Study On Morphological And Electrophysiological Characteristics Of Lamina ? Neurons In Rat Spinal Cord Expressing T-Type Calcium Currents

Objective:T-type calcium channels are low-voltage-activated calcium channels which are a member of voltage-dependent calcium channel family.T-type calcium channels?Cav3?comprise three homologous isoforms?Cav3.1-Cav3.3?and are widely distributed in central and peripheral nervous system including spinal lamina II?also named substantia gelationsa,SG?.SG region is the primary center that integrates the peripheral nociceptive stimulus,mainly includes excitatory and inhibitory interneurons,and closely relates to the transmission and integration of pain information.To better understand the role of Cav3 channels in nociception,we aim to clarify the electrophysiological and morphological characteristics of SG neurons that expressing Cav3 channels in lamina II,which will provide a theoretical basis for revealing the role and mechanism of T-type calcium channels in pain signaling pathways.Methods:First,juvenile male SD rats were used to prepare transversal sections?450?m?for electrophysiology experiments.Whole-cell patch clamp technique was used to record the active membrane properties,passive membrane properties and T-type calcium currents of SG neurons.Using T-type calcium channel blockers,such as nickel chloride,TTA-A2,and mibefradil,to compare the block effect of those drugs on T-type calcium current?I_T?,including the onset time of the drug,the peak time,reversibility,dose-dependent and semi-inhibitory effective concentration(IC₅₀).Next,3-5 weeks SD rats of either gender were selected to prepare 300?m lumbosacral enlargement parasagittal section.For morphological experiments,K⁺-based intracellular solution containing 0.05%neurobiotin 488 was used.After maintaining stable electrophysiological recording for at least 20 min,the slices were transferred to a container filled with 4%PFA at room temperature for 1h and then at 4?overnight for fixation.Neuronal 3D images were reconstructed with a Zeiss LSM 700 confocal microscope.Results:Recording made in transverse spinal cord slices showed that an inward current?I_T?was observed in 44.5%of the SG neurons that was fully blocked by Ni²⁺and TTA-A2.The amplitude of I_T depends on the magnitude and the duration of hyperpolarization pre-pulse.The voltages for eliciting and maximizing I_T were-70mV and-35 mV,respectively.The threshold of action potential?AP?was more negative in neurons expressing T-type current than those lacking it.Consistently,a specific T-type calcium channel blocker TTA-P2 increased the AP threshold and enlarged the difference between AP threshold and resting membrane potential?RMP?.Seven firing patterns recorded in SG neurons of 3-5 weeks old rats include Tonic-firing,Delayed-firing,Single-firing,Initial-burst,Phasic-bursting,Gap-firing and Reluctant-firing.Moreover,we found that most of the I_T-expressing neurons are tonic-firing neurons?76.5%?.In addition,SG neurons can be categorized into various subtypes according to their morphology,including the following five groups:islet,central,vertical,radial,and unclassified.The morphological analysis indicates that most of the I_T-expressing neurons are islet neurons?41.2%?.Conclusions:In conclusion,we identify a cell-type specific distribution and function of T-type calcium channels in SG neurons.These findings may provide new ideas for the study of role of T-type calcium channels in the process of sensory transmission.