The Key Role Of PKC? Interneurons In Spinal "Allodynia-gate" Circuits

Neuropathic pain(NP),such as postherpetic neuralgia and trigeminal neuralgia,is considered to be a chronic disease,and is difficult to cure.It not only seriously endangers human physical and mental health and quality of life,but also leads to a global economic burden.Allodynia,also known as"touch-evoked pain",caused by innocuous stimulation,is a typical symptom of NP.Pain can be induced by daily activities such as dressing and washing,and has become the biggest trouble of patients with NP,so it has important clinical significance to cognize the neural circuits underlying allodynia.The spinal dorsal horn is an important primary center for the integration and sensitization of pain information[1].The neural circuits in dorsal horn are composed of excitatory and inhibitory interneurons and projection neurons.The synaptic plasticity changes of the circuits plays a significant role in the development of chronic pain[2].The interaction between tactile and nocuous information in the spinal cord dorsal horn has been an intense research focus.PKC?neurons,existed only in the central nervous system,are important interneurons in the superficial dorsal horn(SDH)and mainly distributed in the lamina?_i,where mainly accepts inputs from low threshold afferents.In an article published in Science in 1997,it was first proposed that PKC?neurons are associated with mechanical allodynia[10].More recently,a series of studies reported that PKC?neurons were involved in the formation of inflammatory pain and NP[4,5,6,7].Our recent study indicated that PKC?interneurons plays an important role in the“allodynia gate circuit”of the spinal dorsal horn,and the opening of the gate is an important mechanism to realize the transformation of tactile information to pain pathway[8].In this study,we studied the distribution of PKC?neurons in the central nervous system,the basic electrophysiological and morphological properties of spinal PKC?neurons and the types of primary nerve fibers projected to them by using the Prkcg-P2A-Tdtomato fluorescent labeled mice.We further elucidated the role of endocannabinoids in the opening of"Allodynia Gate"[9].The present study will provide a structural and functional basis for finding the drug target for NP treatment.Part 1:The construction and verification of Prkcg-P2A-Tdtomato fluorescent labeled miceObjective:To construct Prkcg-P2A-Tdtomato fluorescent labeled mice and to determine whether the fluorescent labeled mice were successfully made by verifying co-expression of PKC?and fluorescent protein and the distribution of PKC?~+neurons in the central nervous system.Methods:Crispar-cas 9 technique was used to make P2A guided Tdtomato gene knock-in mice;the tail tissue of mice was used to obtain DNA;the genotype was identified by PCR and Southern blot technique;the spinal cord and brain was taken to make frozen slices.The overlay rate of PKC?immunoreactivity and Tdtomato was carried out by immunofluorescence technique.The overlay rates of PKC?and GluR,Gad and GlyT2were obtained by in situ hybridization.A continuous observation of the brain and spinal cord is taken directly by fluoroscopy,and the distribution of PKC?~+neurons in the central nervous system is determined by comparing to mouse atlas(fourth edition).Results:Prkcg-P2A-Tdtomato fluorescence labeled mice were successfully constructed,the inserted gene sequence was expressed stably,the labeling efficiency of Tdtomato to PKC?was more than 95%.The co-expression rate of PKC?and GluR was more than 87%,and there was only a small number of co-expressions with Gad and GlyT2.In conclusion,the spinal PKC?~+neurons mainly belong to excitatory neurons.PKC?~+neurons are widely distributed in the central nervous system,mainly in the hippocampus CA1,CA2 and CA3regions,amygdala,pear cortex,cerebellum lobe and cerebellum nucleus,lamina?i of spinal cord dorsal horn and other functional areas of central nervous system.Discussion:The Prkcg-P2A-Tdtomato fluorescent tool mice successfully labeled PKC?~+neurons,greatly improved the efficiency and accuracy of patch clamp recordings.The PKC?~+neurons are widely distributed in the central nervous system,especially in the hippocampus,amygdala,dorsal angle of the spinal cord lamina?i,indicating that it has a potential connection with learning,memory,emotional regulation and sensory introduction,providing reference and guarantee for the subsequent study concerning neurological functions.Part 2:The basic characteristics of PKC?+neurons in spinal cord were studied by fluorescent labeled mice,which provided a structural basis for further study of the mechanism of allodynia.Objective:To determine the type of receiving nerve fibers,the basic electrophysiological and morphological characteristics of the PKC?~+and PKC?~-neurons by evoked postsynaptic activities.The role of PKC?~+neurons in the“allodynia gate”was also discussed.Methods:Prkcg-P2A-Tdtomato fluorescent labeled mice(3-5 weeks)were used.In a separate experiment,the compound action potentials(AP)of dorsal roots were recorded to determine the stimulation threshold of different types of afferent nerve fibers.The sagittal spinal cord slices with dorsal roots attached were prepared for whole-cell patch clamp recording.The positive and negative membrane prosperities,the evoked excitatory synaptic potentials(eEPSPs)and inhibitory synaptic potentials(eIPSPs)induced by the root stimulation of were recorded in PKC?~+and PKC?~-neurons.The types of fibers received by neurons were determined by stimulation thresholds of different types of nerve fibers.Bicuculline and strychnine(GABA and glycine receptor antagonists)were used to judge the inhibitory components of evoked synaptic responses.The morphology of PKC?~+and PKC?