GABAergic Miniature Inhibitory Postsynaptic Currents Research Articles

Actions of remimazolam on inhibitory transmission of rat spinal dorsal horn neurons

Remimazolam is an ultra-short benzodiazepine that acts on the benzodiazepine site of γ-aminobutyric acid (GABA) receptors in the brain and induces sedation. Although GABA receptors are found localized in the spinal dorsal horn, no previous studies have reported the analgesic effects or investigated the cellular mechanisms of remimazolam on the spinal dorsal horn. Behavioral measures, immunohistochemistry, and in vitro whole-cell patch-clamp recordings of dorsal horn neurons were used to assess synaptic transmission. Intrathecal injection of remimazolam induced behavioral analgesia in inflammatory pain-induced mechanical allodynia (six rats/dose; p < 0.05). Immunohistochemical staining revealed that remimazolam suppressed spinal phosphorylated extracellular signal-regulated kinase activation (five rats/group, p < 0.05). In vitro whole-cell patch-clamp analysis demonstrated that remimazolam increased the frequency of GABAergic miniature inhibitory post-synaptic currents, prolonged the decay time (six rats; p < 0.05), and enhanced GABA currents induced by exogenous GABA (seven rats; p < 0.01). However, remimazolam did not affect miniature excitatory post-synaptic currents or amplitude of monosynaptic excitatory post-synaptic currents evoked by Aδ- and C-fiber stimulation (seven rats; p > 0.05). This study suggests that remimazolam induces analgesia by enhancing GABAergic inhibitory transmission in the spinal dorsal horn, suggesting its potential utility as a spinal analgesic for inflammatory pain.

A phospho-deficient α3 glycine receptor mutation alters synaptic glycine and GABA release in mouse spinal dorsal horn neurons.

Glycine receptors (GlyRs), together with GABAA receptors, mediate postsynaptic inhibition in most spinal cord and hindbrain neurons. In several CNS regions, GlyRs are also expressed in presynaptic terminals. Here, we analysed the effects of a phospho-deficient mutation (S346A) in GlyR α3 subunits on inhibitory synaptic transmission in superficial spinal dorsal horn neurons, where this subunit is abundantly expressed. Unexpectedly, we found that not only were the amplitudes of evoked glycinergic inhibitory postsynaptic currents (IPSCs) significantly larger in GlyRα3(S346A) mice than in mice expressing wild-type α3GlyRs (GlyRα3(WT) mice), but so were those of GABAergic IPSCs. Decreased frequencies of spontaneously occurring glycinergic and GABAergic miniature IPSCs (mIPSCs) with no accompanying change in mIPSC amplitudes suggested a change in presynaptic transmitter release. Paired-pulse experiments on glycinergic IPSCs revealed an increased paired-pulse ratio and a smaller coefficient of variation in GlyRα3(S346A) mice, which together indicate a reduction in transmitter release probability and an increase in the number of releasable vesicles. Paired-pulse ratios of GABAergic IPSCs recorded in the presence of strychnine were not different between genotypes, while the coefficient of variation was smaller in GlyRα3(S346A) mice, demonstrating that the decrease in release probability was readily reversible by GlyR blockade, while the difference in the size of the pool of releasable vesicles remained. Taken together, our results suggest that presynaptic α3 GlyRs regulate synaptic glycine and GABA release in superficial dorsal horn neurons, and that this effect is potentially regulated by their phosphorylation status. KEY POINTS: A serine-to-alanine point mutation was introduced into the glycine receptor α3 subunit of mice. This point mutation renders α3 glycine receptors resistant to protein kinase A mediated phosphorylation but has otherwise only small effects on receptor function. Patch-clamp recordings from neurons in mouse spinal cord slices revealed an unexpected increase in the amplitudes of both glycinergic and GABAergic evoked inhibitory postsynaptic currents (IPSCs). Miniature IPSCs, paired-pulse ratios and synaptic variation analyses indicate a change in synaptic glycine and GABA release. The results strongly suggest that α3 subunit-containing glycine receptors are expressed on presynaptic terminals of inhibitory dorsal horn neurons where they regulate transmitter release.

Lithium Enhances the GABAergic Synaptic Activities on the Hypothalamic Preoptic Area (hPOA) Neurons.

