Neuroscience Letters 397 (2006) 190–195

5-HT1A receptor activation counteracts c-Fos immunoreactivityinduced in serotonin neurons of the raphe nuclei after

immobilization stress in the male rat

Jose Rioja a, Luis J. Santın b, Alicia Dona a, Laura de Pablos b, Francisco J. Minano c,Salvador Gonzalez-Baron a, Jose A. Aguirre a,∗

a Department of Human Physiology, School of Medicine, Campus de Teatinos, Boulevard Louis Pasteur,32, University of Malaga, 29071 Malaga, Spain

b Department of Psychobiology, School of Psychology, University of Malaga, 29071 Malaga, Spainc Department of Pharmacology, School of Medicine, University of Sevilla, 41010 Sevilla, Spain

Received 4 March 2005; received in revised form 4 November 2005; accepted 7 December 2005

Abstract

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The serotoninergic system and the 5-HT1A receptors have been involved in the brain response to acute stress. The aim of our study wasvaluate the role of the 5-HT1A receptors in serotoninergic cells of rostral and caudal raphe nuclei under acute immobilization in rats. Doublemmunocytochemical staining of 5-hydroxy-tryptamine and c-Fos protein and stereology techniques were used to study the specific cell activation inhe raphe nuclei neurons in five groups (control group, immobilization group (immobilization lasting 1 h), DPAT group (8-OH-DPAT 0.3 mg/kg, s.c.),PAT + IMMO group (8-OH-DPAT 0.3 mg/kg, s.c., 30′ prior acute immobilization) and WAY + DPAT + IMMO group (WAY-100635 0.3 mg/kg,

.c. and 8-OH-DPAT 0.3 mg/kg, s.c., 45′ and 30′, respectively, before immobilization). Our results showed an increase in the number of c-Fosmmunoreactive nuclei in serotoninergic cells in both dorsal and median raphe nuclei in the immobilized group. The 8-OH-DPAT pretreatmentounteracted the excitatory effects of the acute immobilization in these brain regions. In addition, WAY-100635 administration reduced the effectf 8-OH-DPAT injection, suggesting a selective 5-HT1A receptor role. Raphe pallidus and raphe obscurus did not show any differences amongxperimental groups. We suggest that somatodendritic 5-HT1A receptors in rostral raphe nuclei may play a crucial role in both mediating theonsequences of uncontrollable stress and the possible beneficial effects of treatment with 5-HT1A receptor agonists.

2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Immobilization stress; Raphe nuclei; c-Fos immunohistochemistry; Serotonin; 5-HT1A receptor; 8-OH-DPAT

eactions related to uncontrollable events but not to control-able ones include expression of conditioned fear [24], defen-ive burying and defeat posturing [43], territorial defense [42]nd increased anxiety observed as decreased social interac-ion [37] and increased passive avoidance tested in elevated Taze [33]. Many efforts to understand the mechanisms medi-

ting these phenomena have focused on serotoninergic neuronsn the dorsal raphe nucleus (DRN) and median raphe nucleusMnRN). Biochemical measurements of serotonin (5-HT) tis-ue levels indicate that midbrain serotoninergic neurons respondo inescapable stress [1,18,20]. Different degrees of responsive-ess appear among serotoninergic neurons under different types

∗ Corresponding author. Tel.: +34 952 134212; fax: +34 952 131650.E-mail address: Jose.Aguirre@uma.es (J.A. Aguirre).

of stress. Furthermore, 5-HT neurons of the DRN and MnRNexhibit a functional heterogeneity [26]. It has been demonstrated[13] that serotoninergic neurons in MnRN, but not serotoniner-gic neurons of DRN or hindbrain raphe nuclei, respond in aselective manner to an inescapable stressor, although activationof 5-HT immunoreactive (5-HT-IR) cells in the DRN in ratsexposed to an uncontrollable stressor has been well demon-strated [10]. Activation of the DRN reduces activity in thedorsolateral periaqueductal gray matter [23] and enhances theactivity in the amygdala which is necessary for fear conditioningdevelopment and increasing anxiety. Since the immediate earlygene products are thought to mediate the metabolic challengeimposed on the cell by strong activation [27], c-Fos immunohis-tochemistry has become a popular technique to assay neuronalactivity [14]. Among the 14 known 5-HT receptor subtypes, the5-HT1A receptor has received most attention mainly because it is

