Nociception-driven decreased induction of Fos protein in ventral hippocampus field CA1 of the rat

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Brain Research 1004 (2004) 167–176

Research report

Nociception-driven decreased induction of Fos protein in ventral

hippocampus field CA1 of the rat

Sanjay Khanna*, Lai Seong Chang, Fengli Jiang, Han Chow Koh

Department of Physiology (MD9), National University of Singapore, 2 Medical Drive, Singapore 117597, Singapore

Accepted 7 January 2004

Abstract

To test the hypothesis that the hippocampus field CA1 is recruited in nociceptive intensity-dependent fashion in the formalin model of

inflammatory pain, we determined the effect of injection of formalin (0.625–2.5%) on the induction of Fos protein along the length of the

hippocampus. Compared to injection of saline, injection of formalin (0.625–2.5%) evoked a concentration-dependent increase in nociceptive

behavior and a significant linear increase in the number of Fos-positive cells in the spinal cord, especially in the deeper laminae. Injection of

saline also increased induction of Fos along the length of hippocampus. On the other hand, injection of formalin decreased the number of

Fos-positive cells in whole CA1, CA3 and dentate gyrus, with a greater significant effect in the posterior–ventral regions of the

hippocampus. Indeed, a formalin concentration-dependent decrease was observed in the ventral CA1. A systematic pattern of change in Fos

induction was not observed in the medial septum region. Of the regions examined, only the formalin-induced changes in Fos cell counts in

the posterior and ventral CA1 were tightly correlated with the changes observed in the spinal cord. The foregoing findings suggest that

nociceptive information is processed in distributed fashion by the hippocampus, and at least the ventral CA1 is implicated in nociceptive

intensity-dependent integrative functions.

D 2004 Elsevier B.V. All rights reserved.

Theme: Sensory systems

Topic: Pain modulation: anatomy and physiology

Keywords: Fos; Formalin model; Spinal cord; Hippocampus; Pyramidal cell layer; Noxious intensity-dependent

1. Introduction Fos [2,11] and Egr1 [24,33] in the hippocampal formation,

The hippocampal formation, which has long been impli-

cated in learning and memory functions, has also been

implicated in affective-motivational response to noxious–

aversive events. For example, microinjection of the local

anaesthetic lidocaine or a glutamate receptor antagonist into

the dorsal hippocampal formation of the rat attenuated the

nociceptive behavior to the unconditioned hind paw injec-

tion of the algogen formalin [18,19]. Moreover, recent

studies have provided evidence that peripheral noxious

stimulation, achieved by manipulations such as hind paw

injections of formalin or high-intensity peripheral electrical

stimulation excited or inhibited the activity of putative

pyramidal cells in anterior hippocampal field CA1 [15,33].

Such manipulations were also found to alter the induction of

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.01.026

* Corresponding author. Tel.: +65-6874-3665; fax: +65-6778-8161.

E-mail address: [email protected] (S. Khanna).

Fos and Egr1 being transcription proteins that are expressed

in neurons following synaptic excitation. As regards Fos, it

is at present contentious whether the induction of this

marker is enhanced [2], decreased [11] or unaffected [24]

with noxious stimulation. The increase was reported with

subcutaneous injection of the noxious agent formalin in

behaving rat [2]. On the other hand, a decrease was

observed with noxious mechanical stimulation of incisor

tooth pulp in anesthetized animal [11]. Such decrease was

stimulus intensity-dependent.

In the present study, we have extended our investigations

into formalin-induced hippocampal CA1 nociceptive

responses to behaving animals so as to explore and re-assess

in the formalin test the change and nociceptive–intensity

relationship in the induction of CA1 Fos over a concentra-

tion range of the algogen (0.625–2.5%). Such stimulation–

intensity relationship has not been tested for formalin in

behaving animal. The change in hippocampal Fos was

S. Khanna et al. / Brain Research 1004 (2004) 167–176168

assessed in relationship to changes in induction of Fos in the

spinal cord. We anticipated that spinal Fos and animal

nociceptive behavior will be, at least partly, proportional

to the concentration of formalin. Such expectation was

based on the previous evidence suggesting that the formalin

concentrations selected for the present study evoke dose-

dependent, morphine-sensitive increases in a variety of

aversive behaviors, including licking, lifting, and shaking

the injured paw [1,9,22]. Indeed, the induction of Fos

protein in the spinal cord parallels the intensity of noxious

stimulation [8,10].

Additionally, in the above experiments we mapped

changes in Fos induction along the anterior–posterior and

the dorsal–ventral axis of the field CA1 whereas previous

investigations have mostly focused on the anterior/dorsal

hippocampus [2,11,24,33]. The reason we analyzed the

changes along the length of the hippocampus was because

the hippocampus exhibits significant regional variations in

connectivity, physiology and function [3,21]. For example,

in the rat, damage to the anterior/dorsal hippocampus affects

spatial memory but damage to the posterior/ventral hippo-

campus does not. Conversely, lesion of the ventral hippo-

campus, but not the dorsal three-quarters decreases rat fear-

related behavior on elevated plus-maze [16].