Lithium (Li+) salt is widely used as a therapeutic agent for treating neurological and psychiatric disorders. Despite its therapeutic effects on neurological and psychiatric disorders, it can also disturb the neuroendocrine axis in patients under lithium therapy. The hypothalamic area contains GABAergic and glutamatergic neurons and their receptors, which regulate various hypothalamic functions such as the release of neurohormones, control circadian activities. At the neuronal level, several neurotransmitter systems are modulated by lithium exposure. However, the effect of Li+ on hypothalamic neuron excitability and the precise action mechanism involved in such an effect have not been fully understood yet. Therefore, Li+ action on hypothalamic neurons was investigated using a whole-cell patch-clamp technique. In hypothalamic neurons, Li+ increased the GABAergic synaptic activities via action potential independent presynaptic mechanisms. Next, concentration-dependent replacement of Na+ by Li+ in artificial cerebrospinal fluid increased frequencies of GABAergic miniature inhibitory postsynaptic currents without altering their amplitudes. Li+ perfusion induced inward currents in the majority of hypothalamic neurons independent of amino-acids receptor activation. These results suggests that Li+ treatment can directly affect the hypothalamic region of the brain and regulate the release of various neurohormones involved in synchronizing the neuroendocrine axis.

Quercetin Enhances Inhibitory Synaptic Inputs and Reduces Excitatory Synaptic Inputs to OFF- and ON-Type Retinal Ganglion Cells in a Chronic Glaucoma Rat Model.

BackgroundGlaucoma is a neurodegenerative disease caused by excitotoxic injury of retinal ganglion cells (RGCs). In our previous model of high intraocular pressure, prepared by injecting magnetic beads into the anterior chamber, we demonstrated that an important natural dietary flavonoid compound (quercetin) can improve RGC function. However, it is unclear whether quercetin can improve the synaptic function of RGCs and how quercetin regulates synaptic transmission in rat models of chronic glaucoma.MethodsA rat model of chronic glaucoma was prepared by electrocoagulation of the superior scleral vein. Electrophysiological electroretinography was used to detect the photopic negative response (PhNR). The whole-cell patch-clamp technique was used to clamp ON- and OFF- type RGCs in sections from normal retinas and from retinas that had been subjected to glaucoma for 4 weeks.ResultsQuercetin can reverse the decrease in PhNR amplitude caused by chronic glaucoma. The baseline frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) in the RGCs of glaucomatous retinal slices was lower than that of the control group. The frequencies of miniature excitatory postsynaptic currents (mEPSCs) were not significantly different between control and glaucomatous RGCs. The baseline frequencies of GABAergic mIPSCs and mEPSCs in OFF-type glaucomatous RGCs were greater than those in ON-type glaucomatous RGCs. Quercetin increased miniature GABAergic inhibitory neurotransmission to RGCs and decreased miniature glutamatergic excitatory neurotransmission, reducing the excitability of the RGCs themselves, thus alleviating the excitability of RGCs in glaucomatous slices.ConclusionQuercetin may be a promising therapeutic agent for improving RGC survival and function in glaucomatous neurodegeneration. Quercetin exerted direct protective effects on RGCs by increasing inhibitory neurotransmission and decreasing excitatory neurotransmission to RGCs, thus reducing excitotoxic damage to those cells in glaucoma.

Perinatal high-fat diet alters development of GABAA receptor subunits in dorsal motor nucleus of vagus.

