Behavioral, immunocytochemical and biochemical studies in rats differing in their sensitivity to pain

Behavioural Brain Research 171 (2006) 189–198

Research report

Behavioral, immunocytochemical and biochemical studies in rats differing in their sensitivity to pain Małgorzata Lehner a , Ewa Taracha a , Anna Sk´orzewska a , Piotr Maciejak a,b , Aleksandra Wisłowska-Stanek b , Małgorzata Zienowicz b , Janusz Szyndler b , Andrzej Bidzi´nski a , Adam Pła´znik a,b,∗ b

a Department of Neurochemistry, Institute of Psychiatry and Neurology, 02-957 Warsaw, 9 Sobieskiego Street, Poland Department of Experimental and Clinical Pharmacology, Medical University, 00-927 Warsaw, 26/28 Krakowskie Przedmie´scie Street, Poland

Received 20 December 2005; received in revised form 13 March 2006; accepted 22 March 2006 Available online 16 May 2006

Abstract The aim of the study was to further explore the anatomical and neurochemical background of differences in response to the conditioned aversive stimuli. The different patterns of behavioral coping strategies (a conditioned freezing response and ultrasonic vocalization) were analyzed in animals differing in their response to the acute painful stimulation, a foot-shock (HS: high sensitivity rats, LS: low sensitivity rats, and MS: medium sensitivity rats, according to their behavior in the flinch-jump pre-test), and correlated with plasma corticosterone levels, expression of c-Fos protein, and distribution of 5-HT innervation, in different brain structures. It was found that HS rats showed significantly more freezing behavior, whereas LS animals vocalized much more intensively. The behavior of LS group (less freezing response and stronger vocalization) was related to activation of prefrontal cortex (PFCX), increased activity of adrenal glands and stronger serotonin immunostaining in the PFCX, in comparison with HS animals. The more passive strategy of coping with the aversive event of HS group was related to increased activity of amygdalar nuclei and some areas of the hippocampus, and stronger 5-HT immunostaining in the baso-lateral nucleus of the amygdala, in comparison with LS rats. The present findings suggest that animals more vulnerable to stress might have innate deficits in the activity of brain systems controlling the hypothalamic-pituitary-adrenal axis that would normally allow them to cope with stressful situations. It appears also that response to pain may determine other patterns of emotional behavior, probably reflecting different activation thresholds of some brain structures controlling anxiety, e.g. prefrontal and secondary motor cortex. © 2006 Elsevier B.V. All rights reserved. Keywords: Individual response to foot-shock pain; Freezing response; Ultrasonic vocalization; c-Fos protein; Corticosterone; 5-HT immunostaining; Rats

1. Introduction The issue of the anatomical and neurochemical basis of differences in the individual sensitivity to aversive stimuli is still unresolved [11,17,23,25,39,49]. The mechanisms regulating individual sensitivity and reactivity to emotional stimuli are important for a variety of physiological and pathological processes, ranging from pain perception to mood and emotions [7,22,23,25,39,45,49,51,52,55]. It was found previously by us that rats subjected to the flinch-jump test, and divided into two groups according to their response to the acute painful stimu∗

Corresponding author. Tel.: +48 22 4582771; fax: +48 22 4582741/64253375. E-mail address: [email protected] (A. Pła´znik). 0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.03.044

lation (high sensitivity and low sensitivity; HS, LS), showed a strong stimulation of brain activity on re-exposure to the shock cage and aversive stimulation (5 foot-shocks, 0.5 mA, 1 s long, repeated every 1 min), on retest 10 days later [32]. A detailed analysis of data revealed a potent enhancement of c-Fos expression in a majority of examined brain structures, including cortical areas, indicating their sensitivity to the direct and indirect (conditioned) aversive stimuli. The only significant difference in c-Fos expression between LS and HS rats was found in the lateral habenular nucleus (LHAB), indicating this brain structure as selectively engaged in processing of the painful stimulation. It was concluded that the reactivity of LHAB may be responsible for the differences in sensitivity to acute pain. The aim of the present study was to further explore the phenomenon of differences in animal response to conditioned

