The Role And Mechanism Of Cannabinoid 1 Receptors In The Ventrolateral Periaqueductal Gray In Mediating Sex-specific Analgesia In Mice

The prevalence of chronic pain is rapidly increasing,which seriously endangers the physical and mental health of people.Although many drugs have been developed to treat pain,the clinical efficacy was still not satisfactory and gender has become an unavoidable factor.Sex difference in analgesic effects has gradually attracted public attention in clinical and pre-clinical studies.At all times and in all over the world,cannabinoid has made breakthrough progress in the treatment of pain.Among them,chronic pain is the most common indication for the application of cannabis in states with medical cannabinoid projects in the United States.Females are more sensitive to cannabinoid antinociception than males in both animals and humans.However,the mechanism of sex-specific analgesia of cannabinoid is still unknown.Cannabinoid 1 receptor（CB₁R）is the most important receptor of exogenous and endogenous cannabinoid,which is widely distributed in the central nervous system.Periaqueductal gray（PAG）,its descending projection to RVM and spinal dorsal horn（PAG-RVM-SDH）circuit constitutes the most classic descending pain regulation system and is the key pathway of endogenous analgesia system.As the key node of this descending inhibitory integration,ventrolateral periaqueductal gray（vlPAG）plays a vital role in the endogenous analgesia of cannabinoid through activating CB₁R.In addition,vlPAG is also regulated by the important brain regions such as upstream medial prefrontal cortex（mPFC）.Although the function of the endocannabinoid system has been explored in central nervous system in both male and female mice.The CB₁R in the vlPAG and mPFC has been demonstrated to participate in antinociceptive effect.However,it is still unclear whether there are sex differences of cannabinoid analgesia and cell type-and sex-specific patterns of vlPAG and mPFC CB₁R expression which affect analgesia.In the current study,we firstly used pain behavior test,pharmacology,immunohistochemistry and fluorescence in situ hybridization（FISH）to detect whether there exists sex difference of pain behavior after CCI,there is sex difference in regulating CB₁R in vlPAG and there are cell type-and sex-specific patterns of vlPAG CB₁R expressions.Secondly,we used transgenic mice combined with virus injection and chemogenetic techniques to verify the cell type-and sex-specific patterns of vlPAG CB₁R expression from behavioral aspect.Then,we used the virus tracing combined with optogenetics to detect the sex difference of CB₁R expression from the synapse aspect.Finally,we used the similar method to detect whether there is sex difference of CB₁R on mPFC-vlPAG pathway.Revealing sex differences in cannabinoid-mediated analgesia can provide a structural and functional basis for exploring more specific therapeutic approaches for chronic pain in females.Part 1 Modulation of CB₁R within vlPAG produces sex difference of pain in mice Objectives:Sex difference in analgesic effects of analgesic drugs has gradually attracted public attention in pre-clinical and clinical studies.Females are more sensitive to cannabinoid than males in both animals and humans.Expression of the CB₁R and the function of the endocannabinoid system in brain of mice have been explored,as well as CB₁R in the vlPAG participated in antinociception.However,whether there is sex difference in female and male mice is not known.The aim of this part of the study was to investigate the role of CB₁R in the vlPAG region for pain and the differences between males and females using the pain behavior test,microinjection method,immunohistochemistry and FISH.Methods:1.The paw withdraw