Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKCepsilon.

Several of the actions of ethanol are mediated by gamma-aminobutyrate type A (GABA(A)) receptors. Here we demonstrated that mutant mice lacking protein kinase C epsilon (PKCepsilon) were more sensitive than wild-type littermates to the acute behavioral effects of ethanol and other drugs that allosterically activate GABA(A) receptors. GABA(A) receptors in membranes isolated from the frontal cortex of PKCepsilon 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 PKCepsilon increases sensitivity of GABA(A) receptors to ethanol and allosteric modulators. Pharmacological agents that inhibit PKCepsilon may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABA(A) receptor function to treat other disorders such as anxiety and epilepsy.

Reduced ethanol withdrawal severity and altered withdrawal-induced c-fos expression in various brain regions of mice lacking protein kinase C-epsilon.

Withdrawal from chronic ethanol consumption can be accompanied by motor seizures, which may be a result of altered GABA(A) receptor function. Recently, we have generated and characterized mice lacking the epsilon isoform of protein kinase C as being supersensitive to the behavioral and biochemical effects of positive GABA(A) receptor allosteric modulators, including ethanol. The aim of the present study was to determine whether protein kinase C-epsilon null mutant mice display altered seizure severity during alcohol withdrawal. In addition, we used c-fos immunohistochemistry immediately following seizure assessment to identify potential brain regions involved in any observed differences in withdrawal severity. Mice were allowed to consume an ethanol-containing or control liquid diet as the sole source of food for 14 days. During the 7-h period following removal of the diet, both ethanol-fed wild-type and protein kinase C-epsilon null mutant mice displayed an overall increase in Handling-Induced Convulsion score versus control-fed mice. However, at 6 and 7h following diet removal, the Handling-Induced Convulsion score was reduced in ethanol-fed protein kinase C-epsilon null mutant mice compared to ethanol-fed wild-type mice. Ethanol-fed protein kinase C-epsilon null mutant mice also exhibited a decrease in the number of Fos-positive cells in the lateral septum, and an increase in the number of Fos-positive cells in the dentate gyrus, mediodorsal thalamus, paraventricular nuclei of the thalamus and hypothalamus, and substantia nigra compared to ethanol-fed wild-type mice. These data demonstrate that deletion of protein kinase C-epsilon results in diminished progression of ethanol withdrawal-associated seizure severity, suggesting that selective pharmacological inhibitors of protein kinase C-epsilon may be useful in the treatment of seizures during alcohol withdrawal. These data also provide insight into potential brain regions involved in generation or suppression of ethanol withdrawal seizures.

The discriminative stimulus properties of self-administered ethanol are mediated by GABA(A) and NMDA receptors in rats.

RATIONALE: The neurobiological systems that mediate the discriminative stimulus effects of self-administered drugs are largely unknown. The present study examined the discriminative stimulus effects of self-administered ethanol. METHODS: Rats were trained to discriminate ethanol (1 g/kg, IP) from saline on a two-lever drug discrimination task with sucrose (10% w/v) reinforcement. Test sessions were conducted with ethanol (0 or 10% v/v) added to the sucrose reinforcement to determine if self-administered ethanol would interact with the discriminative stimulus effects of investigator-administered ethanol, or with the ethanol-like discriminative stimulus effects of the GABAA-positive modulator pentobarbital or the non-competitive NMDA antagonist MK-801. RESULTS: During a saline test session, ethanol (10% v/v) was added to the sucrose reinforcement. Responding by all animals began accurately on the saline-appropriate lever and then switched to the ethanol-appropriate lever after rats self-administered a mean dose of 1.2 +/- 0.14 g/kg ethanol. During cumulative self-administration trials, responding initially occurred on the saline lever and then switched to the ethanol-appropriate lever after ethanol (0.68 +/- 0.13 g/kg) was self-administered. Investigator-administered MK-801 (0.01-1.0 mg/kg, cumulative IP) and pentobarbital (0.3-10.0 mg/kg, cumulative IP) dose-dependently substituted for ethanol. When ethanol (10% v/v) was added to the sucrose reinforcer, MK-801 and pentobarbital dose-response curves were shifted significantly to the left. CONCLUSIONS: Self-administered ethanol substituted for and potentiated the stimulus effects of investigator-administered ethanol, suggesting that the discriminative stimulus effects of self-administered ethanol are similar to those produced by investigator-administered ethanol. Self-administered ethanol enhanced the ethanol-like discriminative stimulus effects of MK-801 and pentobarbital, which suggests that the discriminative stimulus effects of self-administered ethanol are mediated by NMDA and GABAA receptors.

