The amygdala regulates the antianxiety sensitization effect of flumazenil during repeated chronic ethanol or repeated stress

Abstract

Background: The benzodiazepine receptor antagonist flumazenil reduces anxiety-like behavior and sensitization of anxiety-like behavior in various models of ethanol withdrawal in rodents. The mechanism and brain region(s) that account for this action of flumazenil remain unknown. This investigation explored the potential role of several brain regions (amygdala, raphe, inferior colliculus, nucleus accumbens, and paraventricular hypothalamus) for these actions of flumazenil.

Methods: Rats were surgically implanted with guide cannulae directed over the brain region of interest and then treated with an ethanol diet for three 7-day dietary cycles (5 days on ethanol diet followed by 2 days on control diet). At approximately 4 hours, flumazenil was administered intracranially into each of the first 2 withdrawals. Examinations of anxiety-like behavior followed 1 week later during a third withdrawal. In other animals, restraint stress sessions or intra-amygdala DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) injections, preceded by intraperitoneal flumazenil injections, were substituted for the first 2 ethanol treatment cycles to assess the potential anxiety-sensitizing action of stress or a benzodiazepine receptor inverse agonist, respectively.

Results: Flumazenil treatment of the amygdala during the first 2 withdrawals blocked the development of sensitized anxiety seen during a third withdrawal. Similar actions of flumazenil were found when stress sessions substituted for the first 2 cycles of ethanol exposure and withdrawal. Amygdala treatment with DMCM magnified the anxiety response to the single subthreshold chronic ethanol treatment, and prophylactic flumazenil blocked this effect.

Conclusions: Intra-amygdala flumazenil inhibits the development of anxiety sensitized by repeated ethanol withdrawal, stress/ethanol withdrawal, or DMCM/ethanol withdrawal. These actions suggest that site-specific and persistent effects of flumazenil on gamma-aminobutyric acid-modulatory processes in this brain region are relevant to sensitized behavioral effects seen in alcoholism.

Fig. 1

Schematic of treatment and testing strategies (A) and digital photomicrographs (top panel) illustrating targeted areas/needle tracks and schematics (bottom panel) showing representative positive locations of injections into (a) amygdala, (b) dorsal raphe, (c) nucleus accumbens, (d) paraventricular nucleus of the hypothalamus (PVN), (e) inferior colliculus (B). For cannula placements, all measurements are in millimeters, except for the angle of approach, which is measured in degrees from vertical.* All coordinates are relative to Bregma, except for the raphe, which is relative to lambda, and the inferior colliculus where the anterior–posterior (AP) location was defined as 0.2 mm anterior to the interaural line. CD, control diet; ED, 4.5% ethanol diet; ML, medial lateral; DV, dorsoventral. Coordinates and anatomical schematics are from Paxinos and Watson (2005).

Fig. 2

Intra-amygdalar injection of flumazenil selectively counteracts ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the amygdala and then subjected to 3 cycles of 4.5% ethanol diet (ED3). Injections were given 4 hours after ethanol from the first and second cycles of ED was withdrawn. Social-interaction behavior was assessed between 5 and 6 hours after the removal of the third cycle of ethanol. CD, control diet given to unoperated controls; ED3-Veh, ethanol diet with vehicle injections (artificial cerebrospinal fluid); ED3-Flum, Ethanol diet and flumazenil injections (5 μg/μl per side). *p < 0.05 versus CD-Veh.

Fig. 3

Dose–response for intra-amygdala flumazenil pretreatments that selectively counteract ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the amygdala, exposed to 3 cycles of chronic ethanol diet with or without flumazenil pretreatments (2.5, 5, or 10 μg/μl per side) during the first 2 withdrawals. Other groups and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.

Fig. 4

Intra-raphe injection of flumazenil fails to counteract ethanol withdrawal- induced anxiety-like behavior. Rats were implanted with single cannulae above the dorsal raphe nucleus and then subjected to 3 cycles of 4.5% ethanol diet (ED3). Flumazenil treatment was 2.5 μg (0.5 μg/μl). Groups and abbreviations are as per Fig. 2. *p < 0.05 versusCD-Veh.

Fig. 5

Intra-paraventricular nucleus of the hypothalamus injections of flumazenil fails to counteract ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the paraventricular nucleus and then subjected to 3 cycles of 4.5% ethanol diet (ED3). Flumazenil treatment was 2.5 μg (0.5 μg/μl). Groups and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.

Fig. 6

Intra-accumbens injections of flumazenil into the accumbens fail to counteract ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the nucleus accumbens and then subjected to 3 cycles of 4.5% ethanol diet (ED3). Flumazenil treatments, groups, and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.

Fig. 7

Failure of injections of flumazenil in the inferior colliculus to counteract ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the inferior colliculus and then subjected to 3 cycles of 4.5% ethanol diet (ED3). Flum5 and Flum10 injections (5 or 10 μg/μl per side, respectively) were given 4 hours after ethanol from the first and second cycles of ED was withdrawn. Other groups and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.

Fig. 8

Repeated intra-amygdalar injection of methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) sensitizes ethanol withdrawal-induced anxiety-like behavior. Rats were implanted with cannulae above the amygdala, treated with DMCM twice, and then subjected to 1 cycle of 4.5% ethanol diet (ED1). Other groups and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.

Fig. 9

Peripheral pretreatment with flumazenil prevents sensitization of ethanol withdrawal-induced anxiety-like behavior by intra-amygdala injection of methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). Rats were implanted with cannulae above the amygdala, treated with DMCM twice with or without flumazenil pretreatment (5 μg/μl per side), and then subjected to 1 cycle of 4.5% ethanol diet (ED1). Other groups and abbreviations are as per Fig. 2. *p < 0.05 versusCD-Veh.

Fig. 10

Intra-amygdala pretreatment with flumazenil prevents sensitization of ethanol withdrawal-induced anxiety-like behavior by restraint stress. Rats were implanted with cannulae above the amygdala, restraint stressed twice with or without intra-amygdala flumazenil injection (5 μg/μl per side), and then subjected to 1 cycle of 4.5% ethanol diet (ED1). Other groups and abbreviations are as per Fig. 2. *p < 0.05 versus CD-Veh.