Menetrey Et Al 1989

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Expression of c-Fos protein in interneurons andprojection neurons of the rat spinal cord in

response to noxious somatic, articular, and

visceral stimulation

ARTICLE in THE JOURNAL OF COMPARATIVE NEUROLOGY · AUGUST 1989

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THE JOURNAL

OF

COMPARATIVE NEUROLOGY 286177-195 (1989)

Expression of

cfos

Protein in Interneurons

and Projection Neurons of the

Rat

Spinal

Cord in Response to Noxious Somatic,

Articular, and Visceral Stimulation

D . M E ” R E Y , A. GANNON, J.D. LEVINE, ND A.I. BASBAUM

INSERM,

U-161,

Paris, France (D.M.); and Departments of Anatomy, (A.G., A.I.B.),

and Physiology, (J.D.L.), University of California, San Francisco, California 94143

ABSTRACT

This study used immunocytochemistry to examine the pa ttern of nox-

ious-stimulus evoked expression of the proto-oncogene c-fos in the spinal cord

of the ra t. Both noxious somatic and joint stimulation in awake rats evoked

the expression of c-fos protein in similar areas of the lumbar spinal cord. C-

fos-immunoreactive neurons were found in laminae I and outer 11, in the lat-

eral part of the neck

of

the dorsal horn, and in laminae VII, VIII, and

X.

All of

the labelled neurons were located ipsilateral to the injured hindpaw, except for

lamina VIII where bilateral labelling was recorded. The c-fos-immunoreactive

neurons in lamina I extended from the L3 segment to the rostra l sacral cord;

staining in outer lamina I1was only found a t the

L,

segment. The more deeply

located cells, of the dorsal and medioventral horns, had t he most extensive ros-

trocaudal spread; they were found from L, through the rostral sacral seg-

ments.

The pat tern of c-fos-immunoreactivity produced by visceral stimulation,

in anesthetized rats, differed in several ways from that produced by somatic

stimulation. First, there was considerable bilateral, symmetrical labelling of

cells. Second, there was a much more extensive rostrocaudal spread of the

labelling, from cervical through sacral cord. Third, the greatest rostrocaudal

spread was found for neurons in the superficial dorsal horn; labelled cells in

the neck of the dorsal horn and in lamina

X

were restricted to segments a t the

thoracolumbar junction, which is also where the superficial dorsal horn cells

were most concentrated. Fourth, there were very few labelled neurons in the

outer pa rt of the substantia gelatinosa.

To determine whether any neurons that express the c-fos protein in

response to noxious stimulation project t o supraspinal sites, we combined the

immunocytochemical localization of c-fos with the localization of a retro-

gradely transported protein-gold complex that was injected into the thalamic

and brainstem targets of the major ascending spinal pathways. I n rats t ha t

received the somatic noxious stimulus, 9 0

of

all

of

the c-fos projection neu-

rons were recorded in four major areas of the cord: lamina

I (37

),

the lateral

part of the neck of the dorsal horn ( 2 4 ) , aminae VIII ( 9 ) , nd

X

( 2 9 ) .

The remainder were scattered throughout the spinal gray. With the exception

of lamina VIII, which contained c-fos projection neurons contralateral to the

inflamed paw, all of the c-fos projection neurons were located ipsilateral to the

injured paw. Although c-fos-immunoreactive neurons and retrogradely la-

belled cells were found in many other areas of the spinal gray that contain

Accepted February 24,198 9.

Address reprint requests to Allan I. Basbaum, Department of Anatomy,

University of California San Francisco, San Francisco, CA 94143 .

1989 ALAN R. LISS,

INC.



178

D. MENETREY

ET

AL.

nociresponsive neurons, few were double-labelled. Finally, retrogradely la-

belled cells that expressed c-fos in response to visceral stimulation were only

found in the superficial dorsal horn. They were distributed from cervical

through sacral levels; most were at the thoracolumbar junction.

This study demonstrates tha t the c-fos protein can be used

as

a functional

marker to identify the spinal neurons th at are activated by different forms of

noxious stimulation and indicate that in the awake, freely moving animal,

activity in projection neurons of four regions, lamina

I,

the la teral neck

of

the

dorsal horn, laminae VIII and

X ,

contribute to the central transmission

of

nociceptive messages that are probably involved in the conscious appreciation

of pain.

Key words:

c os

prote in, pain mechanisms; immunocytochemistry, retro-

grade tracing, spinal cord ascending tracts

Electrophysiological studies have characterized two

classes of spinal nociceptive neuron: nociceptive specific

cells (Class

31,

which are exclusively driven by noxious

peripheral stimulation, and wide dynamic range cells (Class

21,

which are excited by both nonnoxious and noxious

peripheral stimuli. These nociceptive neurons cluster into

three major regions of the spinal cord gray matter. The

superficial dorsal horn (laminae I and outer 11) contains

both Class

2

and 3 neurons; most of the nociceptive neurons

of the “neck” of the dorsal horn are Class 2; those of the

medioventral horn (including laminae VIII,

X

and medial

VII) are

of

the Class

3

variety (Willis, ’85; Besson and

Chaouch, ’87). Neurons in these areas are at the origin of

five major ascending pathways that are presumed to be

importan t in nociception (Menktrey, ’87). These are the spi-

nosolitary t ract, which terminates in the nucleus

of

the soli-

tary tract; t he medial and lateral components

of

the spinore-

ticular trac ts, which respectively terminate in the medullary

nucleus reticularis gigantocellularis and in the region of the

lateral reticular nucleus; the spinomesencephalic tract,

which terminates in the periaqueductal gray and t he para-

brachial and cuneiform nuclei of the rostral, dorsolateral

pons; and the spinothalamic tract, which terminates in dif-

ferent subnuclei of the thalamus.

Although there

is

extensive information about the anat-

omy, physiology, and pharmacology of the nociceptive mes-

sages transmitted by and the inhibitory controls exerted

upon spinal nociceptive neurons, there are important limi-

tations to these studies. Fir st, most

of

the electrophysiologi-

cal studies that characterized nociceptive neurons were per-

formed in anesthetized, or decerebrate and/or spinalized

animals. The analysis of the properties of dorsal horn neu-

rons in awake animals has been particularly difficult

(Bromberg and Fetz, ’77; Hayes e t al., ’81;Collins,

’87;

Dun-

can et al., ’87;Sorkin et al., ’88).Second, the noxious stimuli

used were usually of short duration, such as pinprick, pinch,

or

noxious heat. Only rarely have recordings been made in

animal models of tonic, or persistent, pain (Menktrey and

Besson,

’82;

Calvin0 et al., ’87; Dickenson and Sullivan, ’87).

Third, sample size is very limited in electrophysiological

studies. Although anatomical techniques are more suited to

studying large populations of neurons, the functional prop-

erties of labelled neurons cannot be appreciated.

