Presynaptic hyperexcitability reversed by positive allosteric modulation of a GABABR epilepsy variant.

GABABRs are key membrane proteins that continually adapt the excitability of the nervous system. These G-protein coupled receptors are activated by the brain's premier inhibitory neurotransmitter GABA. They are obligate heterodimers composed of GABA-binding GABABR1 and G-protein-coupling GABABR2 subunits. Recently, three variants (G693W, S695I, I705N) have been identified in the gene (GABBR2) encoding for GABABR2. Individuals that harbour any of these variants exhibit severe developmental epileptic encephalopathy and intellectual disability, but the underlying pathogenesis that is triggered in neurons, remains unresolved. Using a range of confocal imaging, flow cytometry, structural modelling, biochemistry, live cell Ca2+ imaging of presynaptic terminals, whole-cell electrophysiology of HEK-293T cells and neurons, and two-electrode voltage clamping of Xenopus oocytes we have probed the biophysical and molecular trafficking and functional profiles of G693W, S695I and I705N variants. We report that all three point mutations impair neuronal cell surface expression of GABABRs, reducing signalling efficacy. However, a negative effect evident for one variant perturbed neurotransmission by elevating presynaptic Ca2+ signalling. This is reversed by enhancing GABABR signalling via positive allosteric modulation. Our results highlight the importance of studying neuronal receptors expressed in nervous system tissue and provide new mechanistic insights into how GABABR variants can initiate neurodevelopmental disease whilst highlighting the translational suitability and therapeutic potential of allosteric modulation for correcting these deficits.

Brain carbon monoxide can suppress the rat micturition reflex through brain γ-aminobutyric acid receptors.

OBJECTIVES: To investigate roles of brain carbon monoxide (CO), an endogenous gasotransmitter, in regulation of the rat micturition reflex. METHODS: In urethane-anesthetized (0.8 g/kg, ip) male rats, evaluation of urodynamic parameters was started 1 h before intracerebroventricular administration of CORM-3 (CO donor) or ZnPP (non-selective inhibitor of heme oxygenase, a CO producing enzyme) and continued for 2 h after the administration. We also investigated effects of centrally pretreated SR95531 (GABAA receptor antagonist) or SCH50911 (GABAB receptor antagonist) on the CORM-3-induced response. RESULTS: CORM-3 significantly prolonged intercontraction intervals (ICIs) without changing maximal voiding pressure (MVP), while ZnPP significantly shortened ICI and reduced single-voided volume and bladder capacity without affecting MVP, post-voided residual volume, or voiding efficiency. The ZnPP-induced ICI shortening was reversed by CORM-3. The CORM-3-induced ICI prolongation was significantly attenuated by centrally pretreated SR95531 or SCH50911, respectively. CONCLUSIONS: Brain CO can suppress the rat micturition reflex through brain γ-aminobutyric acid (GABA) receptors.

Emerging modes of regulation of neuromodulatory G protein-coupled receptors.

In the nervous system, G protein-coupled receptors (GPCRs) control neuronal excitability, synaptic transmission, synaptic plasticity, and, ultimately, behavior through spatiotemporally precise initiation of a variety of signaling pathways. However, despite their critical importance, there is incomplete understanding of how these receptors are regulated to tune their signaling to specific neurophysiological contexts. A deeper mechanistic picture of neuromodulatory GPCR function is needed to fully decipher their biological roles and effectively harness them for the treatment of neurological and psychiatric disorders. In this review, we highlight recent progress in identifying novel modes of regulation of neuromodulatory GPCRs, including G protein- and receptor-targeting mechanisms, receptor-receptor crosstalk, and unique features that emerge in the context of chemical synapses. These emerging principles of neuromodulatory GPCR tuning raise critical questions to be tackled at the molecular, cellular, synaptic, and neural circuit levels in the future.

Positive allosteric modulators of the GABAB receptor: a new class of ligands with therapeutic potential for alcohol use disorder.

