GABA-induced Currents Research Articles

Influence of three insecticides targeting GABA receptor on fall armyworm Spodoptera frugiperda: Analyses from individual, biochemical and molecular levels

The fall armyworm (FAW) Spodoptera frugiperda (Lepidoptera: Noctuidae) is a severe agricultural pest, which has invaded into China in 2019 and caused heavy damage to maize. The γ-aminobutyric acid receptor (GABAR)-targeted insecticides including broflanilide, fluralaner and fipronil exhibit high toxicity towards lepidopteran pests. However, whether they could be used for control of FAW and their possible mode of action in FAW remain unclear. In this study, broflanilide, fluralaner and fipronil exhibited high oral toxicity in FAW larvae with median lethal dose (LD50) values of 0.677, 0.711, and 23.577 mg kg−1 (active ingredient/ artificial food), respectively. In the electrophysiological assay, fluralaner and fipronil could strongly inhibit GABA-induced currents of homomeric FAW resistance to dieldrin 1 (RDL1) receptor with median inhibitory concentration (IC50) values of 5.018 nM (95% confidence interval (CI) 2.864–8.789) and 8.595 nM (95% CI 5.105–14.47), respectively, whereas broflanilide could not. In addition, the cytochrome P450 (P450), glutathione-S-transferase (GST) and carboxylesterase (CarE) activities were positively response to broflanilide, P450 and GST to fluralaner, and GST and CarE to fipronil, respectively, compared with those of control. In conclusion, we firstly reported a notable insecticidal activity of three representative GABAR-targeted insecticides to FAW in vivo, and in vitro using electrophysiological assay. The GST is the primary detoxification enzyme for three tested insecticides. Our results would guide the rotational use of GABAR-targeted insecticides in field.

Toxicity and mode of action of the aporphine plant alkaloid liriodenine on the insect GABA receptor

Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20–30 μM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC50 value of about 1 μM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline.

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation

BackgroundGamma-aminobutyric acid A (GABAA) receptors have been implicated in anxiety and epileptic disorders. Hypothesis/PurposeThis study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders. MethodsStigmasterol was evaluated on various recombinant GABAA receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2β2γ2L, α4β3δ, and α4β3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABAA α2β2γ2L, α4β3, and α4β3δ complexes by in silico docking. ResultsStigmasterol enhanced GABA-induced currents at ternary α2β2γ2L, α4β3δ, and binary α4β3 GABAAR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4β3δ was significantly higher compared to the binary α4β3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4β3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of β3 at α4β3δ complex. In in vivo studies, Stigmasterol (0.5–3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism. ConclusionTo our knowledge, this is the first study reporting the positive modulation of GABAA receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.

Epipregnanolone as a Positive Modulator of GABAA Receptor in Rat Cerebellar and Hippocampus Neurons.

Epipregnanolone (3β-hydroxy-5β-pregnan-20-one, Epi) is an endogenous steroid with important physiological effects and high affinity for GABAA receptors. The effect of Epi on GABA-induced chloride current (IGABA) in native neurons has hardly been studied. In this work, we studied the influence of Epi on the IGABA in the Purkinje cells of rat cerebellum and pyramidal neurons of rat hippocampus with the patch clamp technique. We showed that Epi is a positive modulator of the IGABA with EC50 of 5.7 µM in Purkinje cells and 9.3 µM in hippocampal neurons. Epi-induced potentiation of the IGABA was more potent at low vs. high GABA concentrations. Isopregnanolone (3β-hydroxy-5α-pregnan-20-one, Iso) counteracted Epi, reducing its potentiating effect by 2–2.3 times. Flumazenil, a nonsteroidal GABAA receptor antagonist, does not affect the Epi-induced potentiation. Comparison of the potentiating effects of Epi and allopregnanolone (3α-hydroxy-5α-pregnan-20-one, ALLO) showed that ALLO is, at least, a four times more potent positive modulator than Epi. The combined application of ALLO and Epi showed that the effects of these two steroids are not additive. We conclude that Epi has a dual effect on the IGABA increasing the current in the control solution and decreasing the stimulatory effect of ALLO.

