Impaired astrocytic Cx43 syncytial network in lateral habenula affects synaptic connectivity and drives adolescent stress-induced depression

Adolescent depression is a major neuropsychiatric disorder induced by early life stress; the underlying cellular mechanisms instigating pathological circuit malformation remain unclear, and, consequently, effective therapeutic strategy is limited. Here, we analysed the contribution of astrocytes to early life stress-induced adolescent depression using a mouse model of parental separation. We identified a previously unrecognised regional specialisation of lateral habenula (LHb) astrocytes, characterised by compact cellular domains, abundant leaflets, and enhanced Cx43 gap junctional coupling. Parental separation selectively reduced astrocytic Cx43 expression and gap junctional communication in the LHb, which was accompanied by profound astrocytic atrophy and loss of leaflets. Single-cell sequencing further revealed that parental separation induces a broad reorganisation of LHb astrocyte transcriptional states, including metabolic, stress-response, and inflammatory programs; moreover, it specifically reduces astrocyte-neuron communication. Consistent with this, we found that structural remodelling of LHb astrocytes affected tripartite synapse geometry, resulting in impaired excitatory and inhibitory synaptic transmission, and neuronal Ca2+ hyperexcitability, all ultimately associated with depressive-like behaviours. Selective knockdown of astrocytic Cx43 in the LHb recapitulated these pathological changes and translated into depressive-like behaviours. Mechanistically, we found that impaired Cx43 gap junctional communication is associated with excessive astrocytic Ca2+ dynamics, resulting in Rac1-GTPase activation and decreased p-Ezrin levels. In vivo inhibition of LHb astrocytic Ca2+ signalling rescued astrocytic atrophy and depressive-like behaviours following parental separation. Together, our findings establish a Cx43-Ca2+-Rac1-p-Ezrin pathway that links gap junction malfunction to structural remodelling of astrocytes and disruption of neuron-astrocyte interactions in the developing LHb. Our study reveals astrocytic Ca2+ dysregulation as an active downstream effector of Cx43 dysfunction and identifies a regionally specialised astrocytic mechanism through which early-life stress disrupts synaptic transmission and neuronal function during adolescence, providing a potential cellular target for intervention in adolescent depression.