A method for increasing the viability of the external portion of lumbar catheters placed in the spinal subarachnoid space of rats.

A method for direct catheterization of the lumbar subarachnoid space has recently been developed by Storkson et al. (1996) (J Neurosci Methods 1996;65:167-172) that may potentially improve upon the widely used method of Yaksh and Rudy (1976) (Physiol Behav 1976;17:1031-1036). This 'catheter-through-a-needle' technique inserts the catheter between lumbar vertebrae 5 (L5) and 6 (L6), which has been shown to reduce neurological impairment and post-surgical deaths. However, employing this technique allows the external portion of the chronic indwelling catheters to be easily damaged, resulting in approximately 50% attrition within 4 days after surgery. Therefore, we developed an easy and inexpensive method for protecting the external portion of the catheter that enhances catheter viability beyond 14 days after catheter implantation while also maintaining a low injection volume (8 microl). Moreover, this modification does not significantly alter the implantation methods developed by Storkson et al. (1996) (J Neurosci Methods 1996;65:167-172) and allows for more optimal catheter materials to be incorporated. Chronically implanted catheters (n = 70) with the external portion of the catheter protected, resulted in 4% attrition 7 days after surgery and 11% attrition 14 days after surgery. Approximately 5.5% of animals implanted showed very mild and transient neurological impairment.

Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120.

Astrocytes and microglia in the spinal cord have recently been reported to contribute to the development of peripheral inflammation-induced exaggerated pain states. Both lowering of thermal pain threshold (thermal hyperalgesia) and lowering of response threshold to light tactile stimuli (mechanical allodynia) have been reported. The notion that spinal cord glia are potential mediators of such effects is based on the disruption of these exaggerated pain states by drugs thought to preferentially affect glial function. Activation of astrocytes and microglia can release many of the same substances that are known to mediate thermal hyperalgesia and mechanical allodynia. The aim of the present series of studies was to determine whether exaggerated pain states could also be created in rats by direct, intraspinal immune activation of astrocytes and microglia. The immune stimulus used was peri-spinal (intrathecal, i.t.) application of the Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein, gp120. This portion of HIV-1 is known to bind to and activate microglia and astrocytes. Robust thermal hyperalgesia (tail-flick, TF, and Hargreaves tests) and mechanical allodynia (von Frey and touch-evoked agitation tests) were observed in response to i.t. gp120. Heat denaturing of the complex protein structure of gp120 blocked gp120-induced thermal hyperalgesia. Lastly, both thermal hyperalgesia and mechanical allodynia to i.t. gp120 were blocked by spinal pretreatment with drugs (fluorocitrate and CNI-1493) thought to preferentially disrupt glial function.

Modulation of extracellular neurotransmitter levels in the nucleus accumbens by a taurine uptake inhibitor.

Using in vivo microdialysis, we examined the effect of local perfusion of the taurine uptake inhibitor guanidinoethyl sulfonate on extracellular levels of various neurotransmitters in the rat nucleus accumbens. Guanidinoethyl sulfonate (500 microM-50 mM) produced a concentration-dependent increase in extracellular taurine levels. While 500 microM and 5 mM concentrations of guanidinoethyl sulfonate were largely without effect, 50 mM guanidinoethyl sulfonate produced a significant decrease in extracellular levels of aspartate, glutamate and glycine, with no effect on extracellular dopamine levels. These results indicate that guanidinoethyl sulfonate can modulate extracellular amino acid levels in the nucleus accumbens.

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.

Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol in PKCepsilon-deficient mice.

