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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diazepam-withdrawn rats.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,
B R A I N R E S E A R C H 1 1 6 6 ( 2 0 0 7 ) 3 5 – 4 6
⁎ 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
w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s
mailto:[email protected]://dx.doi.org/10.1016/j.brainres.2007.07.007http://dx.doi.org/10.1016/j.brainres.2007.07.007mailto:[email protected]
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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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Fig. 5 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through the
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
Fig. 7 – Photomicrograph of Fos-like immunoreactive cells (dark dots) in coronal sections showing Fos expression through 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).
R E F E R E N C E S
Abercrombie, E.A., Keefe, K.A., DiFrischia, D.A., Zigmond, M.J.,1989. Differential effects of stress on in vivo dopamine releasein striatum, nucleus accumbens, and medial frontal cortex.
J. Neurochem. 52, 1655–1658.
Allison, C., Pratt, J.A., 2003. Neuroadaptative processes inGABAergic and glutamatergic systems in benzodiazepinedependence. Pharmacol. Ther. 98, 171–195.
Andrews, N., File, S.E., Fernandes, C., Gonzalez, L.E., Barnes, N.M.,1997. Evidence that the median raphe nucleus–dorsalhippocampal pathway mediates diazepam withdrawal-inducedanxiety. Psychopharmacology (Berl) 130, 228–234.
Ashton, H., 2005. The diagnosis and management of benzodiazepine dependence. Curr. Opin. Psychiatry 18,249–255.
Azuma, M., Chiba, T., 1996. Afferent projections of the infralimbiccortex (area 25) in rats: a WGA-HRP study. Kaibogaku Zasshi71, 523–540.
Bateson, A.N., 2002. Basic pharmacologic mechanisms involved inbenzodiazepine tolerance and withdrawal. Curr. Pharm. Des. 8,
5–21.Beck, C.H., Fibiger, H.C., 1995. Conditioned fear-induced changes in
behavior and in the expression of the immediate early genec- fos: with and without diazepam pretreatment. J. Neurosci. 15,709–720.
Beckett, S.R., Duxon, M.S., Aspley, S., Marsden, C.A., 1997. Centralc- fos expression following 20 kHz/ultrasound induced defencebehaviour in the rat. Brain Res. Bull. 42, 421–426.
Borlikova, G.G., Merrer, J.L., Stephens, D.N., 2006. Previousexperience of ethanol withdrawal increases withdrawalinduced c- fos expression in limbic areas, but notwithdrawal-induced anxiety and prevents withdrawal-inducedelevations in plasma corticosterone. Psychopharmacology 185,188–200.
Brandão, M.L., Anseloni, V.Z., Pandossio, J.E., De Araujo, J.E.,Castilho, V.M., 1999. Neurochemical mechanisms of thedefensive behavior in the dorsal midbrain. Neurosci. Biobehav.Rev. 23, 863–875.
Brandão, M.L., Troncoso, A.C., de Souza Silva, M.A., Huston, J.P.,2003. The relevance of neuronal substrates of defense in themidbrain tectum to anxiety and stress: empirical andconceptual considerations. Eur. J. Pharmacol. 463, 225–233.
Brandão,M.L.,Borelli, K.G., Nobre, M.J., Santos, J.M., Albrechet-Souza,L., Oliveira, A.R., Martinez, R.C., 2005. Gabaergic regulation of theneural organization of fear in the midbrain tectum. Neurosci.Biobehav. Rev. 29, 1299–1311.
Bullitt, E., 1990. Expression of c- fos -like protein as a marker for neuronal activity following noxious stimulation in the rat.
J. Comp. Neurol. 296, 517–530.Busto, U.E., Sellers, E.M., 1991. Anxiolytics and sedative/hypnotics
dependence. Br. J. Addict. 86, 1647–1652.
44 B R A I N R E S E A R C H 1 1 6 6 ( 2 0 0 7 ) 3 5 – 4 6
8/18/2019 Brainstem Areas Activated by Diazepam Withdrawal and FOS-protein Immunoreactivity in Rats
11/12
Busto, U., Sellers, E.M., Naranjo, C.A., Cappell, H.D., Sanchez-Craig,M., Simpkins, J., 1986. Patterns of benzodiazepine abuse anddependence. Br. J. Addict. 81, 87–94.
Cabral, A., Isoardi, N., Salum, C., Macedo, C.E., Nobre, M.J., Molina,V.A., Brandão, M.L., 2006. Fear state induced by ethanolwithdrawal may be due to the sensitization of the neuralsubstrates of aversion in the dPAG. Exp. Neurol. 200, 200–208.