BACKGROUND: Positive allosteric modulators (PAMs) of the GABAB receptor constitute a new class of GABAB-receptor ligands. GABAB PAMs reproduce several pharmacological effects of the orthosteric GABAB receptor agonist, baclofen, although displaying a better safety profile. AIMS: This paper reviews the reducing or, frequently, even suppressing effects of all GABAB PAMs tested to date on multiple alcohol-related behaviours in laboratory rodents exposed to validated experimental models of human alcohol use disorder. RESULTS: Acute or repeated treatment with CGP7930, GS39783, BHF177, rac-BHFF, ADX71441, CMPPE, COR659, ASP8062, KK-92A, and ORM-27669 reduced excessive alcohol drinking, relapse- and binge-like drinking, operant alcohol self-administration, reinstatement of alcohol seeking, and alcohol-induced conditioned place preference in rats and mice. CONCLUSIONS: These effects closely mirrored those of baclofen; notably, they were associated to remarkably lower levels of tolerance and toxicity. The recent transition of ASP8062 to clinical testing will soon prove whether these highly consistent preclinical data translate to AUD patients.

Action of GABAB receptor on local network oscillation in somatosensory cortex of oral part: focusing on NMDA receptor.

The balance of activity between glutamatergic and GABAergic networks is particularly important for oscillatory neural activities in the brain. Here, we investigated the roles of GABAB receptors in network oscillation in the oral somatosensory cortex (OSC), focusing on NMDA receptors. Neural oscillation at the frequency of 8-10 Hz was elicited in rat brain slices after caffeine application. Oscillations comprised a non-NMDA receptor-dependent initial phase and a later NMDA receptor-dependent oscillatory phase, with the oscillator located in the upper layer of the OSC. Baclofen was applied to investigate the actions of GABAB receptors. The later NMDA receptor-dependent oscillatory phase completely disappeared, but the initial phase did not. These results suggest that GABAB receptors mainly act on NMDA receptor, in which metabotropic actions of GABAB receptors may contribute to the attenuation of NMDA receptor activities. A regulatory system for network oscillation involving GABAB receptors may be present in the OSC.

GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

Cell-Type-Specific Effects of Somatostatin on Synaptic Transmission in the Anterior Cingulate Cortex.

Inhibitory modulation of glutamatergic information processing is a prerequisite for proper network function. Among the many groups of interneurons (INs), somatostatin-expressing interneurons (SOM-INs) play an important role in the maintenance of physiological brain activity. We have previously shown that somatostatin (SOM) causes a reduction in pyramidal cell (PC) excitability. However, the mechanisms of action of the peptide on cortical synaptic circuits are still unclear. To understand the effects of the neuropeptide SOM on cortical synaptic circuits, we performed a detailed side-by-side comparison of its postsynaptic effects on PCs, SOM-INs, and layer 1 interneurons (L1-INs) in the anterior cingulate cortex of male and female mice and found that SOM produced pronounced postsynaptic effects in PCs while having little to no effect on either IN type. This comparison allowed us to link the observed postsynaptic effects to SOM-induced modulations of glutamatergic and GABAergic synaptic transmission and to trace the impact of the neuropeptide on the neuronal circuitry between these three cell types. We show here that SOM depresses glutamatergic synaptic transmission via a presynaptic mechanism while exerting a differential impact on GABAA receptor- and GABAB receptor-mediated transmission at the pre- and postsynaptic level resulting in a shift of inhibition in L2/3 PCs from L1-INs to SOM-INs. In summary, this study unravels a novel aspect by which SOM modulates synaptic signaling between PCs, L1-INs, and SOM-INs.

GABAB receptor-mediated modulation in the developing dorsal nucleus of the lateral lemniscus.

