Acute Hypoxic Effect On NMDA-Evoked Currents In Rat Hippocampal Neurons And GDNF Mechanisms

Cerebrovascular diseases are the third most common cause of death and the most common disabling neurologic disorder.Its incidence increases with age.The underlying pathologic process in cerebrovascular diseases can be either ischemia or hemorrhage, usually from an arterial lesion.Ischemic stroke accounted for about two-thirds of strokes.Ischemic neuronal injury is a complicated pathophysiological process. Lack of glucose and oxygen depletes the cellular energy stores required to maintain membrane potentials and transmembrane ion gradients. Loss of these gradients depolarizes cell membranes, leading to the influx of calcium through voltage-gated calcium channels and triggering the release of neurotransmitters such as glutamate from presynaptic nerve terminals. Glutamate binds to receptors on the postsynaptic neuronal membrane to activate the influx of sodium and calcium.This sets in motion a cascade of biochemical events.This sequence of events has been termed excitotoxicity because of the pivotal role of excitatory amino acids such as glutamate. A variety of neurotrophins and their receptors expression upregulate along with ischemia and protect neurons from ischemic injury.Among them, glial cell line-derived neurotrophic factor(GDNF) protects neurons from ischemic injury through NMD A receptor. NMDA receptor is the most typical excitatory neurotransmitter-gated receptor in central nerves system.In the present experiment, we explored the effect of acute hypoxia on the NMDA-evoked current and GDNF machenisms in primary cultured rat hippocampal neurons by whole-cell patch clamp technique.The main results of the study as follows:1 .The general electrophysiological characteristics of glutamate- or NMDA-evoked current. Both NMDA(100uM) and glutamate( 100uM) can evoke an inward current in primary cultured rat hippocampal neurons in Mg2+-free external solution at a holding protential (Vh) of -60mV. IGLU and INMDA displayed significant properties of desensitizitation and inward rectification.2. An acute hypoxia model was made using 95%N2 mix with 5%CO2 After acute hypoxia for 2 min, the peak of INMDA was enhanced significantly in the primary cultured hippocapal neurons at a holding potential of -60mV.3.Extracellular application of GDNF(0.1,1,10ng/ml) had no effects on the resting membrane conductance and voltage-gated channels. The peaks of INMDA were remarkably inhibited by GDNF when extracellularly applicated NMDA and GDNF(10ng/ml) simultaneously.The inhibitory effect of GDNF on INMDA was reversible when GDNF was washed out.4. When the electrode internal solution was supplemented with a broad spectrum protein kinase inhibitor staurosporine(0.5uM), the peak of INMDA was significantly inhibited, and the inhibition of INMDA induced by GDNF in the presence of staurosporine was little affected.5.A Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-62 (50uM) presented in the internal solution had no significant effects on the peak of current induced by extracellular application of NMDA (100uM), but prevented the inhibitory effect of GDNF on INMDA.Our study confirmed electrophysiologically that NMDA receptor is involved in the excitotoxicity triggered by acute hypoxia and the activities of GDNF. From one hand, acute hypoxia can increased the activity or the number of NMDA receptor in the membrane of hippocampal neurons.From the other, GDNF had inhibitory effects on INMDA in the primary cultured hippocampal neurons and this may be one of the mechanisms to protect the neurons from hypoxic injury. So GDNF can decrease the activity or the number of NMDA receptor and play a important role in the neuroprotection.And the effects maybe associate with PKA/PKC and have something with CaMKII. Although we can not draw a conclusion about the concrete or specific role of NMDA receptor playing in the actions of GDNF, it is not doubt to say that NMDA receptor indeed have something with the action of GDNF neuroprotection.There exhibits a broad space to further probe into the relationship of NMDA recepto...