mechanical threshold（PWMT）,dynamic score and paw withdrawal thermal latency（PWTL）of wide-type（WT）mice were tested using the von Frey,brush and hargreaves respectively at the normal condition and after CCI.2.Microinjection of CB₁R-specific agonist ACEA or CB₁R antagonist NESS0327 into the vlPAG brain region of male and female mice to observe the effects of modulating cannabinoid receptor 1（CB₁R）.3.Immunohistochemistry and FISH were conducted to detect CB₁R expression on inhibitory neurons（GABAergic neurons）as well as excitatory neurons（Ca MKIIα⁺neurons）in the vlPAG region.Results:1.After CCI surgery,mechanical allodynia and heat hyperalgesia was induced in both female and male mice,however there was no significant difference between them.2.Activation of CB₁R through injecting CB₁R agonist ACEA into the vlPAG attenuated the mechanical allodynia induced by CCI to a greater extent compared with the Vehicle group.This effect was more pronounced than that of males,with a statistically significant difference（p<0.05,n=8）.Administration of the CB₁R antagonist NESS0327 in the vlPAG region of mice revealed that female mice also showed more severe mechanical allodynia than males（p<0.05,n=8）.3.Besides,much more CB₁R were found by immunohistochemistry on GABAergic neurons in the vlPAG in female mice compared to male mice（females vs.males,p<0.001,n=4）.The colocalization of Gad2-td Tomato positive neurons with the CB₁R m RNA probe also showed that females had significantly more CB₁R-m RNA in the vlPAG compared to males（female vs.male,p<0.001,n=4）.More than 80%Ca MKIIα⁺neurons were co-localized with CB₁R,with no significant statistically difference（female vs.p=0.1292,n=4）.Conclusion:These results suggest that there were no significant differences between male and female mice in normal and neuropathic pain condition in WT mice and that CB₁R in vlPAG are likely to be involved in sex differences in cannabinoid-induced analgesia.Part 2 GABAergic CB₁R within vlPAG mediated sex difference of analgesia in mice Objectives:The above results preliminarily suggested that sex-specific expression of GABAergic CB₁R may contribute to sex difference of cannabinoid mediated analgesia.In this part,transgenic combined with virus and chemogenetic technique were used to reveal the function of sex-specific CB₁R expression and observe whether this expression contributes to sex difference of cannabinoid mediated analgesia in vivo.Methods:1.Homogenous CB₁R-flox mice were generated and the genotypes were identified by PCR method.2.According to the Cre-lox P system,the r AAV-Dlx5/6-mCherry or r AAV-Dlx5/6-Cre-mCherry virus was injected into the vlPAG of CB₁R-flox mice to specifically label the GABAergic neuron or knockout GABAergic CB₁R in the vlPAG.Three weeks later,the specificity of dlx5/6 promoter was tested and GABAergic CB₁R were demonstrated successfully deleted.Then the rotarod and pain behavioral test were performed followed by CCI model.Finally,the pain behavior after CCI was measured.3.According to the Cre-lox P system,the r AAV-Ca MKIIα-mCherry or r AAV-Ca MKIIα-Cre-mCherry virus was injected into the vlPAG of CB₁R-flox mice to label the glutamatergic neuron or specifically delete CB₁R of Ca MKIIα⁺neuron in the vlPAG.Three weeks later,the specificity of Ca MKIIαpromoter was tested and the Ca MKIIα⁺CB₁R were demonstrated successfully deleted.Then the rotarod and pain behavioral test were performed followed by CCI model.Finally,the pain behavior after CCI was tested.4.Finally,GABA-dependent h M4Di（r AAV-Dlx5/6-h M4D（Gi）-mCherry-WPREs,AAV 2/9）or control virus（r AAV-Dlx5/6-mCherry-WPRE-h GH p A,AAV2/9）was injected into the vlPAG of adult male and female wide type mice.Three weeks later,expression patterns of the DREADD vector were