The Role And Mechanism Of Rostromedial Tegmental Nucleus-substantia Nigra Pars Compacta Circuit In Regulation Of Aversion And Despair Behavior In Mice

The rostromedial tegmental nucleus?RMTg?is a recently identified GABAergic brain region that is termed“the tail of the VTA”and receive major glutamatergic input from the lateral habenula?LHb?,which conveys negative reward and motivation related information,and project intensively to midbrain dopamine neurons,including in the ventral tegmental area?VTA?and substantia nigra pars compacta?SNc?.Anatomically,there are only rare LHb efferents that project directly to the SNc.Therefore,the RMTg is likely to mediate the inhibitory effect of the LHb on the dopaminergic neurons.The RMTg-VTA circiut has been linked to the affective behavior,but the role of the RMTg-SNc circiut in aversion and depression remains to be addressed.The effect of LHb on SNc mediated by the RMTg participating in the pathogenesis of depression remains to be further studied.Methods:?1?To observe the effects of optogenetic exciting the Vgat^RMTg-SNcpathway on aversion/reward and depressive behaviors in mice:AAV encoding the light-sensitive cation channel AAV-Ch R2-DIO-eYFP and AAV-DIO-eYFP as a control were microinjected into the RMTg of Vgat-ires-cre mice,respectively.Optical fibers were implanted above the SNc.A 473 nm bule light stimulation was delivered to selectively activate the Vgat^RMTg-SNcpathway to detect aversion and depression related behaviors in mice,including the real-time place preference test?RPP?,conditioned place preference and aversion test?CPP/CPA?,forced swim test?FST?and sucrose preference test?SPT?.?2?To observe the effects of optogenetic inhibiting the Vgat^RMTg-SNcpathway on aversion/reward and depression behaviors in mice:AAV encoding the light-sensitive cation channel AAV-NPHR3.0-m Cherry and AAV-DIO-eYFP as a control were microinjected into the RMTg of Vgat-ires-cre mice,respectively.Optical fibers were implanted above the SNc.A 589 nm yellow light stimulation was delivered to selectively inhibit the Vgat^RMTg-SNcpathway to detect the aversive behavior in the RPP and CPP.At the same time,optogenetic inhibition of Vgat^RMTg-SNcpathway combined with chronic restraint stress?CRS?depression model to detect the despair behavior in the FST.?3?To observe the changes of SNc activity in activating the Vgat^RMTg-SNcpathway and inhibiting the circuit on CRS depression model mice:Immunohistochemical staining was used to detect the expression of c-Fos positive cells in the SNc.?4?To observe the effect of optogenetic activating Vgat^RMTg-SNcterminals on SNc dopamine neuronal firing in free moving mice:Extracellular recording combined with photogenetic was used and the putative dopaminergic neurons were classified by the electrophysiological characteristics of DA neurons.?5?To observe the effects of optogenetic exciting the Vglut2^LHb-RMTgpathway on aversion/reward and depressive behaviors in mice:AAV encoding the light-sensitive cation channel AAV-Ch R2-DIO-eYFP and AAV-DIO-eYFP as a control were microinjected into the LHb of Vglut2-ires-cre mice,respectively.Optical fibers were implanted above the RMTg.A 473 nm bule light stimulation was delivered to selectively activate the Vglut2^LHb-RMTgpathway to detect aversion and depression related behaviors in the RPP,CPP/CPA,FST and SPT.Results:?1?Exciting the Vgat^RMTg-SNc circuit was sufficient to increase immobility time compared with the eYFP no-CRS group.The percentage of sucrose consumption,total liquid consumption in the SPT and the descent latencies in the pole climb test were not affected by Vgat^RMTg-SNc stimulation.In the RPP and CPA test,Ch R2no-CRS mice spent significantly less time on the side paired with stimulation of the Vgat^RMTg-SNc circuit in the RPP and had lower time ratios that mice spent on the stimulation side of the chamber during the post-stimulation session relative to the time mice spent on the stimulation side of the chamber during the preconditioning session?post/pre?in the CPA compared with the eYFP no-CRS group.?2?Inhibiting the Vgat^RMTg-SNc pathway,NPHR3.0 no-CRS group had noticeably decreased immobility relative to the eYFP no-CRS group in the FST.CRS increased immobility time relative to non-CRS mice and the NPHR3.0 CRS group reversed the increased immobility time relative to the eYFP CRS group in the FST.Following activation of the Vgat^RMTg-SNc circuit,the four groups of mice showed similar latencies to descend in the pole climb test.In the RPP and CPP test,NPHR3.0 no-CRS mice spent more time on the side paired with stimulation of the Vgat^RMTg-SNc circuit in the RPP and had higher time ratios?post/pre?in the CPP compared with the eYFP no-CRS group.?3?Activating the Vgat^RMTg-SNc circuit significantly reduced the number of c-Fos positive cells in the SNc compared with the eYFP no-CRS group.CRS reduced the number of c-Fos positive cells in the SNc compared with the non-CRS mice and inhibiting the Vgat^RMTg-SNc pathway reversed the decreased c-Fos positive cells in the SNc.?4?Extracellular recordings showed that 90%of the SNc putative dopamine neurons were inhibited by activating the Vgat^RMTg-SNcpathway.?5?Exciting the Vglut2^LHb-RMTg circuit was sufficient to increase immobility time compared with the eYFP no-CRS group.The percentage of sucrose consumption,total liquid consumption in the SPT and the descent latencies in the pole climb test were not affected by Vglut2^LHb-RMTg stimulation.In the RPP and CPA test,Ch R2 no-CRS mice spent significantly less time on the side paired with stimulation of the Vglut2^LHb-RMTg circuit in the RPP and had lower time ratios?post/pre?in the CPA compared with the eYFP no-CRS group.Conclusion:?1?The inhibition of SNc putative dopamine neurons by activating the Vgat^RMTg-SNc pathway promoted aversion and despair behavior.?2?Inhibiting the Vgat^RMTg-SNc circuit reversed behavior despair in CRS depression model.?3?The Vglut2^LHb-RMTg circuit regulated aversive and despair behavior,which suggests that the RMTg may mediate the role of LHb glutamatergic neurons in negative behaviors through regulating the activity of SNc neurons.