Role Of T-type Calcium Channel In Lateral Habenular In Chronic Inflammatory Pain-induced Depression

Objective:Clinically,the proportion of chronic pain patients with depression is as high as30%～60%.Moreover,the incidence of chronic pain in patients with severe depression is also high.Each of these two diseases will actively promote the development of the other.The lateral habenular（LHb）serves as a‘hub’that receives convergent information from the limbic system and basal ganglia brain sites.It is a central strategic fortress involved in pain perception and negative emotion formation at the same time.However,it is unclear whether LHb is involved in the regulation of chronic pain and depression.T-type voltage-gated calcium channel（T-VGCC）,which is widely expressed in the central and peripheral nervous system,is a vital ionic basis involved in modulating neuronal excitability.T-VGCC abnormality is closely related to emotional disorder and pain.Recent studies have found that systemic or local injection of T-VGCC blocker into LHb can significantly alleviate the pain or depression-like behavior.So,does chronic pain lead to depression by changing the expression and function of T-VGCC and affecting LHb activity?In or der to answer this question,this experiment explored the central mechanism of chronic inflammatory pain-induced depression through the model of chronic inflammatory pain-induced depression,focusing on the expression and functional changes of T-VGCC in LHb.Methods:Wild-type C57BL/6J mice（male,8 w,20-30g）were injected subcutaneously with 10 ul complete Freund’s adjuvant（CFA）on the left plantar of mice on day 0 and day 10 to establish a chronic inflammatory pain model（CFA group）,respectively.At the same time,the control group was injected with the same dose of normal saline（Sham group）.The weight of mice and the plantar thickness at the injection site were continuously observed.The paw withdrawal threshold（PWT）and paw withdrawal latency（PWL）were measured before the injection and on days 1,3,6,9,12,14,21,28,35,42,49 and 56 after injection.At the same time,the depression-like behavior was evaluated during the existence period（2 w,3 w）and recovery period（8 w）of mechanical pain sensitivity after injection of CFA,so as to clarify the development pattern of chronic inflammatory pain combined with depression induced by CFA.First,during the period of chronic pain-induced depression,immunohistochemistry and electrophysiological techniques were used to observe the expression of C-Fos and the changes in excitability in LHb.Second,on the basis of clarifying the expression of T-VGCC subtypes（Ca V3.1,Ca V3.2 and Ca V3.3）in LHb under physiological state and the regulation of T-VGCC on LHb neuron excitability.Finally,the expression and functional changes of T-VGCC in chronic inflammatory pain-induced depression were determined by western blot and electrophysiological techniques.Results:1.Obvious redness and swelling were observed at the injection site 1 d after CFA injection and lasted for at least 8 w and since 3 w after CFA injection,the weight growth of mice was significantly lowered when compared to that of sham group.CFA resulted in evident mechanical and thermal hyperalgesia,as indicated by cleared lowered PWT and shortened PWL on the ipsilateral paw in response to mechanical and heat stimulation,respectively.Regarding mechanical hyperalgesia,CFA led to a significant decrease in PWT 1h after injection,and the value of PWT began to rise 28 d after injection and returned to the same level as Sham on the 42nd day;The PWL of CFA mice also decreased significantly 1 h after injection,but a significant recovery was observed on the 9th day.And even after the second injection on the 10th day,the thermal pain threshold of CFA mice recovered earlier than the mechanical pain threshold and returned to the sham level on the 21st day.2.CFA injection provoked anxiodepression-like behaviors at 3 w instead of 2 w after inflammatory pain modeling,and such anxiodepression-like consequences could stilled be observed at 8w after CFA treatment.3.During the period of chronic inflammatory pain-induced depression,the C-Fos count in LHb and the excitability of neurons increased.4.Immunofluorescence staining showed that Ca V3.1-Ca V3.3 were expressed in LHb,and the fluorescence intensity of Ca V3.1 and Ca V3.3 were 1.527 and 1.530times higher than that of Ca V3.2,respectively;All three subtypes were expressed on neurons.5.Bath application of T-VGCC blocker Ni Cl₂can significantly reduce the excitability of LHb neurons.6.There was no significant change in the expression of T-VGCC subtypes of LHb in the period of chronic inflammatory pain-induced depression,but the amplitude and current density of I_Twas increased.Conclusions:In conclusion,this study found that the anxiodepression phenotype of CFA chronic inflammatory pain mice is time-dependent,and the manifestations of pain and mood disorders can coexist and separate.This study verified that the up regulation of LHb neuron excitability and the enhancement of T-VGCC function is one of the important central mechanisms involved in the occurrence and development of chronic inflammatory pain-induced depression.