[Show abstract][Hide abstract] ABSTRACT: Rationale:
Benzodiazepines (BZDs) are prescribed to reduce anxiety, agitation, and muscle spasms and for their sedative-hypnotic and anticonvulsant effects. Under specific conditions, BZDs escalate aggression in some individuals. Specific effects of BZDs have been linked to the α-subunit subtype composition of GABAA receptors.
Point-mutated mice rendered selectively insensitive to BZDs at α1-, α2-, or α3-containing GABAA receptors were used to determine which α-subunit subtypes are necessary for BZDs to escalate aggression and social approach and to reduce fear-motivated behavior.
During resident-intruder confrontations, male wild-type (WT) and point-mutated α1(H101R), α2(H101R), and α3(H126R) mice were treated with midazolam (0-1.7 mg/kg, i.p.) and evaluated for aggression in an unfamiliar environment. Separate midazolam-treated WT and point-mutated mice were assessed for social approach toward a female or investigated in a 6-day fear-potentiated startle procedure.
Moderate doses of midazolam (0.3-0.56 mg/kg, i.p.) escalated aggression in WT and α3(H126R) mutants and increased social approach in WT and α1(H101R) mice. The highest dose of midazolam (1.0 mg/kg) reduced fear-potentiated startle responding. All mice were sensitive to the sedative effect of midazolam (1.7 mg/kg) except α1(H101R) mutants.
Midazolam requires BZD-sensitive α1- and α2-containing GABAA receptors in order to escalate aggression and α2- and α3-containing receptors to reduce social anxiety-like behavior. GABAA receptors containing the α1-subunit are crucial for BZD-induced sedation, while α2-containing GABAA receptors may be a shared site of action for the pro-aggressive and anxiolytic effects of BZDs.
[Show abstract][Hide abstract] ABSTRACT: Aversive experiences can lead to complex behavioral adaptations including increased levels of anxiety and fear generalization. The neuronal mechanisms underlying such maladaptive behavioral changes, however, are poorly understood. Here, using a combination of behavioral, physiological and optogenetic approaches in mouse, we identify a specific subpopulation of central amygdala neurons expressing protein kinase C δ (PKCδ) as key elements of the neuronal circuitry controlling anxiety. Moreover, we show that aversive experiences induce anxiety and fear generalization by regulating the activity of PKCδ(+) neurons via extrasynaptic inhibition mediated by α5 subunit-containing GABAA receptors. Our findings reveal that the neuronal circuits that mediate fear and anxiety overlap at the level of defined subpopulations of central amygdala neurons and demonstrate that persistent changes in the excitability of a single cell type can orchestrate complex behavioral changes.
[Show abstract][Hide abstract] ABSTRACT: Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.
[Show abstract][Hide abstract] ABSTRACT: Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with β3 subunits, we tested the hypothesis that etomidate acts through β3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR β3-subunit (β3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by β3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5β1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 μM and below), whereas α5β2γ2L and α5β3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5β3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5β2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons).
[Show abstract][Hide abstract] ABSTRACT: GABAergic inhibition displays rich functional diversity throughout the CNS, which arises from variations in the nature of inputs, subunit composition, subcellular localization of receptors and synapse geometry, or reuptake mechanisms. In the spinal dorsal horn (SDH), GABAA and glycine receptors play a major role in the control of excitability and accuracy of nociceptive processing. Identifying which components shape the properties of the inhibitory synapses in different cell types is necessary to understand how nociceptive information is integrated. To address this, we used transgenic mice where inhibitory interneurons express GAD65-EGFP. We found that GABAA, but not glycine receptor-mediated evoked IPSCs displayed slower kinetics in EGFP+ vs. EGFP- interneurons. GABAA miniature IPSC decay kinetics showed a large variability in both populations, however the distribution of decays differed between EGFP+ and EGFP- interneurons. The range of mIPSC decay kinetics observed was replicated in experiments using rapid application of GABA on outside-out patches taken from SDH neurons in slices. Furthermore, GABAA decay kinetics were not affected by uptake blockers and were not different in mice lacking δ or α5 subunits, indicating that intrinsic channel properties likely underlie the heterogeneity. To identify whether other α subunits shape the various kinetic properties observed we took advantage of knock-in mice carrying point mutations in either the α1, α2 or α3 subunits rendering Ro 15-4513 a selective agonist at the benzodiazepine modulatory site. We found that α1and α2 subunit underlie the fast decaying component of IPSCs while the slow component is determined by the α3 subunit. The differential distribution of GABAA subunits at inhibitory synapses thus sculpts the heterogeneity of the SDH inhibitory circuitry. This diversity of inhibitory elements can be harnessed to selectively modulate different components of the spinal nociceptive circuitry for therapeutic interven
[Show abstract][Hide abstract] ABSTRACT: Background:
Surgical interventions like skin incisions trigger withdrawal reflexes which require motor neurones and local circuit interneurones in the spinal ventral horn. This region plays a key role in mediating immobilizing properties of the GABAergic anaesthetic propofol. However, it is unclear how propofol modulates GABA(A) receptors in the spinal ventral horn and whether tonic or phasic inhibition is involved.