We also mapped the effects of formalin on Fos induction

in regions that strongly influence CA1. These included (a)

hippocampal field CA3, which provides powerful excitatory

drive to CA1 pyramidal neurons [3,4], (b) the dentate gyrus

(DG), which projects to CA3, and (c) the medial septum–

vertical limb of the diagonal band of Broca (MS-VLDBB),

which is reciprocally connected to the hippocampal forma-

tion and influences the neural activity in the region, includ-

ing nociceptive activity in anterior field CA1 [15,35].

2. Experimental procedure

2.1. Animals

Adult male Sprague–Dawley rats (derived from Charles

River stock obtained from Laboratory Animals Center,

National University of Singapore) were used in these

experiments. All efforts were made to minimize animal

suffering and to minimize the number of animals used.

The local animal committee of the National Medical Re-

search Council, Singapore, approved the experimental pro-

cedures. Fos immunocytochemistry was performed on brain

tissue (n = 7 animals per formalin injected group, formalin

concentration injected was either 0% (saline group),

0.625%, 1.25% or 2.5% and in most instances spinal cord

(n = 2 for the saline group, and n = 5 for each formalin-

injected group), obtained after sacrificing the rats (270–295

g) 2 h after injection of formalin (made from 37% formal-

dehyde; Merck). An additional three rats were injected with

saline and the spinal cord immunocytochemically processed

for Fos protein.

The animals were habituated for 7–10 days to the

laboratory and the observers prior to formalin test. During

habituation, the animals were placed in a clear plastic

chamber that also served as the observation chamber during

the day the test was carried out. Formalin (0.1 ml in saline)

or saline (0.1 ml) was injected subcutaneously with 27G

needle into the plantar surface of the right hind paw while

the rat was restrained manually. Injection of formalin

evoked typical nociceptive behavioral responses from the

animal that included lifting, licking and favoring the affect-

ed paw.

2.2. Fos immunocytochemistry

The method of Smith and Day [31] was adapted for Fos

immunocytochemistry. Two hours after injection of formalin

or saline, the animal was deeply anaesthetized with urethane

(1.5 g/kg, i.p.), transcardially perfused with 1% sodium

nitrite solution (Merck), followed by 4% paraformaldehyde

solution (Merck). The two solutions were made in 0.5 and

0.1 M sodium phosphate (Merck) buffer, respectively. The

spinal cord and brain were removed, blocked, post-fixed in

the above fixative for approximately 4 and 24 h, respec-

tively, at 4 jC. Coronal 60-Am sections were taken through

the forebrain and the lumbar spinal cord on vibratome

(Campden Instruments) and collected in 0.05 M Tris-buff-

ered saline (Fisher Scientific).

Alternate sections were immunolabeled for Fos protein

and the ABC technique was applied to detect the antigen.

All procedures were carried out at 4 jC unless otherwise

specified. Briefly, the tissue sections were rinsed with

0.3% hydrogen peroxide (Merck) and then incubated for

2 h at room temperature in the blocking solution of 3%

bovine serum albumin (Sigma) in 0.05 M Tris-buffered

saline with 0.3% Triton X-100 (Merck). Subsequently, the

tissues were rinsed and incubated for 68–69 h with the

primary antibody (1:2000 rabbit anti-Fos polyclonal anti-

body, Ab-5 from Oncogene), followed by overnight

incubation with 1:1000 biotinylated goat anti-rabbit anti-

body (Calbiochem). Finally, the tissues were treated with

avidin–biotin–peroxidase complex for 3 h at room tem-

perature followed by diaminobenzidine treatment (Sigma;

[31]). Once the brown immunolabel developed, the tis-

sues were mounted on chrome alum gelatin-coated slides,

air dried, dehydrated via ethanol and ethanol–xylene and

cover-slipped with DePeX (Merck). The Fos like-immu-

noreactivity (FLI) was visualized as brown reaction

product. Omission of the Fos antibody abolished the

labeling.

2.3. Behavior

The animal nociceptive behavior was assessed by calcu-

lating the duration of licking of the affected paw for 5-min

periods for total duration of 60 min following injection of

formalin. Behavioral observations were carried out in the

Fig. 1. Induction of spinal Fos like-immunoreactivity (FLI) and animal-licking behavior following subcutaneous injection of formalin into the plantar surface of

the right hind paw. (A) is the digital image of the coronal section through the L4 lumbar spinal cord of animal injected with 0.625% formalin (0.1 ml in saline).

The digital image was developed at 600 dpi. Cells expressing FLI stand out darkly stained relative to the background. Note the dense label in the different

laminae of the right spinal cord ipsilateral to the injection. The diagram at top right in (A) illustrates the laminar subdivisions of the lumbar spinal cord. The

plots in (B), (C) and (D) illustrate the formalin concentration-dependent increase in animal licking behavior (B and C) and the number of Fos-positive cells in

spinal cord (D). The data are represented as meanF S.E.M. Note the typical biphasic increase in licking of the injected paw with formalin, but not saline

injection (B). Formalin or saline was injected at time 0 min and the duration of licking (s) was calculated for blocks of 5 min. The histogram plot (C) illustrates

the duration of licking in the second phase (from 16th to 60 min) of animal response following injection of one of the different concentrations of formalin or

saline. The histogram plot (D) illustrates the effect of injection of saline and of different concentrations of formalin on the Fos-positive cell count from the

whole spinal cord ipsilateral to the injection site. Note that the licking behavior, and spinal Fos peaked at formalin concentration of 1.25% (significant

difference * vs. saline group, # vs. 0.625% formalin group).

