Functional Regulation Of Inhibitory Amino Acid Receptors In The Rat Hippocampus

GABA_A receptor(GABA_AR)and glycine receptor(GlyR)are two kinds of important inhibitory amino acid receptor in central nervous system(CNS).According to the differential characteristic of inhibitory conductance mediated by synaptic and extrasynaptic receptors,the inhibition induced by inhibitory amino acid system is divided into two forms:phasic inhibition and tonic inhibition.During synaptic transmission,postsynaptic GABA_ARs or GlyRs are activated by high concentration of GABA or glycine released from presynaptic neurons and then exert inhibitory conductance on postsynaptic neuron.Because vesicle release of presynaptic neuron is not persistent,the inhibition induced by the activation of postsynaptic GABA_AR or GlyR is intermittent.Therefore,this kind of intermittent inhibition is named of phasic inhibition which is strictly controlled by synaptic transmission.But over the last decade,diffusional inhibitory transmission mediated by GABA_ARs or GlyRs located outside the synapses and activated by endogenous ligand(such as GABA,glycine and taurine)present in the extracellular space has triggered a great deal of interest.This form of inhibition is named of tonic inhibition which is characterized by uninterrupted inhibitory conductance and is not strictly restricted by synaptic transmission.Tonic inhibition thus plays a more important role in controlling neuronal excitability than phasic inhibition.In hippocampus,a lot of studies about GABA_AR mediated tonic inhibition were reported.Tonic inhibition is involved in not only the regulation of learning and memory of hippocampus,but also the nosogenesis of diseases such as epilepsy and premenstrual dysphoric disorder.Different GABA_AR subunit,such asδsubunit in dentate gyrus granule cells andα5 subunit in CA1 and CA3 pyramidal cell, contributes to the tonic conductance in different area of hippocampus.And even, receptors devoid of a third type of subunit,i.e.,only containingαandβsubunits that are highly sensitive to Zn²⁺,have been shown to contribute to the tonic current recorded in hippocampal neurons. Although no functional glycinergic synapse was observed in hippocampus,the wide expression of GlyR subunits was revealed by previous studies.Glycine activated current was recorded not only in acutely isolated and cultured hippocampal neurons but also in neurons of hippocampal slices.In addition,endogenous ligand of GlyRs,such as taurine andβ-alanine were highly spread in extracellular space of hippocampus,supporting the possibility of GlyR activation.Recently,some studies have revealed the functional role of GlyR in synaptic transmission and short-term plasticity of hippocampus.The present idea presumes that tonic activation of extrasynaptic GlyR by some endogenous amino acid ligands and modulators exerts the functional role of GlyR in hippocampus.Although tonic inhibition triggered a great deal of interest in last year,few selective modulator of tonic inhibition by far was found.In addition,the functional role of tonic inhibition mediated by GlyR was gradually revealed.Therefore, selective modulator of tonic inhibition will become promising drugs for treating disease such as epilepsy.In present study,we first investigated the modulatory effects of menthol,a representative terpenoid,on the currents mediated by GABA_AR and GlyR.Besides, through site-directed mutagenesis and exogenously expression of GlyR,we investigatedα2 subunit specificity of cyclothiazide(CTZ)inhibition on GlyR.1.Menthol suppresses central neuronal excitation by enhancing tonic GABA inhibitionUsing electrophysiological recording and behavior study of epileptic animals,we found menthol suppressed the excitability of cultured rat hippocampal neurons and the hyper-excitability of CNS neurons induced by pentylenetetrazole(PTZ)injection and electrical kindling in vivo.Menthol enhanced the response of cultured hippocampal neurons to low concentrations of GABA and also activated GABA_AR with higher concentrations of menthol.Furthermore,in the CA1 region of rat hippocampal slices, menthol enhanced tonic GABAergic inhibition while leaving phasic(synaptic) GABAergic inhibition unaffected.These results thus suggest menthol inhibited neuronal excitability through enhancing GABA_AR mediated tonic inhibition in hippocampus.In order to rule out that menthol inhibited neuronal excitability through other molecular target,we firstly investigated the effect of menthol on the current activated by glutamate in hippocampal neurons.No significant modulation was observed in the