menetrey et al 1989
TRANSCRIPT
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 1/20
See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/20651689
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
Impact Factor: 3.23 · DOI: 10.1002/cne.902850203 · Source: PubMed
CITATIONS
476
READS
73
4 AUTHORS, INCLUDING:
Allan I Basbaum
University of California, San Francisco
346 PUBLICATIONS 36,537 CITATIONS
SEE PROFILE
Available from: Allan I Basbaum
Retrieved on: 23 March 2016
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 2/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 3/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 4/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 5/20
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 6/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 7/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 8/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 9/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 10/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 11/20
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-
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 12/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 13/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 14/20
Ank le j o in t u ra te ar t h r i t i s
Figure 10
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 15/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 16/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 17/20
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
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 18/20
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.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 19/20
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.
LITERATURE
CITED
Abram,
S.E.,
and D.R . Kostreva (1986) Spinal cord metabolic response to
noxious radiant heat stimulation of the cat hind footpad. Brain. Res.
385143-147,
Basbaum, A.I., and D. Menktrey (1987) Wheat germ agglutinin-apoHRP
gold: A new retrograde tracer for light- an d electron- m icroscopic single-
and double-label studies. J. Com p Neurol. 261:306-318.
Bennett,
G.J.,
M. Abdelmoumene, H. Hayashi, and R. Dubner (1980). Physi-
ology and morphology of substantia gelatinosa neurons intracellularly
stained with horseradish peroxidase. J. Comp. Neurol. 194t809-827.
Besson, J.M., and A. Chaouch (1987) Peripheral and spinal mechanisms of
nociception. Physiol. R ev. 67:67-186.
Bromberg, M.B., and
E.E.
Fetz (1977) Responses of single units in cervical
spinal cord of alert m onkeys. Exp. Neurol. 55r469482.
Calvino, B., L. Villanueva, and D. LeBars (1987) Dorsal horn (convergent)
neurones in the intact ana esthetized arth ritic rat. I. Segmental excitatory
influence. Pain, 28t81-98.
Cervero F., and L.A. Conn ell(l984 ) Distribution of somatic and visceral pri-
mary afferent fibers within the thoracic spinal cord of the cat.
J.
Comp.
Neurol. 230:8&98.
Cervero, F., and J.H . Wolstencroft (1984) A positive feedback loop between
spinal cord nociceptive pathways and antinociceptive areas of the cat’s
brain stem. Pain, 2Ot125-138.
Ciriello, J., C.V. Rohlicek, R.S. Poulsen, and C. Polosa (1982) Deoxyglucose
uptake in th e rat thoracolumbar cord during activation of a ortic barore-
ceptor afferent fibers. Brain. Res. 231~240- 245.
Coderre,
T.,
and P.D . Wall (1987) Ankle joint urate arthritis in rats: an alter-
native animal model of arthritis to that produced by Freund’s adjuvant.
Pain 28t379-393.
8/19/2019 Menetrey Et Al 1989
http://slidepdf.com/reader/full/menetrey-et-al-1989 20/20
Crfos PROTEIN IN RAT SPINAL CORD
195
Collins, J.G. (1987)A descriptive study of spinal dorsal horn neurons in the
physiologically in tact, awake, drug free cat. Brain Res. 416:3442.
Curran, T., and J.I. Morgan (1985) Superinduction of c-fos by nerve growth
factor in the presence of peripherally active benzodiazepines. Science
229:1265-1268.
Danscher, G. (1981) Localization of gold in biological tissue. A photochemical
method for light and electronmicroscopy. Histochemistry 71331-88.
Devor, M., and D. Claman (1980) Mapping and plasticity of acid phosphatase
afferents in t he r at dorsal horn. Brain Res. 190:17-28.
Dickenson, A.H., and A.F. Sullivan (1987) Subcutaneous formalin-induced
activity of dorsal horn neurons in the rat: differential response to an
intra theca l opiate administered pre or post formal in. Pain 30:349-360.
Duncan. G.H.. C.M. Bushnell. R. Bates. and R. Dubner (1987) Task related
.
responses of monkey medullary dorsal horn neurons.
J.
Neurophysiol.
57:289-310.
Fields, H.L., C.H. Clanton, and S.D. Anderson (1977) Somatosensory proper-
ties of spinoreticular neurones in th e cat. Brain Res. 120:49-66.
Fields, H.L., G.M. Wagner, and S.D. Anderson (1975) Some properties of spi-
nal neurones projecting to the medial brainstem reticular formation. Exp.
Neurol. 47:11%134.
Giesler, G.J. Jr., D. Menetrey, and A.I. Bashaum (1979) Differential origins of
spinothalamic tract projections to medial and lateral th alamus in th e rat.
