M. Foster Olive

Supersensitivity to allosteric GABA A receptor modulators and alcohol in mice lacking PKCε

Several of the actions of ethanol are mediated by γ-aminobutyrate type A (GABAA) receptors. Here we demonstrated that mutant mice lacking protein kinase C epsilon (PKCε) were more sensitive than wild-type littermates to the acute behavioral effects of ethanol and other drugs that allosterically activate GABAA receptors. GABAA receptors in membranes isolated from the frontal cortex of PKCε null mice were also supersensitive to allosteric activation by ethanol and flunitrazepam. In addition, these mutant mice showed markedly reduced ethanol self-administration. These findings indicate that inhibition of PKCε increases sensitivity of GABAA receptors to ethanol and allosteric modulators. Pharmacological agents that inhibit PKCε may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABAA receptor function to treat other disorders such as anxiety and epilepsy.

Elevated extracellular CRF levels in the bed nucleus of the stria terminalis during ethanol withdrawal and reduction by subsequent ethanol intake

Corticotropin-releasing factor (CRF) is widely distributed throughout the brain and has been shown to mediate numerous endocrine and behavioral responses to stressors. During acute ethanol withdrawal, CRF release is increased in the central nucleus of the amygdala (CeA), and there is evidence to suggest that this activation of amygdala CRF systems may mediate the anxiogenic properties of the ethanol withdrawal syndrome. The present study was conducted to determine if another CRF-containing limbic structure, the bed nucleus of the stria terminalis (BNST), we would exhibit similar increases in CRF neurotransmission during ethanol withdrawal. Rats were administered an ethanol-containing (6.7% v/v) or control liquid diet for 2 weeks and subsequently implanted with microdialysis probes into the lateral BNST. A 50-75% increase in dialysate CRF levels was observed following removal of the ethanol-containing diet, while no changes were observed in control animals. When ethanol-withdrawn animals were given subsequent access to the ethanol-containing diet, dialysate CRF levels returned to basal levels. However, when ethanol-withdrawn animals were given subsequent access to the control diet, dialysate CRF levels increased further to 101% above basal levels. These data demonstrate that extracellular CRF levels are increased in the BNST during ethanol withdrawal, and that these increases are reduced by subsequent ethanol intake.

Decreased anxiety-like behavior, reduced stress hormones, and neurosteroid supersensitivity in mice lacking protein kinase Cε

Mice lacking protein kinase Cepsilon (PKCepsilon) are supersensitive to positive allosteric modulators of gamma aminobutyrate type A (GABA(A)) receptors. Since many of these compounds are anxiolytic, we examined whether anxiety-like behavior is altered in these mice. PKCepsilon-null mice showed reduced anxiety-like behavior and reduced levels of the stress hormones corticosterone and adrenocorticotrophic hormone (ACTH). This was associated with increased sensitivity to neurosteroid modulators of GABA(A) receptors. Treatment of PKCepsilon-null mice with the GABA(A) receptor antagonist bicuculline restored corticosterone levels and anxiety-like behavior to wild-type levels. These results suggest that increased GABA(A) receptor sensitivity to neurosteroids contributes to reduced anxiety-like behavior and stress hormone responses in PKCepsilon-null mice. The findings also suggest PKCepsilon as a possible therapeutic target for development of anxiolytics.

The anti-relapse compound acamprosate inhibits the development of a conditioned place preference to ethanol and cocaine but not morphine

The effects of the anti‐relapse compound acamprosate (calcium acetylhomotaurinate) on the conditioned rewarding effects of ethanol, cocaine and morphine were studied using the conditioned place preference (CPP) paradigm. During 3 days of drug conditioning, mice were pretreated with saline or acamprosate (30, 100 or 300 mg kg−1 i.p.) 10 min prior to the administration of ethanol (2 g kg−1 i.p.), cocaine (15 mg kg−1 i.p.) or morphine (10 mg kg−1 i.p.), and subsequently confined to one of two distinct conditioning chambers. On the following day, mice were tested for the expression of CPP. Acamprosate dose‐dependently reduced the development of CPP to ethanol and cocaine but not morphine. When tested as the conditioning drug, acamprosate alone produced neither a conditioned place preference nor aversion. These data suggest that acamprosate can suppress the conditioned rewarding effects of ethanol and certain classes of abused substances.

