Mechanism Underlying The Anterior Insula And Its Related Neural Pathway Involved In The Transmission And Modulation Of Pain

Chronic pain is a common clinical disease,which causes negative emotions including anxiety.Owing to the interaction between intractable pain symptom and emotion disorders,chronic pain exerts tremendous physical and psychological burden on patients.Although scientists have realized the importance of the emotional aspect of pain,the mechanism concerning higher brain centers involved in the emotional dimension of pain remains to be investigated.As one part of brain limbic areas,IC is an important brain node for individuals achieving emotional self-awareness from external stimuli.In recent years,the role of IC in the transmission and modulation of pain gains more and more attention.Based on its anatomical connection feature,it is geneally conceived that anterior insula（aIC）is involved in the emotional aspect of pain,while posterior insula（p IC）is related to the sensory aspect of pain.However,functional evidence is still lacking to support this hypothesis.As one part of the medial pain system,mediodorsal thalamic nucleus（MD）mainly projects to cerebral areas related to emotional modulation of pain,including medial prefrontal cortex（m PFC）,anterior cingulate cortex（ACC）and aIC.The MD-ACC pathway has been clarified to participate in the formation of pain-related aversive emotions.Then,whether the MD-aIC pathway is involved in emotional pain modulation remains to be investigated.Most animal studies concering the role of IC in chronic pain are based on neuropathic pain models.Nerve surgery induces neuroplastic changes in IC,which facilitates the maintenance of chronic pain,and inhibiting insular excitability could alleviate neuropathic pain.Apart from somatosensory pain,IC also receives general visceral inputs from parabrachial nucleus（PBN）and thalamus.However,the role of IC in the progress of chronic visceral pain has been seldom reported yet.Herein,we conducted two major studies to solve aforementioned quentions.In the first study,we began with optogenetics and chemogenetics to investigate the role of insular subregions in the transmission and modulation of pain;then we explored the neuroanatomical features of MD-aIC pathway via tract tracing method;finally,we combined optogenetics and chemogenetics to explore the role of MD-aIC pathway in the transmission and modulation of pain and pain-related negative emotions.In the second part,we combined morphology,molecular biology,electrophysiology and behavioral method to explore the neuroplasticity change of aIC under the condition of chronic pancreatitis（CP）and its underlying molecular mechanisms,as well as the role of aIC in the transmission and modulation of painful CP and related negative emotions.This study can be divided into following four sections:1.Functional study concerning the role of insular subregions in the transmission and modulation of painObjective:To explore the different role of insular subregions in the transmission and modulation of pain.Methods:In this part,spared nerve injury（SNI）induced neuropathic pain model was used.Von Frey test was used to measure paw withdrawl threshold（PWT）,while open field test or elevated plus maze（EPM）test was used to measure anxiety behavior.（1）Immunohistochemistry was used to explore the change of insular FOS expression in SNI mice.（2）Chemogenetic virus,including r AAV2/9-h Syn-h M3D（Gq）-m Citrine or r AAV2/9-h Syn-h M4D（Gi）-m Citrine,was injected into bilateral aIC of C57 mice;CNO was administrated intraperitoneally to unspecifically activate or inhibit aIC and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（3）Chemogenetic virus,including r AAV2/9-h Syn-h M3D（Gq）-m Citrine or r AAV2/9-h Syn-h M4D（Gi）-m Citrine,was injected into bilateral p IC of C57 mice;CNO was administrated intraperitoneally to unspecifically activate or inhibit p IC and the effect on PWT and exploratory behavior was observed in both na（?）ve and SNI mice.