The Role Of Endocannabinoid Dependent Synaptic Plasticity Of Spinal Glycinergic Feed-forward Inhibitory Circuit In The Development Of Neuropathic Allodynia

The pathogenesis of chronic pain has been the research focus of pain medicine for decades.Mechanical allodynia,innocuous stimulus induced pain,is one of the prominent characteristics of chronic pathological pain,and seriously affects patients’physical and mental health and quality of life.The classical Gate Control Theory of Pain has been published for more than 50 years,but the composition and function of the Gate circuit have not been fully elucidated.Our previous study found that there is a“Gate like”feed-forward inhibitory circuit in the spinal dorsal horn,which is formed by glycinergic inhibitory neurons（GlyT2⁺）and PKCγexpressing（PKCγ⁺）excitatory neurons.The Gate open after nerve injury,non-nociceptive information such as touch is uploaded to the pain pathway via PKCγ⁺neurons to induce allodynia,which enriches the gate control theory of pain.Our research group named this“Gate-like”feed-forward inhibitory circuit“Allodynia Gate”.Since the findings were published in the Journal of Clinical Investigation in 2013,some laboratories around the world have used different molecular markers to study neural circuits related to pain in the spinal dorsal horn.The existence of this feed-forward inhibitory circuit is verified and more neural circuits are discovered.Although the neural circuits of the spinal dorsal horn are complex,it is certain that the feed-forward inhibitory circuit is the essential form of the inhibitory circuits.However,the specific mechanism of the reduction of feed-forward inhibition after nerve injury has not been explored.Therefore,the working mechanism of“Allodynia Gate”discovered by our research group is worth further study.It is of great scientific significance in elucidating the neurological mechanism of neuropathic chronic pain and seeking for effective treatment.Synaptic plasticity plays an important role in the development of neuropathic pain.Endocannabinoids（e CBs）,as a typical retrograde neurotransmitter,released from postsynaptic neurons and activated Endocannabinoids-1 receptor（CB1R）on presynaptic neurons,and decreased the release of presynaptic neurotransmitters.The synaptic plasticity mediated by e CBs,known as e CBs-mediated short term depression（STD）and long term depression（LTD）（e CBs-STD/LTD）,plays important roles in learning,memory and pain.Based on the previous research results,we propose the hypothesis of working mechanism of"Allodynia Gate":After peripheral nerve injury,the content of e CBs in the spinal dorsal horn was elevated,and the increased e CBs result in the decrease of glycine release through the activation of CB1R on the axon terminals of GlyT2⁺inhibitory neurons.The feed-forward inhibitory synapses formed between GlyT2⁺neurons and PKCγ⁺neurons express e CB-STD/LTD,and reduce the feed-forward inhibition to PKCγ⁺neurons.Therefore,non-nociceptive information such as tactile sensation is transmitted to the pain pathway via PKCγ⁺neurons,which induces mechanical allodynia.The present project will utilize patch-clamp whole-cell recordings,morphological,molecular biological and behavior techniques to clarify the working mechanisms of“allodynia Gate”and its potential role in the development of neuropathic allodynia induced by peripheral nerve injury in SD rat and 14 strains of cell-specific gene editing mice.The results will provide a structural and functional basis for explaining the neurological mechanism of chronic neuropathic pain and for finding specific drug targets for the treatment of neuropathic pain.Part 1 Changes of e CBs content in spinal dorsal horn after peripheral nerve injuryObjectives:To investigate the changes of e CBs content in the dorsal horn of spinal cord after nerve injury.Methods:Chronic constriction injury（CCI）model was established in rats.The dorsal horn of the spinal cord at the lumbar level was harvested.The changes of e CBs were measured by liquid