The dorsal nucleus of the lateral lemniscus (DNLL) is a GABAergic, reciprocally connected auditory brainstem structure that continues to develop postnatally in rodents. One key feature of the DNLL is the generation of a strong, prolonged, ionotropic, GABAA receptor-mediated inhibition. Possible GABAB receptor-mediated signalling is unexplored in the DNLL. Here, we used Mongolian gerbils of either sex to describe GABAB receptor-mediated modulation of postsynaptic potassium currents and synaptic inputs in postnatal (P) animals of days 10/11 and 23-28. Throughout development, we observed the presence of a Baclofen-activated GABAB receptor-enhanced potassium outward conductance that is capable of suppressing action potential generation. In P10/11, old gerbils GABAB receptor activation enhances glutamatergic and suppresses ionotropic GABAergic synaptic transmission. During development, this differential modulation becomes less distinct, because in P22-28, old animals Baclofen-activated GABAB receptors rather enhance ionotropic GABAergic synaptic transmission, whereas glutamatergic transmission is both enhanced and suppressed. Blocking GABAB receptors causes an increase in ionotropic GABAergic transmission in P10/11 old gerbils that was independent on stimulation frequency but depended on the type of short-term plasticity. Together with the lack of Baclofen-induced changes in the synaptic paired-pulse ratio of either input type, we suggest that GABAB receptor-mediated modulation is predominantly postsynaptic and activates different signalling cascades. Thus, we argue that in DNLL neurons, the GABAB receptor is a post-synaptically located signalling hub that alters signalling cascades during development for distinct targets.

Presynaptic GABAB receptors inhibit vomeronasal nerve transmission to accessory olfactory bulb mitral cells.

Vomeronasal sensory neurons (VSNs) recognize pheromonal and kairomonal semiochemicals in the lumen of the vomeronasal organ. VSNs send their axons along the vomeronasal nerve (VN) into multiple glomeruli of the accessory olfactory bulb (AOB) and form glutamatergic synapses with apical dendrites of mitral cells, the projection neurons of the AOB. Juxtaglomerular interneurons release the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Besides ionotropic GABA receptors, the metabotropic GABAB receptor has been shown to modulate synaptic transmission in the main olfactory system. Here we show that GABAB receptors are expressed in the AOB and are primarily located at VN terminals. Electrical stimulation of the VN provokes calcium elevations in VSN nerve terminals, and activation of GABAB receptors by the agonist baclofen abolishes calcium influx in AOB slice preparations. Patch clamp recordings reveal that synaptic transmission from the VN to mitral cells can be completely suppressed by activation of GABAB receptors. A potent GABAB receptor antagonist, CGP 52432, reversed the baclofen-induced effects. These results indicate that modulation of VSNs via activation of GABAB receptors affects calcium influx and glutamate release at presynaptic terminals and likely balances synaptic transmission at the first synapse of the accessory olfactory system.

Lambert-Eaton Myasthenic Syndrome Complicated by anti-GABAB Receptor Encephalitis.

A 74-year-old man experienced diplopia, generalized muscle weakness, and acute respiratory failure. He was diagnosed with Lambert-Eaton myasthenic syndrome (LEMS) and treated with immunotherapy, but no improvement was observed, and additional symptoms, including central apnea and hallucinations, appeared. Subsequent serum and cerebrospinal fluid (CSF) analyses confirmed the presence of GABAB receptor antibodies, indicating the coexistence of autoimmune encephalitis. Although there were no findings of malignancy, it is highly likely that occult small-cell lung carcinoma was present. When atypical symptoms occur in patients with LEMS, it is important to consider the possibility of concomitant autoimmune encephalitis.

The role of GABAB receptors in the subcortical pathways of the mammalian auditory system.

GABAB receptors are G-protein coupled receptors for the inhibitory neurotransmitter GABA. Functional GABAB receptors are formed as heteromers of GABAB1 and GABAB2 subunits, which further associate with various regulatory and signaling proteins to provide receptor complexes with distinct pharmacological and physiological properties. GABAB receptors are widely distributed in nervous tissue, where they are involved in a number of processes and in turn are subject to a number of regulatory mechanisms. In this review, we summarize current knowledge of the cellular distribution and function of the receptors in the inner ear and auditory pathway of the mammalian brainstem and midbrain. The findings suggest that in these regions, GABAB receptors are involved in processes essential for proper auditory function, such as cochlear amplifier modulation, regulation of spontaneous activity, binaural and temporal information processing, and predictive coding. Since impaired GABAergic inhibition has been found to be associated with various forms of hearing loss, GABAB dysfunction could also play a role in some pathologies of the auditory system.

Discrimination between leucine-rich glioma-inactivated 1 antibody encephalitis and gamma-aminobutyric acid B receptor antibody encephalitis based on ResNet18.