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aversive stimuli using the conditioned fear test (a contextual fear). The rationale for this study was the assumption that reactivity to a simple sensory stimulation may determine animal behavior in response to more complex, e.g. conditioned, anxious stimuli. In other words, it has been assumed that rats with different pain thresholds are characterized also by different sensitivity to emotional stimuli. To elucidate this problem, we have analyzed the behavioral and neurochemical changes in animals divided into groups clearly differing in their sensitivity to pain. To this end, the first threshold of pain, flinch response in the flinch-jump test, was selected to avoid too strong response of animals, and the ceiling-like effects. A conditioned freezing response and aversive ultrasonic vocalization were studied in laboratory rats, divided into groups according to their reactivity to the acute painful stimulation. The different patterns of behavioral coping strategies were analyzed in low sensitivity and high sensitivity rats, and correlated with changes in plasma corticosterone levels, expression of c-Fos protein and distribution of 5-HT immunoreactivity, in different brain structures. It was assumed that these groups of rats should be characterized also by different sensitivity to emotional stimuli, and reactivity of brain structures and system which are involved in processing of emotional input to the central nervous system.

responsiveness of the rat. The flinch threshold was defined as the lowest shock intensity that elicited any detectable response. The jump threshold was defined as the lowest shock intensity that elicited simultaneous removal of at least three paws (both hindpaws) from the grid. To avoid foot damage, the cut-off = 1.2 mA was established. In this way, the flinch and jump thresholds in mA were defined for each rat. The time gap between shocks was 10 s, and each animal was tested only once [57]. Next, all animals were divided into three experimental groups according to the following criterion: low sensitivity animals (LS, flinch threshold above 0.65 mA, n = 15); high sensitivity animals (HS, flinch threshold below 0.45 mA, n = 17); and the medium sensitivity (MS) group with flinch threshold between 0.45 and 0.65 mA (n = 12). The criterion was established in the following way: the mean intensity of a stimulus inducing flinch response ± S.D. (0.55 ± 0.1), i.e. the animals with behavioral response above 0.65 mA, or below 0.45 mA stimulus, were allocated to the appropriate experimental groups. Five animals were eliminated from the study because of their inadequate responses to the painful stimulus (i.e. jumping or immobilization as a first reaction). After 7 days, HS and LS rats were subjected to the conditioned fear test, medium sensitivity rats were subjected to the conditioning box only. Another control group was not subjected to any behavioral testing (C, n = 6) [32]. The procedure has been applied to group animals in two not overlapping populations, in respect of their response to the painful stimulation. It was assumed that these groups of rats should be characterized also by different sensitivity to emotional stimuli, and reactivity of brain structures and system which are involved in processing of emotional input to the central nervous system. The schemes of experimental protocol are shown in Tables 1 and 2.

2.3. Contextual fear conditioning test and ultrasonic vocalization 2. Materials and methods 2.1. Animals The experiment was performed in a cohort of 55 male Wistar rats. The rats (180–200 g), bought from a licensed breeder, were housed in standard laboratory conditions under a 12 h light/dark cycle (lights on at 7 a.m.), in a constant temperature (21 ± 2 ◦ C) and 70% humidity. The experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609 EEC). All experimental procedures using animal subjects were approved by the Local Committee for Animal Care and Use at Warsaw Medical University, Poland.

2.2. Flinch-jump test After 4 days of acclimatization to the vivarium, all rats (n = 49), with the exception of the control group C (n = 6), were subjected to the flinchjump test (Table 1). The test was performed in a box made of Plexiglas (30 cm × 30 cm × 60 cm, w/l/h), with a grid floor made of stainless steel bars wired to a shock generator. The floor of the box was cleaned after each trial with 95% ethanol. The rats were placed individually into the box. Shocks were delivered to the grid floor of the test box through a shock generator. After a 3 min period of habituation to the test box, shocks titrations were continued upwards in a stepwise manner (0.05 mA, 0.05–1.2 mA range) depending upon

The fear conditioning experiment was performed using a computerized fear conditioning system (TSE, Bad Homburg Germany), as described previously [33]. Fear conditioning was performed in the experimental cage (36 cm × 21 cm × 20 cm, w/l/h) under constant white noise condition (65 dB). The experiment was performed during three consecutive days in the same testing box and experimental chamber. On the first day, the animals were placed separately for 2 min in a training box, for adaptation to the experimental conditions. The following day, during a 10 min long session, after 2 min of habituation, the animal received three foot-shocks (stimulus: 0.7 mA, 1 s, repeated every 60 s). On the third day, the freezing response of rats was examined for 10 minlong period, in the testing box without any further stimulation. The conditioned response, a freezing response, was recorded and analyzed by the fear conditioning system. The freezing behavior was measured by a photo beam system (10 Hz detection rate) controlled by the fear conditioning system. Photo beams were spaced 1.3 and 2.5 cm in the direction of the x-axis and the y-axis, respectively. The absolute duration of inactivity was calculated by the fear conditioning system, defined as no interruption of any photo beam over 5 s long periods, and then summarized for the whole 10 min long experimental session (total time of freezing). The fear conditioning system has been validated previously in our laboratory [33,57]. Ultrasonic vocalizations were recorded simultaneously by a microphone, Mini-3 Bat Dector (Noldus Information Technology), attached to the ceiling of the chamber and processed by an interface, Ultravox, Noldus Information Technology, to select the 22 kHz frequency band for aversive vocalization calls (the range: 22 ± 5 kHz, minimum duration of an individual acoustic