Organotypic spinal cord tissue slices were prepared from mice. Whole-cell recordings were performed for quantifying effects of propofol on GABA(A) receptor-mediated phasic transmission and tonic conductance.
Propofol increased GABAergic phasic transmission by a prolongation of the decay time constant in a concentration-dependent manner. The amount of the charge transferred per inhibitory post-synaptic current, described by the area under the curve, was significantly augmented by 1 µM propofol (P<0.01). A GABA(A) receptor-mediated tonic current was not induced by 1 µM propofol but at a concentration of 5 µM (P<0.05).
Propofol depresses ventral horn interneurones predominantly by phasic rather than by tonic GABA(A) receptor-mediated inhibition. However, the present results suggest that the involvement of a tonic inhibition might contribute to the efficacy of propofol to depress nociceptive reflexes at high concentrations of the anaesthetic.
BJA British Journal of Anaesthesia 08/2014; 114(3). DOI:10.1093/bja/aeu269 · 4.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 5-HT2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on serotonin (5-HT) release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5-HT2C receptor-induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5-HT turnover by a 5-HT2C receptor agonist (RO 60-0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA-A or GABA-B receptors in mice. Neither the GABA-B receptor antagonist phaclofen nor the specific genetic ablation of either GABA-B1a or GABA-B1b subunits altered the inhibitory effect of RO 60-0175, although 5-HT turnover was markedly decreased in GABA-B1a knock-out mice in both basal and stress conditions. In contrast, the 5-HT2C receptor-mediated inhibition of 5-HT turnover was reduced by the GABA-A receptor antagonist bicuculline. Because a significant effect of 5-HT2C receptor activation persisted in mutant mice deficient in the α3 subunit of GABA-A receptors, it can be inferred that non α3 subunit-containing GABA-A receptors, but not GABA-B receptors, mediate the 5-HT2C-induced inhibition of stress-induced increase of hippocampal 5-HT turnover in mice.This article is protected by copyright. All rights reserved.
Journal of Neurochemistry 08/2014; 131(5). DOI:10.1111/jnc.12929 · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The enteric nervous system (ENS) provides the intrinsic neural control of the gastrointestinal tract (GIT) and regulates virtually all GI functions. Altered neuronal activity within the ENS underlies various GI disorders with stress being a key contributing factor. Thus, elucidating the expression and function of the neurotransmitter systems, which determine neuronal excitability within the ENS, such as the GABA-GABAA receptor (GABAAR) system, could reveal novel therapeutic targets for such GI disorders. Molecular and functionally diverse GABAARs modulate rapid GABAergic-mediated regulation of neuronal excitability throughout the nervous system. However, the cellular and subcellular GABAAR subunit expression patterns within neurochemically defined cellular circuits of the mouse ENS, together with the functional contribution of GABAAR subtypes to GI contractility remains to be determined. Immunohistochemical analyses revealed that immunoreactivity for the GABAAR gamma (γ) 2 and alphas (α) 1, 2, 3 subunits was located on somatodendritic surfaces of neurochemically distinct myenteric plexus neurons, while being on axonal compartments of submucosal plexus neurons. In contrast, immunoreactivity for the α4-5 subunits was only detected in myenteric plexus neurons. Furthermore, α-γ2 subunit immunoreactivity was located on non-neuronal interstitial cells of Cajal. In organ bath studies, GABAAR subtype-specific ligands had contrasting effects on the force and frequency of spontaneous colonic longitudinal smooth muscle contractions. Finally, enhancement of γ2-GABAAR function with alprazolam reversed the stress-induced increase in the force of spontaneous colonic contractions. The study demonstrates the molecular and functional diversity of the GABAAR system within the mouse colon providing a framework for developing GABAAR-based therapeutics in GI disorders.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2014; 34(31):10361-10378. DOI:10.1523/JNEUROSCI.0441-14.2014 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dorsal raphe nucleus (DRN) provides the major source of serotonin to the central nervous system (CNS) and modulates diverse neural functions including mood. Furthermore, DRN cellular networks are engaged in the stress-response at the CNS level allowing for adaptive behavioural responses, whilst stress-induced dysregulation of DRN and serotonin release is implicated in psychiatric disorders. Therefore, identifying the molecules regulating DRN activity is fundamental to understand DRN function in health and disease. GABAA receptors (GABAARs) allow for brain region, cell type and subcellular domain-specific GABA-mediated inhibitory currents and are thus key regulators of neuronal activity. Yet, the GABAAR subtypes expressed within the neurochemically diverse cell types of the mouse DRN are poorly described. In this study, immunohistochemistry and confocal microscopy revealed that all serotonergic neurons expressed immunoreactivity for the GABAAR alpha2 and 3 subunits, although the respective signals were co-localised to varying degrees with inhibitory synaptic marker proteins. Only a topographically located sub-population of serotonergic neurons exhibited GABAAR alpha1 subunit immunoreactivity. However, all GABAergic as well as non-GABAergic, non-serotonergic neurons within the DRN expressed GABAAR alpha1 subunit immunoreactivity. Intriguingly, immunoreactivity for the GABAAR gamma2 subunit was enriched on GABAergic rather than serotonergic neurons. Finally, repeated restraint stress increased the expression of the GABAAR alpha3 subunit at the mRNA and protein level. The study demonstrates the identity and location of distinct GABAAR subunits within the cellular networks of the mouse DRN and that stress impacts on the expression levels of particular subunits at the gene and protein level.
