Summary Many mammalian neurons release multiple neurotransmitters to activate diverse classes of postsynaptic ionotropic receptors. Entopeduncular nucleus somatostatin (EP Sst+) projection neurons to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear if these are packaged into the same or segregated pools of synaptic vesicles. Here we describe a method combining electrophysiology, spatially-patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA co-release in mouse brain. We find that the properties of postsynaptic currents elicited in LHb neurons by optogenetically activating EP Sst+ terminals are only consistent with co-packaging of glutamate/GABA into individual vesicles. Furthermore, presynaptic neuromodulators that weaken EP Sst+ to LHb synapses maintain the co-packaging of glutamate/GABA while reducing vesicular release probability. Our approach is applicable to the study of multi-transmitter neurons throughout the brain and our results constrain the mechanisms of neuromodulation and synaptic integration in LHb.

Significance It is now established that many neurons can release multiple transmitters. Recent studies revealed that fast-acting neurotransmitters, glutamate and GABA, are coreleased from the same presynaptic terminals in some adult brain regions. The dentate gyrus (DG) granule cells (GCs) are innervated by the hypothalamic supramammillary nucleus (SuM) afferents that corelease glutamate and GABA. However, how these functionally opposing neurotransmitters contribute to DG information processing remains unclear. We show that glutamatergic, but not GABAergic, cotransmission exhibits long-term potentiation (LTP) at SuM-GC synapses. By the excitatory selective LTP, the excitation/inhibition balance of SuM inputs increases, and GC firing is enhanced. This study provides evidence that glutamatergic/GABAergic cotransmission balance is rapidly changed in an activity-dependent manner, and such plasticity may modulate DG activity.

Neurotransmitter co-transmission has become recognized as a fundamental organizing principle of neural communication, challenging the traditional view that individual neurons operate through a single transmitter system. Current evidence demonstrates that many neurons utilize multiple transmitters via distinct synaptic architectures, such as co-packaging within the same vesicle, release from separate vesicle pools within the same terminal, and segregation of transmitters across different boutons or neuronal processes. These organizational modes are not simply structural variants; they impose distinct rules for release, target engagement, and short-term dynamics, thereby shaping circuit function in specific ways. Across neural systems, several common principles have emerged: co-transmission expands signaling across multiple timescales, enhances target specificity, and allows transmitter balance to shift according to firing patterns and circuit state. A major conceptual and technical challenge in the field is that no single method can definitively establish the release mechanism. Consequently, recent advances have relied on integrating molecular profiling, electrophysiology, high-resolution anatomy, optogenetics, and genetically encoded neurotransmitter sensors. Collectively, these approaches are beginning to clarify how multi-transmitter neurons are organized and how their signaling is regulated. Future progress will likely depend on multimodal strategies that connect synaptic

triphosphate (ATP). Any single neuron can release multiple different types of neurotransmitters or neuromodulators … neuromodulators, and also have recep- tors for multiple different receptor types and subtypes, thus making … projections of axons from these monoami- nergic neurons can affect virtually every brain region. In contrast