The deuterated pyrazoloquinolinone targeting α6 subunit-containing GABAA receptor as novel candidate for inhibition of trigeminovascular system activation: implication for migraine therapy.

Introduction: The α6 subunit-containing GABAA receptors (α6GABAARs) are highly expressed in the trigeminal ganglia (TG), the sensory hub of the trigeminovascular system (TGVS). Hypo-GABAergic transmission in the TG was reported to contribute to migraine-related behavioral and histopathological phenotypes. Previously, we found that Compound 6, an α6GABAAR-selective positive allosteric modulator (PAM), significantly alleviated TGVS activation-induced peripheral and central sensitization in a capsaicin-induced migraine-mimicking model. Methods: Here, we tested whether the deuterated analogues of Compound 6, namely DK-1-56-1 and RV-I-29, known to have longer half-lives than the parent compound, can exert a similar therapeutic effect in the same model. The activation of TGVS was triggered by intra-cisternal (i.c.) instillation of capsaicin in male Wistar rats. Centrally, i.c. capsaicin increased the quantity of c-Fos-immunoreactive (c-Fos-ir) neurons in the trigeminal cervical complex (TCC). Peripherally, it increased the calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG, and caused CGRP release, leading to CGRP depletion in the dura mater. Results: DK-I-56-1 and RV-I-29, administered intraperitoneally (i.p.), significantly ameliorated the TCC neuronal activation, TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin, with DK-I-56-1 demonstrating better efficacy. The therapeutic effects of 3 mg/kg DK-I-56-1 are comparable to that of 30 mg/kg topiramate. Notably, i.p. administered furosemide, a blood-brain-barrier impermeable α6GABAAR-selective antagonist, prevented the effects of DK-I-56-1 and RV-I-29. Lastly, orally administered DK-I-56-1 has a similar pharmacological effect. Discussion: These results suggest that DK-I-56-1 is a promising candidate for novel migraine pharmacotherapy, through positively modulating TG α6GABAARs to inhibit TGVS activation, with relatively favourable pharmacokinetic properties.

The de novo missense mutation F224S in GABRB2, identified in epileptic encephalopathy and developmental delay, impairs GABAAR function.

Genetic variants in genes encoding subunits of the γ-aminobutyric acid-A receptor (GABAAR) have been found to cause neurodevelopmental disorders and epileptic encephalopathy. In a patient with epilepsy and developmental delay, a de novo heterozygous missense mutation c.671 T > C (p.F224S) was discovered in the GABRB2 gene, which encodes the ß2 subunit of GABAAR. Based on previous studies on GABRB2 variants, this new GABRB2 variant (F224S) would be pathogenic. To confirm and investigate the effects of this GABRB2 mutation on GABAAR channel function, we conducted transient expression experiments using GABAAR subunits in HEK293T cells. The GABAARs containing mutant ß2 (F224S) subunit showed poor trafficking to the cell membrane, while the expression and distribution of the normal α1 and γ2 subunits were unaffected. Furthermore, the peak current amplitude of the GABAAR containing the ß2 (F224S) subunit was significantly smaller compared to the wild type GABAAR. We propose that GABRB2 variant F224S is pathogenic and GABAARs containing this ß2 mutant reduce response to GABA under physiological conditions, which could potentially disrupt the excitation/inhibition balance in the brain, leading to epilepsy.

Wheel-Running Exercise Alleviates Anxiety-Like Behavior via Down-Regulating S-Nitrosylation of Gephyrin in the Basolateral Amygdala of Male Rats.

Physical exercise has beneficial effect on anxiety disorders, but the underlying molecular mechanism remains largely unknown. Here, it is demonstrated that physical exercise can downregulate the S-nitrosylation of gephyrin (SNO-gephyrin) in the basolateral amygdala (BLA) to exert anxiolytic effects. It is found that the level of SNO-gephyrin is significantly increased in the BLA of high-anxiety rats and a downregulation of SNO-gephyrin at cysteines 212 and 284 produced anxiolytic effect. Mechanistically, inhibition of SNO-gephyrin by either Cys212 or Cys284 mutations increased the surface expression of GABAAR γ2 and the subsequent GABAergic neurotransmission, exerting anxiolytic effect in male rats. On the other side, overexpression of neuronal nitric oxide synthase in the BLA abolished the anxiolytic-like effects of physical exercise. This study reveals a key role of downregulating SNO-gephyrin in the anxiolytic effects of physical exercise, providing a new explanation for protein post-translational modifications in the brain after exercise.