Brain Structure and Function 06/2014; 220(5). DOI:10.1007/s00429-014-0824-7 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transient receptor potential (TRP) channels are abundant in the brain where they regulate transmission of sensory signals. The expression patterns of different TRPC subunits (TRPC1, 4, and 5) are consistent with their potential role in fear-related behaviors. Accordingly, we found recently that mutant mice lacking a specific TRP channel subunit, TRPC5, exhibited decreased innate fear responses. Both TRPC5 and another member of the same subfamily, TRPC4, form heteromeric complexes with the TRPC1 subunit (TRPC1/5 and TRPC1/4, respectively). As TRP channels with specific subunit compositions may have different functional properties, we hypothesized that fear-related behaviors could be differentially controlled by TRPCs with distinct subunit arrangements. In this study, we focused on the analysis of mutant mice lacking the TRPC4 subunit, which, as we confirmed in experiments on control mice, is expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, we found that constitutive ablation of TRPC4 was associated with diminished anxiety levels (innate fear). Furthermore, knockdown of TRPC4 protein in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of constitutive TRPC4-null (TRPC4(-/-)) mouse. Recordings in brain slices demonstrated that these behavioral modifications could stem from the lack of TRPC4 potentiation in neurons in the lateral nucleus of the amygdala through two Gαq/11 protein-coupled signaling pathways, activated via Group I metabotropic glutamate receptors and cholecystokinin 2 receptors, respectively. Thus, TRPC4 and the structurally and functionally related subunit, TRPC5, may both contribute to the mechanisms underlying regulation of innate fear responses.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 03/2014; 34(10):3653-67. DOI:10.1523/JNEUROSCI.2274-13.2014 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABAA receptors (α1GABAARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABAARs and agonistic properties at the other three benzodiazepine-sensitive GABAA receptor subtypes, is self-administered, and that the α2GABAARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to- arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines [α2(H101R) mice] did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABAARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABAARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABAARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior.Neuropsychopharmacology accepted article preview online, 19 February 2014; doi:10.1038/npp.2014.41.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 02/2014; 39(8). DOI:10.1038/npp.2014.41 · 7.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Benzodiazepines (BZs) are safe drugs for treating anxiety, sleep, and seizure disorders, but their use also results in unwanted effects including memory impairment, abuse, and dependence. The present study aimed to reveal the molecular mechanisms that may contribute to the effects of BZs in the hippocampus (HIP), an area involved in drug-related plasticity, by investigating the regulation of immediate early genes following BZ administration. Previous studies have demonstrated that both brain derived neurotrophic factor (BDNF) and c-Fos contribute to memory- and abuse-related processes that occur within the HIP, and their expression is altered in response to BZ exposure. In the current study, mice received acute or repeated administration of BZs and HIP tissue was analyzed for alterations in BDNF and c-Fos expression. Although no significant changes in BDNF or c-Fos were observed in response to twice-daily intraperitoneal (i.p.) injections of diazepam (10 mg/kg + 5 mg/kg) or zolpidem (ZP; 2.5 mg/kg + 2.5 mg/kg), acute i.p. administration of both triazolam (0.03 mg/kg) and ZP (1.0 mg/kg) decreased BDNF protein levels within the HIP relative to vehicle, without any effect on c-Fos. ZP specifically reduced exon IV-containing BDNF transcripts with a concomitant increase in the association of methyl-CpG binding protein 2 (MeCP2) with BDNF promoter IV, suggesting that MeCP2 activity at this promoter may represent a ZP-specific mechanism for reducing BDNF expression. ZP also increased the association of phosphorylated cAMP response element binding protein (pCREB) with BDNF promoter I. Future work should examine the interaction between ZP and DNA as the cause for altered gene expression in the HIP, given that BZs can enter the nucleus and intercalate into DNA directly.