J. Comp. Neurol. 184:107-126.
Giesler, G.J. Jr., R.P. Yezierski, K.D. Gerhart, and W.D. Willis (1981) Spino-
thalamic tract neurons that project to medial and/or lateral thalamic
nuclei: evidence for a physiologically novel population
of
spinal cord neu-
rons. J. Neurophysiol. 46:1285-1308.
Greenberg, M.E., and E.B.
Ziff
(1984) Stimulation of 3T3 cells induces tran-
scription of the c-fos proto-oncogene. Nature 311:433-438.
Greenberg, M.E., L.A. Greene, and E.B. Ziff (1985) Nerve growth factor and
epidermal growth factor induce rapid transient changes in proto-onco-
gene transcription in PC12 Cells. J. Biol. Chem. 26Or14101-14110.
Greenberg, M.E., E.B. Ziff, and L.A. Greene (1986) Stimulation of neuronal
acetylcholine receptors induces rapid gene transcription. Science 234:80-
83.
Hayes, R.L., R. Dubner, and D.S. Hoffman (1981) Neuronal activity in
medullary dorsal horn of awake monkeys trained in thermal discrimina-
tion task.
11
Behavioral modulation of responses to thermal and mechan-
ical stimuli. J. Neurophysiol. 46:428-443.
Hsu., S., L. Raine, and H. Fanger (1981)
A
comparative study of the anti -
peroxidase method and an avidin-biotin complex method for studying
polypeptide hormones with radioimmunoassay antibodies. Am. J. Clin.
Pathol. 75336738.
Hunt,
S.P.,
A.
Pini, and G. Evan (1987) Induction of c-fos-like protein in spi-
nal cord neurons following sensory stimulation. Nature 328:632-634.
Kruijer, W., D. Schubert, and LM. Verma (1985) Induc tion of the proto-onco-
gene fos by nerve growth factor. Proc. Natl. Acad. Sci. (USA) 823330-
7334.
Kruijer, W., J.A. Cooper,T. Hunter, and I.M. Verma (1984) Platelet-derived
growth factor induces rapid but transient expression of the c-fos gene and
protein. Nature 312.711-720.
Leah, J., D. Menetrey, and J. de Pommery (1988) Neuropeptides in long
ascending tract cells in the rat: Evidence for parallel processing of
ascending information. Neuroscience 24r195-207.
Light, A.R., and E.R. Perl (1979) Spinal termination of functionally identi-
fied primary afferent neurons with slowly conducting myelinated fibers.
J.
Comp. Neurol. 186:133-150.
Light, A.R., D.L. Trevino, and E.R. Perl (1979) Morphological features of
functionally identified neurons in the marginal zone and substantia gela-
tinosa of the spinal dorsal horn. J. Comp. Neurol. 186:151-171.
Menetrey, D. (1985) Retrograde tracing of neural pathways with a protein-
gold complex.
I.
Light microscopic detection after silver intensification.
Histochemistry 83:391-395.
Menetrey, D. (1987)
Spina l nociceptive neurons a t the origin of long
ascending pathways in the rat: Electrophysiological, anatomical
and
immunohistochemical approaches. In J.-M. Besson, G. Guilbaud, and M.
Peschanski (eds): Thalamus and Pain. Amsterdam, NY: Excerp ta Med-
ica, pp. 21-34.
Menetrey, D., and A.I. Basbaum (1987) Spinal and trigeminal projections to
the nucleus of the solitary tract: A possible substrate for somatovisceral
and viscerovisceral reflex activation. J. Comp. Neurol. 255r439-450.
Menetrey, D., and J.-M. Besson (1982) Electrophysiologicalcharacteristics
of
dorsal horn cells in rats with cutaneous inflammation resulting from
chronic arthr itis . Pain 13:343-364.
MenBtrey, D.,
J.
De Pommery, and J.-M. Besson (1984a) Electrophysiological
characteristics of lumbar spinal cord neurones backfired from the la teral
reticular nucleus in the rat.
J.
Neurophysiol. 52r595-611.
Menetrey, D., J. De Pommery, and F.Roudier (1984b) Properties of deep spi-
nothalamic tract cells in the rat, with special reference to ventromedial
zone of lumbar dorsal horn. J. Neurophysiol. 52:612-624.
Menktrey, D., F. Roudier, and J.-M. Besson (1983) Spina l neurones reaching
the lateral reticular nucleus as studied in the rat by retrograde transport
of horseradish peroxidase. J. Comp. Neurol. 220:439-452.
Molander, C., and G. Grant (1985) Cutaneous projections from the rat hind-
limb foot to the substantia gelatinosa of th e spinal cord studied by trans-
ganglionic ransport of WGA-HRP conjugate.
J.
Comp. Neurol. 237:476-
484.
Molander, C.,
Q.
u, and G. Grant (1984) The cytoarchitectonic organization
of the spina l cord in the rat. I. Th e lower thoracic and lumbosacral cord.J.
Comp. Neurol. 230:133-141.
