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Research Report

Brainstem areas activated by diazepam withdrawal asmeasured by Fos-protein immunoreactivity in rats

Lucas Baptista Fontanesi, Renata Ferreira, Alicia Cabral, Vanessa Moreno Castilho,Marcus Lira Brandão, Manoel Jorge Nobre⁎

Instituto de Neurociências & Comportamento—INeC, Campus USP, Ribeirão Preto, 14040-901, SP, BrazilLaboratório de Psicobiologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto,14040-901, SP, Brazil

A R T I C L E I N F O

⁎ Corresponding author. Fax: +55 1636024830.E-mail address: mjnes@usp.br (M.J. Nobre

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.07.007

A B S T R A C T

Article history:Accepted 5 July 2007Available online 14 July 2007

In the 1970s, chronic treatment with benzodiazepines was supposed not to causedependence. However, by the end of the decade several reports showed that theinterruption of a prolonged treatment with diazepam leads to a withdrawal syndromecharacterized, among other symptoms, by an exaggerated level of anxiety. In laboratoryanimals, signs that oscillate from irritability to extreme fear-like behaviors and convulsionshave also been reported. In recent years many studies have attempted to disclose the neuralsubstrates responsible for the benzodiazepines withdrawal. However, they have focused ontelencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. Inthis study, we examined the Fos immunoreactivity in brain structures known to beimplicated in the neural substrates of aversion in rats under spontaneous diazepam-withdrawal. We found that the same group of structures that originally modulate thedefensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, thesuperior and inferior colliculus and the dorsal periaqueductal gray, were most labeledfollowing diazepamwithdrawal. It is suggested that an enhanced neural activation of neuralsubstrates of fear in the midbrain tectum may underlie the aversive state elicited indiazepam-withdrawn rats.

© 2007 Elsevier B.V. All rights reserved.

Keywords:WithdrawalDiazepamAnxietyFosBrainstemdPAG

1. Introduction

Benzodiazepines compounds are widely used for the treatmentof anxiety and sleep disorders and are also prescribed for theirsedative, muscle relaxant and anticonvulsant properties(Woods et al., 1987; Bateson, 2002). Initially, chronic treatmentwith benzodiazepines was supposed not to cause dependence.However, it has been demonstrated that, on abrupt withdrawalfrombenzodiazepine, chronic exposure patients can experience

).

er B.V. All rights reserved

a number of symptoms indicative of a dependent state. Amongthe symptoms raised, an exaggerated level of anxiety (reboundanxiety), more intense than that presented during the prescrip-tion of the drug, has been mentioned (Chouinard, 2004; RynnandBrawman-Mintzer, 2004). Additionally, clinical reports havedenounced the appearance of high levels of fear in patientsexperiencing benzodiazepine withdrawal (Rosebush andMazurek, 1996). In the laboratory, similar aspects of thewithdrawalphenomenacanbe reproduced inanimals.Actually,

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Fig. 1 – Percentage of entries in the open arms (A), number ofenclosed arms entries (B) and percentage of time spent in theopen arms (C) of the elevated plus-maze of rats that did notreceive any treatment (No Treat) and rats treated chronicallyp.o. with sucrose or diazepam. As ANOVA showed nosignificant differences between the two control groups usedin this study, all the differences shown in the graphs arerelative to comparisons between the diazepam-treated ratsand those belonging to the sucrose group. Independentgroups of diazepam treated animals were tested at thedependence (30 min after the last drug intake—Dzp DEP) orwithdrawal (48 h after the last drug intake—Dzp W). Data arepresented as mean±S.E.M. One-way ANOVA followed bypost-hoc Newman–Keuls; pbbbb0.05.

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signs that oscillate from irritability to extreme fear behaviorsand convulsions have already been described (Petursson andLader, 1981a,b; Owen and Tyrer, 1983; Emmett-Oglesby et al.,1983; Lacerra et al., 1999; Woods et al., 1987; Ashton, 2005).

Thegreatmajority ofmethodsused in the investigationof theneurobiologicalmechanisms implicated in theproductionof fearand anxiety as, for example, chemical or electrical stimulation orlesions of brain structures, can provide only indirect evidenceson the cerebral operation. In this case, the use of immunohis-tochemical techniques, such as the Fos protein detection, hasbeen employed because it allows a clear visualization of theneurons activated during the presentation of a specific fearstimulus (Dragunow and Faull, 1989; Reiman et al., 1989; Bullitt,1990). In fact, Fos-like immunoreactivity has been used as amarker of the neuronal activation that can be precipitated by awide range of stimuli as, for example, brain electrical stimulation(Sandner et al., 1992; Vianna et al., 2003), local injection ofneurotransmitters (Stone et al., 1991; Richard et al., 1992;Ferreira-Netto et al., 2005), stress induced by immobilization(Imakiet al., 1992;Honkaniemiet al., 1992), pain (Bullitt, 1990) andabstinence of several types of drugs of abuse such as opiates(Hayward et al., 1990), psychostimulants (Persico et al., 1995),alcohol (Moy et al., 2000) and benzodiazepines (Dunworth et al.,2000). Using this technique, it has been shown that someprosencephalic structures are activated by benzodiazepinewithdrawal, such as the shell region of the nucleus accumbens(Dunworth et al., 2000), one of themost important areas involvedwith the expression of motivational factors linked to drugs ofabuse. This type of activation is also produced by anxiogenicstimuli that induce increase in dopamine release and inductionof Fos immunoreactivity in this structure (Abercrombie et al.,1989; Smith et al., 1997). In the same context, a simple exposureto the elevated plus-maze produces great Fos labeling in limbicareas (Silveira et al., 1993; Sandner et al., 1993) related with theproduction of the behavioral and autonomic responses associ-ated with the expression of fear.

