Lactuca sativa L. Extract Enhances Sleep Duration Through Upregulation of Adenosine A1 Receptor and GABAA Receptors Subunits in Pentobarbital-Injected Mice.

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, ß2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1ß2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3ß2 â‰… α3ß4, indicating that ß2/ß4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9', 13', and 16', whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (-) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Involvement of the GABAA Receptor in the Antidepressant-Like Effects Produced by Low and High Doses of the Flavonoid Chrysin in the Rat: A Longitudinal Study.

BACKGROUND: The flavonoid chrysin produces rapid and long-lasting anxiolytic- and antidepressant-like effects in rats. However, it is not known whether low and high doses of chrysin produce differential anti-immobility effects through the Gamma-Aminobutyric Acid sub-type A (GABAA) receptor. The goal of this work was therefore to compare low and high doses of chrysin for their effects on depression-like behavior in a longitudinal study. Moreover, chrysin was compared with the serotonergic fluoxetine and Gamma-Aminobutyric Acid (GABA)ergic allopregnanolone, and its involvement with the GABAA receptor after chronic treatment was also investigated. METHODS: Male Wistar rats were assigned to five groups (n = 8 each): vehicle, 1 mg/kg chrysin, 5 mg/kg chrysin, 1 mg/kg fluoxetine, and 1 mg/kg allopregnanolone. In the first experiment, treatments were injected daily and the effects on locomotor activity and the forced swim test were evaluated at 0, 1, 14, and 28 days of treatment, and 48 h after the final treatment. In the second experiment, similar groups were treated for 28 days with injection of 1 mg/kg picrotoxin to investigate the role of the GABAA receptor. Depending on the experimental design, one- and two-way analysis of variance (ANOVA) tests were used for statistical analysis, with p < 0.05 set as the criteria for significance. RESULTS: In both experiments, the treatments did not alter locomotor activity. However, low and high doses of chrysin, allopregnanolone, and fluoxetine gradually produced antidepressant-like effects in the forced swim test, and maintained this effect for 48 h post-treatment, except with low dose chrysin. Picrotoxin blocked the antidepressant-like effects produced by low dose chrysin, but did not affect those produced by high dose chrysin, allopregnanolone, or fluoxetine. CONCLUSIONS: The differential antidepressant-like effects caused by low and high doses of chrysin are time-dependent. Low dose chrysin produces a rapid antidepressant-like effect, whereas high dose chrysin produces a delayed but sustained the effect, even 48 h after withdrawal. The effect with high dose chrysin was similar to that observed with allopregnanolone and fluoxetine. The mechanism for the antidepressant-like effect of low chrysin appears to be GABAergic, whereas the effect of high dose chrysin may involve other neurotransmission and neuromodulation systems related to the serotonergic system.

Potential of the Blue Calm® food supplement in the treatment of alcohol withdrawal-induced anxiety in adult zebrafish (Danio rerio).

Alcohol use disorder (AUD) is characterized by a set of behavioral, cognitive, nutritional, and physiological phenomena derived from the uncontrolled use of alcoholic beverages. There are cases in which AUD is associated with anxiety disorder, and when untreated, it requires careful pharmacotherapy. Blue Calm® (BC) is a food supplement indicated to aid restorative sleep, which has traces of medicinal plant extracts, as well as myo-inositol, magnesium bisglycinate, taurine, and L-tryptophan as its main chemical constituents. In this context, this study aimed to evaluate the potential of the BC in the treatment alcohol withdrawal-induced anxiety in adult zebrafish (aZF). Initially, BC was submitted to antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl radical. Subsequently, the aZF (n = 6/group) were treated with BC (0.1 or 1 or 10 mg/mL; 20 µL; p.o.), and the sedative effect and acute toxicity (96 h) were evaluated. Then, the anxiolytic-like effect and the possible GABAergic mechanism were analyzed through the Light & Dark Test. Finally, BC action was evaluated for treating alcohol withdrawal-induced anxiety in aZF. Molecular docking was performed to evaluate the interaction of the major chemical constituents of BC with the GABAA receptor. BC showed antioxidant potential, a sedative effect, was not toxic, and all doses of BC had an anxiolytic-like effect and showed potential for the treatment of alcohol withdrawal-induced anxiety in aZF. In addition to the anxiolytic action, the main chemical constituents of BC were confirmed in the molecular docking, thus suggesting that BC is an anxiolytic that modulates the GABAergic system and has pharmacological potential for the treatment of alcohol withdrawal-induced anxiety.