experiments.Secondly,we test the effect of menthol on action potential induced by current injection after blocking excitatory and inhibitory amino acid receptor,and found that the firing characteristic and frequency were not affected by menthol.This result suggests that menthol does not alter the intrinsic neuronal excitability.Lastly,we examine the inhibitory effect of menthol on GABA_AR after chelating extracellular free calcium with BAPTA-AM and observed the similar inhibitory effect with that produced in the absence of BAPTA.Taken together,these results further support that menthol inhibited neuronal excitability through selective enhancement of GABA_AR mediated tonic inhibition.Finally,we investigated the effects of menthol isomers and structure-related agents(menthyl chloride,(-)-isopulegol,and JE207)on the current activated by GABA(1μM)in cultured hippocampal neurons.We found that all menthol isomers significantly enhanced GABA-activated current.No significant enhancement was observed by menthyl chloride and JE207 in which the hydroxyl of menthol is substituted by other group.Therefore,the hydroxyl of menthol plays an important role in menthol enhancement of GABA_AR.2.Menthol preferentially inhibitsα2βheteromeric glycine receptorsUsing whole-cell voltage-clamp recording,we firstly investigated the effect of menthol on GlyRs in cultured hippocampal neurons.Menthol noncompetitively inhibited glycine-activated currents(I_Gly)in a concentration-dependent and voltage-independent manner.The inhibitory effect was not influenced by blocking menthol activation of GABA_AR with bicuculline methiodide(BMI)or chelating intracellular free calcium with BAPTA.By analyzing the differential effect of menthol on I_Glywith different sequences of drug application,we found that open channel block and allosteric modulation were all involved in the inhibition, suggesting that menthol may inhibit GlyRs via at least two different sites. Furthermore,we assessed the subunit selectivity of menthol inhibition on recombinant GlyRs expressed in HEK293T cells.Menthol did not significantly affect I_Glymediated by recombinantα1 homomeric GlyRs.Although menthol showed a relatively high inhibitory potency for the recombinantα2 GlyRs,it displayed markedly higher potency atαβheteromeric receptors.In summary,these results indicated that menthol directly inhibited GlyRs via at least one allosteric site and another blocking site on the GlyRs.The inhibitory effect of menthol was not the result of cross-inhibition between GlyRs and GABA_ARs.Moreover,the change of intracellular free calcium concentration did not contribute to menthol inhibition of GlyRs.In addition,menthol preferentially inhibitedα2 subunit-containing GlyRs, and theβsubunit also significantly increased the efficacy of menthol inhibition.3.Alpha 2 subunit specificity of eyclothiazide inhibition on glyeine receptorsIn cultured rat hippocampal neurons,we found that CTZ,an epilepitogenic agent, potently inhibited I_Gly.The inhibition was glycine concentration-dependent, suggesting a competitive mechanism.Notably,when being heterologously expressed in HEK293T cells,GlyRs containing theα2 but not oil orα3 subunit were inhibited by CTZ,indicating subunit specificity of CTZ action.In addition,the degree of CTZ inhibition on I_Glyin rat spinal neurons declined with time in culture,in parallel with a decline ofα2 subunit expression,which is known to occur during spinal cord development.Furthermore,site-directed mutagenesis indicates that a single amino acid threonine at position 59 near the N-terminal of theα2 subunit confers the specificity of CTZ action.Thus CTZ is a potent and selective inhibitor ofα2-GlyRs, and threonine at position 59 plays a critical role in the susceptibility of GlyR to CTZ inhibition.In conclusion,there are several novelties residing in the present studies:1)For the first time,we find that menthol,a widely used natural chemical in daily life, inhibits neuronal excitability of the hippocampus through modulating tonic inhibition mediated by GABA_AR and GlyR.2)Our results underscore the importance of tonic inhibition by GABA_ARs in regulating neuronal activity and reveal that tonic inhibition may be the promising target of treating disease such as epilepsy.3)For the first time,we find that cyclothiazide preferentially inhibitsα2-GlyR which mainly produces tonic inhibition in hippocampus.In addition,the binding site of cyclothiazide on GlyR was also revealed by site-directed mutagenesis.The selective inhibition of cyclothiazide onα2 GlyR may be helpful to explore the functional role ofα2-GlyR in hippocampus.