Drugwithdrawalmay function as an unconditioned stressor,and as such could activate a particular set of structures involvedwith the organization of fear states, particularly those belongingto thewell-known brain aversion system, suchas the amygdala,dorsal periaqueductal gray (dPAG) (themain effector pathway ofthe defensive behavior responses), superior and inferior colliculi(structures included in the processing of visual and auditoryinformation of aversive nature, respectively) and the medialhypothalamus (mainly tied to the production of the somaticresponses of fear) (Gray, 1982; Graeff, 1990; Brandão et al., 1999,2005). To examine this possibility, in this study we analyzedwhether the expression of fear behaviors observed in ratsabruptly withdrawn from diazepam could lead to a consequentactivation of the same brain structures that modulate the ex-pression of the defensive behavior of animals exposed todangerous situations such as, for example, exposure to theopenarmsof the elevatedplus-maze, amongothers. To thisend,we used the immunohistochemical technique for the detectionof Fos-protein expression in selected structures of diazepam-withdrawn rats.

Thegreatmajorityof studies in the literature that investigatethe chronic effects of a drug of abuse has mainly used intra-peritoneal injections as the method of delivery of the drug.However, it was demonstrated that rats under chronic systemic

injectionsof placebo solutions tend topresent an increase in theanxiety levels when tested in the elevated-plus maze (Griebelet al., 1994). In this case, it could be argued that the aversionpromoted by the exposure to chronic systemic injections couldlead to an increase in the number of Fos-like immunoreactiveneurons in structures that normally modulate this class ofemotional responses. To circumvent this problem, in this work,we used a newmodel of oral drug intake, adapted by Schleimeret al. (2005), in which the animals voluntarily drink the drug orcontrol solutions.

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2. Results

2.1. Plus-maze

One-way ANOVA revealed that the chronic treatment withsucrose, per se, did not promote any effect on the behavior ofrats after the interruption of the long-term treatment, asthere were no observed significant differences in any of theregistered measures on the behavior of rats of this group,when compared with those that did not suffer any inter-vention (No Treat). On the other hand, significant groupdifferences in both the percentage of entries [F (3,72)=5.35;pb0.005] and on the percentage of time spent in the openarms [F (3,72)=18.55; pb0.001] were observed in animalsunder diazepam effects (Dzp DEP) or during withdrawal (DzpW), when compared with those that received sucrose only.Newman–Keuls post-hoc showed that the chronic oral intakeprocedure was effective in producing a significant anxiolytic-like effect in rats tested 30 min after the last oraladministration of diazepam (Dzp DEP); as revealed by the

Table 1 – Mean (±S.E.M.) number of Fos-positive neurons/0.1perfused 2 h after plus-maze exposure

Structures Sucrose

Left Right M

Telencephalon PrL 6.18±1.72 7.20±1.27 6.CG1 21.40±4.43 21.45±4.71 21.CG2 2.86±0.87 2.66±0.79 2.CeA 2.01±0.72 1.15±0.60 1.MeA 8.16±1.60 6.98±1.56 7.BLA 5.15 ±1.26 4.97±1.11 5.CA1 0.61±0.17 1.23±0.39 0.CA2 0.65±0.25 0.46±0.18 0.CA3 1.22±0.33 1.36±0.23 1.AcbC 1.55±0.48 1.92±0.88 1.AcbSh 2.01±0.55 1.94±0.85 1.

Diencephalon PV 19.74±5.41 19.51±5.52 19.PaV 16.18±2.55 17.11±3.05 16.AH 13.77±2.02 15.17±1.87 14.LH 7.84±1.48 8.92±1.11 7.DMH 3.75±0.75 3.57±0.90 3.

Mesencephalon DMPAG 1.35±0.33 1.23±0.31 1.DLPAG 1.25±0.24 1.47±0.32 1.LPAG 2.36±0.31 1.58±0.30 1.VLPAG 5.04±0.77 6.58±0.73 5.DR 6.26±0.70 8.68±0.86 7.MnR 7.79±0.87 8.55±0.95 8.SC 1.37±0.12 1.21±0.13 1.IC 1.91±0.33 2.10±0.40 2.LC 2.67±0.41 3.37±0.37 3.Cun 3.72±0.76 3.40±0.45 3.

