Introduction Axo-axonic synapses can veto, amplify, or synchronize spikes, yet their circuit-scale logic is unknown. Using the complete electron-microscopy connectome of the adult male Drosophila ventral nerve cord (MANC v1.2.1), we charted every axo-axonic input onto the 1,314 descending neurons that carry brain commands to the ventral nerve cord.
Methods A split-Gal4 driver specific to axo-axonic neurons was identified and cross to UAS-CsChrimson construct. Giant Fibers were activated by extracellular stimuli with electrodes placed in the brain. Muscle recordings were obtained from jump and flight muscles. A mouse primary antibody against ChAT was used to confirm cholinergic cells. LIF models were used with acetylcholine synapses, GABA, and glutamate. Simulations of these neurons were made using the BRIAN2 simulator for the ventral nerve cord (VNC) with the entire MANC v1.2.1 connectome. This resulted in 23,437 valid neurons and 1,152,548 connections between them. In the fly, cholinergic receptors are excitatory, whereas glutamatergic and GABAergic receptors are inhibitory.
Results Only 1% of the 861,591 possible descending–descending neuron pairs form such contacts, but when present, synaptic strength grows linearly with partner number regardless of transmitter identity. By definition, any synapse connected to a descending neuron within the cord is axo-axonic. Neurons with many partners (high-degree nodes) integrate into the network without clustering into a ‘rich-club’ of hubs. We identified an octet of ascending neurons whose axo-axonic input to the Giant Fiber descending neurons predicted modulation of the escape circuit. Immunostaining confirms their cholinergic identity, while optogenetic activation confirmed that this excitatory cohort increases Giant Fiber excitability, validating connectome-derived rules.
Discussion By analyzing all 1,314 brain-originating DNs, we move beyond scattered descriptions of AACs and derive circuit-level design rules for presynaptic modulation. The quantitative principles that emerged within this work are (1) extreme sparseness showing that between 0.7 and 1.2% of possible DN-to-DN, AN-to-DN, and IN-to-DN pairs form axo-axonic synapses; (2) a tight linear relation between synaptic strength and partner multiplicity; and (3) a small-world architecture that distributes integration rather than concentrating it in a rich-club core. Together, these features constitute a wiring grammar for axo-axonic control in the adult Drosophila motor system.
References Ceballos, Cesar, Juan Lopez, Ty Roachford, Daniel Sanchez, Sabrina Jara, Kelli Robbins, Casey L. Spencer, Rodney Murphey, and Rodrigo FO Pena. "The Drosophila connectome reveals axo-axonic synapses on descending neurons." iScience 29, no. 5 (2026).
Acknowledgement R.P. was funded by an IBRO Collaborative Research Grants. This material is also based upon work supported by the U.S. Department of Education under Research and Development Infrastructure grant no. P116H230018. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the U.S. Department of Education.
