The Morphological And Functional Investigation On The Nociceptive Inputs Of The Hypothalamic Paraventricular Nucleus

The hypothalamic paraventricular nucleus(PVN)is located in the medial-division of hypothalamic area,close to the dorsal part of the ventral third ventricle.It is an important neuroendocrine nucleus in the animal.There are varied kinds of cells in the PVN,which participate in the regulation of food intake,metabolism,body fluid balance,stress,reproduction,and so on.In addition,it has been reported that PVN can be activated by nociceptive stimulation or pain,and participates in the regulation of pain.However,most of the studies in this field have been focused on the downstream pathway after its activation,but little is known about the nociceptive input pathway of the nucleus.Therefore,in this study,we combineds tracer tracking and pathway modulation to explore the morphological and functional feature of the input pathway of the PVN.Objectives:Screening key nuclei of nociceptive information,Explore the loop of LPB-PVN involved in the regulation of chronic pain and emotional comorbidities.To reveal the upstream pathway mechanism of PVN involved in nociceptive information regulation.It provides a new theoretical basis for the study of pain and analgesia.Methods:(1)In the first part of the study,Fluorescent gold(FG)retrograde tracing and FOS expression induced by nociceptive stimulation were used to investigate the nociceptive input profile of PVN in the brain.The nucleus with the most essential input valence was determined.(2)In the second part of the paper,The pharmacogenetic modulation was used to examine the effects of activating this brain region-PVN pathway on pain sensitivity and the related affective behavior in mice with nerve injury induced neuropathic pain.Results:(1)Intraplanter injection of 5% of formalin induced a significant expression of FOS protein in the PVN region,indicating that PVN is indeed involved in pain processing and regulation.(2)After FG was injected into the PVN and formalin stimuli were applied 1.5 h before animals were killed,the double labelling of FG and FOS in the whole brain were thoroughly mapped.The results showed that brain regions with dense double labeled neurons included area 2 of cingulate cortex(Cg2),intermediate part of lateral septal nucleus(LSI),periaqueductal gray(PAG),posterior hypothalamic area(PH),and lateral part of lateral parabrachial nucleus(LPBE).Among them,the double labelling rates to either single labelling is the highest in the LPBE compared with other brain region,indicating that LPBE is the nucleus with the greatest intensity of transmitting nociceptive information to the PVN.(3)Immunofluorescent triple staining showed that FG/FOS double-labeled neurous in the LPBE were calcitonin gene-related peptide(CGRP)positive,indicating that the LPBE neurons projecting to the PVN may be glutamatergic.(4)r AAV-c FOS-m Cherry virus was injected into the LPBE in mice subjected to spared nerve injury(SNI).A medium intensity of virus-expressing nerve fibers and terminals were observed in the PVN,These projecting fibers formed close axo-dendritic and axo-somatic contacts with oxytocin(OXT)and vasopressin(VP)containing neurons,indicating that LPBE projection terminals may from excitatory synapses with the two types of neuro hormone-containing neurons with analgesic effects.(5)We injected r AAV-DIO-h M3Dq-m Cherry or control virus into the LPBE in Vglu T2-Cre mice.After intraperitoneal injection of Clozapine N-oxide(CNO)to active glutamatergic neurons in the LPBE,we observed significantly improved mechanical allodynia and cold allodynia,reduced total distance,central distance and time in the open field test,and increased feeding latency in the novelty suppressed feeding test,as well as the increased immobility time in the forced swim test.These results suggest that activation of glutamatergic neurons in LPBE can cause analgesia,anxiety and depression like behaviors in mice with SNI(6)The anterograde trans-synaptic Cre virus was injected into the LPBE and DIOh M3Dq-m Cherry virus or control virus was injected into the PVN.The results showed that specific activation of LPBE-PVN pathway caused marked amelioration of the mechanical and cold allodynia in SNI mice,but had no obvious effects on animal performance in the open field test,novelty suppressed feeding test,or forced swim test.These results suggest that LPBE-PVN pathway may mainly affect the processing of sensory pain,but had little roles in the modulation of pain-related affection.Conclusion:PVN receives nociceptive inputs from several brain regions,among which the glutamatergic afferent from LPBE is the most important.Activation of LPBE-PVN projection can alleviate nerve injury induced pain behaviors by up-regulating the activity of VP and OXT containing neurons,but had no significant effects on pain-related affections.This study demonstrates the input nociceptive pathway of PVN and reveals the functional significance of LPBE-PVN projection.The results may provide a new insight for the study of pain and analgesia...