Mechanism Of Glutamatergic Long-term Synaptic Plasticity Of Hypothalamic Paraventricular Nucleus Magnocellular Neurosecretory Cells In Vitro In Rats

[Objective]The hypothalamic paraventricular nucleus(PVN)is a multifunctional nucleus consisting of magnocellular neurosecretory cells,parvocellular neurosecretory cells as well as parvocellular autonomic neurons.It is an important integrated part of hypothalamic-pituitary-adrenal axis to response the stress.The hypothalamic nucleus receives excitatory glutamatergic afferent inputs from different brain regions.These glutamataergic inputs take part in the regulation of MNCs functional activities.These glutamataergic inputs of NMCs could produce long-term plasticity under a certain conditions which generates a long-term regulation of MNCs.In addition,stress stimuli also affect the NMCs glutamataergic synaptic transmission and long-term plasticity.However,the mechanism of glutamate synaptic long-term plasticity in PVN MNCs remains unclear,and the effect of chronic stress on the glutamate synaptic long-term plasticity is unknown.Therefore,we used whole-cell patch-clamp recording combined with SC-RT-mPCR technique,imunohistochemistry,neruopharmacology methods to examine the mechanism of high frequency stimulation(HFS)-induced long-term synaptic plasticity at glutamatergic synapses of PVN MNCs in stress and non-stress rat under in vitro conditions,and to investigate the effect of stress on the long-term synaptic plasticity at glutamatergic synapses of PVN MNCs.[Methods](1)Long-term plasticity induction of MNCs in non-stress rats:Wistar male rats(12-14-day old)were employed in the present study.After the rats were anesthetized,the whole brain was isolated and the PVN included coronal slices were cut by a vibratome.The thickness of these slices was 250 μm.The slices were incubated in 95%O2 and 5%CO2 bubbled ACSF at 24-25℃ for>1 hour.Under the voltage clamp,we used paired-current pulse(0.2 ms,10-100 μA,interval 50 ms)to induce the evoked EPSCs of PVN MNCs,which identified as N1 and N2.We used HFS(100Hz,100 pulses,3 times)to induce long-term plasticity of excitatory inputs in the PVN MNCs.The HFSs were delivered after acquiring a 10-min control baseline.The amplitude and paird pulse ratio of excitatory postsynaptic currents(EPSCs)of MNCs stimulated by electrical stimulation were recorded and analyzed by Axopatch 700B amplifier and Clampex 10.4 software.After the whole-cell patch-clamp recording,the intracellular fluid was collected for single cell RT-mPCR to determine the recorded cell type.The slice was fixed in 4%paraformaldehyde and histological staining was performed to determine the histological morphology of the recorded cells.In order to record the glutamatergic synaptic transmission and plasticity,the ACSF included picrotoxin and cannabinoids type 1(CB1)blocker,AM-251 during all recordings to prevent GABAA and CB1 receptors mediated inhibitory.In the experiments involving KT5720,the application of KT5720 was started at least 30 min before recording.For experiments with L-NNA,the slices were perfused for 1 hour before recording.(2)Long-term plasticity induction of MNCs in stress rats:Wistar male rats(7-10 days old)employed in present study and divided into chronic stress group and non-stress group,Chronic stress was induced by self-restraint cages which made the body unable to move freely for 3 hours per day for 7 consecutive days.No treatment was given in the non-stress group.The weight of two groups was measured before and after the stress,and the weight gain was calculated.After chronic stress,the thymus and adrenal gland were stripped and thymus and adrenal gland index were calculated.Electrophysio logical recording and HFS-induce long-term plasiticity were in the same way as in first part.After electrophysiological recording,the intracellular fluid was collected for RT-mPCR to determine the recorded cell type.The slice was fixed in 4%paraformaldehyde and histological staining was performed to determine the histological morphology of the recorded cells.All the electrophysiological data were analysised by using Clampfit 10.4 software.All data were expressed as mean ± SEM.One-way ANOVA and Mann-Whitney-Wilcoxon test was used to determine the level of statistical significance between groups of data.P-values below 0.05 were considered to indicate a statistically significant difference between experimental groups.[Results]Part I(1)HFS induced presistent potentiation of glutamatergic synaptic transmission,which expresses an over 40-min increase in the N1 amplitude as well as decreased PPR in the PVN MNCs.These results indicate that HFS can induce the LTP of excitatory glutamatergic inputs in the PVN MNCs.(2)In the pesence an mGluR1 antagonist,the LTP was persisted which indicates that HFS-induced LTP in PVN MNCs was not mediated through mGluR1.(3)Blocking NMDA receptors activity abolished the LTP induction in the PVN MNCs,suggesting that induction of LTP in PVN MNCs was dependent on NMDA receptor.(4)HFS-induced LTP in PVN MNCs was blocked by NOS inhibitor,and the LTP can be producd by NO donor,suggesting that HFS-induced LTP was depedent on the production of NO.(5)Inhibition of PKA,HFS failed to induce the LTP in the PVN MNCs,suggesting that PKA signaling pathway mediated HFS-induced LTP in the PVN MNCs.Part Ⅱ(1)After chronic stress,the body weight increment of the stressed rats was significantly lower than the control conditions.Compared with the control,the thymus index of the stress group was significant decreased and the adrenal index was significant increased.In addition,the spontaneous firing activity of CRF mRNA expression neurons increased significantly after chronic stress.(2)HFS induced presistent inhibition of glutamatergic synaptic transmission,which produced an over 40-min depression in the amplitude of N1,as well as increased PPR.These results indicate that HFS can induce the LTD of excitatory glutamatergic inputs in the PVN MNCs.(3)Application of CRF non-selective receptors blocker,HFS failed to induce the LTD,suggesting that CRF receptors were involved the HFS-induced LTD in PVN MNCs of stress rats.(4)In the presence of a selective CRFR1 blocker,the HFS-induced LTD of excitatory glutamategic inputs in the PVN MNCs was enhanced in the stress rats.In contrast,blockade of CRFR2 reversed a HFS-induced LTP in the PVN MNCs of the stress rats,suggesting that CRFR1 and CRFR2 might differently mediate LTP and LTD at glutamatergic synapses of PVN MNCs.(5)Inhibition of PKA,HFS failed to induce LTD of excitatory glutamategic inputs in the PVN MNCs of stress rats.However,blockade of PKC signaling pathway,the HFS-induced LTD was persisted,indicating that HFS-induced LTD in stress rat MNCs occurs via a PKA but not a PKC signaling pathway.[Conclusions](1)HFS can induce an NMDA receptor and NO cascades dependent glutamatergic synaptic LTP in the PVN MNCs via a PKA signaling pathway.(2)Chronic stress can impair glutamatergic LTP and lead to HFS-induced glutamatergic synaptic LTD in the PVN MNCs through CRF receptors and the PKA signaling pathway.