Campeau, S., Falls, W.A., Cullinan, W.E., Helmreich, D.L., Davis, M.,
Watson, S.J., 1997. Elicitation and reduction of fear: behaviouraland neuroendocrine indices and brain induction of theimmediate-early gene c- fos. Neuroscience 78, 1087–1104.
Canteras, N.S., Goto, M., 1999. Fos-like immunoreactivity in theperiaqueductal gray of rats exposed to a natural predator.NeuroReport 10, 413–418.
Canteras, N.S., Swanson, L.W., 1992. The dorsal premammillarynucleus: an usual component of the mammillary body. Proc.Natl. Acad. Sci. U. S. A. 89, 10089–10093.
Canteras, N.S., Chiavegatto, S., Valle, L.E., Swanson, L.W., 1997.Severe reduction of rat defensive behavior to a predator bydiscrete hypothalamic chemical lesions. Brain Res. Bull. 44,297–305.
Charney, D.S., Grillon, C., Bremner, J.D., 1998. The neurobiologicalbasis of anxiety and fear: Circuits, mechanisms and
neurochemical interactions (part I). Neuroscientist 4, 35-44.Chouinard, G., 2004. Issues in the clinical use of benzodiazepines:
potency,withdrawal, andrebound. J. Clin. Psychiatry 65 (Suppl 5),7–12.
Conde, F., Audinat, E., Maire-Lepoivre, E., Crepel, F., 1990. Afferentconnections of the medial frontal cortex of the rat. A studyusing retrograde transport of fluorescent dyes. I. Thalamicafferents. Brain Res. Bull. 24, 341–354.
Dragunow, M., Faull, R., 1989. The use of c- fos as a metabolicmarker in neuronal pathway tracing. J. Neurosci. Methods 29,261–265.
Dunworth, S.J., Mead, A.N., Stephens, D.N., 2000. Previousexperience of withdrawal from chronic diazepam amelioratesthe aversiveness of precipitated withdrawal and reduceswithdrawal-induced c- fos expression in nucleus accumbens.
Eur. J. Neurosci. 12, 1501–1508.Emmert, M.H., Herman, J.P., 1999. Differential forebrain c- fos
mRNA induction by either inhalation or novelty: evidence for distinctive stress pathways. Brain Res. 845, 60–67.
Emmett-Oglesby, M.W., Spencer Jr., D.G., Elmesallamy, F., Lal, H.,1983. The pentylenetetrazol model of anxiety detectswithdrawal from diazepam in rats. Life Sci. 33, 161–168.
Espejo, E.F., 1997. Selective dopamine depletion within the medialprefrontal cortex induced anxiogenic-like effects in rats placedon the elevated plus-maze. Brain Res. 762, 281–284.
Fendt, M., Fanselow, M.S., 1999. The neuroanatomical andneurochemical basis of conditioned fear. Neurosci. Biobehav.Rev. 23, 743–760.
Ferreira-Netto, C., Borelli, K.G., Brandão, M.L., 2005. Neuralsegregation of Fos-protein distribution in the brain following freezing and escape behaviors induced by injections of either glutamate or NMDA into the dorsal periaqueductal gray of rats.Brain Res. 1031, 151–163.
File, S.E., 1990. The history of benzodiazepine dependence: areview of animal studies. Neurosci. Biobehav. Rev. 14, 135–146.
Galpern, W.R., Lumpkin,M., Greenblatt, D.J., Shader, R.I., Miller, L.G.,1991. Chronic benzodiazepine administration. VII. Behavioraltolerance and withdrawal and receptor alterations associatedwith clonazepam administration. Psychopharmacology (Berl)104, 225–230.
Gorman, J.M., Kent, J.M., Sullivan, G.M., Coplan, J.D., 2000.Neuroanatomical hypothesis of panic disorder, revised. Am. J.Psychiatry 157, 493–505.
Graeff, F.G., 1990. Brain defense systems and anxiety. In: Roth, M.,Burrow, G.D., Noyes, R. (Eds.), Handbook of Anxiety. Elsevier,New York, pp. 307–354.
Graeff, F.G., 1994. Neuroanatomy and neurotransmitter regulationof defensive behaviors and related emotions in mammals.Braz. J. Med. Biol. Res. 27, 811–829.
Gray, J.A., 1982. The Neuropsychology of Anxiety. Oxford UniversityPress, London.
Gray, J.A., McNaughton, N., 2000. The neuropsychology of anxiety,An Enquiry into Functions of the Septo-Hippocampal System2nd ed. Oxford University Press, London.
Greenblatt, D.J., Shader, R.I., 1974. Drug therapy. Benzodiazepines(second of two parts). N. Engl. J. Med. 291, 1239–1243.