Number of Fos-positive neurons per region on the left and right sides were4.0, Media Cybernetics). Asterisks indicate the regions in which withdracontrol group (sucrose). Differences between groups were analyzed withand CG2, cingulate areas 1 and 2, respectively; CeA, central nucleus of thnucleus of the amygdala; CA1, CA2 and CA3, hippocampal areas 1, 2 and 3accumbens (shell region); PV, paraventricular thalamic nucleus; PaV, parlateral hypothalamus; DMH, dorsomedial hypothalamus; DMPAG, dorsomLPAG, lateral periaqueductal gray; VLPAG, ventrolateral periaqueductal gracolliculus; IC, inferior colliculus; LC, locus ceruleus; Cun, cuneiform nucle

increase in the percentage of entries (Fig. 1A) and in the total‘open arms’ time (Fig. 1C). An anxiogenic-like effect wasachieved in rats on 48-h withdrawal from diazepam (Dzp W),as these animals stayed less time in the open arms of themaze (Fig. 1C) than the animals of the sucrose group. Thenumber of ‘closed arms’ entries [F (3,72)=3.22; pN0.05]showed a trend towards significance (p=0.055) to thiscondition among the groups tested. As shown in Fig. 1B,this effect was due to a tendency of rats under diazepameffect (Dzp DEP) to increase motor activity, when comparedwith the sucrose control group.

2.2. Fos immunoreactivity

Statistical analysis of the effects of the diazepam withdrawalon Fos-protein expression was conducted in brain areas of 22animals (n=11 for group) randomly chosen from each of thesucrose- or diazepam-withdrawal groups (DzpW) (seeTable 1).The mean number of Fos-protein immunoreactivity in neuro-nal nuclei is shown in Figs. 2, 4 and 6, for each brain regionstudied.

mm2 in brain regions of 48-h diazepam-withdrawn rats

Diazepam pb0.05

ean Left Right Mean

69±1.46 14.70±2.39 18.57±2.51 16.64±2.23 ⁎43±4.04 24.67±2.04 22.44±2.53 23.56±2.1476±0.79 3.23±1.39 4.29±1.71 3.76±1.5558±0.63 1.81±0.60 1.45±0.42 1.63±0.4157±1.41 9.54±1.62 9.49±1.92 9.51±1.7106±1.06 5.36±1.36 5.76±1.16 5.56±1.2192±0.27 1.67±0.32 2.27±0.69 1.97±0.4856±0.20 1.17±0.24 1.19±0.25 1.18±0.1829±0.25 1.36±0.34 1.79±0.43 1.57±03774±0.67 3.04±1.16 3.75±1.61 3.40±1.3598±0.67 3.68±1.32 3.38±1.00 3.53±1.1463±5.43 29.50±6.21 27.74±5.85 28.62±5.8564±2.60 27.57±5.77 31.07±6.68 29.32±6.1547±1.67 21.77±1.97 22.10±2.65 21.93±2.00 ⁎38±1.23 11.97±1.60 13.41±1.53 12.69±1.49 ⁎66±0.75 11.10±1.99 9.99±1.13 10.54±1.14 ⁎29±0.32 11.14±1.25 12.70±1.43 11.92±1.34 ⁎36±0.23 7.50±1.37 8.07±0.98 7.79±1.03 ⁎97±0.27 9.28±1.50 10.14±1.59 9.71±1.42 ⁎81±0.56 11.52±1.32 13.27±2.13 12.39±1.61 ⁎47±0.78 13.45±1.71 13.79±1.75 13.62±1.73 ⁎17±0.91 9.37±0.58 10.93±0.68 10.15±0.6329±0.11 6.65±1.01 6.87±0.57 6.76±0.72 ⁎01±0.35 13.70±1.78 13.62±1.83 13.66±1.36 ⁎02±0.36 1.88±0.28 1.3±0.21 1.60±0.20 ⁎56±0.34 9.21±1.42 10.11±1.16 9.66±1.15 ⁎

counted using a computerized image analysis system (Image Pro Pluswn-rats displayed differential Fos-expression (pb0.05) in relation tothe Student t-test for each region in study. PrL, prelimbic cortex; CG1e amygdala; MeA, medial nucleus of the amygdala; BLA, basolateral, respectively; AcbC, nucleus accumbens (core region); AcbSh, nucleusaventricular hypothalamic nucleus; AH, anterior hypothalamus; LH,edial periaqueductal gray; DLPAG, dorsolateral periaqueductal gray;y; DR, dorsal raphe nucleus; MnR,median raphe nucleus; SC, superiorus.

Fig. 2 – Mean number of Fos-immunoreactive neurons in the telencephalic structures of 48-h diazepam-withdrawn ratssubmitted to the plus-maze. Data are expressed as mean±S.E.M. of Fos-positive cells in 0.1 mm2 area of tissue. The number ofFos-positive neurons per region was bilaterally counted using a computerized image analysis system (Image Pro-Plus 4.0,Media Cybernetics). * Significant differences on the number of Fos-positive cells in each structure studied between diazepam(DzpW) and sucrose groups. The analysis was performedwith the use of the Student t-test. The level of significance was set atpbbbb0.05.

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2.2.1. TelencephalonAs revealed in Figs. 2 and 3 withdrawal from diazepam did notcause important Fos expression in almost all telencephalicregions. In fact, the Student t-test revealed significant dif-ference in the Fos expression only in the pre-limbic cortex(PrL) [t20=3.73; pb0.05] (Fig. 3, upper right). Otherwise, manyother areas mainly involved in withdrawal of drugs of abuse,as the core (AcbC) and shell (AcbSh) regions of the nucleusaccumbens, showed no alterations on Fos-protein immuno-reactivity (Fig. 3, bottom right).