This study aims to discriminate between leucine-rich glioma-inactivated 1 (LGI1) antibody encephalitis and gamma-aminobutyric acid B (GABAB) receptor antibody encephalitis using a convolutional neural network (CNN) model. A total of 81 patients were recruited for this study. ResNet18, VGG16, and ResNet50 were trained and tested separately using 3828 positron emission tomography image slices that contained the medial temporal lobe (MTL) or basal ganglia (BG). Leave-one-out cross-validation at the patient level was used to evaluate the CNN models. The receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) were generated to evaluate the CNN models. Based on the prediction results at slice level, a decision strategy was employed to evaluate the CNN models' performance at patient level. The ResNet18 model achieved the best performance at the slice (AUC = 0.86, accuracy = 80.28%) and patient levels (AUC = 0.98, accuracy = 96.30%). Specifically, at the slice level, 73.28% (1445/1972) of image slices with GABAB receptor antibody encephalitis and 87.72% (1628/1856) of image slices with LGI1 antibody encephalitis were accurately detected. At the patient level, 94.12% (16/17) of patients with GABAB receptor antibody encephalitis and 96.88% (62/64) of patients with LGI1 antibody encephalitis were accurately detected. Heatmaps of the image slices extracted using gradient-weighted class activation mapping indicated that the model focused on the MTL and BG for classification. In general, the ResNet18 model is a potential approach for discriminating between LGI1 and GABAB receptor antibody encephalitis. Metabolism in the MTL and BG is important for discriminating between these two encephalitis subtypes.

A phase 1 randomized, placebo-controlled study to investigate potential interactions between ASP8062, a positive allosteric modulator of the GABAB receptor, and morphine in recreational opioid users.

BACKGROUND: Recent increases in opioid use and subsequent opioid use disorder are a major public health crisis in the United States. AIMS: This phase 1 randomized, placebo-controlled study investigated the safety, tolerability, and pharmacokinetics (PKs) of ASP8062, a γ-aminobutyric acid B receptor-positive allosteric modulator, with and without administration of morphine in participants who used opioids recreationally. METHODS: Participants were randomly assigned (2:1) to daily dosing with ASP8062 25 mg or placebo on days 1-10. On day 10, all participants received morphine as a single oral dose of 45 mg; assessments were performed on days 11-16. The primary end point was safety, evaluated as the nature, frequency, and severity of adverse events, and end-tidal CO2 levels. PK end points were a secondary outcome measure. RESULTS: A total of 24 participants (aged 21-54 years) received ASP8062 (n = 16) or placebo (n = 8). There were no deaths or serious adverse events leading to treatment discontinuation during the study. Most adverse events were mild, with numerically lower absolute number of adverse events reported with ASP8062 plus morphine versus placebo plus morphine. ASP8062 plus morphine did not increase respiratory depression, potential drug abuse- or withdrawal-related adverse events. There were no significant PK interactions. CONCLUSIONS: In this phase 1 study, we did not observe any unexpected safety signals or notable PK interactions with concomitant morphine administration. These data suggest a potentially low risk for an increase in drug abuse- or withdrawal-related adverse events or respiratory distress in participants exposed to ASP8062 and morphine.

Evaluating the effect of R-Baclofen and LP-211 on autistic behavior of the BTBR and Fmr1-KO mouse models.

Introduction: Autism spectrum disorder (ASD) is a persistent neurodevelopmental condition characterized by two core behavioral symptoms: impaired social communication and interaction, as well as stereotypic, repetitive behavior. No distinct cause of ASD is known so far; however, excitatory/inhibitory imbalance and a disturbed serotoninergic transmission have been identified as prominent candidates responsible for ASD etiology. Methods: The GABA B receptor agonist R-Baclofen and the selective agonist for the 5HT7 serotonin receptor LP-211 have been reported to correct social deficits and repetitive behaviors in mouse models of ASD. To evaluate the efficacy of these compounds in more details, we treated BTBR T+ Itpr3 tf /J and B6.129P2-Fmr1 tm1Cgr /J mice acutely with R-Baclofen or LP-211 and evaluated the behavior of animals in a series of tests. Results: BTBR mice showed motor deficits, elevated anxiety, and highly repetitive behavior of self-grooming. Fmr1-KO mice exhibited decreased anxiety and hyperactivity. Additionally, Fmr1-KO mice's ultrasonic vocalizations were impaired suggesting a reduced social interest and communication of this strain. Acute LP-211 administration did not affect the behavioral abnormalities observed in BTBR mice but improved repetitive behavior in Fmr1-KO mice and showed a trend to change anxiety of this strain. Acute R-Baclofen treatment improved repetitive behavior only in Fmr1-KO mice. Conclusion: Our results add value to the current available data on these mouse models and the respective compounds. Yet, additional studies are needed to further test R-Baclofen and LP-211 as potential treatments for ASD therapy.