Griebel, G., Moreau, J.L., Jenck, F., Misslin, R., Martin, J.R., 1994.Acute and chronic treatment with 5-HT reuptake inhibitorsdifferentially modulate emotional responses in anxiety modelsin rodents. Psychopharmacology (Berl) 113, 463–470.
Hayward, M.D., Duman, R.S., Nestler, E.J., 1990. Induction of thec- fos proto-oncogene during opiate withdrawal in the locuscoeruleus and other regions of rat brain. Brain Res. 525,256–266.
Honkaniemi, J., Pelto-Huikko, M., Rechardt, L., Isola, J., Lammi, A.,Fuxe, K., Gustafsson, J.A., Wikstrom, A.C., Hokfelt, T., 1992.Colocalization of peptide and glucocorticoid receptor immunoreactivities in rat central amygdaloid nucleus.Neuroendocrinology 55, 451–459.
Imaki, T., Shibasaki, T., Hotta, M., Demura, H., 1992. Earlyinduction of c- fos precedes increased expression of corticotropin-releasing factor messenger ribonucleic acid inthe paraventricular nucleus after immobilization stress.Endocrinology 131, 240–246.
Jay, T.M., Glowinski, J., Thierry, A.M., 1989. Selectivity of thehippocampal projection to the prelimbic area of the prefrontalcortex in the rat. Brain Res. 505, 337–340.
Jinks, A.L., McGregor, I.S., 1997. Modulation of anxiety-relatedbehaviours following lesions of the prelimbic or infralimbiccortex in the rat. Brain Res. 772, 181–190.
Koob, G.F., 1992. Drugs of abuse: anatomy, pharmacology andfunction of reward pathways. Trends Pharmacol. Sci. 13,177–184.
Koob, G.F., Le Moal, M., 1997. Drug abuse: hedonic homeostatic
dysregulation. Science. 278, 52–58.Lacerra, C., Martijena, I.D., Bustos, S.G., Molina, V.A., 1999.
Benzodiazepine withdrawal facilitates the subsequent onset of escape failures and anhedonia: influence of differentantidepressant drugs. Brain Res. 819, 40–47.
Ladewig, D., 1984. Dependence liability of the benzodiazepines.Drug Alcohol Depend. 13, 139–149.
Lamprea, M.R., Cardenas, F.P., Vianna, D.M., Castilho, V.M.,Cruz-Morales, S.E., Brandão, M.L., 2002. The distribution of fosimmunoreactivity in rat brain following freezing and escaperesponses elicited by electrical stimulation of the inferior colliculus. Brain Res. 950, 186–194.
LeDoux, J.E., 1995. Emotion: clues from the brain. Annu. Rev.Psychol. 46, 209–235.
Lingford-Hughes, A., Nutt, D., 2003. Neurobiology of addiction andimplications for treatment. Br. J. Psychiatry 182, 97–100.
Li, H.Y., Sawchenko, P.E., 1998. Hypothalamic effector neurons andextendedcircuitries activated in neurogenicstress:a comparisonof footshock effects exerted acutely, chronically, and in animalswith controlled glucocorticoid levels. J. Comp. Neurol. 393,244–266.
Lukas, S.E., Griffiths, R.R., 1982. Precipitated withdrawal by abenzodiazepine receptor antagonist (Ro 15-1788) after 7 days of diazepam. Science 217, 1161–1163.
Miller, L.G., Greenblatt, D.J., Roy, R.B., Summer, W.R., Shader, R.I.,1988. Chronic benzodiazepineadministration. II. Discontinuationsyndrome is associated with upregulation of gamma-aminobutyric acidA receptor complex binding andfunction. J. Pharmacol. Exp. Ther. 246, 177–182.
Motzo, C., Porceddu, M.L., Dazzi, L., Sanna, A., Serra, M., Biggio, G.,1997. Enhancement by flumazenil of dopamine release in the
45B R A I N R E S E A R C H 1 1 6 6 ( 2 0 0 7 ) 3 5 – 4 6
8/18/2019 Brainstem Areas Activated by Diazepam Withdrawal and FOS-protein Immunoreactivity in Rats
12/12
nucleus accumbens of rats repeatedly exposed to diazepam or imidazenil. Psychopharmacology 131, 34–39.
Moy, S.S., Knapp, D.J., Duncan, G.E., Breese, G.R., 2000. Enhancedultrasonic vocalization and Fos protein expression following ethanol withdrawal: effects of flumazenil. Psychopharmacology(Berl) 152, 208–215.
Owen, R.T., Tyrer, P., 1983. Benzodiazepine dependence. A reviewof the evidence. Drugs 25, 385–398.
Paxinos, G., Watson, C. 2005. The Rat Brain in StereotaxicCoordinates. First Ed. Third edn. New York: Academic Press.
Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14, 149–167.