2.2.2. DiencephalonHighneural activationwasobserved indiencephalic structures(Figs. 4 and 5). Forty-eight hours of diazepam withdrawal waseffective in promoting a robust increase in Fos expression inthe anterior (AH) [t20=2.86; pb0.05] and lateral hypothalamus(LH) (Fig. 5, upper right) [t20=2.75] and also in the dorsomedial(DMH) [t20=5.03; pb0.05] nuclei of the hypothalamus of theabstinent rats (Fig. 5, bottom right). Student t-test revealed thatthe comparisons between the withdrawal and sucrose groupsreached marginal significance (p=0.056) of the withdrawal onthe expression of Fos positive neuronal nuclei in the para-ventricular region of the hypothalamus (PaV).

2.2.3. MesencephalonStatistical analysis indicated significant differences in Fos ex-pression inalmostallmesencephalicareas studied (Figs. 6and7),as the dorsomedial [DMPAG: t20=7.74; pb0.05], dorsolateral[DLPAG: t20=6.11; pb0.05], lateral [LPAG: t20=5.35; pb0.05] andventrolateral [VLPAG: t20=3.87; pb0.05] columns of the periaque-ductal gray and dorsal raphe nucleus [DR: t20=3.24; pb0.05]

(see Fig. 7, upper right panel). Student t-test also showed sig-nificant differences in the superior [SC: t20=7.51; pb0.05] andinferior [IC: t20=8.28; pb0.05] colliculi (Fig. 7, bottom right) andcuneiform nucleus [Cun: t20=5.07; pb0.05]. Interestingly, of allmesencephalic structures studied, only locus ceruleus showed asignificant decrease in Fos-protein expression, when comparedwith the control group [LC: t20=3.44; pb0.05].

3. Discussion

The interruption of a long period of treatment with benzodiaze-pines produces great probability of developing withdrawalsymptoms in humans or in laboratory animals (Lukas andGriffiths, 1982; Ladewig, 1984). In fact, anxiety-like states havebeen reported as a common symptom in benzodiazepine-withdrawn patients. Avoidance of these unpleasant symptomsseems to negatively-reinforce continuous benzodiazepine useand is one of themain components of craving for the drug (Bustoet al., 1986; Busto and Sellers, 1991). An increase in anxiety-likestates is also observed in rats under spontaneous diazepam-withdrawal and submitted to several types of animal models ofanxiety (Greenblatt and Shader, 1974; Emmett-Oglesby et al.,1983; File, 1990). In our study, the interruption of the chronicregimen of diazepam caused a significant decrease in thepercentage of time spent in the open arms of the plus-maze.This anxiogenic effect promoted by diazepam withdrawal wasaccompanied by significant Fos-positive immunoreactivity intelencephalic, diencephalic and mesencephalic areas includingall columns of the periaqueductal gray, the deep layers of thesuperior colliculus, the central nucleus of the inferior colliculus,

Fig. 3 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through theprelimbic cortex (PrL, above) andon the core (AcbC) and shell (AcbSh) regionsof thenucleusaccumbens (below)of rats submitted tospontaneous diazepam-withdrawal (DzpW, right) and tested 48 h after the interruption of the treatment, when comparedwith itscontrols (Suc, left). Cg1=cingulate cortex, area 1; fmi=forcepsminor of the corpus callosum; Cl=claustrum; IL=infralimbic cortex;LV=lateral ventricle; LacbSh=lateral accumbens shell; aca=anterior commissure, anterior part; ec=external capsule.

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themedial aspects of thedorsal raphenuclei, the anterior, lateraland dorsomedial nuclei of the hypothalamus and the prelimbiccortex, all of them mainly linked to the modulation of theemotional, autonomic and motor expression of the fear-motivated behaviors. For example, the prelimbic cortex, amyg-dala and hypothalamus, in conjunction with other structures,are part of many well-defined anxiety- and fear-related circuitsin the brain, so that anxiogenic drugs that promote high levels ofaversion also significantly activate these circuits (Charney et al.,1998; Gorman et al., 2000; Gray and McNaughton, 2000; Rosenand Schulkin, 1998). Parts of the prefrontal cortex, including theprelimbic region, have been reported to be involved in theproduction of fear- and anxiety-related behaviors (Espejo, 1997;Jinks and McGregor, 1997; LeDoux, 1995). Additionally, manystudies on acute fear have proposed that brain areas such as theamygdala, hippocampus, hypothalamus and periaqueductalgray are putatively involved in fear processing (Fendt andFanselow, 1999; Graeff, 1994; Gray and McNaughton, 2000;LeDoux, 1995). Others have implicated the periaqueductal gray,amygdala, medial hypothalamus, raphe nuclei, superior andinferior colliculusand locusceruleus in theexpressionof the fearbehaviors (Graeff, 1990, 1994; Brandão et al., 1999, 2003, 2005).

