GABA Agonist Research Articles

Conditioned inhibition of amphetamine sensitization

Repeated intermittent exposure to psychostimulants, such as amphetamine, leads to a progressive enhancement of the drug’s ability to increase both behavioral and brain neurochemical responses. The expression of these enhancements, known as sensitization, can be regulated by Pavlovian conditioned stimuli. Cues that are associated with drug experience can facilitate sensitization so that it only occurs in the presence of these stimuli (context-specific sensitization). In contrast, cues that are explicitly related to the absence of drugs (conditioned inhibitors) can prevent the expression of sensitization. We hypothesized that disrupting conditioned inhibition would enable amphetamine sensitization in new contexts. Using male Sprague Dawley rats and a two-context amphetamine conditioning procedure, we found that extinguishing amphetamine experience in one environment led to the loss of conditioned inhibition in a separate context. Thus, amphetamine-induced sensitized locomotion, as well as both enhanced dopamine and glutamate neurotransmission in the nucleus accumbens, were observed in a context where the drug was never experienced before. A similar loss of contextual control of sensitization was seen after using baclofen/muscimol microinjections to transiently inhibit the medial prefrontal cortex, basolateral amygdala, or ventral subiculum of the hippocampus. In other words, compared to control infusions, these intracranial injections of GABA-receptor agonists were able to block conditioned inhibitors from preventing the expression of sensitized locomotion. Together, these findings reveal the importance of conditioned inhibitors for regulating addiction-like behavior. The results suggest that dopaminergic and glutamatergic brain circuitry controls the context-specific expression of amphetamine sensitization.

Decision Making in Mice During an Optimized Touchscreen Spatial Working Memory Task Sensitive to Medial Prefrontal Cortex Inactivation and NMDA Receptor Hypofunction.

Working memory is a fundamental cognitive process for decision-making and is a hallmark impairment in a variety of neuropsychiatric and neurodegenerative diseases. Spatial working memory paradigms are a valuable tool to assess these processes in rodents and dissect the neurobiology underlying working memory. The trial unique non-match to location (TUNL) task is an automated touchscreen paradigm used to study spatial working memory and pattern separation processes in rodents. Here, animals must remember the spatial location of a stimulus presented on the screen over a delay period; and use this representation to respond to the novel location when the two are presented together. Because stimuli can be presented in a variety of spatial configurations, TUNL offers a trial-unique paradigm, which can aid in combating the development of unwanted mediating strategies. Here, we have optimized the TUNL protocol for mice to reduce training time and further reduce the potential development of mediating strategies. As a result, mice are able to accurately perform an enhanced trial-unique paradigm, where the locations of the sample and choice stimuli can be presented in any configuration on the screen during a single session. We also aimed to pharmacologically characterize this updated protocol, by assessing the roles of the medial prefrontal cortex (mPFC) and N-methyl-D-aspartate (NMDA) receptor (NMDAr) functioning during TUNL. Temporary inactivation of the medial prefrontal cortex (mPFC) was accomplished by directly infusing a mixture of GABA agonists muscimol and baclofen into the mPFC. We found that mPFC inactivation significantly impaired TUNL performance in a delay-dependent manner. In addition, mPFC inactivation significantly increased the susceptibility of mice to proactive interference. Mice were then challenged with acute systemic injections of the NMDAr antagonist ketamine, which resulted in a dose-dependent, delay-dependent working memory impairment. Together, we describe an optimized automated touchscreen task of working memory, which is dependent on the intact functioning of the mPFC and sensitive to acute NMDAr hypofunction. With the vast genetic toolbox available for modeling disease and probing neural circuit functioning in mice, the TUNL task offers a valuable paradigm to pair with these technologies to further investigate the processes underlying spatial working memory.

Inactivation of the thalamic paraventricular nucleus promotes place preference and sucrose seeking in male rats