Table 1 Treatment scheme for c-Fos and serotonin immunocytochemistry (groups: C, MS, HS, LS) Days 1–4

Day 5

Days 6–11

Days 12–14

Habituation to the vivarium

Flinch-jump test Animals divided into groups according to the criterion (flinch response) LS (low sensitivity rats) ↑ 0.65 mA (n = 8) HS (high sensitivity rats) ↓ 0.45 mA (n = 9) MS (medium sensitivity rats) 0.45–0.65 mA (n = 6)

Resting time

Contextual fear conditioning test (LS, HS animals) Exposure to the box only (MS animals) (decapitation for c-Fos and serotonin 1.5 h later)

C (control, naive, not disturbed animals) (n = 6)

No exposure to the conditioning box (C animals) (decapitation for c-Fos)

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Table 2 Treatment scheme for corticosterone levels (groups: MS, HS, LS) Days 1–4

Day 5

Days 6–11

Days 12–14

Habituation to the vivarium

Flinch-jump test Animals divided into groups according to the criterion (flinch response) LS (low sensitivity rats) ↑ 0.65 mA (n = 7) HS (high sensitivity rats) ↓ 0.45 mA (n = 8) MS (medium sensitivity rats) 0.45–0.65 mA (n = 6)

Resting time

Contextual fear conditioning test (LS, HS animals) Exposure to the box only (MS animals) (decapitation 10 later)

signal accepted by the Ultravox apparatus as a vocalization event: 300 ms), and to digitize them in an IBM compatible PC. The duration of ultrasonic vocalization (s) in aversive band during 10 min long session was recorded (total time of vocalization, i.e. cumulated time of individual calls in 10 min) [20,57,60].

by Uylings et al. [59]; AP 3.14: baso-lateral amygdala BLA, medial amygdala MeA, CA1, CA3, DG areas of hippocampus, dorsomedial and ventromedial nuclei of hypothalamus HYPTH; AP 8.30: dorsal and median raphe nucleus (DRN, MRN, respectively) (Figs. 1 and 2).

2.4. Corticosterone assay

2.6. Serotonin immunochemistry

Blood samples were collected on the third day of contextual fear conditioning test, 10 min after exposure to the aversive context of a separate group of animals (Table 2). After testing, the rats were transported to the home cage, and 10 min later the blood samples were collected in a different room. Five hundred microlitres samples of blood were collected into heparinised tubes [36]. The samples were immediately centrifuged (2600 × g at 4 ◦ C for 15 ). Plasma was immediately aspirated and stored at −70◦ C, until analysis. After suitable dilution, plasma corticosterone was analyzed by radioimmunoassay [3 H] RIA kit, MP Biomedicals Inc. The concentration range for this assay was 0.025–1 ng/0.5 ml with a six points calibration curve 0.025; 0.05; 0.1; 0.25; 0.5; 1 ng/0.5 ml. Plasma corticosterone averages usually between 100 and 500 ng/1 ml.

The immunocytochemical reaction was performed on slide-mounted brain sections, according to the method described by Amat et al. [2], Staub et al. [54], and Tajudin and Druse [58]. Briefly, coronal 15 ␮m cryostat sections (see above) based on the atlas of Paxinos and Watson [43] were mounted on silancoated slides, and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min. The specimens were then washed twice (2 × 15 min) in 0.01 M PBS solution (pH 7.4), incubated in 3% H2 O2 solution for 30 min to block the activity of endogenous peroxidase, then washed again in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min), and incubated in a 10% normal goat serum blocking solution. Subsequently, slide-mounted brain sections were incubated in rabbit polyclonal serotonin antiserum diluted at 1:100 (Neuromics) in temperature 4–8 ◦ C for 24 h, washed in 0.01 M PBS solution (pH 7.4) three times (3 × 15 min), then incubated with biotynylated anti-rabbit IgGs (Vector Laboratories, CA) for 1 h, rinsed in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min), and incubated with avidine–biotine-peroxydase complex (Vector Laboratories, CA) for 2 h. Finally, after being washed in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min) slide-mounted brain sections were immunoreacted with a solution containing Tris, 0.03% diaminobenzidine hydrochloride and 0.03% H2 O2 . The slides were then dehydrated by serial alcohol rinsing, cleared in xylene, and coverslipped in the histofluid mountant. Serotonin immunoreactivity was assessed by light microscopy (Olympus BX-51 light microscope, Camedia Master C3040 digital camera) at a magnification of ×100 within the plane of the section [58]. The number of serotonin-positive immunoreactive complexes was counted bilaterally with the use of computerized image analysis system (Olympus DPSoft version 3.2 software) from three sections per rat in the following brain areas: AP 1.20: PFCX; AP 3.14: baso-lateral (BLA), and medial amygdala MeA, CA1, CA3, DG areas of the hippocampus; dorsal, medial, ventral areas of the hypothalamus HYPTH; AP 8.30: dorsal and median raphe nucleus (DRN, MRN).

