Functional Responses Of Mormyrid Cerebellar Purkinje Cells To Acute Hypoxia Insult

Hypoxia-mediated brain injury may result from several neonatal diseases, such asasphyxia of newborn, hypoxic-ischemic encephalopathy (HIE) and respiratory distresssyndrome (RDS), being the leading cause of newborn death or chronic disability later.Therefore, the investigations of the processes underlying hypoxic changes of neuronalactivity are required to develop safe and effective drugs that target neuroprotection,helping decrease the mortality of the newborn and the disability of children.The complex multifactorial interactions contribute to the hypoxic brain injury. It iswell accepted that the failure in synaptic transmission accompanied with depolarization isa very early event of oxygen deprivation in mammals and other non-hypoxia-tolerantanimals, leading to the failure of energy-dependent ion pumps and the release of theexcitatory amino-acid neurotransmitters and culminating in excitotoxic cell death withinseveral minutes. Unlike mammals, hypoxia-tolerant vertebrates are adapted to survive under anoxia or hypoxia conditions for several day even months by metabolic depression.Therefore, new advances may arise from a functional analysis of hypoxia-tolerant animalsunder hypoxia condition and will protect mammal from neuronal damage by hypoxiainsult.Numerous studies have proved that in hypoxia condition the most significantneurochemistry changes in hypoxia-tolerant animal is an increased level of inhibitorytransmitter γ-aminobutyric acid (GABA) with a decreased level of excitatory transmitterGlutamate. However, it still remains unclear how the neurons of hypoxia-tolerant animalrespond to hypoxia insult. This study is designed to record the electrophysiologicalfunction of Purkinje cell (PC) and synaptic transmission by patch-clamp method oncerebellar slices of mormyrid fish, to investigate the hypoxia responses of the neuron inhypoxia-tolerant animal.Methods1．The effect of acute hypoxia insult on the excitability of PCThe whole cell patch clamp method was used for the intracellular recording from PCof mormyrid cerebellar slices to evaluate the effect of acute hypoxia insult on theexcitability of PC by comparing the membrane potential, the firing rate of spontaneousspikes and stimulated action potentials of PC before and after a brief hypoxia insultwithout or with pharmacological intervention.2．The effect of acute hypoxia insult on the spontaneous miniature postsynapticcurrentThe spontaneous miniature excitatory postsynaptic current (mEPSC) andspontaneous miniature inhibitory postsynaptic current (mIPSC) were recorded before andafter a brief hypoxia insult, to evaluate the effect of acute hypoxia condition on thesynaptic transmission of mormyrid fish neuronal net.3．The effect of acute hypoxia insult on the synaptic transmission of PF-PCThe excitatory postsynaptic curren (EPSC) of parallel fiber (PF)-PC synapse wasrecorded before and after a brief hypoxia event without or with pharmacological intervention, to investigate the effect of acute hypoxia insult on the synaptic transmissionof PF-PC in mormyrid cerebellar.Results1．PCs show a rapid and significant hyperpolarization with a decreased spontaneousfiring rate following the onset of hypoxia episode and last persistently for more than30minutes. α-amino-3hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptorantagonist6-cyano-7-nitroquinoxaline-2,3-dike-tone (CNQX) did not affect the initiatingof the hyperpolarization, but prevented the hypoxia hyperpolarizaion from long lasting.GABAAreceptor antagonist Bicuculline completely blocks the hypoxia hyperpolarization,and induced a brief hypopolarizaion immediately following hypoxia episode. Meanwhile,the threshold of action potential of PCs was higher, its frequency lower and the amplitudesmaller than in normal condition.2．The frequency of both mEPSC and mIPSC of PCs were increased steadilyfollowing an acute hypoxia insult.3．Acute hypoxia insult induced the long term potentiation (LTP) of PF-PC EPSCwith a decreased PPF of EPSC. CNQX reversed the PF-PC EPSC LTP to long termdepression (LTD), while Bicuculline enhanced the hypoxia LTP of PF-PC EPSC.ConclusionThe results from this study show that the functional responses of mormyrid cerebellarPCs to acute hypoxia insult are quite different from that of mammalian. There are twomain differences as bellow. First, PCs of mormyrid cerebellum respond to hypoxiaepisode with a rapid and significant hyperpolarization, lasting for more than30minuteswith a decreased excitability. Second, both of excitatory synaptic transmission andinhibitory synaptic transmission of mormyrid cerebellar circuitry are enhanced by acutehypoxia event. AMPA receptor mediated excitatory synaptic activity and GABAAreceptormediated inhibitory synaptic activity all contribute to these modifications when oxygenlevels diminish. The results suggests that a proper balance between Glutamatergic andGABAergic synaptic transmission is definitely required for the protective responses of mormyrid neuron under hypoxia condition and probably also for the otherhypoxia-tolerant animal as well. It may be the reason why glutamate receptor antagonist orGABA receptor agonist, which were successful in vitro, were ineffective in humans.