The experience of reward entails both positive affect and motivation. While the brain regions responsible for these distinct aspects of reward are dissociable from each other, the paraventricular nucleus of the thalamus (PVT) may play a role in both. To investigate the role of the PVT in both affect and motivation, and to identify neuropeptides that might mediate these effects. Male rats were tested for conditioned place preference following temporary inactivation of the anterior or posterior PVT with local injections of the GABAB and GABAA agonists, baclofen + muscimol. They were tested for sucrose seeking under a fixed ratio 3 (FR3) schedule of reinforcement and after extinction, following injection into the posterior PVT of baclofen + muscimol or saline vehicle. Finally, quantitative real-time PCR was used to examine local neuropeptide gene expression following injection into the posterior PVT of baclofen + muscimol or saline vehicle. Conditioned place preference was induced by temporary inactivation of the posterior but not anterior PVT. While sucrose seeking under an FR3 schedule of reinforcement was unaffected by inactivation of the posterior PVT, reinstatement of sucrose seeking was promoted by posterior PVT inactivation. Local gene expression of pituitary adenylate cyclase-activating polypeptide (PACAP), but not enkephalin or neurotensin, was reduced following inactivation of the posterior PVT. Temporary inactivation of the posterior PVT affects both affect and motivation as well as local gene expression of PACAP. These results suggest that the posterior PVT is one brain region that may participate in both major aspects of reward.

Feeding behavior elicited by mu opioid and GABA receptor activation in the lateral septum

The lateral septum (LS), a brain region typically associated with behaviors involving reward, anxiety-like behavior, learning, and memory, has recently received increased interest due to its potential role in eating behavior. Our current results showed that morphine (5 μg) microinjected into the LS produced a stable feeding response. Specifically, across five days of repeated injections, there was no increase or sensitization effect, nor a decrease in feeding or tolerance. Additionally, we found that pretreatment with the broad-spectrum opioid receptor antagonist naloxone blocked morphine-elicited feeding, further supporting a role for LS opioid receptors in the activation of feeding behaviors. We had previously found that the GABAA receptor agonist muscimol produces a similar increase in feeding when injected into the LS. Given the involvement of the LS in multiple behaviors, we next evaluated whether other behaviors might be co-occurring with feeding in response to opioid or GABAA receptor agonist injection into the LS. We assessed eating, drinking, grooming, sleeping, activity levels and resting behavior for 3 h after injection of aCSF, DAMGO, morphine, or muscimol. We found that morphine and muscimol both decreased the latency to eat, and all drugs tested increased food intake. The feeding occurred within 30 min of muscimol injection but was delayed after opioid injections. The absence of increases in other goal-oriented behavior like drinking or grooming or behavioral hyperactivity supports a primary effect of muscimol and the opioids on LS mechanisms of feeding control. Significance statementThe LS is interesting because of its role in a wide range of behaviors including defensive behaviors, social behaviors, learning, memory, and motivation. Although the LS was discovered to have a role in feeding stimulation over 30 years ago, only recently has major progress begun to reveal the underlying mechanisms. The present paper contributes by suggesting that LS GABAA and μ-opioid receptors elicit eating by inhibiting LS neurons that themselves inhibit eating. Importantly, this work informs lateral septal research which may shed light on disordered eating included binge eating and anorexia.

Neonatal inflammation via persistent TGF-β1 downregulation decreases GABAAR expression in basolateral amygdala leading to the imbalance of the local excitation-inhibition circuits and anxiety-like phenotype in adult mice

Neonatal inflammation can increase the risk of anxiety disorder in adulthood. The balance between glutamatergic excitatory and GABAergic inhibitory transmissions in the basolateral amygdala (BLA) plays a vital role in controlling anxiety state. Based on the reports that early-life inflammation had adverse effects on GABAergic system, the aim of this study was to investigate whether and how neonatal inflammation affects excitatory-inhibitory circuits in the BLA resulting in anxiety disorder. Neonatal mice received a daily subcutaneous injection of lipopolysaccharide (LPS, 50 μg/kg) or saline on postnatal days 3–5. LPS-treated mice developed anxiety behaviors accompanied by the hyperactivity of adrenal axis in adulthood. Electrophysiological study revealed the increase of postsynaptic neuronal excitability in the cortical-BLA excitatory synapses of LPS mice which could be recovered by bath-application of GABAAR agonist suggesting the impairment of GABAergic system in LPS mice. Compared with controls, GABAARα2 subunit expression and density of GABA-evoked current in BLA principal neurons were reduced in LPS mice. Additionally, neonatal LPS treatment resulted in the down-regulation of transforming growth factor-beta 1 (TGF-β1) expression and PKC signaling pathway in the adult BLA. The local TGF-β1 overexpression in the BLA improved GABAARα2 expression via up-regulating the activity of PKC signaling, which corrected GABAAR-mediated inhibition leading to the abolishment of anxiety-like change in adrenal axis regulation and behaviors in LPS mice. These data suggest the persistent TGF-β1deficit induces the down-regulation of GABAARα2 expression and subsequent disruption of the excitation-inhibition balance in the BLA circuits, which is the important mechanisms of neonatal inflammation-induced anxiety disorder.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