2.5. c-Fos immunocytochemistry 1.5 h after the experiment (conditioned freezing test), the animals were decapitated, their brains removed, frozen in dry-ice cooled isopenthane (2methylbutan), and stored at −70 ◦ C. The immunocytochemical reaction was performed on slide-mounted brain sections, according to the procedure described previously [21,32]. Six slices were taken from each structure, three for c-Fos and three for serotonin immunocytochemistry. Briefly, coronal 15 ␮m cryostat sections based on the atlas of Paxinos and Watson [43] were mounted on silancoated slides, and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min. The specimens were then washed twice (2 × 15 min) in 0.01 M PBS solution (pH 7.4), incubated in 3% H2 O2 solution for 30 min to block the activity of endogenous peroxidase, then washed again in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min), and incubated in a 3% normal goat serum (NGS) blocking solution. Subsequently, slide-mounted brain sections were incubated in rabbit polyclonal c-Fos IgG diluted at 1:1000 (oncogene) in temperature 4–8 ◦ C for 72 h, washed in 0.01 M PBS solution (pH 7.4) three times (3 × 15 min), then incubated with biotynylated anti-rabbit IgGs (Vector Laboratories, CA) for 2 h, rinsed in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min), and incubated with avidine–biotine-peroxydase complex (Vector Laboratories, CA) for 1 h. Finally, after being washed in 0.01 M PBS solution (pH 7.4) twice (2 × 15 min) slidemounted brain sections were immunoreacted with a solution containing Tris, 0.03% diaminobenzidine hydrochloride (DAB) and 0.003% H2 O2 . The slides were then dehydrated by serial alcohol rinsing, cleared in xylene, and coverslipped in the histofluid mountant. It is noteworthy, that control experiments performed without primary and secondary antibodies (to detect nonspecific tissue binding of antibodies and/or endogenous peroxidase activity) yielded negative results. c-Fos-like immunoreactivity was assessed by light microscopy (Olympus BX-51 light microscope, Camedia Master C-3040 digital camera) at a magnification of ×40. The number of c-Fos positive nuclei was counted bilaterally with the use of computerized image analysis system (Olympus DP-Soft version 3.2 software) from three sections per rat in the following subregions: AP 1.20: prefrontal cortex, secondary motor cortex PFCX, within the limits defined

2.7. Statistical analysis The data are shown as mean ± S.E.M. The data on fear conditioning, total time of vocalization, plasma corticosterone concentration, c-Fos immunocytochemistry, were analyzed by one-way ANOVA followed by post hoc Newman–Keuls test. Serotonin immunocytochemical results were calculated by Student’s t-test. For the correlation analysis, a Pearson’s coefficient was calculated. A probability value of P < 0.05 was considered significant in this study.

3. Results 3.1. Flinch-jump test The applied methodology allows to easily distinguish two groups of animals differing in their sensitivity to the painful aversive stimulation. After the test, all animals (n = 49) were

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divided into three experimental groups according to the applied criterion: low sensitivity animals (LS, n = 15); high sensitivity animals (HS, n = 17); and the medium sensitivity group (MS, n = 12). Five animals were eliminated from the study because of their inadequate responses to the painful stimulus (i.e. jumping or immobilization as a first reaction). 3.2. Contextual fear conditioning and ultrasonic vocalization Differences appeared in time of freezing behavior F(2, 21) = 12.58 (P < 0.01), as well in total time of vocalization F(2, 21) = 3.85 (P < 0.05) (Fig. 3). Post hoc analysis showed a significant increase in time of freezing behavior in HS animals in comparison to MS (P < 0.01) and LS (P < 0.01) groups. Post hoc test indicated also an increase in total time of vocalization in LS group (P < 0.05), in comparison to HS group. The difference in total time of vocalization between the HS and MS rats did not reach the level of statistical significance (P = 0.18). 3.3. Radioimmunoassay of coricosterone One-way ANOVA showed a significant difference in plasma concentration of corticosterone F(2, 19) = 4.86 (P < 0.05). Post hoc analysis revealed increased levels of corticosterone in LS group, in comparison to MS (P < 0.05) and HS (P < 0.05) animals (Fig. 4). 3.4. c-Fos

