brainstem areas activated by diazepam withdrawal and fos-protein immunoreactivity in rats

8/18/2019 Brainstem Areas Activated by Diazepam Withdrawal and FOS-protein Immunoreactivity in Rats

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Research Report

Brainstem areas activated by diazepam withdrawal as

measured 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, Brazil

Laboratório 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 A B S T R A C T

Article history:

Accepted 5 July 2007

Available online 14 July 2007

In the 1970s, chronic treatment with benzodiazepines was supposed not to cause

dependence. However, by the end of the decade several reports showed that the

interruption of a prolonged treatment with diazepam leads to a withdrawal syndrome

characterized, among other symptoms, by an exaggerated level of anxiety. In laboratory

animals, signs that oscillate from irritability to extreme fear-like behaviors and convulsions

have also beenreported. In recent years many studies have attempted to disclose the neural

substrates responsible for the benzodiazepines withdrawal. However, they have focused on

telencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. Inthis study, we examined the Fos immunoreactivity in brain structures known to be

implicated in the neural substrates of aversion in rats under spontaneous diazepam-

withdrawal. We found that the same group of structures that originally modulate the

defensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, the

superior and inferior colliculus and the dorsal periaqueductal gray, were most labeled

following diazepamwithdrawal. It is suggested that an enhanced neural activation of neural

substrates of fear in the midbrain tectum may underlie the aversive state elicited in

diazepam-withdrawn rats.

© 2007 Elsevier B.V. All rights reserved.

Keywords:

Withdrawal

Diazepam

AnxietyFos

Brainstem

dPAG

1. Introduction

Benzodiazepines compounds are widely used for the treatment

of anxiety and sleep disorders and are also prescribed for their

sedative, muscle relaxant and anticonvulsant properties

(Woods et al., 1987; Bateson, 2002). Initially, chronic treatment

with benzodiazepines was supposed not to cause dependence.

However, it has been demonstrated that, on abrupt withdrawal

from benzodiazepine,chronic exposure patients can experience

a number of symptoms indicative of a dependent state. Among

the 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; Rynn

and Brawman-Mintzer,2004). Additionally, clinical reports have

denounced the appearance of high levels of fear in patients

experiencing benzodiazepine withdrawal (Rosebush and

Mazurek, 1996). In the laboratory, similar aspects of the

withdrawalphenomena can be reproducedin animals. Actually,

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⁎ Corresponding author. Fax: +55 1636024830.E-mail address: [email protected] (M.J. Nobre).

0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2007.07.007

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

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signs that oscillate from irritability to extreme fear behaviors

and convulsions have already been described (Petursson and

Lader, 1981a,b; Owen and Tyrer, 1983; Emmett-Oglesby et al.,

1983; Lacerra et al., 1999; Woods et al., 1987; Ashton, 2005).

Thegreat majorityof methodsused in theinvestigation of the

neurobiological mechanisms implicatedin theproduction of fear

andanxietyas, for example,chemical or electrical stimulation or

lesions 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, has

been employed because it allows a clear visualization of the

neurons activated during the presentation of a specific fear

stimulus (Dragunow and Faull, 1989; Reiman et al., 1989; Bullitt,

1990). In fact, Fos-like immunoreactivity has been used as a

marker of the neuronal activation that can be precipitated by a

widerange of stimuli as,for example,brain electrical stimulation

(Sandner et al., 1992; Vianna et al., 2003), local injection of

neurotransmitters (Stone et al., 1991; Richard et al., 1992;

Ferreira-Netto et al., 2005), stress induced by immobilization

(Imaki et al.,1992;Honkaniemiet al.,1992),pain(Bullitt,1990)and

abstinence 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 some

prosencephalic structures are activated by benzodiazepine

withdrawal, such as the shell region of the nucleus accumbens

(Dunworth et al.,2000), oneof the most importantareas involved

with the expression of motivational factors linked to drugs of

abuse. This type of activation is also produced by anxiogenic

stimuli that induce increase in dopamine release and induction

of Fos immunoreactivity in this structure (Abercrombie et al.,

1989; Smith et al., 1997). In the same context, a simple exposure

to the elevated plus-maze produces great Fos labeling in limbic

areas (Silveira et al., 1993; Sandner et al., 1993) related with the

production of the behavioral and autonomic responses associ-

ated with the expression of fear.

