Chronic cocaine exposure disrupts cholinergic neuron integrity and function in organotypic slices of the laterodorsal tegmentum

Abstract

BACKGROUND: Cocaine disrupts monoaminergic signaling which leads to neuroadaptive and neurotoxic changes in brain reward circuits. While its effects on mesocorticolimbic regions are well documented, its impact on the laterodorsal tegmentum (LDT), a brainstem cholinergic nucleus that is a key regulator of midbrain dopaminergic activity critical in the reinforcing properties of cocaine, remains unclear. METHODS: We used an organotypic culture model of LDT brain slices combined with a modified flow system enabling controlled cocaine exposure for 14 days in vitro. Slices were exposed to cocaine under continuous or daily 1-hour protocols. Cell viability, morphology, and cytotoxicity were assessed using MTT, DAPI/PI staining, and LDH release. Cholinergic neurons were identified by immunohistochemistry, and their functional responses were evaluated by electrophysiology and calcium imaging following AMPA stimulation. RESULTS: Chronic cocaine exposure induced significant, exposure-dependent reductions in cell viability and structural integrity, with more pronounced effects under continuous exposure. Cholinergic neurons showed decreased number and soma size. Cocaine exposure also altered cytotoxicity and dopamine dynamics. Functionally, cocaine-induced alterations in synaptic activity and calcium signaling were time-dependent, with increased neuronal responses observed at early stages of exposure and reduced responsiveness after prolonged exposure. CONCLUSION: We present a novel and robust in vitro model to study long-term, drug-induced neuroadaptations and show that chronic cocaine exposure disrupts the structural and functional integrity of LDT cholinergic neurons in a time-dependent manner. Our findings suggest that actions of cocaine on altering reward processing that leads to persistence of drug-seeking behaviors likely involve degenerative effects on neurons of the LDT.