Synaptic and circuit functions of multitransmitter neurons in the mammalian brain

Abstract

Neurons in the mammalian brain are not limited to releasing a single neurotransmitter but often release multiple neurotransmitters onto postsynaptic cells. Here, we review recent findings of multitransmitter neurons found throughout the mammalian central nervous system. We highlight recent technological innovations that have made the identification of new multitransmitter neurons and the study of their synaptic properties possible. We also focus on mechanisms and molecular constituents required for neurotransmitter corelease at the axon terminal and synaptic vesicle, as well as some possible functions of multitransmitter neurons in diverse brain circuits. We expect that these approaches will lead to new insights into the mechanism and function of multitransmitter neurons, their role in circuits, and their contribution to normal and pathological brain function.

Keywords: GABA; acetylcholine; co-packaging; corelease; cotransmission; dopamine; glutamate; multitransmitter; serotonin.

Figure 1:. Synaptic diversity of cotransmitting neurons

(A) Three examples of cotransmitting neurons that employ vesicular co-packaging of two neurotransmitters at the presynaptic terminal. (B) Ach/GABA cotransmitting neurons found in cortex (VIP/Chat+) release Ach and GABA at different presynaptic terminals and independently package these neurotransmitters into separate vesicle pools. (C) Many midbrain DA neurons release three neurotransmitters, DA and GABA are co-packaged in the same vesicle, whereas glutamate is independently packaged and released at separate presynaptic sites.

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^,).

Figure 3:. Possible cellular and circuit mechanism of multitransmitter neurons

(A) GABA and glutamate release from midbrain DANs cause fast excitation and slightly longer inhibition of postsynaptic cells (dSPNs) due to the kinetics of each ionotropic receptor. The physiological effects following activation of type 1 DA receptors is delayed by ~500ms and increases firing rate towards the end of the spike train. Arrowheads mark timing of presynaptic action potential. (B) Ach/glutamate release from mHb to the IPN may be sparse but temporally precise when mHb firing rates are low, with synaptic transmission dominated by glutamate and minimal activation of extrasynaptic Ach receptors. During high frequency activity spillover of Ach reaches and activates extrasynaptic Ach receptors on many postsynaptic cells leading to broad, but temporally imprecise activation of IPN. (C) LHb neurons assigning “value” to each presynaptic input from EP based on good or bad outcomes. Because EP inputs cotransmit GABA and glutamate, LHb can tune each synapse positive or negative by insertion of glutamate or GABA receptors, respectively. This process is the equivalent of perceptron-like learning rules that classify contexts as good or bad in artificial neural networks.