Harry L. June

Binge alcohol drinking is associated with GABAA α2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala

Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABAA α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABAA receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABAA α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABAA α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.

Effects of buprenorphine and methadone in methadone-maintained subjects

Buprenorphine, a partial mu opioid agonist, is an experimental medication under development for the treatment of opioid dependence as an alternative to methadone maintenance. The present study examined the relationship between level of opioid physical dependence and response to buprenorphine administration as part of a program to develop procedures for transferring patients from methadone to buprenorphine treatment. This laboratory study characterized the agonist and antagonist effects of acute doses of buprenorphine and methadone in subjects maintained on either 30 (n=7) or 60 (n=6) mg/day oral methadone. Test doses of placebo [sl. and PO), methadone (15, 30, and 60 mg PO) and buprenorphine (2, 4, and 8 mg sl.) were administered to volunteers residing on a closed residential unit. Subjective, physiological, observer-rated, and cognitive/psychomotor measures were collected for 6.5 h after test doses. Test doses of methadone, but not buprenorphine, constricted pupils and produced dose-related increases on subjective report measures reflecting opioid agonist drug effects. Agonist effects of methadone were more prominent in the 30 mg than in the 60 mg methadone maintenance condition. Buprenorphine, but not methadone, precipitated opioid withdrawal signs and symptoms that were more prominent in the 60 mg than in the 30 mg methadone maintenance condition. These findings suggest that abrupt transition from methadone to buprenorphine may produce patient discomfort that is positively related to both methadone maintenance dose and buprenorphine transition dose.

The Reinforcing Properties of Alcohol are Mediated by GABA A1 Receptors in the Ventral Pallidum

It has been hypothesized that alcohol addiction is mediated, at least in part, by specific γ-aminobutyric acidA (GABAA) receptors within the ventral pallidum (VP). Among the potential GABAA receptor isoforms regulating alcohol-seeking behaviors within the VP, the GABAA α1 receptor subtype (GABAA1) appears pre-eminent. In the present study, we developed β-carboline-3-carboxylate-t-butyl ester (βCCt), a mixed agonist–antagonist benzodiazepine (BDZ) site ligand, with binding selectivity at the A1 receptor to explore the functional role of VPA1 receptors in the euphoric properties of alcohol. The in vivo actions of βCCt were then determined following microinfusion into the VP, a novel alcohol reward substrate that primarily expresses the A1 receptor. In two selectively bred rodent models of chronic alcohol drinking (HAD-1, P rats), bilateral microinfusion of βCCt (0.5–40 μg) produced marked reductions in alcohol-reinforced behaviors. Further, VP infusions of βCCt exhibited both neuroanatomical and reinforcer specificity. Thus, no effects on alcohol-reinforced behaviors were observed following infusion in the nucleus accumbens (NACC)/caudate putamen (CPu), or on response maintained by saccharin. Parenteral-administered βCCt (1–40 mg/kg) was equally effective and selective in reducing alcohol-reinforced behaviors in P and HAD-1 rats. Additional tests of locomotor activity revealed that βCCt reversed the locomotor sedation produced by both chlordiazepoxide (10 mg/kg) and EtOH (1.25 g/kg), but was devoid of intrinsic effects when given alone. Studies in recombinant receptors expressed in Xenopus oocytes revealed that βCCt acted as a low-efficacy partial agonist at α3β3γ2 and α4β3γ2 receptors and as a low-efficacy inverse agonist at α1β3γ2, α2β3γ2, and α5β3γ2 receptors. The present study indicates that βCCt is capable of antagonizing the reinforcing and the sedative properties of alcohol. These anti-alcohol properties of βCCt are primarily mediated via the GABAA1 receptor. βCCt may represent a prototype of a pharmacotherapeutic agent to effectively reduce alcohol drinking behavior in human alcoholics.

GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry.

GABA receptors within the mesolimbic circuitry have been proposed to play a role in regulating alcohol-seeking behaviors in the alcohol-preferring (P) rat. However, the precise GABAAreceptor subunit(s) mediating the reinforcing properties of EtOH remains unknown. We examined the capacity of intrahippocampal infusions of an α5 subunit-selective (∼75-fold) benzodiazepine (BDZ) inverse agonist [i.e., RY 023 (RY) (tert-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate)] to alter lever pressing maintained by concurrent presentation of EtOH (10% v/v) and a saccharin solution (0.05% w/v). Bilateral (1.5–20 μg) and unilateral (0.01–40 μg) RY dose-dependently reduced EtOH-maintained responding, with saccharin-maintained responding being reduced only with the highest doses (e.g., 20 and 40 μg). The competitive BDZ antagonist ZK 93426 (ZK) (7 μg) reversed the RY-induced suppression on EtOH-maintained responding, confirming that the effect was mediated via the BDZ site on the GABAA receptor complex. Intrahippocampal modulation of the EtOH-maintained responding was site-specific; no antagonism by RY after intra-accumbens [nucleus accumbens (NACC)] and intraventral tegmental [ventral tegmental area (VTA)] infusions was observed. Because the VTA and NACC contain very high densities of α1 and α2 subunits, respectively, we determined whether RY exhibited a “negative” or “neutral” pharmacological profile at recombinant α1β3γ2, α2β3γ2, and α5β3γ2 receptors expressed in Xenopus oocytes. RY produced “classic” inverse agonism at all α receptor subtypes; thus, a neutral efficacy was not sufficient to explain the failure of RY to alter EtOH responding in the NACC or VTA. The results provide the first demonstration that the α5-containing GABAA receptors in the hippocampus play an important role in regulating EtOH-seeking behaviors.