304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.neulet.2005.12.021

J. Rioja et al. / Neuroscience Letters 397 (2006) 190–195 191

involved in anxiety. Postsynaptic 5-HT1A receptors are found inlimbic regions such as hippocampus, septum and amygdale [32].In addition, administration of partial 5-HT1A receptor agonistssuch as azopirones [4,28,30] and full 5-HT1A receptor agonistssuch as 8-hydroxy-2-(di-n-propilamino)tetralin (8-OH-DPAT)resulted in an anxiolitic-like effect [17]. Presynaptic 5-HT1Areceptors are present in serotoninergic neurons in DRN andMnRN and provide a mechanism for the feedback inhibitionof the 5-HT system, due to the fact that the 5-HT1A receptor isnegatively coupled to adenylyl cyclase [16]. In this way, psy-chopharmacological treatments have shown the crucial role of5-HT1A receptors in the above mentioned emotional disorders[3]. However, the specific role of 5-HT1A receptors in the onsetand development of stress-related disorders remains unclear. Theaim of this study was to contribute to the knowledge of therole of 5-HT1A receptors in serotoninergic cells of rostral andcaudal raphe nuclei under acute immobilization stress (IMMO)in rats.

Specific pathogen-free adult male Sprague–Dawley ratsweighting 250–300 g from central vivarium of the Universityof Malaga supplied by Charles River Lab. Espana (Barcelona,Spain) were used. Rats were housed six per cage in atemperature-controled room (22–24 ◦C) and maintained on a12 h:12 h light:dark cycle (light on at 7:00 am). Food andwater were available ad libitum. The experiments were per-formed following the European Communities Council direc-tatwg(liO(awpp(citsafasffwir

o0

serum (Sigma, Spain) with 0.3% Triton X-100 (10 min, roomtemperature) the sections were incubated with a rabbit poly-clonal antibody (Santa Cruz Biotech. sc-52, USA) raised againstthe c-Fos protein (1:5000). Overnight incubation at 4 ◦C wasperformed in 0.1 M PBS containing 0.3% Triton X-100. Thesections were washed 3 times in PBS and incubated with biotiny-lated specific secondary antibodies. (1:200; Vector Labs Inc,Burlingame, CA) for 1 h at room temperature. The immunos-taining was performed according to the ABC method using theVectastain kit (Vector, Burlingame, CA). The chromogen usedwas 0.03% 3-3′-diaminobenzidine tetrahydrochloride (DAB)(Sigma, Spain) intensified with nickel chloride hexahydrate(Sigma, Spain) 0.04% (w/v) giving a darker black nuclei stain-ing (see Fig. 1) and 0.03% fresh H2O2 in 0.1 M Tris–HCI (pH7.6). 5-HT immunostaining was performed under the same con-ditions as c-Fos but using 0.9% saline Tris buffer 0.1 M (pH 7.6)instead of saline phosphate buffer. The primary antibody wasa monoclonal antibody raised against 5-HT (INCSTAR, USA)(1:50,000). Nickel chloride was not added to the chromogensolution in the second incubation for immunostaining in orderto get a brownish reaction. Sections from every animal fromeach experimental group were processed simultaneously. Aftermounting the sections on gelatin-chromalum coated slides, thesections were dehydrated and coverslipped with DPX (Panreac,Barcelona, Spain). Thus, every section was numbered accord-ing to the rostrocaudal level determined with the Paxinos andW

Fig. 1. Micrograph of dorsal (D) and median (Mn) raphe nuclei (1). Pallidus(Pa) and obscurus (Ob) raphe nuclei (2). Image (3) shows a serotononergicneuron non c-Fos-IR (A), c-Fos-IR non serotonergic neuron (B) and c-Fos-IRserotonergic neuron (C).

ive (86/609/EEC) for animal care and experimental procedurend the experiments were approved by the Ethical Commit-ee for Animal Research of the University of Malaga. Animalsere divided into five experimental groups: (1) saline: controlroup (without acute IMMO but with saline injection) (n = 4);2) IMMO: group submitted to acute IMMO (1 h of immobi-ization in Plexiglas tubes (5.5 cm × 21 cm) 30 min after salinenjection (n = 4)); (3) D + I: group receiving IMMO plus 8-H-DPAT pretreatment 30 min before acute IMMO (n = 4);