PD34-01 SACRAL NEUROMODULATION DEVICES CONSISTENTLY HARBOR BACTERIA AND POSSESS A UNIQUE MICROBE AND METABOLITE PROFILE

You have accessJournal of UrologyCME1 May 2022PD34-01 SACRAL NEUROMODULATION DEVICES CONSISTENTLY HARBOR BACTERIA AND POSSESS A UNIQUE MICROBE AND METABOLITE PROFILE Glenn T. Werneburg, Ava Adler, Daniel Hettel, Sandip Vasavada, Raymond Rackley, Howard Goldman, Jacqueline Zillioux, Bradley Gill, Daniel Shoskes, and Aaron Miller Glenn T. WerneburgGlenn T. Werneburg More articles by this author , Ava AdlerAva Adler More articles by this author , Daniel HettelDaniel Hettel More articles by this author , Sandip VasavadaSandip Vasavada More articles by this author , Raymond RackleyRaymond Rackley More articles by this author , Howard GoldmanHoward Goldman More articles by this author , Jacqueline ZilliouxJacqueline Zillioux More articles by this author , Bradley GillBradley Gill More articles by this author , Daniel ShoskesDaniel Shoskes More articles by this author , and Aaron MillerAaron Miller More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002585.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Sacral neuromodulation (SNM) is an effective therapy for refractory overactive bladder. 25% of explanted SNM devices have been shown to harbor microbes, but only 2% develop infection requiring removal. To understand the transition from the colonized (wherein bacteria are present but not pathological) to infected state, we sought to determine microbial biofilm and metabolite composition on devices, and its association with clinical factors. We hypothesized SNM devices would consistently harbor bacteria, and possess unique bacterial/metabolite profiles. METHODS: Urological patients scheduled to undergo removal or revision of SNM devices were consented per IRB-approved protocol. Devices were swabbed intraoperatively, with safeguards to avoid contamination. Swab samples, alongside controls, were subjected to next-generation sequencing and metabolomics. Association between microbial diversity and clinical variables was then analyzed using t-tests and ANOVA. RESULTS: 11 devices explanted for non-infectious reasons were included, and all harbored microbiota. Common genera included Acinetobacter, Pseudomonas, Corynebacterium, Staphylococcus, among others (Figure left). Diethanolamine (local anesthetic precursor), threonine-butyl-ester (peptide synthesis precursor), and 5-aminovaleric acid (bacterial metabolite of lysine; GABA agonist) were the most commonly detected metabolites (Figure right). There was no significant difference in bacterial diversity based on age, antibiotics, or BMI. SNM devices exhibited a unique microbial (p=0.047) and metabolomic (p=0.045) profile relative to other urologic device types (stents, sphincters, prostheses). CONCLUSIONS: Explanted devices consistently harbored microbiota and possessed a unique microbe and metabolite profile. Some of the most commonly detected microbes in the non-infected devices have been previously implicated in SNM device-associated infections (Staphylococcus, Pseudomonas). Our results open new avenues for investigation of biochemical changes occurring during the transition from colonization to clinical infection, and identify preventive strategies. Future studies will compare SNM devices explanted due to infection to those removed for other reasons to understand this pathophysiology. Source of Funding: N/A © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e559 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Glenn T. Werneburg More articles by this author Ava Adler More articles by this author Daniel Hettel More articles by this author Sandip Vasavada More articles by this author Raymond Rackley More articles by this author Howard Goldman More articles by this author Jacqueline Zillioux More articles by this author Bradley Gill More articles by this author Daniel Shoskes More articles by this author Aaron Miller More articles by this author Expand All Advertisement PDF DownloadLoading ...

