Mechanism Of ANP Regulates Neuronal Activity And Synaptic Transmission In Mouse Hypothalamic PVN CRF-mRNA Expressing Neuron In Vitro

[Objetive]Atrial natriuretic peptide（ANP）is a peptide hormone synthesized and released by atrial myocytes,which regulates various physiological functions such as water and sodium metabolism through its receptors.In recent years,a large number of studies have shown that ANP and its receptors are widely distributed in the central nervous system and are involved in regulating neuronal activity and synaptic transmission.The hypothalamic paraventricular nucleus（PVN）is an important center for the regulation of sympathetic nerve and cardiovascular functional activities,in which corticotropin-releasing factor（CRF）can regulate the functional activities of the hypothalamic-pituitary-adrenal axis（HPA）through the secretion of CRF,and plays a key role in the regulation of body stress and cardiovascular functional activities.Some studies have reported that ANP can regulate the release of CRF in the central nervous system and affect the activity of CRF-ergic neurons of PVN,but the mechanism of ANP regulating the functional activities of PVN CRF-ergic neurons remains unclear.Therefore,this study used patch-clamp recording,molecular biotechnology,immunofluorescence staining and other technical means to observe the effect of ANP on the activity of PVN CRF m RNA expression neurons and synaptic transmission,to study the pharmacological mechanism of ANP on its effect,and to explore the mechanism of changes in the activity of PVN CRF m RNA expression neurons after ANP deletion.[Methods]1.In vitro brain patch clamp recording:C57BL/6 mice aged 4-6 weeks were anesthetized by inhalation of isoflurane,and the brains were severed immediately.Coronal sections containing PVN were prepared by vibration sectioning mechanism.Patch-clamp recordings were acquired using the Axopatch 700 B dual-channel patch-clamp amplifier and via the Clampex 10.6 software and 1500B D/A digital-to-analog converter.Voltage pulses were used to test series resistance,and only recorded cells with series resistance stability were included in the analysis.The effects of ANP on the firing and after-hyperpolarization potential（AHP）activity of PVN CRF-m RNA expressing neurons were studied under the condition of current clamp recording.Under the voltage clamp recording mode,the effect of ANP on the external rectification current（IR）generated by PVN CRF-m RNA expressing neurons was observed,the effect of ANP on the membrane current induced by hyperpolarization voltage of PVN CRF-m RNA expressing neurons was studied,and the effect of ANP on the instant current（I-inst.）,steady current（I-steady）,tail current（IT）and IH current（IH=I-steady-I-inst.）was analyzed.To explore the receptor,channel and signal transduction mechanisms.In addition,the effect of ANP on the spontaneous miniature excitatory postsynaptic currents（m EPSCs）and the excitatory postsynaptic currents（EPSCs）induced by double pulse stimulation（0.05 Hz,0.2 ms）of PVN CRF neurons was studied in voltage clamp recording mode.2.Single-cell PCR and histopathology:After the electrophysiological recording is completed,the intracellular fluid is recovered and the target c DNA fragment is amplified by reverse transcription PCR.The CRF m RNA fragments were amplified by nested PCR,and the recorded neurons were identified as CRF m RNA expressing neurons.After the experiment,the brain slices were fixed in 4%paraformaldehyde solution,then histochemical staining was carried out,and the location,morphology and type of the recorded cells were determined by microphotography.3.Immunofluorescence and western blot:NPPA-specific conditioned knockout mice and negative control mice at 4-6 weeks of age were selected to detect the expression of NPPA protein in the hypothalamus of knockout mice by protein western blot,and the expression of NPR-A receptor in PVN CRF-positive neurons was observed by immunofluorescence staining.4.Statistic analysis:Electrophysiological data were analyzed using Clampfit10.6 software.The Image J software was used to perform gray-scale analysis of the protein blot.The Mann-Whitney test in Graph Pad Prism 6 software or one-way analysis of variance were applied to evaluate the differences between the data groups.P<0.05 was considered statistically significant between the experimental group and the control group.