4) 8-OH-DPAT: group pretreated with 8-OH-DPAT withoutcute IMMO (n = 4); (5) W + D + I: group enduring acute IMMOith N-tetra-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl-)2-henil-propionamide (WAY-100635) 15 min prior 8-OH-DPATlus IMMO treatment. The drugs were provided by SigmaSpain) and were made up in 0.9% saline and administered sub-utaneously with a final concentration of 0.3 mg/kg becauset induces neuroendocrine and behavioral effects [25]. Afterhe administration period, the rats were anaesthetised withodium pentobarbital (Mebumal; 100 mg/kg body weight, i.p.)nd intracardially perfused with 200 ml isotonic ice-cold salineollowed by 200 ml of fixation fluid (4 ◦C) during 8 min. The fix-tive consisted of 4% paraformaldehyde (w/v) in saline 0.1 Modium PB (PBS) (pH 7.4). The brains were removed, postfixedor 2 h in the same fixative and cryoprotected in sucrose (10%or 24 h followed by 30% for 1 week, at 4 ◦C). The brainstemas cut on a cryostat (Micron, HM 500 M, Walldorf, Germany)

n 30 �m coronal sections sampled every eight sections with aandom start.

The sections were processed free-floating. Endogenous per-xidase activity was removed by incubating the sections with.3% H2O2 for 20 min. After blocking with 1% normal goat

atson [31] rat brain atlas.

192 J. Rioja et al. / Neuroscience Letters 397 (2006) 190–195

c-Fos-IR in serotoninergic cell bodies (dark nucleus andbrownish cytoplasm double immunostaining) were counted inthe dorsal raphe nucleus (DRN), median raphe nucleus (MnRN),obscurus raphe nucleus (ObRN) and pallidus raphe nucleus(PaRN). An Olympus BX51 microscope (Olympus, Denmark)was interfaced with a computer and a color JVC digital video-camera. C-Fos-IR nuclei were counted using the CAST-Gridsoftware package (Olympus, Glostrup, Danmark). The quan-tification was done by systematically sampling in each regionusing the following formula: Nv = �(Q−)/�(hafra), where Q− isthe total number of c-Fos-IR nuclei counted in all used frames,afra is the area of the sampling frames used and h is the thicknessof the sections [2,41]. At least two sections of each region werecounted per animal. The number of counting frames used was90–110 per animal in both DRN and MnRN, representing the10% of the whole analyzed volume. Because of the low volumeof the PaRN and ObRN nuclei, the whole volume of each slice(100%) was counted, yielding about 20 sampling frames in thePaRN and 50 sampling frames in the ObRN in each slice. Theresulting densities were averaged in order to obtain the meanof c-Fos-IR (Nv) serotoninergic cells per volume unit (mm3 inrostral raphe nuclei and �m3 in caudal raphe nuclei).

The cell densities were evaluated using the SPSS softwarepackage by one-way ANOVA and post hoc comparison test(Fisher’s least significant difference).

As seen in Fig. 2 acute immobilization produced a sig-n

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IR in serotoninergic cell bodies (5984 ± 1144 cells per mm3;mean ± S.E.M.) in DRN in comparison with c-Fos-IR in sero-toninergic cell bodies density in the control group (1364 ± 396cells per mm3). The 5-HT1A receptor agonist 8-OH-DPAT alonedid not induced any significant effect on c-Fos-IR in serotoner-gic cell bodies (2024 ± 396 cells per mm3), but counteracted theincrease of the density of c-Fos-IR in the experimental group ofIMMO animals pretreated with 8-OH-DPAT, giving a mean den-sity value (2200 ± 352 cells per mm3) similar to the mean valueobtained in the control group (P > 0.05). In addition, pretreat-ment with the specific 5-HT1A antagonist WAY-100635 counter-acted the effect of 8-OH-DPAT in IMMO animals (4840 ± 704cells per mm3 P < 0.05), producing an increase in the number ofc-Fos-IR in serotoninergic cell bodies per mm3. MnRN showeda similar activation pattern of c-Fos-IR in serotoninergic cellnuclei densities. In this way, The IMMO group displayed asignificant seven-fold increase (13,244 ± 2772 cells per mm3,P < 0.05) in the c-Fos-IR target cell bodies density in compari-son with the control group (2024 ± 264 cells per mm3), althoughthe increase in IMMO animals was two-fold in comparison withthe activation observed in the DRN. In the same way as DRN,8-OH-DPAT group did not show any effect if administered with-out IMMO (4312 ± 1452 cells per mm3) but counteracted theeffect of IMMO if administered 30 min before (4136 ± 396 cellsper mm3, P < 0.05). No statistical difference was observed inthe group of animals pretreated with WAY-100635 prior 8-OH-Dtsp1otg

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ificant four-fold increase (P < 0.05) in the density of c-Fos-

ig. 2. Density of c-Fos-IR in serotonergic cell nuclei in dorsal raphe nucleusDRN) (A) and median raphe nucleus (MnRN) (B). CONTROL: saline group;MMO: immobilization group; D + I: group treated with 8-OH-DPAT plus

MMO; 8-OH-DPAT: group only treated with 8-OH-DPAT; W + D + I: groupreated with WAY-100635 plus 8-OH-DPAT plus IMMO. Mean ± S.E.M. (n = 4)ne-way ANOVA with PLSD test, corrected by Bonferoni’s procedure. *P < 0.05or comparison between IMMO group vs. CONTROL and 8-OH-DPAT vs. D + Iroups. +P < 0.05 for comparison between W + D + I group and D + I group.