Persico, A.M., Schindler, C.W., Zaczek, R., Brannock, M.T., Uhl, G.R.,1995. Brain transcription factor gene expression, neurotrans-mitter levels, and novelty response behaviors: alterationsduring rat amphetamine withdrawal and following chronicinjection stress. Synapse 19, 212–227.
Petursson, H., Lader, M.H., 1981a. Withdrawal from long-termbenzodiazepine treatment. Br. Med. J. (Clin. Res. Ed) 283,643–645.
Petursson, H., Lader, M.H., 1981b. Benzodiazepine dependence.Br. J. Addict. 76, 133–145.
Reiman, E.M., Raichle, M.E., Robins, E., Mintun, M.A., Fusselman,M.J., Fox, P.T., Price, J.L., Hackman, K.A., 1989. Neuroanatomicalcorrelates of a lactate-induced anxiety attack. Arch. Gen.Psychiatry 46, 493–500.
Richard, D., Rivest, S., Rivier, C., 1992. The 5-hydroxytryptamineagonist fenfluramine increases Fos-like immunoreactivity inthe brain. Brain Res. 594, 131–137.
Risold, P.Y., Swanson, L.W., 1996. Structural evidence for functionaldomains in the rat hippocampus. Science 272, 1484–1486.
Rosebush, P.I., Mazurek, M.F., 1996. Catatonia afterbenzodiazepinewithdrawal. J. Clin. Psychopharmacol. 16, 315–319.
Rosen, J.B., Schulkin, J., 1998. From normal fear to pathologicalanxiety. Psychol. Rev. 105, 325–350.
Rynn, M.A., Brawman-Mintzer, O., 2004. Generalized anxietydisorder: acute and chronic treatment. CNS Spectr. 9, 716–723.
Sandner, G., Di Scala, G., Rocha, B., Angst, M.J., 1992. C-fosimmunoreactivity in the brain following unilateral electricalstimulation of the dorsal periaqueductal gray in freely moving rats. Brain Res. 573, 276–283.
Sandner, G., Oberling, P., Silveira, M.C., Di Scala, G., Rocha, B.,Bagri, A., Depoortere, R., 1993. What brain structures are activeduring emotions? Effects of brain stimulation elicited aversionon c- fos immunoreactivity and behavior. Behav. Brain Res. 58,9–18.
Schleimer, S.B., Johnston, G.A., Henderson, J.M., 2005. Novel oraldrug administration in an animal model of neuroleptictherapy. J. Neurosci. Methods 146, 159–164.
Senba, E., Matsunaga, K., Tohyama, M., Noguchi, K., 1993.Stress-induced c- fos expression in the rat brain: activationmechanism of sympathetic pathway. Brain Res. Bull. 31,329–344.
Silveira, M.C., Sandner, G., Graeff, F.G., 1993. Induction of Fosimmunoreactivity in the brain by exposure to the elevatedplus-maze. Behav. Brain Res. 56, 115–118.
Silveira, M.C., Sandner, G., Di Scala, G., Graeff, F.G., 1995. c-Fosimmunoreactivity in the brain following electrical or chemicalstimulation of the medial hypothalamus of freely moving rats.Brain Res. 674, 265–274.
Singewald, N., Sharp, T., 2000. Neuroanatomical targets of anxiogenic drugs in the hindbrain as revealed by Fosimmunocytochemistry. Neuroscience 98, 759–770.
Smith, W.J., Stewart, J., Pfaus, J.G., 1997. Tail pinch induces fos
immunoreactivity within several regions of the male rat brain:effects of age. Physiol. Behav. 61, 717–723.
Stone, E.A., Zhang, Y., John, S.M., Bing, G., 1991. c-Fos response toadministration of catecholamines into brain by microdialysis.Neurosci. Lett. 133, 33–35.
Souza-Pinto, L.F., Castilho, V.M., Brandão, M.L., Nobre, M.J., 2007.The blockade of AMPA-kainate and NMDA receptors in thedorsal periaqueductal gray reduces the effects of diazepamwithdrawal in rats. Pharmacol. Biochem. Behav. 87, 250–257.
Toki, S., Saito, T., Hatta, S., Takahata, N., 1996. Diazepam physicaldependence and withdrawal in rats is associated withalteration in GABAA receptor function. Life Sci. 59, 1631–1641.
Vianna, D.M., Borelli, K.G., Ferreira-Netto, C., Macedo, C.E.,Brandão, M.L., 2003. Fos-like immunoreactive neuronsfollowing electrical stimulation of the dorsal periaqueductal
gray at freezing and escape thresholds. Brain Res. Bull. 62,179–189.
Woods, J.H., Katz, J.L., Winger, G., 1987. Abuse liability of benzodiazepines. Pharmacol. Rev. 39, 251–413.
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