GABA(A) and opioid receptors of the central nucleus of the amygdala selectively regulate ethanol-maintained behaviors.

The present study tested the hypothesis that GABAA and opioid receptors within the central nucleus of the amygdala (CeA) regulate ethanol (EtOH), but not sucrose-maintained responding. To accomplish this, βCCt, a mixed benzodiazepine (BDZ) agonist–antagonist with binding selectivity at the α1 subunit-containing GABAA receptor, and the nonselective opioid antagonist, naltrexone, were bilaterally infused directly into the CeA of alcohol-preferring rats. The results demonstrated that in HAD-1 and P rat lines, βCCt (5–60 μg) reduced EtOH-maintained responding by 56–89% of control levels. On day 2, βCCt (10–40 μg) continued to suppress EtOH maintained responding in HAD-1 rats by as much as 60–85% of control levels. Similarly, naltrexone (0.5–30 μg) reduced EtOH-maintained responding by 56–75% of control levels in P rats. βCCt and naltrexone exhibited neuroanatomical and reinforcer specificity within the CeA. Specifically, no effects on EtOH-maintained responding were observed following infusion into the caudate putamen (CPu), a locus several millimeters dorsal to the CeA. Additionally, responding maintained by sucrose, when presented concurrently with ethanol (EtOH) or presented alone, was not altered by βCCt. Naltrexone reduced sucrose-maintained responding only under the 5 μg dose condition when sucrose was presented alone, however, it did not alter sucrose responding when given concurrently with EtOH. These results support the hypothesis that GABAA and opioid receptors within the CeA can selectively regulate EtOH-maintained responding. The CeA may represent a novel target site in the development of prototypical GABAA and opioidergic receptor ligands, which selectively reduce alcohol abuse in humans.

CRF-Amplified Neuronal TLR4/MCP-1 Signaling Regulates Alcohol Self-Administration

Alcohol dependence is a complex disorder that initiates with episodes of excessive alcohol drinking known as binge drinking. It has a 50–60% risk contribution from inherited susceptibility genes; however, their exact identity and function are still poorly understood. We report that alcohol-preferring P rats have innately elevated levels of Toll-like receptor 4 (TLR4) and monocyte chemotactic protein-1 (MCP-1) that colocalize in neurons from the central nucleus of the amygdala (CeA) and ventral tegmental area (VTA). To examine the potential role of a TLR4/MCP-1 signal, we used Herpes Simplex Virus (HSV) vectors (amplicons) that retain in vivo neurotropism. Infusion of amplicons for TLR4 or MCP-1 siRNA into the CeA or VTA from the P rats inhibited target gene expression and blunted binge drinking. A similarly delivered amplicon for scrambled siRNA did not inhibit TLR4 or MCP-1 expression nor reduce binge drinking, identifying a neuronal TLR4/MCP-1 signal that regulates the initiation of voluntary alcohol self-administration. The signal was sustained during alcohol drinking by increased expression of corticotropin-releasing factor and its feedback regulation of TLR4 expression, likely contributing to the transition to alcohol dependence.

Central Opioid Receptors Differentially Regulate the Nalmefene-Induced Suppression of Ethanol- and Saccharin-Reinforced Behaviors in Alcohol-Preferring (P) Rats

The exact opioid-sensitive receptors participating in EtOH-seeking behaviors remains unclear. Previous studies have reported higher densities of μ-opioid receptor binding in the nucleus accumbens (NACC) of P relative to NP rats; however, no differences were seen in δ-receptor binding. In contrast to the NACC, substantially lower levels of μ-receptor binding have been observed in the ventral tegmental area (VTA) of both P and NP rats, albeit no line differences have been observed. In the present study, opioid receptors in the NACC, VTA, and hippocampus were evaluated for their capacity to regulate both EtOH- and saccharin-motivated behaviors in the genetically selected alcohol-preferring (P) rat. To accomplish this, nalmefene, an opiate antagonist with preferential binding affinity for the μ-opioid receptor was unilaterally or bilaterally infused during concurrent availability of 1 h daily EtOH (10% v/v) and saccharin (0.025 or 0.050% w/v) solutions. Rats performed under a two-lever fixed ratio (FR) schedule in which four responses on one lever produced the EtOH solution, and four on a second lever produced the saccharin solution. The results demonstrated that when responding maintained by both EtOH and saccharin are matched at basal levels, unilateral (1–60 μg) or bilateral (0.5–10 μg) microinjections of nalmefene into the NACC produced selective dose-dependent reductions on responding maintained by EtOH. Unilateral (40, 60 μg) and bilateral (10 μg) VTA infusions were also observed to selectively reduced EtOH responding; however, greater nalmefene doses were required and the magnitude of suppression on EtOH responding was markedly less compared with the NACC. The greater sensitivity of nalmefene to suppress EtOH responding in the NACC is likely due to the greater number of opioid receptors in the NACC relative to the VTA. Only bilateral infusion of the 40 μg dose in the NACC and VTA suppressed responding maintained by both EtOH and saccharin. In contrast, intrahippocampal infusions dose dependently suppressed EtOH- and saccharin-maintained responding over a range of doses (1–20 μg). The present study provides evidence that nalmefene suppresses EtOH-motivated behaviors via blockade of opioid receptors within the NACC and VTA, and under various dose conditions both reinforcer and neuroanatomical specificity can be observed.