Perinatal high-fat diet (pHFD) exposure increases the inhibition of dorsal motor nucleus of the vagus (DMV) neurons, potentially contributing to the dysregulation of gastric functions. The aim of this study was to test the hypothesis that pHFD increases the inhibition of DMV neurons by disrupting GABAA receptor subunit development. In vivo gastric recordings were made from adult anesthetized Sprague-Dawley rats fed a control or pHFD (14 or 60% kcal from fat, respectively) from embryonic day 13 (E13) to postnatal day 42 (P42), and response to brainstem microinjection of benzodiazepines was assessed. Whole cell patch clamp recordings from DMV neurons assessed the functional expression of GABAA α subunits, whereas mRNA and protein expression were measured via qPCR and Western blotting, respectively. pHFD decreased basal antrum and corpus motility, whereas brainstem microinjection of L838,417 (positive allosteric modulator of α2/3 subunit-containing GABAA receptors) produced a larger decrease in gastric tone and motility. GABAergic miniature inhibitory postsynaptic currents in pHFD DMV neurons were responsive to L838,417 throughout development, unlike control DMV neurons, which were responsive only at early postnatal timepoints. Brainstem mRNA and protein expression of the GABAA α1,2, and 3 subunits, however, did not differ between control and pHFD rats. This study suggests that pHFD exposure arrests the development of synaptic GABAA α2/3 receptor subunits on DMV neurons and that functional synaptic expression is maintained into adulthood, although cellular localization may differ. The tonic activation of slower GABAA α2/3 subunit-containing receptors implies that such developmental changes may contribute to the observed decreased gastric motility. NEW & NOTEWORTHY Vagal neurocircuits involved in the control of gastric functions, satiation, and food intake are subject to significant developmental regulation postnatally, with immature GABAA receptors expressing slower α2/3-subunits, whereas mature GABAA receptor express faster α1-subunits. After perinatal high-fat diet exposure, this developmental regulation of dorsal motor nucleus of the vagus (DMV) neurons is disrupted, increasing their tonic GABAergic inhibition, decreasing efferent output, and potentially decreasing gastric motility.

The response of vagal motoneurons to brainstem oxytocin stimulation depends on sex and stress levels.

Stress maladaptation plays a pivotal role in the development and exacerbation of functional gastrointestinal disorders (FGID), including impaired gastric emptying and decreased gastric motility, both of which are more prevalent in women. The prototypical anti‐stress hormone, oxytocin (OXY), has been shown to modulate GI tone and motility via vagal pathways. We have shown previously that in naïve male rats, OXY modulates the critical GABAergic synapse projecting to gastric‐projecting preganglionic vagal (DMV) neurons only after pharmacological increase of cAMP levels by forskolin or CRF pretreatment, suggesting a stress‐induced plasticity of brainstem vagal neurocircuits.The aim of this study was to test the hypothesis that the response of vagal motoneurons to brainstem oxytocin application depends on sex and stress adaptation or resilience. Age‐matched male and female SD rats were subdivided in the following groups: i) control; ii) chronic repetitive homotypic (restraint) stress, rats in this group show adaptation to stress; iii) chronic repetitive heterotypic (restraint, forced swim, water avoidance and cold) stress, rats in this group do not show adaptation to stress. Female rats were further subdivided according to their estrogen levels (high: pro‐estrus and estrus; low: meta‐ and di‐estrus). Following 5‐days of stress, whole cell patch clamp recordings were made from thin brainstem slices to examine GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in medial, presumably gastric‐projecting, DMV neurons.Our results, see table, indicate that 1) in male rats OXY modulated inhibitory neurotransmission only in stressed rats; 2) in female rats, OXY modulated inhibitory neurotransmission independently of estrogen or stress load. 3) OXY effects on mIPSC frequency, however, is different when rats are exposed to heterotypic stress load. Taken together, our data support the hypothesis that the response of vagal motoneurons to brainstem oxytocin stimulation depends on sex and stress levels and suggest that, independently of estrogen or stress, female rats have a response similar to that of stressed males.Support or Funding InformationNIH DK 55530This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Presynaptic large‐conductance calcium‐activated potassium channels modulate GABAergic inhibitory synaptic transmission in the superficial dorsal horn of the mouse