Activation of GABA type A receptor is involved in the anti-insomnia effect of Huanglian Wendan Decoction.

Huanglian Wendan Decoction (HWD) is a traditional Chinese medicine (TCM) prescribed to patients diagnosed with insomnia, which can achieve excellent therapeutic outcomes. As positively modulating the γ-aminobutyric acid (GABA) type A receptors (GABAARs) is the most effective strategy to manage insomnia, this study aimed to investigate whether the activation of GABAARs is involved in the anti-insomnia effect of HWD. We assessed the metabolites of HWD using LC/MS and the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and tested the pharmacological activity in vitro and in vivo using whole-cell patch clamp and insomnia zebrafish model. In HEK293 cells expressing α1ß3γ2L GABAARs, HWD effectively increased the GABA-induced currents and could induce GABAAR-mediated currents independent of the application of GABA. In the LC-MS (QToF) assay, 31 metabolites were discovered in negative ion modes and 37 metabolites were found in positive ion modes, but neither three selected active metabolites, Danshensu, Coptisine, or Dihydromyricetin, showed potentiating effects on GABA currents. 62 active metabolites of the seven botanical drugs were collected based on the TCMSP database and 19 of them were selected for patch-clamp verification according to the virtual docking simulations and other parameters. At a concentration of 100 µM, GABA-induced currents were increased by (+)-Cuparene (278.80% ± 19.13%), Ethyl glucoside (225.40% ± 21.77%), and ß-Caryophyllene (290.11% ± 17.71%). In addition, (+)-Cuparene, Ethyl glucoside, and ß-Caryophyllene could also serve as positive allosteric modulators (PAMs) and shifted the GABA dose-response curve (DRC) leftward significantly. In the PCPA-induced zebrafish model, Ethyl glucoside showed anti-insomnia effects at concentrations of 100 µM. In this research, we demonstrated that the activation of GABAARs was involved in the anti-insomnia effect of HWD, and Ethyl glucoside might be a key metabolite in treating insomnia.

Sevoflurane-induced overexpression of extrasynaptic α5-GABAAR via the RhoA/ROCK2 pathway impairs cognitive function in aged mice.

Perioperative neurocognitive disorder (PND) is a serious neurologic complication in aged patients and might be associated with sevoflurane exposure. However, the specific pathogenesis is still unclear. The distribution of α5-GABAAR, a γ-aminobutyric acid type A receptor (GABAAR) subtype, at extrasynaptic sites is influenced by the anchor protein radixin, whose phosphorylation is regulated via the RhoA/ROCK2 signaling pathway and plays a crucial role in cognition. However, whether sevoflurane affects the ability of radixin phosphorylation to alter extrasynaptic receptor expression is unknown. Aged mice were exposed to sevoflurane to induce cognitive impairment. Both total proteins and membrane proteins were extracted for analysis. Cognitive function was evaluated using the Morris water maze and fear conditioning test. Western blotting was used to determine the expression of ROCK2 and the phosphorylation of radixin. Furthermore, the colocalization of p-radixin and α5-GABAAR was observed. To inhibit ROCK2 activity, either an adeno-associated virus (AAV) or fasudil hydrochloride was administered. Aged mice treated with sevoflurane exhibited significant cognitive impairment accompanied by increased membrane expression of α5-GABAAR. Moreover, the colocalization of α5-GABAAR and p-radixin increased after treatment with sevoflurane, and this change was accompanied by an increase in ROCK2 expression and radixin phosphorylation. Notably, inhibiting the RhoA/ROCK2 pathway significantly decreased the distribution of extrasynaptic α5-GABAAR and improved cognitive function. Sevoflurane activates the RhoA/ROCK2 pathway and increases the phosphorylation of radixin. Excess α5-GABAAR is anchored to extrasynaptic sites and impairs cognitive ability in aged mice. Fasudil hydrochloride administration improves cognitive function.

The changing of α5-GABAA receptors expression and distribution participate in sevoflurane-induced learning and memory impairment in young mice.

