The discriminative stimulus effects of baclofen and gamma hydroxybutyrate in C57BL/6J mice.

Baclofen and γ-hydroxybutyrate (GHB) exert γ-aminobutyric acid (GABA)B receptor agonism and have therapeutic utility but possess different pharmacological activities. We examined whether separate groups of mice could be trained to discriminate either baclofen or GHB, and the contribution of GABAB receptors to discriminative stimulus effects. Male C57BL/6J mice were trained to discriminate either baclofen (3.2 mg/kg, intraperitoneal) or GHB (178 mg/kg, intraperitoneal) from saline under a fixed-ratio 10 schedule. The GABAB antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348) was used to pharmacologically assess GABAB receptor involvement. The selectivity of the resulting discriminations was assessed with the opioid agonist morphine and the benzodiazepine midazolam. In baclofen-trained mice, both baclofen and GHB were readily discriminated. Baclofen produced a maximum of 86% baclofen-appropriate responding. CGP 35348 (320 mg/kg, i.p.) produced a 4.7-fold rightward shift in the dose-effect function. GHB produced a maximum of 85.8% baclofen-appropriate responding. In GHB-trained mice, both GHB and baclofen were readily discriminated. In GHB-trained mice, GHB produced a maximum of 85.3% drug-appropriate responding; CGP 35348 (320 mg/kg, i.p.) produced a 1.8-fold rightward shift in the GHB discrimination dose-effect function. Baclofen produced up to 70.0% GHB-appropriate responding. CGP 35348 (320 mg/kg, i.p.) significantly antagonized baclofen discrimination and baclofen produced up to 37% GHB-appropriate responding up to doses that disrupted operant responding. Morphine did not produce substitution for either baclofen or GHB. Midazolam produced partial substitution for both. GHB and baclofen discrimination assays in mice provide a useful approach for examining different receptor types mediating the effects of these two drugs.

Somatostatin interneurons inhibit excitatory transmission mediated by astrocytic GABAB and presynaptic GABAB and adenosine A1 receptors in the hippocampus.

GABAergic network activity has been established to be involved in numerous physiological processes and pathological conditions. Extensive studies have corroborated that GABAergic network activity regulates excitatory synaptic networks by activating presynaptic GABAB receptors (GABAB Rs). It is well documented that astrocytes express GABAB Rs and respond to GABAergic network activity. However, little is known about whether astrocytic GABAB Rs regulate excitatory synaptic transmission mediated by GABAergic network activity. To address this issue, we combined whole-cell recordings, optogenetics, calcium imaging, and pharmacological approaches to specifically activate hippocampal somatostatin-expressing interneurons (SOM-INs), a type of interneuron that targets pyramidal cell dendrites, while monitoring excitatory synaptic transmission in CA1 pyramidal cells. We found that optogenetic stimulation of SOM-INs increases astrocyte Ca2+ signaling via the activation of astrocytic GABAB Rs and GAT-3. SOM-INs depress excitatory neurotransmission by activating presynaptic GABAB Rs and astrocytic GABAB Rs, the latter inducing the release of ATP/adenosine. In turn, adenosine inhibits excitatory synaptic transmission by activating presynaptic adenosine A1 receptors (A1 Rs). Overall, our results reveal a novel mechanism that SOM-INs activation-induced synaptic depression is partially mediated by the activation of astrocytic GABAB Rs.

GABAB receptors constrain glutamate presynaptic release and postsynaptic actions in substantia gelatinosa of rat spinal cord.

The substantia gelatinosa (SG, lamina II of spinal cord gray matter) is pivotal for modulating nociceptive information from the peripheral to the central nervous system. γ-Aminobutyric acid type B receptors (GABABRs), the metabotropic GABA receptor subtype, are widely expressed in pre- and postsynaptic structures of the SG. Activation of GABABRs by exogenous agonists induces both pre- and postsynaptic inhibition. However, the actions of endogenous GABA via presynaptic GABABRs on glutamatergic synapses, and the postsynaptic GABABRs interaction with glutamate, remain elusive. In the present study, first, using in vitro whole-cell recordings and taking minimal stimulation strategies, we found that in rat spinal cord glutamatergic synapses, blockade of presynaptic GABABRs switched "silent" synapses into active ones and increased the probability of glutamate release onto SG neurons; increasing ambient GABA concentration mimicked GABABRs activation on glutamatergic terminals. Next, using holographic photostimulation to uncage glutamate on postsynaptic SG neurons, we found that postsynaptic GABABRs modified glutamate-induced postsynaptic potentials. Taken together, our data identify that endogenous GABA heterosynaptically constrains glutamate release via persistently activating presynaptic GABABRs; and postsynaptically, GABABRs modulate glutamate responses. The results give new clues for endogenous GABA in modulating the nociception circuit of the spinal dorsal horn and shed fresh light on the postsynaptic interaction of glutamate and GABA.