Fig. 1. A scheme showing brain regions analyzed for immunocytochemistry. The areas that were analyzed for c-Fos immunoreactivity are outlined according to Paxinos and Watson [43]. The numbers indicate the distance from bregma (mm, caudal to bregma). PFCX: prefrontal cortex, secondary motor cortex (according to the criterion by Uylings et al. [59]); CA1, CA2, DG area of hippocampus; DRN: dorsal raphe nuclei; MRN: medial raphe nuclei; HYPTH: dorsomedial and ventromedial nuclei of hypothalamus; Cpu: caudate putamen; cc: corpus callosum; PAG: periaqueductal gray matter; BLA: baso-lateral amygdala; MeA: medial amygdala.

One-way ANOVA revealed statistically significant differences in the number of c-Fos positive cells, in the following brain structures examined: PFCX, F(3, 23) = 13.46 (P < 0.01); HYPTH, F(3, 25) = 6.58 (P < 0.01); BLA, F(3, 21) = 5.05 (P < 0.01); MeA, F(3, 21) = 3.51 (P < 0.05); CA1, F(3, 23) = 17.71 (P < 0.01); DG F(3, 22) = 8.35 (P < 0.01); MRN F(3, 16) = 13.61 (P < 0.01). No significant effect was found in the CA3 area of the hippocampus, F(3, 23) = 2.59 (P > 0.05), and DRN, F(3, 22) = 2.24 (P > 0.1) (Table 3, Fig. 2). In PFCX, all experimental groups had higher concentration of c-Fos protein in comparison with control C group (MS, P < 0.05; LS, P < 0.01; HS, P < 0.01). The expression of c-Fos was most potent in LS rats (P < 0.01 versus MS, and P < 0.05 versus HS group). In hypothalamus, there appeared an increase in c-Fos positive nuclei in LS group only (P < 0.01 versus C, P < 0.01 versus MS, and P < 0.05 versus HS). c-Fos labeling was increased exclusively in baso-lateral and medial amygdala in HS rats (P < 0.05 versus C in baso-lateral part, P < 0.05 versus MS in baso-lateral and medial nuclei). In CA1 area in HS rats, c-Fos concentration was significantly higher compared to C, MS and LS groups (P < 0.01). In dentate gyrus, c-Fos was increased in LS rats only (P < 0.01 versus C and HS, P < 0.05 versus MS). In the median raphe nucleus of LS animals, there appeared enhanced c-Fos immunoreactivity in comparison with C (P < 0.01), MS (P < 0.01), and HS rats (P < 0.05).

M. Lehner et al. / Behavioural Brain Research 171 (2006) 189–198

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Fig. 2. Photomicrographs showing representative expression of c-Fos and serotonin immunostaining in PFCX and BLA in two experimental groups: LS and HS rats. The dotted arrow indicates a nucleus immunoreactive for c-Fos protein, the dark arrow indicates serotonin immunoreactive complex. Slices were photographed with lens magnification 20×, and then magnified digitally. Bar indicates 90 ␮m. For more details see Section 2.

3.5. Serotonin immunochemistry

3.6. Correlation analysis results

Student’s t-test showed significant differences between LS and HS group in PFCX (t = 2.64, d.f. = 9, P < 0.05), and in BLA (t = 2.34, d.f. = 14, P < 0.05). In PFCX, serotonin immunoreactivity was elevated in LS group, whereas 5-HT immunostaining in the BLA was stronger in HS group (Table 3, Fig. 2).

Positive correlations were found between total time of vocalization and c-Fos in PFCX, c-Fos in PFCX and 5-HT in PFCX, c-Fos in HYPTH and 5-HT in PFCX, c-Fos in BLA and 5-HT in BLA, c-Fos in MRN and 5-HT in PFCX. On the other hand, negative correlations were found between freezing and c-Fos in

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Fig. 3. Contextual fear conditioning, freezing (A) and aversive ultrasonic vocalization (B). The data are shown as mean ± S.E.M. Rat behavior in the contextdependent fear test was examined 24 h after training. Ordinate: total time of freezing behavior or total time of vocalization (s), respectively. LS: low sensitivity rats (n = 8); HS: high sensitivity rats (n = 9); MS: medium sensitivity group (not conditioned), exposed to the box only (n = 6); (&) differs from LS or HS group, respectively; (#) differs from MS group. & P < 0.05; &&,## P < 0.01. See Section 2 for more details.