There is increasing evidence that individual protein kinase C (PKC) isozymes mediate specific effects of ethanol on the nervous system. In addition, multiple lines of evidence suggest that the mesoaccumbens dopamine reward system is critically involved in the rewarding and reinforcing effects of ethanol. Yet little is known about the role of individual PKC isozymes in ethanol reinforcement processes or in regulation of mesolimbic systems. In this study, we report that mice lacking the epsilon isoform of PKC (PKCepsilon) show reduced operant ethanol self-administration and an absence of ethanol-induced increase in extracellular dopamine levels in the nucleus accumbens. PKCepsilon null mice exhibited a 53% decrease in alcohol-reinforced operant responses under basal conditions, as well as following ethanol deprivation. Behavioural analysis revealed that while both genotypes had the same number of drinking bouts following deprivation, PKCepsilon null mice demonstrated a 61% reduction in number of ethanol reinforcers per bout and a 57% reduction in ethanol-reinforced response rate. In vivo microdialysis experiments showed that, in contrast to wild-type mice, PKCepsilon null mice exhibited no change in extracellular levels of dopamine in the nucleus accumbens following acute administration of ethanol (1 and 2 g/kg i.p.), while mesolimbic dopamine responses to cocaine (20 mg/kg i.p.) or high potassium (100 mM) in these mice were comparable with that of wild-types. These data provide further evidence that increases in extracellular mesolimbic dopamine levels contribute to the reinforcing effects of ethanol, and indicate that pharmacological agents inhibiting PKCepsilon may be useful in the treatment of alcohol dependence.

Allopregnanolone and pentobarbital infused into the nucleus accumbens substitute for the discriminative stimulus effects of ethanol.

BACKGROUND: The discriminative stimulus effects of ethanol are mediated in part by the gamma-aminobutyric acid type A (GABA(A)) receptor system. We have previously shown that microinjections of the competitive GABA(A) agonist muscimol in the nucleus accumbens and amygdala fully substitute for the discriminative stimulus effects of systemic ethanol. However, it is not known whether allosteric binding sites on GABA(A) receptors located within specific limbic brain regions contribute to the discriminative stimulus effects of ethanol. METHODS: Male Long-Evans rats were trained to discriminate between intraperitoneal injections of ethanol (1 g/kg) and saline under a fixed-ratio 10 schedule of sucrose (10% w/v) reinforcement. Injector guide cannulae, aimed at both the nucleus accumbens core and the hippocampus area CA1, were then implanted to allow site-specific infusion of GABA(A)-positive modulators. RESULTS: Infusion of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone, or 3alpha-5alpha-P) in the nucleus accumbens resulted in dose-dependent full substitution for intraperitoneal ethanol (50% effective dose = 0.38 ng/microl per side). Likewise, injection of the barbiturate pentobarbital into the nucleus accumbens also substituted dose-dependently for ethanol (50% effective dose = 1.55 microg/microl per side). However, infusions of either 3alpha-5alpha-P or pentobarbital in the hippocampus failed to substitute for ethanol and produced inverted U-shaped dose-response curves. CONCLUSIONS: These results demonstrate that allosteric positive modulation of GABA(A) receptors in the nucleus accumbens produces full substitution for the stimulus effects of ethanol. This suggests that GABA(A) receptors in the nucleus accumbens may play a more influential role in the discriminative stimulus effects of ethanol than those in the hippocampus.

Alcohol actions on GABA(A) receptors: from protein structure to mouse behavior.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were R. Adron Harris and Susumu Ueno. The presentations were (1) Protein kinase Cepsilon-regulated sensitivity of gamma-aminobutyric acid type A (GABAA) receptors to allosteric agonists, by Robert O. Messing, A. M. Sanchez-Perez, C. W. Hodge, T. McMahon, D. Wang, K. K. Mehmert, S. P. Kelley, A. Haywood, and M. F. Olive; (2) Genetic and functional analysis of a GABAA receptor gamma2 subunit variant: A candidate for quantitative trait loci involved in alcohol sensitivity and withdrawal, by Kari J. Buck and Heather M. Hood; (3) Tryptophan-scanning mutagenesis in GABAA receptor subunits: Channel gating and alcohol actions, by Susumu Ueno; and (4) Can a single binding site account for actions of alcohols on GABAA and glycine receptors? by R. Adron Harris, Yuri Blednov, Geoffrey Findlay, and Maria Paola Mascia.