PLoS ONE 12/2013; 8(12):e84806. DOI:10.1371/journal.pone.0084806 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The γ-aminobutyric acid (GABA) system plays a pivotal role in orchestrating the synchronicity of local networks and the functional coupling of different brain regions. Here we review the impact of the GABAA receptor subtypes on cognitive and emotional behavior, paying particular attention to five disease states: cognitive dysfunction and Down syndrome, anxiety disorders, depression, schizophrenia, and autism. Through the bidirectional modulation of tonic inhibition, α5-subunit-containing GABAA receptors permit the bidirectional modulation of cognitive processes, and a partial inverse agonist acting at the α5-subunit-containing GABAA receptor is in a clinical trial in individuals with Down syndrome. With regard to anxiety disorders, the viability of nonsedative anxiolytics based on the modulation of α2- and α3-subunit-containing GABAA receptors has been established in clinical proof-of-concept trials. Regarding the remaining three disease states, the GABA hypothesis of depression offers new options for antidepressant drug development, cognitive symptoms in schizophrenia are attributed to a cortical GABAergic deficit, and dysfunctional GABAergic inhibition is increasingly understood to contribute to the pathophysiology of autism spectrum disorders. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 54 is January 06, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: Drugs that enhance GABAergic inhibition alleviate inflammatory and neuropathic pain after spinal application. This antihyperalgesia occurs mainly through GABAA receptors (GABAARs) containing α2 subunits (α2-GABAARs). Previous work indicates that potentiation of these receptors in the spinal cord evokes profound antihyperalgesia also after systemic administration, but possible synergistic or antagonistic actions of supraspinal α2-GABAARs on spinal antihyperalgesia have not yet been addressed. Here we generated two lines of GABAAR-mutated mice, which either lack α2-GABAARs specifically from the spinal cord, or, which express only benzodiazepine-insensitive α2-GABAARs at this site. We analyzed the consequences of these mutations for antihyperalgesia evoked by systemic treatment with the novel non-sedative benzodiazepine site agonist HZ166 in neuropathic and inflammatory pain. Wild-type mice and both types of mutated mice had similar baseline nociceptive sensitivities and developed similar hyperalgesia. However, antihyperalgesia by systemic HZ166 was reduced in both mutated mouse lines by about 60% and was virtually indistinguishable from that of global point-mutated mice, in which all α2-GABAARs were benzodiazepine insensitive. The major (α2-dependent) component of GABAAR-mediated antihyperalgesia was therefore exclusively of spinal origin, whereas supraspinal α2-GABAARs had neither synergistic nor antagonistic effects on antihyperalgesia. Our results thus indicate that drugs that specifically target α2-GABAARs exert their antihyperalgesic effect through enhanced spinal nociceptive control. Such drugs may therefore be well-suited for the systemic treatment of different chronic pain conditions.Neuropsychopharmacology advance online publication, 18 September 2013; doi:10.1038/npp.2013.221.
Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 08/2013; 39(2). DOI:10.1038/npp.2013.221 · 7.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABAARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABAARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together, these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking ("endozepine") roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a therapy for epilepsy and other neurological disorders.