Comparison of the negative effect of remimazolam and propofol on cardiac contractility: Analysis of a randomised parallel-group trial and a preclinical ex vivo study.

Remimazolam is a newly developed ultra-short-acting benzodiazepine that exerts sedative effects. This study aimed to clarify the effects of remimazolam on cardiac contractility. In a randomised-parallel group trial, haemodynamic parameters were compared between propofol (n = 11) and remimazolam (n = 12) groups during the induction of general anaesthesia in patients undergoing non-cardiac surgery. In a preclinical study, the direct effects of remimazolam on cardiac contractility were also evaluated using isolated rat hearts. RNA sequence data obtained from rat and human hearts were analysed to assess the expression patterns of the cardiac γ-aminobutyric acid type A (GABAA ) receptor subunits. In a clinical study, the proportional change of the maximum rate of arterial pressure rise was milder during the study period in the remimazolam group (propofol: -52.6 [10.2] (mean [standard deviation])% vs. remimazolam: -39.7% [10.5%], p = 0.007). In a preclinical study, remimazolam did not exert a negative effect on left ventricle developed pressure, whereas propofol did exert a negative effect after bolus administration of a high dose (propofol: -26.9% [3.5%] vs. remimazolam: -1.1 [6.9%], p < 0.001). Analysis of the RNA sequence revealed a lack of γ subunits, which are part of the major benzodiazepine binding site of the GABAA receptor, in rat and human hearts. These results indicate that remimazolam does not have a direct negative effect on cardiac contractility, which might contribute to its milder effect on cardiac contractility during the induction of general anaesthesia. The expression patterns of cardiac GABAA receptor subunits might be associated with the unique pharmacokinetics of benzodiazepines in the heart.

Quercetin increases the antidepressant-like effects of sclareol and antagonizes diazepam in thiopental sodium-induced sleeping mice: A possible GABAergic transmission intervention.

Quercetin is the most common polyphenolic flavonoid present in fruits and vegetables demonstrating versatile health-promoting effects. This study aimed to examine the effects of quercetin (QR) and sclareol (SCL) on the thiopental sodium (TS)-induced sleeping and forced swimming test (FST) mouse models. SCL (1, 5, and 10 mg/kg, p.o.) or QR (50 mg/kg, p.o.) and/or diazepam (DZP) (3 mg/kg, i.p.) were employed. After 30 min of TS induction, individual or combined effects on the animals were checked. In the FST test, the animals were subjected to forced swimming after 30 min of administration of the test and/or controls for 5 min. In this case, immobility time was measured. In silico studies were conducted to evaluate the involvement of GABA receptors. SCL (5 and 10 mg/kg) significantly increased the latency and decreased sleeping time compared to the control in the TS-induced sleeping time study. DZP (3 mg/kg) showed a sedative-like effect in animals in both sleeping and FST studies. QR (50 mg/kg) exhibited a similar pattern of activity as SCL. However, its effects were more prominent than those of SCL groups. SCL (10 mg/kg) altered the DZP-3-mediated effects. SCL-10 co-treated with QR-50 significantly (p < 0.05) increased the latency and decreased sleep time and immobility time, suggesting possible synergistic antidepressant-like effects. In silico studies revealed that SCL and QR demonstrated better binding affinities with GABAA receptor, especially α2, α3, and α5 subunits. Both compounds also exhibited good ADMET and drug-like properties. In animal studies, the both compounds worked synergistically to provide antidepressant-like effects in a slightly different fashion. As a conclusion, the combined administration of SCL and QR may be used in upcoming neurological clinical trials, according to in vivo and in silico findings. However, additional investigation is necessary to verify this behavior and clarify the potential mechanism of action.

4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome.

OBJECTIVE: γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. METHODS: We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA in vitro with recombinant GABAA receptors and in vivo with knockin mice. RESULTS: We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. SIGNIFICANCE: This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.

Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

Schisandrin B, a dual positive allosteric modulator of GABAA and glycine receptors, alleviates seizures in multiple mouse models.

