Investigation Of The Comorbid Mechanisms Of Chronic Pain And Depression Regulated By The Lateral Hypothalamus-lateral Habenula Neural Circuitry

Chronic pain,as a recurrent and intractable chronic disease,significantly impacts individuals’physical and mental health as well as their quality of life.Prolonged pain often elicits mental disorders such as anxiety and depression,further affecting individuals’psychology,sleep,and normal social interactions.The co-occurrence of these symptoms often mutually influences and exacerbates each other,creating immense challenges for clinical treatment.Regrettably,despite significant progress in chronic pain research,the pathogenesis of chronic pain comorbid with mental disorders remains unclear.In recent years,studies have found considerable overlap in neuroplasticity and neurobiological mechanisms of pain and depression,which is crucial for facilitating the occurrence and development of depression caused by chronic pain and vice versa.Therefore,dissecting the central brain regions and neural pathways that link chronic pain to depression and vice versa may provide new research avenues and scientific evidence for understanding the pathogenesis of both.Purpose:This study aims to investigate the regulatory mechanisms underlying chronic pain and depression comorbidity by exploring the role of the parvalbumin（PV）neurons in the lateral hypothalamus（LH）to lateral habenula（LHb）neural circuit using neurotropic viral tracing,tissue immunofluorescence staining,optogenetics in combination with animal behavior detection,whole-cell patch-clamp recording,and fiber photometry techniques.Methods:1、The neuropathic pain and depression animal models were established and their mechanical and thermal pain thresholds,anxiety,and depression-like behaviors were evaluated.2、The morphological relationship of the LH^PV-LHb neural circuit was identified by forward and reverse virus tracing with tissue immunofluorescence staining.Further,the functional connectivity of the LH^PV-LHb neural circuit was confirmed using whole-cell patch-clamp recording techniques.3、The optogenetic combined animal behavior studies were employed to investigate the modulatory role of LH^PV-LHb neural circuit in the chronic pain,anxiety,and depression-like behaviors.4、The fiber photometry technique was utilized to assess calcium activity changes in LHb area under physiological and pathological conditions.Results:1、Mechanical and thermal pain thresholds of mice were evaluated at different time points after SNI and CRS surgery.The results showed that seven days after SNI,mice demonstrated significant and stable mechanical and thermal sensitivity,which persisted throughout the entire testing period.Anxiety-like behavior was observed in mice on day28 after SNI,and depression-like behavior was present on day 56,indicating comorbid psychiatric disorders in mice under a state of prolonged pain sensitization.Meanwhile,after 35 days of modeling,CRS mice showed stable anxiety and depression-like behavior.Furthermore,the CRS mice demonstrated pain sensitization,as evidenced by their decreased mechanical and thermal pain thresholds.2、By injecting r AAV-DIO-mCherry virus into the LH brain region of PV-Cre transgenic mice,the fiber projection of LH^PV neurons to the LHb region was observed,confirming the existence of the LH^PV-LHb neural circuit.Subsequently,the synaptic contacts between different types of neurons in the LH and LHb brain regions were identified using forward trans-synaptic Rabies virus and tissue immunofluorescence staining techniques.Furthermore,the synaptic connectivity between LH^PV neurons and different types of neurons in LHb was confirmed using retrograde trans-synaptic rabies virus and multiple transgenic animals in combination with tissue immunofluorescence staining techniques.Finally,the optogenetic combined whole-cell patch-clamp recording technique was used to confirm the functional and synaptic connectivity between LH^PV and LHb neurons.3、Using optogenetic combined with behavioral assays,it was discovered that the activation of the LH^PV-LHb neural circuit triggered aversive and depressive-like behavior in mice under physiological conditions.Following 35 days of CRS modeling,activating the LH^PV-LHb circuit exacerbated depressive-like behavior in mice,whereas inhibiting it relieved their depressive phenotype.However,whether activating or inhibiting this circuit had no significant effect on the pain behavior of the mice after CRS modeling.Optogenetic modulation of the LH^PV-LHb circuit in mice subjected to SNI modeling for 7 days showed that neither activation nor inhibition of the circuit had a significant impact on their mechanical or thermal pain thresholds.The optogenetic control of LH^PV-LHb neural circuitry for anxiety-like behavior induced by SNI after 28 days also revealed no significant regulatory effect.Similarly,the optogenetic control of the circuit for depressive-like behavior induced by SNI after 56 days showed no significant regulatory effect.4、Using fiber-optic calcium imaging,the calcium activity changes of glutamatergic neurons in the LHb region of mice were detected in physiological and pathological states.The results indicated that,compared to the Sham group,the spontaneous calcium activity of glutamatergic neurons in the LHb region of mice was significantly elevated in response to varying degrees of mechanical and thermal stimuli after CRS modeling.Similarly,in the SNI model,the spontaneous calcium activity of glutamatergic neurons in the LHb region also significantly increased in response to varying degrees of mechanical and thermal stimuli.