Large‐conductance calcium‐dependent potassium (BK) channels are found in various tissues, including nerve, smooth and skeletal muscles, and other cell types. They are potassium‐selective ion channels that are activated in response to increased intracellular calcium concentrations and transmembrane voltages.We previously reported that the BK channels that are located in presynaptic terminals control excitatory synaptic transmission in the superficial dorsal horn (SDH), and that functional downregulation of BK channels accompanies the neuropathic pain induced by peripheral nerve injury.In the present experiments, we attempted to clarify the physiological significance of BK channels in the modulation of GABAergic inhibitory synaptic transmission in the SDH where nociceptive information is processed.Spontaneously occurring GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded from the GFP‐negative neurons located in the SDH of a mouse spinal cord slice by using slice preparations from adult glutamate decarboxylase 67‐green fluorescent protein (GAD67‐GFP) knock‐in mice, and the effects of Iberiotoxin, a BK channel blocker, on the GABAergic mIPSCs were analyzed.Iberiotoxin increased the frequency of GABAergic mIPSCs without affecting the amplitude of GABAergic mIPSCs, suggesting that the activation of BK channels attenuates GABAergic synaptic transmission via presynaptic mechanisms.In order to clarify the role of BK channels in the nociceptive information processing which is modulated by GABAergic transmission, we are currently doing the similar experiments using peripheral nerve‐ligated neuropathic mice.Those results may provide clues for the underlying mechanisms of the nociceptive actions of BK channels in the SDH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin II facilitates GABAergic neurotransmission at postsynaptic sites in rat amygdala neurons

The central nucleus of the amygdala (CeA) is critical in the regulation of sodium appetite. Angiotensin II (Ang II) is important in the generation of sodium appetite and may function as a neurotransmitter or modulator to affect the synaptic transmission and the excitability of neurons. However, the role of Ang II in the CeA remains unclear. In this study, we determined the effects of Ang II on the excitatory and inhibitory synaptic inputs to the CeA neurons in brain slices with whole-cell patch-clamp recordings. Ang II (0.5–5 μM) significantly potentiated the amplitude of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) in a concentration-dependent manner. Ang II (2 μM) significantly increased the amplitude of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) without affecting the frequency. This effect was blocked by Ang II type 1 (AT1) receptor antagonist, losartan. One mM guanosine 5′-O-(-2-thiodiphosphate) (GDP-β-s) in the pipette internal solution eliminated the facilitatory effect of Ang II on GABAergic synaptic transmission. In contrast, Ang II had no effect on the spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) and did not alter the frequency and amplitude of miniature EPSCs at concentrations that facilitated IPSCs. Furthermore, Ang II decreased the firing activity of CeA neurons, and this effect was abolished by losartan and GDP-β-s. In addition, Ang II failed to inhibit CeA neurons in the presence of bicuculline. These data provide substantial new evidence that Ang II inhibits the CeA neurons by facilitation of GABAergic synaptic input efficacy through activation of postsynaptic AT1 receptors.

Alpha7 nicotinic acetylcholine receptor agonist promotes retinal ganglion cell function via modulating GABAergic presynaptic activity in a chronic glaucomatous model

Alpha-7 nicotinic acetylcholine receptor (α7-nAChR) agonists can prevent glutamate-induced excitotoxicity in cultured retinal ganglion cells (RGCs). However, the neuroprotective effects and the mechanism of action of PNU-282987, an α7-nAChR agonist, in a chronic in vivo rat glaucoma model are poorly understood. We found that elevated intraocular pressure (IOP) downregulated retinal α7-nAChR expression. Electroretinography revealed that the amplitude of the photopic negative response (PhNR) decreased in parallel with the loss of RGCs caused by elevated IOP. PNU-282987 enhanced RGC viability and function and decreased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive signals in RGCs. Patch-clamp recordings revealed differences in the baseline frequencies and decay times of the miniature GABAergic inhibitory postsynaptic currents (mIPSCs) of RGCs between control and glaucomatous retinal slices. The results of western blotting and immunostaining showed that glutamic acid decarboxylase 65/67 and GABA deficits persisted in glaucomatous retinas and that these deficits were reversed by PNU-282987. Patch-clamp recordings also showed that PNU-282987 significantly increased the frequency and amplitude of the GABAergic mIPSCs of RGCs. The protective effects of PNU-292987 were blocked by intravitreal administration of selective GABAA receptor antagonists. The modulation of GABAergic synaptic transmission by PNU-282987 causes de-excitation of ganglion cell circuits and suppresses excitotoxic processes.