Drug withdrawal may function as an unconditioned stressor,

and as such could activate a particular setof structures involved

with the organization of fear states, particularly those belonging

to thewell-knownbrain aversion system, such as the amygdala,

dorsal periaqueductal gray (dPAG)(the main effector pathway of

the defensive behavior responses), superior and inferior colliculi

(structures included in the processing of visual and auditory

information of aversive nature, respectively) and the medial

hypothalamus (mainly tied to the production of the somatic

responses of fear) (Gray, 1982; Graeff, 1990; Brandão et al., 1999,

2005). To examine this possibility, in this study we analyzed

whether the expression of fear behaviors observed in rats

abruptly withdrawn from diazepam could lead to a consequent

activation of the same brain structures that modulate the ex-

pression of the defensive behavior of animals exposed to

dangerous situations such as, for example, exposure to the

open arms of theelevated plus-maze, amongothers. Tothis end,

we used the immunohistochemical technique for the detection

of Fos-protein expression in selected structures of diazepam-

withdrawn rats.

Thegreatmajority of studiesin theliterature that investigate

the chronic effects of a drug of abuse has mainly used intra-

peritoneal injections as the method of delivery of the drug.

However, it was demonstratedthat rats under chronic systemic

injections of placebo solutionstendto presentan increase in the

anxiety levels when tested in the elevated-plus maze (Griebel

et al., 1994). In this case, it could be argued that the aversion

promoted by the exposure to chronic systemic injections could

lead to an increase in the number of Fos-like immunoreactive

neurons in structures that normally modulate this class of

emotional responses. To circumvent thisproblem, in this work,

we used a new model of oral drug intake, adapted by Schleimer et al. (2005), in which the animals voluntarily drink the drug or

control solutions.

Fig. 1 – Percentage of entries in the open arms (A), number of

enclosed arms entries (B) and percentage of time spent in the

open arms (C) of the elevated plus-maze of rats that did not

receive any treatment (No Treat) and rats treated chronically

p.o. with sucrose or diazepam. As ANOVA showed no

significant differences between the two control groups used

in this study, all the differences shown in the graphs are

relative to comparisons between the diazepam-treated rats

and those belonging to the sucrose group. Independent

groups of diazepam treated animals were tested at the

dependence (30 min after the last drug intake—Dzp DEP) or

withdrawal (48 h after the last drug intake—Dzp W). Data are

presented as mean± S.E.M. One-way ANOVA followed by

post-hoc Newman–Keuls; p bbbb0.05.

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2. Results

2.1. Plus-maze

One-way ANOVA revealed that the chronic treatment with

sucrose, per se, did not promote any effect on the behavior of

rats after the interruption of the long-term treatment, asthere were no observed significant differences in any of the

registered 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 group

differences in both the percentage of entries [F (3,72)=5.35;

pb0.005] and on the percentage of time spent in the open

arms [F (3,72)=18.55; pb0.001] were observed in animals

under diazepam effects (Dzp DEP) or during withdrawal (Dzp

W), when compared with those that received sucrose only.

Newman–Keuls post-hoc showed that the chronic oral intake

procedure was effective in producing a significant anxiolytic-

like effect in rats tested 30 min after the last oral

administration of diazepam (Dzp DEP); as revealed by the

increase in the percentage of entries (Fig. 1A) and in the total

‘open arms’ time (Fig. 1C). An anxiogenic-like effect was

achieved in rats on 48-h withdrawal from diazepam (Dzp W),

as these animals stayed less time in the open arms of the

maze (Fig. 1C) than the animals of the sucrose group. The

number of ‘closed arms’ entries [F (3,72)=3.22; pN0.05]

showed a trend towards significance ( p =0.055) to this

condition among the groups tested. As shown in Fig. 1B,this effect was due to a tendency of rats under diazepam

effect (Dzp DEP) to increase motor activity, when compared

with the sucrose control group.

2.2. Fos immunoreactivity

Statistical analysis of the effects of the diazepam withdrawal

on Fos-protein expression was conducted in brain areas of 22

animals (n =11 for group) randomly chosen from each of the

sucrose-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 region

studied.