（4）Optogenetic virus r AAV2/9-Ca MKIIa-Ch R2-m Cherry was injected into bilateral aIC of C57 mice;aIC pyramidal neurons were activated via blue light illumination and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（5）Optogenetic virus r AAV2/9-Ca MKIIa-Ch R2-m Cherry was injected into bilateral p IC of C57 mice;p IC pyramidal neurons were activated via blue light illumination and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（6）Chemogenetic virus,including r AAV-h Syn-DIO-h M3D（Gq）-m Cherry or r AAV-h Syn-DIO-h M4D（Gi）-m Cherry,was injected into bilateral aIC of GAD2-cre mice;CNO was administrated intraperitoneally to unspecifically activate or inhibit aIC GABAergic neurons and the effect on PWT and exploratory behavior was observed in both na（?）ve and SNI mice.Results:（1）Comparing to sham mice,SNI mice exhibited elevated expression of FOS in all insular subregions.（2）Unspecifically activating aIC reduced the PWT in na（?）ve mice,as well as center time%instead of total traveling distance in the open field;unspecifically inhibiting aIC activity exerted no effects on PWT and animal behavior in the open field in na（?）ve mice.（3）Unspecifically activating p IC reduced the PWT in na（?）ve mice,with no effect on animal behavior in the open field;unspecifically activating p IC exerted no effect on PWT and open field behavior in SNI mice.（4）Unspecifically inhibiting p IC activity exerted no effect on PWT and open field behavior in na（?）ve mice;it could alleviate pain behavior in SNI mice,with no effect on open field behavior.（5）Specifically activating aIC pyramidal neurons reduced the PWT in na（?）ve mice,as well as open arm time%（OA%）instead of total traveling distance in the EPM.（6）Specifically activating p IC pyramidal neurons reduced the PWT in na（?）ve mice,with no effect on animal behavior in the EPM.（7）Specifically inhibiting aIC GABAergic neurons elicited mechanical pain sensation in na（?）ve mice;specifically activating aIC GABAergic neurons alleviated mechanical hypersensitivity and anxiety in SNI mice.Conclusion:All insular subregions were involved in the transmission or modulation of neuropathic pain.Activating aIC elicits mechanical pain sensation and anxiety while activating p IC only elicits mechanical pain instead of anxiety in na（?）ve mice.The facilitatory role of IC in pain and anxiety is mediated by pyramidal neurons,while GABAergic neurons exert opposite roles.Cortical feedforward inhibition may participate in the processing of pain message within the IC.2.Morphological study of the MD-aIC pathwayObjective:To explore the neuroanatomical features of the MD-aIC pathway.Methods:（1）Whole brain mapping of monosynaptic inputs to pyramidal and GABAergic neurons within both aIC and p IC via rabies virus-based cell-type-specific retrograde tracing system.（2）Retrograde tracing virus was used to confirm that MD VGlu T2 neurons send projects to aIC.（3）Anterograde tracing virus was used to confirm the existence of MD-aIC pathway.（4）Adeno-associated virus（AAV）-based cell-type-specific anterograde tracing system was used to confirm that MD sends direct projections to aIC glutamatergic and GABAergic neurons.Results:（1）Genetically modified RV and helper virus were injected into the aIC of Ca MKIIa-cre and GAD2-cre mice,and no obvious difference in the distribution and amount of whole-brain monosynaptic input neurons of Ca MKIIa and GAD2 neurons were observed.In the telencephalon,a lot of input neurons could be seen in ipsilateral aIC,aIC adjacent cortical areas,piriform cortex（Pir）,and amygdala.In the thalamus,a lot of input neurons could be seen in mediodorsal thalamic nucleus（MD）,ventral posteromedial thalamic nucleus（VPM）,posterior thalamic nucleus（Po）and parafascicular thalamic nucleus（PF）;moderate neurons could be seen in ventral posterolateral thalamic nucleus（VPL）,ventral medial thalamic nucleus（VM）,submedius thalamic nucleus（Sub）and the parvicellular part of ventral posterior thalamic nucleus（VPPC）;a few neurons could be seen in ther midline/intralaminar thalamic nuclei.In the brainstem,input neurons could be seen in the ventral tegmental area（VTA）,substantia nigra pars compacta（SNC）,dorsal raphe cortex（DR）and parabrachial nucleus（PBN）.