COR758, a negative allosteric modulator of GABAB receptors.

Allosteric modulators of G protein coupled receptors (GPCRs), including GABABRs (GABABRs), are promising therapeutic candidates. While several positive allosteric modulators (PAM) of GABABRs have been characterized, only recently the first negative allosteric modulator (NAM) has been described. In the present study, we report the characterization of COR758, which acts as GABABR NAM in rat cortical membranes and CHO cells stably expressing GABABRs (CHO-GABAB). COR758 failed to displace the antagonist [3H]CGP54626 from the orthosteric binding site of GABABRs showing that it acts through an allosteric binding site. Docking studies revealed a possible new allosteric binding site for COR758 in the intrahelical pocket of the GABAB1 monomer. COR758 inhibited basal and GABABR-stimulated O-(3-[35Sthio)-triphosphate ([35S]GTPγS) binding in brain membranes and blocked the enhancement of GABABR-stimulated [35S]GTPγS binding by the PAM GS39783. Bioluminescent resonance energy transfer (BRET) measurements in CHO-GABAB cells showed that COR758 inhibited G protein activation by GABA and altered GABABR subunit rearrangements. Additionally, the compound altered GABABR-mediated signaling such as baclofen-induced inhibition of cAMP production in transfected HEK293 cells, agonist-induced Ca2+ mobilization as well as baclofen and the ago-PAM CGP7930 induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in CHO-GABAB cells. COR758 also prevented baclofen-induced outward currents recorded from rat dopamine neurons, substantiating its property as a NAM for GABABRs. Altogether, these data indicate that COR758 inhibits G protein signaling by GABABRs, likely by interacting with an allosteric binding-site. Therefore, COR758 might serve as a scaffold to develop additional NAMs for therapeutic intervention.

Presynaptic GABAB receptor inhibition sex dependently enhances fear extinction and attenuates fear renewal.

Anxiety and trauma-related disorders are highly prevalent worldwide, and are associated with altered associative fear learning. Despite the effectiveness of exposure therapy, which aims to reduce associative fear responses, relapse rates remain high. This is due, in part, to the context specificity of exposure therapy, which is a form of extinction. Many studies show that fear relapses when mice are tested outside the extinction context, and this is known as fear renewal. Using Pavlovian fear conditioning and extinction, we can study the mechanisms underlying extinction and renewal. The aim of the current experiment was to identify the role of presynaptic GABAB receptors in these two processes. Previous work from our lab showed that genetic deletion or pharmacological inhibition of GABAB(1a) receptors that provide presynaptic inhibition on glutamatergic terminals reduces context specificity and leads to generalization. We therefore hypothesized that inactivation of these presynaptic GABAB receptors could be used to reduce the context specificity associated with fear extinction training and suppress renewal when mice are tested outside of the extinction context. Using CGP 36216, an antagonist specific for presynaptic GABAB receptors, we blocked presynaptic GABAB receptors using intracerebroventricular injections during various time points of extinction learning in male and female mice. Results showed that blocking these receptors pre- and post-extinction training led to enhanced extinction learning in male mice only. We also found that post-extinction infusions of CGP reduced renewal rates in male mice when they were tested outside of the extinction context. In an attempt to localize the function of presynaptic GABAB receptors within regions of the extinction circuit, we infused CGP locally within the basolateral amygdala or dorsal hippocampus. We failed to reduce renewal when CGP was infused directly within these regions, suggesting that presynaptic inhibition within these regions per se may not be necessary for driving context specificity during extinction learning. Together, these results show an important sex-dependent role of presynaptic GABAB receptors in extinction and renewal processes and identify a novel receptor target that may be used to design pharmacotherapies to enhance the effectiveness of exposure therapy.

Reelin signaling modulates GABAB receptor function in the neocortex.

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells.

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors.

