CHARACTERIZATION OF CHANGES IN DOPAMINE P-HYDROXYLASE,

NEUROPEPTIDE Y, SUBSTANCE P AND GROWTH ASSOCXATED PROTEIN43

EXPRESSION IN SPINAL AUTONOMIC NUCLEI AFTER SPINAL CORD

INJURY

Aly K. Cassam Graduate Program in Neuroscience

Submitted in partial fulfillment of the requirements for the degree of

Master of Science

Faculty of Graduate Studies The University of Western Ontario

London, Ontario December 1996

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ABSTRACT

Following spinal cord injury, humans can develop abnormal, Life-threatening

autonomic reflexes. We hypothesized that these reflexes may be caused by abnormal

spinal synapses resulting fiom growth of intraspinal neurons that may contain

catecholamines, substance P or neuropeptide Y. Using immunocytochemistry, increases

in a catecholamine synthesizing enzyme, dopamine P-hydroxylase, and substance P were

detected in spinal interneurons and fibres that did not contain growth associated protein43

(a marker of neurond growth), caudal to a spinal cord transection in rats. This study is

the fist to demonstrate that spinal interneurons caudal to a transection contain

catecholamines, that long term increases occur in dopamine fbhydroxylase in somata but

not fibres and hat long term changes occur in substance P containing spinal internewom.

We concluded that abnormal sympathetic reflexes may result from both morphological and

neurotransmitter changes in intraspinal neurons after cord injury.

KEYWORDS: intemeurons, sprouting, autonomic dysreflexia, catecholamines, substance

P, neuropeptide Y

iii

DEDICATION:

The research presented in this thesis is dedicated to the late Dr. Marilyn Robinson (1945-

1996). Dr. Robinson was a caring and inspirational lecturer who sparked my interest and

subsequent pursuit of the physiological sciences when I was a student in Biology 3 10,

mammalian physiology. I consider myself very fortunate to have been taught by Dr.

Robinson. The research in this thesis is my tribute to her. This thesis is dedicated to you

Marilyn.

ACKNOWLEDGEMENTS:

First and foremost 1 wodd like to thank my s u p e ~ s o r , Dr. Lynne C. Weaver for

her incredible patience, guidance, encouragement and support over the last two years.

Without her Dr. Robinson's early inspiration would likely have gone for nought. 1 am

grateful to Dr. Andrei Krassioukov for his helpW comments and suggestions during the

course of my research. I would also like to thank Dr. Marcia LeVatte for being such a

good listener and for her coostant encouragement over the last couple of years. In

addition, I am fortunate to have received a lot of help and encouragement from Mrs. Barb

Atkioson who taught me the fine art of immunocytochemistry and from Dr. Ida

Llewellyn-Smith our Australian collaborator and co-author on many of my publications

for her insights into immunocytochemistry and research in general. Her visits to our lab

were truly unique and enjoyable experiences. I also thank Joy Mabon for her technical

help and for help in proofreading this thesis, and T a w Atasoy and Michael Columbus

for their technical expertise in imaging and tissue mounting skills respectively. The

antibody against GAP-43 that was used in this research was generously provided by Dr.

David Schreyer. I am grateful to Dr. Canio Palosa for his helpful comments and

criticisms of my first published paper. Finally figures 3,6,7,8 and 9 have been r e p ~ t e d

from NEUROSCIENCE, Cassam, LlewelLyn-Smith and Weaver, "Catecholamine enzymes

and neuropeptides are expressed in fibres and somata in the intermediate grey matter in

chronic spinal rats," 1997, with permission fkom Elsevier Science Ltd., The Boulevard,

Langford Lane, Kidlington OX5 IGB, UK.

On a more personal note, I am gratell to my parents, Sadru and Zahora, my

sister, Hurnaira and my brother-in-law, My, for their constant support and encouragement

over the last few years. A few of my close personal friends also need to be

acknowledged for making life outside the laboratory interesting, exciting and on numerous

occasions, more manageable. To Mary Weil, my long time fiend who long ago

introduced me to Sebastien's, for all the millions of things that she helped me with over

the last two years. My thanks also go to Val Wyatt, whose wedding and constant support

and encouragement are defmite highlight's of my graduate career, to Ian MacPhee for

reigniting my soccer and softball skills and for teaching me how to have a ball under any

condition and to my partner in crime or rather graduate school Jackaha van Kampen for

host of Iittie things too numerous to mention. Last, and certainly not least, I would like

to thank Monique LeBlanc for her sensible advice, encouragement and suggestions over

the past year both in matters personal and professional. I am very grateful to all my

fiends for their help, advice and encouragement, you are all incredible people.

Table of Contents

. * Cer-cate of Examination ....................................................~................... LI

Abstract ........................................................................................................ iii

Dedication ................................................................................................... iv

Acknowledgements ...................................................................................... v

Table of Contents ........................................................................................ vii

List of Tables .......... ............ ........................................................................ ix

List of Figures ............................................................................................. x

Chapter 1: Introduction ............................................................................. Ovemew of project ................................................................

............................................................. List of abbreviations .......................................... ......... Literature review ..............

......................... Sympathetic preganglionic neurons - anatomy Buibospinal control of sympathetic preganglionic neurons ....... Autonomic dysreflexia ..............................................................

........................ Possible mechanisms of autonomic dysreflexia Neurotransmitter inputs to sympathetic preganglioaic neurons . Other protein indicators of intraspinal remodelling - GAP-43 .. Objectives and Hypotheses ................... .... ............................

............................... Chapter 2: Surgical methods and tissue processing ................................................... General overview of methods

.................................... Surgical procedures ........................ ... ................*. .....*....*.............*............ Spinal cord transection ...

................. Identification of sympathetic preganglionic neurons Immunocytochernistry - single colour immunofluorescence .....

. ............ Immunocytoc hemistry dual colour im~unofluorescence Dud colour immunofluorescence . immunocytochemical controls ..................................................................................... 31

...... Analysis of data - single and dual colour immunofluorescence 32 ............. Statistical analyses - dual colour immunofluorescence 34

vii

Chapter 3: Catecholamine enzymes and aeuropeptides are expressed in fibres and somata in the intermediate grey matter in chronic spinal rats ................................................................. 35

3.1 Results ...................................................................................... 36 ................... DBH immunoreactivity in control and spinal rats 36

NPY immunoreactivity in control and spinal rats ................... 47 Substance P immunoreactivity in control and spinal rats ........ 50

3.2 D ~ S C U S S ~ O ~ ................................................................................. 53 Conclusions ............................................................................. 60

Chapter 4: Changes in intraspinal neurotrmsmitters occur independently of changes in GAP-43: a dual colour immpnofIuorescence study ............................................ .. ... 62

4.1 Results .................... ... ............................................................ 64 GAP.43, DBH and substance P immunoreactivity in control rats .......................................................................................... 64 GAP.43, DBH and substance P immunoreactivity in rats one week after cord injury .................................................... 68 GAP.43, DBH and substance P immunoreactivity in rats two weeks after cord injury .................................................. 70 GAP.43, DBH and substance P immunoreactivity in rats six weeks after cord injury ................................................... 78

4.2 Discussion ................................................................................. 84 Conclusions - dual colour immunofluorescence study ............ 94

4.3 Overall Conclusions - single and dual colour immunofluorescence studies ..................................................... 95

References ................................................................................................ 96

.....-*............ ............................--....-..............-.--..**....... Curriculum Vitae ,., 107

viii

List of Tables

Table Description Page

1. Area of immunoreactivity for DBH, NPY and substance P in the intermediolateral cell column of control and spinal rats ............... ........ 40

2. Area of immunoreactivity for GAP-43, DBH and substance P in the intermediolateral cell column of control and spinal rats ... . .. ... . . . 65

Figure

List of Figures

Description Page

Anatomical organization of sympathetic pre- and post- ganglionic ............................................................................................. neurons

................................................ Catecholamine biosynthetic pathway

DBH immunoreactivity in the intermediolateral cell column of ....................................................................................... control rats

Mean numbers of DBH immunoreactive somata in control and ......................................................................................... spinal rats

Stylized, representative distribution of D B R immunoreactive somata in the intermediate grey matter of rats seven days after spinal cord

......................................................................................... transection

DBH immunoreactive fibres in the intermediolateral cell column of rats fourteen days after spinal cord tramection ................ .............

DBH immunoreactive somata in segments TS-8, fourteen days after spinal cord transection ......................................................................

NPY immunoreactive fibres in the intemediolateral cell column of segments TS-8 of a control rat and a spinal rat, fourteen days after spinal cord transection ......................................................................

Substance P immunoreactive fibres in the intermediolateral cell column of segments TS-8 of a control rat and a spinal rat, fourteen

........................ days afker spinal cord transection ................... ..

GAP-43, DBH or substance P immunoreactive fibres in the ....... intermediolateral cell column of segments T1-4 of control rats

GAP-43 immunoreactive somata and fibres in the intermediate, that lack DBH immunoreactivity in segments T5-8 of a rat fourteen days after spinal cord transection ..................................................

DBH immunoreactive somata in segments T5-8 of a rat fourteen days after spinal cord transection that lack GAP43

............................................................................ imrnunoreactivity

13. GAP43 and substance P immunoreactivity are not colocalized in somata or fibres fiom a rat fourteen days after spinal cord transection ........................................................................................ 77

14. GAP-43, DBH or substance P immunoreactive fibres in the intermediolateral cell column of segments TI-4 of rats forty-two

..................................................... days after spinal cord transection 81

15. GAP-43, DBH or substance P immunoreactive fibres and somata in the intermediate grey matter of segments T5-8 of rats forty-two

..................................................... days after spinal cord transection 83

CHAPTER 1 - INTRODUCTION

Overview of project

The overall goal of the project was to search for evidence of neuronal growth

reactions or changes in neurotransmitters in spinal cord neurons involved in the control

of arterial pressure after spinal cord injury. Normally, blood pressure is controlled by

neurons that project fiom the medulla to sympathetic preganglionic neurons in the spinal

cord (Calaresu and YardIey, 1988). These bulbospinal projections are damaged after cord

injury and supraspinal control of sympathetic preganglionic neurons is lost resulting in a

potentially Life threatening condition known as autonomic dysreflexia Autonomic

dysreflexia occurs in ninety percent of quadriplegic and high paraplegic patients (Mathias

and Frankel, 1992). This condition is characterized by extreme increases in arterial

pressure in response to mild visceral or somatic stimulation. For example, urinary bladder

distension can cause severe hypertension or even stroke in people suffering fkom spinal

cord injury. The first objective of this project was to identify the neurotransmitters used

by spinal interneurons to control sympathetic preganglionic neurons after spinal cord

injury. The second objective was to characterize the changes in intraspinal

neurotransmitter expression in the intermediate grey matter of the spinal cord following

spinal cord transection that may contribute to the development of autonomic dysreflexia

after spinal cord transection.

The literature review begins with a brief historical perspective of the anatomy of

autonomic nervous system, which is followed by a description of the anatomical Location

of sympathetic preganglionic neurons in the spinal cord and of the neurons and organs

innervated by these neurons. Next, the role and importance of brainstem neurons in

3

controlling sympathetic preganglionic neurons and ultimately arterial blood pressure are

examined. In addition the role of bulbospinal inputs to sympathetic preganglionic neurons

in inhibiting somato-sympathetic reflexes in the intact spinal cord are also examined. The

symptoms and changes in arterial blood pressure associated with autonomic dysreflexia

in humans are described and compared to the physiologic correlates of this condition in

paraplegic rats. Next, mechanisms that may contribute to the manifestation of autonomic

dysreflexia in humans and rats including an overview of support for the hypothesis that

the development of autonomic dysreflexia relates to changes in the morphology and/or

changes in the neurotransmitter utilized by spinal interneurons that control sympathetic

preganglionic neurons after cord injury. This section includes a description of

neurotransmitter inputs to sympathetic preganglionic neurons in the intact spinal cord,

summarizing experimental evidence for the presence of catecholamines, neuropeptide Y

and substance P. Next, a rationale is presented for examining changes in the distribution

of growth associated protein43 as a marker of growth and morphological changes in

spinal interneurons after spinal cord transection. The introduction concludes with the

objectives and hypotheses for the studies presented in this thesis. A list of all

abbreviations used in this thesis is presented on the next page.

CGRP

DBH

FITC

GABA

GAP43

i.m.

IML

1.p.

LF

MCID

NPY

S.E.M

S.C.

SCT

T

LIST OF ABBREVIATIONS

central canal

calcitoain gene related peptide

dopamine P-hydroxylase

fluorescein isothiocyanate

gamma amino butyric acid

growth associated protein - 43

intramuscular injection

nucleus intermediolateralis, pars principalis

intraperitoneal injection

lateral h i c u l u s

Microcomputer Imaging Device

neuropeptide Y

standard error of mean

subcutaneous injection

spinal cord transection

thoracic

5

Literature review

The autonomic nervous system was fist discovered by the anatomist~physiologist

Galen (AD 130-200) who described the cranial nerves, sympathetic chains and cervical

ganglia that are part of the autonomic nervous system (Ackerknecht, 1974). Based on

physiological studies, Langley (1920) was the fist researcher to divide the autonomic

nervous system into two components consisting of the sympathetic and parasympathetic

nervous systems. With a few exceptions most of the organs and viscera that are regulated

by the autonomic nervous system receive both sympathetic and parasympathetic

innervation. The adrenal chromaffin cells, kidney, spleen and most blood vessels are

innervated exclusively by the sympathetic nervous system (Gilbey and Spyer, 1993). This

branch of the autonomic nervous system is an important regulator of arterial blood

pressure in the body because these organs and blood vessels are important components of

the cardiovascular system which is involved in regulating blood flow throughout the

abdomen and musculature. Therefore, damage to the sympathetic nervous system by

spinal cord injury can have profound effects on blood pressure control that could be fatal.

Sympathetic preganglionic neurons - anatomy

The efferent component of the sympathetic nervous system consists of sympathetic

preganglionic and postganglionic neurons. Sympathetic preganglionic neurons are located

in the thoracic and upper lumbar segments of the spinal cord (Cabot, 1990; Gilbey and

Spyer, 1993). The majority of sympathetic preganglionic neurons are located in the

nucleus intermediolateralis pars principalis (also known as the intermediolateral cell

column) of the thoracic segments of the spinal cord. In addition, sympathetic

6

preganglionic nemoxs are located in the nucleus intercalatus, the central autonomic

nucleus and in the nucleus intermediolateralis pars f'unicularis (also referred to as the

lateral funiculus) (Hong and Weaver, 1993; Cabof 1990; Gilbey and Spyer, 1993).

Sympathetic preganglionic neurons project fiom these nuclei to ganglia located outside

of the cord via the ventral roots of the spinal cord. Within the ganglia, axons projecting

fiom the spinal cord synapse on sympathetic postganglionic neurons. h o s t aU

sympathetic preganglionic neurons synapse on sympathetic postga~giionic neurons in the

ganglia, with one major exception, the chrom&n cells within the adrenal gland, which

are innervated by sympathetic preganglionic neurons that do not synapse within the

peripheral ganglia (Gilbey and Spyer, 1993; Cabot, 1990). Postganglionic neurons project

from the ganglia to al l target cells controlled by the sympathetic nervous system (Fig. 1).

