Treatment with zolpidem after ethanol administration potentiates the expression of ethanol-induced behavioral sensitization in mice

Contradictory findings suggest that the behavioral and abuse-related effects of ethanol are mediated by its action at α1 subunit-containing GABAA (α1GABAA) receptors. In the present study, we investigated the effects of a sub-chronic post-ethanol administration treatment with zolpidem, an α1-preferring positive allosteric modulator at GABAA receptors, on the subsequent expression of ethanol-induced behavioral sensitization in mice. Animals received ethanol (1.8 g/kg, ip) or saline treatments every other day for 15 days (8 treatment sessions) and were subsequently treated with zolpidem (0.5 mg/kg, ip) or vehicle 4 times on alternate days. At the end of the treatment phase, animals were challenged with saline or ethanol on separate days for the evaluation of the expression of conditioned locomotion and behavioral sensitization. Eight-day treatment with ethanol did not lead to the development of ethanol-induced behavioral sensitization. Animals treated with ethanol and subsequently administered vehicle showed similar locomotion frequencies during the last ethanol challenge compared to the control group receiving ethanol for the first time. Animals treated with ethanol and subsequently administered zolpidem expressed behavioral sensitization to ethanol during the ethanol challenge. The present study adds to the literature by providing further evidence of a role of α1GABAA receptors on the behavioral effects of ethanol. Because of the current highly prevalent co-abuse of ethanol and benzodiazepine drugs in humans, the use of zolpidem and other α1GABAA receptor ligands during ethanol withdrawal should be monitored carefully.


Introduction
Ethanol use disorder is a devastating disease, resulting in a series of organic, psychological, social, and economic problems. In the United States alone, nearly 17 million people suffer from ethanol-related disorders, being responsible for 88,000 deaths and costing more than $223.5 billion annually (1). Ethanol is frequently consumed in combination with other drugs of abuse, and the combination of ethanol and sedative-hypnotic drugs, such as zolpidem, has been associated with increased likelihood of being admitted to intensive care units (2,3).
Zolpidem and ethanol share similar mechanisms of action. Ethanol is a central nervous system depressant, and acts by potentiating g-aminobutyric acid (GABA)ergic neurotransmission via action at the GABA A receptor (4). Zolpidem is an a1-preferring positive allosteric modulator at GABA A receptors (5). Although several subtypes of GABA A receptors have been implicated in the abuserelated effects of ethanol (6), the role of a1-containing GABA A (a1GABA A ) receptors on ethanol abuse remains controversial. Previous studies in rodents have shown a reduced preference for ethanol in a1GABA A null mutant mice (7) and reduced ethanol self-administration after treatment with a1GABA A receptor antagonists in rats (8). However, self-administration studies in non-human primates have shown little evidence of a contribution of a1GABA A receptors on the reinforcing effects of ethanol (9), and subject-rated reinforcing effects of zolpidem were not increased by ethanol in humans (10).
In order to further elucidate the role of a1GABA A receptors on ethanol-induced behaviors, the present study was designed to investigate the effects of a sub-chronic post-ethanol administration treatment with zolpidem on the subsequent expression of ethanol-induced behavioral sensitization in mice.

Subjects
Three-month-old Swiss male mice (LaBIO, Universidade Estadual de Santa Cruz, UESC) were used in the experiments. Animals were housed 10 per cage under controlled ventilation, temperature (22-23°C), and lighting conditions (12 h light/dark, lights on at 6:30 am) with free access to water and food. The experiments were in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals (8th edition, revised 2011) and the Brazilian Law No. 11,794, and were approved by the Institutional Ethical Committee of UESC.

