Esa R. Korpi

Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents

The γ-aminobutyric acid (GABA) type A receptor system, the main fast-acting inhibitory neurotransmitter system in the brain, is the pharmacological target for many drugs used clinically to treat, for example, anxiety disorders and epilepsy, and to induce and maintain sedation, sleep, and anesthesia. These drugs facilitate the function of pentameric GABAA receptors that exhibit widespread expression in all brain regions and large structural and pharmacological heterogeneity as a result of composition from a repertoire of 19 subunit variants. One of the main problems in clinical use of GABAA receptor agonists is the development of tolerance. Most drugs, in long-term use and during withdrawal, have been associated with important modulations of the receptor subunit expression in brain-region-specific manner, participating in the mechanisms of tolerance and dependence. In most cases, the molecular mechanisms of regulation of subunit expression are poorly known, partly as a result of neurobiological adaptation to altered neuronal function. More knowledge has been obtained on the mechanisms of GABAA receptor trafficking and cell surface expression and the processes that may contribute to tolerance, although their possible pharmacological regulation is not known. Drug development for neuropsychiatric disorders, including epilepsy, alcoholism, schizophrenia, and anxiety, has been ongoing for several years. One key step to extend drug development related to GABAA receptors is likely to require deeper understanding of the adaptational mechanisms of neurons, receptors themselves with interacting proteins, and finally receptor subunits during drug action and in neuropsychiatric disease processes.

Modifying the Subunit Composition of TASK Channels Alters the Modulation of a Leak Conductance in Cerebellar Granule Neurons

Two-pore domain potassium (K2P) channel expression is believed to underlie the developmental emergence of a potassium leak conductance [IK(SO)] in cerebellar granule neurons (CGNs), suggesting that K2P function is an important determinant of the input conductance and resting membrane potential. To investigate the role that different K2P channels may play in the regulation of CGN excitability, we generated a mouse lacking TASK-1, a K2P channel known to have high expression levels in CGNs. In situ hybridization and real-time PCR studies in wild-type and TASK-1 knock-outs (KOs) demonstrated that the expression of other K2P channels was unaltered in CGNs. TASK-1 knock-out mice were healthy and bred normally but exhibited compromised motor performance consistent with altered cerebellar function. Whole-cell recordings from adult cerebellar slice preparations revealed that the resting excitability of mature CGNs was no different in TASK-1 KO and littermate controls. However, the modulation of IK(SO) by extracellular Zn2+, ruthenium red, and H+ was altered. The IK(SO) recorded from TASK-1 knock-out CGNs was no longer sensitive to alkalization and was blocked by Zn2+ and ruthenium red. These results suggest that a TASK-1-containing channel population has been replaced by a homodimeric TASK-3 population in the TASK-1 knock-out. These data directly demonstrate that TASK-1 channels contribute to the properties of IK(SO) in adult CGNs. However, TASK channel subunit composition does not alter the resting excitability of CGNs but does influence sensitivity to endogenous modulators such as Zn2+ and H+.

Ethanol inhibits glutamate−induced currents in heteromeric NMDA receptor subtypes

Maximal L-glutamate/glycine-evoked currents were inhibited by ethanol in Xenopus laevis oocytes expressing recombinant heteromeric NMDA receptors consisting of NR1-NR2A, NR1-NR2B, and NR1-NR2C subunit combinations. Concentration-dependent inhibition was observed at ethanol concentrations of > or = 50 mM both in Ca(2+)-containing and Ca(2+)-deficient, Ba(2+)-containing Mg(2+)-free media. The NR1-NR2C channels were slightly less sensitive to ethanol inhibition than the other heteromeric channels in Ca(2+)-deficient, Ba(2+)-containing medium. The inhibition was unaffected by the clamping-voltage and by a mutation [NR1-NR2A(N595Q)] that prevents the Mg(2+)-blockade of the channels, indicating that the mechanism of action of ethanol differs from that of Mg2+. The results are consistent with the hypothesis that the NMDA receptor subtypes can mediate many behavioural actions of ethanol.