Recently, Hunt e t al. (’87) demonstra ted tha t

it

is possible

to monitor the “activity”

of

nociceptive neurons of the dor-

sal horn, with single cell resolution, by using immunocyto-

chemical localization of the protein product of the c-fos

proto-oncogene. C-fos is the cellular homologue of the viral

oncogene, v-jos. Expression of the gene is rapid; c-jos mes-

senger RNA can be detected within

15

minutes of presenta-

tion of an appropriate inducing stimulus. The mRNA prod-

uct, the

fos

protein,

is

rapidly synthesized and translocated

to the nucleus (Greenberg and Ziff, ’84;Kruiger et al., ’84,

’85;

Greenberg et al.,

’85, ’86;

Curran and Morgan,

’86;

Ran

et al.,

’86)

where it can be localized with antisera. H unt et al.

(’87) reported that the distr ibution of c-fos-immunoreactive

neurons evoked by different types of peripheral stimulation

was consistent with the known distribution

of

nociceptive

and nonnociceptive neurons in the dorsal horn.

In thi s study we extended those observations, by examin-

ing the expression of c-fos in response to relatively selective

noxious chemical stimulation of somatic, articular and vis-

ceral structures. We also determined whether c-fos is ex-

pressed in spinal neurons tha t project to the brain. Thi s is

important

if

one is

to

use the expression of c-jos to “moni-

tor” the activity of neurons that contribute to the rostrad

transmission of nociceptive messages in the CNS. To ad-

dress thi s question we have used a double-labelling method

to localize noxious stimulus-evoked c-fos expression in spi-

nal cord neurons a t the origin of major ascending pathways.

This method combined immunocytochemical localization of

the expression of c-jos with the localization of a retrogradely

transported protein-gold complex (Basbaum and Menbtrey,

’87).

The tracer was injected into supraspinal terminal sites

of the major ascending spinal pathways that have been

implicated in nociceptive transmission.

EXPERIMENTALPROCEDURES

Experiments were performed on

30

adult, male Sprague

Dawley rats (300 gm). Eleven rats were used to establish

appropriate parameters of noxious stimulation. Five control

animals were studied; two were unst imulated , freely moving

rats, three received an injection of saline into either the paw,

ankle,

or

peritoneal cavity (see below). The remaining 14

rats were used to study c-fos immunoreactive ascending

tract cells. In these double-labelling experiments, we first

injected the retrograde tracer and allowed the animals to

recover several days prior to being exposed to th e somatic

or

visceral noxious stimuli. This experimental design pre-

vented possible contaminating c-jos expression secondary

to the surgical procedure. The rat s were later perfused and

the appropriate sections of spinal cord were processed

so



Cfos

PROTEIN IN RAT SPINAL CORD

179

Fig. 1. This photomontage at the

LSlB

egment of the lumbar cord

illustrates the distribution of immunoreactive c-fos neurons produced

by unilateral injection

of

complete Freund's adjuvant into the plantar

hindpaw. The figure not only illustrates the distr ibution of labelled neu-

rons ipsilateral to the injected paw, in lamina

I

(arrowheads), in the re-

ticular neck of the dorsal horn (Ret DH) and around the central canal

that we could identify both the retrogradely labelled and the

immunoreactive cells.

Injection of the retrograde tracer

The retrograde tracer used was a protein-gold complex

consisting of a wheatgerm agglutinin-apohorseradish perox-

idase (WGA-apoHRP, Sigma L0390) to which colloidal gold

(-10 nm) was conjugated (Basbaum and Menittrey, '87). In

order to label as many of the projection neurons as possible,

multiple injections were made in each rat. The injections

were targeted at the te rminal regions of the five major

ascending pathways that have been implicated in the trans-

mission of nociceptive messages. Although th is approach

did not distinguish between the contribution of the individ-

ual pathways, it allowed us to identify noxious stimulus-

evoked c-fos expression in projection neurons using a mini-

mum number of animals.

Animals were anesthetized with sodium pentobarbi tal (55

mm/kg; ip) and placed in a stereotaxic head holder. Since we

were interested in distinguishing contralateral from ipsi-

lateral projecting pathways, retrograde tracer injections

were made on one side of the brain. We used 20-40 diame-

ter glass micropipettes to unilaterally pressure inject the

tracer (0.5 to 1.0~ 1 )t each of the following sites: the region

(CC),

but also illustrates tha t there is no labelling contralaterally, with

the exception

of

laminae

VIII

(see text).

A t

this level there is no labelling

in the substantia gelatinosa

SG).

A higher power photomicrograph of

the lahelling in lamina

I

can be seen in Figure 3. Calibration bar equals

500

pm.

of the lateral reticular nucleus (LRN), the medial reticular

formation (nucleus reticularis gigantocellularis, Rgc); the

mesencephalon (MES), including the periaqueductal gray,

parabrachial area, and nucleus cuneiformis, and the ventro-

basal complex of the lateral thalamus. These targets were

approached stereotaxically using coordinates from the atlas

of Paxinos and Watson ('86). Since an injection into the

nucleus of the solitary trac t (NTS) typically spreads bilat-

erally,

it

was omitted in the studies involving unilateral,

subcutaneous, or periarticular stimulation. However, since

the NTS is clearly involved in the processing of visceral

information, we included the

NTS

injection, which results

in bilateral labelling in the spinal cord (Menittrey and Bas-

baum, '87), in the visceral studies. The latter was exposed by

ventroflexing the rat's head and incising the dura over the

cisterna magna.

Peripheral stimulation models

Three noxious chemical stimulation models were used to

activate somatic, joint,

or

visceral nociceptors. Since our

preliminary studies demonstrated that general anesthesia

both reduces the expression of c-fos and alters the spat ial

distribution of c-fos immunoreactive neurons a t the spinal

cord level (Presley et al., unpublished observations) most of





Fig.

3.

These photomicrographs illustrate the location of c-fos-

immunoreactive neurons in the superficial layers of the dorsal horn pro-

duced by subcutaneous inflammation of the plantar foot (cf. Fig.

I).

Five

segments are represented, from L, through L5,e Note th at there is a

wider rostrocaudal distribution of labelled cells in lamina

I

than in the

outer part of lamina

11;

the latte r are restricted to the L, segment.

Fig. 2.

These schematics illustrate the distribution of c-fos-immuno-

reactive neurons in the lumbar cord of a rat t ha t received a unilateral

injection of complete Freund’s adjuvant in the right plantar hindpaw, 16

hours prior to being sacrificed. Seven levels are represented, from the L1

through the L6/S1 segments. Each diagram includes all labelled cells in

three

50

Mm sections; each dot represents one labelled cell. Note t ha t

there is a more restricted rostrocaudal distribution

of c-fos

positive cells

in the superficial dorsal horn than in deeper layers of the spinal gray.

The boundary

of

the reticular part of the neck of the dorsal horn (Ret.