Open-field evaluation
Locomotor activity was measured in the open-field apparatus. The apparatus consisted of a circular wooden arena (40 cm in diameter and 50 cm high) with an open top and a floor divided into 19 approximately similar regions delimited by three concentric circles intersected by radial line segments. Animals were exposed to the open-field individually during 10-min sessions. Locomotor activity was tracked using the ANY-maze software (version 5.1, Stoelting, USA) and a webcam was suspended overhead. Figure 1 illustrates the experimental design. Forty male mice were given a 10-min habituation session in the open-field for 3 consecutive days after a saline injection, and locomotor activity was quantified on day 3. Animals were then allocated into 2 groups (N=20 per group). The behavioral sensitization protocol (''ethanol treatment'' phase) was conducted according to a protocol previously used by our research group (11). Animals received treatments of either ethanol (Eth) or saline (Sal) every other day for 15 days (8 treatment sessions, days 4 to 18). Five minutes after injections, animals were individually placed in the open-field for 10 min. Locomotor activity (distance traveled during the session) was measured on the 1st and 15th days of this phase.

Experimental design
Forty-eight hours after the last ethanol session, the zolpidem treatment phase began. Mice received an ip injection of vehicle (Veh) or zolpidem (Zol) every other day for 7 days (4 sessions, days 20 to 26), forming the following groups: Sal-Veh, Eth-Veh, Sal-Zol, and Eth-Zol (N=10 per group). Five minutes after each injection, mice were individually placed in the open-field for 10 min. Locomotor activity was measured on the 1st and last days of this phase.
Four days after the last treatment injection (day 31), all animals were acutely challenged with saline (ip) to evaluate conditioned responses in the open-field apparatus. Forty-eight hours after the saline challenge (day 33), animals were tested for drug-induced expression of behavioral sensitization to ethanol. All animals received an injection of 1.8 g/kg ethanol and were individually placed in the open-field apparatus.

Statistical analysis
Before conducting the parametric tests, all variables were checked for normality (Shapiro-Wilk test) and homogeneity (Levene's test), which validated the use of the parametric test. Data were analyzed by one-or twoway analysis of variance (ANOVA), with or without repeated measures, or Student's t-test for paired samples (within-group comparisons). Post hoc comparisons were performed using Duncan's post hoc test. A P value less than 0.05 was considered to be a statistically significant difference.

Results
Analysis of the 3rd habituation session revealed no significant difference between groups (data not shown).  Post hoc comparisons showed that acute treatment with zolpidem in animals treated with saline, but not ethanol, induced hypolocomotion in mice (Sal-Zol o Sal-Veh, Day 1). Zolpidem-induced hypolocomotion was sensitized after repeated administration (Sal-Zol Day 7 o Sal-Zol Day 1). Although ethanol-treated animals did not express conditioned locomotion to ethanol on the 1st day of the zolpidem treatment phase (Eth-Veh=Sal-Veh), the distance traveled was significantly decreased over time in ethanol-treated animals, indicating an extinction effect ( Figure 2B Post hoc multiple comparisons showed that the locomotor activity of the Eth-Veh group did not change across ethanol treatment/challenge days (Day 1 vs Day 15 vs ethanol challenge), indicating that this group indeed did not express locomotor activity during the ethanol challenge. On the other hand, the Eth-Zol group did show a significantly higher locomotor frequency during the ethanol challenge compared to itself during Days 1 and 15 of the ethanol treatment phase (P=0.02 and P=0.0007, respectively).

