Florence Crestani

Benzodiazepine actions mediated by specific gamma-aminobutyric acidA receptor subtypes

GABAA (γ-aminobutyric acidA) receptors are molecular substrates for the regulation of vigilance, anxiety, muscle tension, epileptogenic activity and memory functions, which is evident from the spectrum of actions elicited by clinically effective drugs acting at their modulatory benzodiazepine-binding site. Here we show, by introducing a histidine-to-arginine point mutation at position 101 of the murine α1-subunit gene, that α1-type GABAA receptors, which are mainly expressed in cortical areas and thalamus, are rendered insensitive to allosteric modulation by benzodiazepine-site ligands, whilst regulation by the physiological neurotransmitter γ-aminobutyric acid is preserved. α1(H101R) mice failed to show the sedative, amnesic and partly the anticonvulsant action of diazepam. In contrast, the anxiolytic-like, myorelaxant, motor-impairing and ethanol-potentiating effects were fully retained, and are attributed to the nonmutated GABAA receptors found in the limbic system (α2, α5), in monoaminergic neurons (α3) and in motoneurons (α2, α5). Thus, benzodiazepine-induced behavioural responses are mediated by specific GABAA receptor subtypes in distinct neuronal circuits, which is of interest for drug design.

General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.

General anesthetics are widely used in clinical practice. On the molecular level, these compounds have been shown to modulate the activity of various neuronal ion channels. However, the functional relevance of identified sites in mediating essential components of the general anesthetic state, such as immobility and hypnosis, is still unknown. Using gene‐targeting technology, we generated mice harboring a subtle point mutation (N265M) in the second transmembrane region of the β3 subunit of the GABAA receptor. In these mice, the suppression of noxious‐evoked movements in response to the intravenous anesthetics etomidate and propofol is completely abolished, while only slightly decreased with the volatile anesthetics enflurane and halothane. β3(N265M) mice also display a profound reduction in the loss of righting reflex duration in response to intravenous but not volatile anesthetics. In addition, electrophysiological recordings revealed that anesthetic agents were significantly less effective in enhancing GABAA receptor‐mediated currents, and in decreasing spontaneous action potential firing in cortical brain slices derived from mutant mice. Taken together, our results demonstrate that a single molecular target, and indeed a specific residue (N265) located within the GABAA receptor β3 subunit, is a major determinant of behavioral responses evoked by the intravenous anesthetics etomidate and propofol, whereas volatile anesthetics appear to act via a broader spectrum of molecular targets.

Decreased GABAA-receptor clustering results in enhanced anxietyand a bias for threat cues

Patients with panic disorders show a deficit of GABAA receptors in the hippocampus, parahippocampus and orbitofrontal cortex. Synaptic clustering of GABAA receptors in mice heterozygous for the γ2 subunit was reduced, mainly in hippocampus and cerebral cortex. The γ2+/– mice showed enhanced behavioral inhibition toward natural aversive stimuli and heightened responsiveness in trace fear conditioning and ambiguous cue discrimination learning. Implicit and spatial memory as well as long-term potentiation in hippocampus were unchanged. Thus γ2+/– mice represent a model of anxiety characterized by harm avoidance behavior and an explicit memory bias for threat cues, resulting in heightened sensitivity to negative associations. This model implicates GABAA-receptor dysfunction in patients as a causal predisposition to anxiety disorders.

GABAA receptor subtypes: dissecting their pharmacological functions

The enhancement of GABA-mediated synaptic transmission underlies the pharmacotherapy of various neurological and psychiatric disorders. GABA(A) receptors are pluripotent drug targets that display an extraordinary structural heterogeneity: they are assembled from a repertoire of at least 18 subunits (alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, rho1-3). However, differentiating defined GABA(A) receptor subtypes on the basis of function has had to await recent progress in the genetic dissection of receptor subtypes in vivo. Evidence that the various actions of allosteric modulators of GABA(A) receptors, in particular the benzodiazepines, can be attributed to specific GABA(A) receptor subtypes will be discussed. Such discoveries could open up new avenues for drug development.

Mechanism of action of the hypnotic zolpidem in vivo

Zolpidem is a widely used hypnotic agent acting at the GABAA receptor benzodiazepine site. On recombinant receptors, zolpidem displays a high affinity to α1‐GABAA receptors, an intermediate affinity to α2‐ and α3‐GABAA receptors and fails to bind to α5‐GABAA receptors. However, it is not known which receptor subtype is essential for mediating the sedative‐hypnotic action in vivo. Studying α1(H101R) mice, which possess zolpidem‐insensitive α1‐GABAA receptors, we show that the sedative action of zolpidem is exclusively mediated by α1‐GABAA receptors. Similarly, the activity of zolpidem against pentylenetetrazole‐induced tonic convulsions is also completely mediated by α1‐GABAA receptors. These results establish that the sedative‐hypnotic and anticonvulsant activities of zolpidem are due to its action on α1‐GABAA receptors and not on α2‐ or α3‐GABAA receptors.