The activation of the amygdala results in behavioral andphysiological responses associated with anxiety and panic-

like behavior. It is intriguing that no changes in Fos-expressionin this region were observed in our study. However, a similarresult was also obtained by Borlikova et al. (2006), who showedthat a single alcohol-withdrawal experience does not inducesignificant Fos expression in this area, contrasting with asignificant Fos expression following repeated withdrawalprocedures. The activation of the paraventricular hypotha-lamic nucleus (PaV) promotes release of a variety of hormonesthat are involved in the neuroendocrine and autonomicresponses to fear and psychological stress, whereas theactivation of the lateral hypothalamus seems to be importantfor the cardiovascular expressions of fear and anxiety. Also, inlaboratory animals, electrical stimulation of the anteriorhypothalamic nucleus elicits hissing and threat posturescharacteristic of defensive behaviors. Electrical or chemicalstimulation of some mesencephalic structures belonging tothe well-known brain aversion system, such as the superiorand inferior colliculus and periaqueductal gray, also promotedefensive behaviors in rats (Brandão et al., 1999, 2005). Theactivation of brainstemstructureswas also observed in severalstudies using Fos-protein expression as a neuronal marker ofanxiety-like states (Senba et al., 1993; Sandner et al., 1993;Silveira et al., 1995; Beck and Fibiger, 1995; Beckett et al., 1997;Canteras and Goto, 1999), and after intraperitoneal injection of

Fig. 4 – Number of Fos-immunoreactive neurons in thediencephalic structures of 48-h diazepam-withdrawn ratssubmitted to the plus-maze. Data are expressed asmean±S.E.M. of Fos-positive cells in 0.1 mm2 area of tissue.The number of Fos-positive neurons per region wasbilaterally counted using a computerized image analysissystem (Image Pro-Plus 4.0, Media Cybernetics). * Significantdifference on the number of Fos-positive cells in eachstructure studied between diazepam (Dzp W) and sucrosegroups. The analysis was performed with the use of theStudent t-test. The level of significance was set at pbbbb0.05.

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drugs that elicit panic-like symptoms (Singewald and Sharp,2000). All these common sets of structures involved in theexpression of the emotional, autonomic and motor compo-nents of fear-related behaviors were also strongly activated inrats suffering withdrawal which were submitted to the plus-maze, showing that the sameneuronal changesverifiedduringthe presentation of fear stimuli seems to be occurring duringdiazepam withdrawal.

The main neurobiological mechanism associated with theeffects of drugs of abuse has been the dopaminergicmesolimbicsystem and its connections with the basal prosencephalon(Koob, 1992; Koob and Le Moal, 1997). In this context, in aprevious experiment, Dunworth et al. (2000) investigatedwhether some diazepam-withdrawal symptoms could belessened by prior withdrawal experience in mice undergoing asingle or repeated flumazenil-precipitated diazepamwithdraw-al. In the third experiment of this study, they evaluated the Fos-protein expression in brain areas that they speculatedwould beimportant in either seizure sensitivity or the expression of anaversive state. The immunohistochemistry results showed asignificant increase in Fos expression in the shell region of thenucleus accumbens of mice submitted to a single withdrawalexperience; contrasting with our findings that showed nosignificant alteration on this measure in the same area. On theother hand, no differences were observed after flumazenil

injection in rats undergoing repeated diazepam-withdrawalexperience.Concerning this discrepancy,wemustpayattentionto some methodological aspects that could clarify the differ-ences obtained between the two studies. Most importantly, asrevealed above, on Dunworth's experiments, Fos immunohis-tochemistry assays were performed in mice through theflumazenil-precipitated diazepam-withdrawal procedure, incontrast with the spontaneous diazepam-withdrawal methodused in our experiments. This difference is seminal to under-stand the increase of Fos expression observed in the formerstudy. Concerning this point, the study of Motzo et al. (1997)demonstrated that a challenge administration of diazepaminhibits dopamine output in the nucleus accumbens. On theother hand, the discontinuation of long-term treatment withdiazepam does not alter the basal dopamine release in thisregion. However, a single intraperitoneal injection of flumazenil(in a dose that has no effect on the basal dopamine levels) iseffective in enhancing the release of this neurotransmitter inthe nucleus accumbens of rats chronically treated withdiazepam and tested after either 6 h or 5 days of withdrawal.In this case, we could argue that the enhanced Fos expressionobserved in the Dunworth's study could be due to a hyperfunc-tioning of the dopaminergic neurons on the accumbens,following flumazenil injection. Besides, the flumazenil concen-tration (20mg/kg)used in theexperiment ofDunworthet al.wasmuch more larger than that noted in the study of Motzo (4 mg/kg). In this case, an increase on the basal activity of thedopaminergic cells that could contribute to promote Fosexpression on the accumbens, could not be disregarded.