Compensatory activation of cannabinoid CB2 receptor inhibition of GABA release in the rostral ventromedial medulla (RVM) in inflammatory pain

The rostral ventromedial medulla (RVM) is a relay in the descending pain modulatory system and an important site of endocannabinoid modulation of pain. Endocannabinoids inhibit GABA release in the RVM but it is not known if this effect persists in chronic pain states. In the present studies, persistent inflammation induced by complete Freund's adjuvant (CFA) increased GABAergic miniature inhibitory postsynaptic currents (mIPSCs). Endocannabinoid activation of cannabinoid (CB1) receptors known to inhibit presynaptic GABA release was significantly reduced in the RVM of CFA‐treated rats compared to naïve rats. The reduction in CFA‐treated rats correlated with decreased CB1 receptor protein expression and function in the RVM. Paradoxically, the non‐selective CB1/CB2 receptor agonist WIN55,212 inhibited GABAergic mIPSCs in both naïve and CFA‐treated rats. However, WIN55,212 inhibition was reversed by the CB1 receptor antagonist rimonabant in naïve rats but not in CFA‐treated rats. WIN55,212‐mediated inhibition in CFA‐treated rats was blocked by the CB2 receptor‐selective antagonist SR144528 indicating that CB2 receptor function in the RVM is increased during persistent inflammation. Consistent with these results, CB2 receptor agonists, AM1241 and GW405833 inhibited GABAergic mIPSC frequency only in CFA‐treated rats and the inhibition was reversed with SR144258. When administered alone, SR144528 and another CB2 receptor‐selective antagonist AM630 increased mIPSC frequency in the RVM of CFA‐treated rats indicating that CB2 receptors are tonically activated by endocannabinoids. Our data provide evidence that CB2 receptor function emerges in the RVM in persistent inflammation and that selective CB2 receptor agonists may be useful for treatment of persistent inflammatory pain.Support or Funding InformationThese studies were supported by the grants from NIH (DA035316 and R56NS093894), and America Heart Association (13SDG14590005, MH.L.).

Interactions between ethanol and the endocannabinoid system at GABAergic synapses on basolateral amygdala principal neurons

The basolateral amygdala (BLA) plays crucial roles in stimulus value coding, as well as drug and alcohol dependence. Ethanol alters synaptic transmission in the BLA, while endocannabinoids (eCBs) produce presynaptic depression at BLA synapses. Recent studies suggest interactions between ethanol and eCBs that have important consequences for alcohol drinking behavior. To determine how ethanol and eCBs interact in the BLA, we examined the physiology and pharmacology of GABAergic synapses onto BLA pyramidal neurons in neurons from young rats. Application of ethanol at concentrations relevant to intoxication increased, in both young and adult animals, the frequency of spontaneous and miniature GABAergic inhibitory postsynaptic currents, indicating a presynaptic site of ethanol action. Ethanol did not potentiate sIPSCs during inhibition of adenylyl cyclase while still exerting its effect during inhibition of protein kinase A. Activation of type 1 cannabinoid receptors (CB1) in the BLA inhibited GABAergic transmission via an apparent presynaptic mechanism, and prevented ethanol potentiation. Surprisingly, ethanol potentiation was also prevented by CB1 antagonists/inverse agonists. Brief depolarization of BLA pyramidal neurons suppressed GABAergic transmission (depolarization-induced suppression of inhibition [DSI]), an effect previously shown to be mediated by postsynaptic eCB release and presynaptic CB1 activation. A CB1-mediated suppression of GABAergic transmission was also produced by combined afferent stimulation at 0.1 Hz (LFS), and postsynaptic loading with the eCB arachidonoyl ethanolamide (AEA). Both DSI and LFS-induced synaptic depression were prevented by ethanol. Our findings indicate antagonistic interactions between ethanol and eCB/CB1 modulation at GABAergic BLA synapses that may contribute to eCB roles in ethanol seeking and drinking.

Interaction of CRF and kappa opioid systems on GABAergic neurotransmission in the mouse central amygdala.