GABA interneurons play a critical role in higher brain functions. Astrocytic glial cells interact with synapses throughout the whole brain and are recognized as regulatory elements of excitatory synaptic transmission. However, it is largely unknown how GABAergic interneurons and astrocytes interact and contribute to stable performance of complex behaviors. Here, we found that genetic ablation of GABAB receptors in medial prefrontal cortex astrocytes altered low-gamma oscillations and firing properties of cortical neurons, which affected goal-directed behaviors. Remarkably, working memory deficits were restored by optogenetic stimulation of astrocytes with melanopsin. Furthermore, melanopsin-activated astrocytes in wild-type mice enhanced the firing rate of cortical neurons and gamma oscillations, as well as improved cognition. Therefore, our work identifies astrocytes as a hub for controlling inhibition in cortical circuits, providing a novel pathway for the behaviorally relevant midrange time-scale regulation of cortical information processing and consistent goal-directed behaviors.

Alterations of GABA B receptors in the APP/PS1 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive decline of memory and cognitive function. The disease is characterized by the presence of amyloid plaques, tau tangles, altered inflammatory signaling, and alterations in numerous neurotransmitter signaling systems, including γ-aminobutyric acid (GABA). Given the extensive role of GABA in regulating neuronal activity, a careful investigation of GABA-related changes is needed. Further, given persistent inflammation has been demonstrated to drive AD pathology, the presence of GABA B receptor expressed on glia that serve a role regulation of the immune response adds to potential implications of altered GABA in AD. There has not previously been a systematic evaluation of GABA-related changes in an amyloid model of AD that specifically focuses on examining changes in GABA B receptors. In the present study, we examined alterations in several GABA-specific targets in the APP/PS1 mouse model at different ages. In the 4-month-old cohort, no significant deficits in spatial learning and memory or alterations in any of the GABAergic targets were observed compared with wild-type controls. However, we identified significant alterations in several GABA-related targets in the 6-month-old cohort that exhibited spatial learning deficits that include changes in glutamic acid decarboxylase 65, GABA transporter type 3, and GABA B receptors protein and mRNA levels. This was the same cohort at which learning and memory deficits and significant amyloid pathology was observed. Overall, our study provides evidence of altered GABAergic signaling in an amyloid model of AD at a time point consistent with AD-related deficits.

An Etiological Foxp2 Mutation Impairs Neuronal Gain in Layer VI Cortico-Thalamic Cells through Increased GABAB/GIRK Signaling.

A rare mutation affecting the Forkhead-box protein P2 (FOXP2) transcription factor causes a severe monogenic speech and language disorder. Mice carrying an identical point mutation to that observed in affected patients (Foxp2+/R552H mice) display motor deficits and impaired synaptic plasticity in the striatum. However, the consequences of the mutation on neuronal function, in particular in the cerebral cortex, remain little studied. Foxp2 is expressed in a subset of Layer VI cortical neurons. Here, we used Ntsr1-EGFP mice to identify Foxp2+ neurons in the mouse auditory cortex ex vivo. We studied the functional impact of the R552H mutation on the morphologic and functional properties of Layer VI cortical neurons from Ntsr1-EGFP; Foxp2+/R552H male and female mice. The complexity of apical, but not basal dendrites was significantly lower in Foxp2+/R552H cortico-thalamic neurons than in control Foxp2+/+ neurons. Excitatory synaptic inputs, but not inhibitory synaptic inputs, were decreased in Foxp2+/R552H mice. In response, homeostatic mechanisms would be expected to increase neuronal gain, i.e., the conversion of a synaptic input into a firing output. However, the intrinsic excitability of Foxp2+ cortical neurons was lower in Foxp2+/R552H neurons. A-type and delayed-rectifier (DR) potassium currents, two putative transcriptional targets of Foxp2, were not affected by the mutation. In contrast, GABAB/GIRK signaling, another presumed target of Foxp2, was increased in mutant neurons. Blocking GIRK channels strongly attenuated the difference in intrinsic excitability between wild-type (WT) and Foxp2+/R552H neurons. Our results reveal a novel role for Foxp2 in the control of neuronal input/output homeostasis.SIGNIFICANCE STATEMENT Mutations of the Forkhead-box protein 2 (FOXP2) gene in humans are the first known monogenic cause of a speech and language disorder. The Foxp2 mutation may directly affect neuronal development and function in neocortex, where Foxp2 is expressed. Brain imaging studies in patients with a heterozygous mutation in FOXP2 showed abnormalities in cortical language-related regions relative to the unaffected members of the same family. However, the role of Foxp2 in neocortical neurons is poorly understood. Using mice with a Foxp2 mutation equivalent to that found in patients, we studied functional modifications in auditory cortex neurons ex vivo We found that mutant neurons exhibit alterations of synaptic input and GABAB/GIRK signaling, reflecting a loss of neuronal homeostasis.