Bulbospinal control of sympathetic pregangiiionic neurons

Sympathetic preganglionic neurons are the final site of integration for brainstem

and intraspinal mechanisms that control systemic arterial blood pressure (Calaresu and

Yardley, 1988). Sympathetic preganglionic neurons in the thoracic and lumbar spinal

cord are innervated by neurons projecting £kom different regions of the brain. Recently,

Strack et al, demonstrated that neurons in the venttomedial and rostra1 ventrolateral

medulla, the caudal raphe nuclei, the A5 cell group of pontine reticular formation and the

paraventricular nucleus of the hypothalamus project to sympathetic preganglionic neurons

in the spinal cord (Gilbey and Spyer, 1993; Strack et a[.. 1989a; Strack et al., 1989b).

Neurons in the medulla are particularly important in regulating the activity of sympathetic

preganglionic neurons involved in controlling arterial pressure. Dampney and Moon

Figure 1. Organhation of the sympathetic nervous system. Sympathetic pregmghon neurons (solid lines) project fkom the thoracic and lumbar spinal cord to their target cells. Dashed lines indicate postganglionic fibres projecting h m the ganglia to their target organs. (Adapted firom DeMyer W Neuroanatnmy. New Y& John Wiley & Sons, 1988.)

8

observed that lesions to the rostral ventrolateral medulla of the brainstem resulted in a

decrease in arterial blood pressure (Dampney and Moon, 1980). Similarly, stimulation

of the r o w ventrolateral medulla resulted in an increase in arterial blood pressure (Ross

et ai.. 1984). These medullary neurons have been shown, using a combination of

electrophysiological and anatomical techniques, to project directly to the intennediolateral

cell column (Caverson et a!., 1983). In addition sympathetic preganglionic neurons were

excited by electrical stimulation of peripheral afferent neurons when bulbospinal inputs

to preganglionic neurons were blocked demonstrating the presence of somato-sympathetic

reflexes within the intact spinal cord (Dembowsky et al., 1980). However, in the intact

spinal cord, these reflexes are inhibited by bulbospinal inputs to sympathetic preganglionic

ne1rons, that prevent fluctuations in blood pressure control in response to visceral and

somatic stimuli (Dembowsky et al., 1980). Therefore, bulbospinal inputs to sympathetic

preganglionic neurons are crucial in controlling sympathetic activity, and in controlling

systemic arterial blood pressure.

A utonomic djsreflexia

When bulbospinal projections to sympathetic pregauglionic neurons are damaged

or destroyed by injury to the spinal cord, the control of arterial blood pressure can become

very volatile. Initially people with spinal cord injuries suffer tiom postural hypotension

due to the loss of bulbospinal excitation of sympathetic preganglionic neurons. The acute

phase following cord injury is characterized by a period of spinal shock during which-

reflexes cannot be initiated caudal to the injury. Thus people in spinal shock following

injury are areflexive and do not exhibit symptoms associated with dysreflexia (Mathias

9

and Frankel, 1993; Lee et a[., 1994). Once this areflexive period has passed, people with

spinal cord injuries may d e r fiom exaggerated autonomic reflexes initiated by stimuli

caudal to the injury (Mathias and Frankel, 1993; Naftchi, 1 WOb; Lee et uL, 1994). For

example, mild visceral and somatic stimuli such as distension of the bladder or colon as

well as muscle spasms can cause episodes of hypertension, bradycardia, sweating and

headaches in quadriplegics or high paraplegics (Mathias and Frankel, 1993; Lee et

al., 1994; Finocchiaro and Hedeld, 1990). These symptoms are characteristic of a

potentially Life threatening condition known as autonomic dysreflexia or autonomic hyper-

reflexia.

Many of the symptoms associated with autonomic dysreflexia were first described

by Hilton in 1860 (Lee et 01.. 1994). However the changes in arterial pressure associated

with autonomic dysreflexia were first reported in 1947 by Guttmann and Whitteridge (Lee

et al., 1994). Approximately ninety percent of quadriplegics and high paraplegics, people

with spinal cord injuries above the sixth thoracic segment, suffer from autonomic

dysreflexia (Mathias and Frankel, 1993; Corbett et al., 1975; Mathias and Frankel, 1992;

Mathias and Frankel, 1983). The level of spinal cord injury is a major factor in

determining whether or not an individual will develop autonomic dysreflexia because the

majority of abdominal blood vessels and organs as well as blood vessels in the muscles

of the lower body are controlled by sympathetic preganglionic neurons located caudal to

the sixth thoracic segment (Torigoe et a[., 1985). As a result, individuals with spinal cord

injuries below T6 are unlikely to develop autonomic dysreflexia because bulbospinal

control of the splanchnic circulation is still intact. Although the symptoms associated with

10

this condition are well characterized in humans, the mechanisms that lead to or cause

autonomic dysreflexia are not well understood.

To attempt to understand the mechanisms underlying autonomic dysreflexia, a

number of researchers have utilized a rat model of spinal cord injury (Maiorov et

u1.. 1997; Krassioukov and Weaver, 1995; Santajuliana et al.. 1995; Osborn ef a[.. 1990).

There are a number of similarities in autonomic dysreflexia between rats and humans.

Rats with complete spinal cord transections rostra1 to T6 develop autonomic dysreflexia

(Maiorov et a1.. 1997; Krassioukov and Weaver, 1995; Santajuliana et al-, 1995; Osborn

et a[.. 1990). Weaver and Krassioukov demonstrated that stimuli such as bladder or colon

distension, that lead to autonomic dysreflexia in humans, also elicit autonomic dysreflexia

in paraplegic rats (Ktassioukov and Weaver, 1995). However, one major difference exists

between the development of autonomic dysreflexia in humans and rats. Unlike humans,

rats do not appear to lose spinal sympathetic reflexes following cord injury (Krassioukov

and Weaver, 1995; Osbom et a[-, 1990). Thus rats initially have dysreflexia as early as

twenty- four hours after cord injury (Maiorov et al.. 1 997; Osbom er al.. 1 990; Krassioukov

and Weaver, 1995), which may be attributed to the loss of bulbospinal inputs to

sympathetic preganglionic neurons after spinal cord injury (Maiorov ef a1.. 1997). Within

the first seven days after cord transection the pressor responses associated with autonomic

dysreflexia are attenuated (Krassioukov and Weaver, 1995), probably due to the transient

atrophy of sympathetic preganglionic neurons that occur within the first week after spinal

cord transection (Maiorov ef aL, 1997; Krassioukov and Weaver, 1995; Krassioukov and

Weaver, 1996). However, by fourteen days after injury sympathetic preganglionic

I 1

neurons have reestablished their dendritic arbour (Krenz and Weaver, 1997) and,

correspondingly, the changes in blood pressure due to autonomic dysreflexia are increased

compared to seven days after spinal cord transection (Maiorov ei aL, 1996). In contrast

to rats, humans may not develop dysreflexia for months or even years after injury (Lee

et al., 1 994). However, because of the similarities between cord-injured rats and humans,

paraplegic rats with complete spinal cord injuries above T6 have been used to understand

the etiology of autonomic dysreflexia.

In a recent study, Maiorov et aZ. (1997) recorded changes in sympathetic nerve

activity and changes in arterial pressure in conscious spinal rats in response to colon

distension. They found in these rats that in response to colon distension there was

increased sympathetic nerve activity and increased arterial pressure characteristic of

autonomic dysreflexia. These r e d & in conscious rats and the results fiom similar

experiments in anaesthetized rats have provided evidence that autonomic dysreflexia may

be due to the hyperactivity of spinal sympathetic reflexes over time after cord injury

(Osbom ef al.. 1990). Finally, the time course of autonomic dysreflexia in rats and the

delayed development of dysreflexia in humans suggest that the mechanisms underlying

this condition may involve changes in intraspinal inputs to sympathetic preganglionic

neurons after spinai cord injury.

Possible mechanisms of autonomic dysteflexia

The intraspinal circuits that form spinal sympathetic reflexes consist of dorsal root

afferents, of somatic or visceral origin and spinal interneurons that synapse on sympathetic

preganglionic neurons. Within the first week after a spinal cord injury, preganglionic

12

neurons caudal to the injury atrophy and retract their dendrites, thus explaining the

decrease in autonomic dysrefleia within seven days after spinal cord injury (Krassioukov

and Weaver, 1995). However, by thirty days after spinal cord injury these degenerative

processes are reversed and sympathetic preganglionic neurons have a normal morphology

with a full dendritic arbour (Krassioukov and Weaver, 1996). Subsequently, intraspinal

neurons may sprout new processes, forming inappropriate inputs on sympathetic

preganglionic neurons that were deafTerented following cord injury and that subsequently

reestablished their dendritic arbour (Krassioukov and Weaver, 1996). Another key finding

of this study was that immunoreactivity for the synaptic vesicle protein, synaptophysin,

increased in the intermediolateral cell column caudal to a complete spinal cord transection.

Increased immunoreactivity for synaptophysin in the intermediolateral cell column may

be indicative of the formation of new synaptic inputs to sympathetic preganglionic

neurons. Alternatively, increased synaptophysin immunoreactivity caudal to the injury

could also be due to the induction of neurotransmitter expression and consequent increase

in the number of neurosecretory vesicles containing synaptophysin, in intraspinal neurons

controlling sympathetic preganglionic neurons caudal to the traasection. If this latter

hypothesis were correct then intraspinal neurons were undergoing changes in gene

expression and not changes in morphology, in response to spinal cord injury.

Nevertheless, changes in synaptophysin irnmunoreactivity caudal to a complete spinal cord

transection provided evidence that intraspinal circuits controlhg sympathetic

preganglionic neurons were being remodelled in response to spinal cord injury.

Neuromsmitter inputs to sympathetic pregunglionic neurons

One approach to ascertain further changes in

injury is to examine changes in the distribution of

13

intraspiaal circuits caudal to the

neurotransmitters caudal to the

transection. The identity of neurotransmitters utilized by intraspinal neurons is difficult

to determine within the intact spinal cord because the neuropil mounding sympathetic

preganglionic neurons is known to contain a number of different neuroactive substances,

derived fiom both supra- and intraspind sources, that could be released from synapses on

sympathetic preganglionic neurons (Gilbey and Spyer, 1993). These potential

neurotransmitters include amino acids such as gamma amino butyric acid (GABA),

glutamate and glycine (Spanswick et a1.. 1994; Spanswick ei al., 1995; Gilbey and Spyer,

1993; Chalmers and Pilowsky, 199 1); monoamines such as serotonin and adrenaline

(Gilbey and Spyer, 1993; Byrum and Guyenet, 1987; Appel et ul.. 1986; Chalmers and

Pilowsky, 1991); neuropeptides such as substance P, neuropeptide Y and enkephalin

(Hong and Weaver, 1993; Llewellyn-Smith er a[., 1990). The sources of these

neurotransmitters include supraspinal neurons within the medulla and hypothalamus

(Gilbey and Spyer, 1993; Calaresu and Yardley, 1988; Chalmers and Pilowsky, 1991),

spinal interneurons and the terminal arbors of dorsal root afferent fibres (Gilbey and

Spyer, 1993; Chalmers and Pilowsky, 1991 ; Laiag et al., 1994; Hong and Weaver, 1993).

However, much of the research to date has focused on identifying the supraspinal sources

of neurotransmitters in the autonomic nuclei within the intact spinal cord (for review see

Chalmers and Pilowsky, 199 1). Following transection of the spinal cord, bulbospinal

inputs to sympathetic preganglionic neurons are lost and the neutopil surrounding

sympathetic preganglionic neurons contains neurotransmitters primarily found in

14

intraspinai neurons. As discussed below, neurotransmitters potentially utilized by

intraspinal neurons to control the activity of sympathetic preganglionic neurons caudal to

a spinal cord injury include catecholamines such as dopamine, adrenafine and

noradrenalhe, neuropeptides, such as neuropeptide Y and substance P, and amino acids

such as glycine and glutamate.

Cutecholurninergic inputs to sympathetic pregangiionic neurons

Catecholamines are found in a large number of neurons in the brainstem that are

involved in controlling sympathetic preganglionic neurons. Armstrong el al. (1982)

demonstrated that medullary neurons were immunoreactive for the catecholamine

synthesizing enzymes tyrosine hydroxylase (found in neurons capable of synthesizing

dopamine, noradrenaline or adrenaline, see Fig. 2), dopamine P-hydroxylase (found in

adrenaline and noradrenaline synthesizing neurons, Fig. 2) and phenylethanolamine-N-

methyltransferase (found only in adrenaline producing neurons, Fig. 2). When adrenergic

neurons in the A5 cell group of the brainstem were injected with the anterograde tracer

horse radish peroxidase conjugated wheat germ agglutinin, Bynun and Guyenet observed

that these neurons projected from the brainstem to the intemediolaterd cell column of

the thoracic spinal cord (Byrum and Guyenet, 1987). In a similar experiment in which

the anterograde tracer phaseolus vulgaris-leucoagglutinin was used in combination with

immunocytochemistry for dopamine P-hydroxylase (DBH), Clark and Proudfit found that

neurons in the A5 cell group were a major source of DBH immunoreactivity in the

intermediolateral cell column (Clark and Proudfit, 1993). Other supraspinal sources of

catecholaminergic inputs to sympathetic preganglionic neurons include the rostra1

Tyrosine

Tvrosine H vdrox ylase

DOPA

Dopamine

Dopamine 13-hvdrox-vlase (DBH) l' O2

Noradrenaline

Phen vlethanolamine-N- meth-vl transferase

Adrenaline Figure 2. Figure illustrating the catecholamine biosynthetic pathway. The rate limiting enymes and the substrates of the enzymes involved in the synthesis of catecholamines are shown above. The catecholamine synthesizing enzymes are underlined on the left side of the figure.

16

ventrolateral medulla and the paraventricular nucleus of the hypothalamus (Gilbey and

Spyer, 1993).

The results from a study by Magnusson (1973), that used biochemical assays,

concluded that fourteen days after s p i d cord transection catecholamines were no longer

present caudal to the injury. Similarly, Haggendal and Dahlstrom examined changes in

catecholamine histofluorescence in the spinal cord after cord injury (Haggendal and

Dahlstrom, 1973). Due to the large decrease in catecholamine histofluorescence caudal

to the transection and the apparent lack of catecholamiaergic cell bodies within the spinal

cord, the authors of both studies concluded that the bulbospinal projections were the only

source of catecholamines in the spinal cord. Although a great deal of experimental

evidence indicates the absence of intraspinal catecholaminergic systems (Haggendal and

Dahlstrom, 1973; Magnusson, 1973; Westlund et al., 198 1 ; Westlund et aL, 1983), later

studies in chronic spinal animals demonstrated activity for DOPA-decarboxylase (Fig. 2),

another enzyme in the catecholamine synthesis pathway (Codssiong, 1985) and low but

measurable levels of noradrenaline and dopamine in the spinal cord using high

performance liquid chromatography (Roudet et al., 1994). In addition, recent studies have

demonstrated the presence of tyrosine hydroxylase positive neurons within the spinal cord

(Diet1 et al., 1985; Mouchet et al., 1986). Therefore, these latter results suggest that spinal

cord circuits involved in autonomic dysreflexia may also contain catecholamiws.

Neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide found in many regions of the

central nervous system (Roddy el al., 1990). In the rostral ventrolateral medulla, NPY has

17

been described in a subpopulation of catecholaminergic neurons that project to the spinal

cord (Tseng et ui., 1993). Studies have also demonstrated that these neurotransmitters are

colocalized in synapses on sympathetic preganglionic neurons in the thoracic spinal cord

(LleweIlyn-Smith et al., 1990). NPY immunoreactive cell bodies have also recently been

identified in the dorsal horn of the spinal cord (Laing et al., 1994). However, the presence

of NPY in the intermediolateral cell column caudal to a chronic spinal cord transection

is uncertain. One study failed to detect NPY caudal to a chronic spinal cord transection

(Hokfelt et al., 1981). However, these experiments were performed using an antibody

against avian pancreatic peptide that is homologous to NPY. Although antibodies against

this peptide are cross reactive with NPY (Hokfelt et al.. 198 1 ; Roddy et ul-. 1990), it is

possible that the antibody used in this earlier study may not have been sensitive enough

to detect low levels of NPY. Therefore this Iimitation warrants some reservation in

accepting the negative results of this study.

Substance P

Another neuropeptide found in bulbspinal and intraspinal inputs to sympathetic

preganglionic neurons is substance P. This neuropeptide is colocaiized with serotonin in

projections horn the rostra1 ventromedial medulla to the intermediolateral cell column

(Chalmers and Pilowsky, 199 1 ; Gilbey and Spyer, 1993). In addition, substance P is also

present in dorsal root afTerents and interneurons within the spinal cord (Hong and Weaver,

1993; De Biasi and Rustioni, 1988; Smith et al.. 1993). Similarly, studies by Davis et al.

and Naftchi provided evidence that substance P is present in the intemediolateral cell

column caudal to a complete spinal cord transection (Davis et a[., 1984; Naftchi, 1990a)

18

and thus may be present in intcaspiaal circuits that control sympathetic preganglionic

neurons. Moreover, following dorsal root rhotomy the area of substance P

immunoreactivity initially decreases in the dorsal horn (Wang et aL, 199 la; Wang et

a1.,1991b), due to the degeneration of terminals of dorsal root afferent fibres.

Interneurons utilizing substance P as a neurotransmitter then sprout new processes and

terminals to innervate deafferented spinal neurons within the dorsal horn, illustrated by

an increase in the area of substance P immunoreactivity within this region over time after

injury (Wmg e l a!., 199 1 a; Wang er al., 199 1 b). Therefore, the results of the studies by

Wang et al. demonstrate that spinal cord circuits utilizing substance P as a

neurotransmitter could innewate deafferented of neurons within the spinal cord that result

fiom injuries to the spinal cord.

Other protein indicators of intraspinal remodelling - G A P 4 3

Changes in the area and distribution of vesicular proteins such as synaptophysin

and neurotransmitters such as noradrenaline, NPY and substance P represent one approach

to assess changes in intraspinal circuits following spinal cord injury. Changes in the

distribution of proteins that are associated with the cytoskeleton or membrane of neuronal

processes andor presynaptic terminals, could also be used to discern changes in intraspinal

circuits following spinal cord injury. Moreover changes in the area and distribution of

structural proteins caudal to the transection would provide evidence of morphologic

changes within intraspinal neurons in response to spinal cord injury. One of the most

commonly used markers of morphoiogic changes in neurons is growth associated protein -

43 (GAP-43), a 43 kilodalton phosphoprotein (Skene, 1989). This protein is an

19

extremely hydrophilic molecule that is bound to the inner plasma membrane of axons,

growth cones, presynaptic nerve terminals and to the membranes of vesicles in the

presynaptic nerve terminals (Benowitz and Penone-Bizzozero, 1 99 1). Within growth

cones, GAP-43 is the single most abundant protein and accounts for 1% of the protein

content of these structures (Skene, 1989). The exact hction of GAP-43 within these

structures is unknown. During the embryonic development of the nervous system,

increased levels of GAP43 are associated with the growth of axons in the spinal cord,

peripheral ganglia and other areas of the central and peripheral nervous systems. As the

fetal nervous system continues to develop, the level of GAP-43 expression decreases in

mature axons and presynaptic nerve terminals after synaptogenesis has occurred (Benowitz

and Perrone-Bizzozero, 1991). Although GAP43 expression is generally very low in

mature axons in the adult central and peripheral nervous systems, some regions of the

central nervous system such as neurons within the dorsal root ganglia and brainstem

(discussed below) continue to constitutively express high levels of GAP-43 mRNA or

protein (Benowitz and Perrone-Bizzozero, 1991; Yao et d, 1993; Kruger et a[.. 1993;

Verge et aL, 1990). Since increased GAP-43 expression is observed in growing axons and

during synaptogenesis during development and in v i m , some researchers have speculated

that GAP043 is involved in axonal pathfinding and membrane adhesiveness (Shea, 1995;

Benowitz and Perrone-Bizzozero, 199 1).

In adults, GAP-43 expression can be reinduced in regenerating neurons. Four to

five days after axotomy, GAP-43 is increased in regenerating toad retinal ganglion and

goldfish optic cells just prior to axonal elongation. However, peak expression of GAP-43

20

in these cells occurs two weeks after axotomy when the regeneration and elongation of

these neurons is M y underway (Skene, 1989). Similarly, GAP43 mRNA is increased

in regenerating sensory neurons in mammals. One day after axotomy of the sciatic nerve,

GAP-43 mRNA increased by 2.5 fold. Elevated levels of GAP-43 mRNA persisted for

up to fourteen days after nerve injury, returning to control levels twenty-eight days later

(Hoflinaa, 1989). Similarly, GAP-43 immunoreactivity is also upregulated in regenerating

rat retinal ganglion cells following optic nerve section (Schaden et aLJ994). These

results show that increases in both mRNA and protein for GAP43 are associated with

axonal regeneration in the central nervous system.

Another phenomenon associated with increased GAP-43 expression in adults is

sprouting. Sprouting is a morphologic response characterized by the extension of neurites

or axonal collaterals by undamaged neurons following neuron deafferentation or tissue

denervation (Aigner et aZ., 1995; Mearow et al., 1994). Mearow ef al. observed that GAP-

43 mRNA expression was increased in undamaged sensory neurons that were sprouting

new processes to reinnewate nearby regions of denervated skin (Mearow et a1.. 1994). In

a recent study, the overexpression of GAP43 in adult transgenic mice resulted in

spontaneous sprouting by nerves at the neuromuscular junction and in the expansion of

the terminal fields of hippocampal mossy fibres (Aigner et al.. 1995). In addition, the

level of sprouting was increased in these mice in response to nerve lesions (Aigner et

a[., 1995). However, these responses were not observed in control mice that did not

overexpress GAP-43. The correlation between increased GAP-43 expression and

sprouting suggests that changes in GAP-43 expression maybe a useN indicator of changes

21

in neuronal morphology, such as sprouting, that involves alterations in neuronal

morphology (ie. process elongation) following deafferentation.

GAP43 expression in the uninjured central nervous system

In the adult central nervous system, GAP43 expression is not restricted to axons

of neurons that are regenerating or sprouting in response to injury. Some regions of the

central nervous system such as the brainstem, s p a cord and dorsal root ganglia

constitutively express GAP-43. In the rat brainstem, high levels of GAP43 mRNA have

been detected in regions such as the substantia nigra pars compacta and the ventral

tegmental area Neurons within these areas are also known to contain monoamines

(Bendotti et al., 1991 ; Yao et a[., 1993; Kruger er a[.. 1993). Following the administration

of 5,7 - dihydroxytryptamine and 6 - hydroxydopamine, neurotoxins specific for

monoaminergic neurons, the hybridization signal for GAP-43 mRNA completely

disappeared in the raphe nuclei, the substantia nigra pars compacta, the ventral tegmental

area and the locus coeruleus. These results provide evidence that monoaminergic neurons

within the adult rat central nervous system constitutively express GAP43 (Bendotti et

a[-. 199 1). Furthermore, the high levels of GAP-43 &A have been observed in

monoamioergic neurons in other species such as the cat and monkey. In both species

Arvidsson et al. observed that monoaminergic neurons within the midiine raphe nucleus

and the nucleus reticularis lateralis of the medulla contained high levels of GAP43

mRNA (Arvidsson et a1..1992). The results from these studies suggest that

monoaminergic neurons in the mammalian central nervous system constitutively express

GAP-43.

In the intact spinal cord, GAP-43 immunoreactivity is present in the

intermediolated cell column (Weaver et a1.. 1997), the dorsal corticospinal tract, around

motoneurons in the ventral horn and within small diameter unmyelinated afferent fibres

within the superficial laminae of the dorsal horn (Curtis et al., 1993; Nacimiento ef

a[.. 1993b; Li et uL, 1993). The latter observations are consistent with the findings of

Verge et al. who observed that GAP43 mRNA was predominantly located in small

diameter unmyelinated sensory neurons in rat dorsal root ganglia (Verge et a/., 1990).

Within the uninjured spinal cord, GAP43 immunoreactivity has been colocalized with

serotonin and DBH (Ching et al.. 1994; Wotherspoon and Priestley, 1995), the latter being

found in fibres around motoneurons within the ventral horn. Because of the correlation

between GAP-43 and plasticity within the nervous system, Curtis et al. hypothesized that

adult neurons constitutively expressing GAP43 retain " a neuroplastic capacity and are

continually being remodelled" (Curtis et a/., 1993).

GAP43 immunoreactivity in the autonomic nuclei

The purpose of a recent study in our laboratory was to examine possible changes

in the morphology of spinal circuits in the autonomic nuclei after spinal cord injury

(Weaver et al., 1997). This objective was accomplished by examining changes in GAP-43

immunoreactivity in the autonomic nuclei in control and spinal rats. In the intact spinal

cord, GAP-43 immunoreactivity is present in fibres oriented in a ladder-like pattern of in

all segments of the thoracic spinal cord. Changes in GAP43 immunoreactivity were also

examined in rats seven to thlay days after spinal cord transection. With time after cord

injury, the ladder-like distribution GAP-43 immunoreactivity deteriorated and was

23

replaced by a reticular pattern of fibres coursing throughout the intermediate grey matter.

In addition, from seven to thirty days after cord injury, an increasing number of spinal

interneurons were immunoreactive for GAP43 caudal but not rostrd to the transection.

This observation demonstrates that spinal interneurons are growing in response to spinal

cord injury. These findings also suggest that intraspinal circuits are changiag and possibly

forming new synapses on sympathetic preganglionic neurons caudal to a complete spinal

cord transection.

Objectives and H M e s e s

The purpose of the studies in this thesis was to identify changes in spinal circuits,

that could contribute to the development of autonomic dysreflexia after spinal cord injury.

The main objective of the fist study was to identify the neurotransmitters present in

intraspinal neurons that could be involved in changing the activity of sympathetic

pregangiionic neurons after spinal cord injury. We hypothesized that dopamine P-

hydroxylase, neuropeptide Y and substance P were present in fibres surrounding

sympathetic preganglionic neurons, or in spinal interneurons located in intermediate grey

matter in regions containing interneurons antecedent to sympathetic preganglionic neurons

(Joshi et al., 1995), caudal to a complete spinal cord transection. This hypothesis was

tested by examining changes in the area of immunoreactivity for this enzyme or these

neuropeptides in the neuropil surrounding sympathetic preganglionic neurons in segments

TI - 12 of control rats

T4.

In a previous

and paraplegic rats, seven or fourteen days after cord transection at

study from our laboratory (Weaver et a[., 1997) we observed that

24

GAP-43, a protein expressed in fibres and somata of sprouting neurons (Mearow et

a[., 1994; Aigner et ai., 1995), was present in spinal interneurons in the intermediate grey

matter caudal to a spinal cord transection. This observation indicates that a population

of spinal internewom may be sprouting after deafferentation of neurons, such as

sympathetic preganglionic neurons, within the spinal cord. The main objective of the

second study was to determine the neurotransmitter phenotype of these spinal interneurons

that were growing in response to cord injury. We hypothesized that increased expression

of dopamine phydroxylase, neuropeptide Y or substance P was present in somata or

fibres immunoreactive for GAP43 caudal to a spinal cord injury. This hypothesis was

tested using dual colour immunocytochemistry to compare changes in GAP-43

immunoreactivity with this enzyme or these peptides in the spinal cord tissue fiom rats

seven, fourteen or forty-two days after cord tramection.

CHAPTER 2:

SURGICAL, METHODS AND TISSUE PROCESSING

26

General overview of methohods

The methods section gives the details of all surgical and immunocytochemical

procedures used to test the hypotheses of each study. The following is a brief summary

of the procedures used in both studies. All of the surgical protocols for these experiments

were approved by the University of Western Ontario animal care cornminee in accordance

with the policies established in the Guide to the Care and Use of experimental Animals

prepared by the Canadian Council on Animal Care. The surgical procedures were

performed on adult male Wistar rats. The hypotheses for both studies were tested using

a rat model of spinal cord injury that involved transecting the fourth thoracic segment of

the spinal cord under anaesthesia In the first study, rats survived for seven or fourteen

days after spinal cord transection. In the second study, rats survived for seven, fourteen

or forty-two days after spinal cord transection, after which the rats were sacrificed and the

spinal cords were fixed using 4% formaldehyde. Controis were rats whose spinal cords

were not injured prior to sacrifice and subsequent fixation in formaldehyde. Spinal cord

tissue fkom the thoracic (T) segments of the spinal cord was then removed in four

segment portions corresponding to TLT4 (rostra1 to the injury site in spinal cord injured

rats), T5T8 (caudal to the injury site in spinal cord injured rats) and T9-12 (caudal to the

transection). A cryostat was used to cut the spinal cords fiom each portion horizontally

or transversely at a thickness of 30 p. In the first study the spinal cord tissues were

processed using fluorescent immunocytochemistry for either DBH, neuropeptide Y or

substance P. In the second study, a dual colour, fluorescent immunocytochemical

procedure was used to visualize either GAP43 and DBH, or GAP43 and substance P in

27

the same sections of the spinal cord In both studies the distribution and area of these

neurotransmitters, or neurotransmitters and GAP43 were analyzed using the

Microcomputer Imaging Device (MCID). A value of P<0.05 was used to determine all

significantly different valws in both studies. Twenty-nine rats were used in the first S ~ Y

and a total of twenty-four rats in the second study. The number of rats processed for each

neurotransmitter or combination of GAP43 and DBH or GAP-43 and substance P are

given in the results section of each study. The details of all surgical protocols,

immunocytochemical procedures and statistical analyses are described below.

Surgical procedures

The following protocols were performed on al l rats used in these studies. Adult

male Wistar rats weighing approximately 350 g were prepared for surgical interventions

by sedation with 2.5 mgkg Diazepam (i.p.) 10 minutes prior to anaesthesia with sodium

pento barbital (3 5mg/kg, i. p .). Supplemental doses of sodium pento barbital (2 mgkg, i. p.)

were given throughout the surgery as necessary.