S. Khanna et al. / Brain Research 1004 (2004) 167–176 169

Fig. 3. Formalin-induced decrease in number of Fos-positive cells in

pyramidal cell layer of posterior field CA1. The posterior region of the

hippocampus, depicted in the diagram at top, is C-shaped in coronal

sections. The posterior CA1 consisted of posterior–dorsal (Dorsal in the

diagram) and ventral (Ventral in the diagram) subdivisions separated by the

intervening field CA2. The panels (middle and bottom rows) are digitized

images (600 dpi) through posterior–dorsal (in the region around the top

arrow in the diagrammatic representation) and ventral (in the region around

the lower arrow in the diagrammatic representation) CA1 pyramidal cell

layer from the coronal brain sections obtained from rat injected with saline

or 0.625% formalin.

Fig. 2. Formalin-induced decrease in number of Fos-positive cells in

pyramidal cell layer of anterior field CA1. At top is the diagrammatic

representation of the anterior hippocampus. The broken lines in the diagram

delineate the pyramidal cell layers of CA1 and CA3, and the dentate gyrus

(DG) granule cell layer. The panels at bottom are digitized images (600 dpi)

through CA1 (in the region around the arrow in the diagrammatic

representation) pyramidal cell layer from the coronal brain sections

obtained from rat injected with saline or 0.625% formalin.


clear plastic chamber that was used to habituate the animal

(see above).

2.4. Data analyses

The spinal and hippocampal Fos-positive cells were

counted manually using an Olympus microscope. For

counting, Fos-positive cells were identified by brown

nucleus that was distinct from background at 40�, and

100�. A grid was inserted into the eyepiece that facili-

tated counting in non-overlapping squares over the region

of interest. The Fos-positive cells were counted for the

spinal cord on the right side (ipsilateral to the injection

site) of the lumbar L4 spinal segments. The total count

was averaged for sections of each animal and then for the

experimental group. In addition, laminar specific counts

were also made for the following: superficial dorsal horn

(laminae I–II), nucleus proprius (laminae III–IV), neck of

dorsal horn (laminae V–VI) and the ventral gray (laminae

VII–X). The counts were averaged as explained above.

The number of spinal sections through L4 per animal that

were analyzed for FLI cells were, on average 24. The

spinal segmental level and the laminar organization were

determined by the configuration of the gray matter (Fig. 1;

[20,23,27]).

Within the hippocampal formation, Fos-positive cells

were counted in the pyramidal cell layer of CA1 and CA3,

and dentate granule cell layer from sections corresponding to

P 1.80 mm to P 6.04 mm [23]. The contours of the cell layers


of the hippocampus and DG stood out in sections due to

dense packing of the principle neurons in these layers as

compared to the surround (e.g. Figs. 2 and 3). As with spinal

cord, the total count for each hippocampal region was

averaged for sections of each animal, and then for the

experimental group. Such averages were also calculated for

anterior (P 2.3 mm to 4.52 mm), and posterior (P 4.8 mm to

6.04 mm) segments. The posterior region was demarcated

from the anterior by the C-shaped organization of the

hippocampal formation in coronal sections (Figs. 2 and 3).

Further, in case of field CA1, the posterior region was

demarcated in frontal sections into dorsal and ventral divi-

sions based on the intervening field CA2 (Fig. 3; [23]).

Averages were also calculated for ventral (P 4.8 mm to 6.04

mm) CA1. Fos-positive cells were also counted in the MS-

VLDBB corresponding to A 1.20 mm to P 0.26 mm based on

the configuration of the region by Paxinos and Watson [23].

Counts were averaged as described above. The numbers of

sections corresponding to whole CA1, CA3, DG and MS-

VLDBB were, on average 21, 23, 24 and 8, respectively.

Fig. 4. Effect of formalin concentrations on the Fos-positive cell counts from differe

are reported as meanF S.E.M. of Fos-positive cells per section of the selected re

formalin evoked an increase in number of Fos-positive cells in laminae I– II (A),

significant effect was observed in laminae (III– IV), although post hoc comparison

for linear trend between means indicated a significant linear increase with injection

trend for the deeper laminae being most dynamic [(V–VI, r2 = 0.59), (VII–X, r2

The results are expressed as meanF S.E.M. The data was

validated for homogeneity of variance using Bartlett’s test

which was followed by statistical comparison using one-way

ANOVA. Subsequently, post hoc comparisons and analysis

of trends were performed using Newman–Keul’s test for

multiple comparisons and test for linear trends, respectively.

Comparison of data from two groups was performed using

two-tailed unpaired t-test. Statistical significance was ac-

cepted at p< 0.05. Spearman’s correlation analysis of data

was used to determine relationship between changes in Fos-

positive cells between regions of interest (see Results).