Role of the GABAa and GABAb receptors of the central nucleus of the amygdala in compulsive cocaine-seeking behavior in male rats.

RATIONALE: Compulsive cocaine use, defined as the continued use despite the dire consequences, is a hallmark of cocaine addiction. Thus, understanding the brain mechanism regulating the compulsive cocaine-seeking and cocaine-taking behaviors is essential to understand cocaine addiction and the key to identification of the molecular targets for the development of medications against this condition. OBJECTIVE: This study aimed to determine how the GABAa and GABAb receptors of the central nucleus of the amygdala (CeA) regulate the compulsive cocaine-seeking behavior. METHODS: Male Wistar outbred rats were trained to self-administer intravenous cocaine (0.4 mg/kg/infusion) under a chained schedule. The compulsive cocaine-seeking behavior was measured as the cocaine-seeking behavior in the face of footshock punishment. The role of the GABA receptors of CeA in the regulation of such behavior was determined by measuring the dose-dependent effects of the GABAa agonist muscimol or the GABAb agonist baclofen bilaterally microinjected into the CeA on the punished cocaine-seeking behavior. RESULTS: The cocaine-seeking behavior was inhibited by footshock punishment in an intensity-dependent manner. Both muscimol and baclofen dose-dependently increased the punished cocaine-seeking behavior. However, the potency of muscimol but not baclofen was negatively correlated with the effects of punishment. CONCLUSION: These data indicate that the CeA GABAa receptors play a key role in the regulation of the compulsive cocaine-seeking behavior and suggest that an increase in the function of the GABAa receptors possibly induced by cocaine or genetic factors may be an important mechanism involved in the development of or vulnerability to the compulsive cocaine use and addiction.

GABAB receptor intracellular signaling: novel pathways for depressive disorder treatment?

Affecting over 320 million people around the world, depression has become a formidable challenge for modern medicine. In addition, an increasing number of studies cast doubt on the monoamine theory of depressive disorder and, worryingly, antidepressant medications only significantly benefit patients with severe depression. Thus, it is not surprising that researchers have shown an increased interest in new theories attempting to explain the pathogenesis of this disease. One example is the excitatory/inhibitory transmission imbalance theory. These abnormalities involve glutamate and γ-aminobutyric acid (GABA) signaling. Studies on GABAB receptors and their antagonists are particularly promising for the treatment of depressive disorders. In this paper, intracellular pathways controlled by GABAB receptors and their links to depression are described, including the impact of ketamine on GABAergic synaptic transmission.

Presynaptic GABAB receptors underlie the antiepileptic effect of low-frequency electrical stimulation in the 4-aminopyridine model of epilepsy in brain slices of young rats.

Low-frequency electrical stimulation (LFES) of the brain is one of the promising methods for helping patients with pharmacoresistant epilepsy. However, the mechanism of the antiepileptic effect of LFES is still unclear. We applied electrophysiological and pharmacological tools and mathematical modeling to investigate it. Using the 4-aminopyridine (4-AP) model of epileptiform activity in juvenile rat brain slices, we found that LFES increased the interval between ictal discharges (IDs) in the entorhinal cortex. The blockade of GABAA, GABAB, AMPA, or NMDA synaptic receptors strongly affected the characteristics of epileptiform discharges in slices. However, only under the blockade of GABAB receptors, LFES becomes entirely ineffective, indicating that the activation of GABAB receptors underlies the main LFES antiepileptic effect. Further experiments allowed us to suggest that LFES activates mostly presynaptic GABAB receptors, which decrease the probability of glutamate release. In line with this hypothesis is the following data: 1) LFES reduces the short-term synaptic depression of excitatory postsynaptic currents similar to the agonist of GABAB receptors SKF-97541; 2) the blockade of excitatory amino acid transporters diminishes the antiepileptic effect of LFES; 3) modeling of the effects of LFES on the probability of glutamate release with a previously proposed mathematical model of epileptiform activity Epileptor-2 also shows the increase of the interval between IDs. Our findings point out a crucial role of presynaptic GABAB receptors in the antiepileptic effect of LFES in the 4-AP model in juvenile rat brain slices.

Haploinsufficiency of Tsc2 Leads to Hyperexcitability of Medial Prefrontal Cortex via Weakening of Tonic GABAB Receptor-mediated Inhibition.