Spinal curd tramection

A laminectomy was performed at the third thoracic to fourth thoracic vertebrae to

expose the mid-thoracic spinal cord The spinal cord was completely transected at the

fourth thoracic (T4) segment. The completeness of the transection was verified by

viewing the entire cut transverse surface at both proximal and distal sides of the

transection. Immediately after surgery, all rats received injections of 20,000 IU antibiotic

(Penlong-XL im., RogadSTB, Canada) and 10 ml of Lactated Ringer solution (Baxter,

s.c.). The rats recovered on a heating pad and were monitored for any signs of distress.

28

Typically they woke 40-50 minutes after surgery and moved in their cages using their

forelimbs within 2 to 3 hours after surgery. The urinary bladder was expressed manually

four times every day until the rats regained automatic bladder hction. The rats were

maintained for seven, fourteen days or forty-two days (dud colour immunofluorescence

study only) after cord transection.

Identijication of sympathetic preganglionic neurons

Intraperitoneal injections of the retrograde tracer Fluoro Gold (0.2ml; 0.25% in

saline) were done one week before sacrifice to label sympathetic preganglionic neurons

within the spinal cord. This method has been shown to label more sympathetic

preganglionic neurons in the intermediolateral cell column than with a single injection of

Fluoro Gold into the adrenal gland (Anderson and Edwards, 1994).

Seven days after injection of Fluoro Gold (control rats), or seven or fourteen days

after spinal cord transection (spinal rats), rats were re-anaesthetized with urethane and

perfused transcardidly with 250 ml of oxygenated Dulbecco's Modified Eagle's Medium

(Sigma, USA) followed by 900 ml of 4% fonnaidehyde in 0.1 M phosphate buffered

saline with pH adjusted to 7.4. Segments o w to twelve of the thoracic spinal cord were

removed after further verification of the level and completeness of the transection in

spinal rats. The segments were divided into three parts containing T1-4, (segments rostra1

to the injury in spinal rats), T5-8 and T9-12. The thoracic segments of the spinal cord

were identitied by their relationship to the dorsal spinal processes of the vertebrae. The

cord segments were postfixed for 24 hours in 4% formaldehyde at 4 degrees celsius and

then cryoprotected in 30% sucrose for 48 hours at 4 degrees celsius. Each four-segment

29

portion of spinal cord was sectioned horizontally or transversely at a thickness of 30 p

in preparation for immunocytochemical processing.

Immunocytochemistry - single colow immunofluorescence

The neurotransmitters were identified in the spinal cords fiom thirty rats using

fluorescent imm~~~ocytochemistry for one of DBH, NPY or substance P. The foilowing

procedures were all performed at room temperature. First the cut sections of spinal cord

were washed three times with Tris-phosphate buffered saline (pH 7.4) containing 0.3%

Triton X-100 detergent. Each of the three washes was ten minutes in length. Next, the

tissues were preincubated in Tris-phosphate buffeted saline containing 0.3% Triton X- 100

and 10% normal horse serum (Cedarlane Laboratories Ltd., Canada) in order to block

non-specific binding of the primary antibody to antigens in the tissue. After forty

minutes, the blocking solution was then removed and the tissue was incubated in

polyclonal primary antisera raised in rabbit against DBH (Incstar, USA, diluted 1 : 10 000)

or NPY (Peninsula, USA, diluted 1:40 000) or substance P (Peninsula, USA, diluted 1:20

000). Primary antibodies were diluted in Tris-phosphate buffered saline containing 1%

n o d horse serum. After twenty-four hours of incubation, the tissue was washed three

times in Tris-phosphate buffered solution. Each wash was ten minutes in duration. The

tissues were then incubated in antiserum against rabbit IgG raised in donkeys and

conjugated to biotin, that was diluted 1:200 to 1 :400 in Tris-phosphate buffered saline

(Jackson ImmuwResearch Laboratories) for twelve hours. The tissues were again washed

three times in Tris-phosphate buffered saline for ten minutedwash. AAer these washes,

the tissue sections were incubated with rhodamine Lissamine conjugated to streptavidin

30

(diluted 1 :200 in Tris-phosphate buffered saline, Jackson ImrnunoResearch Laboratories)

for four hours at room temperature. Finally the tissues were washed three times in 0.1M

phosphate buffer, mounted on gelatinized slides and coverslipped with DPX mounting

medium containing 2% mercaptoethanol. In control incubations, the tissues were

processed as described above, however, primary autiserum was omitted from the

immunocytochemical reaction.

Immunocytochemishy - dual colour immunojuoresceence

AU immunocytochemical procedures were performed at room temperature. Spinal

cord sections were washed three times with Tris-phosphate buffered saline (pH 7.4)

containing 0.3% Triton-X detergent, Each wash was ten minutes in length. Non-specific

binding was blocked by preincubating the tissue for 40 min with a Tns-phosphate

buffered saline-Triton-X solution that contained 10% normal horse serum (Cedarlane

Laboratories Ltd., Canada). The blocking solution was then removed and the tissue was

incubated in a solution of Tris-phosphate buffered saline-Triton-X soiution that contained

1% normal horse serum, polyclonal primary antisem raised in rabbit against DBH

(diluted 1:l 000, Incstar, USA) and a mouse monoclonal antibody against GAP-43

(antibody 9-1E 12 diluted 1:20 000, courtesy of Dr. David Schreyer) (Schreyer and Skene,

1991) or in a similar solution that contained a polyclonal antiserum raised in rabbit

against substance P (diluted 1 : 1 500, Peninsula, USA) and a mouse monoclonal antibody

against GAP43 (Schreyer and Skene, 199 l), in Tris-phosphate buffered saline-Triton-X

containing 1% normal horse serum. After 24 hours of incubation, the tissue was given

three ten minute washes in Tris-phosphate buffered saline. The sections were then

3 1

incubated in a solution of antiserum against mouse IgG raised in donkeys and conjugated

to biotin, that was diluted 1 :200 in Tris-phosphate buffered saline containing 1% normal

horse serum (Jackson Immunoresearch Laboratories) for 12 hours at room temperature.

After fkther washing, the sections were incubated with fluorescein isothiocyanate (FITC)

conjugated to streptavidin and rhodamine conjugated goat anti-rabbit IgG both diluted

1:200 in Tris-phosphate buffered saline with 1% normal horse serum, (Jackson

Immunoresearch Laboratories) for 4 hours at room temperature. The sections were then

washed two times in 0.1M phosphate buffer, mounted on gelatinized slides and

coverslipped with DPX mounting medium containing 2% mercaptoethanol.

Dual d o u r immunojuorescence - immmocytochernistry controls

The following incubations of tissue with primary antisera and/or secondary antisera

were done in order to determine if there was any evidence of cross reactivity between

different antibodies. In one control experiment, both primary antisera, either mouse anti-

GAP-43 and rabbit anti-DBH, or anti-GAP43 and rabbit anti-substance P were omitted

and tissues were incubated in secondary antibodies and fluorochromes as described above.

To determine if there was any cross reactivity between biotinylated donkey anti-mouse

IgG and the rabbit polyclonal antibodies used in this study, tissues were incubated in

primary anti-sera against either DBH or substance P, foliowed by biotinylated donkey

anti-mouse IgG followed by FITC conjugated streptavidin. Similarly, to determine the

extent, if any, of the cross reactivity between m o w anti-GAP-43 and the fluorochrome

used to visualize DBH or substance P, spinal cord tissues from the thoracic segments were

incubated in mouse anti-GAP-43 followed by rhodamine conjugated goat-anti rabbit IgG.

32

Finally the potentiai cross reactivity between biotinylated donkey anti-mouse IgG and

rhodamine conjugated goat anti-rabbit IgG was investigated by incubatiag tissue in a

solution containing mouse anti-GAP-43, followed by biotinylated donkey anti-mouse IgG

and then a solution containing rhodamine conjugated goat anti-rabbit IgG.

Immunocytuchemical controls - r e d s

The results of the immunocytochemicai controls performed in this study were

negative aud did not show any specific cross reactivity between inappropriate secondary

antibodies or fluorochromes, such as mouse anti-GAP43 and rhodamine conjugated goat

anti-rabbit IgG.

Analysis of data - single and dual d o u r imrnuno$ltiorescence studies

Immunofluorescence or Fluoro Gold-labelled sympathetic preganglionic neurons

were detected using a Leitz microscope equipped with an epifluorescence system

containing filters A and N2 (single colour immunoflourescence study) or with filters A,

L2 and N2 (dual colour immunofluorescence study). The distribution and area of

immunoreactivity for DBH, NPY and substance P, or GAP43 and DBH, or GAP43 and

substance P was analyzed in horizontal sections fiom thoracic segments one to four, five

to eight and nine to twelve. Using the Microcomputer Imaging Device system (Imaging

Research, St. Catharines Ontario), a rectangular region of intermediolateral cell column

and adjacent intermediate grey matter medial to the intermediolateral cell column with a

total area of 120,000 p~? was defined. After discriminating the level of immunoreactivity

from background, the number of pixels in each rectangle that fluoresced above the

threshold was summed and converted to pd via a preprogrammed calibration standard.

Ten sample regions

randomly chosen for

33

from each four segment part of spinal cord in each animal were

analysis. Area measurements are reported as mean f standard error

of mean. The observer was blinded to the identity of the tissue being analyzed to

eliminate observer bias.

The numbers of neuronal somata in the single colour immunofluorescence study

(Chapter 3) were counted in the horizontal sections fiom each rat. All somata in each

tissue section fiom a four-segment part of spinal cord were summed and total numbers

reported as mean counts f standard error of mean. Due to the relatively low numbers of

cells, the counts were not corrected for double counting.

Meerhodological limitations - area caZcuIationr

The presence of background or non-specific immunofluorescence is an inherent

obstacle to accurately quantifying changes in proteins identified using

immunocytochemistry. The differences in background staining can vary between animals

processed with different methods (ie. single vs. dual colour immunofluoresceoce), between

tissue fiom different segments of tissue from the same animal and even between different

sections of tissue fiom the same segments of the spinal cord. In ideal conditions, a

constant value for background would be used when comparing immunoreactivity in

different experimental conditions. However, misrepresenting actual background of a

sample would lead to under- or overestimation of specific staining. Although the

variability in background fluorescence was minimal in our studies, we chose to establish

the background level for each slide prior to making each area measurement. This

subjective judgement was unlikely to introduce bias because non-specific

34

immunofluorescence was very diffuse and not confined to any particular neuronal somata

or neuronal process. Conversely, specific staining was clearly confined to distinct

neuronal fibres and/or somata within the grey matter. Once the level of background

fluorescence was determined, the Microcomputer Imaging Device subtracted these vaiues

fiom the total area of fluorescence measured to determine the area of specific

immunofluorescence for a given protein, peptide or enzyme within a specific section of

spinal cord tissue. As a result of the subjective nature of these methods, the area

measurements presented in this thesis are semi-quantitative in nature.

Stutistical analyses - single and dual colour irnmunojuorescen studies

A completely randomized, fixed model, one-way analysis of variance was used to

determine the statistically significant changes in the area of immunoreactivity in the

intemediolateral cell column and to compare the numbers of neuronal somata

immunoreactive for DBH. Tukey's test was used for comparison of mean values

(Snedecor and Cochran, 1989). Differences were considered to be significant at a

probability value less than 0.05.

CHAPTER 3 - RESULTS AND DISCUSSION

CATECHOLAMINI3 ENZYMES AND NEUROPEPTIDES ARE EXPRESSED IN

FIBRES AND SOMATA EN LNTERMEDIAm GREY MATTER IN CHRONIC

SPINAL RATS

3- 1 RESULTS

DBH irnmunoreactivity in contd and spinal rats

DBH imrnunoreactive fibres were found in a rostrocaudal orientation in the

intermediolateral cell column (Fig. 3) of all thoracic spinal segments in three control rats.

These fibres and terminals surrounded Fluoro Gold-labelled sympathetic preganglionic

neurons in this nucleus (see also Fig. 6). In addition, some fibres extended through the

grey matter between the intermediolateral cell column and the central canal (Fig. 3A).

These extensions, when present, often coincided with clusters of sympathetic

preganglionic neurons. The mean area of DBH immunoreactivity in control rats is

presented in Table 1. A small number of DBH immunoreactive somata was found in

laminae V and W medial to the intermediolateral cell column (Fig. 4, see also Fig. 5 and

7)-

One week after cord transection, the area of fibres immunoreactive for DBH in the

intermediolateral cell column of three spinal injured rats decreased slightly but

significantly (Table 1) in segments TI -4 rostra1 to the transection compared to segments

TI-4 in control rats. Caudal to the transection, the area of DBH immunoreactivity in the

intermediolateral cell column decreased markedly in segments TS-12 (Table 1). Fibre

extensions between the intermediolateral cell column and the central canal were no longer

apparent (not shown). The fibres and terminals found in the intermediolateral cell column

surrounded Fluoro Gold-labelled sympathetic preganglionic neurons (not shown).

In contrast to the decrease in DBH immunoreactive fibres, the number of neuronal

somata imrnunoreactive for DBH (Fig. 4) increased in laminae IV, V and MI (Fig. 5 )

37

caudal to the transection, whereas the number remained unchanged in rostral segments

compared to the same segments in control rats. These neurons were distributed

throughout the intermediate grey matter in these laminae (Fig. 5). These somata were 20-

25 pm in diameter and contained bright granular cytoplasmic immunoreactivity that

sometimes extended into proximal dendrites. The number of somata in the caudal

segments of the spinal injured rats was significantly greater than in any thoracic segment

of the control rats (Fig. 4). The number increased three-fold in T5-8 and five-fold in T9-

12 compared to the same segments in control rats.

Two weeks after spinal cord transection in four rats, the mean area of DBH

immunoreactive fibres in segments T1-4 was not different from the same segments in

control rats (Table 1). However, around some of the clusters of sympathetic

preganglionic neurons, this immunoreactivity appeared to increase because the DBH

immtmoreactive fibres were thickly distributed in tangles and clumps. Caudal to the

transection, in segments T5-12, the area of fibres immunoreactive for DBH in the

intermediolateral cell column decreased significantly compared to control rats (Fig. 6A,

Table 1). These fibres surrounded Fluoro Gold-labelled preganglionic neurons (Fig. 6B).

In addition, DBH immunoreactivity became apparent in fine fibres located throughout the

more medial grey matter in laminae V and W. Compared to control rats the number of

DBH immunoreactive neuronal somata (Fig. 7) was increased significantly caudal to the

transection (Fig. 4). In T5-8, the number was four-fold greater than that in the same

segments of control rats and in T9- 1 2, the number was six-fold greater. In segment T5-8,

the number of DBH immunoreactive somata also increased significantly (Fig. 4) compared

Figure 3. DBH immunoreactivity in the intermediolateral cell column of control rats in

horizontal sections of spinal segments TI-4 (A) and T5-8 (B). DBH immunoreactive

fibres were in rostrocaudal orientation in the intermediolateral cell column. Some fibres

project through the more medial grey matter (arrow). Calibration bar in B is 100 pm and

applies to A and B. IML, nucleus intermediolateralis, pars principalis; LF, lateral

filniculus*

Table 1. Area of imrnunoreactivity for DBH, NPY and substance P

in the intermediolateral cell column of control and spinal rats

M'Y

Substance P

One Week SCT (n = 3) 4395 f: 322* I I Tl-4

Control (n = 3)

One Week SCT (n = 3) 3038 + 498 I

5965 + 552

Two Week SCT (n = 3)

Control (n = 3)

5139 +, 79

3430 +, 441

One Week SCT(n= 3) 4751 f 413 I

Two Week SCT (n = 3)

Control (n = 3 )

Two Week SCT (n = 3) 4612 .t 460 I

3327 f 582

4168 5 81 1

DBH, dopamine P-hydroxylase; NPY, neuropeptide Y, SCT, spinal cord transection between

T4K5. Values are mean area (&) f S.E.M. *, significantly different from the same cord

segments in control rats,

TI -4 T5-8 T9-12 Thoracic segments

Figure 4. Mean numbers of somata immmoreactive for DBH in control rats (black bars, n=3), spinal injured rats seven days after cord transection (stippled bars, n=3) and fourteen days after cord tmnsection (striped bars, n=4). Mean counts (f S.E.M.) of somata are shown and each bar represents the total number counted in a four-segment portion of spinal cord. *, significantly different from control tats. t , significantly different fiom rats seven days after cord transection.