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PAT plus IMMO in comparison with 8-OH-DPAT plus IMMOreated animals (P > 0.05). Moreover, the mean c-Fos-IR den-ity in the experimental group WAY-100635 plus 8-OH-DPATlus IMMO (7832 ± 3168 cells per mm3) showed that WAY-00635 had a clear tendency (P = 0.11) to counteract the effectf 8-OH-DPAT on immobilized animals (see Fig. 1), althoughhis effect was not statistically different compared to the controlroup (P = 0.14).

In both DRN and MnRN, the mean percentage of c-Fos-IRerotonergic neurons from the total serotoninergic neurons wasigher in immobilized animals compared to control. In this way,n the DRN, the percentage of c-Fos-IR serotoninergic neuronesn immobilized animals (30%) were two-fold when comparedo control group (15%). In case of MnRN, this percentage inMMO group was also higher (10%) compared to control group2%).

In the other hand, c-Fos-IR in serotoninergic cell bodies den-ities measured in ObRN and PaRN did not show differencesmong the experimental groups (P > 0.05). Thus, 1 h immobi-ization did not produce an increase on c-Fos-IR serotonergicell densities in comparison with the density values obtained inhe control group (P > 0.05). Neither the pretreated 8-OH-DPATroup nor the immobilized 8-OH-DPAT group showed differ-nces (P > 0.05) with the control or IMMO group (Table 1).

In this study, we have reported an increased c-Fos-IR inoth 5-HT MnRN and DRN neurones in rats submitted toncontrollable stress. However, the increase of c-Fos-IR wasreater in the MnRN compared to DRN in response to 1 hf immobilization (Fig. 1). In this way, it was suggested aunctional distinction between the 5-HT neurones responseo stress, in the rostral raphe nuclei [1,10,13]. Different

J. Rioja et al. / Neuroscience Letters 397 (2006) 190–195 193

Table 1Number of target cells × 105/�m3 in pallidus raphe nucleus (PaRN) and obscu-rus raphe nucleus (ObRN)

Experimental groups PaRN (mean ± S.E.M.) ObRN (mean ± S.E.M.)

CONTROL 2.9 ± 2.9 1.4 ± 1.4IMMO 4.3 ± 2.5 1.4 ± 1.4D + I 10.0 ± 10.0 10.1 ± 6.28-OH-DPAT 8.6 ± 8.6 4.3 ± 4.3W + D + I 8.6 ± 4.3 2.9 ± 1.4

CONTROL: saline group; IMMO: immobilized group; D + I: group treated with8-OH-DPAT plus IMMO; 8-OH-DPAT: group only treated with 8-OH-DPAT;W + D + I: group treated with WAY-100635 plus 8-OH-DPAT plus IMMO.

stressors induce a different activation pattern in central 5-HTneurones [21]. In this way, it has been reported that acute swimstress induces a great amount of c-Fos-IR in DRN neurones.Moreover, the great amount of these neurones are GABAergicand about a 4% of them are serotoninergic [21]. In addition,a single immobilization treatment elicited a great tryptophanhydroxylase mRNA increase in MnRN but not in DRN [7].

The reason why 5-HT neurones activation is higher in theMnRN than in the DRN using acute stressors (i.e. swim andimmobilization) remains unclear. Moreover, the noradrenergic[5,6,25,35], glutamatergic/GABAergic and/or CRF [34,39,40]regulation of rostral raphe nuclei could be the neurochemicalbasis underlying the different amount of c-Fos-IR reported in thispaper in those raphe nuclei under acute immobilization stress.Furthermore, our results showed that 1 h of immobilization hasno significant effects in the serotoninergic neurones c-Fos-IRof the PaRN and ObRN (see Table 1). These results contrastto the increase observed in the c-Fos-IR in the serotoninergiccaudal raphe nuclei neurones in animals suffering inescapableshock stress [38]. Because PaRN and ObRN present a verylow volume, randomly error is higher, due to the number ofscores is low. According to this, it is difficult to yield an accu-rate density number of c-Fos-IR serotonergic neurons in theseraphe nuclei at any experimental situation using immunocyto-chemical methods. More experimental approaches need to bedisplayed in order to elucidate a clear effect of acute immobiliza-tn

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nuclei but not to caudal raphe nuclei [12], which are mainlyinvolved in the response to physiological stressor, not emotionalones. According to this, it is reasonable to think that differentactivation patterns can be observed between these two groups ofraphe nuclei.