Morgan, J.I., an d
T.
Curran (1986) Role of ion flux in the control of c-fos
expression. Nature 322552-555.
Morgan, C.,
I.
Nadelhaft, and W. de Groat (1981) The distribution of visceral
primary afferents from th e pelvic nerve to Lissauer’s tract and the spinal
gray matter and its relationship to t he sacral parasympatheticnucleus.
J.
Comp. Neurol. 201:415-440.
Ness, T.J., and G.F. Gebhart (1987) Characterization of neuronal responses
to noxious visceral and somatic stimuli in the medial lumbosacral spinal
cord of the rat. J. Neurophysiol. 57:1867-1892.
Neuhuber, W. (1982) Th e central projections of visceral primary afferent
neurons of the inferior mesenteric plexus and hypogastric nerve and the
location of t he related sensory and preganglionic sympatheti c cell bodies
in the rat. Anat. Emhryol. 164~413425.
Neuhuber, W.L., P.A. Sandoz, and
T.
Fryscak (1986) The central projections
of primary afferent neurons of greater splanchnic and intercostal nerves
in the rat. Anat. Embryol. 174:123-144.
Otsuki, T., H. Nakahama, H. Niizuma, andJ. Suzuki (1986) Evaluation of the
analgesic effects of capsaicin using a new rat model for tonic pain. Brain
Res. 365r235240.
Paxinos, G., and C. Watson (1986) The Ra t Brain in Stereotaxic Coordinates.
Australia: Academic Press.
Presley, R., D. Menetrey, J.D. Levine, and A.I. Basbaum
1988a)
Morphine
suppresses th e noxious stimulation-evoked expression of th e c-fos proto-
oncogene product in spinal neurons. Neurosci. Absts. 18(in press).
Presley, R., D. Menetrey, J.D. Levine, and A.I. Basbaum (1988b)Pentobarbi-
ta l anesthesia suppresses expression of c-fos oncogene in rat spinal cord
neurons. Anesthesia and Analgesia. (abstract) in press.
Ran, W., M. Dean, R.A. Levine, C. Henkle, and
J.
Campisi (1986) Induction
of c-jos and c-myc mRNA by epidermal growth factor or calcium iono-
phore is CAMPdependent. Proc. Natl. Acad. Sci. (U.S.A.) 83:821%3220.
Ruda, M.A., M.J. Iadarola, L.V. Cohen, and W.S. Young, 111 (1988)
In
s tu
hybridization histochemistry and immunocytochemistry reveal an in-
crease in spinal dynorphin biosynthesis in a ra t model of peripheral
inflammation and hyperalgesia. Proc. Natl. Acad. Sci. (USA) 85r622-
626.
Sokoloff, L., M. Reivich, C. Kennedy, M.H. Des Rosiers, C.S. Patlak, K.D.
Pettigrew,
0.
Sakurada, and M. Shinohara (1977) The [“C] deoxyglucose
method for the measurement of local cerebral glucose utilization: Theory,
procedure, and normal values in the conscious and anesthetized albino
rat. J. Neurochem. 28397-916.
Sorkin, L.S.,
T.J.
Morrow, and K.L. Casey (1988) Physiological dentification
of afferent fibers and postsynaptic sensory neurons in the spinal cord of
the intac t, awake cat. Exp. Neurol. 99:412-427.
Sugiura, Y., C.L. Lee, and E.R. Perl (1987) Central projections of identified,
unmyelinated C) afferent fibers innervating mammalian skin. Science
234:35%361.
Sugiura, Y., N. Terui,
Y.
Hosoya, and K. Kohna (1988) Distribution of
unmyelinated primary afferent fibers in the dorsal horn (submitted)
Swett, J.E., and
C.J.
Woolf
(1985)The
somatotopic organization of primary
afferent terminals in the superficial laminae of the dorsal horn of the ra t
spinal cord.
J.
Comp. Neurol. 231:6&77.
Taber , R.I., D.D. Greenhouse, J.K. Rendell, and
S.
Irwin (1969) Agonist an d
antagonist interactions of opioids on acetic acid-induced abdominal
stret ching in mice. J. Pharm. Exp. Ther. 169:29-38.
Takahashi, M., and
T.
Yokota (1983) Convergence of cardiac and cutaneous
afferents onto neurons in th e dorsal horn of the spinal cord in th e cat.
Neurosci. Lett. 38:251-256.
Willis, W.D. (1985) The pain system. T he neural basis of nociceptive trans-
mission in the mammalian nervous system. In P. Gildenherg (ed): Pain
and Headache. Karger, pp. 346.
Wong-Riley, M.T.T., and G.H. Kageyama (1986) Localizationof cytochrome
oxidase in the mammalian spinal cord and dorsal root ganglia with quan-
titat ive analysis of ventral horn cells in monkeys.
J.
Comp. Neurol.
245:41-61.