'ventral Horn

Figure 5. Representative distribution of DBH-immunoreactive neuronal somata in stylized transverse (A) and horizontal @) sections of spinal cord seven days after cord trmsection. Each black dot represents the location of one neuron. The numbers of neurons plotted on the transverse section are those that would be found in one plane at T6; the numbers plotted on the horizontal section are those that would be found in one plane of the intermediolateral cell column (IML) of TS-8.

Figure 6. DBH-immunoreactive fibres in the intermediolateral cell column of segments

TS-8, two weeks after cord transection at T4 (A). DBH immu11oreactive fibres often

surrounded sympathetic preganglionic neurons. Panel B is a photomicrograph of the same

field shown in A to illustrate the location of Fluoro Gold labelled sympathetic

preganglionic neurons in this field. Arrows in A and B are in identical positions.

Calibration bar is 50 pm and applies to A and B. Abbreviations as in Fig. 3.

Figure 7. DBH immunoreactivity in somata in segments T5-8, two weeks aftet cord

transection at T4. Three DBH immunoreactive somata are present in the medial

intermediate grey matter (A, arrows). Details of two somata fiom the centre of the field

are shown at higher magnification in B. Calibration bar on A is 100 p and on B is 50

pm. CC, central canal; other abbreviations as in Fig. 3.

to the same segments fiom rats one week after spinal cord transection.

NPY immunoreactivity in control and spinal rats

In three control rats, NPY immuaoreactivity was primarily observed in fibres

oriented rostrocaudally in the intermediolateral cell column, with some fibres extending

between the intermediolateral cell column and the more medial grey matter (Fig. 8A).

The pattern of NPY immunoreactive fibres was similar to the pattern of DBH

immunoreactive fibres. The mean area of NPY immunoreactivity in segments TI-12 is

presented in Table 1. No NPY immunoreactive cell bodies were found in the thoracic

grey matter of these control rats.

In three rats that survived for one week after cord transeaion, the area of NPY

irmnunoreactivity rostrd to the transection was unchanged (Table 1). However, caudal

to the transection, the area of NPY immunoreactivity decreased significantly in segments

TS- 12. The fibres in the intermediolateral cell column surrounded Fluoro Gold-labelled

sympathetic preganglionic neurons. Somata ixnmunoreactive for NPY were not observed

in these rats.

The distribution and area of NPY immunoreactive fibres found in segments TI-4

remained unchanged two weeks after transection in five rats compared to control rats.

Caudal to the transection, the area of immunoreactive fibres decreased sijpificantly

compared to control rats (Table 1). The NPY immunoreactive fibres that were still

present in the intermediolaterd cell column (Fig. 8B) surrounded Fluoro Gold labelled

sympathetic preganglionic neurons (not shown). NPY immunoreactive somata were not

found in the spinal cords of these rats.

Figure 8. NPY immunoreactivity in the iatennediolateral cell column of spinal segments

T5-8 in a control rat (A) and a spinal injured rat two weeks after cord transection at T4

(B). Several Fluoro Gold labelled sympathetic preganglionic neurons are apparent in the

intermediolateral cell column shown in panel A. Immunoreactivity for NPY caudal to

the cord transection (B) is decreased, but remains concentrated in the intermediolateral cell

column (arrows). Calibration bar is 100 pm and applies to both A and B. Abbreviations

as in Fig. 3.

50

Substance P immunoreactivity in control and spinal rats

Fibres immunoreactive for substance P in three control rats also lay in a

rostrocaudal orientation dong the intermediolateral cell column (Fig. 9A). In addition,

substance P immunoreactive fibres and terminals were sparsely distributed throughout the

intermediate grey matter medial to the intermediolateral cell column. The mean area of

substance P immunoreactivity in segments TI-12 is presented in Table 1. Substance P

was not found in somata in the thoracic spinal cord in the control rats.

In contrast to the changes in DBH and NPY immunoreactivity, the area of

substance P immunoreactive fibres in the intermediolateral cell column and adjacent grey

matter rostrd and caudal to the transection did not decrease in three spinal injured rats

one week after injury. Rostra1 to the transection the area of substance P immunoreactive

fibres was unchanged compared to control rats (Table 1). However, in T5-8 caudal to the

transection, the area of immunoreactivity tended to increase and in T9-12 the area was

significantly increased. Substance P immunoreactivity was not apparent in somata in the

thoracic cord of the spinal injured rats.

In three rats two weeks after spinal cord transection, the area of substance P

immunoreactive fibres in the intermediolateral cell column and adjacent intermediate grey

matter was unchanged rostra1 to the transection (Table 1). Caudal to the injury, in T5-8

the area tended to be increased and in segments T9-12 the area was significantly increased

as had been observed one week after injury. Furthermore, the substance P

immunoreactive fibres extended even more widely into the more medial intermediate grey

matter than they had at seven days (Fig. 9B), creating an intensely immunoreactive

Figure 9. Substance P irnrnunoreactivity in the intermediolateral cell column of spinal

segments TS-8 in a control rat (A) and a spinal injured rat two weeks after cord

transection at T4 (B). In the control rat, fibres and terminals immunoreactive for substance

P are heavily distributed in the region of the intermediolaterai cell column (arrow) and

a less dense distribution is present in the more medial grey matter. Caudal to the cord

transection fibres and terminals immunoreactive for substance P are distributed more

densely throughout the intermediate grey matter (arrow). Calibration bar is 100 pn and

applies to both A and B. Abbreviations as in Fig. 3.

53

reticular pattern of fibres. Substance P immunoreactive somata were not apparent in any

thoracic segments at this time.

3.2 D I S C U S S .

The catecholamine-synthesizing enzyme DBH and the peptide NPY were found

in fibres mounding sympathetic preganglionic neurons caudal to a cord transection at

seven and fourteen days after the transection. DBH and NPY immunoreactive fibres were

particularly apparent in the intermediolateral cell column, revealing a selective targeting

of the fibres to autonomic areas in the spinal animal. Since, caudal to a cord transection,

degeneration of terminals of bulbospinal fibres in the intermediolateral cell column is

complete by seven days (Llewellyn-Smith et al., 1995) aud, at this time, no more

degenerating histofluorescent monoaminergic fibres are found (Haggendai and Dahlstrom,

1973), these data suggest that the DBH and NPY immunoreactive fibres caudal to a cord

transection are present within spinal cucuits. Therefore DBH and NPY immunoreactive

fibres in the spinal cord caudal to the transection may originate from dorsal root afferent

neurons or from spinal interneurons. Although a few neuronal somata immunoreactive

for DBH were found in the spinal cords of control rats, a markedly greater number

appeared in the cord caudal to a trausection and the numbers increased with time after

transection. This increase in the number of DBH immunoreactive somata may reflect

either upregulation of enzyme synthesis in a pre-existing population of catecholaminergic

interneurons or a switch to a catecholaminergic phenotype by a group of spinal

interneurons. Finally, in accord with a previous report (Davis et aL, 1984), substance P

immunoreactive fibres remained in the intermediolateral cell column of the chronic spinal

54

injured rats. However, cord transection caused an increase in substance P expression in

spinal circuits utilizing substance P because the area of fibres immunoreactive for this

peptide spread more widely through the intermediate grey matter caudal to the transection.

This new distribution of substance P immunoreactive fibres also evolved with time after

cord transection.

Changes in DBH immunoreacrivty afer spinal cord h.unsection

Despite investigations of central nervous system rnonoamines for more than thirty

years, evidence for spinal circuits containing adrenergic or noradrenergic neurons has not

emerged prior to the present study. The robust and intense immunoreactivity of the DBH

facilitated our detection of these catecholaminergic spinal interneurons and fine fibres in

the intermediolateral cell column, perhaps because the vesicular location of DBH yields

a concentrated store of antigen (Olschowka et a1.. 1981). However, if previous work is

scrutinized carefully, hints of catecholamines within spinal circuits can be found. Others

have shown decreases in, but not complete loss of, noradrenaline in segments caudal to

midthoracic spinal cord transection. These assessments were made within seven days of

transection in rabbits and within fifteen days in rats (Anden el al., 1964; Magnusson, 1973;

McNicolas er ul., 1980; Haggendal and Dahlstrom, 1973). Roudet et al. (1994) described

a few fibres immunoreactive for DBH in the intermediolateral cell column of the lumbar

cord one week after cord transection at T8 and, using liquid chromatography, they also

demonstrated very low levels of noradrenaline and dopamine in the lumbar segments of

these rats. However, in a later study, Roudet ef al. (1995) were unable to detect

noradrenaline using an antibody against this catecholamine, perhaps because of inadequate

55

sensitivity of the antibody. Finally, the low levels of catecholamines measured in earlier

studies are consistent with our results as the numbers of DBH immunoreactive neurons

and fibres that we observed would not yield high concentrations of catechoiamiws in

whole cord homogenates.

What are the sources of catecholamines within the spinal cord? Potential sources

are sympathetic postganglionic fibres innervating spinal cord blood vessels, afferent fibres

from dorsal root ganglia or intemeurons. Although sympathetic postganglionic fibres

contain DBH, these are not likely to be the source of DBH immunoreactive fibres that we

observed in the spinal injured rats because the localized clustering of DBH fibres in the

intemediolateral cell column did not parallel the vascular supply to the cord, the main

target of sympathetic postganglionic innervation of the cord (McNicolas et a[., 1980). In

this study we focused primarily on the intermediate grey matter. However, we also

nweyed sections of dorsal horn and found no DBH immunoreactive fibres in the more

dorsal laminae that contain primary afferent terminals. Although sensory neurons in

dorsal root ganglia are immunoreactive for tyrosine hydroxylase, these neurons lack DBH

and also demonstrate no histo fluorescence for catecholamines (Kummer et a[. ,1990; Vega

et a/., 199 1). Therefore the terminal arbours of dorsal root affietents probably were not

the source of DBH within the spinal cord. Thus, intemeurons are the Likely source of

DBH immunoreactive fibres in the intemediolateral cell column caudal to the transection.

However, fuaher experiments must be done, using transynaptic tract tracers such as herpes

virus, to establish that the DBH immunoreactive fibres surrounding sympathetic

preganglionic neurons originate from DBH immunoreactive interneurons antecedent to

56

sympathetic preganglionic neurons. These methods have been used previously to

demonstrate interneurons antecedent to sympathetic preganglioaic neurons (Joshi et

a[., 1995) and these interneurons were found in the same laminae as the DBH

immunoreactive somata in the present study.

Spinal interneurons containing tyrosine hydroxylase have been described previously

in intact rats (Dieti es ai.. 1985; Mouchet et a[., 1986) and in primary cultures of rat and

mouse spinal cord (Mariani et al., 1986). The location and size of the tyrosine hydroxylase

immunoreactive neurons described in rats (Dietl et ai.. 1985; Mouchet et a[., 1986) are

similar to the size and location of the DBH immunoreactive neurons that we found in our

experiments. However, because the tyrosine hydroxylase immunoreactive neurons did not

contain DBH, the authors concluded that they were dopaminergic. Our results do not

rehte the possibility that some spinal neurons are dopaminergic, but the presence of DBH

strongly suggests that the neurons we observed were noradrenergic or adrenergic. Indeed,

spinal catecholaminergic systems may contain both dopaminergic and adrenergic neurons.

The area of DBH immunoreactive fibres caudal to the transection decreased in the

intermediolateral cell column, despite the increase in the number of DBH immunoreactive

somata at seven and fourteen days after cord injury. These observations are consistent

with the fact that the majority of catecholaminergic inputs to sympathetic preganglionic

neurons in the intact cord originate from supraspinal sources, with very few originating

£?om spinal interneurons. After cord transection, very fine fibres immunoreactive for

DBH were observed in the medial grey matter only caudal to the transection, in regions

where none were found prior to transection. These fibres may have been sprouting

57

processes fiom interneurons. In the absence of DBH immuooreactive interneu1:ons, we

probably would have seen no DBH immunoreactivity in the intermediolateral cell column

caudal to a complete cord transection.

The increased number of DBH immunoreactive somata after spinal cord injury

may be due to induction of pre-existing low levels of gene traoscription or increased

enzyme synthesis or may reflect a change in neurotransmitter phenotype of the neurons.

Much is known about control of another catecholamine synthesizing enzyme, tyrosine

hydroxylase. Transcription and translation of this enzyme is highly regulated, dependent

upon activity of the neurons and stimulated by growth factors, such as nerve growth factor

(Gizang-Ginsberg and Z B , 1990; Zigmond et a[.. 1989). In contrast, control of DBH

synthesis has not been studied in the same depth and we can only speculate about the

trigger for upregulation of DBH after cord injury. One potential stimulus is the massive

reactive gliosis that occurs throughout the grey matter after cord transection (Krassioukov

and Weaver, 1996). Since astrocytes can produce and potentially release a host of

neurotrophic factors (Gray and Patei, 1992; Rudge et a[., 1992; Ho et a[., 1995), the

environment in the neuropil after cord injury could contain a multiplicity of trophic

factors that could stimulate upregulation of DBH in pre-existing catecholaminergic

neurons. Alternatively, growth factors might initiate a switch in transmitter phenotype of

spinal interneurons. Rat GABAergic striatd neurons, when placed in tissue culture and

exposed to "dopamine differentiation factor", express a dopaminergic as well as a

GABAergic phenotype (Max et a[. ,1996). The transmitter phenotypes of developing

peripheral neurons in vivo are well known to depend upon the trophic substances derived

58

fiom their targets (Rao and Landis, 1992). For example some of the sympathetic

postganglionic neurons innervating eccrine sweat glands in footpad tissue switch fkom an

adrenergic phenotype to a cholinergic phenotype after being exposed to a so called

"cholinergic differentiation factor" released by these sweat glands. Spinal interneurons

may react to neurotrophins released after cord injury in the same ways as during

development,

Changes in NPY immmoreactivity afier spinal cord trmection

In the intact spinal cord, NPY is known to be contained in axons originating fiom

catecholaminergic neurons in the ventrolateral medulla that project to the intermediolateral

cell column (Minson et al., 1994; Tseng er a[., 1993; Halliday and McLachlan, 1991;

Blessing et al.. 1986). The pattern of NPY immunoreactive fibres that we observed

throughout the thoracic cord in control rats and in the upper thoracic segments of spinal

injured rats corresponds to previous descriptions of the distribution of fibres

immunoreactive for this peptide (Fuxe er d.1990; Gibson ef a!., 1984). To our

knowledge, we are the first to show NPY immunoreactive fibres caudal to a cord

transection in one and two week spinal cord injured rats, suggesting a spinal source of this

peptide. In contrast, one study reported complete loss of NPY immunoreactivity in the

intermediolateral cell column caudal to a cord transection (Hokfielt et al., 198 1 ) . However

the antibody used in that study may not have been specific enough to detect NPY in the

small number of fibres immunoreactive for NPY that we were able to detect in spinal cord

injured rats. The source of the NPY immunoreactive fibres in the intemediolateral cell

column was not apparent in our study. We found no evidence for NPY immunoreactive

59

primary afferent fibres in the dorsal horn. Although we detected no NPY immunoreactive

somata using imrnunofluorescence in this study, we have seen NPY immunoreactive

neuronal somata in the intermediate grey of control and spinal injured rats using very

sensitive immunoperoxidase methods in preparation for electron microscopy. Likewise,

others have shown NPY immunoreactive cells in lamina I-IJI of the dorsal horn (Laing

et aL. 1994). Thus NPY immunoreactive fibres in the intemediolateral cell column of the

chronic spinal injured rats probably originate from somata in the spinal grey matter that

were below threshold for detection by the immunofluorescent method that we used.

