神经内分泌调节肽对大鼠下丘脑室旁核神经元活动及突触传递的影响

Neuroendocrine regulatory peptides（NERP-1 and NERP-2）are recently identified novel amidated peptides which are separated from human medullary thyroid carcinoma TT cells. NERPs are abundant in the paraventricular nuclei （PVN）and supraoptic nuclei of rat hypothalamus, which are involved in the regulation of vasopressin (VP) release, hypothalamic neuroendocrine, water-electrolyte metabolism balance, and energy homeostasis. We recently found that intracerebroventricular (i.c.v) administration of NERP-1 induced c-fos over expression in the rat PVN neurons. Patch-clamp recording from PVN neurons of rat hypothalamic slices showed that NERP-1 induced a depolarization of membrane potential and an increase in spike firing in a subpopulation of the neurons. In order to investigate the mechanisms of NERPs affect the neuronal activity and synaptic transmission of the PVN neurons in vitro in rats, applicant will use the whole-cell patch-clamp recording, histochemistry，single-cell reverse transcription-multiplex polymerase chain reaction (scRT-Multi-PCR) and neuropharmacology methods to study the effects of NERP-1 and NERP-2 on the neuronal activity, membrane currents (potential) and synaptic transmission of PVN VP, OT (oxytocin), CRH (corticotrophin-release hormone),TRH (thyrotropin-releasing hormone) and NMU (neuromedine U) mRNA expressing neurons in vitro in rats. We aim to understand the mechanisms of NERPs on PVN affect endocrine neuronal activity，synaptic transmission and plasticity. Our results will provide theoretical evidence for elucidating the cellular and molecular mechanisms of NERPs involved in regulation of the PVN neuroendocrine, water-electrolyte metabolism balance, energy metabolism and stress responses.

神经内分泌调节肽(NERP-1和 -2) 是从人甲状腺髓样癌TT细胞中发现的新酰胺肽，在下丘脑室旁核（PVN）和视上核中含量丰富，影响血管升压素（VP）的分泌，参与下丘脑神经内分泌、水盐代谢和能量平衡调节。最近，我们研究发现大鼠侧脑室注射NERP-1引起c-fos在PVN中表达增加，脑片膜片钳记录研究发现NERP-1可使部分PVN神经元去极化,增加自发性放电频率。为了进一步探讨NERPs对PVN神经元活动和突触传递的影响机制，申请人拟应用脑片膜片钳记录、组织化学、单细胞RT-Multi-PCR和药理学手段，研究NERP-1和-2对大鼠PVN中VP、OT、CRH、TRH、NMU mRNA表达神经元活动、膜电流（电位）和突触传递的影响，明确NERPs对PVN分泌神经元活动、突触传递及可塑性的影响机制，为阐明NERPs参与调节神经内分泌、水盐平衡、能量代谢和应激反应的细胞与分子机制提供理论依据。

本项目主要应用电生理学、组织化学、单细胞RT-mPCR和药理学在大鼠幼崽下丘脑切片上研究了下丘脑室旁核（PVN）大神经分泌细胞（MNCs）谷氨酸能突触产生长时程增强（LTP）的机制以及神经内分泌调节肽（NERPs）、尼古丁对大鼠下丘脑室旁核神经元活动及突触传递的影响。探讨了高频刺激诱发PVN神经元突触可塑性的类型及其机制，NERP-1、NERP-2、尼古丁对PVN神经元活动及其突触传递的影响及其机制，明确了高频刺激能够诱发幼年大鼠PVN的OT mRNA表达MNCs谷氨酸能突触产生LTP；明确了NERP-1能够抑制PVN中OT mRNA表达MNCs的活动，而NERP-2则对大鼠PVN中CRH mRNA表达小细胞产生兴奋作用，以及NERP-1能够影响下丘脑室旁核神经元的突触传递，这将为神经内分泌信息在PVN中的传递与整合机制的建立提供很好的理论依据。具体结果如下： .（1）发现高频刺激（HFS，100Hz）能够诱发幼年大鼠PVN的OT mRNA表达MNCs产生谷氨酸能突触长时程增强（LTP），并伴有PVN MNCs成对脉冲比（PPR）降低。（2）发现高频刺激诱导下丘脑室旁核中OT mRNA表达MNCs 的LTP是NMDA受体和一氧化氮（NO）级联反应依赖性的LTP。（3）发现NERP-1抑制PVN中I型细胞的放电频率，NERP-2增强PVN中II型细胞的放电频率。（4）发现NERP-1通过影响突触前的PKA通路来调节PVN中I型细胞的电活动。（5）发现NERP-1能够影响PVN中MNCs谷氨酸能突触传递，并且能够弱化该突触的信息传递。（6）发现尼古丁通过增强GABAergic的抑制作用使PVN OT mRNA表达神经元的自发放电频率降低。