chromatography-mass spectrometry（lc-ms）.The rats were divided into two groups:Experimental Group（CCI Group）and Control Group（Sham Operation Group）.CCI was performed in the experimental group under isoflurane anesthesia,while the sham operation was performed in the control group under isoflurane anesthesia.The sham operation is identical with experimental group without ligation of the sciatic nerve.After 1d,3d,5d,7d and 14 days,the lumbar enlargement of the spinal cord was taken for the detection of e CBs.Results:The content of AEA increased significantly on the 1st（CCI group vs.sham group,189.0±37.6 vs.56.6±16.9,two-tailed Student’s unpaired t test,P=0.01）and 7th（CCI group vs.sham group,146.5±13.1 vs.72.5±10.4,P=0.001）day after surgery.2-AG（CCI group vs.sham group,18049.3±1919.7 vs.11397.0±433.3,P=0.007）and 1-AG（CCI group vs.sham group,3029.6±773.2 vs.1587.5±147.9,P=0.002）increased significantly on the 7th day after nerve injury.Conclusion:The content of e CBs in the spinal dorsal horn increased on the first day after peripheral nerve injury and reached its peak on the 7th day after injury.At the same time,we also used the neurotransmitter fluorescence probe technology to further verify the changes of e CBs content,the results were consistent with the experiment（un-published data）.The results of this study provided a theoretical foundation for the further study of the role of e CBs in the pathogenesis of allodynia.Part 2 Construction and identification of GlyT2-P2A-i Cre transgenic miceObjectives:Construction of GlyT2-P2A-i Cre mice to specifically label GlyT2⁺neurons or knock out CB1R from GlyT2⁺neurons,to provide tools for the study of the effect of CB1R activation on GlyT2⁺neurons to the feed-forward inhibitory circuit composed of GlyT2⁺neurons and PKCγ⁺neurons（GlyT2⁺-PKCγ⁺inhibitory circuit）.Methods:Using CRISPR/Cas9 technology,GlyT2-P2A-icre mice were constructed and were crossed with ACTB-td Tomato-EGFP and Rosa26 SOR td Tomato（AI9）reporter mice to generate the Glyt2-m EGFP and GlyT2-i Cre-td Tomato mice respectively.The Cre enzyme activity of GlyT2-P2A-i Cre was verified.The basic electrophysiological properties of GlyT2⁺neurons and the types of synaptic transmission mediated by primary afferent fibers were recorded by in vitro patch-clamp technique.The distribution of CB1R on GlyT2⁺neurons was revealed by in situ hybridization.Results:GlyT2-i Cre-td Tomato mice were successfully obtained by hybridizing GlyT2-i Cre with ACTB-td Tomato-EGFP reporter mice.The GlyT2-i Cre-td Tomato labeled neurons were stained with the GlyT2m RNA probe,the percentage of GlyT2m RNA to GlyT2-i Cre-td Tomato was 89.86%（727/809）and GlyT2-i Cre-td Tomato to GlyT2m RNA was 93.11%（784/842）,indicating that the GlyT2-i Cre-td Tomato labeled almost all of the GlyT2⁺neurons.We also found that GlyT2⁺neurons were mainly located in the deep layer of lamina IIi marked by PKCγ⁺lineage neurons.Then the GlyT2-i Cre-td Tomato was co-labeled with CNR1m RNA probe,the percentage of CNR1m RNA to GlyT2-i Cre-td Tomato and GlyT2-i Cre-td Tomato to CNR1m RNA was 46.39%（98/393）and 24.94%（135/291）respectively.It was also found that GlyT2 neurons mainly presented tonic discharge pattern,with Central and Islet dominant morphology,and mainly accepted the excitatory synaptic transmission mediated by primary Ab fibers.Conclusion:The GlyT2-i Cre-td Tomato fluorescent mice were successfully constructed by using CRISPR/CAS9 gene editing technique,which provided a useful tool for further electrophysiological and behavioral studies.The tool mice can label almost all of the GlyT2⁺neurons,greatly improving the efficiency and accuracy of electrophysiological experiments.CNR1m RNA was expressed on 50%of GlyT2⁺neurons,and 70%of GlyT2⁺neurons were innervated by monosynaptic excitatory primary afferent mediated by Ab fibers.This study provides a structural and functional basis for further study of the role of CB1R on GlyT2⁺neurons proposed in the scientific hypothesis.Part 3 The role of e CBs in the development of STD/LTD