Ruth Keist

Trace fear conditioning involves hippocampal α5 GABAA receptors

The heterogeneity of γ-aminobutyric acid type A (GABAA) receptors contributes to the diversity of neuronal inhibition in the regulation of information processing. Although most GABAA receptors are located synaptically, the small population of α5GABAA receptors is largely expressed extrasynaptically. To clarify the role of the α5GABAA receptors in the control of behavior, a histidine-to-arginine point mutation was introduced in position 105 of the murine α5 subunit gene, which rendered the α5GABAA receptors diazepam-insensitive. Apart from an incomplete muscle relaxing effect, neither the sedative, anticonvulsant, nor anxiolytic-like activity of diazepam was impaired in α5(H105R) mice. However, in hippocampal pyramidal cells, the point mutation resulted in a selective reduction of α5GABAA receptors, which altered the drug-independent behavior. In line with the role of the hippocampus in certain forms of associative learning, trace fear conditioning, but not delay conditioning or contextual conditioning, was facilitated in the mutant mice. Trace fear conditioning differs from delay conditioning in that the conditioned and unconditioned stimulus are separated by a time interval. Thus, the largely extrasynaptic α5GABAA receptors in hippocampal pyramidal cells are implicated as control elements of the temporal association of threat cues in trace fear conditioning.

Pharmacology of recombinant γ‐aminobutyric acidA receptors rendered diazepam‐insensitive by point‐mutated α‐subunits

Amino acids in the α‐ and γ‐subunits contribute to the benzodiazepine binding site of GABAA‐receptors. We show that the mutation of a conserved histidine residue in the N‐terminal extracellular segment (α1H101R, α2H101R, α3H126R, and α5H105R) results not only in diazepam‐insensitivity of the respective αxβ2,3γ2‐receptors but also in an increased potentiation of the GABA‐induced currents by the partial agonist bretazenil. Furthermore, Ro 15‐4513, an inverse agonist at wild‐type receptors, acts as an agonist at all mutant receptors. This conserved molecular switch can be exploited to identify the pharmacological significance of specific GABAA‐receptor subtypes in vivo.

Specific subtypes of GABAA receptors mediate phasic and tonic forms of inhibition in hippocampal pyramidal neurons

The main inhibitory neurotransmitter in the mammalian brain, GABA, mediates multiple forms of inhibitory signals, such as fast and slow inhibitory postsynaptic currents and tonic inhibition, by activating a diverse family of ionotropic GABA(A) receptors (GABA(A)Rs). Here, we studied whether distinct GABA(A)R subtypes mediate these various forms of inhibition using as approach mice carrying a point mutation in the alpha-subunit rendering individual GABA(A)R subtypes insensitive to diazepam without altering their GABA sensitivity and expression of receptors. Whole cell patch-clamp recordings were performed in hippocampal pyramidal cells from single, double, and triple mutant mice. Comparing diazepam effects in knock-in and wild-type mice allowed determining the contribution of alpha1, alpha2, alpha3, and alpha5 subunits containing GABA(A)Rs to phasic and tonic forms of inhibition. Fast phasic currents were mediated by synaptic alpha2-GABA(A)Rs on the soma and by synaptic alpha1-GABA(A)Rs on the dendrites. No contribution of alpha3- or alpha5-GABA(A)Rs was detectable. Slow phasic currents were produced by both synaptic and perisynaptic GABA(A)Rs, judged by their strong sensitivity to blockade of GABA reuptake. In the CA1 area, but not in the subiculum, perisynaptic alpha5-GABA(A)Rs contributed to slow phasic currents. In the CA1 area, the diazepam-sensitive component of tonic inhibition also involved activation of alpha5-GABA(A)Rs and slow phasic and tonic signals shared overlapping pools of receptors. These results show that the major forms of inhibitory neurotransmission in hippocampal pyramidal cells are mediated by distinct GABA(A)Rs subtypes.

