高胆红素致耳蜗核神经元超兴奋的机制研究

The Role of Synaptic Transmission and Intrinsic Firing in Bilirubin-induced Hyperexcitability in Cochlear Nucleus

目的探讨胆红素引起耳蜗核(cochlear nuclear,CN)神经元超兴奋性的机制。方法选用出生后1~9天(P1~9)C57Bl/6J小鼠60只,制备含有CN的脑片。利用膜片钳技术,用细胞贴附式或者全细胞式记录神经元自发性动作电位、超极化激活环磷腺苷依赖性阳离子通道(hyperpolarization-activated and cyclic nucleotide-gated channels,HCN)电流、自发性抑制性突触后电流(spontaneous inhibitory postsynaptic current,sIPSC)和自发性兴奋性突触后电流(spontaneous excitatory postsynaptic current,sEPSC)。神经元在人工脑脊液中自发性放电或者阻断突触的内源性放电记为对照组,随后在脑脊液中灌流加6μm胆红素作用9 min记为胆红素组,最后回归至人工脑肾液中记为洗脱组。加药过程由一个多阀门控制,依靠重力单向输出的灌流装置完成,平均流速在2 ml/min。结果胆红素可以提高CN神经元自发性放电频率(胆红素组较对照组升高了250%±31.2%;洗脱组降为对照组的162%±21.4%);胆红素可以促进sEPSC(胆红素组增加至对照组的193.2%±26.4%)和sIPSC(胆红素组增加至对照组的135.3%±16.4%)频率增大,说明胆红素可以促进谷氨酸和GABA/甘氨酸能突触传递。CN神经元存在不依赖于突触传递的自发性内源性放电,并且这种放电是由HCN离子通道起搏介导的。HCN通道的抑制剂CsCl和ID7288可以明显降低自发性内源性放电的频率(CsCl:降至对照组的48.75%±9.23%,ZD7288:降至对照组的68.45%±10.39%)。胆红素作用后,HCN通道电流的激活曲线右移(V_(0.5):对照组-104.9±1.5 mV,胆红素组:-95.4±2.2 mV)。抑制HCN通道电流后,高胆红素不再发挥超兴奋作用。结论胆红素通过促进神经突触和HCN通道介导的内源性放电发挥超兴奋作用。

Objective To explore the mechanism of bilirubin-induced hyperexcitability in cochlear nucleus(CN).Methods C57Bl/6J mice between postnatal day 1 and 9 were used to prepare for brain slice of CN.We used patch-clamp technique to record spontaneous firing,current of hyperpolarization-and cyclic nucleotide-gated(HCN)channel,spontaneous inhibitory postsynaptic current(sIPSC)and spontaneous excitatory postsynaptic current(sEPSC)in whole-cell or cell-attach configuration.Control group means spontaneous or intrinsic firing frequencies of neurons in ACSF(artificial cerebral spinal fluid).Then bilirubin were diluted to 6μm in ACSF and perfused about 9 min,which were recorded as bilirubin group.Finally,the recorded neuron went back to aboved-mentioned control perfusate,which were recorded as wash group.Application of drugs was achieved by switchinga multi-valve,single-output gravity perfusion system at a speed about 2 ml/min.Results Bilirubin significantly facilitated the frequency of spontaneous firing in CN neurons(bilirubin group:250%±31.2%of control group;wash group:162%±21.4%of control group).Bilirubin increased the frequency of sEPSC(193.2%±26.4%of control group)and sIPSC(135.3%±16.4%of control group),which meaned GABA/glycinergic transmission specifically contribute to bilirubin-induced hyperexcitability in the early stage of development.In CN neurons,spontaneous firing were reduced but not eliminated by blockers for excitatory and inhibitory synaptic inputs,implicating the involvement of intrinsic pacemaker channels.Next,we demonstrated intrinsic firing was driven by hyperpolarization-and cyclic nucleotide-gated(HCN)channel.Inhibitor of HCN channel significantly decreased frequency of intrinsic firing(CsCl:48.75%±9.23%of control group;ZD7288:68.45%±10.39%of control group).Moreover,we found that HCN activation curve was shifted toward more depolarized potentials in bilirubin perfusion(V_(0.5):control group:-104.9±1.5 mV;bilirubin group:-95.4±2.2 mV).If we pretreated CN with inhibitor of HCN channel,the frequency of spontaneous intrinsic firing during bilirubin perfusion was similar to the control level.Conclusion Bilirubin-induced enhancement of synaptic transmission and HCN-evoked intrinsic firing may play a critical role in mediating neuronal hyperexcitation associated with jaundice.