In the other hand, the pharmacological modulation of the5-HT1A receptor used in our study, showed that the adminis-tration of 8-OH-DPAT in the quiet condition (absence of stressstimulation), induced no changes in the c-Fos-IR in any raphenuclei. Previously, electrophysiological studies had reported thatthe firing of the serotoninergic neurons, is reduced in the ros-tral [15] and the caudal [22] raphe nuclei by the activationof 5-HT1A receptors. In contrast, in our study, the pharma-cological stimulation of the 5-HT1A receptors in both nucleiwas not able to induce changes in the c-Fos-IR (see Fig. 2and Table 1). However, in the acute IMMO condition, the 8-OH-DPAT pretreatment counteracted the increased c-Fos-IRinduced by 1 h IMMO in rostral raphe nuclei (Fig. 2) but notin the caudal raphe nuclei studied (Table 1). The serotonergicneurones in both rostral and caudal raphe can be differen-tially activated by different stressors. In this way, our resultsshowed a different activation pattern between both raphe nucleigroups in response to acute immobilization stress. However,it has been shown that inescapable electric shock induces astrong activation in serotoninergic neurones in all raphe nuclei[38].

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ion stress and 5HT1A receptor treatment on these caudal rapheuclei.

Conversely, we found no c-Fos-IR changes in the serotoniner-ic caudal raphe nuclei associated to acute immobilization stresssee Table 1). These results suggest a functional dissociationetween the caudal and rostral raphe neurones. The differentnvolvement between the serotoninergic neurones in the ros-ral and the caudal raphe nuclei in the acute immobilizationtress could be related to differences in the neurochemical con-rol during the immobilization stress. In this way, caudal rapheuclei neurons show low or very low �1 adrenoceptor mRNAevels [11]. The noradrenergic stimulation of those receptorsight not be enough to induce a significant neuronal activation

nder acute immobilization stress. In addition, the immobiliza-ion stress has been described as an emotional model of stresshich is related to the activation mainly in limbic areas in the

orebrain and midbrain [8]. The main serotoninergic afferentrojections and control of these nuclei belong to rostral raphe

The ability of the 8-OH-DPAT to counteract the increase of-Fos-IR induced by acute immobilization in the rostral rapheuclei dependent on the stimulation of the 5-HT1A receptors.n this sense, WAY-100635 pretreatment reverted the effect of-OH-DPAT in both rostral raphe nuclei. However, the potencyf the WAY-100635 to blockade the 8-OH-DPAT is greater inhe DRN than in the MnRN.

This differential and unexpected effect of the WAY-100635etween the rostral raphe nuclei, would be explained by at leastwo possible mechanisms: a greater 5-HT1A receptor reservend/or a greater 8-OH-DPAT binding to other 5-HT receptorsn the MnRN. In addition, unfortunately, the density of 5-HT1Aeceptors observed in the MnRN is lower than in the DRN [32]hich discard the first mechanism. In this way, the 8-OH-DPAT

n presence of WAY-100635 would be able to binding to the2 adrenoceptor [5], which is able to inhibit the 5-HT synthe-is, metabolism and liberation in both DRN [9] and MnRN [1].owever, to our knowledge, no differences have been describedetween MnRN and DRN in the density and/or 8-OH-DPATffinity to the �2 adrenoceptor that allow us to support thisypothesis.

In summary, our results suggest a strong involvement of the-HT1A serotonergic receptors in both MnRN and DRN to coun-eract c-Fos-IR activation in response to acute IMMO stress.reviously, several studies have reported long-term alterations

nductions impairments related to acute stress in emotional [29],ognitive [36] and autonomic [19] responses. In addition, 5-T1A receptor activation before acute stress counteracts induced

ong-term behavioral effects [33]. These beneficial effects of 5-T1A receptor agonists could be associated to reduced c-Fos-IR

n the caudal raphe nuclei reported in our study.

194 J. Rioja et al. / Neuroscience Letters 397 (2006) 190–195

Acknowledgement

This work has been supported by the Spanish DGICYT(PM99-0159).

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