The corticotropin-releasing factor (CRF) and kappa-opioid receptor (KOR) systems are both implicated in stress-related behaviors and drug dependence. Although previous studies suggest that antagonism of each system blocks aspects of experimental models of drug dependence, the possible interaction between these systems at the neuronal level has not been completely examined. We used an in vitro brain slice preparation to investigate the interaction of these two peptide systems on inhibitory neurotransmission in the central nucleus of the amygdala (CeA). Application of exogenous CRF increased the mean frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSC) by 20.2%, suggesting an increase in presynaptic GABA release. Although the pharmacological blockade of KORs by norBNI alone did not significantly affect mIPSC frequency, it significantly enhanced the effect of CRF (by 43.9%, P = 0.02). Similarly, the CRF effects in slices from KOR knockout (KO) mice (84.0% increase) were significantly greater than in wild-type (WT) mice (24.6%, P = 0.01), although there was no significant difference in baseline mIPSC frequency between slices from KOR KO and WT mice. The increase in CRF action in the presence of norBNI was abolished by a CRF-1 receptor antagonist but was unaffected by a CRF-2 receptor antagonist. We hypothesize that CRF facilitates the release of an endogenous ligand for KORs and that subsequent activation of KOR receptors modulates presynaptic effects of CRF in CeA. These results suggest that potential pharmacotherapies aimed at neurobehavioral and addictive disorders may need to involve both the KOR/dynorphin and the CRF systems in CeA.

Activation of mu-opioid receptors in the ventrolateral orbital cortex inhibits the GABAergic miniature inhibitory postsynaptic currents in rats

Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5μM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1μM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

Glutamatergic mechanisms associated with stress-induced amygdala excitability and anxiety-related behavior

The neural factors underlying individual differences in susceptibility to chronic stress remain poorly understood. Preclinical studies demonstrate that mouse strains vary greatly in anxiety-related responses to chronic stress in a manner paralleled by differential stress-induced changes in glutamatergic signaling in the basolateral amygdala (BLA). Previous work has also shown that alterations in the amygdala gene expression of the GluN1 NMDA and the GluK1 kainate receptors are associated with stress-induced alterations in anxiety-like behavior in the C57BL/6J mouse strain. Using in vivo behavioral pharmacological and ex vivo physiological approaches, the aim of the current study was to further elucidate changes in glutamate neurotransmission in the BLA caused by stress and to test the functional roles of GluN1 and GluK1 in mediating stress-related changes in behavior. Results showed that stress-induced alterations in anxiety-like behavior (light/dark exploration test) were absent following bilateral infusion of the GluK1 agonist ATPA into the BLA. Intra-BLA infusion of the competitive NMDA antagonist AP5 produced a generalized behavioral disinhibition/locomotor hyperactivity, irrespective of stress. Slice electrophysiological recordings showed that ATPA augmented BLA GABAergic neurotransmission and that stress increased the amplitude of network-dependent spontaneous excitatory postsynaptic currents and amplitude of GABAergic miniature inhibitory postsynaptic currents in BLA. These findings could indicate stress-induced BLA glutamatergic neuronal network hyperexcitability and a compensatory increase in GABAergic neurotransmission, suggesting that GluK1 agonism augmented GABAergic inhibition to prevent behavioral sequelae of stress. Current data could have implications for developing novel therapeutic approaches, including GluK1 agonists, for stress-related anxiety disorders.

Dihydromyricetin Prevents Fetal Alcohol Exposure-Induced Behavioral and Physiological Deficits: The Roles of GABAA Receptors in Adolescence