Acute physical dependence: Time course and relation to human plasma morphine concentrations

To characterize the postmorphine time course of precipitated withdrawal responses in comparison with the time course of opioid agonist effects and of plasma morphine concentrations.

The novel benzodiazepine inverse agonist RO19-4603 antagonizes ethanol motivated behaviors: neuropharmacological studies

The novel imidazothienodiazepine inverse agonist RO19-4603 has been reported to attenuate EtOH intake in home cage drinking tests for at least 24 h post-drug administration after systemic administration. In the present study, selectively bred alcohol-preferring (P) rats were trained under a concurrent (FR4-FR4) operant schedule to press one lever for EtOH (10% v/v) and another lever for saccharin (0.05% or 0.75% g/v), then dose-response and timecourse effects of RO19-4603 were evaluated. Systemic RO19-4603 injections (0.0045-0.3 mg/kg; i.p.) profoundly reduced EtOH responding by as much as 97% of vehicle control on day 1. No effects were seen on saccharin responding except with the highest dose level (0.3 mg/kg). In a second experiment, microinjections of RO19-4603 (2-100 ng) directly into the nucleus accumbens (NA) suppressed EtOH responding on day 1 by as much as 53% of control: Control injections dorsal to the NA or ventral tegmental area did not significantly alter EtOH or saccharin responding. On day 2, rats in both experiments received no RO19-4603 treatments; however, all 7 of the i.p. doses, and all 3 of the intra-NA infusions continued to significantly suppress EtOH responding by 43-85% of vehicle control levels. In addition, i.p. injections of RO19-4603 produced a dose-dependent decrease in the slope of the cumulative record for EtOH responding, while concomitantly producing a dose-dependent increase in the slope for saccharin responding. RO19-4603s actions appear to be mediated via recognition sites at GABAA-BDZ receptors which regulate EtOH reinforcement, and not via mechanisms regulating ingestive behaviors. Based on recent in situ hybridization studies in our laboratory, we hypothesize that occupation of alpha4 containing GABAA diazepam insensitive (DI) receptors in the NA, may mediate in part, the RO19-4603 suppression of EtOH responding in EtOH-seeking P rats.

Blockade of GABAA receptors within the extended amygdala attenuates D2 regulation of alcohol-motivated behaviors in the ventral tegmental area of alcohol-preferring (P) rats

The dopamine (DA) mesolimbic pathway, which originates from DA cell bodies within the ventral tegmental area (VTA), has been shown by various studies to play a role in the mediation of various drugs of abuse including alcohol (EtOH). It has been suggested that the VTAs control of EtOH reward is mediated in part by the D2 receptors within the VTA. These receptors may be under the regulation of reciprocal GABAergic inputs from forebrain components of the mesolimbic path such as the nucleus accumbens (NAcc), a classic EtOH reward substrate, and the bed nucleus of the stria terminalis, a substrate recently implicated in EtOH reinforcement, forming a self-regulating feedback loop. To test this hypothesis, D2 regulation of EtOH self-administration (SA) was evaluated by the microinfusion of the D2 antagonist eticlopride into the VTA of P rats, which produced profound reductions in EtOH SA in the highest (20.0 and 40.0microg) doses tested in both BST/VTA and NAcc/VTA implanted P rats. To determine the role of GABA in the mediation of EtOH SA, a 32.0ng dose the non-selective GABA antagonist SR 95531 was microinfused into the BST producing no effect on responding for EtOH and into the NAcc which lead to a reduction in EtOH responding. Finally, the hypothesis that GABA innervation of the VTA from the mesolimbic forebrain may influence EtOH SA was examined by the simultaneous infusion of eticlopride (40.0microg) into the VTA and SR 95531 (32.0ng) into either the BST or NAcc. This combination infusion completely attenuated the reduction in EtOH SA observed with the 40.0microg dose of eticlopride alone in both groups of animals. These results suggest that while the D2 receptors within the VTA regulate EtOH-motivated behaviors, this is modulated by GABAergic input from the mesolimbic forebrain, specifically from the BST and NAcc.