Histamine H1-receptor-mediated modulation of NMDA receptors signaling responses.

This study attempted to clarify the role of histamine H1 receptors in epilepsy by exploring the effects of agonists and inverse agonists on the rundown of the current induced by iterative applications of NMDA or GABA in primary neuronal culture. Mepyramine, a classical H1-receptor antagonist/inverse agonist, increased the NMDA current by about 40% during the first minutes of recording. This effect was concentration-dependent, maximal at 10 nM, and mimicked by triprolidine, another antagonist/inverse agonist. No endogenous histamine was detected in the cultures by a selective immunoassay; both compounds were acting as inverse agonists. Indicating a high constitutive activity of the H1 receptor in this system, histamine did not affect the NMDA rundown, including its settlement, but significantly reversed the effect of mepyramine. A similar pattern was obtained with 2,3 bromophenyl histamine, a selective H1-receptor agonist. The initial increase induced by the two inverse agonists was followed by the same rundown as in controls. H1- and NMDA receptors are colocalized in most cultured neuronal cells. Mepyramine and histamine did not affect the GABA rundown. Our findings suggest an interaction between H1- and NMDA receptors. Inactivation of the H1-receptor by its inverse agonists delays the settlement of the NMDA rundown, which may underlie their proconvulsant effect reported in clinics. Therefore, H1-receptor constitutive activity and the effect of histamine revealed in its absence, tend to facilitate the initiation of the rundown, which is consistent with the anticonvulsant properties of histamine via activation of H1-receptors reported in many studies.

Chronic Nasal Administration of Kisspeptin-54 Regulates Mood-Related Disorders Via Amygdaloid GABA in Hemi-Parkinsonian Rats

Background: Depression and anxiety, the most prevalent neuropsychiatric manifestations in Parkinson's disease (PD), negatively impact their quality of life. Aims: To determine whether the chronic nasal administration of kisspeptin-54 (KP-54) could. Alleviate symptoms of anxiety and depression in hemi-Parkinsonian rats. Study Design: Experimental study. Methods: This study included adult Sprague Dawley male rats who were administered either a vehicle (artificial cerebrospinal fluid) or 6-hydroxydopamine (6-OHDA) unilaterally into the medial forebrain bundle. The vehicle, or KP-54 (3 nmol/kg, applied topically to the rhinarium), was administered daily for a seven-day period. The sucrose preference test (SPT), elevated plus maze test (EPMT), and open field test (OFT) were implemented to evaluate depression- and anxiety-like behaviors, respectively, seven days following the lesion surgery. Gamma-aminobutyric acid (GABA) concentrations in the amygdala were quantified using mass spectrometry. Tyrosine hydroxylase in substantia nigra was analyzed using immunohistochemistry. Results: The nasal delivery of KP-54 significantly reduced depressionand anxiety-like behaviors that were induced by 6-OHDA, as indicated by the results of the SPT, OFT, and EPMT. Moreover, it was observed that nasal KP-54 effectively mitigated 6-OHDA-induced motor deficits and the loss of nigral dopaminergic neurons. The nasal administration of KP-54 augmented the decline in GABA levels in the amygdala induced by 6-OHDA. Furthermore, effective correlations were established between GABA concentrations and behavioral parameters. Conclusion: The nasal delivery of KP-54 could function as a viable therapeutic alternative for treating mood-related disorders in PD.

Inhibitory Actions of Potentiating Neuroactive Steroids in the Human α1ß3γ2L γ-Aminobutyric Acid Type A Receptor.

The γ-aminobutyric acid type A (GABAA) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3ß-hydroxy steroids inhibit, while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1ß3γ2L GABAA receptor by the endogenous neurosteroid 3α-hydroxy-5ß-pregnan-20-one (3α5ßP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17ß-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5ßP and ACN potentiate the GABAA receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC50s of ∼13 µM and maximal inhibitory effects of 70-90%. Receptor inhibition by 3α5ßP was sensitive to substitution of the α1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5ßP inhibit the GABAA receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and ß3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3α5ßP and ACN. SIGNIFICANCE STATEMENT: The heteromeric GABAA receptor is inhibited by sulfated steroids and 3ß-hydroxy steroids, while 3α-hydroxy steroids are considered to potentiate the receptor. We show here that 3α-hydroxy steroids have inhibitory effects on the α1ß3γ2L receptor, which are observed in specific experimental settings and are expected to manifest under different physiological conditions.