Epilepsy is a prevalent and severe neurological disorder and approximately 30% of patients are resistant to existing medications. It is of utmost importance to develop alternative therapies to treat epilepsy. Schisandrin B (SchB) is a major bioactive constituent of Schisandra chinensis (Turcz.) Baill and has multiple neuroprotective effects, sedative and hypnotic activities. In this study, we investigated the antiseizure effect of SchB in various mouse models of seizure and explored the underlying mechanisms. Pentylenetetrazole (PTZ), strychnine (STR), and pilocarpine-induced mouse seizure models were established. We showed that injection of SchB (10, 30, 60 mg/kg, i.p.) dose-dependently delayed the onset of generalized tonic-clonic seizures (GTCS), reduced the incidence of GTCS and mortality in PTZ and STR models. Meanwhile, injection of SchB (30 mg/kg, i.p.) exhibited therapeutic potential in pilocarpine-induced status epilepticus model, which was considered as a drug-resistant model. In whole-cell recording from CHO/HEK-239 cells stably expressing recombinant human GABAA receptors (GABAARs) and glycine receptors (GlyRs) and cultured hippocampal neurons, co-application of SchB dose-dependently enhanced GABA or glycine-induced current with EC50 values at around 5 µM, and application of SchB (10 µM) alone did not activate the channels in the absence of GABA or glycine. Furthermore, SchB (10 µM) eliminated both PTZ-induced inhibition on GABA-induced current (IGABA) and strychnine (STR)-induced inhibition on glycine-induced current (Iglycine). Moreover, SchB (10 µM) efficiently rescued the impaired GABAARs associated with genetic epilepsies. In addition, the homologous mutants in both GlyRs-α1(S267Q) and GABAARs-α1(S297Q)ß2(N289S)γ2L receptors by site-directed mutagenesis tests abolished SchB-induced potentiation of IGABA and Iglycine. In conclusion, we have identified SchB as a natural positive allosteric modulator of GABAARs and GlyRs, supporting its potential as alternative therapies for epilepsy.

Ginger (Zingiber Officinale Roscoe) ameliorates ethanol-induced cognitive impairment by modulating NMDA and GABA-A receptors in rat hippocampus.

Brain damage caused by ethanol abuse may lead to permanent damage, including severe dementia. The aim of this study was to investigate the effects of ginger powder on ethanol-induced cognitive disorders by examining oxidative damage and inflammation status, and the gene expression of N-methyl-D-aspartate (NMDA) and γ-Aminobutyric acid (GABA)-A receptors in the hippocampus of male rats. 24 adult male Sprague-Dawley rats were allocated randomly to four groups as follows control, ethanol (4g/kg/day, by gavage), ginger (1g/kg/day, by gavage), and ginger-ethanol. At the end of the study, memory and learning were evaluated by the shuttle box test. Moreover, to explore mechanisms involved in ethanol-induced cognitive impairment and the protective effect of ginger, the expression of Nuclear factor kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), NMDA receptor, and GABA-A receptor was measured along with inflammatory and oxidative biomarkers in the hippocampus tissue. The results showed that ethanol could induce cognitive impairment in the ethanol group, while pretreatment with ginger could reverse it. The gene expression of the NF-κB/ Tumor necrosis factor (TNF)-α/Interleukin (IL)-1ß pathway and NMDA and GABA-A receptors significantly increased in the ethanol group compared to the control group. While pretreatment with ginger could significantly improve ethanol-induced cognitive impairment through these pathways in the ginger-ethanol group compared to the ethanol group (P < 0.05). It can be concluded that ginger powder could ameliorate ethanol-induced cognitive impairment by modulating the expression of NMDA and GABA-A receptors and inhibiting oxidative damage and the NF-κB/TNF-α/IL-1ß pathway in the rat hippocampus.

Interleukin 6 (IL-6) Regulates GABAA Receptors in the Dorsomedial Hypothalamus Nucleus (DMH) through Activation of the JAK/STAT Pathway to Affect Heart Rate Variability in Stressed Rats.