3. Results

3.1. Effect of injection of formalin on animal licking

behavior, and spinal Fos

Injection of saline into the right hind paw evoked little or

no licking of that paw (Fig. 1B). However, injection of

nt laminar regions of the spinal cord ipsilateral to the injection site. The data

gion averaged for the group. Compared to injection of saline, injection of

V–VI (C), VII–X (D) (significant difference * vs. saline group). A main

indicated that the various groups were not different from each other. The test

of saline and different formalin concentrations in the above laminae with the

= 0.51), (I – II, r2 = 0.41) and (III – IV, r2 = 0.34)].


formalin evoked the typical biphasic pattern of licking of the

injected paw (Fig. 1B). The first phase of intense licking

behavior was observed in the first 5-min period following

formalin injection. Thereafter, licking decreased in the

second 5-min period and rose after about 15 min. The

duration of licking in the second phase (from 16th to 60

min following injection of formalin) was formalin concen-

tration-dependent with plateau at formalin concentrations of

1.25–2.5% (ANOVA p < 0.0003; Fig. 1C).

Injection of formalin also induced Fos in the spinal cord.

In general, the induction of Fos-positive cells in the gray

matter along rostro-caudal and medial-lateral axis of the

spinal cord following injection of formalin was consistent

with that reported by Presley et al. [27]. The highest count

of Fos-positive cells was obtained at L4 level of the spinal

cord ipsilateral to the injection site (Fig. 1). Little or no FLI

was observed in the contralateral spinal cord (Fig. 1) and

was not included in the following analysis (see below).

Very few Fos-positive cells were observed in the spinal

cord of saline-injected animals (Figs. 1D and 4). In compar-

ison, the number of Fos-positive cells in the ipsilateral spinal

Fig. 5. The effect of injection of saline (A) or formalin (B, C, D) into hind paw of

limb of diagonal band of Broca (MS-VLDBB), pyramidal cell layer of hippocampa

section represents the bilateral meanF S.E.M. of Fos-positive cells per section of

Fos-positive cell count in handled, but non-injected (basal) animals vs. saline injec

differences between groups in (B), (C) and (D) were calculated using ANOVA follo

Note that the number of Fos-positive cells in whole CA1, CA3 and DG were low

injected animals.

cord was increased following injection of formalin (Fig. 1D)

with significant linear trend (r2 = 0.53, p< 0.0003). Laminar-

specific analysis also indicated that laminae I–II, V–VI and

VII–X displayed increase in Fos-positive cell count follow-

ing injection of formalin (Fig. 4) with significant linear

trends with corresponding r2 values of 0.41 ( p < 0.002),

0.59 ( p < 0.0001) and 0.51 ( p < 0.0005). A significant effect

of treatment was also observed for the counts from laminae

II–III (ANOVA < 0.05), though post hoc analysis indicated

that the various treatment groups were not different from

each other. The linear trend was also the shallowest

(r2 = 0.34, p < 0.01) for the counts from these laminae.

3.2. Changes in Fos induction in whole CA1, CA3, DG and

MS-VLDBB

The change in number of Fos-positive cells following

injection of formalin was evaluated along the length of

fields CA1, CA3, DG and MS-VLDBB. The Fos-positive

cell counts were bilaterally symmetrical. Indeed, the differ-

ences in counts between the left and the right halves of the

behaving rat on the number of Fos-positive cells in medial septum-vertical

l fields CA1 and CA3, and dentate gyrus (DG) granule cell layer. The # Fos/

the selected region averaged for the group. Significant difference between

ted animals in (A) was calculated using two-tail unpaired t-test. Significant

wed by Newman–Keul’s test for multiple comparisons (* vs. saline group).

er at all formalin concentrations when compared to the count from saline-


above regions were less than 20% (data not shown). Thus,

the counts from the left and the right halves were combined

and the composite value reported (see below). In basal

(control) animals, that were handled but not injected,

relatively few Fos-positive cells were present in each region

of interest (Fig. 5A). On the other hand, in animals injected

with saline in the right-hind paw a relatively high count of

Fos-positive cells was observed in all the regions evaluated

(Figs. 5 and 6).

Compared to the injection of saline, injection of formalin

evoked a decrease in Fos-positive cells in whole CA1

(ANOVA p < 0.0002; Fig. 5B), CA3 (ANOVA p < 0.0003;

Fig. 5C) and dentate gyrus (ANOVA p < 0.007; Fig. 5D). In

all the three regions, the cell counts for 0.625%, 1.25% and

2.5% formalin groups were comparable and were not

statistically different from each other.

A significant effect of formalin was not observed on Fos

induction in MS-VLDBB region (ANOVA p>0.09), though

the count from the 0.625% and 1.25% formalin group

tended to be lower than that from the saline injected group.

