The Role Of Hyperpolarization-Activated Nucleotide-Gated Channels In The Lateral Habenula Underlying Comorbid Depressive Symptoms In Chronic Pain

Objective:Many patients with chronic pain are often accompanied by negative emotions such as anxiety and depression,and the breeding of negative emotions can aggravate the pain feeling of patients,the two interactions,forming a vicious circle,seriously affect the prognosis of chronic pain patients;At present,Comorbid depressive symptoms in chronic pain(CDS)has become one of the difficult problems should be solved in the clinical diagnosis and treatment of pain.Lateral habenula(LHb)is an important brain region that not only participates in the regulation of pain,but also plays a key role in the modulation of emotional information.Overexcitation of the Lateral habenula(LHb)has been proved to be one of the core mechanisms of depression.However,it is not clear how the excitability of neurons in LHb changes under the CDS state.As an important factor affecting the electrical activity of neurons in multiple brain regions of the peripheral and central nervous system,Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels are highly expressed in LHb.Does the expression and function of the HCN channel change during CDS formation? In view of these uncertainties,in this study,we used Spared Nerve Injury(SNI)to build a mouse model of CDS.According to combine with behavioral assays,electrophysiology,immunohistochemistry,and molecular biology techniques,we determined the changes in neuron excitability in the LHb region in chronic pain accompanied by depression.HCN channel was used to reveal its cellular ionic mechanism,to provide a new theoretical basis for the clinical prevention and treatment of CDS.Methods:All wild-type C57BL/6J male mice(8-12 weeks)were randomly selected for modeling.The experiment was divided into the SNI model group and the Sham group.After modeling,the following tests were carried out:1.Behavioral tests:(1)Mechanical pain threshold was measured 1 day before modeling and 1,3,7,14,21,28,35 and 42 days after modeling.(2)Depression-like behavior of mice was observed 6 weeks after modeling,the base of preference test(SPT),Forced swimming test(FST),and Tail suspension test(TST);The anxiety-like behavior and motor ability of mice were observed by Open field test(OFT)and Elevated Plus maze(EPM).2.Immunohistochemical test: The brain slices containing LHb were cut with a frozen microtome for HCN staining,to observe the distribution of HCN channels in LHb.3.In vitro electrophysiological: Isolated brain slices which containing LHb were prepared and patch-clamp experiment was performed to record LHb neurons and observed the changes in electrophysiological characteristics of LHb neurons and Ih current under chronic pain and depression comorbidity.4.After the behavioral test,LHb tissues were taken and proteins were extracted to observe the expression of HCN channels in the LHb region under chronic pain and depression comorbidity.Results:1.At 6 weeks,the mice showed anxious-depression-like behaviors after SNI surgery,which were mainly reflected in the following aspects,compared to Sham,in SNI:(1)the preference value of sucrose decreased,and the immobility time in FST and TST increased;(2)the open arm entries and the time in open arms decreased,so does time in center in the OFT.2.The excitability of LHb neurons in mice under the comorbidity of chronic pain and depression is increased.Compared with the Sham group,the results showed in the SNI group:(1)the percentage of Burst discharge was increased,(2)the active discharge frequency was increased,(3)the Tonic discharge frequency was increased(4)higher m EPSC amplitude,and lower m IPSC frequency3.The four subtypes of the HCN channel were all distributed in LHb,and neurons with Ih current had higher intrinsic excitability than neurons without Ih current.The excitability of LHb neurons could be reduced by ZD7288,an HCN channel blocker.4.The expression and function of HCN channels in LHb changed under CDS,that is,the expression of HCN2 increased,but the expression of HCN1,HCN3,and HCN4 did not change significantly.Meanwhile,the amplitude and current density of Ih mediated by HCN channels increased significantly,the activation time constant decreased significantly,and half of the effective activation voltage moved to the direction of depolarization.Conclusions:In conclusion,after 6 weeks of chronic neuropathic pain,the mice showed typical depression-anxiety behavior.Increased excitability of LHb neurons,up-regulated expression of HCN channels in LHb region(mainly HCN2 subtype)and enhanced function of HCN channels may be one of the key central mechanisms of CDS formation.