V) is outlined for orientation.



182

D. MENETREY

ET AL.

Sub cutan eous Inflammation (Plantar Foot)

Tracer Contralateral t o Stimulated Side

1 rnrn

L5/6s

J

6 S 1

Fig.4.

These schematics illustrate the distribu tion of c-fos-immuno-

reactive ascending tract cells in the lumbar cord of a r at with unilateral

subcutaneous inflammation of the plantar hindpaw (cf. Fig.

1).

The

tracer was unilaterally injected into the thalamus, mesencephalon, lat-

era1 reticular nucleus and reticular formation, contralateral

(A)

or ipsi-

lateral

(B)

to the inflamed paw. Each diagram includes double-labelled

cells from

five

50

pm

sections. Each dot represents one double-labelled

neuron.



Cgos PROTEIN IN RAT SPINAL CORD

183

S u b c u t a n e o u s I n f l am m a t io n P l a n t a r F o o t )

T r a c e r l p s i l a t e r a l t o S t i m u l a t e d S i d e

1 mm

Figure

4



184

D.

MENETREY

ET

A

S t

f r om :

L a m i n a

Re t DH

L am i n a

x T

i r nu l a t ed

s i d e

St

A

f r o m :

Lam ina V l l l

i r n u l a t e d

i d e

1

Fig.

5.

Schematic representation of the spinal gray matter origin of

the c-10s positive ascending projection neurons. The overall regional

source of doubIe-labelled cells is indicated by stippling; t he cell bodies

represent the ou tput from this region, not the out put from a particular

lamina. Th e inflamed paw (stimula ted side) is on the left. The upper

diagram illustrates projecting cells th at originate in lamin a I, the reticu-

lar neck of the dorsal horn (Ret. DH) and lamina X; the lower diagr

illustrates projecting cells th at originate in lamina VIII. Th e thickness

th e arrows is proportional to t he numbers of contralaterally and ip

laterally projecting cells that originate from the particular source. N

th at all double-labelled cells in lamina

VIII

are located contralatera

the inflamed paw.

the stud ies were performed in awake, freely moving animals.

Th e somatic and joint stimulation protocols were based on

established chronic inflammation models in awake animals.

These protocols were evaluated an d approved by the Com-

mittee on Animal Research at UCSF. Somatic stimulation

involved unilateral subcutaneous injection of 150

pl

com-

plete Freund's adjuvant int o the planta r foot (Ruda e t al.,

'88). Th e injection was made under brief halothane anes-

thesia. Periarticular noxious stimulation was produced by

unilaterally placing 150

pg

of ur ate crystals throu gh a skin

incision close to the ank le joint (Otsuki et al., '86; Coderre

and Wall, 87)- The crystals were implanted under pento-

barbital anesthesia

(40

mg/kg; ip). The animals were awake

within 2 hours. Periarticular urate crystal injection pro-

duces some inflammation

of

somatic tissue, bu t there

is

a

significant inflammation of the joint.

For

both somatic a

joint stimulation protocois, th e rat s were perfused

16

hou

aft er injection.

The visceral stimulation protocol is based on the acet

acid stretching test (Taber e t al., 69) and involves injecti

of 0.5 ml of

9%

acetic acid into the peritoneal cavity, jus t

the midline. These studies were performed under gene

anesthesia

(55

mg/kg; i.p.).* Ra ts were perfused 1 hour af

injection of the acetic acid. In light of the above commen

'Committee on Animal Research approval to

perform

the

visceral

stim

tion studies in awake animals is pending.



Cfos PROTEIN

IN

RAT

SPINAL

CORD

185

Tissue processing

Animals were deeply anesthetized and perfused through

the aorta with 200 ml of phosphate-buffered saline (PBS)

followed by 500 ml of 4% paraformaldehyde. The brain and

spinal cord were then removed, and postfixed for 4 hours in

the same fixative at

4 °C

before they were cryoprotected in

phosphate-buffered 30 sucrose solution overnight. Serial

frozen sections of the spinal cord

40

hm)

or

brain (100 pm)

were cut and collected in phosphate buffer. Since the dense

gold deposit at the injection site can be visualized without

silver intensification, brain sections were mounted, stained,

and coverslipped for localizing injection sites. The spinal

cord sections were processed as free-floating sections. They

first underwent a silver intensification procedure to make

the gold visible at the light microscopic level (Danscher, 81)

and then were immunostained with the avidin-biotin proce-

dure of Hsu et al. ('81) by using commercial kits (Vector

Labs, Burlingame, CA). Details of this double-labelling

procedure have been described previously (Menktrey, '85;

Basbaum and MenBtrey, '87). After the DAB reaction was

completed, the sections were air dried, mounted, and cover-

slipped. The location

of

retrogradely labeled cells (contain-

ing silver particles) and c-fos-immunoreactive neurons were

plotted with a camera lucida attachment. The location of

cells described

is

based on the spinal cord cytoarchitectonic

atlas of Molander et al. ('84).

Four different primary antisera were tested. Most of the

studies were performed with a rabbit polyclonal antiserum,

directed against an in vitro translated c-fos gene product,

kindly provided by Dr. Dennis Slamon (Department of

Medicine at UCLA). We also used three monoclonal anti-

sera (Microbiological Associates Inc., Bethesda) that were

raised against synthetic peptide fragments from different

regions of the c-fos protein, residues, 4-17 (N terminal) ,

132-154 (mid) and 359-378 (C terminal). Good results were

only obtained with the polyclonal antiserum and the N ter-

minal directed monoclonal antiserum. Both of these anti-

sera were used at a 1/5,000 dilution, with the polyclonal

antiserum preabsorbed against acetone-dried liver powder

prior to use. The two antisera revealed the same pattern of

staining; however, the immunoreactivity was always more

intense with the polyclonal antiserum.

The c-fos protein is not available in sufficient quantities

and thus absorption controls could not be performed with

the polyclonal antiserum, which was directed against the

enti re c-fos gene product. Preabsorption of the N-termina l

directed monoclonal antiserum with synthetic N-te rminal

peptide, (1 wg/ml diluted antiserum), however, completely

abolished the staining. The staining pattern was not

changed when the N-terminal monoclonal antiserum was

absorbed with either the synthetic C-terminal peptide or

peptide from the midportion of the c-fos protein.

Fig. 6. This brightfield photomicrograph illustrates c-fos-immuno-

reactive and retrogradely labelled neurons in a 40-pm thick horizontal

section through lamina

I of

the lumbar dorsal horn.

C-fos

positive cells

have a uniformly stained nucleus (arrowhead). Projection neurons con-

tain punctate cytoplasmic label, which denotes the silver precipitate; the

nuclei of single labelled projection neurons are unstained (open arrow).