Involvement of GABAA-rho Receptor in a Trigeminal Neuralgia Model

The antiepileptic drug carbamazepine is effective in a subpopulation of trigeminal neuralgia patients, in particular those suffering from pain associated with vascular compression of a trigeminal nerve. However, carbamazepine has failed in clinical trials for other neuropathic pain conditions, even those associated with peripheral nerve compression injuries. Carbamazepine is both a GABAA receptor (GAR) agonist and a blocker of voltage-gated Na+ channels (VGSC), and our recent data suggest that differences between trigeminal and somatic nerves with respect to the VGSC present are not sufficient to account for the therapeutic selectivity of carbamazepine. Therefore, we hypothesized that these observations reflected a differential involvement of GARs in somatic and trigeminal nerves. To test this, we employed a combination of electrophysiological and behavioral experiments in rat models of somatic (sciatic nerve, SN) and trigeminal (infraorbital nerve, ION) nerve compression (CCI). We observed that the potency of carbamazepine-induced compound action potential (CAP) block was increased by CCI of the ION but not the SN, and this increase in potency was reduced by the GAR blocker picrotoxin. The potency of the GAR agonist muscimol-induced block of the ION was greater than the SN, and CCI produced a further increase in efficacy. The GABAA-rho subunit mRNA and protein was increased by CCI of the ION, but not SN. The GABAA-rho receptor selective antagonist TPMPA reduced carbamazepine-induced CAP block of the ION. CCI was also associated with an increase in the potency of the GABAA-rho receptor preferring agonist TACA-induced block of the ION, but not SN. Finally, local administration of TACA to the ION but not SN, dose-dependently reversed CCI-induced mechanical sensitivity. Taken altogether, our results suggest GABAA-rho receptors are present and functional in ION, but not SN. This differential distribution of GABAA receptors could account for the therapeutic selectivity of carbamazepine for the treatment of TN. The work was supported by generous donations from TN patients and family members as well as NIH grant R01NS064988.

Changes in GABAA Receptor Function Is Dependent on Osteoarthritis Pain State

Osteoarthritis pain is a heterogeneous pain state. Mid-stage osteoarthritis is associated with pain during joint use that abates during rest and can be managed with non-steroidal anti-inflammatory agents (NSAIDs). Advanced osteoarthritis is characterized by development of persistent ongoing pain that is resistant to NSAIDs. Mechanistic differences underlying these different osteoarthritis pain states are poorly understood. This study investigated the hypothesis that changes in spinal GABAA receptor signaling is dependent on the characteristics of the chronic pain state. A murine model of monosodium iodoacetate (MIA)-induced knee joint osteoarthritis (OA) in males and females was used. Mice with mid-stage OA demonstrate weight asymmetry without a persistent ongoing pain state. Mice with advanced OA develop both weight asymmetry and persistent ongoing pain. Control mice received intra-articular saline. Alterations in GABAA receptors were investigated using spinal administration of the GABAA agonist (muscimol) and antagonist (bicuculline). In mice with mid-stage OA, spinal muscimol normalized weight asymmetry whereas spinal bicuculline did not alter weight asymmetry. Conversely, in mice with advanced OA, spinal muscimol failed to alter weight asymmetry whereas bicuculline normalized weight bearing. Further, in mice with advanced OA, muscimol induced conditioned place aversion, indicated worsening of joint pain, whereas bicuculline induced conditioned place preference, indicating pain relief. Restoring KCC2 function through spinal administration of CLP290 reversed weight asymmetry in mice with advanced but not mid-stage OA. These data indicate that GABAA receptors induce net inhibition in mid-stage OA, a chronic pain state characterized by intermittent joint pain. In contrast, GABAA receptors induce net excitation in advanced OA, a chronic pain state characterized by persistent ongoing joint pain. This switch is likely mediated by diminished KCC2 function in the spinal cord. Grant support from NIH Centers of Biomedical Research Excellence (COBRE) Grant P20GM103643

Perinatal High Fat Diet and Acute Stress alters the Chloride Gradient in Dorsal Motor Nucleus of the Vagus Neurons Controlling Gastric Function