Loss-of-function mutation in one of the tumor suppressor genes TSC1 or TSC2 is associated with several neurological and psychiatric diseases, including autism spectrum disorders (ASDs). As an imbalance between excitatory and inhibitory neurotransmission, E/I ratio is believed to contribute to the development of these disorders, we investigated synaptic transmission during the first postnatal month using the Tsc2+/- mouse model. Electrophysiological recordings were performed in acute brain slices of medial prefrontal cortex. E/I ratio at postnatal day (P) 15-19 is increased in Tsc2+/- mice as compared with wildtype (WT). At P25-30, facilitated GABAergic transmission reduces E/I ratio to the WT level, but weakening of tonic GABAB receptor (GABABR)-mediated inhibition in Tsc2+/- mice leads to hyperexcitability both at single cell and neuronal network level. Short (1 h) preincubation of P25-30 Tsc2+/- slices with baclofen restores the GABABR-mediated inhibition and reduces network excitability. Interestingly, the same treatment at P15-19 leads to weakening of GABABR-mediated inhibition. We hypothesize that a dysfunction of tonic GABABR-mediated inhibition might contribute to the development of ASD symptoms and suggest that GABABR activation within an appropriate time window may be considered as a therapeutic target in ASD.

GABAB receptor-mediated tonic inhibition of locus coeruleus neurons plays a role in deep anesthesia induced by isoflurane.

Noradrenergic neurons in the locus coeruleus referred to as locus coeruleus neurons, provide the major supply of norepinephrine to the forebrain and play important roles in behavior through regulation of wakefulness and arousal. In a previous study using brain slice preparations, we reported that locus coeruleus neurons are subject to tonic inhibition mediated by γ-aminobutyric acid B receptors (GABABRs) and that the extent of tonic inhibition varies with ambient GABA levels. Since ambient GABA in the locus coeruleus was reported to fluctuate during the sleep-wakefulness cycle, here we tested whether GABABR-mediated tonic inhibition of locus coeruleus neurons could be a mechanism underlying changes in brain arousal. We first demonstrated that GABABR-mediated tonic inhibition of locus coeruleus neurons also exists in vivo by showing that local infusion of CGP35348, a GABABR antagonist, into the locus coeruleus increased the firing rate of locus coeruleus neurons in anesthetized rats. We then showed that this manipulation accelerated the behavioral emergence of rats from deep anesthesia induced by isoflurane. Together, these observations show that GABABR-mediated tonic inhibition of locus coeruleus neurons occurs in vivo and support the idea that this effect may be important in regulating the functional state of the brain.

Modulation of Cough Reflex by Gaba-Ergic Inhibition in Medullary Raphé of the Cat.

We studied the effects of GABA receptor agonists microinjections in medullary raphé on the mechanically induced tracheobronchial cough response in anesthetized, unparalyzed, spontaneously breathing cats. The results suggest that GABA-ergic inhibition significantly contributes to the regulation of cough reflex by action of both GABA(A) and GABA(B) receptors. The data are consistent with inhomogeneous occurrence of GABA-ergic neurons in medullary raphé and their different involvement in the cough reflex control. Cells within rostral nucleus raphéobscurus with dominant role of GABA(A) receptors and neurons of rostral nucleus raphépallidus and caudal nucleus raphémagnus with dominant role of GABA(B) receptors participate in regulation of cough expiratory efforts. These cough control elements are distinct from cough gating mechanism. GABA-ergic inhibition in the raphé caudal to obex had insignificant effect on cough. Contradictory findings for GABA, muscimol and baclofen administration in medullary raphé suggest involvement of coordinated activity of GABA on multiple receptors affecting raphé neurons and/or the local neuronal circuits in the raphé modulating cough motor drive.

The organizing principle of GABAB receptor complexes: Physiological and pharmacological implications.

GABAB receptors (GBRs), the G protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA), regulate synaptic transmission at most synapses in the brain. Proteomic approaches revealed that native GBR complexes assemble from an inventory of ~30 proteins that provide a molecular basis for the functional diversity observed with these receptors. Studies with reconstituted GBR complexes in heterologous cells and complementary knockout studies have allowed to identify cellular and physiological functions for obligate and several non-obligate receptor components. It emerges that modular association of receptor components in space and time generates a variety of multiprotein receptor complexes with different localizations, kinetic properties and effector channels. This article summarizes current knowledge on the organizing principle of GBR complexes. We further discuss unanticipated receptor functions, links to disease and opportunities for drug discovery arising from the identification of novel receptor components.