Characterization of commercially available murine fibrosarcoma NCTC-2472 cells both in vitro and as a model of bone cancer pain in vivo.

For many cancer patients tumor burden negatively impacts quality of life due to associated pain onset. Neuropathic pain is commonly associated with late cancer stages, and is resultant of tumor metastasis to bone, herein referred to as cancer-induced bone pain. Given the severe impact on quality of life and clinical treatment strategies focusing on symptom management, novel therapeutics are needed to alleviate cancer-induced bone pain and/or reduce cancer burden. In the current study we characterized a commercially available murine fibrosarcoma cell line, NCTC-2472 in vitro, which can be used to assess the capacity of novel compounds to impact cellular viability. We found that dimethyl sulfoxide, a known cytotoxic agent and common drug preparation compound, significantly decreased cell viability in a dose-related manner. We then characterized the in vivo tumor development and associated pain behavior characteristics following implantation of NCTC-2472 fibrosarcoma into male and female C3H/HeJ mice. The C3H/HeJ strain was utilized as these mice are syngeneic with NCTC-2472 fibrosarcoma and their use reduces potential implantation failure. We found that tumor development in mice resulted in the development of mechanical allodynia but not thermal hyperalgesia. Gabapentin, a clinically relevant analgesic, produced dose-related mechanical allodynia reversal. These studies provide further characterization of a cancer-induced bone pain model that can be used to examine novel compounds as anti-cancer and analgesic therapeutics.

Gastroprotective Effect of GABA in Metabolic Stress.

We studied the effect of enteral administration of GABA on the gastric mucosa in male Wistar rats (n=47) with modeled metabolic stress (food deprivation for 9 days with free access to water). The relative weights of the adrenal glands and thymus were determined, and histological examination of the stomach was performed. In control rats, modeling the metabolic stress was accompanied by the development of erosive damage to the gastric mucosa related to blood supply disturbances. Administration of GABA prevented erosions and exhibited a pronounced gastroprotective effect. Thus, administration of GABA can be a promising method for the prevention and treatment of erosive gastric lesions associated with metabolic stress.

Gabapentin improves neuropathic pain in Minamata disease model rats.

BACKGROUND: Methylmercury (MeHg), the causative agent of Minamata disease, damages the cranial nervous system and causes specific sensory disturbances, especially hypoesthesia, in the extremities. However, recent reports demonstrate that patients with chronic Minamata disease conversely develop neuropathic pain in the lower extremities. Studies on our established Minamata disease model rats showed that MeHg-mediated neurodegeneration might induce neuropathic pain by over time through inducing rewiring with neuronal activation in the somatosensory cortex via microglial activation in the spinal dorsal horn. METHODS: In this study, the effects of gabapentin, a potentially effective treatment for neuropathic pain, was evaluated using this Minamata disease model rats. To further elucidate the mechanism of its medicinal effects, histochemical and biochemical analyses of the nervous system of Minamata disease model rats were conducted. RESULTS: Gabapentin treatment restored the reduction in the pain threshold caused by MeHg exposure in rats. Histochemical and biochemical analyses revealed that gabapentin showed no effect on MeHg-induced neurodegeneration in entire nervous system and microglial activation in the spinal dorsal horn. However, it was shown that gabapentin may reduce excessive synaptogenesis through its antagonist action on the alpha2-delta-1 subunit of calcium channels in the somatosensory cortex. CONCLUSIONS: These results indicate that gabapentin may alleviated neuropathic pain in MeHg poisoning, as typified by Minamata disease, by reversibly modulation synaptic rewiring in the somatosensory cortex.

Effects of psoralidin on the expression of glutamate decarboxylases and inhibitory synapse development.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter required for excitation/inhibition balance is synthesized by the glutamic acid decarboxylases (GADs) in GABAergic neurons. The levels and activity of GADs are strongly correlated with GABA and neural transmission. Dysregulation of GADs and GABA is associated with various neurological disorders. The study used psoralidin, found in the seeds of Psoralea corylifolia, to investigate its effect on GAD levels and regulatory mechanisms in primary cortical neurons. Psoralidin reduced GAD67 through transcriptional regulation. The reduction was not mediated by the N-methyl-D-aspartate receptor. Additionally, psoralidin attenuated the formation of inhibitory synapses in primary hippocampal neurons.