The US is currently facing an obesity epidemic, though the concurrent rise in maternal obesity rates receives less attention. Maternal obesity is an important factor that influences intrauterine and early postnatal environment and is associated with developmental changes in the offspring, including alterations in metabolic function and susceptibility to anxiety and depression, all of which are associated with abnormal gastrointestinal (GI) tract function. Previous work from this lab has shown that perinatal high fat diet (pHFD) leads to developmental changes to vagal neurocircuits, including changes to GABAA receptor subunits on dorsal motor nucleus of the vagus (DMV) neurons. However, the manner of neuronal recordings (whole‐cell patch clamp) disrupted internal chloride (Cl‐) concentration, preventing accurate assessment of the extent of GABA‐mediated inhibition. KCC2, a Cl‐ transporter, is important in determining GABAergic inhibition and stress outcomes in hypothalamic neurons, however its role in regulating these factors in DMV neurons is unknown. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently collapsing the Cl‐ gradient and leading to altered gastric output.Sprague‐Dawley rats were fed either a control (14% kcal from fat) or high fat (60% kcal from fat) diet starting on embryonic day 13. After weaning on postnatal day 28, brainstem slices were made to conduct patch clamp recordings on DMV neurons. This was done in both naïve (unstressed) or stressed conditions, in which rats underwent a 10 minute forced swim (FS). To determine the Cl‐ reversal, perforated patch clamp recordings were performed in order to preserve the internal Cl‐ concentration. Then, DMV neurons were voltage clamped from ‐90 to ‐40 mV and the amplitude of the current response to picospritz of the GABAAagonist muscimol (100 μM) was measured. These events were plotted on an I‐V curve, with the x‐intercept representing the reversal of Cl‐. To determine a potential mediator in the shift in the Cl‐ reversal, changes in KCC2 levels following pHFD exposure were evaluated immunohistochemically. Lastly, gastric emptying (GE) assays were performed using the 13C octanoic acid breath assessment.In both pHFD and control diet + FS DMV neurons, there was a depolarizing shift in the Cl‐ reversal compared to naïve rats (‐43.05 mV and ‐44.58 mV respectively, compared to ‐64.56 mV in naïve conditions; p < 0.0001). It was also found that there is a significant reduction in KCC2 expression in pHFD DMV region compared to controls (2.237% area fluorescence in pHFD compared to 5.335% in control diet; p=0.0147). Lastly, pHFD rats had significantly delayed GE rates compared to those fed a control diet (83.7 minutes for half‐decay in pHFD compared to 66.7 minutes in controls; p < 0.05), resembling previously published data which showed a delay in GE following an acute stress. These results demonstrate that both a pHFD and acute stress lead to depolarizing shifts in the Cl‐ reversal and that this may be through a mechanism involving a reduction in KCC2 expression. As the gastric emptying rates demonstrate, these changes could lead to a maladaptive GI stress response in pHFD rats.Determining the mechanism in which pHFD and acute stress modulate DMV function, as well as the impact of their intersection on the gastric stress response, will better inform our understanding of the reduced stress resiliency seen in pHFD offspring.

A Novel Theanine Complex, Mg-L-Theanine Improves Sleep Quality via Regulating Brain Electrochemical Activity.

L-Theanine is commonly used to improve sleep quality through inhibitory neurotransmitters. On the other hand, Mg2+, a natural NMDA antagonist and GABA agonist, has a critical role in sleep regulation. Using the caffeine-induced brain electrical activity model, here we investigated the potency of L-theanine and two novel Mg-L-theanine compounds with different magnesium concentrations on electrocorticography (ECoG) patterns, GABAergic and serotonergic receptor expressions, dopamine, serotonin, and melatonin levels. Furthermore, we evaluated the sleep latency and duration in the pentobarbital induced sleep model. We herein showed that L-theanine, particularly its various complexes with magnesium increases the expression of GABAergic, serotonergic, and glutamatergic receptors, which were associated with decreased ECoG frequency, increased amplitude, and enhanced delta wave powers. Besides increased dopamine, serotonin, and melatonin; decreased MDA and increased antioxidant enzyme levels were also observed particularly with Mg-complexes. Protein expression analyses also showed that Mg-L-theanine complexes decrease inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) levels significantly. In accordance with these results, Mg complexes improved the sleep latency and duration even after caffeine administration. As a result, our data indicate that Mg-L-theanine compounds potentiate the effect of L-theanine on sleep by boosting slow-brain waves, regulating brain electrical activity, and increasing neurotransmitter and GABA receptor levels.

KCC2 receptor upregulation potentiates antinociceptive effect of GABAAR agonist on remifentanil-induced hyperalgesia.