Table 1 – Mean (±S.E.M.) number of Fos-positive neurons/0.1 mm2 in brain regions of 48-h diazepam-withdrawn ratsperfused 2 h after plus-maze exposure

Structures Sucrose Diazepam pb0.05

Left Right Mean Left Right Mean

Telencephalon PrL 6.18 ±1.72 7.20 ±1.27 6.69 ±1.46 14.70 ±2.39 18.57 ±2.51 16.64 ±2.23 ⁎

CG1 21.40 ± 4.43 21.45 ± 4.71 21.43 ±4.04 24.67 ± 2.04 22.44 ± 2.53 23.56 ±2.14

CG2 2.86 ±0.87 2.66 ±0.79 2.76 ±0.79 3.23 ±1.39 4.29 ±1.71 3.76 ±1.55

CeA 2.01 ±0.72 1.15 ±0.60 1.58 ±0.63 1.81 ±0.60 1.45 ±0.42 1.63 ±0.41

MeA 8.16 ±1.60 6.98 ±1.56 7.57 ±1.41 9.54 ±1.62 9.49 ±1.92 9.51 ±1.71

BLA 5.15 ±1.26 4.97 ±1.11 5.06 ±1.06 5.36 ±1.36 5.76 ±1.16 5.56 ±1.21CA1 0.61 ±0.17 1.23 ±0.39 0.92 ±0.27 1.67 ±0.32 2.27 ±0.69 1.97 ±0.48

CA2 0.65 ±0.25 0.46 ±0.18 0.56 ±0.20 1.17 ±0.24 1.19 ±0.25 1.18 ±0.18

CA3 1.22 ±0.33 1.36 ±0.23 1.29 ±0.25 1.36 ±0.34 1.79 ±0.43 1.57 ±037

AcbC 1.55 ±0.48 1.92 ±0.88 1.74 ±0.67 3.04 ±1.16 3.75 ±1.61 3.40 ±1.35

AcbSh 2.01 ±0.55 1.94 ±0.85 1.98 ±0.67 3.68 ±1.32 3.38 ±1.00 3.53 ±1.14

Diencephalon PV 19.74 ± 5.41 19.51 ± 5.52 19.63 ±5.43 29.50 ± 6.21 27.74 ± 5.85 28.62 ±5.85

PaV 16.18 ± 2.55 17.11 ± 3.05 16.64 ±2.60 27.57 ± 5.77 31.07 ± 6.68 29.32 ±6.15

AH 13.77 ± 2.02 15.17 ± 1.87 14.47 ±1.67 21.77 ± 1.97 22.10 ± 2.65 21.93 ±2.00 ⁎

LH 7.84 ±1.48 8.92 ±1.11 7.38 ±1.23 11.97 ±1.60 13.41 ±1.53 12.69 ±1.49 ⁎

DMH 3.75 ±0.75 3.57 ±0.90 3.66 ±0.75 11.10 ±1.99 9.99 ±1.13 10.54 ±1.14 ⁎

Mesencephalon DMPAG 1.35 ± 0.33 1.23 ± 0.31 1.29 ± 0.32 11.14 ± 1.25 12.70 ± 1.43 11.92 ± 1.34 ⁎

DLPAG 1.25 ±0.24 1.47 ±0.32 1.36 ±0.23 7.50 ±1.37 8.07 ±0.98 7.79 ±1.03 ⁎

LPAG 2.36 ±0.31 1.58 ±0.30 1.97 ±0.27 9.28 ±1.50 10.14 ±1.59 9.71 ±1.42 ⁎

VLPAG 5.04 ± 0.77 6.58 ± 0.73 5.81 ±0.56 11.52 ± 1.32 13.27 ± 2.13 12.39 ±1.61 ⁎

DR 6.26 ±0.70 8.68 ±0.86 7.47 ±0.78 13.45 ±1.71 13.79 ±1.75 13.62 ±1.73 ⁎MnR 7.79 ±0.87 8.55 ±0.95 8.17 ±0.91 9.37 ±0.58 10.93 ±0.68 10.15 ±0.63

SC 1.37 ±0.12 1.21 ±0.13 1.29 ±0.11 6.65 ±1.01 6.87 ±0.57 6.76 ±0.72 ⁎

IC 1.91 ±0.33 2.10 ±0.40 2.01 ±0.35 13.70 ±1.78 13.62 ±1.83 13.66 ±1.36 ⁎

LC 2.67 ±0.41 3.37 ±0.37 3.02 ±0.36 1.88 ±0.28 1.3 ±0.21 1.60 ±0.20 ⁎

Cun 3.72 ±0.76 3.40 ±0.45 3.56 ±0.34 9.21 ±1.42 10.11 ±1.16 9.66 ±1.15 ⁎

Number of Fos-positive neurons per regionon the left and right sides were countedusing a computerized image analysis system (Image ProPlus