The renoprotective activity of amikacin-gamma-amino butyric acid-chitosan nanoparticles: a comparative study.

The development of nanoparticles (NPs) with active components with upgraded stability, and prolonged release helps in enhanced tissue regeneration. In addition, NPs are feasible strategy to boost antibiotic effectiveness and reduce drug side effects. Our study focuses on the use of amikacin (AMK) and gamma amino butyric acid (GABA) unloaded combinations or loaded on chitosan nanoparticles (CSNPs) for kidney protection. The AMK-GABA-CSNPs were prepared with the ionic gelation method, the morphology was studied using transmission electron microscopy (TEM), zetasizer and the Fourier transform-infrared spectroscopy (FT-IR) spectrum of the synthesized NPs was observed. The average size of AMK-GABA-CSNPs was 77.5 ± 16.5 nm. Zeta potential was + 38.94 ± 2.65 mV. AMK-GABA-CSNPs revealed significant in vitro antioxidant, anti-coagulation, non-hemolytic properties and good cell compatibility. To compare the effects of the unloaded AMK-GABA combination and AMK-GABA-CSNPs on the renal tissue, 42 healthy Sprague-Dawley rats were divided into seven groups. G1: normal control (NC), normal saline; G2: low-dose nephrotoxic group (LDN), AMK (20 mg/kg/day; i.p.); G3: unloaded AMK (20 mg/kg/day; i.p.) and GABA (50 mg/kg/day; i.p.); G4: AMK-GABA-CSNPs (20 mg/kg/day; i.p.); G5: high-dose nephrotoxic group (HDN), AMK (30 mg/kg/day; i.p.); G6: unloaded AMK (30 mg/kg/day; i.p.) and GABA (50 mg/kg/day; i.p.) and G7: AMK-GABA-CSNPs (30 mg/kg/day; i.p.). The results showed that AMK-GABA-CSNPs formulation is superior to unloaded AMK-GABA combination as it ameliorated kidney functions, oxidative stress and displayed a significant homeostatic role via suppression of inflammatory cytokines of Th1, Th2 and Th17 types. Hence, AMK-GABA-CSNPs could afford a potential nano-based therapeutic formula for the management of AMK-nephrotoxicity.

Cell-specific extracellular vesicle-encapsulated exogenous GABA controls seizures in epilepsy.

BACKGROUND: Epilepsy affects ∼60 million people worldwide. Most antiseizure medications in the market act on voltage-gated sodium or calcium channels, indirectly modulating neurotransmitter GABA or glutamate levels or multiple targets. Earlier studies made significant efforts to directly deliver GABA into the brain with varied success. Herein, we have hypothesized to directly deliver exogenous GABA to the brain with epilepsy through extracellular vesicles (EVs) from human GABA-producing cells and their progenitors as EVs largely mimic their parent cell composition. METHODS: Human neural stem cells (NSCs), medial ganglionic eminence (MGE) cells, and GABAergic interneurons (INs) were generated from induced pluripotent stem cells (iPSCs) and characterized. EVs were isolated from NSCs, MGE cells, and INs and characterized for size and distribution, morphological features, and molecular markers. Exogenous GABA was passively loaded to the isolated EVs as a zwitterion at physiological pH, and the encapsulated dose of GABA was quantified. Epilepsy was developed through status epilepticus induction in Fisher rats by administration of repeated low doses of kainic acid. The extent of the seizures was measured for 10 h/ day for 3-6 months by video recording and its evaluation for stage III, IV and V seizures as per Racine scale. EVs from INs, MGE cells, and NSCs encapsulated with exogenous GABA were sequentially tested in the 4th, 5th, and 6th months by intranasal administration in the rats with epilepsy for detailed seizure, behavioral and synapse analysis. In separate experiments, several controls including exogenic GABA alone and EVs from INs and MGE cells were evaluated for seizure-controlling ability. RESULTS: Exogenic GABA could enter the brain through EVs. Treatment with EVs from INs and MGE cells encapsulated with GABA significantly reduced total seizures, stage V seizures, and total time spent in seizure activity. EVs from NSCs encapsulated with GABA demonstrated limited seizure control. Exogenic GABA alone and EVs from INs and MGE cells individually failed to control seizures. Further, exogenic GABA with EVs from MGE cells improved depressive behavior while partially improving memory functions. Co-localization studies confirmed exogenous GABA with presynaptic vesicles in the hippocampus, indicating the interaction of exogenous GABA in the brain with epilepsy. CONCLUSION: For the first time, the study demonstrated that exogenous GABA could be delivered to the brain through brain cell-derived EVs, which could regulate seizures in temporal lobe epilepsy. It is identified that the cellular origin of EVs plays a vital role in seizure control with exogenous GABA.

GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABAA receptors.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABAARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABAAR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABAAR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABAAR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABAARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

Effect of GABAa-receptors on neuronal discharge and ion activity in focal seizures.

Focal seizures are a type of epileptic event that has plagued the medical community for a long time, and the existing drug treatment is mainly based on the modulation of ${GABA}_a$-receptors to affect GABAergic signaling to achieve the therapeutic purpose. The majority of research currently focuses on the impact of ${GABA}_a$-receptors on neuronal firing, failing to analyze the molecular and ionic mechanisms involved. Specifically, the research on deeper-level mechanisms on how ${GABA}_a$-receptors affect neuronal firing by altering ion activity has not been addressed. This research aimed to study the effects of different ${GABA}_a$-receptor structures on ion activity in focal seizures model by adjusting parameters of the ${GABA}_a$-receptors: the rise time constant (${tau}_1$) and decay time constant (${tau}_2$). The research indicates that as the values of ${tau}_1$ and ${tau}_2$ of the ${GABA}_a$-receptor change, the ion concentration will vary based on the change of the ${GABA}_a$-receptor potential. To a certain extent, the duration of epileptic activity will also be affected to a certain extent. In conclusion, the alteration of ${GABA}_a$-receptor structure will affect the inhibitory effect of interneurons on pyramidal neurons, and different parameters of the ${GABA}_a$-receptor will directly impact the therapeutic effect.

Effects of New Psychoactive Substance Esketamine on Behaviors and Transcription of Genes in Dopamine and GABA Pathways in Zebrafish Larvae.

Esketamine (ESK) is the S-enantiomer of ketamine racemate (a new psychoactive substance) that can result in illusions, and alter hearing, vision, and proprioception in human and mouse. Up to now, the neurotoxicity caused by ESK at environmental level in fish is still unclear. This work studied the effects of ESK on behaviors and transcriptions of genes in dopamine and GABA pathways in zebrafish larvae at ranging from 12.4 ng L- 1 to 11141.1 ng L- 1 for 7 days post fertilization (dpf). The results showed that ESK at 12.4 ng L- 1 significantly reduced the touch response of the larvae at 48 hpf. ESK at 12.4 ng L- 1 also reduced the time and distance of larvae swimming at the outer zone during light period, which implied that ESK might potentially decrease the anxiety level of larvae. In addition, ESK increased the transcription of th, ddc, drd1a, drd3 and drd4a in dopamine pathway. Similarly, ESK raised the transcription of slc6a1b, slc6a13 and slc12a2 in GABA pathway. This study suggested that ESK could affect the heart rate and behaviors accompanying with transcriptional alterations of genes in DA and GABA pathways at early-staged zebrafish, which resulted in neurotoxicity in zebrafish larvae.

Gut microbiota-derived gamma-aminobutyric acid from metformin treatment reduces hepatic ischemia/reperfusion injury through inhibiting ferroptosis.

Hepatic ischemia/reperfusion injury (HIRI) is a common and inevitable factor leading to poor prognosis in various liver diseases, making the outcomes of current treatments in clinic unsatisfactory. Metformin has been demonstrated to be beneficial to alleviate HIRI in recent studies, however, the underpinning mechanism remains unclear. In this study, we found metformin mitigates HIRI-induced ferroptosis through reshaped gut microbiota in mice, which was confirmed by the results of fecal microbiota transplantation treatment but showed the elimination of the beneficial effects when gut bacteria were depleted using antibiotics. Detailedly, through 16S rRNA and metagenomic sequencing, we identified that the metformin-reshaped microbiota was characterized by the increase of gamma-aminobutyric acid (GABA) producing bacteria. This increase was further confirmed by the elevation of GABA synthesis key enzymes, glutamic acid decarboxylase and putrescine aminotransferase, in gut microbes of metformin-treated mice and healthy volunteers. Furthermore, the benefit of GABA against HIRI-induced ferroptosis was demonstrated in GABA-treated mice. Collectively, our data indicate that metformin can mitigate HIRI-induced ferroptosis by reshaped gut microbiota, with GABA identified as a key metabolite.

Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of 'Zaosu' pear fruit by L-glutamate treatment.

'Zaosu' pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of 'Zaosu' pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and ß-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbß-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of 'Zaosu' pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

Commentary: Fifty years exploring pharmacology with Sam Enna.

The passing of Sam Enna in June of 2023 is major loss to the world of pharmacology. While best known for his extensive research activities in the area of γ-aminobutyric acid (GABA) pharmacology, Sam devoted much of his professional time to teaching and as an Editor in Chief for the legacy journals - the Journal of Pharmacology and Experimental Therapeutics (JPET - 1998-2003); Pharmacology & Therapeutics (P & T - 2003-2023) and Biochemical Pharmacology (BCP -2003-2023) - increasing the volume of submissions for all three journals and their Impact Factors while decreasing the time for peer review and publication. Sam was a well-respected consultant in the CNS area for the biopharmaceutical industry and served as Secretary General and President of the International Union of Basic and Clinical Pharmacology where his efforts were focused on sustaining research integrity, particularly in the areas of data reproducibility and fraud. This Commentary provides a personal overview of Sam's 50-year career in pharmacology and briefly updates topics that were of keen interest to Sam including: developments on the continuing reproducibility crisis where systematic fraud continues to proliferate now reaching industrial scale proportions, aided and abetted by paper mills, AI and the erosion of meritocratic norms; and the fall and rise of CNS drug discovery.

T-Type Ca2+ Channels Mediate a Critical Period of Plasticity in Adult-Born Granule Cells.

Adult-born granule cells (abGCs) exhibit a transient period of elevated synaptic plasticity that plays an important role in hippocampal function. Various mechanisms have been implicated in this critical period for enhanced plasticity, including minimal GABAergic inhibition and high intrinsic excitability conferred by T-type Ca2+ channels. Here we assess the contribution of synaptic inhibition and intrinsic excitability to long-term potentiation (LTP) in abGCs of adult male and female mice using perforated patch recordings. We show that the timing of critical period plasticity is unaffected by intact GABAergic inhibition such that 4-6-week-old abGCs exhibit LTP that is absent by 8 weeks. Blocking GABAA receptors, or partial blockade of GABA release from PV and nNos-expressing interneurons by a µ-opioid receptor agonist, strongly enhances LTP in 4-week-old GCs, suggesting that minimal inhibition does not underlie critical period plasticity. Instead, the closure of the critical period coincides with a reduction in the contribution of T-type Ca2+ channels to intrinsic excitability, and a selective T-type Ca2+ channel antagonist prevents LTP in 4-week-old but not mature GCs. Interestingly, whole-cell recordings that facilitate T-type Ca2+ channel activity in mature GCs unmasks LTP (with inhibition intact) that is also sensitive to a T-type Ca2+ channel antagonist, suggesting T-type channel activity in mature GCs is suppressed by native intracellular signaling. Together these results show that abGCs use T-type Ca2+ channels to overcome inhibition, providing new insight into how high intrinsic excitability provides young abGCs a competitive advantage for experience-dependent synaptic plasticity.

Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4ß2, α3ß4, and α7) and muscarinic (M1-M5) AChRs. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

Factors promoting the release of picrotoxin from the trap in the GABA(A) receptor pore.

Picrotoxin (PTX), a convulsant of plant origin, has been used in many studies as research tool. PTX is the open channel blocker of the GABAA receptor (GABAAR). Being in the pore, PTX initiates transfer of the channel to the closed state and thus it falls into the "trap". The consequence of this PTX trapping is so-called aftereffect, i.e. continuation of the blockade of the GABA-induced chloride current (IGABA) after removal of PTX from the external solution. The present work shows that the positive allosteric modulators (PAMs) of the GABAA receptor, allopregnanolone (Allo) and zolpidem (Zolp) as well as a high concentration of GABA shortened the PTX aftereffect. Experiments were carried out on isolated Purkinje neurons of the rat cerebellum using the whole-cell patch-clamp method. IGABA was induced by applications of 5 µM GABA (EC30) for 1 s with 30 s intervals. 50 µM PTX completely blocked IGABA, and recovery upon PTX washout occurred with a time constant (τrec) of 20.2 min. 1 µM Allo reduced the blocking effect of PTX by 30% and accelerated the recovery of IGABA by almost 10 times (τrec = 2.4 min). 0.5 µM Zolp did not change the IGABA block in the presence of PTX but accelerated the recovery of IGABA by more than 3 times (τrec = 5.6 min). Increasing the GABA concentration to 20 µM did not change the blocking effect of PTX, but accelerated recovery by 6 times (τrec = 3.3 min). The mechanism of the shortening of the PTX aftereffect is presumably the expansion of the GABAAR pore in the presence of PAMs and a high concentration of the agonist and, as a consequence, the escape of PTX from the "trap". The work describes new pharmacological properties of Allo and Zolp.