From synapse to behavior: Rapid modulation of defined neuronal types with engineered GABAA receptors

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Reduced aggression in AMPA-type glutamate receptor GluR-A subunit-deficient mice

The importance of AMPA‐type glutamate receptors has been demonstrated in neuronal plasticity and in adaptation to drugs of abuse. We studied the involvement of AMPA receptors in social interaction and anxiety and found that in several paradigms of agonistic behavior naïve male mice deficient for the GluR‐A subunit‐ containing AMPA receptors are less aggressive than wild‐type littermates. GluR‐A deficient mice and wild‐type littermates exhibited similar basic behavior and reflexes as monitored by observational Irwins test, but they tended to be less anxious in elevated plus‐maze and light‐dark tests. Maternal aggression or male‐female encounters were not affected which suggests that male hormones are involved in the expression of suppressed aggressiveness. However, testosterone levels and brain monoamines can be excluded and found to be similar between GluR‐A deficient and wild‐type littermates. The reduced AMPA receptor levels caused by the lack of the GluR‐A subunit, and measured by a 30% reduction in hippocampal [3H]‐S‐AMPA binding, seem to be the reason for suppressed male aggressiveness. When we analyzed mice with reduced number of functional AMPA receptors mediated by the genomic introduced GluR‐A(Q582R) channel mutation, we observed again male‐specific suppressed aggression, providing additional evidence for GluR‐A subunit‐containing AMPA receptor involvement in aggression.

Biological function of GABAA/benzodiazepine receptor heterogeneity

gamma-Aminobutyric acid (GABA) is the most prominent of the inhibiting neurotransmitters in the brain. It exerts its main action through GABAA receptors. The receptors respond to the presence of GABA by the opening of an intrinsic anion channel. Hence, they belong to the molecular superfamily of ligand-gated ion channels. There exist in the brain multiple GABAA receptors that show differential distribution and developmental patterns. The receptors presumably form by the assembly of five proteins from at least three different subunits (alpha 1-6, beta 1-3 and gamma 1-3). The regulation of functional properties by benzodiazepine (BZ) receptor ligands, neurosteroids, GABA and its analogs differs dramatically with the alpha variant present in the complex. Additional variation of the GABAA receptors comes with the exchange of the gamma subunits. No clear picture exists for the role of the beta subunits, though they may play an important part in the sensitivity of the channel-receptor complex. The effects of BZ receptor ligands on animal behavior range from agonist effects, e.g. anxiolysis, sedation, and hypnosis, to inverse agonist effects, e.g. anxiety, alertness, and convulsions. The diversity of effects reflects the ubiquity of the GABAA/BZ receptors in the brain. Recent data provide some insight into the mechanism of action of BZ ligands, but no clear delineation can be drawn from a single ligand to a single behavioral effect. This may be due to the fact that intrinsic efficacies of the ligands differ between receptor subtypes, so that the diversity of native receptors is further complicated by the diversity of the mode the ligands act on GABAA receptor subtypes. The behavioral actions of alcohol (ethanol) are similar to those produced by GABAA receptor agonists. In agreement, alcohol-induced potentiation of GABAergic responses has often been observed at behavioral, electrophysiological and biochemical levels. Thus, there is clearly a GABAA-dependent component in the actions of alcohol. However, the site and mode of action of ethanol on GABAA/BZ receptors remain controversial.

Effect of GABAergic drugs on motor impairment from ethanol, barbital and lorazepam in rat lines selected for differential sensitivity to ethanol

The effect of GABAergic drugs on the motor-impairing effects of ethanol, barbital, and lorazepam were studied in the ethanol-sensitive ANT (Alcohol Nontolerant) and ethanol-insensitive AT (Alcohol Tolerant) rat lines, selected for differential ethanol-induced motor impairment on the tilting plane. The basic population from which these rat lines were derived, the mixed (M) line, was also included in the study. The ANT rats were more sensitive to the intoxicating effects of ethanol, barbital, and lorazepam than the AT and M rats at the dose ranges tested. Picrotoxin antagonized motor impairment from all three drugs. Flumazenil (Ro 15-1788) antagonized only the effects of lorazepam, and isoniazid did not modify motor impairment induced by any of the three drugs. These results confirm that the selection of AT and ANT lines has not been specific to ethanol, and that it has increased sensitivity to ethanol, barbital, and lorazepam in the ANT rats rather than decreasing it in the AT rats relative to the M rats. The finding that picrotoxin counteracted motor impairment from ethanol, barbital, and lorazepam support the view that the GABAA receptor complex is important in mediating the intoxicating effects of these drugs. These results also suggest that the genetically-determined difference in sensitivity to ethanol between the rat lines involves GABAergic mechanisms, but it remains to be determined whether any part of the GABAA receptor itself has been affected by the selection program.