The Fos-positive cell counts per section for the saline group

Fig. 6. Formalin-induced decrease in number of Fos-positive cells in anterior (A),

CA1. The dorsal CA1 comprised of the anterior CA1 and the posterior–dorsal sub

subdivision of posterior CA1. The # Fos/section represents the bilateral meanF S.

group. A formalin concentration-dependent effect was observed in ventral CA1.

and 0.625%, 1.25%, and 2.5% formalin groups were

40.11 + 8.15 (n = 7) and 30.76F 7.15 (n= 7), 24.39F 2.86

(n = 7), 45.07F 5.55 (n = 7), respectively.

3.3. Changes in Fos-positive cell counts along anterior–

posterior CA1, CA3, DG and along dorsal–ventral axis of

CA1

Compared to injection of saline, injection of formalin

decreased the number of Fos-positive cells along the ante-

rior–posterior axis of CA1 (Figs. 2 3 6). In contrast, a

suppressive effect of formalin on Fos-positive cell count

was observed in posterior CA3 (ANOVA p < 0.0001) and

dentate gyrus (ANOVA p < 0.02), but not anterior CA3

(ANOVA p>0.07; data not illustrated) and dentate gyrus

(ANOVA p>0.09; data not illustrated). In context of poste-

rior CA3, the Fos-positive cell counts corresponding to

saline group and 0.625%, 1.25%, and 2.5% formalin groups

were 245.9F 16.70 (n = 7), and 132.5F 7.71 (n = 7),

123.3F 8.01 (n = 7), and 127.0F 15.51 (n = 7), respective-

ly. Post hoc comparison using Newman–Keul’s analysis

posterior (B), dorsal (C) and ventral (D) regions of the hippocampus fields

division of the posterior CA1 (Fig. 3), while the ventral CA1 was the ventral

E.M. of Fos-positive cells per section of the selected region averaged for the

Significant difference: * vs. saline group, § vs. other formalin groups.


indicated that the Fos-positive cell counts from the various

formalin groups was low as compared to the count from the

corresponding saline group. In context of posterior dentate

gyrus, the Fos-positive cell counts corresponding to saline

group and 0.625%, 1.25%, and 2.5% formalin groups were

81.67 F 11.45 (n = 7) , and 55.92 F 6.18 (n = 7) ,

50.09F 4.73 (n = 7), and 50.63F 3.30 (n = 7), respectively.

Post hoc analysis indicated that the Fos-positive cell counts

from the various formalin groups was low as compared to

the count from the corresponding saline group.

The Fos-positive cell count for the posterior–dorsal CA1

of the posterior CA1 (Fig. 3) was significantly low in

formalin groups as compared to the corresponding count

in the saline group (data not illustrated). The anterior CA1

and posterior–dorsal CA1 Fos-positive cell counts were

grouped as dorsal CA1 and presumably represented the

anterior three-quarters or so of the hippocampus. The

ventral CA1 region of the posterior CA1 presumably

represented the remaining one-quarter or so of the hippo-

campus. Analysis indicated a strong decrease in Fos induc-

tion in dorsal CA1 at all concentration of formalin, whereas

in case of ventral CA1, a significant suppression of Fos-

positive cell count was obtained at the higher formalin

concentrations of 1.25% and 2.5%, but not at 0.625%,

although the count from 0.625% group tended to be lower

than that from the saline group (Fig. 6). Further, a formalin

concentration-dependent effect was observed on the count

from ventral CA1 (Fig. 6D).

3.4. Correlation between Fos-positive cell counts in spinal

cord and septo-hippocampal regions

Spearman’s correlation indicated that formalin-induced

total spinal Fos-positive cell counts varied inversely with

Fos-positive cell count in whole CA1 (r =� 0.58, p < 0.03).

No correlation was found between total spinal Fos-positive

cell count and the corresponding counts in whole CA3

( p>0.8), DG ( p>0.8), and MS-VLDBB ( p>0.1). Correla-

tion analysis between spinal laminar specific Fos-positive

cell counts and the corresponding count in whole CA1

indicated a significant inverse relationship of the latter with

the counts in laminae V–VI (r =� 0.60, p< 0.02) and VII–

X (r=� 0.58, p < 0.03), but not with the Fos-positive cell

count in laminae I–II ( p>0.09).

Along the anterior–posterior axis of the CA1, a signif-

icant inverse correlation was observed between the Fos-

positive cell counts in posterior CA1 vs. the counts in spinal

laminae V–VI (r=� 0.7, p < 0.004) and VII–X (r =� 0.81,

p < 0.0004). The Fos-positive cell count in anterior CA1 was

not correlated with spinal laminar counts. Along the dorsal–

ventral axis of CA1, the ventral Fos-positive cell count was

significantly correlated with the counts in the laminae V–VI

(r =� 0.80, p < 0.0005) and VII–X (r =� 0.87, p < 0.0001).

The Fos-positive cell counts in dorsal CA1, anterior and

posterior CA3, DG, and MS-VLDBB was not correlated

with spinal laminar counts.

4. Discussion

The objective of the present study was to investigate the

change in induction of FLI in the hippocampus field CA1 in

nociceptive framework of the formalin model of inflamma-

tory pain. In this context, we report that injection of the

algogen formalin evoked a decreased induction of CA1 Fos

in the framework of formalin concentration-dependent in-

crease in nociceptive behavior and induction of spinal Fos.

