The Specific Distribution Of Hyperpolarization-activated Cyclic Nucleotide-gated Cation(HCN)channels In The Dorsal Horn Of Rat Spinal Cord

Objective:There are four isoforms of Hyperpolarization-activated cyclic nucleotide-gated cation(HCN)channels in mammalian cells,which are mainly expressed on the cell membrane of nerve cells and cardiomyocytes.HCN channels are strongly associated with the development of epilepsy,depression,pain as well as cardiac disorders.The spinal dorsal horn(SDH)is a primary portal for the central transmission of peripheral nociceptive information,and HCN channels in the superficial layer of SDH have an important role in the development and progression of chronic pain.Although studies have shown that HCN channels are widely expressed in the superficial layer of SDH,the specific expression profile of HCN channels in neurons and glial cells,and even their distribution in neuronal subcellular structures is still unknown.Therefore,we observed the specific distribution of the four subtypes of HCN channels in the SDH of SD rats,in order to provide a theoretical basis that reveal the role of HCN channels of SDH in the pain signaling pathway.Methods:Healthy sprague dawley(SD)rats aged 3-5 weeks were randomly selected,which contained both female and male.Rats were anesthetized with an intraperitoneal bolus of urethane and transcardially perfused with 100 ml of 0.9% Na Cl and 150 ml of 4% paraformaldehyde(PFA).L4-L5 spinal cord segments were left for immunofluorescence(IHC)staining.All spinal cord slices were observed under a confocal microscope and analyzed with Zen software.First,spinal cord slices were immunochemically stained with antibodies specific for the marker HCN1-4 to observe the gross distribution of HCN channels in the dorsal horn of the spinal cord;Cells were then stained with HCN1-4 antibodies in combination with the neuronal marker neuronal nuclei(Neu N)and the astrocyte marker glial fibrillary acidic protein(GFAP)as well as with a microglial specific marker(anti integrinαM[CD11b] antibody,OX-42)were co-stained to observe the distribution of HCN channels on neurons and glial cells in the spinal dorsal horn;Finally,HCN1-4 antibodies were used in conjunction with antibodies against calcitonin gene-related peptide(CGRP),Isolectin B4(IB4),a marker of interneuron dendrites,microtubule associated protein 2(MAP2)and the axonal marker axonal neurofilament(Pan axonal neurofilament,SMI-312)as well as the interneuron axonal terminal marker vesicle γ aminobutyric acid transporter(VGAT)was co-stained to observe the distribution of HCN channels on the neuronal subcellular of spinal dorsal horn.Results:1.Four subtypes of HCN channel were widely distributed throughout the spinal cord gray matter,and HCN2-4 were mainly concentrated in laminae I-II of the spinal dorsal horn.In addition,HCN1-4 were faintly expressed in spinal cord white matter;2.Distribution of HCN channels on neurons and glia in the dorsal horn of the spinal cord: co-expression with the astrocyte marker GFAP was mainly HCN1(5.2±0.8),followed by HCN4(3.0±0.5),HCN2(2.3±0.4)and HCN3(0.4±0.1);Co-expression with the microglial marker OX-42 increased from high to low levels for HCN1(3.5±0.6)>HCN4(1.6±0.2)>HCN2(1.3±0.2)>HCN3(0.4±0.1);Co-staining with the neuronal marker Neu N was predominant for HCN2 and HCN3 and ranged from high to low for HCN3(64.7±3.1)>HCN2(45.6±5.0)>HCN1(12.6±2.9)>HCN4(2.2±0.5).HCN1 was mainly distributed on astrocytes and microglia,HCN2 and HCN3 were mainly distributed on neurons.3.Distribution of HCN channels on primary afferent nerve terminals in the spinal dorsal horn: co-expression with CGRP,a marker of peptidergic primary afferent terminals,was mainly HCN2(6.3±0.7),followed by HCN1(2.8±0.5)and HCN4(2.5±0.3).HCN3(0.7±0.1)was less co-expressed with CGRP.Co-expression with IB4,a marker of non peptidergic primary afferent nerve terminals,was mainly HCN4(9.5±0.2).HCN1(1.9±0.3),HCN2(0.8±0.1)and HCN3(0.2±0.1)were less co-expressed with IB4.HCN2 and HCN4 were the predominant types distributed on nociceptive primary afferent terminals;4.Distribution of HCN channels in dendrites and axons of interneurons in the dorsal horn of the spinal cord.HCN1 and HCN4 were mainly co-expressed with the neuronal dendritic marker MAP2,and the co-expression degree from high to low was HCN4(8.9±0.7)>HCN1(7.4±0.7)>HCN2(2.9±0.3)>HCN3(0.6±0.0).Neuronal axon markers SMI-312 and HCN1-4 were less co-expressed,HCN1(2.0±0.3),HCN2(1.5±0.3),HCN3(0.1±0.0),and HCN4(1.1±0.2),respectively.HCN1 and HCN4 were the predominant isoforms distributed in dendrites,and HCN1-4 expression were significantly higher in dendrites than in axons;5.Distribution of HCN channels at the axonal terminals of spinal dorsal horn interneurons: co-expression with VGAT,a marker for axonal terminals of inhibitory interneurons,was mainly HCN4(14.7±3.2),whereas HCN1(1.5±0.2),HCN2(0.6±0.1)and HCN3(0.3±0.0)were less co-expressed with VGAT.HCN4 is the predominant isoform distributed in axon terminals of inhibitory interneurons.Conclusions:HCN channels are specifically distributed in SDH neurons,glia and neuronal subcellular structures.These specific distribution characteristics of HCN channels are important for studying their specific cellular and molecular mechanisms underlying disease-related pathophysiology.