（2）Genetically modified RV and helper virus were injected into the p IC of Ca MKIIa-cre and GAD2-cre mice,and no obvious difference in the distribution and amount of whole-brain monosynaptic input neurons of Ca MKIIa and GAD2 neurons were observed.The distribution of monosynaptic input neurons into p IC was similar with that of aIC in the telencephalon and brainstem,but not in the thalamus:a lot of neurons could be seen in the VPPC and VPM;moderate neurons could be seen in the VPL,Po and PF;few neuron could be seen in the MD.（3）Retrograde tracing virus retro AAV2-h Syn-DIO-Ch R2-EYFP was injected into the aIC of VGlu T2-cre mice,and a lot of EYFP-labeled fibers within all layers of aIC and some EYFP-labeled neurons within the MD were observed.（4）Anterograde tracing virus r AAV2/9-Ca MKIIa-m Cherry was injected into MD of C57 mice,and a lot of fibers could be seen in the rostral agranular insular cortex（RAIC）.（5）Anterograde transsynaptic tracing virus sc AAV2/1-h Syn-FLEX-Flpo was injected into the MD of VGlu T2-cre and GAD2-cre mice,and r AAV2/9-h EF1a-f DIO-EYFP was injected into the aIC.GFP-labeled neurons could be seen in the RAIC in both VGlu T2-cre and GAD2-cre mice.Conclusion:No obvious difference in the distribution and amount of whole-brain monosynaptic input neurons of Ca MKIIa and GAD2 neurons were observed in the same insular subregion.Different insular subregions differ in the distribution of input neurons,especially in the thalamus:MD mainly projects to aIC instead of p IC.Glutamatergic neurons within MD provide direct inputs into both pyramidal and GABAergic neurons within the aIC.3.Functional study concerning the role of MD-aIC pathway in the transmission and modulation of painObjective:To explore the role of MD-aIC pathway in the transmission and modulation of pain.Methods:（1）Chemogenetic virus,including r AAV2/9-h Syn-h M3D（Gq）-m Citrine or r AAV2/9-h Syn-h M4D（Gi）-m Citrine,was injected into MD of C57 mice;CNO was administrated intraperitoneally to unspecifically activate or inhibit MD and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（2）Optogenetic virus r AAV2/9-Ca MKIIa-Ch R2-m Cherry was injected into MD of C57 mice and optic fiber was implanted into ipsilateral aIC.The MD-aIC pathway was activated via blue light illumination and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（3）Retrograde tracing virus retro AAV2/2-CMV-b Globin-cre was injected into the aIC of C57 mice and cre-dependent chemogenetic virus r AAV-h Syn-DIO-h M3D（Gq）-m Cherry was injected into ipsilateral MD.CNO was administrated intraperitoneally to unspecifically activate aIC-projecting MD neurons and the effect on PWT and exploratory behavior was observed in na（?）ve mice.（4）Genetically modified RV containing Ch R2 and helper virus were injected into the aIC of Ca MKIIa-cre and GAD2-cre mice which received SNI treatment,and optic fiber was implanted upon MD to activate MD neurons which project to aIC GABAergic neurons.The effect on mechanical pain hypersensitiviry and anxiety was observed in SNI mice.Results:（1）Activating MD reduced the PWT in na（?）ve mice,as well as center time%instead of total traveling distance in the open field;Inhibiting MD activity exerted no effect on PWT and animal behavior in the open field in na（?）ve mice,but alleviated pain behavior in SNI mice.（2）Optogenetically activating MD-aIC pathway reduced the PWT in na（?）ve mice,as well as OA%instead of total traveling distance in the EPM.（3）Chemogenetically activating MD-aIC pathway reduced the PWT in na（?）ve mice,as well as center time%instead of total traveling distance in the open field.（4）Specifically activating MD neurons projecting to aIC GABAergic neurons alleviated pain hypersensitivity and anxiety in mice,with no effect on total traveling distance in the EPM.Conclusion:Activating MD or MD-aIC pathway elicits pain hypersensitivity and anxiety in na（?）ve mice.Thus,MD-aIC pathway may mediate the transmission of emotional pain message.Within this pathway,aIC GABAergic neurons could exert inhibitory effects on pain processing within the aIC via feedforward inhibition.4.Functional study concerning the role of aIC in the transmission and modulation of chronic pancreatitis painObjective:To explore the role of aIC in the transmission and modulation of chronic pancreatitis pain.Methods:（1）Chronic pancreatitis rat model was established by trinitro-benzene-sulfonic acid（TNBS）administration into pancreatic duct.The measurement of serum indicators and animal behavior was conducted to confirm that the model was successfully established.