~-neurons was determined by fluorescence staining of the recorded neurons with biocytin injection.Results:(1)The rheobase,threshold and resting membrane potential of PKC?~+neurons are higher than those of PKC?~-neurons,and both types of neurons showed multiple firing patterns.The PKC?~+neurons are dominated by phasic and initial discharge patterns,while the PKC?~-neurons are mainly tonic and delay types.(2)The stimulation intensity threshold of eEPSP induced by A?fiber is 0.1-0.3V,and the conduction velocity(CV)is 3.33-4.54 m/s;the threshold of A?fiber is 0.4-1V,and the CV is 1.43-1.79 m/s;the stimulation threshold of C fiber is 1.4-6V,and the CV is 0.49-0.98m/s.(3)The PKC?~+neurons mainly receive A?fiber mediated biphasic eEPSP-eIPSP inputs,and a small number of neurons receive A?and C fiber inputs.PKC?~-neurons mainly accepted A?and C fiber mediated excitatory inputs.(4)There are four morphological types(central,island,radiant,vertical)in both types of neurons,PKC?~+neurons are mainly central type.Discussion:The discharge patterns and morphology of PKC?~+neurons in the spinal dorsal horn have much in common with the PKC?~-neurons,suggesting that it is difficult to accurately judge the category of neurons based solely on discharge patterns and neuronal morphology.The main difference between the two types of neurons is the type of primary afferent nerve fibers.PKC?~+neurons mainly receive low threshold innocuous information mediated by A?fibers.However,under normal conditions,PKC?~+neurons is regulated by feedforward inhibition of inhibitory interneurons,which is not easy to be excited,thus constituting the basic circuit structure of"allodynia gate".Part 3:The PKC?~+neurons in the spinal dorsal horn is involved in the formation of allodyniaObjective:To explore the changes of excitability of PKC?~+neurons in dorsal horn mediated by eCB,as one of the mechanisms involved in the formation of allodynia.Methods:Prkcg-P2A-Tdtomato fluorescent labeled mice(3 weeks)were selected.Firstly,the changes of electrophysiological characteristics,such as resting membrane potential,rheobase,threshold,and the amplitude of evoked inhibitory synaptic potential(eIPSPs)induced by A?stimulation were observed after the formation of NP,and compared with that of normal mice.The same assessment indexes were compared in mice given 2-AG intrathecal injection.The effect of endocannabinoid AEA and CB1 receptor antagonists(AM251)perfusion on eIPSPs of PKC?~+neurons was recorded by selecting the normal Prkcg-P2A-Tdtomato fluorescent labeled mice at 3-5 weeks of birth.The long-term depression(LTD)of eIPSPs was induced by low frequency electrical stimulation(LFS),and the effect of pre-perfusion of CB1 receptor antagonist(NESS0327)on LTD was observed.Then the effect of endocannabinoids on the short term and long-term changes of eIPSPs of PKC?~+neurons was evaluated.The GlyT2-CB1-KO mice(3-5 weeks)were selected to record the effect of AEA perfusion on the eIPSPs of PKC?~+neurons.The LTD of eIPSPs induced by LFS was evaluated in GlyT2-CB1-KO mice.Results:(1)Compared with normal mice,the proportion of PKC?~+neurons with exploding AP increased,the resting membrane potential,the rheobase,the threshold and the amplitude of eIPSPs of PKC?~+neurons decreased after the formation of NP,which indicated that the excitability of PKC?~+neurons increased after the NP.(2)Compared with normal mice,the proportion of PKC?~+neurons with exploding AP was increased,the rheobase,the threshold,the resting membrane potential and the amplitude of eIPSPs of PKC?~+neurons decreased after intrathecal injection of endocannabinoid2-AG,which indicated that the endocannabinoids increased the excitability of PKC?~+neurons,which was similar to the results after CCI molding.(3)The perfusion of endocannabinoids AEA in vitro spinal cord slices can reduce the amplitude of eIPSPs of PKC?~+neurons mediated by A?fiber,which is similar with the changes of PKC?~+neurons after the formation of NP.This process can be reversed by AM251,and partially reversed by knock-out the CB1 receptor on the glycine neurons.This result informed that the activation of CB1 receptors in glycine neurons might be involved in the occurrence of allodynia.(4)LFS can lead to LTD of A?induced eIPSPs in PKC?~+neurons,which can be blocked by NESS0327.Knockout the CB1 receptors on glycine neurons can also prevent the occurrence of LTD,indicating that the endocannabinoids can participate in the LTD occurrence of eIPSPs in PKC?~+neurons.Discussion:Both allodynia formation and 2-AG intrathecal injection can enhance the excitability of PKC?~+neurons in the spinal dorsal horn.Pharmacological inhibition or knockout of CB1 receptors can antagonize the inhibitory effect of endogenous cannabinoids on eIPSPs,implying that the endocannabinoids are involved in the occurrence of allodynia associated with PKC?~+neurons.Summary:(1)The Prkcg-P2A-Tdtomato fluorescent labeled mice were successfully constructed.The PKC?~+neurons in spinal cord were labeled efficiently,which provided a powerful tool to study the role of PKC?~+neurons in the development of mechanical allodynia.(2)PKC?~+neurons in the spinal cord had lower excitability,mainly received low-threshold inputs,and were not easy to burst action potentials under normal physiological conditions.It constitutes the structural foundation of“allodynia gate”.(3)The excitability of PKC?~+neurons was enhanced after CCI induced neuropathic pain or intrathecal administration of eCB,indicating that PKC?~+neurons play an important role in the formation of mechanical allodynia.By decreasing the amplitude of eIPSPs induced by A?fiber in PKC?~+neurons,eCB can participate in the formation of LTD induced by LFS to enhance the excitability of neurons,which provides an important functional basis for further elucidating the occurrence and maintenance mechanism of neuropathic pain.