Basmisanil, a highly selective GABAA-\u03b15 negative allosteric modulator: preclinical pharmacology and demonstration of functional target engagement in man

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Competitive antagonism of housefly γ-aminobutyric acid receptors by iminopyridazine butanoic acids

The competitive antagonistic activities of a series of gabazine (3) based 3-substituted iminopyridazine butanoic acid analogs [4-(3-aryl/heteroaryl-1,6-dihydro- 6-iminopyridazin-1-yl) butanoic acid hydrochlorides] 4a-4k (Figure 1) were examined at housefly (Musca domestica) GABA receptors expressed in Xenopus oocytes using two-electrode voltage-clamp (TEVC) technique. The 4-biphenylyl analog 4e exhibited the highest inhibition (approximately 68%) of GABA-induced currents at 100 μM with an IC50 value of 40.81 ± 3.89 μM. The 2-naphthyl analog 4h was the second most active compound with approximately 48% inhibition. The 4-biphenylyl analog 4e demonstrated competitive antagonism of housefly GABA receptors. Ligand docking studies into the binding site of housefly GABA receptor homology model predicted that the aromatic 3-substituents are tolerable in the pyridazine ring. The results presented in this paper about GABA receptor competitive antagonists may helpful for design and development of GABA receptor related insecticides.
 Bangladesh J. Sci. Ind. Res.56(1), 9-16, 2021

Humulone Modulation of GABAA Receptors and Its Role in Hops Sleep-Promoting Activity.

Humulus lupulus L. (hops) is a major constituent of beer. It exhibits neuroactive properties that make it useful as a sleeping aid. These effects are hypothesized to be mediated by an increase in GABAA receptor function. In the quest to uncover the constituents responsible for the sedative and hypnotic properties of hops, recent evidence revealed that humulone, a prenylated phloroglucinol derivative comprising 35–70% of hops alpha acids, may act as a positive modulator of GABAA receptors at low micromolar concentrations. This raises the question whether humulone plays a key role in hops pharmacological activity and potentially interacts with other modulators such as ethanol, bringing further enhancement in GABAA receptor-mediated effects of beer. Here we assessed electrophysiologically the positive modulatory activity of humulone on recombinant GABAA receptors expressed in HEK293 cells. We then examined humulone interactions with other active hops compounds and ethanol on GABA-induced displacement of [3H]EBOB binding to native GABAA receptors in rat brain membranes. Using BALB/c mice, we assessed humulone’s hypnotic behavior with pentobarbital- and ethanol-induced sleep as well as sedation in spontaneous locomotion with open field test. We demonstrated for the first time that humulone potentiates GABA-induced currents in α1β3γ2 receptors. In radioligand binding to native GABAA receptors, the inclusion of ethanol enhanced humulone modulation of GABA-induced displacement of [3H]EBOB binding in rat forebrain and cerebellum as it produced a leftward shift in [3H]EBOB displacement curves. Moreover, the additive modulatory effects between humulone, isoxanthohumol and 6-prenylnaringenin were evident and corresponded to the sum of [3H]EBOB displacement by each compound individually. In behavioral tests, humulone shortened sleep onset and increased the duration of sleep induced by pentobarbital and decreased the spontaneous locomotion in open field at 20 mg/kg (i.p.). Despite the absence of humulone effects on ethanol-induced sleep onset, sleep duration was increased dose-dependently down to 10 mg/kg (i.p.). Our findings confirmed humulone’s positive allosteric modulation of GABAA receptor function and displayed its sedative and hypnotic behavior. Humulone modulation can be potentially enhanced by ethanol and hops modulators suggesting a probable enhancement in the intoxicating effects of ethanol in hops-enriched beer.

Molecular cloning and pharmacology of Min-UNC-49B, a GABA receptor from the southern root-knot nematode Meloidogyne incognita.