observed,followed by CCI and pain behavior test.Results:1.The homogenous CB₁R-flox mice were successfully generated.2.After injecting r AAV-Dlx5/6-mCherry or r AAV-Dlx5/6-Cre-mCherry virus into vlPAG of CB₁R-flox mice,results demonstrated that 68.0±1.2%dlx5/6-mCherry neurons overlay with GAD2m RNA probe and 67.0±0.8%GAD2m RNA positive neurons colocalize with dlx5/6-mCherry positive neurons.Then 46.53%and 57.63%GABAergic CB₁R in female and male mice were deleted respectively,which did not influence sensorimotor coordination in females（Dlx5/6-cre-mCherry vs.Dlx5/6-mCherry,p=0.96,n=10）or in males（Dlx5/6-cre-mCherry vs.Dlx5/6-mCherry,p=0.88,n=10）,and there was no sex difference.Besides,it did not affect the punctate mechanical allodynia,dynamic mechanical allodynia and thermal hyperalgesia.However,in the Dlx5/6-Cre-mCherry group,which had lower expression of GABAergic CB₁R in the vlPAG,females had greater punctate mechanical allodynia than males at 5 days（female vs.male,p=0.0439,n=8）,10 days（female vs.male,p=0.0395,n=8）,and 14 days（female vs.male,p=0.0238,n=8）after CCI surgery.Besides,females had greater dynamic mechanical allodynia than males at 10 days（female vs.male,p<0.01）and 14 days（p<0.05）.3.After injecting r AAV-Ca MKIIα-mCherry or r AAV-Ca MKIIα-Cre-mCherry virus into vlPAG of CB₁R-flox mice,92.0±0.6%Ca MKIIα-mCherry overly with v Glut2m RNA probe and 89.0±0.9%v Glut2m RNA positive neurons colocalize with Ca MKIIα-mCherry positive neurons.Then,53.7%and 38.17%Ca MKIIα⁺CB₁R in female and male mice were deleted respectively,which did not affect sensorimotor coordination in both females（p=0.8340,n=10）and males（p=0.6379,n=10）.Under chronic pain conditions,CB₁R on Ca MKIIα⁺neuron did not contribute to analgesia in females（PWMT:p=0.5813;Dynamic score:p=0.7044,n=8）or males（PMWT:p=0.0952;Dynamic score:p=0.7991,n=8）.4.Ten days after CCI surgery,we acutely inhibited GABA^vlPAG via systemic administration of clozapine-N-oxide（CNO）.We observed that this CNO-dependent inhibition of vlPAG GABAergic neurons led to higher PWMT threshold in the filament mechanical stimuli test and lower dynamic scores to brush stimuli compared with mCherry controls after CNO administration in both females（PWMT:p<0.001;Dynamic score:p<0.001,n=6）and males（CNO vs.Vehicle,PWMT:p<0.001;Dynamic score:p<0.001,n=6）.No sex differences were found in both groups（female CNO vs.male CNO,PWMT:p=0.0763;Dynamic score:p=0.5591,n=6）.Similarly,21 days after CCI surgery,chemogenetic inhibition of GABAergic neurons also led to amelioration of mechanical allodynia in females（PWMT:p<0.0001;Dynamic score:p<0.0001,n=5）and males（PWMT:p<0.0001;Dynamic score:p<0.0001,n=5）compared with the control groups.No sex difference was found（PWMT:p=0.4273;Dynamic score:p=0.5317,n=6）.Conclusion:Specific knockout of CB₁R on GABAergic neuron not glutamatergic CB₁R in vlPAG produced more severe mechanical allodynia in female mice and ruled out the possibility that GABAergic neurons themselves in female and male mice mediating cannabinoid-induced sex difference in analgesia.These findings suggest that sex specific GABAergic CB₁R expression may play a key role in mediating cannabinoid-induced sex difference in analgesia.Part 3 Activation of CB₁R results in sex difference on synaptic transmission ofGABA-v Glut2 inhibitory circuit in miceObjectives:The previous results confirmed that the sex specific GABAergic CB₁R expression through the perspective of behavior and morphology.In this part,transgenic technology,electrophysiology combined with optogenetic and pharmacology method were used to modulate the presynaptic GABAergic CB₁R and explore whether the above CB₁R-dependent sex difference in behavior was due to difference in CB₁R-mediated disinhibition of the GABA-v Glut2 inhibitory