Study On Mechanism Of HCN Channels On Regulating Excitatory Inputs Of Spinal Dorsal Substantia Gelatinosa Neurons

Objective:Hyperpolarization-activated cyclic nucleotide-gated(HCN)channels are voltagegated ion channels that are associated with pathological pain.They are widely expressed in central nervous system including spinal dorsal lamina II(also named substantia gelatinosa,SG).SG region is the primary center that integrates the peripheral nociceptive stimulus.During the generation of neuropathic pain,the enhancement of glutamatergic inputs in SG neurons often leads to the hypersensitivity of pain(also caled the central sensitization),which is the main pathophysiological basis of clinical symptoms such as spontaneous pain and alodynia,etc.Here,we examined the distribution of HCN channels in excitatory presynaptic terminals as well as their role in the modulation of synaptic transmission in SG neurons from juvenile animals,which will provide new theoretical basis for the clinical treatment of children’s pathologic pain.Methods:First,juvenile male SD rats were used for immunohistochemistry experiments.The expression of four HCN isoforms in the excitatory synaptic terminals of SG region was observed by using anti-HCN1-4 and anti-VGLUT2(the marker of the excitatory axonal terminals).Next,transverse slices(400-500 mm thickness)of spinal cords of SD rats were prepared in vitro.Using the electrophysiological technique,we recorded the spontaneous or miniature excitatory postsynaptic currents(EPSCs),and the effects of HCN channel blockers(ZD7288 and CsCl)or activator(forskolin,FSK)on the frequency and amplitude of these currents were observed.Finally,GAD67-GFP C57/BL6 transgenic mice were used to identify the green fluorescent protein(GFP)positive(GABAergic neurons)and GFP negative(glutamatergic neurons).The effect of the above HCN channel modulators on sEPSCs in different target cells were further investigated.Results:In immunohistochemistry experiments,we found that HCN1-4 are all expressed in the SG region.Among them,the HCN4 isoform showed the highest level of expression in the excitatory axonal terminals of SG neurons(23 ± 3%),followed by HCN1(15 ± 2%)and HCN2(14 ± 2%).However,HCN3 isoform is mostly located in the intracellular region and is not detected in these terminals.In 53% of the SG neurons examined in SD rats,electrophysiological experiments indicated that the HCN channel blocker,ZD7288(10 mM),decreased the frequency of sEPSCs and mEPSCs to 63 ± 4% and 67 ± 4%,respectively.In contrast,FSK significantly increased the frequency of mEPSCs to 325 ± 34%,which are partially inhibited by ZD7288.Moreover,in the juvenile GAD67-GFP mice,the effects of ZD7288 and FSK on sEPSC frequency were replicated in GFP negative glutamatergic excitatory neurons,but not in GFP positive GABAergic inhibitory neurons.Conclusions:In summary,our results found a novel celular mechanism that underlying the regulation of HCN channels on pain neural circuit in spinal SG.Presynaptic HCN4 isoform modulates the presynaptic release of glutamate,whose postsynaptic targets are excitatory but not inhibitory neurons.