Modulation Of GABA And Glu Neurotransmitter Systems By Succinate In Rat Hippocampal CA1 Neurons

Succinate is a major active component in amber which is a traditional anticonvulsive drug. Succinate exerts many beneficial effects,including sedation and analgesia,anticonvulsive and antiepileptic effect.It is also a key intermediate metabolites of tricarboxylic acid.The metabolic bypass of Glu-GABA-succinate,is similar with aminoethylsulfonic acid, neurotransmitter GABA and Glu,and it participates in not only energy metabolism but have some effect on balance between excitatory and inhibitory transmitters.GABA,Glu and Taurine are active intraencephal substances.Succinate content of brain changes in a direct impact on brain energy metabolism and the conversion of the GABA and the Glu. This shows that succinate may be a natural endogenous neuromodulator.Kindling model for neurophysiological and pharmacological research,enhanced postsynaptic long-term(LTP) and brain plasticity provides a better model of animal studies,the amygdala and the hippocampus involved in kindling model of epilepsy, mental illness,addiction and memory mechanisms,GABA and Glu are involved in a major neurotransmitter.Previous study in our laboratory showed that succinate inhibited the brain amygdala electrical kindling,chemical PTZ kindling and rapid hippocampal kindling in rats.It proved that succinate had a central inhibitory effect.However,there is no direct evidence to demonstrate the effects of SA on GABAergic and Gluergic systems in the brain.,The study was to determine the modulation of GABA-A receptor-mediated inhibitory postsynaptic currents(IPSCs) by succinate through the infrared visual whole cell patch clamp recording techniques before and after treated with succinate in rat hippocampal CA1 neurons.Different subtypes of specific antagonist and agonist of L,N and P/Q type calcium channels,Thapsigargin—an agonist of intracellular Ca²⁺ library(inositol 1,4,5-trisphosphate) IP₃ receptor,Ryanodine—an agonist of intracellular Ca²⁺ library Ryanodine receptor,KT5720-an inhibitor of PKA,KN62-an inhibitor of Ca²⁺/CaM-Kâ…¡, and Flumazcnil-an inhibitor of the binding site of Benzodiazepine(BZ) in CABA-A receptor,all of them were observed the effect on the modulation by succinate.The modulation of the Glu receptor mediated tiny excitatory postsynaptic currents(mEPSCs) and spontaneous excitatory postsynaptic currents(sEPSCs) by succinate were observed. We observed the effects of succinate on currents induced by Glu and its ionotropic Glu receptor agonists-AMPA,KA and NMDA by the traditional whole-cell patch-clamp technique.We also observed the effects of succinate on the high-voltage activated(HVA) and low-voltage activated(LVA) calcium channel currents role by the traditional whole-cell patch clamp technology and nystatin(Nystatin) perforated patch-clamp techniques.The results were as follows:1.Succinate could remarkbaly increase mIPSCs and sIPSCs frequency in hippocampal CA1 region,but had no effects on IPSCs amplitude.After perfusion of 1μM succinate, the mIPSCs frequency was 1.97±0.20 Hz,and sIPSCs 4.76±0.76 Hz,which had statistical significance compared with the control group(P＜0.01).2.To determine the possible mechanisms of succinate causing the increase in the mIPSC frequency,we observed the effects of an antagonist of N-type calcium channelω-Cg-GVIA,N-and P/Q-type channel antagonistω-Cm-MVIIC,L-type calicium channel agonist BAY K-8644 and antagonist Nicardipine.When we usedω-Cg-GVIA in ACSF, the frequency of mIPSCs was not changed,whileω-Cm-MVIIC decreased it,BAY K-8644 increased it,while Nicardipine blocked BAY K-8644 action definitely.When used together,ω-Cg-GVIAω-Cm-MVIIC and Nicardipine had no influence on the increasement of the mIPSCs frequency caused by succinate,while they did not change the mIPSCs amplitude either.Thapsigargin(an agonist of intracellular Ca²⁺ library IP₃ receptor) could obviously increase the frequency of mIPSCs,while the enhancement on frequency of mIPSCs caused by succinate no more appeared.Ryanodine,KT5720,and KN62 did not affect the frequency increace induced by succinate.3.Succinate prolonged the half-decay time of mIPSCs and sIPSCs.After perfusion of 1μM succinate,the half-decay time of mIPSCs was 8.73±0.50 ms,and sIPSCs 8.14±0.55 ms,which had statistical significance compared with the control group(P＜0.01).4.To determine the possible mechanisms of succinate causing prolongation of mIPSCs decay time,we also observed the effect of Flumazcnil,a specific antagonist for BZ bingding point and KN62,a specific antagonist for Ca²⁺/CaM-Kâ…¡.Zolpidem as a positive control,it prolonged mIPSCs decay time significantly,which could be blocked by Fhimazcnil,but the latter had no effect on mIPSCs decay time by succinate.KN62 totally blocked the effect caused by succinate.5.Succinate had no effect on the frequency and decay time of sEPSCs and mEPSCs,but it significantly decreased their amplitudes(P＜0.01). 6.Glu and its ionic receptor agonists-AMPA,KA,NMDA could elicit inward currents in hippocampal CA1 neurons.Succinate inhibited the currents elicited by Glu,AMPA and KA.7.Different concentrations of succinate inhibited concentration-dependently the high-voltage activated calcium currents.After perfusing with 1μM succinate,the high voltage-activated calcium currents decreased from 581.04±6.13 pA to 562.74±5.89 pA,which was statistical significance(P＜0.01).These results suggested that succinate increased presynaptic GABA release in rat hippocampal CA1 area,and increased chloride channel open time or frequency,producing hyperpolarizing effect on the hippocampal CA1 neurons.The release of Ca²⁺ from IP₃-sensitive intracellular Ca²⁺ library is the main factor that succinate enhanced the release of presynaptic GABAergic neurotransmitter.Activation of the Ca²⁺/CaM-Kâ…¡path was one of the ways through which succinate regulated the function of GABAA receptor. Succinate had the hyperpolarizing action on the hippocampal CA1 neurons which may be the main mechanism of succinate to inhibit the development of epilepsy.Succinate reduced the excitatory response in hippocampal CA1 neurons by accelerating the desensitization of AMPA- and KA-type receptors,performing its pharmacological effects.To sum up,succinate has the modulation effect on both GABAergic and Gluergic neurotransmitter systems in rat hippocampal CA1 area,and it is probably through Ca²⁺ as the core mechanism in the regulation of GABA system,which provides the evidence that succinate is a brain neuromodulator.