Double-labelled cells (arrow) have a densely stained nucleus and cyto-

plasmic silver precipitate.

concerning the effects of general anesthetic on c-fos immu-

noreactivity in the spinal cord,

it

is likely th at t he numbers

and distribution

of

cells observed in the visceral stimulat ion

studies underestimate what would be generated in the

awake animal.

Several control studies were performed. Two unstimu-

lated, freely moving animals were perfused and the spinal

cord examined. Consistent with the report of Hunt et al.

('87), with the exception of a few lightly labelled neurons in

laminae

I11

and IV, we found almost no c-fos immunoreac-

tive neurons in the spinal cord. In three additional animals,

we studied the effect of injecting the appropriate volume of

saline, into either the plantar foot, the ankle oint (af ter skin

incision),

or

the peritoneal cavity. Two of the rats (plan tar

foot and joint) were perfused 16 hours later; the third was

perfused

1

hour after the intraperitoneal injection. The

same number of sections was studied in control and experi-

mental animals; in none of these animals was there signifi-

cant c-fos expression in the spinal cord. Importantly,

although the skin incision (for urate crystal injection)

evokes the expression of c-fos in the spinal cord, the control

study established that by 16 hours almost no cell labelling

persisted.

R SULTS

Characteristics of C@x-immunoreactive

neurons

As described above, with the exception of a few very

lightly labelled cells in laminae I11 and IV, there were no c-

fos-immunoreactive spinal neurons in control rats. C-fos-

immunoreactive cells were found only in the spinal cord of

rats that experienced a peripheral noxious stimulus (Fig. 1).

In this respect, the spinal cord can be distinguished from

certain brainstem and forebrain areas where we have



186

D.

MENETREY ET AL.

Fig.

7.

These photomicrographs illustrate c-fos-immunoreactive

and retrogradely labelled neurons in a 40-gm thick horizontal section

through the reticular neck of the dorsal horn. With darkfield illumina-

tion

1)

he longitudinal bundles

of

fibers in the reticular par t

of

the dor-

sal horn (Ret

V)

can be distinguished from the

more

medial dorsal horn

(mDH). There are large numbers of retrogradely labelled cells (bright

recorded

a

baseline level of neuronal c-fos-immunoreactiv-

ity. In the case of rats with hindpaw injury, the c-fos positive

neurons were found almost exclusively ipsilateral to the

injured paw and at rostrocaudal levels of the spinal cord

that receive afferent fibers from the injured limb (Devor and

Clayman, '80; Molander and Grant, '85; Swett and Woolf,

'85).

Importantly, since the retrogradely labelled neurons

were found at all levels

of

the spinal cord, but the c-fos

expression was restricted to the lumbosacral cord,

it

is clear

that the c-fos-immunoreactivity was not secondary to the

neuron having transported the retrograde tracer. Moreover,

with the same noxious stimulus, the pattern of c-fos immu-

noreactivity was the same, whether the an imal received the

retrograde tracer or not; thus the pattern of c-fos expression

is not a function of some interaction between the retrograde

tracer and the noxious stimulus.

The c-fos-immunoreactive material was uniformly dis-

tributed in the nucleus of lahelled neurons; nucleoli were

not labelled. This staining patte rn and the overall distribu-

tion of labelled cells was the same whether we used the poly-

clonal

or

the N-terminal directed monoclonal antisera. Al-

though the monoclonal antibody only stained neuronal cell

nuclei, there was some additional staining seen with the

polyclonal antiserum. Specifically, we sometimes noted a

filamentous, cytoplasmic staining in some small cells that

surround the central canal; less commonly, we noted cyto-

plasmic staining in a few cells

of

the substantia gelatinosa

dots) in the reticular neck;some are immediately adjacent to the dorso-

lateral funiculus (DLF). The dorsal columns (DC) are on the left. The

higher magnification brightfield photomicrographs 2 and 3 illustrate

the two double-labelled neurons th at

are

indicated by arrows in 1.Cali-

bration bars equal

50

pm.

and in the latera l spinal nucleus of the dorsolateral funicu-

lus. Immunostained terminals were occasionally recorded in

the superficial dorsal horn. This presumed crossreactivity

was readily distinguished from the diffuse, nuclear c-fos-

immunoreactivity.

Distribution

of

ascending tract cells in the

spinal cord

Consistent with the known cells of origin of the brainstem

and thalamic targets injected with retrograde tracer, the ret -

rogradely labelled spinal projection neurons clustered into

several distinct populations (see references in Leah et al.,

'88). At all lumbar segments, projection neurons were

located in lamina I of th e dorsal horn, in the reticular part of

the neck of the dorsal horn, in laminae VII, VIII, and

X,

and

in t he lateral spinal nucleus of the dorsolateral funiculus.

Two additional clusters of cells were labelled only at upper

lumbar segments. One cluster was at the medial border of

the reticular part of the neck of the dorsal horn; this is

another source of spinal projections to the region of the lat-

eral reticular nucleus (Menktrey et al., '83). The second clus-

ter was located in the most ventromedial part of the dorsal

horn , abutting the dorsal columns; this region contains cells

at the origin of spinothalamic t ract axons (Giesler et al.,

'79;

Menktrey et al., '84b). The majority of the retrogradely

labelled cells were located contralateral to the injection site;

however, a substantial number (30%) were located ipsi-



Cf os

PROTEIN IN RAT SP INAL CORD

187

Fig. 8.

These photomicrographs were taken from a 40-pm horizontal

section that was located slightly dorsal to the section in Figure 6. They

illustrate retrograde labelling in the medial neck

of

the dorsal horn

(mDH). This cluster of retrogradely cells has a very restricted dorsoven-

tra l and rostrocaudal distribution; neurons in this region primarily pro-

ject t o the region of the lateral reticular nucleus (MenBtrey et al.,

'83).

Although c-fos-immunoreactive neurons are found in this region

l ) ,

double-labelled cells were rarely found. The higher magnification

hrightfield photomicrograph

2)

illustrates the double-labelled neuron

that is indicated hy an arrow in 1. The cell is located laterally, in the

reticular neck (Ret

V f

the dorsal horn.

DLF

and DC identify the dor-

solateral funiculus and dorsal columns, respectively. Calibration bar

equals 50 pm.

lateral. The latter are primarily cells at t he origin of the

spinoreticular projections, which are bilaterally projecting

systems (Menbtrey, '87). This widespread distribution

indicates that there was significant transport from all sites

injected with the retrograde tracer; however, since not all

terminal sites could have been injected, the numbers of re t-

rogradely labelled cells, and thus the number of double-

labelled neurons (see below) is certainly underestimated.

