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 GABA(A) 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 gamma2 subunit of GABA(A) receptors exhibit a modest functional deficit in mainly postsynaptic GABA(A) 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 gamma2 subunit gene to further analyze the mechanism and brain substrate underlying this phenotype. Heterozygous deletion of the gamma2 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 gamma2 subunit-containing GABA(A) receptors. In contrast, a comparable forebrain-specific GABA(A) receptor deficit induced selectively in mature neurons during adolescence lacked neurogenic and behavioral consequences. These results suggest that modestly reduced GABA(A) 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.

Sleep deprivation and daily torpor impair object recognition in Djungarian hamsters.

Sleep has been shown to play a facilitating role in memory consolidation, whereas sleep deprivation leads to performance impairment both in humans and rodents. The effects of 4-h sleep deprivation on recognition memory were investigated in the Djungarian hamster (Phodopus sungorus). Because sleep during the first hours after daily torpor has many similarities to recovery from sleep deprivation, the effects of spontaneous torpor on object recognition were also assessed. A 4-h sleep deprivation, starting immediately after an object learning task, diminished the ability of the hamsters to: (1) discriminate between an already encountered object (target) and a novel object presented in a novel context, (2) retrieve a target within a complex spatial scene, and (3) detect a spatial rearrangement of familiar objects in a familiar context. Plasma stress hormone levels were similar in sleep-deprived and control hamsters. The occurrence of a daily torpor episode during retention was associated with impaired old-new object discrimination performance in the more effortful complex spatial scene task only, and in a two-object choice situation in a novel context no torpor-induced deficit was found. Our results show that post learning sleep deprivation and daily torpor induce a deficit in familiar object retrieval performance in a complex spatial scene, while sparing familiarity-based recognition and novelty processing. Sleep deprivation during the first 4 h of memory consolidation hampered also recency memory for discrete objects. Stress was not a factor contributing to the sleep deprivation-induced impairment.

Astrogliosis in epilepsy leads to overexpression of adenosine kinase, resulting in seizure aggravation.

Adenosine kinase (ADK) is considered to be the key regulator of the brain's endogenous anticonvulsant, adenosine. In adult brain, ADK is primarily expressed in a subpopulation of astrocytes and striking upregulation of ADK in these cells has been associated with astrogliosis after kainic acid-induced status epilepticus (KASE) in the kainic acid mouse model of temporal lobe epilepsy. To investigate the causal relationship between KASE-induced astrogliosis, upregulation of ADK and seizure activity, we have developed a novel mouse model [the Adktm1(-/-)-Tg(UbiAdk) mouse] lacking the endogenous astrocytic enzyme due to a targeted disruption of the endogenous gene, but containing an Adk transgene under the control of a human ubiquitin promoter. Mutant Adktm1(-/-)-Tg(UbiAdk) mice were characterized by increased brain ADK activity and constitutive overexpression of transgenic ADK throughout the brain, with particularly high levels in hippocampal pyramidal neurons. This ADK overexpression was associated with increased baseline levels of locomotion. Most importantly, two-thirds of the mutant mice analysed exhibited spontaneous seizure activity in the hippocampus and cortex. This was the direct consequence of transgene expression, since this seizure activity could be prevented by systemic application of the ADK inhibitor 5-iodotubercidin. Intrahippocampal injection of kainate in the mutant mice resulted in astrogliosis to the same extent as that observed in wild-type mice despite the absence of endogenous astrocytic ADK. Therefore, KASE-induced upregulation of endogenous ADK in wild-type mice is a consequence of astrogliosis. However, seizures in kainic acid-injected mutants displayed increased intra-ictal spike frequency compared with wild-type mice, indicating that, once epilepsy is established, increased levels of ADK aggravate seizure severity. We therefore conclude that therapeutic strategies that augment the adenosine system after astrogliosis-induced upregulation of ADK constitute a neurochemical rationale for the prevention of seizures in epilepsy.

Modulation of anxiety and fear via distinct intrahippocampal circuits.

Recent findings indicate a high level of specialization at the level of microcircuits and cell populations within brain structures with regards to the control of fear and anxiety. The hippocampus, however, has been treated as a unitary structure in anxiety and fear research despite mounting evidence that different hippocampal subregions have specialized roles in other cognitive domains. Using novel cell-type- and region-specific conditional knockouts of the GABAA receptor α2 subunit, we demonstrate that inhibition of the principal neurons of the dentate gyrus or CA3 via α2-containing GABAA receptors (α2GABAARs) is required to suppress anxiety, while the inhibition of CA1 pyramidal neurons is required to suppress fear responses. We further show that the diazepam-modulation of hippocampal theta activity shows certain parallels with our behavioral findings, suggesting a possible mechanism for the observed behavioral effects. Thus, our findings demonstrate a double dissociation in the regulation of anxiety versus fear by hippocampal microcircuitry.

Requirement of alpha5-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 alpha1-GABA(A) 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 thealpha1-,alpha2-,alpha3-, oralpha(5)-GABA(A) receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg x kg(-1) x d(-1)) and tested for motor activity. Wild-type, alpha2(H101R), and alpha3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, alpha5(H105R) mice failed to display any sedative tolerance. alpha1(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 alpha5-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 alpha5-GABA(A) receptors in the dentate gyrus. Thus, the chronic activation of alpha(5)-GABA(A) receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with alpha1-GABA(A) receptors.

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 beta3 subunit of the GABA(A) 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. beta3(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 GABA(A) 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 GABA(A) receptor beta3 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.