Root-knot nematodes are plant-parasitic nematodes that cause immense damage to a broad range of cultivated crops by forming root galls, resulting in yield losses in crops. To facilitate the development of faster-acting selective nematicides, we cloned three cDNAs encoding UNC-49B proteins from the southern root-knot nematode Meloidogyne incognita and examined their functional and pharmacological properties by two-electrode voltage clamp electrophysiology using a Xenopus oocyte expression system. The three cloned cDNAs encoded Min-UNC-49B, Min-UNC-49B-L and Min-UNC-49B-XL; the last two proteins have longer N-terminal regions than the first protein. When expressed in Xenopus oocytes, these proteins responded to γ-aminobutyric acid (GABA) to activate currents with high-micromolar or low-millimolar half-maximal effective concentration (EC50 ) values, indicating the formation of functional homo-pentameric GABA receptors. Fipronil and picrotoxinin inhibited GABA-induced currents with high-nanomolar and low-micromolar half-maximal inhibitory concentration (IC50 ) values, respectively, in oocytes expressing Min-UNC-49B. The G2'A and T6'M mutations in the second transmembrane domain of Min-UNC-49B enhanced and reduced the sensitivity of Min-UNC-49B to these two antagonists, respectively. Samaderine B and SF-14 inhibited GABA responses in oocytes expressing Min-UNC-49B with low-micromolar and high-nanomolar IC50 values, respectively. Ivermectin, α-terpineol, thymol and methyl eugenol exerted dual effects on Min-UNC-49B by potentiating currents induced by low concentrations of GABA and inhibiting currents induced by high concentrations of GABA. We have shown that structurally diverse compounds act at Min-UNC-49B GABA receptors. Our results may serve as a starting point to decipher the molecular function of native GABA receptors of plant-parasitic nematodes, which could aid in the structure-based design of novel nematicides. © 2020 Society of Chemical Industry.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

Positive allosteric modulation of GABAA receptors by a novel antiepileptic drug cenobamate

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABAA-mediated currents and GABAA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (IGABA) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC50, 164 μM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABAA currents (Itonic), which is defined as a holding current shift by the GABAA receptor antagonist bicuculline (EC50, 36.63 μM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated Itonic and Iphasic in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced IGABA mediated by GABAA receptors. This effect was similar for all tested hGABAA receptors containing six different alpha subunits (α1β2γ2 or α2-6β3γ2), with EC50 values ranging from 42 to 194 μM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABAA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABAA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances Itonic inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.

Neurosteroids as Selective Inhibitors of Glycine Receptor Activity: Structure-Activity Relationship Study on Endogenous Androstanes and Androstenes.

The ability of androstane and androstene neurosteroids with modifications at C-17, C-5, and C-3 (compounds 1-9) to influence the functional activity of inhibitory glycine and γ-aminobutyric acid (GABA) receptors was estimated. The glycine- and GABA-induced chloride current (IGly and IGABA) were measured in isolated pyramidal neurons of the rat hippocampus and isolated rat cerebellar Purkinje cells, correspondingly, using the patch-clamp technique. Our results demonstrate that all the nine neurosteroids display similar biological activity, namely, they strongly inhibited IGly and weakly inhibited IGABA. The threshold concentration of neurosteroids inducing effects on IGly was 0.1 μM, and for effects on IGABA was 10–50 μM. Moreover, our compounds accelerated desensitization of the IGly with the IC50 values varying from 0.12 to 0.49 μM and decreased the peak amplitude with IC50 values varying from 16 to 22 μM. Interestingly, our study revealed that only compounds 4 (epiandrosterone) and 8 (dehydroepiandrosterone) were able to cause a significant change in IGABA in 10 μM concentration. Moreover, compounds 3 (testosterone), 5 (epitestosterone), 6 (dihydroandrostenedione), and 9 (etiocholanedione) did not modulate IGABA up to the concentration of 50 μM. Thus, we conclude that compounds 3, 5, 6, and 9 may be identified as selective modulators of IGly. Our results offer new avenues of investigation in the field of drug-like selective modulators of IGly.

Acute toxicity, bioconcentration, elimination, action mode and detoxification metabolism of broflanilide in zebrafish, Danio rerio