Indeed, the number of Fos-positive cells in the whole CA1

and, strikingly, the posterior–ventral CA1 was inversely

correlated with that in the spinal cord, especially the deeper

laminae V–VI and VII–X. In comparison, the decrease in

Fos-positive cell counts observed in CA3 and dentate gyrus,

including the posterior regions was generalized and did not

correlate with the Fos-positive cell count in spinal cord.

Interestingly, injection of saline per se increased the

number of Fos-positive cells in the hippocampus and

dentate gyrus as compared to non-injected basal animal.

In situ hybridization and immunocytochemical techniques

suggest that procedures that are mildly stressful and/or

arousing such as intraperitoneal injection of saline, or

exposure to novel environment induces c-fos mRNA and

Fos in pyramidal cell layer in CA1 and CA3 and dentate

gyrus granule cell layer [13,29,30,36]. The above attributes,

i.e. mild stress and/or novelty with hind paw injection of

saline might also explain the increased Fos-positive cell

count to saline injection in the present study. On the other

hand, the relatively low level of Fos-positive cell count in

hippocampus and dentate gyrus following injection of the

different concentrations of formalin into the hind paw of

animals suggests that the aversive–noxious nature of the

stimulus precluded the facilitation by injection of induction

of Fos in the hippocampal neurons, perhaps by evoking an

inhibition of the activity of these neurons. Here it is notable,

that sensory stimulation, including non-noxious and noxious

stimulation activates both excitatory and inhibitory inputs to

the hippocampus [7,15,33]. Furthermore, there is evidence

to suggest that the strength and/or duration of noxious

stimulus affects the pattern of excitation– inhibition in

anterior field CA1. For example, hind paw injection of

formalin, which induces persistent nociception, excited

around 25% of CA1 pyramidal neurons while suppressing

the discharge of the remaining population. Brief noxious

heat stimulus applied to the periphery, however excited

about 50% of the population of CA1 pyramidal cells [15].

Correspondingly, in the current study in behaving animal,

the number of Fos-positive cells, especially in CA1, was also

inversely linked to the aversive strength of the injection.

The finding that strongly aversive stimulation with injec-

tion of formalin precludes the induction of FLI in CA1 is not

incompatible with previous evidence from anaesthetized rat

[11,24]. In this context, Funahashi et al. [11] reported a

decrease in FLI in CA1 to tooth pulp stimulation. Pearse et

al. [24], while did not report a decrease in FLI in CA1 also

did not observe an increase of FLI cells in CA1 to sciatic


nerve stimulation. This group did not observe a decrease,

presumably because the baseline level of Fos-positive cells

in CA1 of hippocampus was close to zero in their study.

Contrary to above, another study emphasized an increase in

hippocampal number of Fos-positive cells following injec-

tion of formalin in behaving animals [2]. The difference

between present study and the previous study [2] is partly

because the latter compared change in Fos-positive cell

counts following injection of formalin to that observed in

non-injected animal. The basal induction of FLI in undis-

turbed, relatively non-stressed animals is generally low and,

additionally, does not take into account the facilitatory effect

of injection per se on induction of hippocampal FLI.

Collectively, the above argues for the notion that the

aversive–noxious stimulation promotes the suppression of

induction of Fos in hippocampus. This contrasts to noxious

stimulus-induced facilitation of Egr1 induction observed in

the anterior hippocampus [24,33]. The divergent changes in

the two transcription factors are intriguing. Although un-

certain, the increase in Egr1 and the decrease in Fos may, in

part, reflect cellular changes in the population of pyramidal

cells that are excited and inhibited, respectively, following

noxious stimulation [15,33]. The noxious stimulus-induced

changes in level of transcription protein may have implica-

tion for long-term cellular excitability and synaptic plasticity

of affected population of neurons in the hippocampus. For

example, Egr1 is linked to facilitation of long-term poten-

tiation of synaptic efficacy in the anterior hippocampus [33]

that is related to mnemonic processes and animal adaptive

behavior. Similarly, the induction of Fos affects the cellular

levels of brain-derived nerve growth factor in the hippo-

campus [34].

The current findings also suggest that nociceptive signal

is processed in a distributed fashion throughout the length of

the hippocampus and dentate gyrus. However, unlike the

unitary-like response to mildly aversive injection of saline,

the noxious–aversive stimulus differentially recruits the

posterior–ventral regions. This is compatible with the view

that the hippocampus is not a unitary structure; rather the

ventral part is distinct from dorsal two-thirds in terms of

connectivity, physiology and effects of lesion [21]. The

contribution of hippocampus, especially the posterior–ven-

tral region to nociception remains unclear, though such

nociception-related decrease in hippocampal Fos was bilat-

eral and symmetrical suggesting that it is related to affec-

tive-motivational component of nociception. On the other

hand, changes in the spinal cord were predominantly ipsi-

lateral reflecting the topographic organization of the sensory

inputs to the region. The nociceptive recruitment of CA1,

and other hippocampal regions, may be juxtaposed as part a

wider recruitment of limbic forebrain regions in pain and

nociception. In this context, the other limbic regions that

have gained prominence include anterior cingulate cortex,

amygdala and also the other regions of the hippocampal

formation and interlinked structures such as nucleus accum-

bens [5,6,12,14,17–19,25,26,28,32].