Behavioral functions of GABAA receptor subtypes--the Zurich experience.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. They are heteropentamers that are typically classified according to their α subunits. By rendering each of the benzodiazepine-sensitive α subunits (α1, α2, α3, and α5) insensitive to modulation by classical benzodiazepines by His to Arg point mutations in knock-in mice, we were able to identify behavioral functions mediated by different GABAA receptor subtypes, which led to the development of novel therapeutic strategies. In this chapter, we provide a largely chronological overview on behavioral studies on GABAA receptor mutant mice at the Institute of Pharmacology and Toxicology at the University of Zurich.

Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects.

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

Dissecting the role of diazepam-sensitive γ-aminobutyric acid type A receptors in defensive behavioral reactivity to mild threat.

Moderate reductions in synaptic γ-aminobutyric acid(A) receptors (GABA(A)Rs) have been associated with an enhanced defensive behavioral reactivity to mild threat, sensitive to diazepam. We here tested whether a deficit in α2 subunit-containing GABAergic synapses is sufficient to cause this anxiety-related phenotype and to prevent its attenuation by the benzodiazepine. Wild type (α2+/+), heterozygous (α2+/-) and homozygous (α2-/-) knock-out littermates were tested in the free-choice exploratory (FCE) and the light/dark choice (LDC) paradigms. α2-/- mice, double mutant α1H101Rα2-/- and α3H126Rα2-/- mice, which combine a lack of α2-GABA(A)Rs with point-mutated diazepam-insensitive either α1H101R or α3H126R-GABA(A)Rs, and double point-mutated α1H101Rα2H101R and α1H101Rα3H126R mice were used to uncover the GABA(A)R subtype(s) mediating the drug effects. Data show that in the FCE, α2-/- mice exhibited more retractions (i.e. risk assessment) and longer latencies to first occurrence into the novel compartment and less transitions and time spent inside it in comparison to α2+/- and α2+/+ mice. In the LDC, α2-/- mice visited and spent less time in the lit box and stayed longer in the tunnel than the other two groups. Minor differences were found between α2+/- and α2+/+ mice in the two paradigms. Diazepam (1.5mg/kg per os) normalized retractions and latencies in the FCE in α2-/- and α3H126Rα2-/- mice, but not in α1H101Rα2-/- mice. The same drug treatment failed to attenuate behavioral aversion in both paradigms in all mutants with impaired α2-GABA(A)R function. These results reveal α2-containing GABA(A)Rs as key molecular determinants in the regulation of anxiety-related responses elicited by exposure to relative novelty and mild threat. In the absence of these receptors, diazepam through activation of α1-GABA(A)Rs remains effective in reducing risk assessment, but not behavioral aversion.

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

Ionotropic gamma-aminobutyric acid (GABA(A)) 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 GABA(A) 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 (10mg/kg, i.p.), but fully retained in GABA(A) receptor alpha1 point-mutated mice, which were resistant against the sedative action of diazepam. Experiments carried out in mice with two diazepam-insensitive subunits (alpha1/alpha2, alpha1/alpha3 and alpha1/alpha5 double point-mutated mice) allowed addressing the contribution of alpha2, alpha3 and alpha5 subunits to systemic diazepam-induced antihyperalgesia in the absence of sedation. The relative contributions of these subunits were alpha2 approximately alpha3>alpha5, and thus very similar to those found for intrathecal diazepam (0.09 mg/kg). Accordingly, SL-651498 (10mg/kg, p.o.), an "anxioselective" benzodiazepine site agonist with preferential activity at alpha2/alpha3 subunits, significantly reduced formalin-induced flinching in wild-type mice. We conclude that systemic diazepam exerts a genuine antihyperalgesic effect, which depends on spinal GABA(A) receptors containing alpha2 and/or alpha3 subunits.

alpha2-gamma-Aminobutyric acid (GABA)A receptors are the molecular substrates mediating precipitation of narcosis but not of sedation by the combined use of diazepam and alcohol in vivo.

Classical benzodiazepines such as diazepam are widely used tranquillisers and hypnotics in various neuropsychiatric diseases including alcohol-related disorders. One of the major drawbacks of benzodiazepine therapy, however, is an exacerbation of the sedative and hypnotic effects associated with alcohol intake, even at low doses. Even though the gamma-aminobutyric acid (GABA)A receptor complex is a common target for the actions of both classes of drugs, the molecular mechanisms underlying the enhanced pharmacological properties of the combined use of benzodiazepines and alcohol remain to be identified. The present experiments aimed at clarifying which of the GABAA receptor subtypes mediate the augmented hypnotic-like and sedative effects of combined diazepam and alcohol using the righting reflex and motor activity assays, respectively, in histidine-to-arginine point mutated mice that possess diazepam-insensitive alpha1-, alpha2-, alpha3- or alpha5-GABAA receptors. The combination of diazepam (2 or 3 mg/kg) and ethanol (3 g/kg) induced loss of righting reflex with a significantly dose-dependent increase of the latency to its full recovery in wild-type, alpha1(H101R), alpha3(H126R) and alpha5(H105R) but not in alpha2(H101R) mice. A combined treatment with diazepam (1 mg/kg) and ethanol (2.5 g/kg) precipitated motor inhibition similarly in wild-type and alpha2(H101R) mice. Responsiveness of the alpha2(H101R) mice to ethanol alone was similar to that of wild-type mice. These results demonstrate that induction of loss of righting reflex by combined diazepam and alcohol is closely dependent on the activation of the alpha2-GABAA receptors by the benzodiazepine whereas precipitation of sedation involves GABAA receptors other than the alpha2-GABAA receptors.