circuit,which ultimately participated in the analgesic effects of cannabinoids elicited by CB₁R activation.Methods:1.Firstly,v Glut2-Cre were crossed with Ai9 reporter mice to generate v Glut2-td Tomato mice.Genotype was identified with PCR and the heterozygote was screened for experiment.2.Secondly,in vitro vlPAG slice of v Glut2-td Tomato mice was prepared and v Glut2⁺neurons in vlPAG were patched.The spontaneous inhibitory postsynaptic currents（s IPSC）received by v Glut2⁺neurons were recorded before and after perfusion of CB₁R agonist ACEA.3.Finally,3-4 weeks Gad2-Cre mice were selected and the vlPAG was injected with a mixture of r AAV-DIO-Ch R2-EGFP and r AAV-Ca MKIIα-mCherry viruses（1:1）.After 3weeks,vlPAG brain slices expressing the mixture were prepared and Ca MKIIα-mCherry neurons were pathed.The opto-evoked inhibitory postsynaptic currents（o IPSC）and（opto-evoked excitatory postsynaptic currents,o EPSC）received by Ca MKIIα-mCherry neurons was recorded before and after perfusion of ACEA.Results:1.The v Glut2-td Tomato mice were successfully constructed and screened for homozygous using PCR method.2.The CB₁R agonist ACEA（10μM）significantly reduced the frequency of s IPSCs by 74.08%in females（Control vs.ACEA,p=0.0003,n=6）.The inhibitory input to td Tomato-positive（v Glut2⁺）neurons was less sensitive to ACEA in males,with an average reduction of only42.98%（Control vs.ACEA,p=0.0187,n=6）.The effect of ACEA on s IPSC frequency produced a large statistically significant difference between males and females（female vs.male,p=0.0147,n=6）.In contrast,application of the CB₁R agonist ACEA did not affect the amplitude of s IPSCs recorded on the v Glut2⁺positive neurons and there was no difference between male and female mice.3.The application of ACEA significantly reduced the amplitude of o IPSC compared to controls in females（Control vs.ACEA,p<0.001 n=6）and males（Control vs.ACEA,p<0.01,n=6）.There was no difference in the amplitude of o IPSC between female and male mice before perfusion of the ACEA.Activation of CB₁R induced more robust reduction of amplitude of o IPSC in the females than in males（p<0.001,n=6）.Conclusion:Activation of CB₁R in the GABA-v Glut2 inhibitory circuit within vlPAG resulted in v Glut2-positive neurons receiving less GABAergic inhibitory components and being relatively more excitable.Hence,the sex-specific CB₁R expression on GABAergic neuron within vlPAG did produce more pronounced presynaptic inhibition in females,resulting in v Glut2⁺output neurons within vlPAG more excitable and thus producing a more potent analgesic effect.Part 4 Sex-specific effects of CB₁R activation in Ca MKIIα^mPFC-GABA^vlPAGcircuit on the synaptic transmission efficiency of GABAergic neurons within vlPAGObjectives:The above study revealed modulation of CB₁R on the presynaptic GABAergic neuron terminal in GABA-v Glut2 feedforward inhibitory circuit in vlPAG produced stronger analgesia in females.However,whether the GABAergic neurons,as the core upstream of v Glut2⁺projection neurons,are affected by the long projection of other brain regions is worthy of further investigation in this part experiment.Methods:1.Firstly,r AAV-Ef1α-DIO-EGFP-TVA and r AAV-Ef1α-DIO-RVG（1:1 mixture）were injected into the vlPAG region of Gad2-cre mice.Three weeks later,RV-ENVA-ΔG-Ds Red virus were re-injected into the vlPAG region,followed by frozen sections being observed for tracing one week later.2.The brain slices from mPFC area were stained with GABA（anti-mouse）or Ca MKIIα（anti-mouse）antibody.The co-labeling with retrograde tracer-labeled neurons was observed and statistically analyzed.3.The brain slices from mPFC area were stained with Ca MKIIα（anti-mouse）and CB₁R（anti-goat）,and the co-labeling of Ca MKIIαwith CB₁R was observed