Broflanilide, a novel meta-diamide insecticide, shows high insecticidal activity against agricultural pests and is scheduled to be launched onto the market in 2020. However, little information about its potential toxicological effects on fish has been reported. In this study, broflanilide showed low toxicity to the zebrafish, Danio rerio, with LC50 > 10 mg L−1 at 96 h and also did not inhibit GABA-induced currents of the heteromeric Drα1β2Sγ2 GABA receptor. Broflanilide showed medium bioconcentration level with a bioconcentration factor at steady state (BCFss) of 10.02 and 69.40 in D. rerio at 2.00 mg L−1 and 0.20 mg L−1, respectively. In the elimination process, the concentration of broflanilide rapidly decreased within two days and slowly dropped below the limit of quantification after ten days. In the 2.00 mg L-1 broflanilide treatment, CYP450 activity was significantly increased up to 3.11-fold during eight days. Glutathione-S- transferase (GST) activity significantly increased by 91.44 % within four days. In conclusion, the acute toxicity of broflanilide was low, but it might induce chronic toxicity, affecting metabolism. To our knowledge, this is the first report of the toxicological effects of broflanilide on an aquatic organism, which has the potential to guide the use of broflanilide in the field.

Intravenous lipid emulsion modifies synaptic transmission in hippocampal CA1 pyramidal neurons after bupivacaine-induced central nervous system toxicity.

Local anesthetics can cause severe toxicity when absorbed systemically. Rapid intravenous administration of lipid emulsion (LE) is the standard of care for severe local anesthetic systemic toxicity which can cause cardiovascular and central nervous system (CNS) injury. The biological mechanism by which LE alleviates CNS toxicity remains unknown and understudied. Previous research has suggested that local anesthetics cause an imbalance of excitatory and inhibitory transmission in the brain. Therefore, this study aimed to observe the effect of LE on glutamate‐ and GABA‐induced currents in CA1 pyramidal neurons after bupivacaine‐induced CNS toxicity. We further characterized post‐synaptic modifications in these cells to try to elucidate the mechanism by which LE mediates bupivacaine‐induced CNS toxicity. Sprague–Dawley rats received intravenous bupivacaine (1 mg kg−1 min−1) in either normal saline or LE (or LE without bupivacaine) for 5 min. An acute brain slice preparation and a combination of whole‐cell patch clamp techniques and whole‐cell recordings were used to characterize action potential properties, miniature excitatory, and inhibitory post‐synaptic currents, and post‐synaptic modifications of excitatory and inhibitory transmission in CA1 hippocampal pyramidal neurons. The expression level of GABAA receptors were assessed with western blotting, whereas H&E and TUNEL staining were used to assess cytoarchitecture and apoptosis levels respectively. Bupivacaine treatment significantly increased the number of observed action potentials, whereas significantly decreasing rheobase, the first interspike interval (ISI), and hyperpolarization‐activated cation currents (Ih) in CA1 pyramidal neurons. LE treatment significantly reduced the frequency of miniature inhibitory post‐synaptic currents and enhanced GABA‐induced paired pulse ratio with 50 ms interval stimulation in bupivacaine‐treated rats. Regulation of GABAA levels is a promising mechanism by which LE may ameliorate CNS toxicity after systemic absorption of bupivacaine.

Spinal gastrin releasing peptide receptor expressing interneurons are controlled by local phasic and tonic inhibition

Dorsal horn gastrin-releasing peptide receptor (GRPR) neurons have a central role in itch transmission. Itch signaling has been suggested to be controlled by an inhibitory network in the spinal dorsal horn, as increased scratching behavior can be induced by pharmacological disinhibition or ablation of inhibitory interneurons, but the direct influence of the inhibitory tone on the GRPR neurons in the itch pathway have not been explored. Here we have investigated spinal GRPR neurons through in vitro and bioinformatical analysis. Electrophysiological recordings revealed that GRPR neurons receive local spontaneous excitatory inputs transmitted by glutamate and inhibitory inputs by glycine and GABA, which were transmitted either by separate glycinergic and GABAergic synapses or by glycine and GABA co-releasing synapses. Additionally, all GRPR neurons received both glycine- and GABA-induced tonic currents. The findings show a complex inhibitory network, composed of synaptic and tonic currents that gates the excitability of GRPR neurons, which provides direct evidence for the existence of an inhibitory tone controlling spontaneous discharge in an itch-related neuronal network in the spinal cord. Finally, calcium imaging revealed increased levels of neuronal activity in Grpr-Cre neurons upon application of somatostatin, which provides direct in vitro evidence for disinhibition of these dorsal horn interneurons.

