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

Many neurons in the brain are known to be "multi-transmitter," meaning they release more than one chemical messenger. A long-standing question in neurobiology is whether these transmitters are segregated into distinct pools of synaptic vesicles within the same terminal or co-packaged into the same individual vesicles. The authors investigated this question in Entopeduncular nucleus (EP) neurons that express somatostatin (Sst+), which project to the lateral habenula (LHb) and are known to co-release glutamate and GABA.

To determine the mechanism of co-release, the researchers utilized a multidisciplinary approach. They employed array tomography to visualize the molecular machinery (vesicular transporters) within single synaptic boutons. To differentiate the two models (independent versus co-packaging) of release, they developed a computational model that predicts distinct statistical signatures for each. They then tested these predictions using "DMOS" (DMD-based minimal optogenetic stimulation), which allowed for high-resolution, trial-by-trial analysis of neurotransmitter release from individual synapses. Finally, they used pharmacological manipulation with serotonin to alter release probability and test the stability of their observations.

The authors found strong evidence that glutamate and GABA are co-packaged into individual vesicles. Their statistical analysis of unitary synaptic responses showed that glutamate and GABA release events are highly correlated in time and amplitude, consistent with the co-packaging model. Furthermore, when serotonin was applied to reduce the overall release probability, the correlated release of the two transmitters was maintained, providing further evidence that they originate from the same vesicular pool. Sites that appeared to suggest independent release were strongly associated with lower signal-to-noise ratios and higher baseline noise, suggesting that technical limitations, rather than biological reality, were responsible for those outliers.