Effects of acute acamprosate and homotaurine on ethanol intake and ethanol-stimulated mesolimbic dopamine release

The purpose of the present study was to determine the acute effects of the anticraving compound acamprosate (calcium acetylhomotaurinate) and the closely related compound homotaurine on ethanol intake and ethanol-stimulated dopamine release in the nucleus accumbens. Male rats were treated with acamprosate (200 or 400 mg/kg intraperitoneally, i.p.) or homotaurine (10, 50, or 100 mg/kg i.p.) 15 min prior to access to 10% ethanol and water for 1 h in a two-bottle choice restricted access paradigm. A separate group of rats was implanted with microdialysis probes in the nucleus accumbens and given an acute injection of ethanol (1.5 g/kg i.p.) that was preceded by saline, acamprosate, or homotaurine. Acamprosate and homotaurine dose-dependently reduced ethanol intake and preference. These compounds also delayed or suppressed ethanol-stimulated increases in nucleus accumbens dopamine release, suggesting that acamprosate and homotaurine may reduce ethanol intake by interfering with the ability of ethanol to activate the mesolimbic dopamine reward system.

Effect of the mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) on the acute locomotor stimulant properties of cocaine, D-amphetamine, and the dopamine reuptake inhibitor GBR12909 in mice

RationaleRecent evidence suggests that, in addition to ascending monoaminergic systems, glutamate systems also play a role in psychostimulant-induced locomotor activity. The present study was conducted to examine the effects of the selective type-5 metabotropic glutamate receptor (mGluR5) antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP) on the acute locomotor stimulant effects of cocaine, d-amphetamine, and the dopamine reuptake inhibitor GBR12909.MethodsMale DBA/2J mice were treated with saline or MPEP (1, 5, 20 or 30xa0mg/kg i.p.) 10xa0min prior to the administration of cocaine (15xa0mg/kg or 30xa0mg/kg i.p.), d-amphetamine (3xa0mg/kg or 5xa0mg/kg i.p.) or GBR12909 (10xa0mg/kg or 20xa0mg/kg i.p.). Locomotor activity was then monitored in an open-field environment for 30xa0min. The effects of MPEP alone (1, 5, 20 and 30xa0mg/kg i.p.) on locomotor activity were also examined.ResultsMPEP dose dependently inhibited the acute locomotor stimulant effects of cocaine, d-amphetamine, and the 10-mg/kg dose of GBR12909. However, MPEP had no effect on the locomotor stimulant effects of the higher (20xa0mg/kg) dose of GBR12909. When tested alone, MPEP increased locomotor activity at doses of 5xa0mg/kg and 20xa0mg/kg.ConclusionsOur data suggest that mGluR5 receptors not only mediate spontaneous locomotor activity in DBA/2J mice but also the acute locomotor stimulant effects of cocaine, d-amphetamine and lower doses of GBR12909. However, the fact that MPEP did not attenuate the locomotor stimulant effects of the high (20xa0mg/kg) dose of GBR12909 suggests complex interactions between metabotropic glutamate receptors, dopamine transporters and possibly other monoamines in the regulation of psychostimulant-induced locomotor activity.

The glucocorticoid receptor antagonist mifepristone reduces ethanol intake in rats under limited access conditions

There is a substantial amount of evidence indicating control over ethanol intake by steroid hormones, particularly adrenal glucocorticoids. Thus far, however, studies employing pharmacological methods have failed to find effects of glucocorticoid receptor blockade on voluntary ethanol consumption. Since length of ethanol access period can influence ethanol consumption levels as well as potential pharmacological effects in such studies, the present study was conducted to determine the effects of acute administration of the glucocorticoid receptor (GR) antagonist mifepristone on voluntary ethanol intake under limited access conditions. Rats were fluid restricted and given concurrent access to 10% ethanol and water in a two-bottle choice paradigm for 1 h/day, 5 days a week. Both fluids were available ad libitum during the remaining 2 days per week. Administration of mifepristone (1, 5 and 20 mg/kg i.p.) immediately prior to the limited access two-bottle access period dose-dependently suppressed ethanol intake (maximum 40% at 20 mg/kg). The mineralcorticoid receptor (MR) antagonist spironolactone (10, 25 and 50 mg/kg i.p.) was without effect on ethanol intake, and neither compound had an effect on water intake. These data confirm an active role of GRs in modulating voluntary ethanol consumption, particularly under conditions of limited access.