4.0, Media Cybernetics). Asterisks indicate the regions in which withdrawn-rats displayed differential Fos-expression ( pb0.05) in relation to

control group (sucrose). Differences between groups were analyzed with the Student t-test for each region in study. PrL, prelimbic cortex; CG1

and CG2, cingulate areas 1 and 2, respectively; CeA, central nucleus of the amygdala; MeA, medial nucleus of the amygdala; BLA, basolateral

nucleus of the amygdala; CA1, CA2 and CA3, hippocampal areas 1, 2 and 3, respectively; AcbC, nucleus accumbens (core region); AcbSh, nucleus

accumbens (shell region); PV, paraventricular thalamic nucleus; PaV, paraventricular hypothalamic nucleus; AH, anterior hypothalamus; LH,

lateral hypothalamus; DMH, dorsomedial hypothalamus; DMPAG, dorsomedial periaqueductal gray; DLPAG, dorsolateral periaqueductal gray;

LPAG, lateralperiaqueductal gray; VLPAG, ventrolateral periaqueductal gray; DR,dorsal raphe nucleus; MnR,median raphe nucleus; SC,superior

colliculus; IC, inferior colliculus; LC, locus ceruleus; Cun, cuneiform nucleus.

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2.2.1. Telencephalon

As revealed in Figs. 2 and 3 withdrawal from diazepam did not

cause important Fos expression in almost all telencephalic

regions. In fact, the Student t-test revealed significant dif-

ference in the Fos expression only in the pre-limbic cortex(PrL) [t20=3.73; p b0.05] (Fig. 3, upper right). Otherwise, many

other areas mainly involved in withdrawal of drugs of abuse,

as the core (AcbC) and shell (AcbSh) regions of the nucleus

accumbens, showed no alterations on Fos-protein immuno-

reactivity (Fig. 3, bottom right).

2.2.2. Diencephalon

Highneuralactivation wasobservedin diencephalicstructures

(Figs. 4 and 5). Forty-eight hours of diazepam withdrawal was

effective in promoting a robust increase in Fos expression in

the 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 that

the comparisons between the withdrawal and sucrose groups

reached marginal significance ( p =0.056) of the withdrawal on

the expression of Fos positive neuronal nuclei in the para-

ventricular region of the hypothalamus (PaV).

2.2.3. Mesencephalon

Statistical analysis indicated significant differences in Fos ex-

pressionin almost allmesencephalicareas 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] and

ventrolateral [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] and

inferior [IC: t20=8.28; pb0.05] colliculi (Fig. 7, bottom right) and

cuneiform nucleus [Cun: t20=5.07; p b0.05]. Interestingly, of all

mesencephalic structures studied, only locus ceruleusshowed asignificant decrease in Fos-protein expression, when compared

with 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 withdrawal

symptoms in humans or in laboratory animals (Lukas and

Griffiths, 1982; Ladewig, 1984). In fact, anxiety-like states have

been reported as a common symptom in benzodiazepine-

withdrawn patients. Avoidance of these unpleasant symptoms

seems to negatively-reinforce continuous benzodiazepine use

and isone of the maincomponentsof cravingfor the drug(Busto

et al., 1986; Busto and Sellers, 1991). An increase in anxiety-like

states is also observed in rats under spontaneous diazepam-

withdrawal and submitted to several types of animal models of

anxiety (Greenblatt and Shader, 1974; Emmett-Oglesby et al.,

1983; File, 1990). In our study, the interruption of the chronic

regimen of diazepam caused a significant decrease in the

percentage of time spent in the open arms of the plus-maze.

This anxiogenic effect promoted by diazepam withdrawal was

accompanied by significant Fos-positive immunoreactivity in

telencephalic, diencephalic and mesencephalic areas including

all columns of the periaqueductal gray, the deep layers of the

superior colliculus, the central nucleus of the inferior colliculus,

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 of

Fos-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

(Dzp W) and sucrose groups. The analysis was performed with the use of the Student t -test. The level of significance was set at

p bbbb0.05.