Changes in substance P immunoreactbity afer spinal cord iransection

Unlike DBH and NPY, the area of substance P immunoreactive fibres in the

intermediolateral cell column did not decrease caudal to a cord transection, consistent with

previous work suggesting that it is contained within spinal circuits (Davis et al., 1984;

Faden et a[., 1985; Naf3ch.i et al., 1978). Seven to fourteen days after cord transection, the

area of substance P immunoreactive fibres increased throughout the intemediolateral cell

column and intermediate grey matter, with the greatest increases occurring in segments

T9-12. During this time a reticular network of substance P immunoreactive fibres

developed within the intermediate grey matter. Although some substance P is contained

within bulbospinal neurons projecting to the intemediolateral cell column (Pilowsky er

aL, 1986; Fuxe et aL, 1990; Heke et a[., 1982), much of the substance P in the spinal cord

appears to derive fiom afferent and interneuronal sources (Davis et uZ., 1984; Hong and

Weaver, 1993; Naftchi et a[., 1978; Sharkey et a[., 1987). The increased area of substance

P immunoreactive fibres may be due to an increase in synthesis of this peptide in spinal

60

intemeurons that have processes distributed through the medial grey matter or to sprouting

of dorsal root afferents or spinal interneurons that contain substance P, leading to a more

extensive network of immunoreactive fibres. Wang et al. (1991a,b) also have

demonstrated that increases in spinal cord substance P immunoreactive fibres can be

attributed to reactions of intemeurons. These investigators reported initial decreases and

then later increases in this peptide following dorsal rhizotomy in rats. An increase in

trophic factors in the injured cord could stimulate substance P synthesis as well as

sprouting (Lindsay and Harmar, 1989). Since immunoreactivity for substance P appears

in new areas of the grey matter after cord transection, some substance P immunoreactive

fibres in the intermediolateral cell column might originate fiom sprouting of spinal

interneurons, as a response of the cord to injury, whereas other fibres might have been

part of spinal circuits already present in the intact cord.

Conclusions

This study has provided evidence that the catecholamine synthesizing enzyme DBH

is upregulated in spinal interneurons and is present in fibres coursing near sympathetic

preganglionic neurons caudal to the cord transection in chronic spinal rats. This

observation suggests that a spinal catecholaminergic system has its gene expression

upregulated in response to cord injury. NPY immunoreactive fibres were also present in

the intermediolateral cell column of spinal injured rats and the distribution of NPY

immunoreactive fibres closely resembled that of DBH immunoreactive fibres, suggesting

that they are colocalized in the same spinal system. The expansion of a system of

substance P immunoreactive fibres also appears to be part of the spinal cord's response

61

to transection. Our data provide no distinction between the possibility that new fibres

appear in the intermediolateral cell column, after cord transection, forming new synapses,

or that fibres are part of pre-existing spioal circuits. Finally, because the effects of

catecholamines, NPY and substance P on sympathetic preganglionic neurons can be

excitatory, a spinal source of catecholamines and these peptides could contribute to the

development of autonomic dysreflexia, a condition of hyperexcitability that develops after

spinal cord injury and causes abnormal regulation of arterial pressure.

CEIAPTER 4 - RESULTS AND DISCUSSION

CHANGES IN INTRASPINAL NEUROTRANSMITTERS OCCUR

INDEPENDENTLY OF CHANGES IN GAP-43: A DUAL COLOUR

IMlMUNOFLUORESCENCE STUDY

63

Chapter 4 - Objective and Hyporhsis

In a previous study from our laboratory (Weaver ef al., 1997) we observed that

GAP-43, a protein expressed in fibres and somata of sprouting neurons (Mearow el

a[., 1994; Aigner et a[., 1999, was present in spinal interneurons in the intermediate grey

matter caudal to a spinal cord transection. This observation indicates that a popdahon

of spinal interneurons may be sprouting after deaffierentation of neurons, such as

sympathetic preganglionic neurons, within the spinal cord. The objective of this study

was to determine the neurotransmitter phenotype of these spinal interneurons that were

growing in response to cord injury. FoIlowing spinal cord transection, the number of

DBH immunoreactive somata and the area of substance P immunoreactive fibres increased

caudal to the transection. Therefore we hypothesized that immunoreactivity for dopamine

P-hydroxylase, and substance P was increased in somata or fibres immunoreactive for

GAP-43 caudal to a spinal cord injury indicating that spinal interneurons containing this

enzyme or this neuropeptide were sprouting in response to cord injury. This hypothesis

was tested using dual colour imrnunocytochemistry to search for coloc31i7ation of

immunoreactivity for GAP-43 with that for DBH or substance P in the spinal cord tissue

from rats seven, fourteen or forty-two days after cord transection.

-/, I RESULTS

GAP-13, DBH and substance P immunoreactivily in control rats

In six control rats, GAP43 immunoreactivity was observed in bundles of fibres

lying in a rostrocaudd orientation in the intennediolateral cell column (Fig. 10 A,C). In

addition, numerous GAP43 immunoreactive fibres extended through the grey matter

between the intermediolateral cell column and the central canal. The mean area of fibres

immunoreactive for GAP43 in the intermediolateral cell column is presented in Table 2.

In control rats, GAP43 immunoreactive neuronal somata were not observed anywhere in

the grey matter of the thoracic segments of the spinal cord.

In three control rats, the distribution of DBH immunoreactive fibres was similar

to the distribution of GAP43 immunoreactive fibres in the intermediolateral cell column

and adjacent intermediate grey matter (Fig. 10 A,B). The mean area of DBH

imrnunoreactive fibres was less than the area of GAP-43 immunoreactive fibres in

segments TI-4 (Table 2). In thoracic segments 5-12, the mean area of DBH

immunoreactivity was similar to the mean area of GAP-43 immunoreactivity in the

intermediolateral cell column. In control rats, most DBH immunoreactive fibres in the

intermediolateral cell column were immunoreactive for GAP-43 (Fig. 10 A,B) throughout

the thoracic spinal cord. Single, small, DBH immunoreactive fibres that were not

immunoreactive for GAP-43 (Fig. 10B) were observed in the intermediate grey matter

medial to the intermediolateral cell column. Not all GAP-43 immunoreactive fibres were

DBH immunoreactive (Fig. 10 A,B). Within the intermediate grey matter, DBH

immunoreactivity was observed in punctate speckles of irnrnunoreactivity suggestive of

Table 2- Area of immunoreactivity for GAP-43, DBH and substance P in

the intermedlolateral cell column of control and spinal rats

DBH

L

Substance P

Control (n = 6)

One Week SCT (n = 6)

Two Week SCT (a = 6) Six Week SCT (n = 4)

Control (n = 3)

One Week SCT (n = 3)

Two Week SCT (n = 3) Six Week SCT (n = 3)

Control (n = 3)

One Week SCT (n = 3)

Two Week SCT (n = 3)

S i x Week SCT (n = 3)

4788 k 136

797 +, 8SC

291 t 57 * Below Threshold

f 928 + 182

2568 + 324 3651 + 373'

4472 k 229'

T9-I2 2980 + 455

1202 f 74*

2644 f 576 4175 2 284

3621 + 192

804 +, 5 t *

244 4 37' Below Threshold

1628 e 163

2322 ,+ 155'

3549 k 170*

3891 f 199*

GAP-43, growth associated protein-43; DBH, doparnine P-hydroxylase; SCT, spinal cord

transection between T4/T5. Values are mean area (pm2) f S.E.M. *, significantly

different from the same cord segments in control rats.

Figure 10. GAP-43 immunoreactivity (A) in segments T 1-4 of a control rat processed for

GAP-43 (A) and, in the same field DBH (B). GAP43 immunoreactivity (A, solid

arrows) was observed in almost all DBH immunoreactive fibres (B, solid arrows). Not all

GAP-43 immunoreactive fibres (A, open arrow) were immtmoreactive for DBH (By open

arrowhead). Some DBH immunoreactive fibres (B, open arrow) within the grey matter

medial to the intermediolateral cell column lacked GAP-43 immunoreactivity (A, open

arrowhead). In segments TI-4 from another control rat, GAP43 immunoreactivity (C,

solid arrow) was observed in almost all substance P immunoreactive fibres (D, solid

arrow). Not all GAP43 immunoreactive fibres (C, open arrows) were immunoreactive

for substance P (B, open arrowheads). Single fibres immunoreactive for substance P @,

open arrow) were obsewed that lacked GAP43 immunoreactivity @, open arrowhead).

Calibration bar in D is 25 pm and applies to A-D. IML, nucleus intermediolateralis, pars

principalis; LF, lateral funiculus.

68

terminals and varicosities within the intermediolaterd cell column, whereas G A P 4

immunoreactivity was primarily observed in fibres. A few DBH immunoreactive somata

were observed in all segments of the thoracic spinal cord, however none of these somata

were immunoreactive for GAP43 (not shown).

The distribution of substance P immunnreactive fibres in the intermediolateral cell

column and intermediate grey matter in three control rats, processed for GAP43 and

substance P, was similar to the distribution of fibres immunoreactive for GAP-43. In

comparison to GAP43 and DBH, fewer substance P imrnunoreactive fibres were observed

in a rostrocaudal orientation in the intermediolateral cell column and in the grey matter

between the intermediolateral cell column and central canal (Fig. 10D). Throughout the

intermediolateral cell column of the thoracic cord, the area of substance P immunoreactive

fibres was smaller than the area of GAP-43 immunoreactive fibres (Table 2). Most, but

not all, substance P imrnunoreactive fibres in the intermediolateral cell column were

immunoreactive for GAP-43 (Fig. 10 C,D). In the dorsal horn all substance P

imrnunoreactive fibres were immunoreactive for GAP43 (not shown). However, most

but not all GAP43 immunoreactive fibres in the dorsal horn were immunoreactive for

substance P.

GAP-43, DBH and substance P immunoreactivity in rats one week after cord injury

In six rats, the mean area and pattern of GAP-43 immunoreactive fibres in the

intermediolateral cell column rostra1 to the transection did not change significantly

compared to the same segments in control rats (Table 2). In tissue fiom three of these

rats, processed for GAP43 and DBH, the area of fibres immunoreactive for DBH were

69

decreased significantly in the intermediolateral cell column of segments T1-4 compared

to control rats (Table 2). Nevertheless almost all DBH immunoreactive fibres were

immunoreactive for GAP43 r o d to the site of injury. In contrast to DBH, the mean

area of substance P innnunoreactive fibres did not change significantly rostral to the

transection in three rats processed for both substance P and GAP-43. In these segments,

most but not all substance P immunoreactive fibres were also immunoreactive for GAP-

43.

Caudal to the transection the area of fibres imrnunoreactive for GAP-43 or DBH

decreased significantly (Table 2). Fibre extensions immunoreactive for GAP43 or DBH,

in the intermediate grey matter between the intermediolateral cell column and central

canal, were rarely observed. In addition, caudal but not rostral to the transection, a few

GAP-43 immunoreactive somata were observed in the thoracic grey matter. Caudal, to

the transection approximately three DBH immunoreactive somata were observed per field

(250X magnification). Conversely in the same segments of control rats, a single DBH

irnmunoreactive soma was sometimes observed per field. None of the GAP43

immunoreactive fibres or somata caudal to the t rmct ion contained immunoreactivity for

DBH. Similarly none of the DBH immunoreactive somata or fibres were immunoreactive

for GAP-43.

In contrast to GAP-43 and DBH, the area of substance P imrnunoreactive fibres

caudal to the transection did not decrease (Table 2). In segments T5-8 the area of

substance P immunoreactive fibres in the intermediolateral cell column did not change

significantly compared to the same segments in control rats (Table 2). Moreover, in

70

segments T9-12 the area of fibres immunoreactive for substance P was siflcntly

increased compared to control rats (Table 2). Unlike GAP-43 or DBH, substance P

immunoreactivity could not be detected in any somata in the thoracic grey matter.

Substance P and GAP-43 immunoreactivity were never colocalized in the same fibres or

within GAP43 immunoreactive somata within the intermediolateral cell column and

intermediate grey matter caudal to the transection.

GAP-43, DBH and substance P immunoreactivity in ruts two week afier cord injury

Fourteen days after cord transection the mean area and distribution of GAP43

immunoreactive fibres in the intermediolateral cell column of six rats did not change

significantly rostral to the injury compared to control rats (Table 2). In three rats, whose

spinal cords were processed for GAP43 and DBH, the mean area of DBH

immunoreactivity was not significantly different rostral to the transection compared to the

same segments in control rats (Table 2). Similarly the area of substance P

immunoreactive fibres rostral to the injury in three rats, processed for both GAP43 and

substance P, was not significantly different fiom the same segments in control rats. In

TI-4, GAP-43 immunoreactivity was observed in almost ali DBH or substance P

immunoreactive fibres in the intermediolateral cell column.

In thoracic segments 5-12, the area of fibres immunoreactive for GAP-43 increased

significantly compared to the same segments seven days after injury, but not compared

to the corresponding segments in control rats (Table 2). Caudal to the tramection GAP-

43 immunoreactive fibres were distributed in a reticular pattern of fibres in the

intermediate grey matter between the central canal and the interrnediolateral cell column,

71

compared to the pattern of immunoreactivity observed in control rats and in segments

rostra1 to the transection. The numbers of GAP43 immuuoreactive somata increased to

approximately three somata per field (250X magoification) in segments T5-12 compared

to two GAP-43 immunoreactive somata per field in the same segments from rats seven

days after injury. In contrast to GAP-43, the area of DBH irnmunoreactive fibres in the

intemediolateral cell column (Table 2) continued to decrease caudal to the injury.

Although the numbers of GAP43 immunoreactive somata appeared to increase in

segments T5-12, none of these somata contained immunoreactivity for DBH and none of

the fibres immunoreactive for GAP43 were immunoreactive for DBH (Fig. 1 I). Fourteen

days after cord transection, the number of DBH immunoreactive somata increased to

approximately six somata per field (250X magnification) caudal to the injury. Despite the

increase in DBH immunoreactive somata caudal to the transection, none of these somata

or fibres were immunoreactive for GAP43 (Fig. 12).