Discussion
In the present study, treatment with zolpidem after subchronic administration of ethanol (sensitization phase) promoted the expression of ethanol-induced behavioral  sensitization. While animals in the Eth-Veh group exhibited similar locomotion frequency compared to control animals receiving ethanol for the first time (Sal-Veh), animals previously sensitized with ethanol and treated with zolpidem expressed higher locomotion frequencies than all other groups. Of note, animals receiving ethanol for the first time (Sal-Veh and Sal-Zol groups) showed similar locomotor activity levels during the ethanol challenge, suggesting that zolpidem did not simply potentiate the acute locomotor effects of ethanol and, instead, promoted the expression of ethanol-induced behavioral sensitization. Zolpidem-induced cross sensitization was only evident during the ethanol challenge after a prolonged drug-free period. According to Lessov and Phillips (12), repeated ethanol administration associated with the test apparatus can promote modifications in neural pathways that mediate locomotor activity so that these pathways become more sensitive and responsive to a subsequent ethanol challenge. In fact, previous studies from our group have also shown that a drug-free interval is necessary for the expression of ethanol-induced behavioral sensitization (13). These neuroadaptations would explain the expression of behavioral sensitization after a drug-free period following a sensitization protocol and zolpidem treatment, and suggest that zolpidem might be modulating or further contributing to ethanol-induced neuroadaptations.
Ethanol interacts with several neurotransmitter systems (14,15), and ethanol-induced activation of the mesolimbic dopaminergic pathway seems to be the main mechanism underlying acute ethanol-induced locomotor stimulation (16,17). The ventral tegmental area (VTA) is one of the major regions in this pathway, and is predominantly comprised of dopamine neurons (B70%) and GABA interneurons (B20%) (18,19). Of note, ethanolinduced firing of dopamine neurons in the VTA seems to be modulated by ethanol-induced decreased firing of VTA GABAergic interneurons (20).
GABA is the main inhibitory neurotransmitter of the central nervous system, with presynaptic and post-synaptic action at inotropic (GABA A ) and metabotropic (GABA B ) receptors. Each GABA A receptor is composed of five subunits, and the final composition of each receptor determines its distinct physiological and pharmacological properties (21). GABAergic interneurons located in the VTA selectively express a1-containing GABA A receptors in rats (22,23). Thus, studies have focused on the investigation of the role of a1GABA A receptors on ethanol-induced behaviors, with contradictory findings. While rodent studies show favorable evidence for a potential role of a1GABA A receptors on ethanol preference (7) and ethanol drinking (8), nonhuman primate studies show little to no evidence of a contribution of this receptor to the abuse-related effects of ethanol (9).
The present study adds to the literature by providing further evidence of a role of a1GABA A receptors in the behavioral effects of ethanol in rodents. More specifically, while 8 intermittent treatments with ethanol alone did not lead to the expression of behavioral sensitization in mice, animals treated with ethanol and subsequently administered zolpidem expressed ethanol-induced behavioral sensitization under our experimental conditions. These findings suggest that zolpidem extended the effects of ethanol when administered in the open-field apparatus. It is important to note, however, that treatment with zolpidem induced hypolocomotion in mice, indicating that the dose of zolpidem used in the present study might have induced a sedative effect in mice, opposed to the stimulant dose of ethanol. However, in the absence of zolpidem, when animals in the Zol-vs Veh-treated groups no longer showed differences in locomotor activity (saline challenge), the long-lasting effects of sub-chronic treatment with zolpidem after ethanol sensitization became evident. Our findings are in agreement with a previous study demonstrating that a 7-day treatment with zolpidem led to the development of mesolimbic dopamine-dependent neural plasticity in mice (24). It is important to note, however, that a previous study did not observe locomotor-enhancing effects of zolpidem in animals sensitized with ethanol. In the study by Linsenbardt and Boehm (25), the authors use a strain of mice known to be particularly susceptible to the development of ethanol-induced behavioral sensitization. In contrast to the present study, the authors did observe the development of behavioral sensitization to ethanol within the 15-day sensitization period. After the ethanol sensitization period, zolpidem did not potentiate the locomotor activity of mice. These data suggest that the cross-sensitization observed in the present study was, perhaps, only present because animals did not express sensitization to ethanol during the sensitization protocol in the first place.
Studies on the interaction between GABA A receptor ligands and the abuse-related effects of ethanol become extremely relevant in light of the current highly prevalent co-abuse of ethanol and benzodiazepine drugs in humans (26). Because behavioral sensitization is thought to share neuroadaptations with drug craving in humans (27), data from the present study suggest that the use of zolpidem during ethanol withdrawal, such as in the context of hypnotic drug use due to Eth withdrawal-induced sleep impairment (28,29), may favor the installation of compulsive behavior.
Our findings suggested that a1GABA A receptors play an important role on ethanol-induced behaviors, and that the use of zolpidem and other GABA A receptor ligands in the context of ethanol abuse should be monitored carefully.