Modulatory neurons project widely throughout the brain, dynamically altering network processing based on an animal's physiological state. The connectivity of individual modulatory neurons can be complex, as they often receive input from a variety of sources and are diverse in their physiology, structure, and gene expression profiles. To establish basic principles about the connectivity of individual modulatory neurons, we examined a pair of identified neurons, the "contralaterally projecting, serotonin-immunoreactive deutocerebral neurons" (CSDns), within the olfactory system of Drosophila Specifically, we determined the neuronal classes providing synaptic input to the CSDns within the antennal lobe (AL), an olfactory network targeted by the CSDns, and the degree to which CSDn active zones are uniformly distributed across the AL. Using anatomical techniques, we found that the CSDns received glomerulus-specific input from olfactory receptor neurons (ORNs) and projection neurons (PNs), and networkwide input from local interneurons (LNs). Furthermore, we quantified the number of CSDn active zones in each glomerulus and found that CSDn output is not uniform, but rather heterogeneous, across glomeruli and stereotyped from animal to animal. Finally, we demonstrate that the CSDns synapse broadly onto LNs and PNs throughout the AL but do not synapse upon ORNs. Our results demonstrate that modulatory neurons do not necessarily provide purely top-down input but rather receive neuron class-specific input from the networks that they target, and that even a two cell modulatory network has highly heterogeneous, yet stereotyped, pattern of connectivity.SIGNIFICANCE STATEMENT Modulatory neurons often project broadly throughout the brain to alter processing based on physiological state. However, the connectivity of individual modulatory neurons to their target networks is not well understood, as modulatory neuron populations are heterogeneous in their physiology, morphology, and gene expression. In this study, we use a pair of identified serotonergic neurons within the Drosophila olfactory system as a model to establish a framework for modulatory neuron connectivity. We demonstrate that individual modulatory neurons can integrate neuron class-specific input from their target network, which is often nonreciprocal. Additionally, modulatory neuron output can be stereotyped, yet nonuniform, across network regions. Our results provide new insight into the synaptic relationships that underlie network function of modulatory neurons.

Animals use vision to perform such diverse behaviors as finding food, interacting socially with other animals, choosing a mate, and avoiding predators. These behaviors are complex and the visual system must process color, motion, and pattern cues efficiently so that animals can respond to relevant stimuli. The visual system achieves this by dividing visual information into separate pathways, but to what extent are these parallel streams separated in the brain? To answer this question, we recorded intracellularly in vivo from 105 morphologically identified neurons in the lobula, a major visual processing structure of bumblebees (Bombus impatiens). We found that these cells have anatomically segregated dendritic inputs confined to one or two of six lobula layers. Lobula neurons exhibit physiological characteristics common to their respective input layer. Cells with arborizations in layers 1-4 are generally indifferent to color but sensitive to motion, whereas layer 5-6 neurons often respond to both color and motion cues. Furthermore, the temporal characteristics of these responses differ systematically with dendritic branching pattern. Some layers are more temporally precise, whereas others are less precise but more reliable across trials. Because different layers send projections to different regions of the central brain, we hypothesize that the anatomical layers of the lobula are the structural basis for the segregation of visual information into color, motion, and stimulus timing.

Serotonergic neurons project widely throughout the brain to modulate diverse physiological and behavioral processes. However, a single-cell resolution understanding of the connectivity of serotonergic neurons is currently lacking. Using a whole-brain EM dataset of a female Drosophila, we comprehensively determine the wiring logic of a broadly projecting serotonergic neuron (the CSDn) that spans several olfactory regions. Within the antennal lobe, the CSDn differentially innervates each glomerulus, yet surprisingly, this variability reflects a diverse set of presynaptic partners, rather than glomerulus-specific differences in synaptic output, which is predominately to local interneurons. Moreover, the CSDn has distinct connectivity relationships with specific local interneuron subtypes, suggesting that the CSDn influences distinct aspects of local network processing. Across olfactory regions, the CSDn has different patterns of connectivity, even having different connectivity with individual projection neurons that also span these regions. Whereas the CSDn targets inhibitory local neurons in the antennal lobe, the CSDn has more distributed connectivity in the LH, preferentially synapsing with principal neuron types based on transmitter content. Last, we identify individual novel synaptic partners associated with other sensory domains that provide strong, top-down input to the CSDn. Together, our study reveals the complex connectivity of serotonergic neurons, which combine the integration of local and extrinsic synaptic input in a nuanced, region-specific manner.SIGNIFICANCE STATEMENT All sensory systems receive serotonergic modulatory input. However, a comprehensive understanding of the synaptic connectivity of individual serotonergic neurons is lacking. In this study, we use a whole-brain EM microscopy dataset to comprehensively determine the wiring logic of a broadly projecting serotonergic neuron in the olfactory system of Drosophila Collectively, our study demonstrates, at a single-cell level, the complex connectivity of serotonergic neurons within their target networks, identifies specific cell classes heavily targeted for serotonergic modulation in the olfactory system, and reveals novel extrinsic neurons that provide strong input to this serotonergic system outside of the context of olfaction. Elucidating the connectivity logic of individual modulatory neurons provides a ground plan for the seemingly heterogeneous effects of modulatory systems.