The mean area of substance P immunoreactive fibres increased significantly in the

intemediolateral cell column and intermediate grey matter of segments T5-12 (Table 2)

fourteen days after cord injury compared to control rats. In these segments GAP-43

immunoreactive somata and fibres were not irnmunoreactive for substance P (Fig. 13A).

Similarly, substance P immuooreactive fibres in the intemediolateral cell column and

adjacent grey matter lacked GAP43 immunoreactivity (Fig. 13B). In contrast to both

GAP-43 and DBH, substance P imrnunoreactivity could not be detected in any somata in

the grey matter caudal to the injury.

Figure 1 1. High power photomicrographs of the intermediate grey matter, medial to the

grey matter, from segments T5-8, caudal to a complete cord transection, from a rat

fourteen days after spinal cord traosection at T4. GAP-43 immunoreactive fibres and cells

(A, open arrows) were not immunoreactive for DBH (B, open arrowheads). The same

field is shown in both panels. Calibration bar is 25 pn and applies to A and B.

Figure 12. High power photomicrographs of the intermediate grey matter, medial to the

grey matter, from segments T5-8, caudal to a compiete cord transection, fiom a rat

fourteen days after spinal cord transection at T4. GAP-43 immunoreactivity (A, open

arrowheads) was not observed in DBH immunoreactive somata (B, open arrows).

Calibration bar is 25 pm and applies to A and B.

Figure 13. High power photomicrographs of the intermediate grey matter, medial to the

grey matter, fiom segments T5-8, caudal to a compiete cord transection, fiom a rat

fourteen days after spiaal cord transection at T4. GAP43 irnmunoreactive somata and

fibres (A, open arrows) were not immunoreactive for substance P (B, open arrowheads).

GAP-43 immunoreactivity (A, open arrowheads) was not observed in substance P

immunoreactive fibres (B, open arrows) caudal to the injury. Calibration bar is 25 pm

and applies to A and B.

78

GAP-43, DBH and mbstmce P irnmunoreactivity in rats six weeks afer cord injtiry

In four rats that survived for forty-two days after spinal cord transectioa the area of

GAP-43 irnmunoreactive fibres in the intermediolateral cell column did not differ

significantly in segments T1-4 compared to the same segments in control rats (Table 2).

In these segments of the cord, there was little change in the distribution of GAP43

imrnunoreactive fibres (Fig. 14 A$). Rostra1 to the injury, GAP-43 continued to be

expressed in almost al i fibres immunoreactive for DBH (Fig. 14 A,B) or substance P (Fig.

14 C,D). The area (Table 2) and distribution (Fig. 14B) of fibres immunoreactive for

DBH in three rats was not significantly different rostral to the injury compared to conrrol

rats. Conversely, in three rats, the area of substance P immunoreactive fibres increased

rostral to the injury compared to control rats (Table 2). In segments T1-4, the number

of substance P immunoreactive fibres and terminals increased in the intermediate grey

matter (Fig. 14D).

Caudal to the transection, the reticular distribution of GAP43 irnmunoreactivity

persisted in the intermediate grey matter (Fig. MA) forty-two days after cord injury. In

segments T9-12, the number of GAP-43 immunoreactive fibres in the intermediate grey

matter appeared to increase compared to 14 days after transection, resulting in a small but

insignificant increase in the area of GAP43 immunoreactive fibres (Table 2). Compared

to fourteen days after transection, the same number of GAP-43 immunoreactive somata

were observed caudal to the injury (approximately three GAP-43 immunoreactive somata

per field). In segments T5-12, DBH immunoreactive fibres could not be detected in the

intermediolateral cell column or adjacent intermediate grey matter. However in the same

79

segments, DBH immunoreactive somata were observed in the intermediate grey matter

adjacent to the intermediolateral cell column (Fig. 1SB). At this time, approximately six

DBH immunoreactive somata were observed per field (250X magnification) and the

number of somata immunoreactive for DBH did not appear to change compared to

fourteen days after injury. None of the GAP43 immunoreactive somata or fibres caudal

to the transection were immunoreactive for DBW and none of the DBH immunoreactive

somata contained GAP-43 immunoreactivity forty-two days after cord transection.

In contrast to GAP-43 and DBH, the area of substance P immunoreactivity in the

intermediolateral cell column and intermediate grey matter increased significantly

throughout the thoracic grey matter caudal to the transection compared to control rats

(Table 2). In thoracic segments 5-12 the number of substance P immunoreactive fibres

and varicosities increased in the intermediate grey matter between the intermediolateral

cell column and central canal (Fig. 15C). Caudal to the transection substance P

immunoreactive fibres were not immunoreactive for GAP-43 and GAP43 immunoreactive

somata were devoid of substance P immunoreactivity. At this time substance P

immunoreactivity could not be detected in somata in the grey matter rostral or caudal to

the injury.

Figure 14. GAP-43 immunoreactivity in segments TI-4 of a rat forty-two days after cord

transection at T4, processed for GAP43 (A) and, in the same field DBH (B). GAP43

immunoreactivity (A, solid arrows) was observed in almost all DBH immunoreactive

fibres (B, solid arrows). Not all GAP-43 immunoreactive fibres (A, open arrows) were

immunoreactive for DBH (B, open arrowheads). Some fibres within the intermediate grey

matter were immunoreactive for DBH (B, open arrow) but not GAP43 (A, open

arrowhead). In segments T1-4, fiom another rat, forty-two days after spinal cord

transection at T4, GAP-43 immunoreactivity (C, solid arrows) was observed in almost all

substance P immunoreactive fibres (D, solid arrows). Not all fibres immunoreactive for

GAP-43 (C, open arrow) were immtmoreactive for substance P (B, open arrowhead).

Single fibres immunoreactive for substance P @, open arrow) were not always

immunoreactive for GAP-43 @, open arrowhead). Calibration bar in D is 25 pm and

applies to A-D. Abbreviations as in Fig. 10.

Figure 15. GAP-43 (A), DBH (B) and substance P (C) immunoreactivity in the

intermediate grey matter fioom segments T5-8 of rats that survived for forty-two days after

spinal cord tramection at T4. GAP43 immmoreactivity was observed in a reticular

network of fibres and in neuronal somata (A, open arrows) caudal to the transection.

DBH immunoreactivity was observed in numerous wuronal somata medial to the

intermediolateral cell column (B, open arrows), whereas substance P immunoreactivity

was observed in numerous fibres but not somata in the intermediolateral cell column and

intermediate grey matter (C). Calibration bar is 100 pm and applies to A-C.

Abbreviation as in Fig. 10

84

4.2 DISCUSSION

The purpose of these experiments was to use dual colour immunofluorescence to

compare changes in the distribution of fibres and terminals immunoreactive for GAP-43

and DBH or GAP43 and substance P in the intennediolateral cell column between control

and spinal cord injured rats. GAP43 is upregulated in growing neurons in development

or after injury (Mearow et a[., 1994; Aigner et ul., 1995; Benowitz and Perrone-Bizzozero,

1991). However the function of GAP43 expression in the adult nervous system is

unknown although it is constitutively expressed in some descending spinal monoaminergic

pathways (Bendotti et al., 1991 ; Yao et aL, 1993; Ching et al., 1994). Accordingly this

study demonstrated that, in the intact spinal cord and rostral to a complete transection,

almost all DBH and substance P immunoreactive fibres and terminals in the

intermediolateral cell column of control rats were immunoreactive for GAP-43. In control

rats and rostral to the injury in spinal rats GAP43 immunoreactivity could not be detected

in any somata We hypothesized that DBH immunoreactive somata or fibres caudal to

the transection would be immunoreactive for GAP43 if the spinal interneurons expressing

DBH neurotransmitters were sprouting and subsequently growing new processes. In

addition, we hypothesized that GAP43 and substance P immunoreactivity would be

colocalized in the same fibres and/or somata caudal to the transection, if the neurons

expressing substance P were sprouting and subsequently growing new processes to replace

degenerated bulbospinal inputs to sympathetic preganglionic neurons caudal to the

transection. This hypothesis must be rejected because caudal to the transection, GAP43

immunoreactivity was not colocalized with immunoreactivity for either DBH or substance

85

P ir! fibres m somata in segments T5-12, seven, fourteen or forty-two days after cord

injury. Forty-two days after injury DBH immunoreactive somata but not fibres were

detected in the intermediate grey matter medial and dorsal to the intermediolateral cell

column caudal to the injury. Background levels of fluorescence were higher in this study

because tissue was processed with dual colour immunocytochemistry (see methods). As

a result the area of substance P, and DBH immunoreactive fibres in control rats, was less

than the area of fibres immunoreactive for this enzyme and neuropeptide in the previous

chapter. Nevertheless, seven to forty-two days after injury, the area of substance P

immunoreactive fibres increased caudal to the transection. However, somata

immunoreactive for substance P could not be detected rostra1 or caudal to the transection

at any of the time points examined. The results from these experiments demonstrated that

intraspinal neurons, whose neurotransmitter expression was altered by cord transection,

were different from intraspinal neurons undergoing morphologic changes associated with

changes in GAP-43.

Colocalization of GAP-43 and DBH or substance P in the intact spinal cord

In the intact spinal cord, the colocaLization of GAP43 and DBH immunoreactivity

has previously been reported in fibres and tetminds around motoneurons in the ventral

horn (Wotherspoon and Priestley, 1995). The most likely source of these fibres are

neurons in the locus coeruleus that are a major source of catecholaminergic innewation

in the ventral horn (Foote et al., 1983) and have been shown to constitutively express

GAP-43 (Bendotti et a[., 1991). In the present study, GAP43 immunoreactivity was

observed in all DBH immunoreactive fibres in the intermediolateral cell column of control

86

rats and rostral to the bansection in cord injured rats. Two of the main sources of DBH

immunoreactive fibres and terminals in the intermediolateral cell column are neurons in

the A5 cell group of the medulla and the rostral ventrolaterd medulla (Gilbey and Spyer,

1993; Tseng et a', 1993; Clark and Proudfi~ 1993; Chalmers and Pilowsky, 1991). High

levels of GAP43 mRNA have also been detected within these regions of the brainstem

using in situ hybridization (Yao et al., 1993; Kruger et al., 1993). Therefore these results

suggest that DBH and GAP-43 are colocalized in bulbospinal projections to the

intermediolateral ceU column in control rats and rostra1 to the injury in spinal cord injured

rats. High levels of GAP43 mRNA have also been demonstrated in neurons within the

caudal raphe nucleus known to be the source of serotonin and substance P innervation

within the intermediolateral cell column W g e r et a/., 1993; Bendotti et al.. 199 1 ) .

Consistent with these findings GAP43 immunoreactivity has been found in all

serotonergic fibres and terminals within the intermediolateral cell column (Ching et

aL, 1994). In the present study, the observation of GAP43 immunoreactivity in almost

all substance P immunoreactive fibres, is consistent with a previous study that

demonstrated that all serotonergic synapses on sympathetic preganglionic neurons

contained substance P (Appel et u1..1986), but not all substance P immunoreactive

synapses contained serotonin. Since GAP43 expression in the adult central nervous

system was often observed in monoarninergic neurons ( b g e r et a/., 1993; Ching et

a[., 1994; Bendotti er a/., 199 I), the lack of GAP-43 immunoreactivity observed in some

substance P imrnuuoreactive fibres in the intermediolateral cell column suggests that they

originate from supraspinal or intraspinal sources that are not monoaminergic and therefore

lack catecholamines or serotonin.

Possible ficnctions of GAP-43 in monodnergic neurons

What is the functional significance of constitutive GAP43 expression in

monoaminergic neurons in the adult central nervous system? The cellular l o c ~ t i o n of

GAP-43 in membranes surrounding synaptic vesicles (Arvidsson et al., 1992) and on the

inner plasma membrane of the presynaptic nerve terminal (Benowitz and

Perrone-Bizzozero, L991), suggests that GAP-43 may be involved in neurotransmitter

release, specifically monoamine release from neurons in the adult central nervous system.

Dekker el al. observed that treatment of cortical synaptosomes with antibodies that

prevented GAP43 phosphorylation prevented calcium induced noradrenaline release fiom

these cells (Dekker et aL, 1989). Since GAP43 is phosphorylated by protein kinase C,

the authors speculated that GAP43 may be a signal transduction molecule involved in the

release of monoamines fiom these cells. Similarly, when PC 12 cells were tradected

with a recombinant vector that expressed antisense GAP43 RNA, that decreased GAP43

expression within these cells, calcium induced dopamine release from these cells was

blocked (Ivins et a[., 1993). These results demonstrated that GAP-43 was involved in the

release of monoamines in vitro. Similarly, constitutive GAP-43 expression may be

required in neurons that synthesize and release monoamines. However, to date, there is

no evidence to confirm or disprove these findings in vivo.

The constitutive expression of GAP43 in adult monoaminergic neurons may not

simply be related to the release of neurotransmitters from these neurons. During

development and following neural injury, high levels of GAP-43 are associated with

88

axonal elongation, synaptogenesis growth cone guidance, sprouting and regeneration

(S kene, 1989; Schaden et aL, 1994; Mearow et al., 1994; Benowitz and Perrone-Bizzozero,

199 1 ; Aigner et al., 1995; Shea, 1995). Therefore it has been hypothesized that neurons

constitutively expressing GAP43 have a predisposition, or ability to alter their synaptic

connectivity in the adult (Kmger et al., 1993; Yao et a[.. 1993; Arvidsson et a[., 1992;

Alonso et ai., 1995; Bendotti et a', 1991) either in response to injury, or under normal

conditions. Following transection of the anterior portion of the medial forebrain bundle,

Alonso et a[. observed GAP43 expression in sprouting monoaminergic neurons. h

addition, high levels of GAP43 mRNA have been linked with changes in the synaptic

organization of monoaminergic neurons within the hippocampus that may underlie the

condition known as long term potentiation (Yao et a[., 1993; Kruger et al., 1993; Benowitz

and Perrone-Bizzozero, 199 1 ).

Intraspinal changes within the spinal cord caudal to the transection - DBH and GAP43

Seven to fourteen days after spinal cord transection, the number of neuronal somata

immunoreactive for GAP43 or DBH increased caudal to the transection. Increased GAP-

43 expression is associated with sprouting phenomena in both the central and peripheral

nervous system (Mearow et a[., 1994; Mearow and Kril, 1995; Alonso et a[., 1995;

Ho&an, 1989; Skene, 1989; Aigner et 01.. 1995). However, now of the DBH

imrnunoreactive somata or fibres caudal to the transection were immunoreactive for GAP-

43. Therefore these resuits suggest that the changes in the number of DBH

immunoreactive neurons and fibres caudal to the aansection are not due to sprouting

within intraspinal catecholarninergic circuits. Increased expression of the gene encoding

89

&A for DBH, or increased translation of DBH mRNA may result in increased levels

of DBH within the perikarya of neurons expressing low levels of this enzyme, to a level

detectable by the methods used in this study. This hypothesis may account for the

increased numbers of DBH somata observed caudal to the transection. The ectopic

expression of DBH foilowing spinal cord transection could also be due to the induction

of this gene in interneurons that express another neurotransmitter phenotype. Trophic

factors present in the neuropil around neurons may influence the neurotransmitter

phenotype of a given neuron. For example, avian chromaffin cells are a plwipotential cell

line capable of expressing catecholamines as well as different neuropeptides such as

somatostatin, enkephalin or neuropeptide Y (Ramirez-Ordonez and Garcia-Arraras, 1 995).