Contribution of chemical and electrical transmission to the low delta-like intrinsic retinal oscillation in mice: A role for daylight-activated neuromodulators.

Basal electroretinogram (ERG) oscillations have shown predictive value for modifiable risk factors for type 2 diabetes. However, their origin remains unknown. Here, we seek to establish the pharmacological profile of the low delta-like (δ1) wave in the mouse because it shows light sensitivity in the form of a decreased peak frequency upon photopic exposure. Applying neuropharmacological drugs by intravitreal injection, we eliminated the δ1 wave using lidocaine or by blocking all chemical and electrical synapses. The δ1 wave was insensitive to the blockade of photoreceptor input, but was accelerated when all inhibitory or ionotropic inhibitory receptors in the retina were antagonized. The sole blockade of GABAA, GABAB, GABAC, and glycine receptors also accelerated the δ1 wave. In contrast, the gap junction blockade slowed the δ1 wave. Both GABAA receptors and gap junctions contribute to the light sensitivity of the δ1 wave. We further found that the day light-activated neuromodulators dopamine and nitric oxide donors mimicked the effect of photopic exposure on the δ1 wave. All drug effects were validated through light flash-evoked ERG responses. Our data indicate that the low δ-like intrinsic wave detected by the non-photic ERG arises from an inner retinal circuit regulated by inhibitory neurotransmission and nitric oxide/dopamine-sensitive gap junction-mediated communication.

Excitatory action of low frequency depolarizing GABA/glycine synaptic inputs is prevalent in prenatal spinal SOD1G93A motoneurons.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease characterized by progressive motor neuron degeneration and muscle paralysis. Recent evidence suggests the dysfunction of inhibitory signalling in ALS motor neurons. We have shown that embryonic day (E)17.5 spinal motoneurons (MNs) of the SOD1G93A mouse model of ALS exhibit an altered chloride homeostasis. At this prenatal stage, inhibition of spinal motoneurons (MNs) is mediated by depolarizing GABAergic/glycinergic postsynaptic potentials (dGPSPs). Here, using an ex vivo preparation and patch clamp recording from MNs with a chloride equilibrium set below spike threshold, we report that low input resistance (Rin ) E17.5 MNs from the SOD1G93A ALS mouse model do not correctly integrate dGPSPs evoked by electrical stimulations of GABA/glycine inputs at different frequencies. Indeed, firing activity of most wild-type (WT) MNs with low Rin was inhibited by incoming dGPSPs, whereas low Rin SOD1G93A MNs were excited or exhibited a dual response (excited by low frequency dGPSPs and inhibited by high frequency dGPSPs). Simulation highlighted the importance of the GABA/glycine input density and showed that pure excitation could be obtained in SOD-like MNs by moving GABA/glycine input away from the cell body to dendrites. This was in agreement with confocal imaging showing a lack of peri-somatic inhibitory terminals in SOD1G93A MNs compared to WT littermates. Putative fast ALS-vulnerable MNs with low Rin are therefore lacking functional inhibition at the near-term prenatal stage. KEY POINTS: We analysed the integration of GABAergic/glycinergic synaptic events by embryonic spinal motoneurons (MNs) in a mouse model of the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. We found that GABAergic/glycinergic synaptic events do not properly inhibit ALS MNs with low input resistance, most probably corresponding to future vulnerable MNs. We used a neuron model to highlight the importance of the GABA/glycine terminal location and density in the integration of the GABAergic/glycinergic synaptic events. Confocal imaging showed a lack of GABA/glycine terminals on the cell body of ALS MNs. The present study suggests that putative ALS vulnerable MNs with low Rin lack functional inhibition at the near-term stage.