RGS6 negatively regulates inhibitory G protein signaling in dopamine neurons and positively regulates binge-like alcohol consumption in mice.

BACKGROUND AND PURPOSE: Drugs of abuse, including alcohol, increase dopamine in the mesocorticolimbic system via actions on dopamine neurons in the ventral tegmental area (VTA). Increased dopamine transmission can activate inhibitory G protein signalling pathways in VTA dopamine neurons, including those controlled by GABAB and D2 receptors. Members of the R7 subfamily of regulator of G protein signalling (RGS) proteins can regulate inhibitory G protein signalling, but their influence on VTA dopamine neurons is unclear. Here, we investigated the influence of RGS6, an R7 RGS family memberthat has been implicated in the regulation of alcohol consumption in mice, on inhibitory G protein signalling in VTA dopamine neurons. EXPERIMENTAL APPROACH: We used molecular, electrophysiological and genetic approaches to probe the impact of RGS6 on inhibitory G protein signalling in VTA dopamine neurons and on binge-like alcohol consumption in mice. KEY RESULTS: RGS6 is expressed in adult mouse VTA dopamine neurons and it modulates inhibitory G protein signalling in a receptor-dependent manner, tempering D2 receptor-induced somatodendritic currents and accelerating deactivation of synaptically evoked GABAB receptor-dependent responses. RGS6-/- mice exhibit diminished binge-like alcohol consumption, a phenotype replicated in female (but not male) mice lacking RGS6 selectively in VTA dopamine neurons. CONCLUSIONS AND IMPLICATIONS: RGS6 negatively regulates GABAB - and D2 receptor-dependent inhibitory G protein signalling pathways in mouse VTA dopamine neurons and exerts a sex-dependent positive influence on binge-like alcohol consumption in adult mice. As such, RGS6 may represent a new diagnostic and/or therapeutic target for alcohol use disorder.

The GABA and GABA-Receptor System in Inflammation, Anti-Tumor Immune Responses, and COVID-19.

GABA and GABAA-receptors (GABAA-Rs) play major roles in neurodevelopment and neurotransmission in the central nervous system (CNS). There has been a growing appreciation that GABAA-Rs are also present on most immune cells. Studies in the fields of autoimmune disease, cancer, parasitology, and virology have observed that GABA-R ligands have anti-inflammatory actions on T cells and antigen-presenting cells (APCs), while also enhancing regulatory T cell (Treg) responses and shifting APCs toward anti-inflammatory phenotypes. These actions have enabled GABAA-R ligands to ameliorate autoimmune diseases, such as type 1 diabetes (T1D), multiple sclerosis (MS), and rheumatoid arthritis, as well as type 2 diabetes (T2D)-associated inflammation in preclinical models. Conversely, antagonism of GABAA-R activity promotes the pro-inflammatory responses of T cells and APCs, enhancing anti-tumor responses and reducing tumor burden in models of solid tumors. Lung epithelial cells also express GABA-Rs, whose activation helps maintain fluid homeostasis and promote recovery from injury. The ability of GABAA-R agonists to limit both excessive immune responses and lung epithelial cell injury may underlie recent findings that GABAA-R agonists reduce the severity of disease in mice infected with highly lethal coronaviruses (SARS-CoV-2 and MHV-1). These observations suggest that GABAA-R agonists may provide off-the-shelf therapies for COVID-19 caused by new SARS-CoV-2 variants, as well as novel beta-coronaviruses, which evade vaccine-induced immune responses and antiviral medications. We review these findings and further advance the notions that (1) immune cells possess GABAA-Rs to limit inflammation in the CNS, and (2) this natural "braking system" on inflammatory responses may be pharmacologically engaged to slow the progression of autoimmune diseases, reduce the severity of COVID-19, and perhaps limit neuroinflammation associated with long COVID.

