The Effect Of Paeoniflorin On The Functional Responses Of Rat Cerebellar Purkinje Cells To Acute Hypoxia Insult

Many diseases may cause hypoxic brain injury, such as perinatal asphyxia, cardia-cerebrovascular disease, respiratory disease, head trauma, altitude stress and so on. Hypoxic brain injury is the main reason which may lead to death in acute phase or produce adverse outcome in future life. Therefore, it is very important and urgent to investigate the mechanisms and therapeutic medicines for hypoxic brain injury.Neurons in mammalian CNS are highly sensitive to hypoxia or anoxia. Several minutes of hypoxia may lead to neuronal dysfunction or neuronal injury and apotosis. Prominent neuronal functional responses induced by hypoxia include substantial membrane depolarization and rapid failure of synaptic transmission in mammals. Both Glutamatergic and γ-aminobutyric acid(GABA) ergic synaptic transmissions play important roles in hypoxia-induced functional responses of neurons. Numerous studies have demonstrated that glutamate shift in role from neurotransmitter to neurotoxin under hypoxic conditions, however GABA could inhibit electrical activities through activating GABAA receptors and exert neuroprotective effects during hypoxia.The basic mechanisms of hypoxic brain injury have been studied primarily in slice preparations from hippocampus and cortex in rat. However, cerebellum, which is one of the most sensitive areas, did not draw enough attentions. Cerebellar Purkinje cells(PCs) receive excitatory inputs from parallel fibres(PF) and the climbing fibres(CF), and strong inhibitory inputs from interneurons and neighbouring PCs, which makes cerebellum an ideal model to investigate the mechanism of hypoxic brain injury and hypoxic neuronal protection.Paeony radix, one of the traditional Chinese crude drugs, has been widely used as a component of traditional Chinese prescriptions for relaxing abdominal spasm, alleviating pain, and improving blood circulation. Paeoniflorin(Pae), the main bioactive ingredient of Paeony radix, has been proved to possess potent neuroprotective effects. Since Pae is able to pass blood brain barrier, it may be a candidate to treat nervous system diseases. Previous studies found that Pae could inhibit the increase of excitatory amino acid during hypoxia, inhibit the overload of intracellular calcium, block sodium channel and activate adenosine A1 receptor. However, it has been unclear whether Pae affect the hypoxia-induced functional responses of neurons. The present study employ whole-cell patch-clamp to record the hypoxic changes of cerebellar PCs in membrane potential and synaptic transmission, and then investigate the modulation of Pae on these hypoxia-induced functional responses of PCs.1. Effects of Pae on hypoxic changes in membrane potential of rat cerebellar PCsObjective: To investigate the effects of Pae on hypoxia-induced membrane potential changes in rat cerebellar PCs and the possible mechanisms.Methods: The whole-cell patch-clamp was used to record membrane potential under current-clamp mode. When the recording was steady, hypoxia insult(2 min of hypoxic perfusion) was carried out to observe the effects of hypoxia on membrane potential of PCs. Glutamate AMPA receptor antagonist CNQX,GABAA receptor antagonist Bicuculline and Pae were added to the hypoxic perfusate respectively during hypoxia episode to exam the effects of these pharmacological compounds on hypoxic changes in the membrane potential of PCs.Result: A brief episode of hypoxia induced triphasic changes on membrane potential of PCs: an early brief hyperpolarization followed by a large depolarization and a stable posthypoxic hyperpolarization companied with increased firing. CNQX blocked the early and posthypoxic hyperpolarization and had no significant effect on the hypoxic depolarization. Bicuculline blocked the early hyperpolarization, increased the amplitude and duration of the hypoxic depolarization and had no significant effects on the posthypoxic hyperpolarization. Pae not only blocked both the early and posthypoxic hyperpolarization, but also decreased the duration and amplitude of the hypoxic depolarization significantly compared with hypoxia alone.Conclusion: Acute hypoxic insult induced tripahsic changes in PCs membrane potential. AMPA receptor and GABAA receptor played different roles in hypoxia-induced tripahsic changes in membrane potential of PCs. Pae significantly inhibited hypoxic tripahsic changes of PCs membrane potential and may mimic the effects of Glutamate AMPA receptor antagonist and GABAA receptor agonist.2. Effects of Pae on the excitability changes of rat cerebellar PCs upon acute hypoxiaObjective: To investigate the effects of Pae on hypoxia-induced changes in PCs excitability.Methods: The action potentials were evoked by injecting depolarized current into PCs under current-clamp mode. Hypoxia insult was applied to observe the effects of hypoxia on the firing rate of PCs and the threshold stimuli for evoking action potentials. The effect of Pae on the changes above was investigated by perfusion with hypoxic episode..Result: The frequency of action potentials of PCs increased and the threshold valuedecreased after hypoxia. Pae did not show significant effect on these changes.Conclusion: Hypoxia up-regulated the excitability of PCs. Pae did not affect the hypoxiainduced high excitability of PCs.3. Effects of Pae on hypoxic depression of rat cerebellar PF-PC synaptic transmissionObjective: To investigate the effects of Pae on hypoxia-induced depression of cerebellar PF-PC synaptic transmission.Methods: The PC excitatory postsynaptic currents(EPSCs) were recorded by stimulating PF under voltage-clamp mode. CNQX, Bicuculline and Pae were used respectively during hypoxia insult to investigate the effects of these pharmacological compounds on hypoxia-induced depression in cerebellar PF-PC EPSCs.Result: Hypoxia depressed the amplitude of PF-PC EPSCs and lasted for more than 30 min, showing long term depression(LTD). CNQX enhanced the depression of PF-PC EPSCs. When using Bicuculline together with hypoxic episode, the amplitude of PF-PC EPSCs increased at the beginning of the treatment and then decreased gradually. PaeConclusion: Hypoxia induced LTD of PF-PC EPSCs. Blocking the AMPA receptor did not prevent the hypoxic inhibition in PF-PC EPSCs. GABAA receptor mediated the early inhibition of PF-PC EPSCs. Pae significantly decreased the depression of PF-PC EPSCs and may be helpful on maintaining the PF-PC synaptic function.