Interleukin-17 Regulates Neuron-Glial Communications, Synaptic Transmission, and Neuropathic Pain after Chemotherapy

The proinflammatory cytokine interleukin-17 (IL-17) is implicated in pain regulation. However, the synaptic mechanisms by which IL-17 regulates pain transmission are unknown. Here, we report that glia-produced IL-17 suppresses inhibitory synaptic transmission in the spinal cord pain circuit and drives chemotherapy-induced neuropathic pain. We find that IL-17 not only enhances excitatory postsynaptic currents (EPSCs) but also suppresses inhibitory postsynaptic synaptic currents (IPSCs) and GABA-induced currents in lamina IIo somatostatin-expressing neurons in mouse spinal cord slices. IL-17 mainly expresses in spinal cord astrocytes, and its receptor IL-17R is detected in somatostatin-expressing neurons. Selective knockdown of IL-17R in spinal somatostatin-expressing interneurons reduces paclitaxel-induced hypersensitivity. Overexpression of IL-17 in spinal astrocytes is sufficient to induce mechanical allodynia in naive animals. In dorsal root ganglia, IL-17R expression in nociceptive sensory neurons is sufficient and required for inducing neuronal hyperexcitability after paclitaxel. Together, our data show that IL-17/IL-17R mediate neuron-glial interactions and neuronal hyperexcitability in chemotherapy-induced peripheral neuropathy.

Brimonidine enhances inhibitory postsynaptic activity of OFF- and ON-type retinal ganglion cells in a Wistar rat chronic glaucoma model

Glaucoma is a multifactorial disease in which retinal ganglion cells (RGCs) undergo excitotoxic damage, leading to their degeneration. The α2-adrenoceptor (α2-AR) agonist brimonidine exerts a neuroprotective effect by regulating postsynaptic excitatory N-methyl-D-aspartate (NMDA) receptor activity in RGCs. However, researchers have not clearly determined whether or how brimonidine regulates inhibitory synaptic transmission in rat models of chronic glaucoma. Whole-cell voltage-clamp and current-clamp recordings were performed in ON- and OFF-type RGCs in retinal slices. Brimonidine directly and acutely enhanced γ-aminobutyric acidergic (GABAergic) transmission mediated by ionotropic GABAA receptors in ON- and OFF-type RGCs in rat retinal slices; this effect occurred at the synaptic terminals and was independent of action potentials and multi-synaptic connections. The highly selective α2-AR antagonist yohimbine blocked the effects of brimonidine. Regarding the postsynaptic GABA receptor sensitivity, brimonidine also increased the amplitude of the GABA-induced current. Additionally, compared to RGCs from the control group, the frequencies and amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) did not change after brimonidine gravity perfusion. Brimonidine significantly decreased the spontaneous firing frequency of rat RGCs with intact synaptic inputs and decreased the resting membrane potential of RGCs, changes that were blocked by the highly selective GABAA receptor antagonist SR95531. SR95531 alone increased spontaneous action potentials and the resting membrane potential. Based on these findings, an α2-AR agonist facilitated the frequency of the GABAergic inhibitory postsynaptic currents (IPSCs), directly increased the amplitude of the postsynaptic GABA-induced current (GABA receptor reactivity/sensitivity), suppressed the firing frequency of spontaneous action in RGCs with intact synaptic inputs and decreased the resting membrane potential of RGCs, thus deactivating RGCs from the neural network level and reducing the excitotoxic damage occurring during the pathological process of chronic glaucoma.

Action of citral on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in juvenile mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is admitted as a pivotal site of integrating and regulating orofacial nociceptive inputs. Although citral (3,7-dimethyl-2,6-octadienal) is involved in antinociception, the action mechanism of citral on the SG neurons of the Vc has not been fully clarified yet. In this study, we examined the direct membrane effects of citral and how citral mediates responses on the SG neurons of the Vc in juvenile mice using a whole-cell patch-clamp technique. Under high chloride pipette solution, citral showed repeatable inward currents that persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, the citral-induced inward currents were partially blocked by picrotoxin, a gamma-aminobutyric acid (GABAA)-receptor antagonist, or by strychnine, a glycine receptor antagonist. Further, the citral-induced responses were almost blocked by picrotoxin with strychnine. We also found that citral exhibited additive effect with GABA-induced inward currents and glycine-induced inward currents were potentiated by citral. In addition, citral suppressed the firing activities by positive current injection on the SG neurons of the Vc. Taken together, these results demonstrate that citral has glycine- and/or GABA-mimetic actions and suggest that citral might be a potential target for orofacial pain modulation by the activation of inhibitory neurotransmission in the SG area of the Vc.