A phase 1b study to investigate the potential interactions between ASP8062 and buprenorphine/naloxone in patients with opioid use disorder.

BACKGROUND: There is an unmet need for therapeutics with greater efficacy and tolerability for the treatment of opioid use disorder (OUD). ASP8062 is a novel compound with positive allosteric modulator activity on the γ-aminobutyric acid type B receptor under development for use with standard-of-care treatment for patients with OUD. AIMS: To investigate the safety, tolerability, interaction potential, and pharmacokinetics (PK) of ASP8062 in combination with buprenorphine/naloxone (B/N; Suboxone®). METHODS: In this phase 1, randomized, double-masked, placebo-controlled study, patients with OUD began B/N (titrated to 16/4 mg/day) treatment upon enrollment (induction, Days 1-4; maintenance, Days 5-18; downward titration, Days 19-26; and discharge, Day 27). On Day 12, patients received a single dose of ASP8062 60 mg or placebo with B/N and underwent safety and PK assessments. Primary endpoints included frequency and severity of treatment-emergent adverse events (TEAEs), clinical laboratory tests, respiratory depression, and suicidal ideation. Secondary endpoints investigated the impact of ASP8062 on B/N PK. RESULTS: Eighteen patients were randomized and completed the study (ASP8062, n = 12; placebo, n = 6). With this sample size typical for phase 1 drug-drug interaction studies, ASP8062 was well tolerated; most TEAEs were mild in severity, and none led to treatment withdrawal. ASP8062 did not enhance substance use-related TEAEs, respiratory depression, or suicidal ideation and did not have a clinically significant impact on the PK of B/N. CONCLUSIONS: In this phase 1 study, ASP8062 was safe, well tolerated, and did not enhance respiratory suppression induced by buprenorphine. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04447287.

Targeting the interaction of GABAB receptors with CaMKII with an interfering peptide restores receptor expression after cerebral ischemia and inhibits progressive neuronal death in mouse brain cells and slices.

Cerebral ischemia is the leading cause for long-term disability and mortality in adults due to massive neuronal death. Currently, there is no pharmacological treatment available to limit progressive neuronal death after stroke. A major mechanism causing ischemia-induced neuronal death is the excessive release of glutamate and the associated overexcitation of neurons (excitotoxicity). Normally, GABAB receptors control neuronal excitability in the brain via prolonged inhibition. However, excitotoxic conditions rapidly downregulate GABAB receptors via a CaMKII-mediated mechanism and thereby diminish adequate inhibition that could counteract neuronal overexcitation and neuronal death. To prevent the deleterious downregulation of GABAB receptors, we developed a cell-penetrating synthetic peptide (R1-Pep) that inhibits the interaction of GABAB receptors with CaMKII. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions restored cell surface expression and function of GABAB receptors. R1-Pep did not affect CaMKII expression or activity but prevented its T286 autophosphorylation that renders it autonomously and persistently active. Moreover, R1-Pep counteracted the aberrant downregulation of G protein-coupled inwardly rectifying K+ channels and the upregulation of N-type voltage-gated Ca2+ channels, the main effectors of GABAB receptors. The restoration of GABAB receptors activated the Akt survival pathway and inhibited excitotoxic neuronal death with a wide time window in cultured neurons. Restoration of GABAB receptors and neuroprotective activity of R1-Pep was verified by using brain slices prepared from mice after middle cerebral artery occlusion (MCAO). Treatment with R1-Pep restored normal GABAB receptor expression and GABA receptor-mediated K+ channel currents. This reduced MCAO-induced neuronal excitability and inhibited neuronal death. These results support the hypothesis that restoration of GABAB receptor expression under excitatory conditions provides neuroprotection and might be the basis for the development of a selective intervention to inhibit progressive neuronal death after ischemic stroke.