GABAergic system disinhibition played an important role in the pathogenesis of remifentanil-induced hyperalgesia (RIH). K+-Cl--cotransporter-2 (KCC2) has the potential to enhance the strength of GABAergic signaling function. However, few reports have focused on the additive analgesic effect of KCC2 enhancer and GABAA receptor agonist on the spinal dorsal horn. Therefore, we evaluated the role of GABA type A receptor (GABAAR) agonist (muscimol), KCC2 enhancer (CLP257) in remifentanil-induced hyperalgesia, as well as GABA and KCC2 receptors responses in the dorsal spinal horn. Remifentanil started to reduce paw withdrawal mechanical thresholds at postoperative 4 h and lasted to 72 h. The RIH associated decreases in spinal GABA release was transient. The amount of spinal GABA transmitter by microdialysis was observed to be decreased at the beginning and reached bottom at 150 min, then returned to the baseline level at 330 min. The synthesis and transportation of GABA transmitter were inhibited, characterized as spinal GAD67 and GAT1 downregulation after the establishment of RIH model. The effect of RIH on GABA receptor downregulation was linked to the reduced expression of spinal KCC2 receptor. This decrease in KCC2 expression has coincided with an early loss of GABA inhibition. KCC2 enhancer, which is reported to lead to a reduction in intracellular Cl−, can enhance GABA-mediated inhibitory function. Both muscimol and CLP257 could dose-dependently inhibit mechanical hypersensitivity caused by remifentanil-induced downregulation of GABAAα2R and KCC2, respectively. Compared with muscimol acting alone, the joint action of CLP257 and muscimol showed a higher pain threshold and less c-fos expression via upregulation of KCC2 and GABAAα2R. Taken together, these findings suggested that the RIH was initiated by decreased GABA release. Downregulation of GABAAα2R and KCC2 receptor contributed to spinally mediated hyperalgesia in RIH. KCC2 enhancer was proved to potentiate antinociceptive effect of GABAAR agonist in RIH.

GABAA receptor agonist suppresses pediatric medulloblastoma progression by inhibiting PKA-Gli1 signaling axis

The Sonic hedgehog-activated subgroup of medulloblastoma (SHH-MB) is one of the most common malignant pediatric brain tumors. Recent clinical studies and genomic databases indicate that GABAA receptor holds significant clinical relevance as a therapeutic target for pediatric MB. Herein, we report that "moxidectin," a GABAA receptor agonist, inhibits the proliferation of Daoy, UW426, UW228, ONS76, and PFSK1 SHH-MB cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy demonstrated that moxidectin significantly induced GABAA receptor expression and inhibited cyclic AMP (cAMP)-mediated protein kinase A (PKA)-cAMP response element-binding protein (CREB)-Gli1 signaling in SHH-MB. Gli1 and the downstream effector cancer stem cell (CSC) molecules such as Pax6, Oct4, Sox2, and Nanog were also inhibited by moxidectin treatment. Interestingly, moxidectin also inhibited the expression of MDR1. Mechanistic studies using pharmacological or genetic inhibitors/activators of PKA and Gli1 confirmed that the anti-proliferative and apoptotic effects of moxidectin were mediated through inhibition of PKA-Gli1 signaling. Oral administration of 2.5mg/kg moxidectin suppressed the growth of SHH-MB tumors by 55%-80% in subcutaneous and intracranial tumor models in mice. Exvivo analysis of excised tumors confirmed the observations made in the invitro studies. Moxidectin is an FDA-approved drug with an established safety record, therefore any positive findings from our studies will prompt its further clinical investigation for the treatment of MB patients.

Efficacy and Safety of Remimazolam Tosilate Combined With Esketamine for Analgesic Sedation in Mechanically Ventilated ICU Patients: A Single-Arm Clinical Study Protocol.

IntroductionPatients in the intensive care unit (ICU) frequently experience increased heart rate, blood pressure, and respiration rate as a product of anxiety and restlessness about their condition and treatments. Analgesia and sedation commonly involve benzodiazepines or opioids that lead to respiratory suppression and other adverse reactions. Remimazolam tosilate is a short-acting GABAA receptor agonist with reduced cardiovascular and respiratory inhibition compared to other commonly used benzodiazepines. Esketamine is a non-competitive N-methyl-D-aspartic acid (NMDA) receptor inhibitor that inhibits hyperalgesia and prolongs postoperative analgesia. It also reduces postoperative pain, delirium, and the use and acute tolerance of opioids. This study aims to assess the efficacy and safety of remimazolam tosilate combined with esketamine and sufentanil for sedation and analgesia in mechanically ventilated ICU patients.Methods and AnalysisThis prospective, single-arm, single-center, open-label clinical trial will be conducted from January 2022 to December 2023. The study will include 200 adult patients (≥ 18 years) from Shandong Provincial Hospital (affiliated with Shandong First Medical University) who are mechanically ventilated and admitted to the ICU between 24 and 72 h from the time of ventilation and who are administered analgesia and sedatives. Patients will undergo arterial blood gas analysis before administration. Remimazolam tosilate (0.2 mg/kg) will be injected intravenously within 30 s, followed by continuous infusion at a rate of 0.1 to 0.3 mg/kg/h via micropump. Esketamine (0.25 mg/kg) will be injected intravenously and maintained at 0.15 mg/kg/h, while sufentanil will be maintained at the rate of 0.1 to 0.2 μg/kg/h. The primary study outcome is the overall time required to maintain sedation. Secondary outcomes will include the total dosage used to reach the target sedation level, total mechanical ventilation time, awakening time, length of hospital stay, and incidence of cardiorespiratory-related adverse events and delirium. Adverse events (AEs) will be reported regardless of their relationship to the experimental drugs. AEs associated with adverse drug reactions will be classified as “affirmative correlation,” “possible relevance,” and “unable to determine.” A paired t-test or Wilcoxon signed-rank test will be used to compare the changes of observed indexes before and after treatment. A P < 0.05 will be considered statistically significant.Ethics and DisseminationThis study was approved by the local ethics committee at Shandong Provincial Hospital affiliatied to Shandong First Medical University. The results of this trial will be disseminated in peer-reviewed journals and at scientific conferences.Trial RegistrationThe trial is registered at the Chinese Clinical Trial Registry: ChiCTR2100053106; date of registration: 2021-11-10.