Fetal alcohol exposure (FAE) can lead to a variety of behavioral and physiological disturbances later in life. Understanding how alcohol (ethanol, EtOH) affects fetal brain development is essential to guide the development of better therapeutics for FAE. One of EtOH's many pharmacological targets is the γ-aminobutyric acid type A receptor (GABAAR), which plays a prominent role in early brain development. Acute EtOH potentiates inhibitory currents carried by certain GABAAR subtypes, whereas chronic EtOH leads to persistent alterations in GABAAR subunit composition, localization and function. We recently introduced a flavonoid compound, dihydromyricetin (DHM), which selectively antagonizes EtOH's intoxicating effects in vivo and in vitro at enhancing GABAAR function as a candidate for alcohol abuse pharmacotherapy. Here, we studied the effect of FAE on physiology, behavior and GABAAR function of early adolescent rats and tested the utility of DHM as a preventative treatment for FAE-induced disturbances. Gavage administration of EtOH (1.5, 2.5, or 5.0g/kg) to rat dams on day 5, 8, 10, 12, and 15 of pregnancy dose-dependently reduced female/male offspring ratios (largely through decreased numbers of female offspring) and offspring body weights. FAE (2.5g/kg) rats tested on postnatal days (P) 25-32 also exhibited increased anxiety and reduced pentylenetetrazol (PTZ)-induced seizure threshold. Patch-clamp recordings from dentate gyrus granule cells (DGCs) in hippocampal slices from FAE (2.5g/kg) rats at P25-35 revealed reduced sensitivity of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and tonic current (Itonic) to potentiation by zolpidem (0.3μM). Interestingly, potentiation of mIPSCs by gaboxadol increased, while potentiation of Itonic decreased in DGCs from FAE rats. Co-administration of EtOH (1.5 or 2.5g/kg) with DHM (1.0mg/kg) in pregnant dams prevented all of the behavioral, physiological, and pharmacological alterations observed in FAE offspring. DHM administration alone in pregnant rats had no adverse effect on litter size, progeny weight, anxiety level, PTZ seizure threshold, or DGC GABAAR function. Our results indicate that FAE induces long-lasting alterations in physiology, behavior, and hippocampal GABAAR function and that these deficits are prevented by DHM co-treatment of EtOH-exposed dams. The absence of adverse side effects and the ability of DHM to prevent FAE consequences suggest that DHM is an attractive candidate for development as a treatment for prevention of fetal alcohol spectrum disorders.

Modulation of GABAergic Synaptic Currents and Current Responses by α-Thujone and Dihydroumbellulone

α-Thujone (1a), a constituent of wormwood, has been suspected to cause adverse psychoactive reactions in addicted drinkers of absinthe. While the content of 1a in absinthe is too low for such effects, at higher doses it can indeed induce seizures and inhibit GABA(A) receptors (GABA(A)Rs). The effect of 1a on GABAergic synaptic currents and the mechanisms by which it modulates GABA(A)Rs remain unknown. To address these issues, cultured hippocampal neurons were used to investigate the action of 1a on GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and on responses to exogenous GABA applications. Since lipophilic compounds often show nonspecific actions related to their hydrophobicity, the action of 1a was compared to that of dihydroumbellulone (2), a configurationally pseudoenantiomeric constitutional isomer. α-Thujone (1a) reduced mIPSC frequency and amplitude and also moderately affected their kinetics, indicating both pre- and postsynaptic mechanisms. Analysis of current responses to exogenous GABA revealed that 1a reduced their amplitude, affecting their onset, desensitization, and deactivation, suggesting an effect on receptor gating. In contrast, 2 caused only a weak or negligible effect on GABAergic currents, supporting the effects of 1a on GABAergic inhibition as being due to specific interactions with GABA(A)Rs.

Dopamine modulates neuronal excitability pre- and post-synaptically in the rat subfornical organ

The aims of this study were to investigate the involvement of dopamine (DA) in drinking behaviour related to body fluid balance. All experiments were performed in rats. Water intake induced by intracerebroventricular injection of angiotensin II (ANGII) was suppressed by co-injection of DA in a dose-dependent manner. RT-PCR revealed the presence of mRNAs for all known DA receptors, D1-D5, in the subfornical organ (SFO), a brain region that plays a key role in regulating drinking behaviour. Extracellular recordings and whole-cell patch-clamp recordings from SFO neurons showed that DA or the D4 selective agonist PD168077 inhibited spontaneous electrical activity. The D4 antagonist L745870 blocked DA-induced inhibition of spontaneous electrical activity in SFO neurons. Under conditions of synaptic blockade, the inhibitory effects of DA and PD168077 still remained, but the D2/D3 agonist quinpirole and the D1/D5 agonist SKF38393 had almost no effect on electrical activity. While DA induced excitation in a small number of neurons, these excitatory responses almost disappeared following synaptic blockade. All neurons with firing rates that were suppressed by DA were excited by ANGII. In voltage clamp mode, we found that DA and quinpirole, but not SKF38393, suppressed GABAergic miniature inhibitory post-synaptic currents. These results suggest that DA inhibits neuronal activity in ANGII-sensitive SFO neurons primarily through the postsynaptic D4 receptor subtype. This may be a cause of the suppression of ANGII-induced water intake by DA. In addition, the inhibitory DA responses in SFO neurons may be modulated by presynaptic suppression of GABAergic inhibitory inputs through D2/D3 receptor subtypes.