Pharmacological targeting of the GABAB receptor alters Drosophila's behavioural responses to alcohol.

When exposed to ethanol, Drosophila melanogaster display a variety of addiction-like behaviours similar to those observed in mammals. Sensitivity to ethanol can be quantified by measuring the time at which 50% of the flies are sedated by ethanol exposure (ST50); an increase of ST50 following multiple ethanol exposures is widely interpreted as development of tolerance to ethanol. Sensitivity and tolerance to ethanol were measured after administration of the gamma-aminobutyric acid receptor B (GABAB ) agonist (SKF 97541) and antagonist (CGP 54626), when compared with flies treated with ethanol alone. Dose-dependent increases and decreases in sensitivity to ethanol were observed for both the agonist and antagonist respectively. Tolerance was recorded in the presence of GABAB drugs, but the rate of tolerance development was increased by SKF 97451 and unaltered in presence of CGP 54626. This indicates that the GABAB receptor contributes to both the sensitivity to ethanol and mechanisms by which tolerance develops. The data also reinforce the usefulness of Drosophila as a model for identifying the molecular components of addictive behaviours and for testing drugs that could potentially be used for the treatment of alcohol use disorder (AUD).

Long-lasting changes in hippocampal GABAB-receptor mediated inhibition following early-life seizures in kindling-prone but not kindling-resistant rats.

The hypotheses that hippocampal GABAB receptor dysfunction is a long-lasting consequence of early-life seizures, and its dependence on genetic background, were tested. Two strains of rats bred to be prone (FAST) or resistant (SLOW) to amygdala kindling were used. On postnatal day (PND) 10, control rats were injected with saline, and seizure rats with kainic acid to induce status epilepticus (SE) for 2 h. A significantly lower dose of kainic acid was found to induce SE in FAST as compared to SLOW rats. Population excitatory postsynaptic potentials (pEPSPs) and population spikes (PSs) were recorded in CA1 of hippocampal slices of adult rats in vitro, following stimulation of stratum radiatum. Input-output relation of the single-pulse pEPSP and PS did not show a significant difference between seizure and control rats, sex, or strain (FAST and SLOW). Paired-pulse PSs were significantly enhanced at 10-50 ms interpulse intervals, in FAST seizure male rats compared to FAST male controls, but not in other groups. In adult FAST but not SLOW rats, significantly lower suppression of pEPSPs at 250-300 ms following heterosynaptic burst stimulation was found in seizure rats compared to control rats; the heterosynaptic suppression of the pEPSP was blocked by selective GABAB receptor antagonist CGP55845A. The results provide evidence that an early-life SE has a long-lasting effect in decreasing GABAB receptor-mediated presynaptic inhibition in the hippocampus, in FAST but not in SLOW rats.

Enhancement of mouse diaphragm contractility in the presence of antagonists of GABAA and GABAB receptors.

NEW FINDINGS: What is the central question of this study? Do GABA receptors play any role at the neuromuscular junction? What is the main finding and its importance? In the presence of either ionotropic or metabotropic GABA receptor antagonists, diaphragm muscle force production elicited by stimulating the motor nerve at ≥50 Hz was increased. Our data indicate the presence of GABAergic signalling at the neuromuscular junction. ABSTRACT: Despite the signalling role of GABA in the brain and spinal cord, the role of this molecule in the peripheral nervous system and, in particular, at the neuromuscular junction remains practically unexplored. In the present work, the force of mouse diaphragm contractions was measured in the presence of blockers of metabotropic GABAB receptors (CGP 55845) and ionotropic GABAA receptors (picrotoxin) with various patterns of indirect and direct stimulation of muscle by trains of 40 pulses delivered at 10, 20, 50 and 70 Hz. It was found that neither blocker affected the diaphragm contractility caused by indirect stimulation through the motor nerve at 10 and 20 Hz. However, when the stimulation frequency was increased to 50 or 70 Hz, the force of subsequent contractions in the train (when compared with the amplitude of contraction in response to the first pulse) was increased by both CGP 55845 and picrotoxin. With direct stimulation of the diaphragm, no significant changes in the contraction force were detected at any frequency used. The results obtained support the following conclusions: (i) pharmacological inhibition of GABA receptors increases the contractile activity of skeletal muscle; and (ii) frequency-dependent enhancement of GABA receptor activation takes place in the region of the neuromuscular junction.