The absence of Fos expression in the nucleus accumbens inthe present study may be related to the notion that benzodiaz-epine withdrawal recruits neural substrates different from thedopaminergic one (Lingford-Hughes and Nutt, 2003) to promoteits effects. Indeed, changes in the GABA system have beenpostulated to underlie diazepam-withdrawal syndrome (Milleret al., 1988; Galpern et al., 1991; Toki et al., 1996). Secondaryalterations in other neurotransmitter systems as, for example,the glutamatergic and serotoninergic mechanisms have alsobeen considered (Allison and Pratt, 2003; Andrews et al., 1997)Additionally, we have found that the inhibition of the glutama-tergic neurotransmission through local injections of AMPA-kainate or NMDA antagonists in the dPAG reduce the fearbehaviors in diazepam-withdrawn rats, implicating the excit-atory amino acids of the dPAG in themodulation of the aversivestates induced by diazepam withdrawal (Souza-Pinto et al.,2007). As the mechanisms that modulate fear are primarilylocated in the brainstem, it is possible that these changes takeplace initially in the mesencephalon. This is supported by thepronounced Fos-positive immunoreactivity in structures basi-cally involved in the modulation of fear behaviors, such as theperiaqueductal grayand thesuperior and inferior colliculi of ratsunder diazepam withdrawal. Thus, it is likely that the fearelicited by diazepamwithdrawal is the result of the activation ofthe neural substrates of aversion in the brainstem, such as thedefense reaction elicited by stimulation of the dPAG (Brandãoet al., 1999, 2005). On the other hand, prosencephalic structures,such as amygdala, are involved in themodulation of the anxietypromoted by associative learning, such as that generated inaversive conditioning. In support of thepresent results, wehavefound that rats under ethanol withdrawal had an increase in

Fig. 5 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through theparaventricular (PvA), anterior (AH) and lateral (LH) regions of the hypothalamus (above), and the dorsomedial hypothalamicnucleus (below) of rats submitted to spontaneous diazepam-withdrawal (DzpW, right) and tested 48 h after the interruption ofthe treatment, when compared with its control (Suc, left). f=fornix; 3V=third ventricle; VMH=ventromedial hypothalamus;opt=optic tract; mt=mamillothalamic tract; ic=internal capsule.

Fig. 6 – Number of Fos-immunoreactive neurons in themesencephalic structures of 48-h diazepam-withdrawn rats submittedto the plus-maze. Data are expressed asmean±S.E.M. of Fos-positive cells in 0.1mm2 area of tissue. The number of Fos-positiveneurons per region was bilaterally counted using a computerized image analysis system (Image Pro-Plus 4.0, MediaCybernetics). * Significant difference on the number of Fos-positive cells in each structure studied between diazepam (Dzp W)and sucrose groups. The analysis was performed with the use of the Student t-test. The level of significance was set at pbbbb0.05.

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Fig. 7 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through theregions of the periaqueductal gray (above) and inferior collicullus (below) of rats submitted to spontaneousdiazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of the treatment, when compared with its control(Suc, left). DMPAG=dorsomedial periaqueductal gray; DLPAG=dorsolateral periaqueductal gray; LPAG: lateral periaqueductalgray; VLPAG: ventrolateral periaqueductal gray; Aq=Aqueduct of Sylvius; DR=dorsal raphe; DCIC=dorsal cortex of theinferior colliculus; ECIC=external cortex of the inferior colliculus; CIC=central nucleus of the inferior colliculus.

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reactivity to the electrical stimulation of the dPAG along with areduction in their ultrasonic vocalizations (a phenomenonusually observed during exposure to an intense uncontrollablestressor), showing that ethanol withdrawal sensitizes thesubstrates of fear at the level of dPAG (Cabral et al., 2006).

In summary, neural substrates of fear seem to be recruitedby thewithdrawal fromdiazepam intake in rats, as revealed bythe Fos-protein immunohistochemistry assay. In this context,this is the first report showing that an increaseof the activationof the neural substrates of fear in the midbrain tectum mayunderlie the aversive states elicited by diazepam withdrawal.

4. Experimental procedures

4.1. Animals

Wistar rats, weighing 100–110 g at the beginning of the treat-ment, from the animal house of the campus of Ribeirão Preto,University of São Paulo, were used. Theywere housed in groupsof four in Plexiglas-walled cages, lined with wood shavingschanged every 3 days, maintained in a 12:12 dark/light cycle(lights on 07:00 h) at 24±1 °C, and given free access to food and

water. Before the beginningof the treatments, theanimalshadathree-day habituation period to the lodging conditions. Theexperiments reported in this article were performed in compli-ance with the recommendations of SBNeC (Brazilian Society forNeuroscience and Behavior), which are in accordance with therules of the National Institutes of Health Guide for Care and Useof Laboratory Animals.

4.2. Behavioral procedure

In the first part of our study,we tested theefficacyof theperorally(p.o.) drug intake procedure in producing signs of abstinence 48 hafter the interruption of the diazepam regimen,which had lastedfor 18 days, by means of the elevated plus-maze test. On day 18,theanxiolyticeffectsofdiazepamwerealso tested.Two indicesofanxietywereused: thepercentageofentriesand timespent in theopenarmsof themaze.Wealso recorded thenumberof entries inthe closed arms as a measure of the animal's motor activity.

4.3. Administration of drugs by oral route

The self-administration of diazepam p.o. in rats suffers the biasof thegustative and temporal factors in functionofadelay in the

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production of its reinforcing effects. In this case, waterdeprivation and sucrose were two elements added to ourexperimental design that helpedus to circumvent this problem.In fact, the use of deprivation for the establishment ofmotivational drives has been very successful when emotionalaspects of the behavior are evaluated. On the other hand, it iswell known that sucrose, by itself, has high reinforcing effectsand it has been found that sucrose-withdrawn animals haveincreased anxiety levels. In our study, as we shall see, themaximum dose of 100 μg of sucrose did not have any con-sequenceonthemotivational driveof theanimalsasneither thepercentage of entries nor the time spent in the open arms of theplus maze were changed in these animals, in comparison withthe no-treatment group.

