Biophysical demonstration of co-packaging of glutamate and GABA in individual synaptic vesicles in the central nervous system

Multi-transmitter neurons are common, but determining whether they co-release neurotransmitters from the same synaptic vesicles or from distinct, segregated pools remains challenging. Researchers investigated whether somatostatin-expressing (Sst+) neurons in the entopeduncular nucleus (EP), which project to the lateral habenula (LHb), co-package glutamate and GABA. To address this, the authors combined array tomography (to visualize protein localization), electrophysiology (to measure synaptic currents), and computational modeling. They developed a novel method using digital micromirror device (DMD)-based optogenetics to activate individual presynaptic boutons and record unitary postsynaptic currents (uPSCs) with high temporal precision.

Immunohistochemical analysis demonstrated that the proteins necessary for both glutamatergic (Vglut2) and GABAergic (Vgat) transmission are colocalized within individual EP Sst+ axon terminals. Through electrophysiological stimulation of individual boutons, the researchers observed biphasic PSCs (excitatory AMPAR-mediated and inhibitory GABAR-mediated components). Statistical analysis of these trial-by-trial responses—specifically looking at joint probability, correlation of amplitude peaks, and conditional distributions—revealed that these currents were strongly correlated in a manner consistent with co-packaging in a single vesicle. Further experiments showed that serotonin, a neuromodulator known to weaken these synapses, reduced the overall probability of release but did not disrupt the co-packaging mechanism. This suggests that the same vesicle contains both neurotransmitters regardless of the instantaneous release probability.

This work provides definitive evidence for a co-packaging mechanism in a specific neural circuit, moving beyond the simple observation of co-transmission. By identifying that these EP Sst+ terminals use a shared vesicular pool, the study clarifies the mechanisms of synaptic integration in the LHb. This is particularly relevant for understanding how maladaptive changes in the balance of excitatory and inhibitory inputs contribute to conditions like chronic stress and depression, where synaptic plasticity at these specific synapses is thought to be involved.