BACKGROUND: Sevoflurane is a superior agent for maintaining anesthesia during surgical procedures. However, the neurotoxic mechanisms of clinical concentration remain poorly understood. Sevoflurane can interfere with the normal function of neurons and synapses and impair cognitive function by acting on α5-GABAAR. METHODS: Using MWM test, we evaluated cognitive abilities in mice following 1 h of anesthesia with 2.7%-3% sevoflurane. Based on hippocampal transcriptome analysis, we analyzed the differential genes and IL-6 24 h post-anesthesia. Western blot and RT-PCR were performed to measure the levels of α5-GABAAR, Radixin, P-ERM, P-Radixin, Gephyrin, IL-6, and ROCK. The spatial distribution and expression of α5-GABAAR on neuronal somata were analyzed using histological and three-dimensional imaging techniques. RESULTS: MWM test indicated that partial long-term learning and memory impairment. Combining molecular biology and histological analysis, our studies have demonstrated that sevoflurane induces immunosuppression, characterized by reduced IL-6 expression levels, and that enhanced Radixin dephosphorylation undermines the microstructural stability of α5-GABAAR, leading to its dissociation from synaptic exterior and resulting in a disordered distribution in α5-GABAAR expression within neuronal cell bodies. On the synaptic cleft, the expression level of α5-GABAAR remained unchanged, the spatial distribution became more compact, with an increased fluorescence intensity per voxel. On the extra-synaptic space, the expression level of α5-GABAAR decreased within unchanged spatial distribution, accompanied by an increased fluorescence intensity per voxel. CONCLUSION: Dysregulated α5-GABAAR expression and distribution contributes to sevoflurane-induced partial long-term learning and memory impairment, which lays the foundation for elucidating the underlying mechanisms in future studies.

Basmisanil, an α5-GABAAR negative allosteric modulator, produces rapid and sustained antidepressant-like responses in male mice.

There is a critical need for safer and better-tolerated alternatives to address the current limitations of antidepressant treatments for major depressive disorder. Recently, drugs targeting the GABA system via α5-containing GABAA receptors (α5-GABAAR) as negative allosteric modulators (α5-NAMs) have shown promise in alleviating stress-related behaviors in preclinical studies, suggesting that α5-NAMs may have translational relevance as novel antidepressant medications. Here, we evaluated the efficacy of Basmisanil, an α5-NAM that has been evaluated in Phase 2 clinical studies as a cognitive enhancer, in a battery of behavioral tests relevant to coping strategies, motivation, and aversion in male mice, along with plasma and brain pharmacokinetic measurements. Our findings reveal that Basmisanil induces dose-dependent rapid antidepressant-like responses in the forced swim test and sucrose splash test without promoting locomotor stimulating effects. Furthermore, Basmisanil elicits sustained behavioral responses in the female urine sniffing test and sucrose splash test, observed 24 h and 48 h post-treatment, respectively. Bioanalysis of plasma and brain samples confirms effective blood-brain barrier penetration by Basmisanil and extrapolation to previously published data suggest that effects were observed at doses (10 and 30 mg/kg i.p.) corresponding to relatively modest levels of α5-GABAAR occupancy (40-65 %). These results suggest that Basmisanil exhibits a combination of rapid and sustained antidepressant-like effects highlighting the potential of α5-NAMs as a novel therapeutic strategy for depression.

The suppressive effect of the specific KCC2 modulator CLP290 on seizure in mice.

Epilepsy is a chronic neurological disorder characterized by episodic dysfunction of central nervous system. The most basic mechanism of epilepsy falls to the imbalance between excitation and inhibition. In adults, GABAA receptor (GABAAR) is the main inhibitory receptor to prevent neurons from developing hyperexcitability, while its inhibition relies on the low intracellular chloride anion concentration ([Cl-]i). Neuronal-specific electroneutral K+-Cl- cotransporter (KCC2) can mediate chloride efflux to lower [Cl-]i for GABAAR mediated inhibition. Our previous study has revealed that the coordinated downregulation of KCC2 and GABAAR participates in epilepsy. According to a high-throughout screen for compounds that reduce [Cl-]i, CLP290 turns out to be a specific KCC2 functional modulator. In current study, we first confirmed that CLP290 could dose-dependently suppress convulsant-induced seizures in mice in vivo as well as the epileptiform burst activities in cultured hippocampal neurons in vitro. Then, we discovered that CLP290 functioned through preventing the downregulation of the KCC2 phosphorylation at Ser940 and hence the KCC2 membrane expression during convulsant stimulation, and consequently restored the GABA inhibition. In addition, while CLP290 was given in early epileptogenesis period, it also effectively decreased the spontaneous recurrent seizures. Generally, our current results demonstrated that CLP290, as a specific KCC2 modulator by enhancing KCC2 function, not only inhibits the occurrence of the ictal seizures, but also suppresses the epileptogenic process. Therefore, we believe KCC2 may be a suitable target for future anti-epileptic drug development.

Elucidating the binding mechanisms of GABA and Muscimol as an avenue to discover novel GABA-mimetic small molecules.