Of the other regions examined, a significant effect of

formalin was not observed on induction of Fos in the MS-

VLDBB region; whereas, the pattern of change observed in

field CA3 and dentate gyrus paralleled that in CA1. Thus,

injection of saline evoked an increased induction in all three

hippocampal regions while injection of formalin decreased

induction in these regions. Given the links amongst the three

regions, the change in Fos-positive cell count in CA3 and

dentate gyrus is suggestive that these region channel, at least

partly, noxious stimulus-induced changes to CA1 to influ-

ence this area. However, the differences in concentration-

dependent effect of formalin on Fos-positive cell count in

posterior and ventral CA1 vs. CA3 and DG suggests that the

formalin concentration-dependent decrease in the former be

not entirely linked to Fos-related neural processing in CA3

and DG.

In summary, the present study using Fos-mapping tech-

nique indicates that injection of formalin differentially

recruited the posterior and ventral regions of hippocampus

and dentate gyrus, and points to the possibility that at least

the posterior–ventral CA1 is recruited in the framework of

formalin concentration-dependent increase in nociception.

The present findings also indicate that persistent nociception

alters synaptic transmission along the dentate–CA3–CA1

axis and this might influence CA1 response to injection of

formalin.

Acknowledgements

This work was supported by research grants from

National Medical Research Council, Singapore and Aca-

demic Research Fund, National University of Singapore

to SK.

References

[1] F.V. Abbott, K.B.J. Franklin, R.F. Westbrook, The formalin test: scor-

ing properties of the first and second phases of the pain response in

rats, Pain 60 (1995) 91–102.

[2] A.M. Aloisi, M. Zimmermann, T. Herdegen, Sex-dependent effects of

formalin and restraint on c-Fos expression in the septum and hippo-

campus of the rat, Neuroscience 81 (1997) 951–958.

[3] D.G. Amaral, M.P. Witter, The three-dimensional organization of the

hippocampal formation: a review of anatomical data, Neuroscience 31

(1989) 571–591.

[4] P. Andersen, T.V.P. Bliss, K.K. Skrede, Lamellar organization of hip-

pocampal excitatory pathways, Exp. Brain Res. 13 (1971) 222–238.

[5] J.F. Bernard, H. Bester, J.M. Besson, Involvement of the spino-para-

brachio-amygdaloid and -hypothalamic pathways in the autonomic

and affective emotional aspects of pain, in: G. Holstege, R. Saper,

C.B. Saper (Eds.), Progress in Brain Research, vol. 107. Elsevier,

Amsterdam, The Netherlands, 1996, pp. 243–255.

[6] U. Bingel, M. Quante, R. Knab, B. Bromm, C. Weiller, C. Buchel,

Subcortical structures involved in pain processing: evidence from

single-trial fMRI, Pain 99 (2002) 313–321.

[7] J. Brankack, G. Buzsaki, Hippocampal responses evoked by both

tooth-pulp and acoustic stimulation: depth profiles and effect of be-

havior, Brain Res. 378 (1986) 303–314.


[8] J. Buritova, J.M. Besson, J.F. Bernard, Involvement of the spino-

parabrachial pathway in inflammatory nociceptive processes: a c-

Fos protein study in the awake rat, J. Comp. Neurol. 397 (1998)

10–28.

[9] T.J. Coderre, M.E. Fundytus, J.E. McKenna, S. Dalal, R. Melzack,

The formalin test: a validation of the weighted-scores method of

behavioral pain rating, Pain 54 (1993) 43–50.

[10] C.A. Doyle, S.P. Hunt, Substance P receptor (neurokinin-1)-express-

ing neurons in lamina I of the spinal cord encode for the intensity of

noxious stimulation: a c-Fos study in rat, Neuroscience 89 (1999)

17–28.

[11] M. Funahashi, Y.-F. He, T. Sugimoto, R. Matsuo, Noxious tooth pulp

stimulation suppresses c-fos expression in the rat hippocampal forma-

tion, Brain Res. 827 (1999) 215–220.

[12] R.W. Gear, J.D. Levine, Antinociception produced by an ascending

spino-supraspinal pathway, J. Neurosci. 15 (1995) 3154–3161.

[13] U.S. Hess, G. Lynch, C.M. Gall, Regional patterns of c-fos mRNA

expression in rat hippocampus following exploration of a novel en-

vironment versus performance of a well-learned discrimination,

J. Neurosci. 15 (1995) 7796–7809.

[14] J.P. Johansen, H.L. Fields, B.H. Manning, The affective compo-

nent of pain in rodents: direct evidence for contribution of the

anterior cingulate cortex, Proc. Natl. Acad. Sci. U. S. A. 98

(2001) 8077–8082.

[15] S. Khanna, Dorsal hippocampus field CA1 pyramidal cell responses

to a persistent versus an acute nociceptive stimulus and their septal

modulation, Neuroscience 77 (1997) 713–721.