Requirement of α5-GABAA Receptors for the Development of Tolerance to the Sedative Action of Diazepam in Mice

Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via α1-GABAA receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which theα1-,α2-,α3-, orα5-GABAA receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg · kg-1 · d-1) and tested for motor activity. Wild-type, α2(H101R), and α3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, α5(H105R) mice failed to display any sedative tolerance. α1(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [3H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in α5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of α5-GABAA receptors in the dentate gyrus. Thus, the chronic activation of α5-GABAA receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with α1-GABAA receptors.

GABAA receptors containing the α5 subunit mediate the trace effect in aversive and appetitive conditioning and extinction of conditioned fear

A reduction in α5 subunit‐containing γ‐aminobutyric acid (GABA)A receptors has been reported to enhance some forms of learning in mutant mouse models. This effect has been attributed to impaired α5 GABAA receptor‐mediated inhibitory modulation in the hippocampus. The introduction of a point mutation (H105R) in the α5 subunit is associated with a specific reduction of α5 subunit‐containing GABAA receptors in the hippocampus. The present study examined the modulation of associative learning and the extinction of conditioned response in these animals. The strength of classical conditioning can be weakened when a trace interval is interposed between the conditioned stimulus and unconditioned stimulus. Here we report that this ‘trace effect’ in classical conditioning was absent in the mutant mice − they were insensitive to the imposition of a 20‐s trace interval. This effect of the mutation was most clearly in the female mice using an aversive conditioning paradigm, and in the male mice using an appetitive conditioning paradigm. These gender‐specific phenotypes were accompanied by a resistance to extinction of conditioned fear response in the mutant mice that was apparent in both genders. Our results identify neuronal inhibition in the hippocampus mediated via α5 GABAA receptors as a critical control element in the regulation of the acquisition and expression of associative memory.

Spatiotemporal specificity of GABAA receptor-mediated regulation of adult hippocampal neurogenesis

GABAergic transmission regulates adult neurogenesis by exerting negative feedback on cell proliferation and enabling dendrite formation and outgrowth. Further, GABAergic synapses target differentiating dentate gyrus granule cells prior to formation of glutamatergic connections. GABAA receptors (GABAARs) mediating tonic (extrasynaptic) and phasic (synaptic) transmission are molecularly and functionally distinct, but their specific role in regulating adult neurogenesis is unknown. Using global and single‐cell targeted gene deletion of subunits contributing to the assembly of GABAARs mediating tonic (α4, δ) or phasic (α2) GABAergic transmission, we demonstrate here in the dentate gyrus of adult mice that GABAARs containing α4, but not δ, subunits mediate GABAergic effects on cell proliferation, initial migration and early dendritic development. In contrast, α2‐GABAARs cell‐autonomously signal to control positioning of newborn neurons and regulate late maturation of their dendritic tree. In particular, we observed pruning of distal dendrites in immature granule cells lacking the α2 subunit. This alteration could be prevented by pharmacological inhibition of thrombospondin signaling with chronic gabapentin treatment, shown previously to reduce glutamatergic synaptogenesis. These observations point to homeostatic regulation of inhibitory and excitatory inputs onto newborn granule cells under the control of α2‐GABAARs. Taken together, the availability of distinct GABAAR subtypes provides a molecular mechanism endowing spatiotemporal specificity to GABAergic control of neuronal maturation in adult brain.

Presynaptic α2-GABAA Receptors in Primary Afferent Depolarization and Spinal Pain Control

Spinal dorsal horn GABAA receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution of a major subpopulation of GABAA receptors (those containing the α2 subunit) to spinal pain control in mice lacking α2-GABAA receptors specifically in primary nociceptors (sns-α2−/− mice). sns-α2−/− mice exhibited GABAA receptor currents and dorsal root potentials of normal amplitude in vitro, and normal response thresholds to thermal and mechanical stimulation in vivo, and developed normal inflammatory and neuropathic pain sensitization. However, the positive allosteric GABAA receptor modulator diazepam (DZP) had almost completely lost its potentiating effect on PAD and presynaptic inhibition in vitro and a major part of its spinal antihyperalgesic action against inflammatory hyperalgesia in vivo. Our results thus show that part of the antihyperalgesic action of spinally applied DZP occurs through facilitated activation of GABAA receptors residing on primary nociceptors.