Fig. 4. Plasma corticosterone concentration (ng/ml). The data are shown as mean ± S.E.M. LS: low sensitivity rats (n = 7); HS: high sensitivity rats (n = 8); MS: medium sensitivity group (not conditioned), exposed to the box only (n = 6); (&) differs from HS; (#) differs from MS; #,& P < 0.05. For more details see Section 2.

HYPTH, total time of vocalization and c-Fos in BLA, and c-Fos in PFCX and c-Fos in BLA (Table 4). 4. Discussion It was found that the two groups of animals selected according to their sensitivity to the acute painful stimulation (high sensitivity and low sensitivity; HS and LS group, respectively), revealed a complex and different pattern of behavioral, immunocytochemical and biochemical changes, in response to the conditioned aversive stimuli. HS rats showed significantly more freezing behavior, whereas LS animals vocalized much more intensively. These different behavioral coping strategies were accompanied by different changes in plasma corticosterone levels, as well as different expression of c-Fos protein and different distribution of 5-HT immunoreactivity in the brain structures. HS rats showed a selective increase in c-Fos positive cells in the baso-lateral nucleus of amygdala BLA, medial nucleus of amygdala MEA and CA1 area of the hippocampus (versus MS group), a decreased density of 5-HT positive innervation in the PFCX, and increased density of 5-HT immunostaining in the BLA, in

comparison to LS rats, and the absence of changes in plasma corticosterone concentration. On the other hand, in LS animals, the highest increase of c-Fos positive cells was observed in the PFCX, as well as an increase of this immunocytochemical reaction in the hypothalamus, and dentate gyrus of the hippocampus. In the median raphe nucleus, the density of c-Fos positive staining was significantly enhanced in LS group only. In comparison to HS group, they had a lower number of 5-HT-like positive complexes in the BLA, and enhanced 5-HT immunostaining in the PFCX. Also, a significant increase in plasma corticosterone levels, following conditioned aversive stimulus, was observed in LS rats. These findings are new, and important for the recognition of central mechanisms which are responsible for differences in the reactivity to affective stimuli [17,22,23,25,29,39,49]. Both strategies to cope with an aversive stimulation (increased freezing versus intensified vocalization) are very well known and described by ethologists, and fulfill the criterion of an effective tactic, for the individual’s survival in a threatening situation. It appears also that they have different neurochemical backgrounds. The data on c-Fos protein expression confirm previous results obtained in different laboratories, and indicate the involvement of amygdalar nuclei, hippocampal subregions, hypothalamus, and prefrontal cortex, in processing of conditioned stimuli related to aversive context [49,53,56]. In our previous study, it was shown that animals subjected to the flinch-jump test retained a strong emotional response on re-exposure to the shock cage with aversive stimulation on retest, 10 days later, as revealed by the widespread expression of c-Fos protein in the majority of examined brain structures [32]. The only significant difference in c-Fos expression between LS and HS rats was found in the lateral habenular nucleus, indicating that this brain structure is selectively engaged in processing of the emotional-cognitive component of a painful stimulation. It was concluded that the reactivity of LHAB may be responsible for the differences in sensitivity to pain. In the present study, many more differences in c-Fos expression were found between both experimental groups, LS and HS

72.7 94.9 148.0 98.2 90.3 113.1 108.4 104.3 ± ± ± ± ± ± ± ± 20.9**&##

The data represent mean ± S.E.M. Number of rat in the group varied from 6 to 9; (*) differs from C; (&) differs from HS; (#) differs from MS; *&# P < 0.05; **&&## P < 0.01.

19.9& 6.5 24.1 13.7 4.0 34.3

40.6 54.2 206.0 151.4 56.8 260.4 51.6 284.3 55.8 ± 57.1+4.8 139.1 ± 154.2 ± 62.09 ± 229.1 ± 47.6 ± 272.5 ± 81.3 57.5 126.3 144.1 164.6 47.8 88.8 139.1 55.7 122.1 140.0 101.7 99,0 64.2 22.5 52.5 96.7 76.9 36.5**&## 4.7 9.1 3.4&& 5.4**&&# 10.2

150.5 ± 243.3 ± 80.5 ± 68.7 ± 39.0 ± 47.7 ± 59.1 ± 69.5+16.0 138.0 ± 23.5 5.5 5.1 1.4 5.9 7.2 6.3 1.6

106.6 100.8 66.6 48.3 32.4 30.6 37.1 34.6 27.7 Prefrontal cortex secondary motor cortex Hypothalamus dorsomedial and ventromedial nuclei Baso-lateral amygdala Medial amygdala CA1 Dentate gyrus CA3 Dorsal raphe nucleus Median raphe nucleus