Alcohol drinking is reduced by a μ1- but not by a δ-opioid receptor antagonist in alcohol-preferring rats

To assess the roles of opioid receptor subtypes in voluntary alcohol drinking, alcohol-preferring AA (Alko, Alcohol) rats, non-deprived of food or water, were used in a paradigm where access to 10% alcohol solution was limited to 1–4-h sessions on every 2nd working day. The δ-opioid receptor antagonist naltrindole (1–5 mg/kg i.p. 15 min before the session) had no effect on alcohol drinking, while it attenuated the δ-opioid receptor agonist [d-Pen2,d-Pen5]enkephalin-induced locomotor stimulation. The μ1-opioid receptor antagonist naloxonazine (1–15 mg/kg i.p. 20 h before the session), at the largest dose, decreased alcohol drinking. It also decreased food intake. When naltrindole (1 mg/kg) and naloxonazine (15 mg/kg) were given prior to 3 consecutive sessions, the former had no effects at any session. Naloxonazine decreased alcohol consumption only in the 1st session, although the reduction of daily water intake became stronger during repeated administration. 4 days after the last drug administration, naloxonazine-treated animals consumed alcohol nearly twice as much as in the control session before any drug treatment. These data suggest that δ-opioid receptors are not involved in the regulation of alcohol drinking in AA rats. μ1-Opioid receptors may be involved in alcohol drinking, although the data suggest that even their prolonged blockade alone is insufficient to induce a sustained decrease in alcohol drinking.

Diazepam-insensitive [3H]Ro 15-4513 binding in intact cultured cerebellar granule cells.

[3H]Ro 15-4513, a partial inverse agonist at the benzodiazepine receptor, binds to two sites in the rat cerebellum, only one of which is sensitive to diazepam. The diazepam-insensitive component, whose identity is unknown, is unique to this brain area. We studied the binding of [3H]Ro 15-4513 in cultured cerebellar granule cells to characterize its binding sites in a specified neuronal cell population and to determine the effects of ethanol on the binding. We also compared the properties of [3H]Ro 15-4513 binding in washed membranes of cultured cells and 14-day-old rat cerebella. [3H]Ro 15-4513 had two binding components in intact granule cells, one sensitive to diazepam, that probably represents binding to the benzodiazepine agonist site, the other sensitive to an antagonist (Ro 15-1788) and two inverse agonists (ethyl-beta-carboline-3-carboxylate, beta CCE and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) of the benzodiazepine receptor (diazepam-insensitive binding). This diazepam-insensitive binding was stimulated by GABA; the maximal increase in binding was about 60% with an EC50 of 0.3 microM. The effect of GABA (10 microM) on the diazepam-insensitive binding was unaffected by 100 microM nipecotic acid but was partially inhibited by 100 microM bicuculline. The stimulation by GABA was also seen at 37 degrees C with washed membranes of 14-day-old rat whole cerebella in the presence of micromolar diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Voluntary ethanol drinking increases locomotor activity in alcohol-preferring AA rats

This study explored, first, whether voluntary ethanol consumption produces locomotor stimulation in ethanol-preferring AA rats. Rats had continual access to water but access to a second bottle containing 10% ethanol, 0.1% saccharin, or water only for 10 min/day. Locomotor activity was significantly increased after the drinking in the ethanol group. Second, we compared the locomotor responses of AA and ANA (ethanol-avoiding) rats to IP ethanol (0.6 and 1.0 g/kg). Rats habituated to test cages showed no effects, but on a modified open field novel to animals there was a short increase in activity without any rat line difference. This activity increase might have resulted from a weak anxiolytic action of ethanol, indicated by the finding in the elevated plus-maze where IP ethanol (1.0 g/kg) increased the number of crosses from a closed arm to another in both AA and ANA rats. The results suggest that ethanol has reinforcing effects in AA rats when drunk but not when injected IP.