Whole-brain connections of glutamatergic neurons in the mouse lateral habenula in both sexes

The lateral habenula (LHb) is an epithalamus nucleus that is evolutionarily conserved and involved in various physiological functions, such as encoding value signals, integrating emotional information, and regulating related behaviors. The cells in the LHb are predominantly glutamatergic and have heterogeneous functions in response to different stimuli. The circuitry connections of the LHb glutamatergic neurons play a crucial role in integrating a wide range of events. However, the circuitry connections of LHb glutamatergic neurons in both sexes have not been thoroughly investigated. In this study, we injected Cre-dependent retrograde trace virus and anterograde synaptophysin-labeling virus into the LHb of adult male and female Vglut2-ires-Cre mice, respectively. We then quantitatively analyzed the input and output of the LHb glutamatergic connections in both the ipsilateral and contralateral whole brain. Our findings showed that the inputs to LHbvGlut2 neurons come from more than 30 brain subregions, including the cortex, striatum, pallidum, thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum with no significant differences between males and females. The outputs of LHbvGlut2 neurons targeted eight large brain regions, primarily focusing on the midbrain and pons nuclei, with distinct features in presynaptic bouton across different brain subregions. While correlation and cluster analysis revealed differences in input and collateral projection features, the input-output connection pattern of LHbvGlut2 neurons in both sexes was highly similar. This study provides a systematic and comprehensive analysis of the input and output connections of LHbvGlut2 neurons in male and female mice, shedding light on the anatomical architecture of these specific cell types in the mouse LHb. This structural understanding can help guide further investigations into the complex functions of the LHb. The inputs to LHbvGlut2 neurons encompass over 30 brain subregions, mainly arising from the pallidum, hypothalamus, and midbrain. The outputs of LHbvGlut2 neurons involve eight large brain regions, mainly targeting midbrain and pons nuclei, displaying distinct features in presynaptic boutons across each subregion. The pattern of input-output connections of LHbvGlut2 neurons is remarkably similar in both males and females.