Physiologic and nanoscale distinctions define glutamatergic synapses in tonic vs phasic neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts - a "phasic" pattern. Synapses formed by tonic vs phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge towards illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction (NMJ), most muscle fibers are co-innervated by two motor neurons, the tonic "MN-Ib" and phasic "MN-Is". Here, we employed selective expression of a newly developed botulinum neurotoxin (BoNT-C) transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca 2+ imaging demonstrated ∼two-fold greater Ca 2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca 2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca 2+ influx collaborate to differentially tune glutamate release at tonic vs phasic synaptic subtypes. SIGNIFICANCE STATEMENT: "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how the input-specific synaptic diversity is achieved, which could have significant implications for the development of therapeutic interventions for neurological disorders that involve changes in synaptic function.