The dorsomedial hypothalamus nucleus (DMH) is an important component of the autonomic nervous system and plays a critical role in regulating the sympathetic outputs of the heart. Stress alters the neuronal activity of the DMH, affecting sympathetic outputs and triggering heart rate variability. However, the specific molecular mechanisms behind stress leading to abnormal DMH neuronal activity have still not been fully elucidated. Therefore, in the present study, we successfully constructed a stressed rat model and used it to investigate the potential molecular mechanisms by which IL-6 regulates GABAA receptors in the DMH through activation of the JAK/STAT pathway and thus affects heart rate variability in rats. By detecting the c-Fos expression of neurons in the DMH and electrocardiogram (ECG) changes in rats, we clarified the relationship between abnormal DMH neuronal activity and heart rate variability in stressed rats. Then, using ELISA, immunohistochemical staining, Western blotting, RT-qPCR, and RNAscope, we further explored the correlation between the IL-6/JAK/STAT signaling pathway and GABAA receptors. The data showed that an increase in IL-6 induced by stress inhibited GABAA receptors in DMH neurons by activating the JAK/STAT signaling pathway, while specific inhibition of the JAK/STAT signaling pathway using AG490 obviously reduced DMH neuronal activity and improved heart rate variability in rats. These findings suggest that IL-6 regulates the expression of GABAA receptors via the activation of the JAK/STAT pathway in the DMH, which may be an important cause of heart rate variability in stressed rats.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

OBJECTIVE: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

[Effects of electroacupuncture pretreatment on GABAA receptor of fastigial nucleus and sympathetic nerve activity in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effects of electroacupuncture (EA) pretreatment on cardiac function, sympathetic nerve activity, indexes of myocardial injury and GABAA receptor in fastigial nucleus in rats with myocardial ischemia reperfusion injury (MIRI), and to explore the neuroregulatory mechanism of EA pretreatment in improving MIRI. METHODS: A total of 60 male SD rats were randomly divided into a sham operation group, a model group, an EA group, an agonist group and an agonist+EA group, 12 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery. EA was applied at bilateral "Shenmen" (HT 7) and "Tongli" (HT 5) in the EA group and the agonist+EA group, with continuous wave, in frequency of 2 Hz and intensity of 1 mA, 30 min each time, once a day for 7 consecutive days. After intervention, the MIRI model was established. In the agonist group, the muscone (agonist of GABAA receptor, 1 g/L) was injected in fastigial nucleus for 7 consecutive days before modeling, 150 µL each time, once a day. In the agonist+EA group, the muscone was injected in fastigial nucleus 30 min before EA intervention. The data of electrocardiogram was collected by PowerLab standard Ⅱ lead, and ST segment displacement and heart rate variability (HRV) were analyzed; the serum levels of norepinephrine (NE), creatine kinase isoenzyme MB (CK-MB) and cardiac troponin I (cTnI) were detected by ELISA; the myocardial infarction area was measured by TTC staining; the morphology of myocardial tissue was observed by HE staining; the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were detected by immunohistochemistry and real-time PCR. RESULTS: Compared with the sham operation group, in the model group, ST segment displacement and ratio of low frequency to high frequency (LF/HF) of HRV were increased (P<0.01), HRV frequency domain analysis showed enhanced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were increased (P<0.01), the percentage of myocardial infarction area was increased (P<0.01), myocardial fiber was broken and interstitial edema was serious, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were increased (P<0.01). Compared with the model group, in the EA group, ST segment displacement and LF/HF ratio were decreased (P<0.01), HRV frequency domain analysis showed reduced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were decreased (P<0.01), the percentage of myocardial infarction area was decreased (P<0.01), myocardial fiber breakage and interstitial edema were lightened, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were decreased (P<0.01). Compared with the EA group, in the agonist group and the agonist+EA group, ST segment displacement and LF/HF ratio were increased (P<0.01), HRV frequency domain analysis showed enhanced sympathetic nerve excitability, the serum levels of NE, CK-MB and cTnI were increased (P<0.01), the percentage of myocardial infarction area was increased (P<0.01), myocardial fiber breakage and interstitial edema were aggravated, the positive expression and mRNA expression of GABAA receptor in fastigial nucleus were increased (P<0.01). CONCLUSION: EA pretreatment can improve the myocardial injury in MIRI rats, and its mechanism may be related to the inhibition of GABAA receptor expression in fastigial nucleus, thereby down-regulating the excitability of sympathetic nerve.