Melatonin Modulates the GABAergic Response in Cultured Rat Hippocampal Neurons

In the present study, we investigated the effect of melatonin on the GABA-induced current (IGABA) and GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in cultured rat hippocampal neurons using the whole-cell patch-clamp technique. We found that melatonin rapidly and reversibly enhanced IGABA in a dose-dependent manner, with an EC50 of 949 μM. Melatonin markedly enhanced the peak amplitude of a subsaturating IGABA but not that of a saturating IGABA. Interestingly, melatonin was effective only when GABA and melatonin were applied together. Furthermore, the effect of melatonin on IGABA was voltage-independent and did not change the ion selectivity of the GABAA receptor. The melatonin enhancement on IGABA can not be blocked by luzindole, a melatonin receptor antagonist, indicating that melatonin-induced IGABA enhancement was not via activation of its own membrane receptors. However, this enhancement may be mediated via high-affinity benzodiazepine sites as it was inhibited by the classical benzodiazepine antagonist flumazenil, suggesting an allosteric modulation of melatonin by binding to the sites of GABAA receptors. In addition, melatonin increased both amplitude and frequency of GABAergic mIPSCs, indicating that melatonin enhances GABAergic inhibitory transmission. Hence, our observation that melatonin has an enhancing effect on the GABAergic system may implicate a potential pathway for the neuroprotective effects of melatonin.

GABAergic synaptic response and its opioidergic modulation in periaqueductal gray neurons of rats with neuropathic pain

BackgroundNeuropathic pain is a chronic and intractable symptom associated with nerve injury. The periaqueductal gray (PAG) is important in the endogenous pain control system and is the main site of the opioidergic analgesia. To investigate whether neuropathic pain affects the endogenous pain control system, we examined the effect of neuropathic pain induced by sacral nerve transection on presynaptic GABA release, the kinetics of postsynaptic GABA-activated Cl- currents, and the modulatory effect of μ-opioid receptor (MOR) activation in mechanically isolated PAG neurons with functioning synaptic boutons.ResultsIn normal rats, MOR activation inhibited the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) to 81.3% of the control without any alteration in their amplitude. In neuropathic rats, the inhibition of mIPSC frequency by MOR activation was 82.4%. The frequency of GABAergic mIPSCs in neuropathic rats was 151.8% of normal rats without any difference in the mIPSC amplitude. Analysis of mIPSC kinetics showed that the fast decay time constant and synaptic charge transfer of mIPSCs in neuropathic rats were 76.0% and 73.2% of normal rats, respectively.ConclusionsThese results indicate that although the inhibitory effect of MOR activation on presynaptic GABA release is similar in both neuropathic and normal rats, neuropathic pain may inhibit endogenous analgesia in the PAG through an increase in presynaptic GABA release.

Activation of serotonin 1A receptors in ventrolateral orbital cortex depresses persistent nociception: A presynaptic inhibition mechanism

The present study examined the effect of serotonin 1A (5-HT 1A) receptor activation in the ventrolateral orbital cortex (VLO) upon formalin-evoked flinching behavior and spinal Fos expression, and further determined whether activation of 5-HT 1A receptors affected the spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in rat VLO slice by pharmacologically separated neurons to understand the possible mechanism underlying this effect. Microinjection of the 5-HT 1A receptors agonist 8-OH-DPAT (8-hydro-2-(di-n-propylamino) tetralin) into the VLO depressed the formalin-evoked nociceptive behavior flinching response and the Fos expression in the lumbar spinal cord dorsal, which was antagonized by pre-treatment with 5-HT 1A receptors antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide). Furthermore, application of 8-OH-DPAT into VLO slice inhibited GABAergic mIPSC frequency in a dose-dependent manner without effects on amplitude of the GABAergic mIPSCs, this effect was blocked by NAN-190. These results provide evidence for the involvement of 5-HT 1A receptors in VLO in the modulation of persistent inflammatory nociception, and suggest that a presynaptic inhibition of the GABA release may contribute to the 5-HT 1A receptor-mediated descending antinociception.