of the GlyT2⁺-PKCγ⁺feed-forward inhibitory synapses of spinal dorsal hornObjectives:Patch clamp electrophysiological technique was used to reveal the origin,synthesis and release mechanism of e CBs after nerve injury,and how the elevated e CBs affects the GlyT2⁺-PKCγ⁺feed-forward inhibitory circuit and induces synaptic plasticity changes in this circuit（STD and LTD）,and eventually lead to neuropathic allodynia.Methods:（1）In our recent study,we have developed Prkcg-P2A-td Tomato knock-in mice based on CRISPR/Cas9 technology,in which the PKCγ⁺interneurons were genetically labeled by td Tomato fluorescent protein.Patch-clamp whole cell recordings were made from PKCγ⁺fluorescent neurons,dorsal root stimulation was used to evoke synaptic responses on PKCγ⁺neurons.Ab fiber mediated biphasic synaptic responses（Aβ-e EPSP-e IPSP,Gate Model,see our recent publication,Anesthesiology,2020）were selected.The holding potential was held at rest membrane potential（RMP）level.The effects of e CBs（2-AG or JZL195）on burst action potential（AP）,IPSP amplitude,and time course of synaptic response were recorded.（2）The PKCγ⁺neurons receiving Aβmediated EPSP-IPSP or only IPSP synaptic response were selected,and the holding potential was held at the 0m V.Then the amplitude of IPSP were recorded before and after perfusion of the 2-AG or JZL195 to analyze its effect on the feed-forward inhibitory synapses on PKCγ⁺neurons.（3）Using the Cre-loxp system,triple transgenic mice GlyT-CB₁R-KO;Rosa26-td Tomato^mut/+and GlyT-CB1R-WT;Rosa26-td Tomato^mut/+were constructed to knockout the CB1R on GlyT2⁺neurons.Then,the constructed triple transgenic mice GlyT2-CB₁R-KO;Rosa26 td Tomato^mut/+and GlyT2-CB₁R-WT;Rosa26-td Tomato^mut/+were given low-frequency stimulation（LFS）to observe the induction of long-term depression（LTD）.（4）Similarly,the holding potential of PKCγ⁺neuron was held at 0m V,and m Glu R5 antagonist MPEP and Ca²⁺chelator BAPTA were perfused respectively before and after perfusion of JZL195 to observe the effect on IPSP.（5）After clamping GlyT2-i Cre-td Tomato cells,the dorsal root evoked EPSPs mediated by Ab fibers were selected,the membrane potential was clamped to RMP,and the EPSP amplitude was recorded as Control,the effect of JZL195 on the EPSP received by GlyT2⁺neurons was observed.Results:（1）After perfusion of 100μM 2-AG for 10 minutes,31%（4/13）of PKCγ⁺neurons directly fired burst action potentials,while 46%（6/13）neurons only showed IPSP amplitude decreasing and EPSP amplitude increasing.2-AG did not affect the rest of 23%（3/13）neurons.After perfusion of 100μM JZL195 for 15 min,23%（3/13）of PKCγ⁺neurons directly erupted action potentials,62%（8/13）of neurons decreased IPSP and increased EPSP amplitude,while 15%（2/13）of neurons did not respond.In addition,all the effects of the drug can be eluted within 30 minutes.（2）When the membrane potential was held at 0 m V,perfusion of 100μM 2-AG and JZL195 could reduce the IPSP amplitude in a concentration dependent manner and induced the short-term depression（STD）effect,which could be reversed by CB1R antagonists.（3）LFS was given to the dorsal roots of GlyT-CB₁R-KO;PKCγ-td Tomato^mut/+and GlyT-CB1R-WT;PKCγ-td Tomato^mut/+mice.The wild type mice can be induced LTD,while the CB1R-KO mice could not be induced.（4）When the membrane potential was held at 0 m V,m Glu R5 antagonists could significantly reversed the IPSP reduction of JZL195（MPEP vs.JZL195,1.0±0.07%vs.0.70±0.05%,one way ANOVA,P=0.01,n=7）.Perfusion of Ca²⁺chelator BAPTA could not change the IPSP amplitude（BAPTA vs.JZL195,0.79±0.07%vs.0.70±0.05%,one way ANOVA,P=0.29,n=7-9）.