Heukharang lettuce (Lactuca sativa L.) leaf extract displays sleep-promoting effects through GABAA receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Although lettuce is traditionally known to have hypnotic and sedative effects, to date, only a few studies have documented its sleep-promoting effects and elucidated the related mechanisms. AIM OF THE STUDY: We aimed to investigate the sleep-promoting activity of Heukharang lettuce leaf extract (HLE) with increased lactucin content, known as a sleep-promoting substance in lettuce, in animal models. MATERIALS AND METHODS: To evaluate the effect of HLE on sleep behavior, analysis of electroencephalogram (EEG), gene expression of brain receptors, and activation mechanisms using antagonists were investigated in rodent models. RESULTS: High-performance liquid chromatography analysis showed that HLE contained lactucin (0.78 mg/g of extract) and quercetin-3-glucuronide (1.3 mg/g of extract). In the pentobarbital-induced sleep model, the group administered 150 mg/kg of HLE showed a 47.3% increase in sleep duration time as compared to the normal group (NOR). The EEG analysis showed that the HLE significantly increased non-rapid eye movement (NREM), where delta waves were improved by 59.5% when compared to the NOR, resulting in increased sleep time. In the caffeine-induced arousal model, HLE significantly decreased the awake time increased by caffeine administration (35.5%) and showed a similar level to NOR. In addition, HLE increased the gene and protein expression of gamma-aminobutyric acid receptor type A (GABAA), GABA type B, and 5-hydroxytryptamine (serotonin) receptor 1A. In particular, in comparison to the NOR, the group administered 150 mg/kg HLE showed an increase in expression levels of GABAA and protein by 2.3 and 2.5 times, respectively. When the expression levels were checked using GABAA receptor antagonists, HLE showed similar levels to NOR, as the sleep duration was reduced by flumazenil (45.1%), a benzodiazepine antagonist. CONCLUSIONS: HLE increased NREM sleep and significantly improved sleep behavior due to its action on the GABAA receptors. The collective findings suggest that HLE can be used as a novel sleep-enhancing agent in the pharmaceutical and food industries.

Profiling GABA(A) Receptor Subunit Expression in the Hippocampus of PMDD Rat Models Based on TCM Theories.

γ-Aminobutyric acid type A receptors (GABAARs) play an important role in cognitive and emotional regulation and are related to the hippocampus. However, little is known regarding patterns of hippocampal GABAAR subunit expression in rat models of premenstrual dysphoric disorder (PMDD). This study investigated the above changes by establishing two PMDD rat models based on Traditional Chinese Medicine (TCM) theories, namely, PMDD liver-qi invasion syndrome (PMDD-LIS) and PMDD liver-qi depression syndrome (PMDD-LDS). Behavioral tests were used to detect depression and irritability emotion. Western blot analysis was used to investigate protein levels of GABAAR α1, α2, α4, α5, ß2, ß3, and δ subunits, whereas ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis was performed to determine gamma-aminobutyric acid (GABA) and glutamate (Glu) levels in the hippocampus across each group. Concurrently, behavioral data indicated that the PMDD-LDS and PMDD-LIS rat models had been successfully established. GABAAR α2, α5, ß2, and δ subunit was significantly upregulated, whereas α4 was significantly downregulated (P < 0.05) in PMDD-LDS rat models relative to controls. On the other hand, GABAAR α1, α2, and ß3 were significantly downregulated while α4 and ß2 were significantly upregulated in PMDD-LIS rat models relative to the control group (P < 0.05). Moreover, GABA levels significantly decreased, while Glu and the ratio of glutamate to GABA increased in PMDD-LIS rat models (P < 0.05). Conversely, GABA and Glu levels significantly decreased, whereas the ratio of glutamate to GABA increased in PMDD-LIS rat models (P < 0.05). Conclusively, our results revealed differential expression of GABAAR α1, α2, α4, α5, ß2, ß3, and δ subunits between PMDD-LIS and PMDD-LDS rat models, suggesting that they may be biomarkers in the pathogenesis of PMDD.

Identification of a GABAergic neural circuit governing leptin signaling deficiency-induced obesity.