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themedial aspectsof thedorsal raphe nuclei,theanterior,lateral

and dorsomedial nuclei of the hypothalamus and the prelimbic

cortex, all of them mainly linked to the modulation of the

emotional, 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 circuits

in the brain,so that anxiogenic drugs that promote high levelsof

aversion also significantly activate these circuits (Charney et al.,

1998; Gorman et al., 2000; Gray and McNaughton, 2000; Rosen

and Schulkin, 1998). Parts of the prefrontal cortex, including the

prelimbic region, have been reported to be involved in the

production of fear- and anxiety-related behaviors (Espejo, 1997;

Jinks and McGregor, 1997; LeDoux, 1995). Additionally, many

studies on acute fear have proposed that brain areas such as the

amygdala, hippocampus, hypothalamus and periaqueductal

gray are putatively involved in fear processing (Fendt and

Fanselow, 1999; Graeff, 1994; Gray and McNaughton, 2000;

LeDoux, 1995). Others have implicated the periaqueductal gray,

amygdala, medial hypothalamus, raphe nuclei, superior and

inferiorcolliculusand locusceruleus in theexpression of thefear

behaviors (Graeff, 1990, 1994; Brandão et al., 1999, 2003, 2005).

The activation of the amygdala results in behavioral and

physiological responses associated with anxiety and panic-

like behavior.It is intriguing thatno changes in Fos-expression

in this region were observed in our study. However, a similar

result was also obtained by Borlikovaet al. (2006), who showed

that a single alcohol-withdrawal experience does not induce

significant Fos expression in this area, contrasting with a

significant Fos expression following repeated withdrawal

procedures. The activation of the paraventricular hypotha-

lamic nucleus (PaV) promotes release of a variety of hormones

that are involved in the neuroendocrine and autonomic

responses to fear and psychological stress, whereas the

activation of the lateral hypothalamus seems to be important

for the cardiovascular expressions of fear and anxiety. Also, in

laboratory animals, electrical stimulation of the anterior

hypothalamic nucleus elicits hissing and threat postures

characteristic of defensive behaviors. Electrical or chemical

stimulation of some mesencephalic structures belonging to

the well-known brain aversion system, such as the superior

and inferior colliculus and periaqueductal gray, also promote

defensive behaviors in rats (Brandão et al., 1999, 2005). The

activation of brainstem structures was alsoobservedin several

studies using Fos-protein expression as a neuronal marker of

anxiety-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. 3 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through the

prelimbic cortex (PrL, above)and on thecore (AcbC) and shell (AcbSh) regions of the nucleusaccumbens (below)of ratssubmitted to

spontaneous diazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of the treatment, when compared with its

controls (Suc, left). Cg1= cingulate cortex, area 1; fmi= forceps minor 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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drugs that elicit panic-like symptoms (Singewald and Sharp,

2000). All these common sets of structures involved in the

expression of the emotional, autonomic and motor compo-

nents of fear-related behaviors were also strongly activated in

rats suffering withdrawal which were submitted to the plus-

maze, showingthat thesame neuronalchanges verifiedduring

the presentation of fear stimuli seems to be occurring during

diazepam withdrawal.

The main neurobiological mechanism associated with the

effects of drugs of abuse hasbeen thedopaminergic mesolimbic

system and its connections with the basal prosencephalon

(Koob, 1992; Koob and Le Moal, 1997). In this context, in a

previous experiment, Dunworth et al. (2000) investigated

whether some diazepam-withdrawal symptoms could be

lessened by prior withdrawal experience in mice undergoing a

single or repeated flumazenil-precipitated diazepam withdraw-

al. In thethird experiment of this study, they evaluated theFos-

protein expression in brain areas that they speculated would be

important in either seizure sensitivity or the expression of an

aversive state. The immunohistochemistry results showed a

significant increase in Fos expression in the shell region of the

nucleus accumbens of mice submitted to a single withdrawal

experience; contrasting with our findings that showed no

significant alteration on this measure in the same area. On the

other hand, no differences were observed after flumazenil

injection in rats undergoing repeated diazepam-withdrawal

experience. Concerningthis discrepancy, we mustpay attention

to some methodological aspects that could clarify the differ-

ences obtained between the two studies. Most importantly, as

revealed above, on Dunworth's experiments, Fos immunohis-

tochemistry assays were performed in mice through the

flumazenil-precipitated diazepam-withdrawal procedure, in

contrast with the spontaneous diazepam-withdrawal methodused in our experiments. This difference is seminal to under-

stand the increase of Fos expression observed in the former

study. Concerning this point, the study of Motzo et al. (1997)

demonstrated that a challenge administration of diazepam

inhibits dopamine output in the nucleus accumbens. On the

other hand, the discontinuation of long-term treatment with

diazepam does not alter the basal dopamine release in this

region. However, a singleintraperitonealinjection of flumazenil

(in a dose that has no effect on the basal dopamine levels) is

effective in enhancing the release of this neurotransmitter in

the nucleus accumbens of rats chronically treated with

diazepam and tested after either 6 h or 5 days of withdrawal.