GABAergic Control of Adult Hippocampal Neurogenesis in Relation to Behavior Indicative of Trait Anxiety and Depression States

Stressful experiences in early life are known risk factors for anxiety and depressive illnesses, and they inhibit hippocampal neurogenesis and the expression of GABAA receptors in adulthood. Conversely, deficits in GABAergic neurotransmission and reduced neurogenesis are implicated in the etiology of pathological anxiety and diverse mood disorders. Mice that are heterozygous for the γ2 subunit of GABAA receptors exhibit a modest functional deficit in mainly postsynaptic GABAA receptors that is associated with a behavioral, cognitive, and pharmacological phenotype indicative of heightened trait anxiety. Here we used cell type-specific and developmentally controlled inactivation of the γ2 subunit gene to further analyze the mechanism and brain substrate underlying this phenotype. Heterozygous deletion of the γ2 subunit induced selectively in immature neurons of the embryonic and adult forebrain resulted in reduced adult hippocampal neurogenesis associated with heightened behavioral inhibition to naturally aversive situations, including stressful situations known to be sensitive to antidepressant drug treatment. Reduced adult hippocampal neurogenesis was associated with normal cell proliferation, indicating a selective vulnerability of postmitotic immature neurons to modest functional deficits in γ2 subunit-containing GABAA receptors. In contrast, a comparable forebrain-specific GABAA receptor deficit induced selectively in mature neurons during adolescence lacked neurogenic and behavioral consequences. These results suggest that modestly reduced GABAA receptor function in immature neurons of the developing and adult brain can serve as a common molecular substrate for deficits in adult neurogenesis and behavior indicative of anxious and depressive-like mood states.

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-receptor subtypes: a new pharmacology

The GABA(A) receptor is a pluripotent drug target mediating anxiolytic, sedative, anticonvulsant, muscle relaxant and amnesic activity. These drug actions have now been attributed to defined receptor subtypes. Thus, precise guidelines are available for the development of novel drugs with more selective action and less side effects than those currently in clinical use.

Genuine antihyperalgesia by systemic diazepam revealed by experiments in GABAA receptor point-mutated mice.

ABSTRACT Ionotropic γ‐aminobutyric acid (GABAA) receptors control the relay of nociceptive signals at several levels of the neuraxis. Experiments with systemically applied benzodiazepines, which enhance the action of GABA at these receptors, have suggested both anti‐ and pronociceptive effects. The interpretation of such experiments has been notoriously difficult because of confounding sedation. Here, we have used genetically engineered mice, which carry specific benzodiazepine‐insensitive GABAA receptor subunits, to test whether diazepam, a frequently used classical benzodiazepine, exerts antihyperalgesia after systemic administration in the formalin test, a model of tonic nociception. In wild‐type mice, systemic diazepam (3–30 mg/kg, p.o.) dose‐dependently reduced the number of formalin‐induced flinches during both phases of the test by about 40–70%. This antinociception was reversed by the benzodiazepine site antagonist flumazenil (10 mg/kg, i.p.), but fully retained in GABAA receptor α1 point‐mutated mice, which were resistant against the sedative action of diazepam. Experiments carried out in mice with two diazepam‐insensitive subunits (α1/α2, α1/α3 and α1/α5 double point‐mutated mice) allowed addressing the contribution of α2, α3 and α5 subunits to systemic diazepam‐induced antihyperalgesia in the absence of sedation. The relative contributions of these subunits were α2 ≈ α3 > α5, and thus very similar to those found for intrathecal diazepam (0.09 mg/kg). Accordingly, SL‐651498 (10 mg/kg, p.o.), an “anxioselective” benzodiazepine site agonist with preferential activity at α2/α3 subunits, significantly reduced formalin‐induced flinching in wild‐type mice. We conclude that systemic diazepam exerts a genuine antihyperalgesic effect, which depends on spinal GABAA receptors containing α2 and/or α3 subunits.

Resolving differences in GABAA receptor mutant mouse studies.

1059 TO THE EDITOR—The validity of genetically altered mice as models for disease or for drug target identification relies on the reproducibility of behavioral test results. In a recent paper in Nature Neuroscience1, behavioral tests were described on α1(H101R) mice carrying a histidine-toarginine point mutation in the α1 subunit of the GABAA receptor. An accompanying News and Views article2 discussed discrepancies between results of behavioral experiments for this study1 and our study of α1(H101R) mice3. The two lines of mice seemed to differ in their drug-induced behavior, although both had been constructed with the same point mutation. Now we report that these discrepencies were caused by differences in the behavioral protocols used by the two groups, not by differences in the mouse lines. Both studies compared the behavioral effects of diazepam in wild-type and pointmutated α1(H101R) mice whose α1 GABAA receptor subtype was made insensitive to diazepam by a histidine-to-arginine substitution. We assessed the sedative action of diazepam on spontaneous motor activity of mice familiar with the testing environment3. Diazepam produced sedation in wild-type mice, as measured by the dose-dependent decrease in the extent of spontaneous motor activity, whereas diazepam failed to impair the spontaneous motor activity in α1(H101R) mice up to 30 mg per kg (ref. 3). These results led us to conclude that the sedative action of diazepam as measured by this protocol is mediated by α1 GABAA receptors. In contrast, McKernan and colleagues measured locomotor activity in mice exploring an unfamiliar environment1. The authors reported that diazepam (3 mg per kg orally) did not affect wild-type mice, but induced a significant increase in locomotor activity in the α1(H101R) mice. This has been interpreted as “paradoxical hyperactivity”2 and “reduction in neophobia”1 or “reduction in GABA-mediated neuronal inhibition”1. Following transfer to a new testing room 30 minutes before drug treatment, our α1(H101R) mice also exhibit hyperactivity (Fig. 1). These findings, which correspond to McKernan and colleagues’ results1, suggest that the failure of diazepam between the α1(H101R) mice generated by the two laboratories1,3. This shows that taking environmental and technical details of test procedures into account may help to resolve interlaboratory differences in results.