The hormone leptin is known to robustly suppress food intake by acting upon the leptin receptor (LepR) signaling system residing within the agouti-related protein (AgRP) neurons of the hypothalamus. However, clinical studies indicate that leptin is undesirable as a therapeutic regiment for obesity, which is at least partly attributed to the poorly understood complex secondary structure and key signaling mechanism of the leptin-responsive neural circuit. Here, we show that the LepR-expressing portal neurons send GABAergic projections to a cohort of α3-GABAA receptor expressing neurons within the dorsomedial hypothalamic nucleus (DMH) for the control of leptin-mediated obesity phenotype. We identified the DMH as a key brain region that contributes to the regulation of leptin-mediated feeding. Acute activation of the GABAergic AgRP-DMH circuit promoted food intake and glucose intolerance, while activation of post-synaptic MC4R neurons in the DMH elicited exactly opposite phenotypes. Rapid deletion of LepR from AgRP neurons caused an obesity phenotype which can be rescued by blockage of GABAA receptor in the DMH. Consistent with behavioral results, these DMH neurons displayed suppressed neural activities in response to hunger or hyperglycemia. Furthermore, we identified that α3-GABAA receptor signaling within the DMH exerts potent bi-directional regulation of the central effects of leptin on feeding and body weight. Together, our results demonstrate a novel GABAergic neural circuit governing leptin-mediated feeding and energy balance via a unique α3-GABAA signaling within the secondary leptin-responsive neural circuit, constituting a new avenue for therapeutic interventions in the treatment of obesity and associated comorbidities.

Electroacupuncture-induced activation of GABAergic system alleviates airway inflammation in asthma model by suppressing TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND: Electroacupuncture (EA) has been shown to attenuate airway inflammation in asthmatic mice; however, the underlying mechanism is not fully understood. Studies have shown that EA can significantly increase the inhibitory neurotransmitter γ-aminobutyric acid (GABA) content in mice, and can also increase the expression level of GABA type A receptor (GABAAR). Furthermore, activating GABAAR may relieve inflammation in asthma by suppressing toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. Therefore, this study aimed to investigate the role of GABAergic system and TLR4/MyD88/NF-κB signaling pathway in asthmatic mice treated with EA. METHODS: A mouse model of asthma was established, and a series of methods including Western blot and histological staining assessment were employed to detect the level of GABA, and expressions of GABAAR and TLR4/MyD88/NF-κB in lung tissue. In addition, GABAAR antagonist was used to further validate the role and mechanism of GABAergic system in mediating the therapeutic effect of EA in asthma. RESULTS: The mouse model of asthma was established successfully, and EA was verified to alleviate airway inflammation in asthmatic mice. The release of GABA and the expression of GABAAR were significantly increased in asthmatic mice treated with EA compared with untreated asthmatic mice ( P  < 0.01), and the TLR4/MyD88/NF-κB signaling pathway was down-regulated. Moreover, inhibition of GABAAR attenuated the beneficial effects of EA in asthma, including the regulation of airway resistance and inflammation, as well as the inhibitory effects on TLR4/MyD88/NF-κB signaling pathway. CONCLUSION: Our findings suggest that GABAergic system may be involved in mediating the therapeutic effect of EA in asthma, possibly by suppressing the TLR4/MyD88/NF-κB signaling pathway.

Efficacy and safety of lemborexant as an alternative drug for patients with insomnia taking gamma-aminobutyric acid-benzodiazepine receptor agonists or suvorexant.

BACKGROUND: Although gamma-aminobutyric acid-benzodiazepine (GABA-BZ) receptor agonists are used to treat insomnia, their long-term or high-dosage use causes adverse events. Nevertheless, evidence regarding the discontinuation and replacement of GABA-BZ receptor agonists with alternative agents is lacking. Suvorexant (SUX), an existing orexin receptor antagonist, is effective in preventing nocturnal awakening in 70%-75% of patients with insomnia. METHODS: The novel dual orexin receptor antagonist lemborexant (LEM) has fewer adverse effects than GABA-BZ receptor agonists. Therefore, in this retrospective study, we categorised patients taking GABA-BZ receptor agonists and SUX into LEM-treated (switched) and non-treated (non-switched) groups and compared their outcomes over a 12-week period. RESULTS: The GABA-BZ group (N = 59) comprised 34 'switched' and 25 'non-switched' and the SUX group (N = 14) comprised 6 'switched' and 8 'non-switched' patients. A mixed model showed a significant diazepam equivalence reduction in patients taking GABA-BZ receptor agonists and improved Athens Insomnia Scale score in those taking SUX. The safety and tolerability of GABA-BZ receptor agonists and SUX were high, and no serious adverse effects were observed after switching to LEM. CONCLUSIONS: Lemborexant may be a useful alternative for long-term GABA-BZ receptor agonist users. For SUX, the number of cases (N = 6) was insufficient to draw definite conclusions.