[16] K.G. Kjelstrup, F.A. Tuvnes, H.-A. Steffenach, R. Murison, E.I.

Moser, M.-B. Moser, Reduced fear expression after lesions of

the ventral hippocampus, Proc. Natl. Acad. Sci. U. S. A. 99 (2002)

10825–10830.

[17] B.H. Manning, A lateralized deficit in morphine antinociception after

unilateral inactivation of the central amygdala, J. Neurosci. 18 (1998)

9453–9470.

[18] J.E. McKenna, R. Melzack, Analgesia produced by lidocaine micro-

injection into the dentate gyrus, Pain 49 (1992) 105–112.

[19] J.E. McKenna, R. Melzack, Blocking NMDA receptors in the hippo-

campal dentate gyrus with AP5 produces analgesia in the formalin

pain test, Exp. Neurol. 172 (2001) 92–99.

[20] C. Molander, Q. Xu, G. Grant, The cytoarchitectonic organization of

the spinal cord in the rat: I. The lower thoracic and lumbosacral cord,

J. Comp. Neurol. 230 (1984) 133–141.

[21] M.-B. Moser, E.I. Moser, Functional differentiation in the hippocam-

pus, Hippocampus 8 (1998) 608–619.

[22] K. Okuda, C. Sakurada, M. Takahashi, T. Yamada, T. Sakurada,

Characterization of nociceptive responses and spinal release of nitric

oxide metabolites and glutamate evoked by different concentrations

of formalin in rats, Pain 92 (2001) 107–115.

[23] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates,

Academic, New York, 1982.

[24] D. Pearse, A. Mirza, J. Leah, Jun, Fos and Krox in the hippocampus

after noxious stimulation: simultaneous-input-dependent expression

and nuclear speckling, Brain Res. 894 (2001) 193–208.

[25] A. Ploghaus, I. Tracey, S. Clare, J.S. Gati, J.N.P. Rawlins, P.M. Mat-

thews, Learning about pain: the neural substrate of the prediction

error for aversive events, Proc. Natl. Acad. Sci. U. S. A. 97 (2000)

9281–9286.

[26] A. Ploghaus, C. Narain, C.F. Beckmann, S. Clare, S. Bantick, R.

Wise, P.M. Matthews, J.N.P. Rawlins, I. Tracey, Exacerbation of pain

by anxiety is associated with activity in a hippocampal network,

J. Neurosci. 21 (2001) 9896–9903.

[27] R.W. Presley, D. Menetrey, J.D. Levine, A.I. Basbaum, Systemic

morphine suppresses noxious stimulus-evoked Fos protein-like im-

munoreactivity in the rat spinal cord, J. Neurosci. 10 (1990)

323–335.

[28] P. Rainville, Brain mechanisms of pain affect and pain modulation,

Curr. Opin. Neurobiol. 12 (2002) 195–204.

[29] A.E. Ryabinin, K.R. Melia, M. Cole, F.E. Bloom, M.C. Wilson, Al-

cohol selectively attenuates stress-induced c-fos expression in rat hip-

pocampus, J. Neurosci. 15 (1995) 721–730.

[30] F.R. Sharp, S.M. Sagar, K. Hicks, D. Lowenstein, K. Hisanaga, c-fos

mRNA, Fos, and Fos-related antigen induction by hypertonic saline

and stress, J. Neurosci. 11 (1991) 2321–2331.

[31] D.W. Smith, T.A. Day, Neurochemical identification of Fos-positive

neurons using two-color immunoperoxidase staining, J. Neurosci.

Methods 47 (1993) 73–83.

[32] B.A. Vogt, R.W. Sikes, L.J. Vogt, Anterior cingulate cortex and the

medial pain system, in: B.A. Vogt, M. Gabriel (Eds.), Neurobiology

of Cingulate Cortex and Limbic Thalamus: a Comprehensive Hand-

book, Birkhauser, Boston, 1993, pp. 313–344.

[33] F. Wei, Z.C. Xu, Z. Qu, J. Milbrandt, M. Zhuo, Role of Egr1 in

hippocampal synaptic enhancement induced by tetanic stimulation

and amputation, J. Cell Biol. 149 (2000) 1325–1333.

[34] J. Zhang, D. Zhang, J.S. McQuade, M. Behbehani, J.Z. Tsien, M. Xu,

c-fos regulates neuronal excitability and survival, Nat. Genet. 30

(2002) 416–420.

[35] F. Zheng, S. Khanna, Selective destruction of medial septal choliner-

gic neurons attenuates pyramidal cell suppression, but not excitation

in dorsal hippocampus field CA1 induced by subcutaneous injection

of formalin, Neuroscience 103 (2001) 985–998.

[36] X.O. Zhu, B.J. McCabe, J.P. Aggleton, M.W. Brown, Differential

activation of the rat hippocampus and perirhinal cortex by novel

visual stimuli and a novel environment, Neurosci. Lett. 229 (1997)

141–143.

Nociception-driven decreased induction of Fos protein in ventral hippocampus field CA1 of the rat

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