Gamma-aminobutyric acid (GABA) signaling is the principal inhibitory pathway in the central nervous system. It is critical in neuronal cell proliferation and fate determination. Any aberration in GABA inhibition results in psychiatric and neurological diseases. Thus, modulating GABAergic neurotransmission has become the basis of drug therapy for psychiatric and several neurological diseases. Though GABA and muscimol are classical inhibitors of GABA receptors, the search for novel inhibitors continues unabated. In this study, the binding mechanism of GABA and muscimol was elucidated and applied in the search for small molecule GABAergic inhibitors using comprehensive computational techniques. It was revealed that a high-affinity binding of GABA and muscimol was mediated by a water molecule involving α1Thr129 and then stabilized by strong interactions including salt bridges with ß2Glu155 and α1Arg66 amidst hydrogen bonds, π-π stacking, and π -cation interactions with other residues. The binding of GABA and muscimol was also characterized by stability and deeper penetration into the hydrophobic core of the protein which resulted in conformational changes of the binding pocket and domain, by inducing correlated motions of the residues. Thermodynamics analysis showed GABA and muscimol exhibited total binding free energies of -19.85 ± 8.83 Kcal/mol and -26.55 ± 3.42 Kcal/mol, respectively. A pharmacophore model search, based on the energy contributions of implicating binding residues, resulted in the identification of ZINC68604167, ZINC19735138, ZINC04202466, ZINC00901626, and ZINC01532854 as potential GABA-mimetic compounds from metabolites and natural products libraries. This study has elucidated the binding mechanisms of GABA and muscimol and successfully applied in the identification of GABA-mimetic compounds.Communicated by Ramaswamy H. Sarma.

The Activation of GABAAR Alleviated Cerebral Ischemic Injury via the Suppression of Oxidative Stress, Autophagy, and Apoptosis Pathways.

Ischemic stroke is a devastating disease leading to neurologic impairment. Compounding the issue is the very limited array of available interventions. The activation of a γ-aminobutyric acid (GABA) type A receptor (GABAAR) has been reported to produce neuroprotective properties during cerebral ischemia, but its mechanism of action is not yet fully understood. Here, in a rat model of photochemically induced cerebral ischemia, we found that muscimol, a GABAAR agonist, modulated GABAergic signaling, ameliorated anxiety-like behaviors, and attenuated neuronal damage in rats suffering cerebral ischemia. Moreover, GABAAR activation improved brain antioxidant levels, reducing the accumulation of oxidative products, which was closely associated with the NO/NOS pathway. Notably, the inhibition of autophagy markedly relieved the neuronal insult caused by cerebral ischemia. We further established an oxygen-glucose deprivation (OGD)-induced PC12 cell injury model. Both in vivo and in vitro experiments demonstrated that GABAAR activation obviously suppressed autophagy by regulating the AMPK-mTOR pathway. Additionally, GABAAR activation inhibited apoptosis through inhibiting the Bax/Bcl-2 pathway. These data suggest that GABAAR activation exerts neuroprotective effects during cerebral ischemia through improving oxidative stress and inhibiting autophagy and apoptosis. Our findings indicate that GABAAR serves as a target for treating cerebral ischemia and highlight the GABAAR-mediated autophagy signaling pathway.

Enhancing GABAergic Tonic Inhibition Reduces Seizure-Like Activity in the Neonatal Mouse Hippocampus and Neocortex.

Approximately one-third of neonatal seizures do not respond to first-line anticonvulsants, including phenobarbital, which enhances phasic inhibition. Whether enhancing tonic inhibition decreases seizure-like activity in the neonate when GABA is mainly depolarizing at this age is unknown. We evaluated if increasing tonic inhibition using THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, gaboxadol], a δ-subunit-selective GABAA receptor agonist, decreases seizure-like activity in neonatal C57BL/6J mice (postnatal day P5-8, both sexes) using acute brain slices. Whole-cell patch-clamp recordings showed that THIP enhanced GABAergic tonic inhibitory conductances in layer V neocortical and CA1 pyramidal neurons and increased their rheobase without altering sEPSC characteristics. Two-photon calcium imaging demonstrated that enhancing the activity of extrasynaptic GABAARs decreased neuronal firing in both brain regions. In the 4-aminopyridine and the low-Mg2+ model of pharmacoresistant seizures, THIP reduced epileptiform activity in the neocortex and CA1 hippocampal region of neonatal and adult brain slices in a dose-dependent manner. We conclude that neocortical layer V and CA1 pyramidal neurons have tonic inhibitory conductances, and when enhanced, they reduce neuronal firing and decrease seizure-like activity. Therefore, augmenting tonic inhibition could be a viable approach for treating neonatal seizures.

