Effect Of NERP-1 On The Activity Of Pvn Magnocellular Neurosecretory Cells In Vitro In Rats

[Objective]To study the effect of neuroendocrine regulatory peptide-1(NERP-1)on the activity of hypothalamic paraventricular nucleus(PVN)magnocellular endocrine cells(MNCs),using whole-cell patch-clamp recording technique,imunohistochemistry and pharmacological methods in vitro in rats.We aimed to explore the effect of NERP-1 on the activity of the PVN MNCs,and to indicate the synaptic mechanism of NERP-1 regulating the activity of PVN MNCs in rats.[Methods]The male Wistar rats of 2-3 weeks were selected as experimental animals.After inhalation anesthetized by isoflurane,the heads were cut off and brains of rats were put into ice-cold artificial cerebral spinal fluid(ACSF)quickly.Then,the hypothalamus sections with PVN of 250 μm in thickness were prepared with vibration microtome.At room temperature(about 24-25℃),the slices of hypothalamus were incubated in ACSF filled with 95%O2 and 5%CO2 for more than 60 minutes.The ACSF was consisted of(mM):118 NaCl,3 KCl,1 NaH2PO4.2H2O,1 MgCl2.6H2O,10 D-Glucose,25 NaHCO3,2 CaCl2;PH value was 7.25-7.35,and osmolarity was 295-300 mOsM.The recording electrodes were filled with 10 μl internal solution,which consisted by following(mM):120 Potassium Gluconate,8 biocytin,5 KCl,4 Na2ATP,1 EGTA,4 NaCl,10 HEPES,0.2 Na2GTP,3.5 MgCl2.PH value was adjusted to 7.3 with KOH.Patch pipette resistances were 5-7 MΩ in the bath.The spontaneous spike firing of PVN neurons was recorded by patch clamp amplifier(Axopatch-200B)and data acquisition software.NERP-1 was perfused around the recording neurons by peristaltic pump(Gilson Minipulse 3).At the end of electrophysiological recording,the brain slices used in the experiment were fixed with 4%paraformaldehyde solution for 24 hours,then immunohistochemical staining and micrography were carried out to determine the type,location,morphological characteristics and projection of the recorded cells.Clampfit 10.4 software and MiniAnalysis(6.0)software were used for data analysis of electrophysiological experiments.All data were expressed by Mean ± S.E.M.The data were analyzed by Student’s paired t-test using SPSS software.P<0.05 was considered statistically significant.[Results](1)Under current-clamp conditions,PVN MNCs were exhibited a mean membrane potential of 54.8 ± 6.2 mV,and a mean membrane resistance of 232.7±9.3 MΩ.In addition,these MNCs were sensitive to the injection of depolarization currents,and they exhibit outwardly rectification currents.(2)Application of NERP-1 induced a significant decrease in spike firing and membrane hyperpolarization rate of PVN MNCs,which were recovery after washout for 20 min.(3)Non-selective ionotropic glutamate receptors blocker(kynurenic acid,KYC)can block perfusion application of NERP-1-induced the frequency of spike firing decrease and membrane hyperpolarization of PVN MNCs.(4)Blocking GABAA receptor,NERP-1 still can inhibit the activity of neurons and membrane hyperpolarization.(5)In the presence of voltage dependent sodium channel blocker TTX,NERP-1 significantly reduced the frequency of miniature postsynaptic currents(mEPSCs),but had no significant effect on the amplitude of mEPSCs.(6)NERP-1 significantly decreased the amplitude of the paired stimulation evoked excitatory postsynaptic currents(eEPSCs),and increased the paired-pulse radio(P2/P1).(7)NERP-1-induced inhibition of PVN MNCs can be blocked by bath application of protein kinase A inhibitor KT5720.(8)Blocking the intracellular PKA has no significant effect on NERP-1-induced inhibition of PVN MNCs.[Conclusion]NERP-1 can down regulate glutamate release from excitatory glutamatergic afferent terminals through presynaptic PKA pathway,and reduce the excitability of hypothalamus PVN MNCs,suggesting that NERP-1 can indirectly inhibit the activity of PVN MNCs in vitro in rats.