[Results]PartⅠ:Mechanism of ANP modulates the activity of PVN CRF-m RNA expressing neurons in mouse hypothalamusWe used C57BL/6 mouse hypothalamus sections,whole-cell patch clamp recording,single-cell RT-PCR and pharmacological methods to study the effect of ANP on mouse PVN CRF-m RNA expressing neurons.ANP significantly inhibited the membrane excitability of PVN CRF-m RNA expressing neurons under current clamp,and the ANP receptor（NPR-A）antagonist A71915 could completely block the inhibitory effect of ANP on PVN CRF-m RNA expressing neurons.In addition,ANP can significantly increase the amplitude of afterhyperpolarization potential（AHP）and rectification current（IR）in CRF-m RNA expressing neurons.Under voltage clamp,ANP significantly inhibited the hyperpolarization-activated cationic currents（IH）of CRF-m RNA expressing neurons,and the inhibitory effect of ANP on IH channel of PVN CRF-m RNA expressing neurons could be blocked by NPR-A receptor antagonists.Application of protein kinase A（PKA）inhibitor,KT5720,completely blocked the inhibitory effect of ANP on the IH channel of CRF-m RNA expressing neurons.These results indicate that ANP inhibits the IH channel activity of PVN CRF-m RNA expressing neurons through NPR-A receptor and PKA signaling pathway.PartⅡ:Mechanism of changes in neuronal activity and synaptic transmission in PVN CRF-m RNA expression in NPPA gene knockout miceThe excitability of PVN CRF-m RNA expressing neurons in NPPA^-/-mice was significantly higher than that in wild-type(NPPA^+/+)mice,which showed an increase in firing frequency and a decrease in input resistance,but there was no significant difference in the AHP and half-width values of the action potentials of PVN CRF-m RNA expressing neurons in the two types of mice.Blocking GABA_Areceptor mediated inhibitory synaptic transmission had no significant effect on the excitability of PVN CRF-m RNA expressing neurons in NPPA^-/-mice,but blocking Glutaminergic synaptic transmission significantly reduced the excitability of PVN CRF-m RNA expressing neurons in NPPA^-/-mice.Compared with wild type mice,NPPA^-/-mice showed no change in inhibitory synaptic transmission and enhanced excitatory synaptic transmission.Under voltage-clamp,the frequency of PVN CRF-m RNA expression neuron m EPSC in NPPA^-/-mice was significantly higher than that in wild-type mice.In the presence of GABA_Areceptor blockers,the amplitude,area under the curve（AUC）and half-width of excitatory postsynaptic current（EPSCs）P1in PVN CRF-m RNA expressing neurons of NPPA^-/-mice induced by double pulse stimulation were significantly higher than those of wild-type mice,accompanied by a decrease in paired-pulse ratio（PPR）.These results suggest that presynaptic glutaminergic synaptic transmission of PVN CRF-m RNA expressing neurons in NPPA^-/-mice is significantly enhanced compared with wild type.Blocking the metabolic glutamate receptor（m Glu R1）had no significant effect on the evoked PVN CRF-m RNA expressing neurons EPSCs in NPPA^-/-and wild-type mice.However,blocking the NMDA receptor resulted in a significant decrease in the amplitude and AUC of EPSCs P1 induced by paired-pulse stimulation of PVN CRF-m RNA expression neurons in two types of mice,while the PPR increased significantly.There was no significant difference between the two types of mice.These results indicated that the presynaptic NMDA receptor function was enhanced in PVN CRF-m RNA expressing neurons of NPPA^-/-mice.Specifically blocking the NMDA receptor containing Glu N2A subunit,the amplitude and AUC of EPSCs induced by double pulse stimulation of NPPA^-/-and wild-type PVN CRF-m RNA expressing neurons significantly decreased,and the PPR increased,but there was no significant difference between them.The amplitude,AUC and half-width of EPSCs P1 induced by double-pulse stimulation of PVN CRF-m RNA expressing neurons in NPPA^-/-mice and wild-type mice significantly increased,accompanied by a decrease in PPR.There was no significant difference between the two mice.It is suggested that the function of NMDA receptors containing Glu N2A subunits in presynaptic sites of PVN CRF-m RNA expressing neurons in NPPA^-/-mice have been enhanced.Under voltage-clamp,ANP significantly decreased the IH current of PVN CRF-m RNA expressing neurons in NPPA^+/+mice,but had no significant effect on the IH current of PVN CRF-m RNA expressing neurons in NPPA^-/-mice.Immunohistochemistry showed that NPR-A receptor expression was missing in PVN CRF-m RNA expressing neurons of NPPA^-/-mice,suggesting NPR-A receptor degradation,indicating that ANP affects IH channel activity of PVN CRF-m RNA expressing neurons through its receptors.[Conclusions]1.ANP inhibits the activity of IH channel by activating NPR-A receptor in PVN CRF-m RNA expressing neurons through PKA signaling pathway,thus inhibiting the excitability of PVN CRF-m RNA expressing neurons.2.NPPA gene knockout enhances the function of presynaptic NMDA receptors containing Glu N2A subunits in mouse PVN CRF-m RNA expressing neurons,thereby increasing the release of presynaptic glutamate and enhancing glutaminergic synaptic transmission.3.NPPA gene knockout leads to the loss of NPR-A receptor expression in mice PVN CRF-m RNA expressing neurons,and the enhancement of postsynaptic IH channel activity,which leads to the increase of neuronal excitability.