Unique association of anti-GABAA receptor encephalitis and myasthenia gravis in a patient with type A thymoma.

Association between anti-GABAAR encephalitis and myasthenia gravis is extremely rare with few reported cases. Herein, we report a case of a female patient diagnosed with anti-GABAAR encephalitis and thymoma at the first admission. She was administered glucocorticoids for long-term immunotherapy, and thymectomy with biopsy demonstrated a type A thymoma. After 4 months, the symptoms of encephalitis were relieved, but she then developed post-thymectomy myasthenia gravis with anti-AChR and anti-titin dual positivity. Antibodies to connective tissue (anti-ANA, anti-PCNA) and those characteristics of paraneoplastic syndrome (anti-Ma2/Ta) were also positive. She received oral glucocorticoids and tacrolimus as immunosuppressive therapy, and myasthenic symptoms were stable during a 2-year follow-up. Our case revealed that anti-GABAAR encephalitis and myasthenia gravis can appear in patient with type A thymoma at different periods, which alerts physicians to take long-term follow-up for anti-GABAAR encephalitis with thymoma, even after thymectomy. Concurrent positivity for more than one antibody after thymectomy is rarely observed, and their contribution to the clinical course and treatment decision remains to be further investigated.

Knockdown of SIK3 in the CA1 Region can Reduce Seizure Susceptibility in Mice by Inhibiting Decreases in GABAAR α1 Expression.

Imbalance between excitation and inhibition is an important cause of epilepsy. Salt-inducible kinase 1 (SIK1) gene mutation can cause epilepsy. In this study, we first found that the expression of SIK3 is increased after epilepsy. Furthermore, the role of SIK3 in epilepsy was explored. In cultured hippocampal neurons, we used Pterosin B, a selective SIK3 inhibitor that can inhibit epileptiform discharges induced by the convulsant drug cyclothiazide (a positive allosteric modulator of AMPA receptors, CTZ). Knockdown of SIK3 inhibited epileptiform discharges and increased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). In mice, knockdown of SIK3 reduced epilepsy susceptibility in a pentylenetetrazole (a GABAA receptor antagonist, PTZ) acute kindling experiment and increased the expression of GABAA receptor α1. In conclusion, our results suggest that blockade or knockdown of SIK3 can inhibit epileptiform discharges and that SIK3 has the potential to be a novel target for epilepsy treatment.

Dynamical changes in the expression of GABAergic and purinergic components occur during the polarization of THP-1 monocytes to proinflammatory macrophages.

The monocytes are key components of innate immunity, as they can differentiate into phagocytic cells or macrophages with proinflammatory or anti-inflammatory phenotypes. The gamma-aminobutyric acid (GABA) and adenosine triphosphate (ATP), two known neurotransmitters, are two environmental signals that contribute to the differentiation of monocytes into macrophages and their subsequent polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. Although monocytes and macrophages express proteins related to GABA and ATP-mediated response (GABAergic and purinergic systems, respectively), it is unknown whether changes in their expression occur during monocyte activation or their differentiation and polarization into macrophages. Therefore, we evaluated the expression levels of GABAergic and purinergic signaling components in the THP-1 monocyte cell line and their changes during monocyte activation, differentiation, and polarization to M1 proinflammatory macrophages. Our results showed that activated monocytes are characterized by increased expression of two GABAergic components, the GABA transporter 2 (GAT-2) and the glutamic acid decarboxylase (GAD)-67, an enzyme involved in GABA synthesis. Also, monocytes showed a pronounced expression of the purinergic receptors P2X4 and P2X7. Interestingly, during differentiation, monocytes increased the expression of the ß2 subunit of GABA A-type receptor (GABA-AR), while the purinergic receptors P2X1 and P2X1del were reduced. In contrast, proinflammatory M1 macrophages showed a reduced expression in the α4 subunit of GABA-AR and GAD67, while P2X4 and P2X7 were overexpressed. These results indicate that dynamical changes in the GABAergic and purinergic components occur during the transition from monocytes to macrophages. Since GABA and ATP are two neurotransmitters, our results suggest that monocytes and macrophages respond to neurotransmitter-induced stimulation and may represent a path of interaction between the nervous and immune systems during peripheral inflammation and neuroinflammation development.

