Synaptotagmin isoforms differentially regulate glutamate and GABA release in the lateral habenula

Neurotransmitter co-transmission contributes to diverse physiological processes throughout the mammalian brain, including sensory integration, motivational control, and social behaviors. Projections from the globus pallidus internus (GPi; the entopeduncular nucleus, EPN, in rodents) to the lateral habenula (LHb) are well-characterized by the co-transmission of both GABA and glutamate. These dual-release inputs modulate behavioral states in chronically learned helpless (cLH) rats, influencing both the onset and recovery of pathological phenotypes. Here, we employed confocal 3D reconstructions that confirmed the presence of both vesicular transporters VGAT and VGLUT2 in EPN axon terminals within the LHb. Further investigation revealed that GABA and glutamate are packaged in distinct vesicle populations within individual presynaptic terminals. Notably, the calcium (Ca²⁺) sensors Synaptotagmin-2 (Syt2) and Synaptotagmin-3 (Syt3) are highly expressed in the EPN whereas expression of the canonical Ca²⁺ sensor, Synaptotagmin 1 (Syt1), is downregulated. Additionally, using confocal microscopy, we observed selective spatial correlations of Syt2 and VGLUT2 and between Syt3 and VGAT in LHb axon terminals. These observations strongly suggested that Syt2 serves as the predominant Ca²⁺ sensor for glutamatergic vesicle fusion, and Syt3 serves as the predominant Ca²⁺ sensor for GABAergic vesicle fusion in the LHb. To test this hypothesis, we employed targeted antisense oligonucleotide (ASO) knockdown of Syt2 and Syt3 in EPN neurons and measured LHb glutamatergic and GABAergic currents. Syt2 knockdown resulted in an increase in mEPSC frequency, amplitude, half-width and decay, suggesting increased glutamate vesicle release probability and increased glutamate vesicle packing. However, Syt2 knockdown had no influence on mIPSCs amplitude or frequency. On the other hand, Syt3 knockdown had no apparent effect on glutamate release but caused an increase in mIPSC frequency suggesting increased quantal release probability of GABA. Together, these findings identify a molecular mechanism by which synaptotagmin isoforms govern differential glutamate and GABA release at EPN dual-transmitter terminals in the LHb. These results provide evidence for presynaptic mechanisms regulating excitatory-inhibitory balance within this brain structure and importantly provide molecular targets for pharmacological intervention.