The Role Of PKCγ Neurons In The Development Of Neuropathic Allodynia

Neuropathic pain,as one of clinical common refractory chronic pain,has brought great physical and mental sufferings to patients.It is defined as the pain arising as a direct consequence of a lesion or disease affecting the somatosensory system.Allodynia is the most agonizing feature of it,which refers to the sensation of pain caused by non-painful stimuli such as touch.Although many breakthroughs have been made by the basic research of pain mechanisms,there is still a lack of effective clinical treatments for neuropathic pain.PKCγneurons are kinds of excitatory neurons specifically distributed throughout the central nervous system.In 1997,Basbaum first proposed the opinion that PKCγneurons were associated with mechanical allodynia in chronic pain,which had been confirmed by a number of basic studies.However,those researches mainly focused on PKCγneurons in the spinal cord.My research at master stage is mainly about clarifying the fact that the feedforward inhibition circuit composed of PKCγand Gly T2 neurons in the spinal cord is the structural basis of neuropathic allodynia by electrophysiological methods.Now few studies can confirm whether or not the PKCγneurons in the spinal cord are directly regulated by the descending control system,and whether or not the PKCγneurons in the brain are involved in the development of neuropathic allodynia.Therefore,with the help of mutant mice,neural circuit tracing techniques,optogenetics techniques,chemical genetics techniques,and patch clamp electrophysiology techniques,we can specially modulate and record the activity of PKCγneurons in normal or neuropathic pain model mice and observe their relationship with pain behavior,investigate the regulation of pain descending control system on PKCγneurons in spinal cord and its contribution to neuropathic allodynia,and further explore the relationship between PKCγneurons in brain and neuropathic allodynia.Then PKCγneurons would be used as the entry point to reveal and perfect the mechanism of PKCγneurons involved neuropathic allodynia,providing a theoretical basis for the prevention and treatment of neuropathic allodynia.Part I:The modulation of 5-HT control system to PKCγneurons in the spinal cord Objective:To investigate the effect of 5-HT control system on PKCγneurons in spinal cordMethods:1.The PKCγand non-PKCγneurons in spinal cord were all perfused with 5-HT.The changes of membrane potential were recorded underⅠ-clamp.2.The m IPSC of PKCγneurons in the spinal cord were recorded in V-clamp to compare the changes in frequency and amplitude.Immunofluorescence staining was performed on PKCγneurons with biocytin after 5-HT perfusion.Morphological characteristics of PKCγneurons evaluated by sholl analysis were compared with those without 5-HT perfusion.3.The non-PKCγneurons including GABA neurons（GAD2/Vgad-td Tomato mice）and Glycine neurons（Gly T2-td Tomato mice）were recorded in spinal cord,administered with5-HT.The changes of membrane potential were obtained under I-clamp.4.Sixteen adult C57 wild-type mice were selected and divided into sham group and CCI model group on average.On the 10th day after surgery,the spinal dorsal horn tissues were taken out for single-cell sequencing to examine the expression of 5-HT receptor subtypes in GABA and Glycine neurons.5.PKCγneurons in spinal cord were clamped and m IPSC was recorded under V-clamp,perfused with 5-HT and 5-HT_2C receptor antagonists,the frequency and amplitude of m IPSC were recorded.Results:1.The membrane potentials of PKCγneurons inⅠ-clamp had no change before and after 5-HT perfusion,as well as the morphological characteristics.However,the 5-HT significantly reduced the m IPSC frequency of PKCγneurons,with no effect on their amplitude.2.After 5-HT perfusion,nearly half of non-PKCγneurons（GABA neurons and Glycine neurons）changed a lot in membrane potential,with an increase in the absolute value of it,and the spontaneous discharge of neurons disappeared.3.Single-cell sequencing results showed that GABA and Glycine neurons in spinal dorsal horn mainly expressed the 5-HT_2C receptor,which was highly expressed in CCI model mice.The 5-HT_2C receptor antagonist significantly reversed the effect of 5-HT on m IPSC frequency of PKCγneurons.Conclusion:5-HT has a presynaptic regulation effect on PKCγneurons in spinal cord,rather than affecting PKCγneurons directly or changing their morphology.It can stimulate PKCγneurons indirectly by activating 5-HT_2C to reduce the inhibitory interneurons effect,resulting in pain facilitation.Part II:The neural connections between PKCγneurons in the spinal cord and neurons of brain centersObjective:To determine whether brain centers regulate pain through descending control system acting on spinal PKCγneuronsMethods:1.Prkcg-icre mice were injected with retrograde across single synapse tracing virus in spinal dorsal horn,looking for upstream projection sites of PKCγneurons in the spinal cord.2.In order to study