Figure 2: Methods for investigating individual cotransmitting neurons and synapses.

(A) Methods to evaluate gene expression in single neurons such as single-cell whole transcriptome sequencing (shown) can examine expression levels of many genes in single neurons to determine if the genetic constituents required for neurotransmitter corelease are present (dots circled in purple represent individual Sst+ GABA/Glutamate coreleasing neurons isolated from EP, color represents gene expression level for vGluT2 (left) or VGAT (right)). (B) Fluorescent in situ hybridization (FISH) allows for confirmation of Sc-seq results in tissue without losing spatial patterns of expression. (C) Confocal image of tissue section from the LHb containing axons labeled from Sst+ EP neurons (YFP) and stained for synaptic proteins. Examining protein expression in synaptic terminals using high resolution methods such as array tomography (shown), electron microscopy, and super-resolution imaging is critical for examining the distribution/localization of pre and postsynaptic vesicular transporters, receptors, and synaptic organizers. (D) (Top) Zoomed image of area highlighted in (C) showing overlapping expression of VGAT and VGlut2 in synaptic terminals. (Bottom) Enrichment of each protein within a terminal over scrambled expression patterns demonstrates high concentrations of VGAT and VGluT2 in presynaptic terminals. (E) Diagram of optical components required for stimulation of individual synapses in acute brain slices. (F) Illustration of viral targeting of optogenetic activators to specific genetically defined cotransmitting neurons in the entopeduncular nucleus (EP) and activation of their axons using light guided by a DMD (digital micromirror device) while performing whole cell recordings in LHb. (G) Optical stimulation can be targeted to a grid of many small spots that overlay the recorded neuron and allow of stimulation of single axons. Action potentials are blocked (TTX/4-AP) to restrict spreading of optical axonal stimulation to multiple synapses on the same axon. (H) Careful calibration of optical stimulus parameters is required to enter into a minimal stimulation regime where stimulation of individual synapses is ensured, and quantal analysis can be performed. (I) When the neuron is voltage clamped at an intermediate potential both GABAergic (blue dot) and glutamatergic (red dot) post-synaptic currents and be observed simultaneously on single trials. (J) Scatterplot of the peak amplitudes for all trials shown in (I) to highlight strong correlation between GABAergic and glutamatergic responses, and providing strong evidence for co-packaging of the two neurotransmitters into the same synaptic vesicle (Figure modified from^25,39).