TrkA-cholinergic signaling modulates fear encoding and extinction learning in PTSD-like behavior

Recent studies have suggested that the use of cognitive enhancers as adjuncts to exposure-based therapy in individuals suffering from post-traumatic stress disorder (PTSD) may be beneficial. Brain cholinergic signaling through basal forebrain projections to the hippocampus is an established pathway mediating fear response and cognitive flexibility. Here we employed a genetic strategy to enhance cholinergic activity through increased signaling of the NGF receptor TrkA. This strategy leads to increased levels of the marker of cholinergic activation, acetylcholine synthesizing enzyme choline acetyltransferase, in forebrain cholinergic regions and their projection areas such as the hippocampus. Mice with increased cholinergic activity do not display any neurobehavioral abnormalities except a selective attenuation of fear response and lower fear expression in extinction trials. Reduction in fear response is rescued by the GABA antagonist picrotoxin in mutant mice, and, in wild-type mice, is mimicked by the GABA agonist midazolam suggesting that GABA can modulate cholinergic functions on fear circuitries. Importantly, mutant mice also show a reduction in fear processing under stress conditions in a single prolonged stress (SPS) model of PTSD-like behavior, and augmentation of cholinergic signaling by the drug donepezil in wild-type mice promotes extinction learning in a similar SPS model of PTSD-like behavior. Donepezil is already in clinical use for the treatment of dementia suggesting a new translational application of this drug for improving exposure-based psychotherapy in PTSD patients.

Molecular Modelling of 1H-Benzo [b] [1,5] Diazepine-2(3H)-one Derivatives and Docking Studies Against Receptor Associated Protein

In the present investigation, some N1-benzoyl/ N1-(1,3,4-thiadiazol-2-yl amino acetyl) -7-substituted- 4-methyl-1,5-benzodiazepine-2-one were designed and docked at active site of cavity 1# of GABA-A receptor associated protein (1KJT) to distinguish from their hypothetical binding mode. The X-ray crystal structure of mammalian GABA-A receptor associated protein (1KJT) obtained from protein data bank was used as target protein. In this investigation the comparative analysis of the docking experiments of modelled compounds with known GABA agonist, Lofendazam was carried out. The dock scores calculated for Lofendazam was -4.7373. Among the modelled compounds, following conformation were found to have lower dock scores as indicated in bracket in comparison to other confermers; N1-benzoyl-7- bromo- 4-methyl-1,5-benzodiazepine-2-one, conformer_C3 (-5.0915), N1-(1,3,4-thiadiazol-2-yl amino acetyl) -7-chloro-4-methyl-1,5-benzodiazepine-2-one, Conformer_C2 (-4.6532). These conformers have more affinity for active site of GABA-A receptor associated protein than other molecules.

Antispasmodic activity of carnosic acid extracted from rosmarinus officinalis: Isolation, spectroscopic characterization, DFT studies, and in silico molecular docking investigations

This research work focuses on the isolation, spectroscopic characterization (FT-IR, UV–vis, and NMR), density functional theory (DFT) studies, and the potential application of carnosic acid as an antispasmodic agent drug evaluated by molecular docking with various spasmolytic receptors proteins and the results compared with diazepam (DZP) and toleterodine (TTD) as standard drugs. All computational calculations of the isolated and spectroscopic determined structure was performed at the B3LYP/6–311++G(d,p) theoretical method within the framework of DFT approach. The theoretical vibrational assignments of the various functional groups along with the potential energy distributions (PEDs) were performed using the vibrational energy distribution analysis (VEDA) program and carefully compared with the experimental data. The natural bond orbital (NBO), nonlinear optics (NLO), and frontier molecular orbital (FMO) molecular electronic properties were also investigated and reported. While the binding affinity showed that carnosic acid has better interaction with the nicotinic acetylcholine receptor when compared to DZP and TTD, however, it is not a GABAa receptor agonist but a much better nicotinic acetylcholine receptor agent when juxtaposed with a similar agent like DZP and TTT as an antispasmodic agent as a smooth muscle relaxant.