Subcutaneous inflammation

C-fos expression was examined

16

hours after unilateral

injection of

150

pl complete Freund's adjuvant in the plan-

tar surface of one hindpaw. The subcutaneous inflammation

that resulted extended over the plantar surface of the paw,

the toes, and the tissue surrounding he ankle. Figure 2 illus-

trates the distr ibution of c-fos-immunoreactive neurons in

the spina l cord of a rat injected with adjuvant. Labelled neu-

rons were found from the

L,

to L, spinal segments and in the

rostral sacral cord. All but a few were ipsilateral to the adju-

vant injection. The density of staining varied over the ros-

trocaudal extent of the cord, with the most densely labelled

nuclei located close to the entry zone (L4and L5)of the affer-

ent fibers that innervate the stimulated area. Lightly

labelled neurons were most commonly found rostral and

caudal to the L4,5segments. They were also found intermin-

gled with the heavily labelled cells of the L, and L, seg-

ments. The greatest concentration of ipsilateral c-fos-

immunoreactive neurons was in the superficial layers of the

dorsal horn, laminae 1and outer 11, in the reticular part of

the neck of the dorsal horn, around the base

of

the dorsal

horn, and in laminae

VII, VIII,

and

X.

Some cells were

found in the lateral spinal nucleus. In some rats, a few cells

were found in the nucleus proprius, laminae I11 and IV, in

the inner par t of the subs tantia gelatinosa, in the medial

part of the neck of the dorsal horn (i.e., medial lamina V),

or

in lamina IX.

Significant differences exist in the rostral caudal extent of

the labelling in these different laminae (Fig.

2 .

The labelled

neurons in outer lamina I1 were the most restricted rostro-

caudally; they were only found at the L4 segment (Fig.

3).

The staining in lamina I, however, extended from the L, seg-

ment to the rostral sacral cord. Labelled cells in lamina I of

the rostral lumbar segments were concentrated medially;

they shifted laterally in more caudal sections. Cells of the

reticular part of the dorsal horn and in laminae VII, VIII,

and

X

had the most extensive rostrocaudal spread, from all

levels of the lumbar cord to the most rostral sacral segments.

Finally, some c-fos-immunoreactive neurons were found in

lamina VIII contralateral to the side of the adjuvant injec-

tion.

C-fos-immunoreactive

projection

neurons. The

distributionof contralaterally and ipsilaterally ascending c-

fos-immunoreactive projection neurons was studied in rats

that received unilateral tracer injections, either contralat-

era1

or

ipsilateral to the inflamed paw. Although

it

is

of

interest to determine the relative proportion of contralater-

ally and ipsilaterally projecting c-fos-immunoreactive cells

in the same animal, we avoided th is approach since it would

have required that th e rats be injected bilaterally with adju-

vant. The tracer injections were made in the thalamus, mid-

brain, nucleus reticularis gigantocellularis, and the region of

the lateral reticular nucleus. As described above, since injec-

tions into the nucleus of the solitary tract typically spread

bilaterally, that injection was omitted for these studies. The

counts of single and double-labeled cells were made in t he



188

D.

MENETREY ET

AL.

Fig. 9. This brightfield photomicrograph is from a 40-pm transverse

section through lamina

X

of the lumbar spinal cord. Arrowheads point

to c-fos immunoreactive cells (with uniform, densely stained nucleus).

Retrogradely labelled cells have punctate cytoplasmic silver precipitate

L,-L,

segments, which contained the majority of the double-

labelled cells.

Figure

4

llustrates cases in which the retrograde tracer

was injected contralateral (A) or ipsilateral

B)

to the

inflamed hindpaw. Double-labelled cells were commonly

observed in all rats that received tracer injections. Figure

schematizes the distribution of these contralaterally and

ipsilaterally projecting c-fos-immunoreactive neurons. All

but some in lamina VIII were located ipsilateral to the

injected hindpaw. The rostrocaudal location of the double-

labelled cells included all lumbar spinal segments; the maxi-

mum concentration ( 8 2 ) was found at segments

L,-L5.

The double-labelled cells constituted only a small propor-

tion of the total population of c-fos-immunoreactive neu-

rons and the total populat,ion of ascending tract cells. The

mean values were 6% and 8 % , espectively. Despite the

wide distribution of both c-fos positive and ascending tract

cells, however,

90%

of the double-labelled cells were clus-

tered in four discrete areas: laminae

I

and outer

11,

the lat-

eral, reticular part of the neck of the dorsal horn, lamina

VIII, and lamina X.

Double-labelled cells

were most commonly recorded in the superficial dorsal

horn. They constituted

37

of all double-labelled cells in

the spinal cord. Almost all were located in segments

L,-Ls.

Very few were found at

L,

and

L,,

where the c-fos positive

cells were located medial to the bulk of the retrogradely

labelled neurons. All but a few of the superficial dorsal horn

double-labelled neurons were in lamina I; the remainder

were located in outer

I1

and were confined to the

L4,6

unc-

tion. Approximately 16% of all the retrogradely labelled

cells and 19% of all the c-fos positive cells in the superficial

dorsal horn at segments

L,-L,

were double-labelled. Figure

6 illustrates examples of double-labelled cells in lamina I.

C-fos-immunoreactive neurons in lamina

I

contribute to

The superBcia1 dorsal horn.

and a clear, unstained nucleus. The open arrows point to retrogradely

labelled, but not immunoreactive neurons. T he arrows point

to

double-

labelled cells that are located just dorsal to the central canal (CC) and

ventral to the border (dotted line) of the dorsal columns (DC).

tive to the total population of contralaterally or ipsilaterally

projecting cells, we found a greater number of superficial

dorsal horn neurons that projected contralaterally. Thus

46 of all c-fos positive contralaterally projecting tract

cells, but only 25% of all c-fos positive ipsilaterally pro-

jecting tract cells were located in the superficial dorsal

horn.

C-fos-immu-

noreactive projection neurons were also common in the lat-

eral, reticular, par t of the neck of the dorsal horn (Figs.

7 , 8 ) .

All double-labelled cells in thi s par t of the cord were located

ipsilateral to the inflamed paw; most were located in the

La-

L,

segments. These cells constituted 24 of all double-

labelled cells recorded in the spinal cord. In contrast to the

cells located in the superficial dorsal horn, they were less

common in contralateral than in ipsilateral projecting pa th-

ways (17% of all c-fos positive contralateral tract cells vs.

32 of all c-fos positive ipsilateral tract cells). Approxi-

mately 10% of all the retrogradely labelled cells and 11 of

all the c-fos positive cells in the lateral part of the neck of

the dorsal horn were double-labelled. The clustering of dou-

ble-labelled cells in this region distinguishes

i t

from the

medially and ventrally adjacent grey matter tha t contains

both c-fos and retrogradely labelled neurons, but very few

double-labelled cells.

The reticular part of the dorsal horn.

Fig.

10.

These schematics illustrate t he distribution of c-fos-immu-

noreactive cells in the lumbar cord of a rat that experienced periarticu-

lar inflammation after implant

of

urate crystals close to the ankle. The

rat was perfused 16 hours after the crystals were implanted. Seven levels

of the cord are represented, from the

L1

hrough the

Ls/S,

segments.