In this case, we could argue that the enhanced Fos expression

observed 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 of Dunworth et al.was

much more larger than that noted in the study of Motzo (4 mg/

kg). In this case, an increase on the basal activity of the

dopaminergic cells that could contribute to promote Fos

expression on the accumbens, could not be disregarded.

The absence of Fos expression in the nucleus accumbens in

the present study may be related to the notion that benzodiaz-

epine withdrawal recruits neural substrates different from the

dopaminergic one (Lingford-Hughes and Nutt, 2003) to promote

its effects. Indeed, changes in the GABA system have been

postulated to underlie diazepam-withdrawal syndrome (Miller

et al., 1988; Galpern et al., 1991; Toki et al., 1996). Secondary

alterations in other neurotransmitter systems as, for example,

the glutamatergic and serotoninergic mechanisms have also

been 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 fear

behaviors in diazepam-withdrawn rats, implicating the excit-

atory amino acids of the dPAG in the modulation of the aversive

states induced by diazepam withdrawal (Souza-Pinto et al.,

2007). As the mechanisms that modulate fear are primarily

located in the brainstem, it is possible that these changes take

place initially in the mesencephalon. This is supported by the

pronounced Fos-positive immunoreactivity in structures basi-

cally involved in the modulation of fear behaviors, such as the

periaqueductal gray andthe superior andinferiorcolliculiof rats

under diazepam withdrawal. Thus, it is likely that the fear

elicited by diazepam withdrawal is theresult of theactivation of

the neural substrates of aversion in the brainstem, such as the

defense reaction elicited by stimulation of the dPAG (Brandão

et al., 1999, 2005). On the other hand, prosencephalic structures,

such as amygdala,are involved in themodulation of theanxiety

promoted by associative learning, such as that generated in

aversive conditioning. In supportof thepresent results, we have

found that rats under ethanol withdrawal had an increase in

Fig. 4 – Number of Fos-immunoreactive neurons in the

diencephalic structures of 48-h diazepam-withdrawn rats

submitted 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 of Fos-positive neurons per region was

bilaterally counted using a computerized image analysis

system (Image Pro-Plus 4.0, Media Cybernetics). * 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 p bbbb0.05.

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paraventricular (PvA), anterior (AH) and lateral (LH) regions of the hypothalamus (above), and the dorsomedial hypothalamic

nucleus (below) of rats submitted to spontaneous diazepam-withdrawal (Dzp W, right) and tested 48 h after the interruption of

the 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 the mesencephalic structures of 48-h diazepam-withdrawn rats submitted

to theplus-maze. Dataare expressedas mean± S.E.M.of Fos-positive cells in 0.1 mm2 area of tissue. Thenumber of Fos-positive

neurons per region was bilaterally counted using a computerized image analysis system (Image Pro-Plus 4.0, Media

Cybernetics). * 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 p bbbb0.05.

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reactivity to the electrical stimulation of the dPAG along with a

reduction in their ultrasonic vocalizations (a phenomenon

usually observed during exposure to an intense uncontrollable

stressor), showing that ethanol withdrawal sensitizes the

substrates of fear at the level of dPAG (Cabral et al., 2006).

In summary, neural substrates of fear seem to be recruited

by thewithdrawalfrom diazepam intakein rats, as revealed by

the Fos-protein immunohistochemistry assay. In this context,

this isthe firstreport showingthatan increaseof the activation

of the neural substrates of fear in the midbrain tectum may

underlie 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. They werehoused in groups

of four in Plexiglas-walled cages, lined with wood shavings

changed 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.Beforethe beginningof thetreatments, theanimals hada

three-day habituation period to the lodging conditions. The

experiments reported in this article were performed in compli-

ance with the recommendations of SBNeC (Brazilian Society for

Neuroscience and Behavior), which are in accordance with the

rules of the National Institutes of Health Guidefor Care and Use

of Laboratory Animals.

4.2. Behavioral procedure

In the firstpartof our study,we testedthe efficacy ofthe perorally

(p.o.) drug intake procedure in producing signs of abstinence 48 h

after theinterruption of thediazepam regimen, which hadlasted

for 18 days, by means of the elevated plus-maze test. On day 18,

theanxiolyticeffects of diazepam were alsotested.Two indicesof

anxiety wereused:the percentageof entriesand time spentin the

open arms ofthe maze.We also recordedthe numberof entriesin

the 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 bias

of thegustativeand temporal factors in function of a delay in the


regions of the periaqueductal gray (above) and inferior collicullus (below) of rats submitted to spontaneous

diazepam-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 periaqueductal

gray; 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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production of its reinforcing effects. In this case, water

deprivation and sucrose were two elements added to our

experimental design that helpedus to circumvent this problem.