Stiripentol inhibits spike-and-wave discharges in animal models of absence seizures: A new mechanism of action involving T-type calcium channels.

SummaryObjectiveStiripentol (STP; Diacomit®) is an antiepileptic drug indicated for Dravet syndrome that has been identified as a γ‐aminobutyric acid (GABAergic) positive allosteric modulator. Dravet syndrome is characterized by multiple seizure types: generalized tonic–clonic, focal, myoclonic, and absence seizures. In addition to its antiepileptic effects on tonic–clonic seizures, STP has also been reported to reduce the frequency of atypical absence seizures in patients. Our study focused on STP potential effects on absence seizures, to better characterize its full spectrum of mechanisms of action.MethodsSTP effects on absence seizures were quantified by electroencephalographic recording in two animal models: rats treated with a low dose of pentylenetetrazol (20 mg/kg ip) and rats from the WAG/Rij strain. In addition, we characterized STP effects on T‐type calcium channel activity. Peak currents were recorded with manual patch clamp on cells transfected with cDNA encoding for the human isoform for Cav3.1, Cav3.2, and Cav3.3.ResultsSTP administered before pentylenetetrazol almost completely abolished the generation of spike‐and‐wave discharges (SWDs) at the dose of 300 mg/kg. At this dose, STP also statistically significantly decreased SWD cumulated duration and number in WAG/Rij rats. Its antiepileptic effect was maintained in WAG/Rij rats, whose seizures were aggravated by the GABA agonist THIP (gaboxadol hydrochloride). Furthermore, electrophysiological recordings showed that STP inhibits T‐type calcium channel peak activity, with a higher specificity for the Cav3.3 subtype.SignificanceIn addition to its previously characterized anticonvulsive properties, these data highlight a new mechanism of action of STP on abnormal thalamocortical activity. This strong antiabsence effect on seizures is correlated with an inhibition of T‐type calcium channels. This new mechanism of action could be implicated in the specificity of STP therapeutic effects in Dravet syndrome.

Detection of pentylenetetrazol-induced seizure activity in the 19-21 Hz beta range using a magnetic coil induction method.

A force transducer or automatic scoring system is not sufficient to detect small or fine seizure activity. To improve previous assessments of epileptic behavior, a novel coil method was developed to detect an early behavioral marker for epileptic seizures. The present study used the γ-aminobutyric acid (GABA) receptor antagonist pentylenetetrazol (PTZ) to induce seizure activity and epileptic behavior in mice. A coil method was used to detect motor seizures consisting of small amplitude 19-21 Hz muscle contractions. Seizure activity in the 19-21 Hz range detected by the coil method was positively correlated with generalized clonic seizures with a kangaroo posture after PTZ administration. GABA receptor agonist valproic acid and ethosuximide decreased PTZ-induced 19-21 Hz seizure activity. The pattern of the amplitude ratio (%) of 19-21 Hz seizure activity after administration of the GABAA/C receptor antagonist picrotoxin was similar to the group that was treated with PTZ but different from the group that was treated with the nonselective muscarinic receptor agonist pilocarpine. The coil method detected 19-21 Hz seizure activity after PTZ administration. However, the force transducer method did not detect 19-21 Hz seizure activity. The coil method was more sensitive than the force transducer method for detecting epileptic behaviors. The findings may indicate a novel behavioral marker that can be detected by the coil method to reveal epileptic seizures, thus improving our understanding of the brain mechanisms of action and specific brain waves that are associated with PTZ-induced 19-21 Hz seizure activity.

Dexmedetomidine and sleep quality in mechanically ventilated critically ill patients: study protocol for a randomised placebo-controlled trial

IntroductionSleep deprivation, which is a common complication in the intensive care unit (ICU), is associated with delirium and increased mortality. Sedation with gamma-aminobutyric acid agonists (propofol, benzodiazepine) results in significant...