Each diagram includes all labelled cells in three 50-um sections: each dot

both contralateral and ipsilateral ascending pathways. Rela-

represeni$ one labelled cell.



Ank le j o in t u ra te ar t h r i t i s

Figure 10



19

D. MENETREY ET AL.

V isce ra l S t imu la t i o n

a n e s t h e t i z e d )

L6/S1

Figure

11



Thor

V i s c e ra l S t i mu l a t i on

a n e s t h e t i z e d )

L 4

0

1

mm

L6/S1

Fig. 12. These schematics illustrate the distribution of c-fos-immunoreactive ascending tract cells in

response to visceral stimulation. The retrograde tracer was injected into the thalamus, mescencephalon, lat-

eral reticular nucleus, and reticular formation unilaterally and into the nucleus of the solitary tract; the l at-

ter injection spread bilaterally. Each diagram includes double-labelled cells from five 50 pm sections (cells

from both sides of the cord are plotted). Each dot represents one double-labelled neuron.

Fig. 11.

These schematics illustrate the cervical

(C,)

through lumbo-

sacral (LJS,) distribution of c-fos-immunoreactive cells in response to

noxious visceral stimulation (intraperi toneal injection of 0.5 ml of

9%

acetic acid). The experiment was performed under general anesthesia

and the rat was perfused 1hour after visceral stimulation. Each diagram

includes all labelled cells in three 50-pm sections; each dot represents

one labelled cell. Since we found bilaterally symmetric labelling after

visceral stimulation, we have only illustrated the cell distribution on one

side of the cord. The boundary of the reticular par t of the neck of the

dorsal horn is outlined for orientation. Note tha t the densest staining is

at the thoracolumbar junction (T13-LJ and the most extensive rostro-

caudal dist ribution of labelled cells was found in the superficial dorsal

horn.



192

The are surrounding the central cana l.

C-fos-

immunoreactive projection neurons were numerous in the

area surrounding the central canal. They were found in lam-

ina X throughout the lumbar enlargement and at the LZw3

levels, in a region located just dorsolateral to lamina

X.

The

double-labelled cells in the region of the central canal con-

stituted approximately

20%

of all c-fos positive projection

neurons recorded in the lumbar cord. All of these double-

labelled cells were located ipsilateral to the inflamed paw

and were approximately equally divided between contralat-

erally and ipsilaterally projecting pathways. Figure 9 illus-

trates examples of double-labelled cells in lamina

X.

Double-labelled cells in lamina VIII ac-

counted for 9 % of all c-fos positive projection cells recorded

in the lumbar spinal cord. As in the region of the central

canal, contralaterally and ipsilaterally projecting c-fos posi-

tive cells were equally divided. In contrast to the other dou-

ble-labelled cells, however, all c-fos positive projection neu-

rons in lamina VIII were located contralateral to the

inflamed paw. None of the c-fos-immunoreactive neurons in

lamina VIII ipsilateral to the inflamed paw were double-

labelled. Thus the ipsilaterally projecting c-fos positive lam-

ina VIII cells terminated in the brain contralateral to the

injured paw; those projecting contralaterally terminated ip-

silateral to the injured paw. The presence of th e latte r pa th-

way suggests that information from the inflamed paw may,

in part , access the brain ipsilateral t o the injury, via a neural

network th at crosses the cord a t least twice.

Double-labelled cells in the re-

maining areas of the spinal cord accounted for a t most 10%

of

all the spinal c-fos positive projection neurons. These

double-labelled cells were scattered along the lumbar en-

largement in several areas, including the medial part of the

neck of the dorsal horn , the inte rmediate gray between the

dorsal and ventral horns, and the lateral spinal nucleus of

the dorsolateral funiculus.

Periarticdar inflammation

C-fos expression was examined 16 hours af ter unilaterally

implanting 150 kg of urate crystals close to the ankle join t.

The inflammation that resulted extended over the tissue

surrounding the ankle as well as the proximal part of the

dorsum of the foot. The severity and exten t of the inflam-

mation was much less than was seen with adjuvant injection.

Figure 10 illustrates the resulting pattern of c-fos-immuno-

reactivity in these animals. C-fos labelled cells were found

from segments

L1

through L,; almost all were located ipsi-

lateral to the inflamed paw, in lamina I, the lateral neck of

the dorsal horn, a t the base of the dorsal horn, and in lami-

nae VII, VIII, and

X.

Very few labelled cells were recorded

in the outer par t of the substantia gelatinosa or in contralat-

era1 lamina VIII. Thus with the exception of minor differ-

ences in the superficial dorsal horn and the minimal contra-

lateral neuronal labelling, the pat tern of staining after urate

crystal implantation was comparable to that seen after

injection of Freund's adjuvant. The major difference was

quantitative; that is, many more neurons were found after

adjuvant injection, which suggests that the numbers of cells

labelled is related to the severity of the inflammation.

We used the protocol described above to study the distri-

bution of contralaterally and ipsilaterally projecting c-fos

positive ascending tract cells. Double-labelled neurons were

seen in both series of experiments and were concentrated in

lamina I, the reticular neck of the dorsal horn, and to a lesser

extent in lamina

X.

This distribution differed from that

Lamina WII.

Other spinal areas.

D.

MENETREY ET AL.

found afte r adjuvant injection only in t hat fewer double-

labelled cells were recorded.

Visceral noxious stimulation

The entire visceral stimulation protocol, which lasted 1

hour, was carried out under general anesthesia. The pattern

of c-fos staining (Fig.

11)

was significantly different from

that found after subcutaneous or periarticular inflamma-

tion in the awake rat. First, visceral stimulation produced a

much more bilaterally symmetric distribution of c-fos-

immunoreactive neurons, perhaps because of the bilateral

nature of the stimulus (intraperitoneal injection). Second,

the c-fos labelling was not restr icted to t he lumbar enlarge-

ment, but extended from cervical to sacral cord. The

greatest density and largest number of labelled cells was,

however, found a t the thoraco-lumbar junction, segments

T,,-L,. Thi rd, the number of labelled cells in the superficial

dorsal horn was much greater than in deeper areas (77 vs.

23

of all c-fos- immunoreactive cells, respectively).

Fourth, there was a significant difference in the rostrocau-

dal extent of the labelled cells located superficially from

those located in deeper areas. The labelled neurons in the

superficial dorsal horn were the only ones located a t cervical

through sacral levels; the c-fos-immunoreactive neurons in

the neck of the dorsal horn and in lamina

X

were confined to

the thoracolumbar junction. At most levels the marginal

cells spanned the whole mediolateral extent of the gray mat-

ter; at cervical levels, the labelled neurons were located

along the lateral border of the gray matter.

