Neuronal wiring diagram of an adult brain

The completeness of the FlyWire brain connectome enables tracing complete pathways from sensory inputs to motor outputs. We demonstrate this capability by examining circuits that emanate from the ocellar ganglion and leveraging cell-type information. In addition to the large compound eyes, flying insects have smaller visual sensory organs, including the three ocelli on the dorsal surface of the head cuticle (Fig. 7a). The ocelli project a blurry image of light-level changes in the UV and blue region of the spectrum and are thought to be useful for flight control and orientation relative to the horizon. Notably, although the role of the ocelli has been hypothesized (for example, light-level differences between the eyes when the fly is shifted off axis should quickly drive righting motions of the head, wings and body to stabilize gaze and re-orient the body), little is known about the circuitry downstream of this sensory organ that would mediate this function.

We find that photoreceptor axons (n = 273) from the three ocelli innervate three distinct regions of the ocellar ganglion separated by glial sheets (Fig. 7a,b). The ocellar ganglion additionally contains 63 neurons that we categorized into four broad groups (Fig. 7c and Extended Data Fig. 10a): local neurons (n = 16), 2 types of interneurons, divided on the basis of their arborizations and caliber (OCG01 (n = 12), OCG02 (n = 8)), descending neurons (DNp28, n = 2), and centrifugal or feedback neurons (n = 25). Ocellar local neurons are small and connect sparsely with photoreceptors from all ocelli.

Twelve OCG01 interneurons and two descending neurons (DNp28, one per lateral ocellus) represent the main pathway from the ocellar ganglion to the central brain. DNp28 projects to the intermediate, haltere, wing and neck tectula of the ventral nerve cord. In each ocellus, half of the OCG01s were inferred to express glutamate (likely inhibitory), and the other half were inferred to express acetylcholine (likely excitatory). There are four OCG01s per ocellus (Fig. 7d). OCG01s tile the ocellar ganglion, indicating that their receptive fields tile the visual fields of the ocelli (Extended Data Fig. 10b,c). OCG02 axons are much thinner than OCG01 axons, and likely transmit signals more slowly. Two OCG02 subgroups (a and b) innervate similar neuropils to the OCG01s (inferior posterior slope (IPS) and superior posterior slope (SPS)), and OCG02c neurons target the PLP, a brain region that also receives input from VPNs from the compound eyes.

Neurons downstream from OCG01s in the IPS, SPS and GNG receive inhibitory input from the ipsilateral ocellus and excitatory input from the contralateral ocellus (Fig. 7d, left), and the amount of synaptic input from each ocellus is tightly correlated (Fig. 7e, R = 0.78, P < 10⁻²⁶)—this balance is likely to be a key ingredient in how signals are integrated (the descending circuits are activated by a signal difference between the eyes). We found that 15 different descending neurons each receive more than 200 synapses from the OCG01 neurons. For example, 2 descending neurons in each hemisphere received more than 30% of their synaptic inputs in the brain from ocellar projection neurons: DNp20/DNOVS1 (left: 57%, right: 44%) and DNp22/DNOVS2 (left: 36%, right: 33%). DNOVS1 and other descending neurons with strong input from OCG01s generally also receive strong input from ipsilateral VPNs (neurons that connect the optic lobe to the central brain) (Extended Data Fig. 10d). For example, DNOVS1 is known to be activated by rotational optic flow fields across the compound eye, and projects to the neck motor system. A handful of glutamatergic (putative inhibitory) VPNs also sparsely innervate descending neurons in both hemispheres. As the ocelli transmit mainly information about light levels, the dense integration with motion direction signals from the compound eyes was not previously appreciated, but should aid in precision adjustments of head and body movements for gaze stabilization and flight control.

There is also extensive feedback from the brain directly to the ocellar ganglion via 25 ocellar centrifugal neurons (OCC). We found striking targeting specificity of two OCC subgroups (OCC01a and OCC01b, predicted to be cholinergic) which synapse onto all OCG01 and DNp28 neurons with strong connections compared with their overall synaptic budget (Extended Data Fig. 10e). The OCC01s receive input in a wide range of neuropils, notably the SEZ, as well as IPS and SPS, the same neuropils that receive inputs from the OCG projection neurons (Extended Data Fig. 10f). The role of the OCCs in gating visual information and potentially driving the OCGs in the absence of photoreceptor activity remains to be determined.

On the basis of our analysis of connectivity, we hypothesize how the pathways from the ocelli to descending neurons function (Fig. 7f). As in a Braitenberg vehicle for phototaxis, excitation and inhibition are organized so that the head and body of the fly should roll around the anteroposterior axis to orient the ocelli towards light. In this compact example, the whole-brain connectome, which extends from brain inputs to outputs, uncovers new pathways and facilitates the generation of testable hypotheses for circuit mechanisms of sensorimotor behaviour.