and statistical analysis was performed.4.The r AAV-Ca MKIIα-Cre-mCherry virus was injected into mPFC region of CB₁R-flox mice to specifically knock out the CB₁R on the Ca MKIIα⁺neuron.Three weeks later,baseline of the pain behavior was tested at the normal condition.Then,CCI surgery was performed and pain behaviors were measured at 1,3,5,7,10,14,and 21 days after CCI.5.The r AAV2/R-h Syn-FLP-EGFP and r AAV2/9-h Syn-f DIO-Cre-mCherry virus were injected into vlPAG and mPFC region of WT or CB₁R-flox mice respectively to label Ca MKIIα⁺neurons or ablate the CB₁R on the Ca MKIIα⁺neurons.Three weeks later,baseline of the pain behavior was tested at the normal condition.Then,CCI surgery was performed and pain behaviors were measured at 1,3,5,7,10,14,and 21 days after CCI.6.The r AAV-Ca MKIIα-Ch R2-EYFP virus was injected into the mPFC region of Gad2-Cre-td Tomato,and virus expression was verified after 3 weeks by immunohistochemistry.The mPFC as well as vlPAG brain slices expressing the virus were prepared after successful virus transfection.The Ca MKIIα-EYFP neurons were clamped in the mPFC and action potential was recorded after the optical light to verify the virus function normally.Then the o IPSC and o ESPC were recorded on the Gad2-Cre-td Tomato in the vlPAG.Results:1.RV-Ds Red-labeled neurons were observed in several brain regions,such as Ce M,mPFC,M1 and M2,in both female and male mice,with no sex difference between them.2.RV-Ds Red positive neurons within the mPFC were mainly co-labeled with Ca MKIIαbut not GABA.Besides,RV-Ds Red was co-labeled with Ca MKIIαas well as v Glut1.3.In the normal condition,specifically knocking out CB₁R on all Ca MKIIαneuron or the Ca MKIIαneuron projecting to vlPAG had no effect on pain behavior.After CCI,mechanical allodynia was significantly aggravated in knockout group compared with control group in both female and male mice.Besides,female mice showed more severe pain than male mice.4.Action potential was evoked on the Ca MKIIα-EYFP neurons within the mPFC when stimulated by the blue light,demonstrating that the viral expression was successful.Mainly o EPSC was recorded on the Gad2-td Tomato neuron in the vlPAG and perfusion of 10μM ACEA significantly reduced the amplitude of o EPSC in female mice to a much extent.However,o EPSC was not evoked in the male mice.Conclusion:The retrograde projection of GABA^vlPAG into the mPFC is predominantly Ca MKIIα⁺excitatory neurons and abundant CB₁Rs were distributed in the Ca MKIIα^mPFC-GABA^vlPAG circuit.Activation of CB₁R on this circuit reduced excitatory synaptic responses to the GABAergic neurons in vlPAG.Finally,Ca MKIIα^mPFC-GABA^vlPAG and GABA^vlPAG-v Glut2^vlPAG inhibitory circuit synergistically promote the analgesia effects of cannabinoids.Overall conclusion:Our results showed that activation or inhibition of CB₁R in the vlPAG produce greater analgesia or mechanical allodynia respectively in females compared to males.Specific ablation of GABAergic CB₁R in the vlPAG caused greater mechanical allodynia.Sex-specific expression of GABAergic CB₁R in the vlPAG resulted in behavioral sex differences.Furthermore,GABAergic inputs to td Tomato-positive（v Glut2⁺）neurons are less sensitive to CB₁R agonists in males compared to females.Activation of CB₁R in Ca MKIIα^mPFC-GABA^vlPAGcircuit reduces excitatory synaptic responses,and GABA neurons within vlPAG are relatively inhibited.Finally,GABA^vlPAG-v Glut2^vlPAG inhibitory and Ca MKIIα^mPFC-GABA^vlPAG excitatory circuit synergistically promoted the analgesic effect of cannabinoids.The above findings revealed sex differences in cannabinoid-mediated analgesia,which provided a structural and functional basis for exploring more specific therapeutic approaches for chronic pain in females.