Protein kinase C isozymes and addiction

Protein kinase C (PKC) has long been recognized an important family of enzymes that regulate numerous aspects of neuronal signal transduction, neurotransmitter synthesis, release and reuptake, receptor and ion channel function, neuronal excitability, development, and gene expression. Much evidence has implicated PKCs in the effects of several drugs of abuse, and in behavioral responses to these drugs. The present review summarizes the effects of both acute and chronic exposure to various drugs of abuse on individual PKC isozymes in the brain. In addition, we summarize recent studies utilizing mice with targeted deletions of the genes for PKCγ and PKCɛ. These studies suggest that individual PKC isozymes play a role in the development of drug dependence and addiction.

Ethanol consumption patterns and conditioned place preference in mice lacking preproenkephalin

There is a great deal of evidence suggesting that endogenous opioid systems are involved in the control of ethanol-seeking behavior and reward. To ascertain the role of the enkephalinergic opioid peptide system in these processes, we examined voluntary ethanol consumption patterns in mice lacking the preproenkephalin (Penk) gene using a two-bottle choice paradigm with free access to water and increasing concentrations of ethanol (2, 4, 8, and 10% v/v). We also examined the ability of ethanol (2 g/kg i.p.) to establish a conditioned place preference in these mice. No differences in ethanol consumption or preference were observed between wildtypes and Penk null mutant mice. In addition, both genotypes displayed a similar conditioned place preference to ethanol. These data suggest that the preproenkephalin system is not involved in voluntary ethanol consumption patterns or ethanol reward.

Microdialysis in the mouse nucleus accumbens: a method for detection of monoamine and amino acid neurotransmitters with simultaneous assessment of locomotor activity

Microdialysis has been extensively used to characterize the effects of drugs of abuse on extracellular levels of various neurotransmitters in nucleus accumbens (NAc) of the rat brain. However, recent advances in mouse genetics have prompted the need for studying the in vivo neurochemical correlates of drug intake in genetically engineered mice. While an earlier study has shown the feasibility of measuring monoamines in the NAc of behaving transgenic mice [I. Sillaber, A. Montkowski, R. Landgraf, N. Barden, F. Holsboer, R. Spanagel, Enhanced morphine-induced behavioural effects and dopamine release in the nucleus accumbens in a transgenic mouse model of impaired glucocorticoid (type II) receptor function: influence of long-term treatment with the antidepressant moclobemide, Neuroscience, 85 (1998) 415-425 [16] ], in this protocol we demonstrate a method for measuring both monoamine and amino neurotransmitters from the NAc of freely moving mice combined with open field locomotor activity monitoring. Mice were implanted with guide cannulae aimed at the NAc and allowed 4 days of recovery before being implanted with microdialysis probes equipped with 1-mm cuprophane membranes. On the following day, mice were placed in plexiglass chambers equipped with infrared photobeams, where microdialysis samples and locomotor activity data were collected in 10-min intervals. Immediately after collection, microdialysis samples were split into two equal aliquots for separate analysis of monoamine and amino acid neurotransmitter content. High performance liquid chromatography (HPLC) analysis revealed that norepinephrine, dopamine, serotonin, aspartate, glutamate, glycine, taurine, and gamma-aminobutyric acid (GABA) could be detected in each microdialysis sample. Thus, we have shown it is feasible to monitor extracellular levels of multiple neurotransmitters with simultaneous measurement of locomotor behavior in the mouse, making this model suitable for studying differential neurochemical and behavioral responses to drugs of abuse in genetically engineered mice.