Figure

12

illustrates t he distribution of c-fos positive

ascending tract cells in the spinal cord of a rat that received

a unilateral tracer injection in the thalamus, midbrain,

nucleus reticularis gigantocellularis, and lateral reticular

nucleus contralateral to the side of the acetic acid injection

and into the nucleus of the solitary tract. As described

above, the NTS injection typically spreads bilaterally. Al-

though the c-fos-positive projection cells were located bilat-

erally, we plotted all double-labelled cells on one side. Dou-

ble-labelled neurons were found at upper thoracic through

sacral spinal levels, but were most concentrated at the tho-

raco-lumbar junction. No double-labelled cells were re-

corded a t cervical levels. All but a few of the c-fos-immuno-

reactive projection neurons were located in lamina

I

of the

superficial dorsal horn; occasional cells were recorded in the

white matter adjacent to the neck of the dorsal horn.

Although c-fos-immunoreactive and retrogradely labelled

neurons were found in lamina

X,

none of those were double-

labelled.

DISCUSSION

Consistent with the original report of Hunt et al. ('87), we

found that some spinal neurons express c-fos in response to

noxious peripheral stimulation. We also demonstra ted tha t

different pat terns of c-fos-immunoreactivity are produced

by stimuli targeted at different peripheral structures and

tha t some of those neurons t ha t express c-fos are a t the ori-

gin of major ascending spinal pathways, many of which have

been implicated in the rostra1 transmission of nociceptive

messages.

To study noxious stimulus-evoked expression of c-fos, we

chose two subacute inflammation models an d one acute vis-

ceral stimulation model. These stimulation protocols gener-

ated considerable labelling in laminae I and outer 11, the

neck of the dorsal horn, and in laminae VII, VIII and

X

of



Cf os PROTEIN IN RAT SPINAL CORD

193

the ventromedial gray. Electrophysiological studies have es-

tablished t ha t all of these spinal areas contain nociceptive

cells, many of which are strongly driven under conditions of

inflammation (Menbtrey and Besson, '82; Calvino et al., '87;

Dickenson and Sullivan, '87). It was particularly relevant

that we found dense labelling of neurons in laminae I and

outer 11, bu t rarely in the inner part of the substantia gelati-

nosa, a region that predominantly is activated by nonnox-

ious stimulation (Light et al., '79; Bennett et al.,

'82).

Thus

the distribu tion of c-fos-immunoreactive neurons was es-

sentially restricted to the known distribution of nociceptive

spinal neurons. However, since neither the factors that

induce c-fos expression nor the consequences of expression

of the c-fos protein are known, caution must be taken in

interpreting these data. We cannot conclude that all

labelled cells are nociceptive nor can we conclude that a par-

ticular neuron was transmitting a nociceptive message. The

absence of induction of c-fos in a spinal neuron also does not

mean that the neuron was not nociceptive. The neuron may

not synthesize c-fos, or may only do

so

in amounts too low to

be detec ted by immunocytochemistry. In other words, the

c-fos protein is merely a marker tha t tells us tha t something

happened to that particular neuron in response to the

applied peripheral stimulus. In spite of these caveats, we

believe that this technique allows one to monitor, with sin-

gle cell resolution, t he responsiveness of large populations of

neurons to noxious stimulat ion in awake animals, i.e., in s it-

uations in which the pain behavior of the animal can simul-

taneously be evaluated. Although the 2-deoxyglucose

method (Sokoloff et al., '77) has been used to monitor func-

tional changes in the spinal cord (Ciriello et al., '82; Abram

and Kostreva, '86), the resolution of the technique is poor

and the approach cannot be combined in double-labelling

(i.e., projection neuron) studies. The cytochrome oxidase

method offers cellular resolution but has yet to be used to

map noxious-stimulus evoked changes in metabolic activity

of spinal cord neurons (Wong-Riley and Kageyama,

'86).

Baseline levels of cytochrome oxidase activity are high,

which may make it difficult to detect noxious stimulus-

evoked increases in enzyme activity.

This study revealed several important features of the spa-

tial organization of spinal nociceptive transmission systems.

Firs t, there is a very widespread rostrocaudal distribution of

neurons that respond to a relatively localized peripheral

noxious stimulus. Second, there

is

a differential laminar dis-

tribution of responsive neurons a t different rostrocaudal

levels, and thi rd, there is a differential pattern

of

responsive

neurons produced by subacute somatic or periarticular in-

flammation and acute, visceral stimulation.

Subcutaneous

or

periarticular inflammation produced

neuronal staining through all lumbar segments into the ros-

tral sacral cord;

i t

thus included several segments rostral to

the region (L3-L5),which receives the densest primary affer-

ent fiber input from the stimulated paw. The laminar distr i-

bution of labelling was extensive and included the superfi-

cial dorsal horn, which contained the densest concentration

of cells, the lateral par t of the neck of the dorsal horn, the

intermedia te gray (i.e., laminae VI and VII), and laminae

VIII and

X. It

is of interest that there were nonrandom vari-

ations in the intensity of staining throughout the lumbar

cord. In general the most densely stained cells were located

in the L,-L, segments. The paler staining of neurons located

rostrally suggests that the density of staining is proportional

to the amount of afferent drive.

The most restricted rostrocaudal distribution

of

labelled

cells was found in the superficial laminae. I t appears tha t

the segmental pattern of labelling in laminae

I

and outer

I1

follows the central somatotopic organization of the cuta -

neous afferent fibers that innervate the inflamed area (De-

vor and Claman, '80; Molander and Grant,

'85;

Swett and

Woolf,

'85).

Specifically, the progressive medial shift in the

location of c-fos-immunoreactive neurons in lamina I and

their dropout at more rostral lumbar segments parallelled

the central projection of small diameter primary afferents

from the hindpaw. These data suggest tha t the cells of the

superficial dorsal horn that express c-fos receive a mono-

synaptic input from the small diameter afferent fibers tha t

innervate the inflamed area. The most widespread rostro-

caudal d istr ibution of labelled cells, however, was found in

laminae VII and VIII. Since small diameter , nociceptive pri-

mary afferents do not terminate in these regions (Light and

Perl , '79; Suguira et al., '87), it is likely that the nociceptive

input to cells in laminae VII and VIII is polysynaptic. Fur-

thermore, since single cells in laminae VII and VIII have

rather large cutaneous receptive fields (Fields et al., '75, '77;

Giesler et al.,

'81;

Cervero and Wolstencroft, '84; Men6trey

et al., '84a), the polysynaptic input probably derives from

relatively large regions of the body. Taken together, these

data could account for the widespread rostrocaudal distri-

bution of c-fos immunoreactive neurons in deeper parts of

the spinal cord.