GABAA Receptor α5 Subunits Contribute to GABAA,slow Synaptic Inhibition in Mouse Hippocampus

gamma-Aminobutyric acid type A (GABA(A)) receptor alpha5 subunits, which are heavily expressed in the hippocampus, are potential drug targets for improving cognitive function. They are found at synaptic and extrasynaptic sites and have been shown to mediate tonic inhibition in pyramidal neurons. We tested the hypothesis that alpha5 subunits also contribute to synaptic inhibition by measuring the effect of diazepam (DZ) on spontaneous and stimulus-evoked inhibitory postsynaptic currents (IPSCs) in genetically modified mice carrying a point mutation in the alpha5 subunit (alpha5-H105R) that renders those receptors insensitive to benzodiazepines. In wild type mice, DZ (1 microM) increased the amplitude of spontaneous IPSCs (sIPSCs) and stimulus-evoked GABA(A,slow) IPSCs (eIPSCs) and prolonged the decay of GABA(A,fast) sIPSCs. In alpha5-mutant mice, DZ increased the amplitude of a small-amplitude subset of sIPSCs (<50 pA) and eIPSCs (<300 pA) GABA(A,slow) and prolonged the decay of GABA(A,fast) sIPSCs, but failed to increase the amplitude of larger sIPSCs and eIPSCs GABA(A,slow). These results indicate that alpha5 subunits contribute to a large-amplitude subset of GABA(A,slow) synapses and implicate these synapses in modulation of cognitive function by drugs that target alpha5 subunits.

Antidepressant-like properties of α2-containing GABAA receptors

Growing evidence suggests that altered function of the GABAergic system can contribute to the pathophysiology of depression. Many GABAergic effects are mediated via ionotropic GABA(A) receptors, which are functionally defined by their α subunit (α1-α6). Although it remains unknown which specific GABA(A) receptor population mediates depressive-like effects, we posit that α2-containing GABA(A) receptors, which are highly expressed in limbic regions, may underlie these behaviors. We hypothesized that genetic inactivation of α2-containing GABA(A) receptors would generate a depressive-like phenotype in mice. Male and female wild type, α2 heterozygous, and α2 homozygous knockout mice generated on the 129X1/SvJ background were examined in the novelty-suppressed feeding (NSF) test, the forced swim test (FST) and the tail suspension test (TST). Male α2 knockout mice took longer to eat in the NSF test and became immobile faster and remained immobile longer when challenged in the FST and the TST compared to wild types. In females significant genotypic differences were only observed in the FST. We conclude that GABAergic inhibition acting via α2-containing GABA(A) receptors has an antidepressant-like effect in vivo and that these receptors represent a specific molecular substrate that can regulate depressive-like states. α2-containing GABA(A) receptors may therefore represent a novel target for the development of more effective antidepressants.

Diazepam-induced changes on sleep and the EEG spectrum in mice: role of the α3-GABAA receptor subtype

Benzodiazepines reduce EEG slow‐wave activity in non‐REM sleep by potentiating GABAergic neurotransmission at GABAA receptors via a modulatory binding site. However, the mechanisms of action underlying the effects of benzodiazepines on sleep and the sleep EEG are still unknown. Slow waves during sleep are generated by the corticothalamic system and synchronized by the inhibitory GABAergic neurons of the reticular thalamic nucleus. This region contains exclusively α3‐containing GABAA receptors. We investigated the role of these receptors in the mediation of diazepam effects on the sleep EEG by studying point‐mutated mice in which the α3‐GABAA receptor is diazepam‐insensitive [α3(H126R)]. Sleep was recorded for 12 h after i.p. injection of 3 mg/kg diazepam or vehicle at light onset in α3(H126R) and wild‐type controls (n = 13–17 per genotype). The main effect was a marked reduction of slow‐wave activity (EEG power density in 0.75–4.00 Hz) in non‐REM sleep and a concomitant increase in frequencies above 15.00 Hz in non‐REM sleep and waking in both genotypes. Neither effect of diazepam differed significantly between the genotypes. Despite the exclusive expression of α3‐containing GABAA receptors in the reticular thalamic nucleus, these receptors do not seem to be critical for the mediation of the effects of diazepam on the sleep EEG.