the types of upstream neurons of PKCγneurons in spinal cord,neurons in spinal cord were stained with Gly T2 and PV antibody,and neurons in RVM were stained with Vglu T2,GABA,5-HT antibody,and the rates of co-staining were recorded.3.The influences of Vglu T2 neurons in RVM on pain:adult male Prkcg-icre mice were divided into CCI group and sham group.The viruses with h Ch R2 and e Np HR3.0elements were injected into the RVM region.CCI Surgery was carried out 3 weeks later,and the behaviors of the mice would be tested one week after surgery.4.To explore function of the connection between Vglu T2 neurons in RVM and PKCγneurons in spinal cord.The AAV2/9 virus with Ca MKⅡa and h Ch R2 elements was injected into RVM of Prkcg-icre mice（3-week）.Three weeks after virus injection,spinal cord slices were obtained for electrophysiological recording.Results:1.Retrograde tracing virus was successfully expressed in the spinal cord.The labeled neurons were mainly distributed in the deep layer of the spinal cord.In the brain,they mainly located in Gi of RVM,RMC,Pn O,M.We choose RVM as study object for it had the greatest number of labeled neurons.2.In the spinal cord,the co-expressed ratio of labeled neurons and Glycine neurons,PV neurons was 57.69±12.875%and 43.077±6.904%respectively;In the RVM,the co-location ratio of the labeled neurons and Vglu T2 neurons,GABA neurons,5-HT neurons were 56.723±1.765%,33.878±2.144%,23.503±4.692%respectively.Vglu T2 neurons were the most likely the neurons which would project to PKCγneurons in spinal cord.3.When blue light activated fibers of Vglu T2 neurons in spinal cord,the mice showed mechanical and thermal pain hypersensitivity.When yellow light inhibited,the mechanical and thermal pain hypersensitivity of CCI mice were significantly improved.4.When blue light activated fibers of Vglu T2 neurons in spinal cord,the synaptic activity between Vglu T2 neurons in RVM and PKCγneurons in spinal cord was absent,and the electrophysiological properties of PKCγneurons were not changed.Conclusion:The direct projection from Vglu T2 neurons of descending control system to PKCγneurons in spinal cord is hardly found,and the regulation of pain by Vglu T2neurons in RVM is not directly realized by PKCγneurons.PartⅢ:PKCγneurons in ACC are involved in neuropathic allodyniaObjective:To determine whether PKCγneurons in ACC are involved in neuropathic allodynia.Methods:1.8 adult male Prkcg-Tdtomato mice were randomly divided into Sham and CCI model groups with 4 in each group.Immunofluorescence staining was performed 10days after surgery to observe the expression of c-fos in ACC and then to evaluate the activation of PKCγneurons in ACC of neuropathic pain mice.2.Whole-cell patch clamp recording was used to get the basic electrophysiological characteristics,discharge patterns and frequency of PKCγneurons in ACC of normal and neuropathic pain mice to determine the changes of excitability of PKCγneurons in ACC.3.After electrophysiological recordings,the PKCγneurons with biocytin were stained,and the morphological characteristics got by Sholl analysis of the PKCγneurons in Sham and CCI model groups were compared.4.To observe whether the activity of PKCγneurons in ACC influences the pain behavior of mice.Prkcg-icre mice were divided into CCI group and sham group.Viruses with h M3D（Gq）or h M4D（Gi）element were injected into bilateral ACC respectively.The surgery was operated after 3 weeks later,then the behavioral test of mice was carried out one week later before and after CNO at 30min,1h,2h,4h,6h,and 8h.5.To find the neural pathway of PKCγneurons in ACC,anterograde and retrograde tracing virus across single synapse were injected unilateral into ACC of Prkcg-icre mice to look for upstream and downstream projections of PKCγneurons in ACC.Results:1.The expression of c-fos in CCI mice was significantly increased compared with normal mice,and the activation proportion of PKCγneurons in ACC was also increased.2.Compared with normal mice,the rheobase of PKCγneurons in ACC of CCI mice decreased and the firing frequency increased,thus the excitability of neurons increased.3.Morphological characteristics of PKCγneurons in ACC of normal mice were not significantly different from that of CCI mice.4.Activation of PKCγneurons in ACC of normal mice can induce mechanical allodynia and pain-related aversion,whereas inhibition of PKCγneurons in ACC of CCI mice can alleviate that.5.The fibers of neurons in the claustrum nucleus（CL）projected to PKCγneurons of ACC,and the fibers of PKCγneurons can be seen densely distributed in the cerebral peduncle（CP Cerebral Pedunculus）.In view of the fact that CP fibers can descend directly to the spinal cord,it is suggested that PKCγneurons in ACC can directly project to the spinal cord.Conclusion:During neuropathic pain,PKCγneurons in ACC can be largely activated,and participate in influencing pain-related mechanical and emotional behavior.The PKCγneurons involved CL-ACC-CP pathway may be the structural basis of neuropathic pain.