（2）Immunohistochemistry was performed to measure the change in insular FOS expression of CP rats;western blot was performed to examine the content of VGlu T2,NR2B and Glu R1.（3）Electrophysiological indicators of excitatory synaptic transmission,synaptic plasticity and neural activity of aIC in CP rats were examined by extracellular field EPSP recordings and whole-cell patch clamp recordings.（4）AMPAR antagonist CNQX or NMDAR antagonist AP-5 was injected into bilateral aIC and the effect on abdomen withdrawl threshold（AWT）in CP rats was examined.（5）Chemogenetic virus r AAV2/9-m Ca MKIIa-h M4D（Gi）-m Cherry was injected into bilateral aIC of CP rats and CNO was administrated intraperitoneally to inhibit aIC pyramidal neruons.The effect on AWT and anxiety in CP rats was observed.Results:（1）TNBS-treated rats exhibited acinar atrophy and inflammatory infiltration in pancreatic tissue as well as increased serum lipase,amylase and total bilirubin;TNBS-treated rats exhibited abdomen mechanical hypersensitivity,hypolocomotion and anxiety along the course of CP.（2）Comparing to sham group,FOS expression within all insular subregions was increased in CP rats.（3）Comparing to sham group,the input（stimulating intensity）-output（the number of activated channels）curve of aIC shifted to the left in CP rats.LTP was occluded in CP rats after theta burst stimulation.（4）Comparing to sham group,the input（injected current）-output（spike number）curve of aIC pyramidal neurons in layers Ⅱ-Ⅲ shifted to the left in CP rats;the rheobase current was decreased and the resting membrane potential was elevated;the frequency and amplitude of s EPSC in aIC pyramidal neurons from layers II-III were increased in CP rats,together with decreased paired-pulse ratio;Input（stimulation intensity）-output（EPSC amplitude）curves of both AMPAR and NMDAR-mediated currents significantly shifted to the left after TNBS treatment;The rectification index(I_{-40m V}/I_{+50m V})of AMPAR-EPSCs,instead of NMDAR-EPSCs,was significantly increased in CP rats.（5）Immunohistochemistry data showed that the expression of VGlu T2 within aIC was significantly increased in CP rats;Western blot data showed that the amount of NR2B and Glu R1 significantly increased,especially its membrane content,in CP rats.CP rats also showed increased phosphorylation of NR2B and Glu R1 in the aIC.（6）Blocking insular excitatory transmission via CNQX and AP-5alleviated abdomen hypersensitivity in CP rats.（7）Specifically inhibiting aIC pyramidal neurons via chemogenetics alleviated abdomen hypersensitivity,hypolocomotion and anxiety in CP rats.Conclusion:Chronic pancreatitis induced robust abdomen mechanical hypersensitivity and anxiety in rats.Under the condition of CP,the excitatory synaptic transmission within the aIC underwent long-term potentiation changes.Both presynaptic and postsynaptic mechanisms were involved in this process,which facilitated behavior hypersensitivity.Inhibiting the excitatory synaptic transmission within the aIC could alleviate visceral pain.In addition,pyramidal neurons within the aIC exhibit hyperactivity under the condition of CP,and inhibiting the neural hyperactivity could alleviate both visceral pain and anxiety.Concluding remarks:（1）aIC is related to both the emotional and sensory aspects of pain,while p IC is related to the sensory aspect of pain;（2）MD is the specific thalamic nucleus projecting to aIC instead of p IC.MD directly projects to both pyramidal and GABAergic neurons within the aIC;（3）MD-aIC pathway mediates the transmission of emotional pain message to aIC.Within this pathway,insular GABAergic neurons inhibit pain transmission within aIC via feedforward inhibition;（4）Excitatory synaptic transmission within aIC underwent long-term potentiation under the condition of chronic pancreatic pain,and inhibiting these changes could alleviate CP-induced abdomen mechanical allodynia and anxiety.