Indole Alkaloids of Rauvolfia ligustrina and Their Anxiolytic Effects in Adult Zebrafish.

Rauvolfia species are well known as producers of bioactive monoterpene indole alkaloids, which exhibit a broad spectrum of biological activities. A new vobasine-sarpagan-type bisindole alkaloid (1: ) along with six known monomeric indoles (2, 3/4, 5: , and 6/7: ) were isolated from the ethanol extract of the roots of Rauvolfia ligustrina. The structure of the new compound was elucidated by interpretation of their spectroscopic data (1D and 2D NMR and HRESIMS) and comparison with published data for analog compounds. The cytotoxicity of the isolated compounds was screened in a zebrafish (Danio rerio) model. The possible GABAergic (diazepam as the positive control) and serotoninergic (fluoxetine as the positive control) mechanisms of action in adult zebrafish were also evaluated. No compounds were cytotoxic. Compound 2: and the epimers 3: /4: and 6: /7: showed a mechanism action by GABAA, while compound 1: showed a mechanism action by a serotonin receptor (anxiolytic activity). Molecular docking studies showed that compounds 2: and 5: have a greater affinity by the GABAA receptor when compared with diazepam, whereas 1: showed the best affinity for the 5HT2AR channel when compared to risperidone.

In silico molecular modeling, neuro-behavioral profile, and toxicity assessment of the essential oil of Ferula gummosa Boiss. as an anti-seizure agent.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferula gummosa Boiss., known in Persian as "Baridje," belongs to the Apiaceae family. All parts of this plant, especially the root, contain galbanum. Galbanum, the oleo-gum resin of F. gummosa, is one of the essential traditional herbal medicines in Iran, which is used as a tonic for epilepsy and chorea, memory enhancement, gastrointestinal diseases, and wound healing. AIM OF THE STUDY: We investigated the toxicity, anticonvulsant effects, and molecular modeling of the essential oil (EO) distilled from the oleo-gum resin of F. gummosa. MATERIALS AND METHODS: Gas chromatography-mass spectrometry was used to identify the EO components. The cytotoxicity of EO on HepG2 cell lines was assessed by the MTT method. Male mice were arranged as follows: negative control groups (sunflower oil (10 ml/kg, i.p.) or saline (10 ml/kg, p.o.)), EO groups (0.5, 1, 1.5, and 2.5 ml/kg, p.o.), and positive control groups (ethosuximide (150 mg/kg, p.o.) or diazepam (1.0 or 2 mg/kg, i.p.)). The motor coordination and neurotoxicity of EO were studied using the rota-rod test. Open-field, novel object recognition, and passive avoidance learning tests were used to investigate the effect of EO on locomotor activity and memory function. An acute pentylenetetrazole-induced seizure model was utilized to evaluate the anticonvulsant properties of the EO. The interaction of the EO main components with the GABAA receptor was investigated by coarse-grained molecular dynamics simulations. RESULTS: ß-pinene, sabinene, α-pinene, and ρ-cymene were the main components of EO. The IC50 of the EO at 24, 48, and 72 h was found to be 59.90, 12.96, and 3.93 µl/ml, respectively. No adverse effects were observed in memory, motor coordination, and locomotor activity in mice treated with EO. Administration of EO (1, 1.5, and 2.5 ml/kg) improved survival rates in mice receiving pentylenetetrazole (PTZ; to induce an epileptic seizure). Sabinene was able to bind to the binding site of benzodiazepines at the GABAA receptor. CONCLUSIONS: Acute treatment with the EO of F. gummosa caused antiepileptic effects and could effectively increase the survival rate in PTZ-treated mice with no significant toxicity.