Striatal Neurodegeneration that Mimics Huntington's Disease Modifies GABA-induced Currents.

Huntington’s Disease (HD) is a degenerative disease which produces cognitive and motor disturbances. Treatment with GABAergic agonists improves the behavior and activity of mitochondrial complexes in rodents treated with 3-nitropropionic acid to mimic HD symptomatology. Apparently, GABA receptors activity may protect striatal medium spiny neurons (MSNs) from excitotoxic damage. This study evaluates whether mitochondrial inhibition with 3-NP that mimics the early stages of HD, modifies the kinetics and pharmacology of GABA receptors in patch clamp recorded dissociated MSNs cells. The results show that MSNs from mice treated with 3-NP exhibited differences in GABA-induced dose-response currents and pharmacological responses that suggests the presence of GABAC receptors in MSNs. Furthermore, there was a reduction in the effect of the GABAC antagonist that demonstrates a lessening of this GABA receptor subtype activity as a result of mitochondria inhibition.

Interaction of insecticides with heteromeric GABA-gated chloride channels from zebrafish Danio rerio (Hamilton).

The ionotropic GABAA receptor (GABAAR) is the main fast inhibitory post-synaptic receptor and is also an important insecticidal target. Effect of insecticides on fish has attracted intensive attention. However, no systematic study on heteromeric zebrafish GABAAR expressed in oocytes has been reported to date. In this study, the α1 subunit, the β2S subunit lacking 47 amino acid residues compared with the β2L subunit, and the γ2 subunit having five transmembrane domains were isolated from zebrafish Danio rerio. The responses of homomeric and heteromeric (α1, β2S and γ2) channels to agonists and GABAAR-targeted compounds were detected with two-electrode voltage clamp. Dose-dependent responses were observed in heteromeric α1β2S, β2Sγ2, and α1β2Sγ2 GABAR channels with EC50 values at 21.75, 6291, and 33.69 μM for GABA-induced current and 3.28, 155.5, and 3.79 mM for β-alanine-induced current, respectively. However, no response was induced by benzamidine in all GABAR channels. Abamectin, dieldrin, fluralaner and fipronil could strongly inhibited GABA-induced inward current ≥50% at 10-6 M, while α-endosulfan, flufiprole and ethiprole only inhibited GABA-induced current <50%. This study has clarified the interaction of insecticides with the heteromeric GABAAR channel, which could help us further explore the potential function and toxicological importance of GABAARs from D. rerio.

Design, Synthesis, and Pharmacological Evaluation of Novel β2/3 Subunit-Selective γ-Aminobutyric Acid Type A (GABAA) Receptor Modulators.

Subunit-selective modulation of γ-aminobutyric acid type A receptors (GABAAR) is considered to exert fewer side effects compared to unselective clinically used drugs. Here, the β2/3 subunit-selective GABAAR modulators valerenic acid (VA) and loreclezole (LOR) guided the synthesis of novel subunit-selective ligands with simplified structures. We studied their effects on GABAARs expressed in Xenopus laevis oocytes using two-microelectrode voltage clamp technique. Five compounds showed significantly more efficacious modulation of GABA-evoked currents than VA and LOR with retained potency and selectivity. Compound 18 [( E)-2-Cyano-3-(2,4-dichlorophenyl)but-2-enamide] induced the highest maximal modulation of GABA-induced chloride currents ( Emax: 3114 ± 242%), while 12 [( Z)-3-(2,4-dichlorophenyl)but-2-enenitrile] displayed the highest potency (EC50: 13 ± 2 μM). Furthermore, in hippocampal neurons 12 facilitated phasic and tonic GABAergic inhibition, and in vivo studies revealed significantly more potent protection against pentylenetetrazole (PTZ)-induced seizures compared to VA and LOR. Collectively, compound 12 constitutes a novel, simplified, and subunit-selective GABAAR modulator with low-dose anticonvulsant activity.