（5）When the membrane potential was clamped at resting potential level,100μm JZL195 had no effect on the EPSP recorded on GlyT2⁺neurons（JZL195 vs.Control,1.06±0.09 vs.1.01±0.01,One-way ANOVA,P=0.8,n=6）.Conclusion:Using patch clamp recordings,it was found that e CBs could decrease the amplitude of Ab-e IPSP recorded on PKCγ⁺neurons by activating CB1R on GlyT2⁺neurons.The e CBs induced STD in GlyT2⁺-PKCγ⁺feed-forward inhibitory synapses is concentration-dependent.LFS to the dorsal root（simulate the inputs of nociceptive information）evoked LTD in GlyT2⁺-PKCγ⁺feed-forward inhibitory synapses.Specifically knockout of the CB1R gene on GlyT2⁺neurons blocked the production of LTD.The results showed that e CBs could induce LTD in GlyT2⁺-PKCγ⁺feed-forward inhibitory synapses by activating CB1R on GlyT2⁺neurons,and impaired the function of GlyT2⁺-PKCγ⁺feed-forward inhibitory circuit.In addition,m Glu R on PKCγ⁺neurons participates in the synthesis and release of e CBs in PKCγ⁺neurons,but Ca²⁺does not.e CBs did not affect the Ab primary afferent to GlyT2⁺neurons.These results provided direct electrophysiological evidence for the validation of scientific hypotheses.Part 4 The role of e CB-STD/LTD of feed-forward inhibitory circuit in the development of allodynia induced by nerve injuryObjectives:A serial behavioral tests were used to examine whether nerve injury or intrathecal injection of e CBs induce a cascade event,that is,elevated e CBs,activation of CB1R on GlyT2⁺neurons,decreased inhibitory transmitter release,and impaired the function of GlyT2⁺-PKCγ⁺feed-forward inhibitory circuit,eventually lead to the occurrence of allodynia caused by nerve injury.Methods:（1）SD rats were divided into three groups,the control group（vehicle+CCI group）,6μg NESS0327+CCI group and 60μg NESS0327+CCI group.The CB1R antagonist NESS0327 was injected intrathecally in advance of the CCI model.（2）The rats with positive lidocaine test were divided into four groups.The 1μg,10μg,60μg 2-AG group were intrathecally injected with 2-AG,and the control group was given vehicle.At the3rd,5th,7th and 14th after drug injection,dynamic or punctate mechanical allodynia were tested.NESS0327 was given before intrathecal injection of 1μg 2-AG to observe whether the effect of 2-AG could be reversed.At the same time,the effect of intraperitoneal injection of 2-AG on pain behavior was also observed.（3）AEA was given in the same way as（2）and the effect on pain behavior was observed.（4）Similarly,JZL195 was injected intrathecally and intraperitoneally to observe its effect on pain behavior.Results:（1）After perfusion of 100μM 2-AG for 10 minutes,31%（4/13）of PKCγ⁺neurons directly fired burst action potentials,while 46%（6/13）neurons only showed IPSP amplitude decreasing and EPSP amplitude increasing.2-AG did not affect the rest of 23%（3/13）neurons.After perfusion of 100μM JZL195 for 15 min,23%（3/13）of PKCγ⁺neurons directly erupted action potentials,62%（8/13）of neurons decreased IPSP and increased EPSP amplitude,while 15%（2/13）of neurons did not respond.In addition,all the effects of the drug can be eluted within 30 minutes.（2）When the membrane potential was held at 0 m V,perfusion of 100μM 2-AG and JZL195 could reduce the IPSP amplitude in a concentration dependent manner and induced the short-term depression（STD）effect,which could be reversed by CB1R antagonists.（3）LFS was given to the dorsal roots of GlyT-CB₁R-KO;PKCγ-td Tomato^mut/+and GlyT-CB₁R-WT;PKCγ-td Tomato^mut/+mice.The wild type mice can be induced LTD,while the CB1R-KO mice could not be induced.（4）When the membrane potential was held at 0 m V,m Glu R5 antagonists could significantly reversed the IPSP reduction of JZL195（MPEP vs.JZL195,1.0±0.07%vs.0.70±0.05%,one-way ANOVA,P=0.01,n=7）.Perfusion of Ca²⁺chelator BAPTA could not change the IPSP amplitude（BAPTA vs.JZL195,0.79±0.07%vs.0.70±0.05%,one-way ANOVA,P=0.29,n=7-9）.