78.0 82.0 65.0 58.0 24.9 17.2 77.8 61.2 37.0

± ± ± ± ± ± ± ± ±

2.0 9.9 6.1 12.1 5.5 2.3 13.6 16.0 8.0

MS

± ± ± ± ± ± ± ± ±

5.5*

LS

9.5**&##

HS

± ± ± ± ± ± ± ± ±

8.2**

21.3 9.4*# 14.5# 4.7**## 3.2 7.0 21.2 10.0

HS

3.7& LS C

% HS/LS

Serotonin c-Fos Brain region

Table 3 c-Fos and serotonin-like immunoreactivity

195

Table 4 Correlation between behavior, c-Fos and serotonin immunoreactivity

4.4 3.7 19.7 17.0 15.3 24.3 6.8 38.5

% HS/LS

M. Lehner et al. / Behavioural Brain Research 171 (2006) 189–198

Parameters

Pearson’s coefficient

P-value

Freezing/c-Fos HYPTH Vocalization/c-Fos PFCX Vocalization/c-Fos BLA c-Fos PFCX/5-HT PFCX c-Fos HYPTH/5-HT PFCX c-Fos BLA/5-HT BLA c-Fos MRN/5-HT PFCX c-Fos PFCX/c-Fos BLA

(−)0.84 (+)0.87 (−)0.76 (+)0.76 (+)0.79 (+)0.95 (+)0.78 (−)0.77

0.02 0.01 0.04 0.04 0.03 0.01 0.04 0.04

(see above). Conceivably, this is due to the different behavioral reactions examined. In the former experiment, after the pretest, the animals were divided into experimental groups, and exposed to the testing cage and aversive stimulation on retest 10 days later (HS and LS groups, given 5 mild foot-shocks 1.5 h before immunocytochemical part of the experiment). Thus, in that study, the effect of an acute painful stimulation in the aversively conditioned context on c-Fos protein, was examined in different brain structures. In the present experiment, which tested the response to the aversive context only, HS rats showed significantly more freezing behavior and a selective increase in c-Fos positive cells in BLA, MeA and CA1 area of the hippocampus, in comparison to LS group. This finding points to the enhanced sensitivity of the amygdalar complex and CA1 subregion of the hippocampus in the HS rats to the context-related, conditioned aversive stimuli. Indeed, in the literature, there is a large number of papers indicating an important role of the amygdalar complex and hippocampus, in processing of emotional input [9,10,12,14,18,19,27,31,35,45,56]. The present data also confirm a significant contribution of amygdalar serotonin in this respect, as HS rats had higher density of serotonin immunostaining in BLA. In the LS group, a selective enhancement of c-Fos expression occurred in the PFCX, HYPTH, DG, and median raphe nucleus (in comparison to C, MS and HS animals), along with a less potent freezing response. These data seem unexpected and counter-intuitive, at the first sight, as weaker freezing response was accompanied by enhanced cortical activity. However, it has become gradually more clear that serotoninergic innervation of the medial prefrontal cortex plays an important, inhibitory, role in the regulation of conditioned fear processing linked to the aversive conditioning [3,9,13,18,30,37,38,40,41,44,48]. In the rat, both the medial and lateral prefrontal cortical lesions substantially increased freezing behavior in different phases of the fear conditioning [30,41]. Rats that froze the least had the greatest increase in medial prefrontal cortex conditioned fear stimulus responses [37]. In vivo microdialysis showed that conditioned fear stress increased extracellular 5-HT levels in the medial prefrontal cortex, and this serotonin increase was followed by a resolution of the freezing behavior [15]. Furthermore, citalopram, a 5-HT reuptake inhibitor, administered before exposure to the conditioned fear stress, immediately and potently increased extracellular 5-HT concentration, and reduced freezing behavior [15]. The physiological stimulation of the serotoninergic raphe nuclei excited