In fact, the use of deprivation for the establishment of

motivational drives has been very successful when emotional

aspects of the behavior are evaluated. On the other hand, it is

well known that sucrose, by itself, has high reinforcing effects

and it has been found that sucrose-withdrawn animals haveincreased anxiety levels. In our study, as we shall see, the

maximum dose of 100 μg of sucrose did not have any con-

sequenceon themotivational drive of theanimalsas neither the

percentage of entries nor the time spent in the open arms of the

plus maze were changed in these animals, in comparison with

the no-treatment group.

Theoral voluntaryingestion of diazepam started 3 days after

the arrival of the animals at the laboratory's animal house. The

chronic administration of a placebo solution by daily injections

can, by itself,induce an increase in theanxiety levelsin animals

tested in the elevated plus-maze (Griebel et al., 1994). To avoid

these caveats we used an oral procedure, adapted by Schleimer

et al.(2005), in which the animals were submitted daily to 14h of

water deprivation (19:00 to 9:00), followed by 10 h of water ad

libitum (9:00 to 19:00). This procedure began 2 days before the

start of the 18 treatment days, as a period of habituation of the

animals to the drinking procedure. Diazepam was separately

dissolved in a concentration of 10 mg/ml of saline plus

propilenoglycol (5%) and offered, once daily, in a volume of

1 ml/kg diluted ina solutionof 2 mlof tap water added tosucrose

(5%) plus propilenoglycol (5%). The solutions were available to

the animals, in glass pipettes of 5 ml, at the end of the daily

periodof water deprivation,deliveredduringeach of the18 days

of treatment. Except for the first 2 days of treatment, in which

control and experimental solutions were available for 30 min,

the animals that did not drink for 10 min were discarded from

the experiments. This was necessary to avoid prolonging the

deprivationperiod. Still,to evaluate the possible aversive effects

of water deprivation or reinforcing effects of the sucrose

solution per se, a second control group was included in which

the animals had food and water ad libitum during the whole

period of the experiment. Thus, four groups of animals were

formed: 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 withdrawal

condition (Dzp W, n =19).

4.4. Elevated plus-maze test

The elevated plus-maze was made of wood and consisted of

four arms of equal dimensions (50 cm×12 cm). Two of the

arms were enclosed by 40-cm-high walls and were arranged

perpendicularly to two opposite open arms. The apparatus

was elevated 50 cm above the floor, with a 1 cm Plexiglas rim

surrounding theopen arms to prevent falls (Pellowet al., 1985).

The apparatus was located inside an isolated room with 20 lx

of luminosity at the end the open arms. The recording of the

behaviors of the animals in the plus-maze was made through

a camera (Everfocus, USES) linked to a monitor and videocas-

sette, external to the experimental room. The tests were

conducted 30 min (we named this situation the dependence

condition, 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 the

drug). The tests lasted 5 min. The animals were placed in the

center of the plus-maze facing one of the closed arms. Each

animal was tested just once.

4.4.1. Statistical analysis

The data are presented as mean±S.E.M. The behavioral datawere analyzed by means of a one-way ANOVA. The dependent

factor 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, diazepam

dependent (Dzp DEP) or diazepam withdrawal (Dzp W) groups.

Newman–Keuls's post-hoc comparisonswere carried out when-

ever significant overall F-values were obtained. In all cases a

probability 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 of

diazepam-withdrawal. Eleven animals from each of the

sucrose and diazepam oral regimens were randomly chosen

for immunohistochemical protocols. Two hours afterthe plus-

maze test, these animals were deeply anaesthetized with

urethane (1.25 g/kg, i.p., Sigma, USA) and intracardially

perfused with 0.1 M phosphate-buffered saline followed by

4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were

removed and immersed (4 °C) for 2 h in paraformaldehyde and

then stored for at least for 48 h in 30% sucrose in 0.1 M PBS

cryoprotection. They were then quickly frozen in isopentane

(−40°C) and slicedby the use of a cryostat (−19 °C). To keep the

experimental conditions similar for all animals of both

sucrose and diazepam-withdrawal groups, all incubations

had brain slices of each structure analyzed in this study.