The oral voluntary ingestion of diazepamstarted 3 days afterthe arrival of the animals at the laboratory's animal house. Thechronic administration of a placebo solution by daily injectionscan, by itself, induce an increase in the anxiety levels in animalstested in the elevated plus-maze (Griebel et al., 1994). To avoidthese caveats we used an oral procedure, adapted by Schleimeret al. (2005), inwhich the animalswere submitteddaily to 14h ofwater deprivation (19:00 to 9:00), followed by 10 h of water adlibitum (9:00 to 19:00). This procedure began 2 days before thestart of the 18 treatment days, as a period of habituation of theanimals to the drinking procedure. Diazepam was separatelydissolved in a concentration of 10 mg/ml of saline pluspropilenoglycol (5%) and offered, once daily, in a volume of1ml/kgdiluted ina solutionof2mlof tapwateradded tosucrose(5%) plus propilenoglycol (5%). The solutions were available tothe animals, in glass pipettes of 5 ml, at the end of the dailyperiodofwater deprivation, delivered during eachof the 18daysof treatment. Except for the first 2 days of treatment, in whichcontrol and experimental solutions were available for 30 min,the animals that did not drink for 10 min were discarded fromthe experiments. This was necessary to avoid prolonging thedeprivationperiod. Still, to evaluate thepossible aversive effectsof water deprivation or reinforcing effects of the sucrosesolution per se, a second control group was included in whichthe animals had food and water ad libitum during the wholeperiod of the experiment. Thus, four groups of animals wereformed: a) No treatment group (No Treat, n=20), b) Sucrose p.o.intake (n=21), c) diazepam p.o. intake dependence condition(Dzp DEP, n=16) and d) diazepam p.o. intake withdrawalcondition (DzpW, n=19).

4.4. Elevated plus-maze test

The elevated plus-maze was made of wood and consisted offour arms of equal dimensions (50 cm×12 cm). Two of thearms were enclosed by 40-cm-high walls and were arrangedperpendicularly to two opposite open arms. The apparatuswas elevated 50 cm above the floor, with a 1 cm Plexiglas rimsurrounding the open arms to prevent falls (Pellow et al., 1985).The apparatus was located inside an isolated room with 20 lxof luminosity at the end the open arms. The recording of thebehaviors of the animals in the plus-maze was made througha camera (Everfocus, USES) linked to a monitor and videocas-sette, external to the experimental room. The tests wereconducted 30 min (we named this situation the dependencecondition, in which the animals were tested during the effect

of the drug) or 48 h after the last oral drug intake (the with-drawal condition, in which the animals were tested free of thedrug). The tests lasted 5 min. The animals were placed in thecenter of the plus-maze facing one of the closed arms. Eachanimal was tested just once.

4.4.1. Statistical analysisThe data are presented as mean±S.E.M. The behavioral datawere analyzed by means of a one-way ANOVA. The dependentfactor was the number of entries in the closed arms and per-centage of entries and time spent in the open arms; the inde-pendent factor was no treatment (No Treat), sucrose, diazepamdependent (Dzp DEP) or diazepam withdrawal (Dzp W) groups.Newman–Keuls's post-hoc comparisonswere carried outwhen-ever significant overall F-values were obtained. In all cases aprobability level of pb0.05 was considered to be significant.

4.5. Fos protein immunoreactivity

We evaluated the Fos-protein expression in rats under 48 h ofdiazepam-withdrawal. Eleven animals from each of thesucrose and diazepam oral regimens were randomly chosenfor immunohistochemical protocols. Two hours after the plus-maze test, these animals were deeply anaesthetized withurethane (1.25 g/kg, i.p., Sigma, USA) and intracardiallyperfused with 0.1 M phosphate-buffered saline followed by4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains wereremoved and immersed (4 °C) for 2 h in paraformaldehyde andthen stored for at least for 48 h in 30% sucrose in 0.1 M PBScryoprotection. They were then quickly frozen in isopentane(−40 °C) and sliced by the use of a cryostat (−19 °C). To keep theexperimental conditions similar for all animals of bothsucrose and diazepam-withdrawal groups, all incubationshad brain slices of each structure analyzed in this study.