Exposing these cells to brain derived neurotrophic factor increased the synthesis of

noradrenaline and decreased the expression of neuropeptide Y, without affecting the

expression of enkephalin or somatostatin (Ramirez-Ordonez and Garcia-Arraras, 1995).

Conversely when these cells were placed in media containing transforming growth factor

beta-1, noradrenaline synthesis decreased whereas somatostatin synthesis increased.

Therefore the increased immunoreactivity for DBH caudal to the transection could reflect

changes in the trophic environment, resulting in ectopic DBH expression in non-

catecholaminergic spinal intemeurons.

In this study, DBH immunoreactivity was observed in somata but not fibres or

terminals in the intermediolateral cell column forty-two days after spinal cord transection.

In fibres and presynaptic nerve terminals DBH is bound to the plasma membrane of

secretory vesicles that store noradrenaline (Feng and Sabban, 1995). Within neuronal

90

perikarya, DBH has been detected within the endoplasmic reticulum, a possible site of

posttranslational modification of newly synthesized DBH and also within the Golgi

apparatus (Feng et al., 1992). The lack of DBH immunoreactivity in fibres and terminals

but not cell bodies forty-two days after cord transection suggests that although DBH is

being produced in the cell, catecholaminergic neurosecretory vesicles are not being formed

and transported out of the perikarya The presence of DBH within these cells does not

necessarily imply that dopamine, which is converted by DBH to noradrenalhe, is also

present in these cells. The mechanisms underlying the formation of catecholamine

secretory vesicles are not known. However, it may be hypothesized that the lack of

dopamine within these somata may prevent the formation and incorporation of DBH into

neurosecretory vesicles that are subsequently transported to the nerve terminal. The

potential lack of dopamine within these cells may be due to a lack of tyrosine hydroxylase

(the catecholamine synthetic pathway is illustrated in Figure 2), an enzyme that converts

tyrosine to DOPA, or due to a lack of su£€icient concentrations of tyrosine or DOPA

within these spinal interneurons (Commissiong, 1985). The results from research by

Commissiong and colleagues suggests that the lack of DOPA is a limiting factor in the

production of catecholamines caudal to a midthoracic transection (Commissiong, 1985).

Using gas chromatographic-mass spectrometry, Commissiong was unable to detect

monoamines within the caudal segments of the spinal cord one hundred days after a mid-

thoracic spinal cord transection (Commissiong, 1985). After injecting cord injured rats

with L-DOPA, he was able to detect dopamine within the spinal cord caudal to the

transection (Cornmissiong, 1985). Similarly in a related study Commissiong was unable

91

to detect catecholaminergic cell bodies within the developing rat spinal cord using

catecholamine histofluorescence alone without any other intervention. However, on fetal

day IS, following the injection of L-DOPA into the mother, he was able to observe

clumps of cells within the thoracoiumbar spinal cord that expressed catecholamine

histofluorescence (Commissiong, 1983). Therefore Cornmissiong concluded that during

development and after spinal cord injury the enzymes required for the production of

catecholamines, but not the enzyme substrates, were present within the spinal cord,

possibly within spinal intemeurons. However in these studies, he did not examine the

distribution of catecholamine synthesizing enzymes to determine if they were present in

somata and fibres and terminals, or in somata alone.

Changes in substance P caudal to a complete ~a?m?c t i~n

The area of substance P immunoreactivity was increased seven days after spinal cord

transection in the intennediolateral cell column and adjacent grey matter and was fuaher

increased forty-two days after cord transection. The two intraspinal sources of substance

P within the spinal cord are terminal arbors of primary afferent fibres and interneurons

(Hong and Weaver, 1993; Faden et al., 1985; Davis et ai., 1984; Faden et ai., 1985; De

Biasi and Rustioni, 1988). There is no evidence of monosynaptic connections between

primary afferent neurons and sympathetic preganglionic neurons (Shen et aL, 1990;

Spanswick et al., 1994). Therefore the changes in substance P that were observed in the

intermediate grey matter may be the result of changes in gene expression and/or mRNA

translation within spinal intemeurons. In the present study none of the substance P

immunoreactive fibres within the intermediolateral cell column were double labelled for

92

GAP43 although substance P and GAP43 were colocalizpd in the dorsal horn rostrai and

caudal to the tratl~ection. Therefore it is unlikely that spinal interneurons that utilize

substance P as a neurotransmitter are sprouting new processes in response to the loss of

bulbospinal inputs to sympathetic preganglionic neurons.

Mechanisms of substance P accumulation caudal to a spinal cord tramection

What is the cause of increased substance P immunoreactivity caudal to the

transection? The elevation of substance P immunoreactivity may be due to increased gene

expression, or increased translation of substance P mRNA resulting in increased levels

of substance P within spinal intemeurons. Therefore, the apparent appearance of "new"

substance P immunoreactive fibres in the grey matter adjacent to the intennediolateral cell

column, is Likely due to the elevation of substance P to a level detectable by the

immunocytochemical techniques used in this study (Isaacson et al., 1995). Similarly,

following a bilateral ganglionectomy of the superior cervical ganglion, lsaacson and

colleagues observed an apparent increase in the number of nonsympathetic fibres

immunoreactive for calcitonin-gene-related-peptide (CGRP) that innervate inmdwal blood

vessels within the brain using light microscopy. However, using electron microscopy

these researchers were unable to detect an increase in the number of CGRP

immunoreactive axoas ultrastructurally. Furthermore, within these axons they detected

an increase in the number of granular vesicles known to transport CGRP to the

presynaptic terminal. Therefore they concluded that the apparent increase in CGRP fibres

at the light microscopic level was due to the upregulation of neurotransmitter content

within the presynaptic nerve terminal (Isaacson et a/., 1995). The same conclusions were

93

made by Cosette et ul. who observed an increase in cboiine acetyltcaasferase activity

within the cerebral cortex, afier lesioning the nucleus basalis, that could not be attributed

to the sprouting ofudesioned cholinergic neurons (Cosette er al., 1993). Therefore these

results illustrate the need to correlate increases in neurotransmitters following neuronal

deaffierentation with changes in morphologic markers proteins, such as GAP-43, or the use

of ultrastructural studies to accurately determine if the increases are the result of

morphologic changes such as sprouting, or are due to the upregulation of neurotransmitter

within previously undetected populations of fibres.

In addition to changes in gene expression, alterations in substance P release and

metabolism (Galeazza et aiJ995) could affect the amount of substance P

immunoreactivity caudal to the injury. For example, if the activity of spinai interneurons

that utilized substance P decreased, then these neurons would release less substance P after

spinal cord injury. Assuming the rate of substance P synthesis within these neurons did

not change, then increased amounts of substance P would be stored in vesicles located in

the fibres and terminals of these neurons. As a result, the area of substance P

immunoreactivity would increase in the spinal cord without any change in gene expression

or morphology within spinal interneurons utilizing substance P. Similarly, if the rate of

substance P metabolism decreased, due to a decrease in the number of proteolytic

enzymes within the spinal cord, then the area of substance P immunoreactivity would

appear to increase following cord injury. Thus changes in the storage and degradation of

substance P could also result in increased substance P immunoreactivity after spinal cord

transection.

Conclusions - dual colour immunofluorescence study

The results of this study indicate that there are likely two distinct reactions by spinal

internewom after spinal cord transection. First, the increased GAP-43 immtmoreactivity

in somata, fibres and terminals caudal to the injury, suggests that a population of

interneurons are undergoing morphological changes, possibly sprouting, in response to

injury. Second, the induction of DBH in somata and substance P in fibres caudal to the

transection that are not immunoreactive for GAP43 suggests that changes in

proteirdpeptide synthesis are occurring within distinct populations of spinat interneurons.

The results from this study do not exclude the possibility that substance P is present in

spinal interneurons that do not increase GAP-43 expression. Future studies could examine

changes in other cytoskeletal proteins associated with axonal growth to determine whether

or not substance P is present within sprouting interneurons. For example, class I1 beta

tubulin is a cytoskeletal protein that is expressed in neurons during development and also

in regenerating neurons (Hohan, 1989). Therefore fiture studies could use in situ

hybridization to detect class I1 beta tubulin, and immunocytochemistry to visualize

substance P, to determine whether or not the two proteins are colocalized in growing

neurons after spinal cord injury that do not increase GAP43 expression. Moreover, these

responses persist for at least forty-two days after spinal cord transection indicating that

these changes in intraspinal circuits are permanent and ongoing and are not transient

consequences of spinal cord injury. The changes in these proteins and this enzyme

provide evidence that intraspinal neurons are growing and changing neurotransmitter

expression after spinal cord transection. Finally, these changes in intraspinal neurons may

contribute to the development of autonomic dysreflexia after spinal cord injury.

4.3 OVERALL C02VCLUSIONS

The r e d t s of these studies demonstrated the potential of spinal neurons, caudal to

the site of injury, to change in response to spinal cord transection thereby changing

intraspinal circuits. Two potential forms of intraspinal adaptation were 1) changes in

neuropeptides and proteins associated with neurotransmitter synthesis and 2) changes in

a membrane bound marker of axonal growth. Increased expression of neuropeptides and

enzymes associated with neurotransmitter synthesis occurred within a distinct population

of interneurons that were not undergoing changes in morphology. Similarly, increased

expression of a membrane bound protein was observed in a distinct population of

interneurons independent of changes in intraspiad neurotransmitters. Therefore, the view

that neuronal growth, such as sprouting, does not occur in the injured spinal cord

(Nacimiento et uL, 1995; Nacimiento et a[., 1993a) is inaccurate. Finally, further research

is requited to identify the stimuli responsible for these intraspinal changes, so that

strategies can be designed to minimize the abnormal reorganization of intraspinal circuits,

without compromising the regeneration of supraspinal neurons within the injured spinal

cord. The changes in intraspinal circuits may be deleterious resulting in the development

of abnormal reflexes that underlie the development of conditions such as autonomic

dysreflexia and muscle spasticity following spinal cord injury.

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ANDEN, N.-E., J. HAGGENDAL, T. MAGNUSSON and E. ROSENGFEN (1964). The time course of disappearance of noradrenaline and 5 - h y d r o x y a y p ~ e in the spinal cord after tramection. Acta Physiologia Scandinmica 62, 1 15- 1 18.

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CURRICULUM VITAE

NAME: Aly Khan Cassam

PLACE OF BIRTH: Lumsbya, Zambia

YEAR OF BIRTH: 1971

POST-SECONDARY The University of Western Ontario EDUCATION AND London, Ontario, Canada DEGREES: 1990-1994 BSc. Honours Physiology

The University of Western Ontario 1994-1 997 MSc. Neuroscience

HONOURS AND AWARDS: Faculty of Science Dean's Honour List 1993, 1994

Ontario Heart and Stroke Foundation John D. Schultz Summer Science Student Scholarship Summer I994

Faculty of Graduate StudieslGraduate Program in Neuroscience University of Western Ontario Graduate Teaching Award 1994-1995; 1995-1996

Society of Graduate Students, University of Western Ontario Certihate of Teaching Excellence, 1996

RELATED WORK EXPERIENCE:

Summer Science Student The J.P. Robarts Research Institute Sumer 1994

Teaching Assistant, Mammalian Physiology Department of Physiology, University of Western Ontario 1 994- 1996

108

P UBLK ATIONS:

Cassam, A.K, Llewellyn-Smith, LJ. and L.C. Weaver (1997). Catecholamine enzymes and neuropeptides are expressed in fibres and somata in the intermediate grey matter in chronic spinal rats. Neurosci., in press.

Weaver,L.C., Cassam, A.K, Krassioukov, A.V., and LJ. LlewelIyn-Smith (1997). Changes in immunoreactivity for growth associated protein43 suggest reorganization of synapses on spinal sympathetic preganglionic neurons after cord transection. Neurosci., in press.

Llewellyn-Smith, I.J., Cassam, A.K., Krenz, N.R, Krassioukov, A.V. and L.C. Weaver (1 996). Glutamate- and GABA-immunoreactive synapses on sympathetic preganglionic neurons caudal to a complete spinal cord transection in rats. Neurosci., submitted.

Cassam, A.K., and L.C. Weaver (1996). Changes in immunoreactivity for intraspinal neurotransmitters occur independently of intraspinal changes in gap43 immunoreactivity in spinal autonomic nuclei after spinal cord injury. In preparation.

Llewellyn-Smith, I.$., Cassam, A.K., Krenz, N.R Krassioukov, A.V. and L.C. Weaver (1 996). Tyrosine hydroxylase-immunoreactive synapses in rat intermediolateral cell column caudal to a cord transection. Neurosci., in preparation.

ABSTRACTS AND POSTERS:

Cassam, A.K, Krassioukov, A.V., and L.C. Weaver (1994). Growth associated protein43 in spinal autonomic nuclei after cord injury. Twelfth Annual Neurotrauma Symposium.

Llewellyn-Smith, I.J., Cassam, A.K., Krenz, N.R, Krassioukov, A.V. and L.C. Weaver (1995). Tyrosine hydroxylase immunoreactive nerve fibres in transected rat spinal cord. J. Neurotrauma 1 2:US.

Llewellyn-Smith, I.J., Cassam, A.K., Krenz, N.R., Krassioukov, A.V., Minson, J.B., Pilowsky, J.B., Amolda, L.F., Chalmers, J.P. and Weaver, L.C. (1995). Do sympathetic pregangiionic neurons receive catecholaminergic synapses from intraspinal catecholamine neurons? Neurosci. Abst. 2 1 : 140 1.

Cassam, A.K. and L.C. Weaver (1995). Intraspinal neurotransmitters potentially involved in autonomic dysreflexia after cord injury. Neurosci. Abst. 2 1 : 1404.

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ABSTRACTS AND POSTERS (continued):

Weaver, L.C., Krenz, N., Cassam, AD, Korkola,M. and A. Krassioukov (1995). Sprouting of inputs to sympathetic preganglionic neurons after cord injury in rats. Proceedings of the Australian Neuroscience Society. 6: 1 1 1.

Weaver, L.C., Krassioukov, A-V., Cassam, A.K., Krenz, N.R, Korkola, M.L., and B.A. Atkinson (1995). Disordered cardiovascular control after spinal cord injury: mechanisms for autonomic dysreflexia L Neurotrauma 12:359.

Cassam, A.K., Krassioukov, A-V., and L.C. Weaver (1995). Growth associated protein-43 in spinal autonomic nuclei in rats seven to thirty days after cord injury. J. Neurotrauma 12:415.

Llewellyn-Smith, I.L, Cassam, A K , Krenz, N.Ry Krassioukov, A.V., Minson, 4 Arnolda, L.F. and L.C. Weaver (1997). Glutamate and GABA- immunoreactive synapses on sympathetic preganglionic neurons (SPN) caudal to a spind cord transection in rats. Australian Neuroscience Society Meeting