population may not have been overlooked in previous studies.

Instead, PACS of both the trkA-positive and trkA-negative populations would not have

occurred.







-92-


It is now clear that a new population of afferents has been demonstrated to

participate in PACS. With that comes the likelihood that some factor other then NGF

could be capable of controlling PACS. As a result, any future research regarding

attempts to manipulate PACS should include considerations for this new factor, which is

likely to be GDNF. Of particular note, work attempting to "encourage PACS as a

means for recovery or improvement of function in response to peripheral or central

injuries, or attempting to suppress PACS in an attempt to alleviate any number of

pathological sensory processes in which PACS, or CS in general, may have played a role,

should be concerned with these findings.




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