The pattern of c-fos expression produced by visceral stim-

ulation differs significantly from that seen after subacute

inflammation of the paw. The injection into the peritoneal

cavity produced extensive rostrocaudal labelling, from the

cervical through sacral cord. Since there was probably

spread of the injection volume,

it

is possible tha t some

of

the

labelling in the cervical cord reflected activation of dia-

phragmatic afferents. The extensive pattern of labelling

may, however, also relate to t he fac t tha t small diameter pri-

mary afferents that innervate visceral structures arborize

over more segments than do cutaneous or muscle afferents

(Neuhuber, '82; Neuhuber et al., '86; Suguira, submitted).

Interestingly, this extensive d istribution was found

only

for

cells in the superficial dorsal horn; deeper cells (in the neck

of the dorsal horn and in lamina

X)

were confined to the

thoracolumbar junction. Importantly, although the anes-

thet ic conditions under which the visceral experiments were

run may have restricted the c-fos labelling pattern (Presley

et al., unpublished observations), our results are consistent

with anatomical and electrophysiological studies that have

implicated neurons in laminae I and

X

in the central trans-

mission of visceral nociceptive information in the r at (Taka-

hashi and Yokota,

'83;

Ness and Gebhart , '87). In fact, the

very limited expression of c-fos of neurons in the substantia

gelatinosa after acetic acid injection is consistent with the

sparse projection of visceral afferents to lamina I1 (Cervero

and Connell, '84; Morgan et al.,

'81).

The great advantage of a functionally oriented anatomi-

cal approach is that the activity

of

large numbers of neu-

rons can be readily identified. Although electrophysiological

studies can unequivocally characterize the nociceptive

properties of spinal cord neurons, it is both difficult and

time-consuming to locate a large sample of physiologically

characterized projection neurons tha t can be antidromically

activated. Monitoring the noxious stimulus-evoked expres-

sion of the c-fos protein in spinal cord projection neurons

thus powerfully complements electrophysiological studies.



194

We found that t he c-fos positive ascending tract cells con-

stituted a small percentage of all spinal neurons that

expressed c-fos and of all neurons that were retrogradely

labelled. These data suggest that most of the cells which

express c-fos in response to noxious stimulat ion make short

intraspinal connections. These may be interneurons

or

pro-

priospinal neurons. One explanation for the relatively low

percentage of double-labelled neurons is that none of the

ascending pathways studied are purely nociceptive. In fact,

in the basal part of the dorsal horn and medioventral horn of

the rat spinal cord there are many nonnociceptive spino-

thalamic and spinoreticular neurons that transmit cuta-

neous or proprioceptive messages (Menktrey et al., ’84a,b;

Ness and Gebhart,

’87).

These are intermingled with the

nociceptive projection neurons and might be retrogradely

labelled, but would probably not express c-fos under the

experimental conditions we used. It is also certain that only

a part of all the nociceptive afferents were activated with the

stimulation parameters used. Thus, for example, any spinal

neurons selectively responsive to noxious stimulation of

muscles or joints would have been only minimally activated

by subcutaneous injection of adjuvant into the hindpaw.

It

was highly significant that the double-labelled c-fos

cells were concentrated in very discrete spinal areas. In fact,

the regional distribu tion of nociceptive cells, defined in

electrophysiological studies, and of ascending t rac t cells,

defined in anatomical studies (refs. in Menktrey, ’87 , is

considerably larger than the regional distribution of c-fos

positive ascending tract cells. Thi s indicates that among the

many regions of the cord that contain nociceptive neurons,

the following regions, the superficial dorsal horn, the late ral

neck of the dorsal horn, and laminae VIII and

X,

may be

particularly relevant to the transmission of nociceptive mes-

sages to supraspinal levels via long ascending tracts.

A major goal of pain research studies is to understand the

relative contribution of the different types of nociceptive

neurons, in the different regions of the cord, to the conscious

appreciation of pain. With rare exception, electrophysiolog-

ical studies are performed in anesthetized or decerebrate-

spinal preparations. Thus the spinal cord neuronal activity

in an animal that experiences pain cannot be evaluated. By

monitoring noxious stimulus-evoked c-fos expression in

projection neurons in awake animals, we can draw some con-

clusions about the overall pattern of activity generated

by

pain-producing stimuli. The superficial dorsal horn con-

tains wide dynamic and nociceptive specific neurons (class

2

and 3, respectively). Most of these have relatively restricted

receptive fields, which are somatopically organized within

the cord. Nociceptive neurons of the lateral part of the neck

of the dorsal horn are predominantly wide dynamic range

cells and have a higher degree of spatial and/or modality

convergence. Neurons in both regions are believed to con-

tribute to the detection and localization of noxious stimuli.

On the other hand, the class 3, nociceptive-specific neurons

of deeper parts of the cord (laminae VII and VIII) have com-

plex excitatory and inhibitory receptive fields tha t are much

larger and are often bilateral. These neurons are thus proba-

bly not involved in the location and discrimination of the

noxious stimulus (as are the dorsal horn nociceptive neu-

rons), but rather have been implicated in the escape reac-

tions and motor responses produced by noxious stimuli.

Since we have shown that ascending projection neurons in

all of these regions are activated under conditions of tonic

pain, the present data indicate that a wide variety of noci-

ceptive projection neurons come into play when an animal

D.

MENETREY

ET AL.

experiences a tonic noxious stimulus. Since different neu-

rons within t he four major regions in which double-labelled

cells were located project to brainstem and thalamus, we

cannot be certain of the specific targets of the projection

neurons that express c-fos. This, however,

is

the first case in

which it has been demonstrated that these pathways are

indeed activated by noxious stimuli in the awake animal; we

are presently studying the projection of c-fos-immunoreac-

tive neurons to particular brainstem and thalamic loci.

In conclusion, this study provides evidence that c-fos

is

expressed in subpopulations of spinal cord cells and that it

can be used as a functional marker for populations of noci-

ceptive cells, including those a t the origin of long ascending

tracts. Th e findings of this st udy emphasize the contribu-

tion of neurons in the superficial laminae, in the lateral neck

of

the dorsal horn, laminae VIII and X to the central trans-

mission of nociceptive information in animals tha t experi-

ence pain. The ability to monitor the “activity” of large

numbers of nociceptive neurons provides a comprehensive

anatomical basis for studies of the mechanisms through

which these neurons are controlled. For example, by moni-

toring the changes in noxious stimulus-evoked expression

of

c-fos that a re produced by systemic, intracerebral, or spinal

administrat ion of narcotics, it should be possible to better

understand the relative contribution

of

spinal and supraspi-

nal mechanisms to opiate analgesia (Presley et al., ’88a).

ACKNOWLEDGMENTS

We thank Mme. Annie Menktrey and Ms. Simona Ikeda

for excellent help with graphics and photography. We also

thank Dr. Jan Tuttleman for providing some of the antisera

and for her helpful suggestions as to the ir use. This work was

supported by PHS grants NS14627, NS21445 and

AM32634. D. Menktrey is supported by the Cent re National

de la Recherche Scientifique, France, and a fellowship from

NATO.

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