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local neurons in the medial prefrontal cortex in vivo [46,47]. These findings indicate that consolidation of extinction learning is potentiated by serotonin-related medial prefrontal cortex activity, which inhibits fear during subsequent encounters with fear stimuli. More recently, it was shown that stimulation of the infralimbic subregion of the medial prefrontal cortex inhibited freezing if given immediately after the onset of a conditioned stimulus [38]. It is also noteworthy that both the dorsal and medial raphe nuclei were found to control the activity of PFCX pyramidal neurons in a complex manner [42,47]. These findings fit very well with the present data. LS animals showed less freezing behavior, enhanced activity of PFCX and stronger serotonin immunostaining in the same structure, in comparison to HS rats. Thus, it can be concluded that serotoninrelated activity of the medial prefrontal cortex is important for mediating or modulating central states of fear and anxiety, and extinction learning is potentiated by infralimbic activity, which inhibits fear during subsequent exposure to the fear stimuli. It should be remembered that our immunochemical method on serotonin concentration in the brain structures is semiquantitative, and it does not allow for a precise recognition of intrastructural localization of immunopositive complexes. Nevertheless, this method is highly sensitive, permits for estimation of a total serotonin immunoreactivity in the examined brain areas, and is often used for examining the activity of central serotonin system [2,54,58]. Moreover, strong statistically significant correlations found between the density of serotonin immunostaining in the brain structures and behavioral and biochemical variables (see below), also substantiate usefulness of this neurochemical procedure. In LS rats, there appeared also an increase in plasma concentration of a stress hormone, corticosterone, measured immediately after the contextual freezing test, in comparison to HS rats. This finding is also unexpected and counter-intuitive, as enhanced activity of the hypothalamic-adrenal axis has been linked to the stronger response to fear [6,49]. It is noteworthy, however, that in the LS group, there occurred also a selective and significant increase in c-Fos activity in the HYPTH, the brain area where CRF is synthesized, in comparison to other experimental groups. This may be considered an evidence of stronger activation by aversive context of this brain structure in LS rats, accompanied by enhanced activity of stress axis, and elevated plasma levels of corticosterone. It is also striking that in LS rats, c-Fos protein expression was selectively increased in DG of the hippocampus. This finding may be important for explaining the mechanism of a hyperactivity of stress axis in the LS group, given the assumed role of hippocampus in the control of hypothalamic CRF synthesis and release [26]. The biological significance of this phenomenon is not clear, however, it can be considered as another coping mechanism which, along with activation of PFCX, could help to better prepare the organism to handle a threatening stimulus [22]. Such interpretation can be inferred from a very well known role of glucocorticoids, released during stress, in the modification of different functions of the organism which are crucial for survival in the demanding situation (e.g. an increase in blood pressure and respiration, hyperglycaemia, etc.) [6,8,26,35,49]. Accordingly, LS rats

were less inhibited behaviorally and had higher concentration of corticosterone in the blood. It is also conceivable that when stress is too strong or chronic, such adaptive processes may fail, and disturbances in behavioral and physiological processes may emerge [35,51,53]. It is also noteworthy, that it was not possible to perform the assays of c-Fos and serotonin immunocytochemistry, and plasma corticosterone at the same time point, because of a long latency of induction of c-Fos protein, on the one hand, and a short duration of plasma corticosterone elevation after stress, on the other hand (1.5 h and 10 min, respectively) [16,32]. The analysis of correlations among different behavioral and biochemical measures examined in the present study provides more support for some of the above conclusions (Table 4). These data support the assumption about the role of serotonin originating from MRN in the stimulation of cortical neurons activity, controlling concurrently with HYPTH rat emotional behavior expressed as ultrasonic vocalization [1,4,5]. On the other hand, serotoninergic innervation of BLA appears be related more selectively to the regulation of a conditioned freezing response. In sum, the present data indicate that both strategies of coping with an aversive event (increased freezing versus vocalization) are under control of different central mechanisms [11,20,22,29,60]. The behavior of LS group (less freezing response and stronger vocalization) was related to stimulation of prefrontal cortex activity, increased activity of adrenal glands and stronger serotonin immunostaining in the PFCX, in comparison with HS animals. The more passive strategy of coping with the aversive event of HS group was related to increased activity of amygdalar nuclei and some areas of the hippocampus, and stronger 5-HT immunostaining in the baso-lateral nucleus of the amygdala, in comparison with LS rats. The present findings suggest that animals more vulnerable to stress might have innate deficits in the activity of brain systems controlling the hypothalamic-pituitary-adrenal axis that would normally allow them to cope with stress. It appears also, that response to pain may determine other patterns of emotional behavior, probably reflecting different activation thresholds of some brain structures controlling anxiety, e.g. prefrontal and secondary motor cortex [24,28,34,50,59]. Acknowledgements The study was supported by Grant no. 61/2005 from the Institute of Psychiatry and Neurology in Warsaw. The authors express their sincere gratitude to Mrs. Ala Biegaj for her excellent technical support. References [1] Adell A, Casanovas JM, Artigas F. Comparative study in the rat of the actions of different types of stress on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacology 1997;36:735–41. [2] Amat J, Tamblyn JP, Paul ED, Bland ST, Amat P, Foster AC, et al. Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala. Neuroscience 2004;129:509–19.

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