Twoadjacent seriesof 40μmthickbrainsliceswereobtained.One series was Nissl stained and used for neuroanatomicalcomparison purposes and the other series was collected for theimmunohistochemical studies. Tissue sections were collectedin 0.1MPBS and subsequently processed free-floating accordingto the avidin–biotin procedure, using the Vecstatin ABC Eliteperoxidase rabbit IgG kit (Vector, USA, ref. PK 6101). All reactionswere carried out under agitation at room temperature. Thesliceswere first incubatedwith 1%H2O2 for 10min, washed fourtimeswith 0.1M PBS (5min each) and then incubated overnightat room temperature with the primary Fos rabbit polyclonal IgG(Santa Cruz, USA, SC-52) at a concentration of 1:2000 in PBS+(0.1 M PBS enriched with 0.2% Triton-X and 0.1% Bovine SerumAlbumin, BSA). Sections were again washed three times (5 mineach) with 0.1 M PBS and incubated for 1 h with secondary Fosbiotinylated anti-rabbit IgG (H+L) (Vecstatin, Vector Laborato-ries) at concentration of 1:400 in PBS+. After another series ofthree 5-min washings in 0.1 M PBS the sections were incubatedfor 1 h with the avidin–biotin–peroxidase complex in 0.1 M PBS(A and B solution of the kit ABC, Vecstatin, Vector Laboratories)at concentration of 1:250 in 0.1 M PBS, and then were againwashed three times in 0.1 M PBS (5 min per wash). Fos im-munoreactivity was revealed by the addition of the chromogen3,3′-di-aminobenzidine (DAB, 0.02%, Sigma) to which hydrogenperoxide (0.04%) was added just prior to use. Finally the tissuesections were washed twice with 0.1 M PBS.

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4.5.1. Quantification of Fos-positive cellsTissue sections were mounted on gelatin-coated slides, dehy-drated for observation and cell counting under bright-fieldmicroscopy. The nomenclature and nuclear boundaries utilizedwere based on the atlas of Paxinos and Watson (2005). Cellscontaining a nuclear brown–black reaction product with areasbetween 10 and 80 μm2 were identified and automaticallycounted as Fos-positive neurons by a computerized imageanalysis system (Image Pro Plus 4.0, Media Cybernetics, USA).Sections of 26 different regions at different levels in the brainwere collected, according to a method used in previous studies(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,2005).Mounted sectionsof the tissuewereobservedusinga lightmicroscope (Olympus BX-50) equipped with a video-cameramodule (Hamatsu Photonics C2400) and coupled to a comput-erized image analysis system indicated above. Counting of Fos-positive cells was performed under a ×10 objective at amagnification of ×100 in one field per area encompassing theentire brain region included in quantification. An area of thesame shape and size per brain regionwas used for each rat. Thesystem was calibrated to ignore background staining. All brainregions were bilaterally counted for each rat. The analyzedencephalic regions and their respective AP coordinates frombregma (Paxinos and Watson, 2005) were as follows: theprelimbic cortex (PrL) (AP: 3.72 mm to 3.24 mm), the cingulateareas 1 (CG1) and 2 (CG2) (AP: 1.56 mm to 1.44 mm), the central(CeA), medial (MeA) and basolateral nuclei of the amygdala(BLA) (AP: −2.40 mm to −2.52 mm), the CA1, CA2 and CA3hippocampal areas (AP: −2.64 mm to −2.92 mm), the dorsalaspects of the nucleus accumbens core (AcbC) and shell (AcbSh)(AP: 1.68mm to 1.44mm), the paraventricular thalamic nucleus(PV) (AP: −2.16 mm to −2.40 mm), the paraventricular (PaV, allsubnuclei), anterior (AHC, central and posterior regions) and thelateral hypothalamic nuclei (LH, peduncular part) (AP: −1.80 to−1.92mm), the dorsomedial hypothalamus (DMH, all subnuclei)(AP: −3.00 mm to −3.24 mm), the dorsomedial (DMPAG), dor-solateral (DLPAG), lateral (LPAG) and ventrolateral (VLPAG) partsof the intermediate portions of the periaqueductal gray(AP: −6.48 mm to −6.96 mm), the dorsal (DR) and medianraphe nucleus (MnR) (AP: −7.68 mm to −7.80 mm), the deeplayersof the superior colliculus (SC) (AP:−7.44mmto−7.68mm),the ventral part of the central nucleus of the caudal inferiorcolliculus (IC) (AP: −8.16 mm to −8.52 mm), the locus ceruleus(LC) (−9.48 to −9.96 mm) and the cuneiform nucleus (CnF)(AP: −7.80 mm to −8.04 mm). Nuclei were counted individuallyand expressed as number of Fos-positive cells per 0.1 mm2

(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,2005), as shown in Table 1. The choice of the areas sampled inthis study took into consideration the importance of theseregions in the modulation of the emotional, autonomic andmotor expression of the fear-motivated behaviors (Azumaand Chiba, 1996; Beck and Fibiger, 1995; Brandão et al., 1999,2003, 2005; Campeau et al., 1997; Canteras et al., 1997;Canteras and Swanson, 1992; Charney et al., 1998; Condeet al., 1990; Emmert and Herman, 1999; Espejo, 1997; Fendtand Fanselow, 1999; Graeff, 1990, 1994; Gorman et al., 2000;Gray and McNaughton, 2000; Jay et al., 1989; Jinks andMcGregor, 1997; LeDoux, 1995; Li and Sawchenko, 1998; Risoldand Swanson, 1996; Rosen and Schulkin, 1998; Silveira et al.,1993), although we are aware that many other brain regions,

not specified here, also contribute to the modulation/expres-sion of behaviors elicited by fear stimuli.

4.5.2. Statistical analysisFos-protein expression was analyzed only in withdrawalanimals (sucrose and Dzp W) through Student t-test for eachregion in study. The significance level was set at pb0.05.

Acknowledgment

This work was supported by grants from FAPESP (04/02859-0).

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