（5）When the membrane potential was clamped at resting potential level,100μm JZL195 had no effect on the EPSP recorded on GlyT2⁺neurons（JZL195 vs.Control,1.06±0.09 vs.1.01±0.01,One-way ANOVA,P=0.8,n=6）.Conclusion:The results of in vivo behavioral experiments show that the CB1R dependent allodynia（dynamic or punctate）can be induced by intrathecal injection of e CBs or e CBs hydrolase inhibitors.Intrathecal pre-injection of CB1R antagonist can effectively prevent the formation of dynamic or punctate allodynia.These results indicated that the elevated e CBs in the spinal cord after nerve injury activated CB1R receptor and resulted in dynamic and punctate allodynia.Part 5 Specifically ablation of the CB1R of spinal GlyT2⁺neurons alleviate the formation of allodyniaObjectives:In the fourth part of the experiments,the key role of CB1R receptor in the development of allodynia was determined.In this further study,mice in which the CB1R was knock-out（whole body or local）from GlyT2⁺or GABAergic neurons were used to verify whether CB1R on GlyT2⁺neurons or GABAergic neurons is involved in the formation of allodynia.Methods:（1）Using cell-specific knockout method,the CB1R on GlyT2⁺neurons was knocked out and the mice were divided into knockout group（GlyT-CB₁R-KO）and control group（GlyT-CB₁R-WT）.Firstly,the knock-out rate of CB1R on GlyT2⁺neurons of the two groups was revealed,and then the pain behavior under the normal condition was measured.Finally,the pain behavior after nerve injury was measured.（2）Using the virus injection method,the CB1R on GlyT2⁺neurons in the dorsal horn of spinal cord was specifically knocked out locally.The mice were divided into r AAV-sh RNA knockout group and sh RNA control group.Firstly,the knock-out rate of CB1R on GlyT2⁺neurons was tested.Then the pain behavior was measured after nerve injury.（3）Constructing GAD2-CB₁R-KO/WT mice,and the knock-out rate was revealed.The pain behavior in the normal condition and after nerve injury was tested respectively.Results:（1）After ablation of the CB1R on the GlyT2⁺neurons,there was a significant statistical difference in the ablation rate between the knockout group and the control group（GlyT-CB₁R-KO vs.GlyT-CB₁R-WT,27%vs.60%,chi square test,P=0.01）.Before the CCI model,there was no difference in the basic pain behavior between the two groups（GlyT-CB₁R-KO vs.GlyT-CB₁R-WT,two tailed student’s unpaired t test,P>0.05,n=10-12）.On the 3rd,5th,7th and 14th day after CCI,there was significant difference in the paw withdraw threshold and brush score between the two groups（GlyT-CB₁R-KO vs.GlyT-CB₁R-WT,two-way ANOVA,P<0.05）.The punctate and dynamic allodynia were relieved in KO mice.（2）Locallly specific knockout of CB1R on GlyT2⁺could also significantly alleviate the punctate and dynamic allodynia（r AAV-sh RNA（CNRA）vs.sh RNA,two-way ANOVA,P<0.05）.（3）Ablation of CB1R on the GAD2⁺neuron did not affect the punctate and dynamic allodynia（GAD2-CB₁R-KO vs.GAD2-CB₁R-WT,two-way ANOVA,P<0.05）.Conclusion:In the fourth part,the role of CB1R in the development of allodynia was revealed by direct intrathecal injection of e CBs or administration of CB1R antagonists before CCI.In this study,CB1R gene was deleted from GlyT2⁺neurons directly in whole body or in spinal cord using transgenic technique or virus microinjection technique,further demonstrated the key role of CB1R of spinal GlyT2⁺neurons in the formation of neuropathic allodynia induced by nerve injury using behavioral analysis.Overall conclusion:This study used patch-clamp electrophysiology,morphology,molecular biology and behavioral techniques;14 strains of cell-specific gene editing mice were constructed as research platform;the project hypothesis is verified by five series of experiments.After peripheral nerve injury,the content of e CBs in the spinal dorsal horn was elevated,and the increased e CBs result in the decrease of glycine release through the activation of CB1R on the axon terminals of GlyT2⁺inhibitory neurons.The feed-forward inhibitory synapses formed between GlyT2⁺neurons and PKCγ⁺neurons express e CB-STD/LTD,and reduce the feed-forward inhibition to PKCγ⁺neurons.Therefore,non-nociceptive information such as tactile sensation is uploaded to the pain pathway via PKCγ⁺neurons,which induces mechanical allodynia.The present study indicated that the activation of CB1R of spinal GlyT2⁺neurons is the core mechanism of nerve injury induced allodynia,and provides a structural and functional basis for elucidating the neurological mechanism of chronic neuropathic allodynia and for finding specific drug targets for the treatment of neuropathic pain.