Twoadjacentseries of 40μm thick brain sliceswereobtained.

One series was Nissl stained and used for neuroanatomical

comparison purposes and the other series was collected for the

immunohistochemical studies. Tissue sections were collected

in 0.1M PBSand subsequentlyprocessed free-floating according

to the avidin–biotin procedure, using the Vecstatin ABC Elite

peroxidase rabbitIgG kit(Vector, USA, ref. PK 6101).All reactions

were carried out under agitation at room temperature. The

sliceswere first incubated with 1% H2O2 for10 min, washed four

times with 0.1 M PBS (5 min each) and then incubated overnight

at 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 Serum

Albumin, BSA). Sections were again washed three times (5 min

each) with 0.1 M PBS and incubated for 1 h with secondary Fos

biotinylated anti-rabbit IgG (H+ L) (Vecstatin, Vector Laborato-

ries) at concentration of 1:400 in PBS+. After another series of

three 5-min washings in 0.1 M PBS the sections were incubated

for 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 again

washed three times in 0.1 M PBS (5 min per wash). Fos im-

munoreactivity was revealed by the addition of the chromogen

3,3′-di-aminobenzidine (DAB, 0.02%, Sigma) to which hydrogen

peroxide (0.04%) was added just prior to use. Finally the tissue

sections were washed twice with 0.1 M PBS.

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4.5.1. Quantification of Fos-positive cells

Tissue sections were mounted on gelatin-coated slides, dehy-

drated for observation and cell counting under bright-field

microscopy. The nomenclature and nuclear boundaries utilized

were based on the atlas of Paxinos and Watson (2005). Cells

containing a nuclear brown–black reaction product with areas

between 10 and 80 μm2 were identified and automatically

counted 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 brain

were collected, according to a method used in previous studies

(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,

2005). Mounted sections of thetissuewere observed usinga light

microscope (Olympus BX-50) equipped with a video-camera

module (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 a

magnification of ×100 in one field per area encompassing the

entire brain region included in quantification. An area of the

same shape and size per brain region was used foreachrat. The

system was calibrated to ignore background staining. All brain

regions were bilaterally counted for each rat. The analyzed

encephalic regions and their respective AP coordinates from

bregma (Paxinos and Watson, 2005) were as follows: the

prelimbic cortex (PrL) (AP: 3.72 mm to 3.24 mm), the cingulate

areas 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 CA3

hippocampal areas (AP: −2.64 mm to −2.92 mm), the dorsal

aspects of the nucleus accumbens core(AcbC) andshell (AcbSh)

(AP: 1.68 mm to 1.44 mm), the paraventricular thalamic nucleus

(PV) (AP: −2.16 mm to −2.40 mm), the paraventricular (PaV, all

subnuclei), anterior (AHC, central andposterior regions) andthe

lateral hypothalamic nuclei (LH, peduncular part) (AP: −

1.80 to

−1.92mm), the dorsomedialhypothalamus (DMH,all subnuclei)

(AP: − 3.00 mm to −3.24 mm), the dorsomedial (DMPAG), dor-

solateral (DLPAG), lateral (LPAG) and ventrolateral (VLPAG) parts

of the intermediate portions of the periaqueductal gray

(AP: −6.48 mm to −6.96 mm), the dorsal (DR) and median

raphe nucleus (MnR) (AP: −7.68 mm to −7.80 mm), the deep

layersof thesuperior colliculus (SC) (AP: −7.44mmto−7.68mm),

the ventral part of the central nucleus of the caudal inferior

colliculus (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 individually

and expressed as number of Fos-positive cells per 0.1 mm2

(Lamprea et al., 2002; Vianna et al., 2003; Ferreira-Netto et al.,

2005), asshownin Table 1. The choice of the areas sampledin

this study took into consideration the importance of these

regions in the modulation of the emotional, autonomic and

motor expression of the fear-motivated behaviors (Azuma

and 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; Conde

et al., 1990; Emmert and Herman, 1999; Espejo, 1997; Fendt

and Fanselow, 1999; Graeff, 1990, 1994; Gorman et al., 2000;

Gray and McNaughton, 2000; Jay et al., 1989; Jinks and

McGregor, 1997; LeDoux, 1995; Li and Sawchenko, 1998; Risold

and 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 analysis

Fos-protein expression was analyzed only in withdrawal

animals (sucrose and Dzp W) through Student t-test for each

region 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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brainstem areas activated by diazepam withdrawal and fos-protein immunoreactivity in rats

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