Impact of Elevated Brain IL-6 in Transgenic Mice on the Behavioral and Neurochemical Consequences of Chronic Alcohol Exposure.

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of the neuroimmune system is known to dysregulate the production of neuroimmune factors, such as the cytokine IL-6, thereby changing the neuroimmune status of the brain, which could impact the actions of alcohol. The consequences of neuroimmune-alcohol interactions are not fully known. In the current studies we investigated this issue in transgenic (TG) mice with altered neuroimmune status relative to IL-6. The TG mice express elevated levels of astrocyte-produced IL-6, a condition known to occur with alcohol exposure. Standard behavioral tests of alcohol drinking and negative affect/emotionality were carried out in homozygous and heterozygous TG mice and control mice to assess the impact of neuroimmune status on the actions of chronic intermittent alcohol (ethanol) (CIE) exposure on these behaviors. The expressions of signal transduction and synaptic proteins were also assessed by Western blot to identify the impact of alcohol-neuroimmune interactions on brain neurochemistry. The results from these studies show that neuroimmune status with respect to IL-6 significantly impacts the effects of alcohol on multiple levels.

Dexmedetomidine relieves inflammatory pain by enhancing GABAergic synaptic activity in pyramidal neurons of the anterior cingulate cortex.

Pyramidal neuron (Pyn) hyperactivity in the anterior cingulate cortex (ACC) is involved in the modulation of pain. Previous studies indicate that the activation of α2 adrenoceptors (α2-ARs) by dexmedetomidine (DEX) is a safe and effective means of alleviating multiple types of pain. Here, we showed that systemically administered DEX can ameliorate the inflammatory pain induced by hindpaw injection of formalin (FA) and further examined the molecular and synaptic mechanisms of this DEX-elicited antinociceptive effect. We found that FA caused an increase in c-Fos expression in contralateral layer 2/3 (L2/3) ACC, and that intra-ACC infusion of DEX could also relieve phase 2 inflammatory pain behavior. DEX elicited an increase in the amplitude and frequency of miniature inhibitory post-synaptic currents (mIPSCs) and evoked IPSC amplitude, as well as a reduction in the hyperexcitability and both paired-pulse and excitation/inhibition ratios in contralateral L2/3 ACC Pyns of FA mice. These electrophysiological effects were associated with the upregulation of GABA A receptor (GABAAR) subunits. The interaction of phosphorylated Akt (p-Akt) with GABAAR subunits increased in the ACC following administration of DEX. These results suggest that DEX treatment reduces hyperactivity and enhances GABAergic inhibitory synaptic transmission in ACC Pyns, which produces analgesic effects by increasing GABAAR levels and activating the Akt signaling pathway.

Influence of Inflammatory Pain and Dopamine on Synaptic Transmission in the Mouse ACC.

Dopamine (DA) inhibits excitatory synaptic transmission in the anterior cingulate cortex (ACC), a brain region involved in the sensory and affective processing of pain. However, the DA modulation of inhibitory synaptic transmission in the ACC and its alteration of the excitatory/inhibitory (E/I) balance remains relatively understudied. Using patch-clamp recordings, we demonstrate that neither DA applied directly to the tissue slice nor complete Freund's adjuvant (CFA) injected into the hind paw significantly impacted excitatory currents (eEPSCs) in the ACC, when recorded without pharmacological isolation. However, individual neurons exhibited varied responses to DA, with some showing inhibition, potentiation, or no response. The degree of eEPSC inhibition by DA was higher in naïve slices compared to that in the CFA condition. The baseline inhibitory currents (eIPSCs) were greater in the CFA-treated slices, and DA specifically inhibited eIPSCs in the CFA-treated, but not naïve group. DA and CFA treatment did not alter the balance between excitatory and inhibitory currents. Spontaneous synaptic activity revealed that DA reduced the frequency of the excitatory currents in CFA-treated mice and decreased the amplitude of the inhibitory currents, specifically in CFA-treated mice. However, the overall synaptic drive remained similar between the naïve and CFA-treated mice. Additionally, GABAergic currents were pharmacologically isolated and found to be robustly inhibited by DA through postsynaptic D2 receptors and G-protein activity. Overall, the study suggests that CFA-induced inflammation and DA do not significantly affect the balance between excitatory and inhibitory currents in ACC neurons, but activity-dependent changes may be observed in the DA modulation of presynaptic glutamate release in the presence of inflammation.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) causes seizure activity in larval zebrafish via antagonism of γ-aminobutyric acid type A receptor α1ß2γ2.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1ß2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (ß2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1ß2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.