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 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. 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 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.

Serotonergic neurons modulate diverse physiological and behavioral processes in a context-dependent manner, based on their complex connectivity. However, their connectivity has not been comprehensively explored at a single-cell resolution. Using a whole-brain EM dataset we determined the wiring logic of a broadly projecting serotonergic neuron (the "CSDn") in Drosophila. Within the antennal lobe (AL; first-order olfactory region), the CSDn receives glomerulus-specific input and preferentially targets distinct local interneuron subtypes. Furthermore, the wiring logic of the CSDn differs between olfactory regions. The CSDn innervates the AL and lateral horn (LH), yet does not maintain the same synaptic relationship with individual projection neurons that also span both regions. Consistent with this, the CSDn has more distributed connectivity in the LH relative to the AL, preferentially synapsing with principal neuron types based on presumptive transmitter content. Lastly, we identify protocerebral neurons that provide abundant synaptic input to the CSDn. Our study demonstrates how an individual modulatory neuron can interact with local networks and integrate input from nonolfactory sources.

Serotonin plays different roles across networks within the same sensory modality. Previously, we used whole-cell electrophysiology in Drosophila to show that serotonergic neurons innervating the first olfactory relay are inhibited by odorants (Zhang and Gaudry, 2016). Here we show that network-spanning serotonergic neurons segregate information about stimulus features, odor intensity and identity, by using opposing coding schemes in different olfactory neuropil. A pair of serotonergic neurons (the CSDns) innervate the antennal lobe and lateral horn, which are first and second order neuropils. CSDn processes in the antennal lobe are inhibited by odors in an identity independent manner. In the lateral horn, CSDn processes are excited in an odor identity dependent manner. Using functional imaging, modeling, and EM reconstruction, we demonstrate that antennal lobe derived inhibition arises from local GABAergic inputs and acts as a means of gain control on branch-specific inputs that the CSDns receive within the lateral horn.

The circadian rhythm is a fundamental process that regulates the sleep–wake cycle. This rhythm is regulated by core clock genes that oscillate to create a physiological rhythm of circadian neuronal activity. However, we do not know much about the mechanism by which circadian inputs influence neurons involved in sleep–wake architecture. One possible mechanism involves the photoreceptor cryptochrome (CRY). In Drosophila, CRY is receptive to blue light and resets the circadian rhythm. CRY also influences membrane potential dynamics that regulate neural activity of circadian clock neurons in Drosophila, including the temporal structure in sequences of spikes, by interacting with subunits of the voltage-dependent potassium channel. Moreover, several core clock molecules interact with voltage-dependent/independent channels, channel-binding protein, and subunits of the electrogenic ion pump. These components cooperatively regulate mechanisms that translate circadian photoreception and the timing of clock genes into changes in membrane excitability, such as neural firing activity and polarization sensitivity. In clock neurons expressing CRY, these mechanisms also influence synaptic plasticity. In this review, we propose that membrane potential dynamics created by circadian photoreception and core clock molecules are critical for generating the set point of synaptic plasticity that depend on neural coding. In this way, membrane potential dynamics drive formation of baseline sleep architecture, light-driven arousal, and memory processing. We also discuss the machinery that coordinates membrane excitability in circadian networks found in Drosophila, and we compare this machinery to that found in mammalian systems. Based on this body of work, we propose future studies that can better delineate how neural codes impact molecular/cellular signaling and contribute to sleep, memory processing, and neurological disorders.

19Serotonergic neurons modulate diverse physiological and behavioral processes in a 20 context-dependent manner, based on their complex connectivity. However, their 21 connectivity has not been comprehensively explored at a single-cell resolution. Using a 22 whole-brain EM dataset we determined the wiring logic of a broadly projecting 23 serotonergic neuron (the "CSDn") in Drosophila. Within the antennal lobe (AL; first-order 24 olfactory region), the CSDn receives glomerulus-specific input and preferentially targets 25 distinct local interneuron subtypes. Furthermore, the wiring logic of the CSDn differs 26 between olfactory regions. The CSDn innervates the AL and lateral horn (LH), yet does 27 not maintain the same synaptic relationship with individual projection neurons that also 28 span both regions. Consistent with this, the CSDn has more distributed connectivity in 29 the LH relative to the AL, preferentially synapsing with principal neuron types based on 30 presumptive transmitter content. Lastly, we identify protocerebral neurons that provide 31 abundant synaptic input to the CSDn. Our study demonstrates how an individual 32 modulatory neuron can interact with local networks and integrate input from non-33 olfactory sources. 35Introduction 36 Every neural network receives modulatory input from a variety of sources [1, 2], 37 and in some cases from heterogeneous populations of neurons that release the same 38 modulatory transmitter [3][4][5]. In mammals, one ubiquitous neuromodulator, serotonin, is 39 released by tens of thousands to hundreds of thousands of neurons which originate in 40 the raphe nuclei and project throughout the brain [6, 7]. Serotonergic raphe neurons are 41 highly diverse in their projections, connectivity, and electrophysiological properties, and 42 2 are implicated in a wide breadth of behaviors and physiological processes [4,[8][9][10][11][12][13][14][15][16]. 43 Further, the raphe system receives monosynaptic input from up to 80 anatomical areas 44 [8, 9]. As a result, a significant amount of work has focused on disentangling the 45 functional and behavioral roles of serotonergic neurons. Several recent studies have 46 suggested that serotonergic raphe neurons may be organized into functional 47 subpopulations based on neuroanatomy, electrophysiology, and behavior [4, 12,[17][18][19][20][21]. 48For example, two parallel sub-systems of serotonergic raphe neurons collateralize 49 complimentarily and are both activated by reward, yet have opposing responses to 50 aversive stimuli and promote distinct behaviors [18]. Sparse neuron reconstructions in 51 mice show that a single serotonin neuron can interconnect the olfactory bulb, piriform 52 cortex, and anterior olfactory nucleus [19], demonstrating that a single serotonergic 53 neuron can arborize several processing stages within the same sensory modality. Thus, 54 determining the precise patterns of connectivity of single serotonergic neurons within 55 and across the brain regions will be critical for understanding t...

SignificanceThe centrifugal innervation of neuronal circuits is ubiquitous across centralized nervous systems. Such inputs often arise from modulatory neurons that arborize broadly throughout the brain. How information is integrated in such cells and how release from their distant terminals is regulated remains largely unknown. We show that a serotonergic neuron that innervates multiple stages of odor processing in Drosophila has distinct activity throughout its neurites, including opposite polarity responses in first and second order olfactory neuropils. Disparate activity arises from local interactions within each target region. Our results show that such neurons exhibit dendritic computation rather than somatic integration alone, and that examining local interactions at release sites is critical for understanding centrifugal innervation. AbstractAll centralized nervous systems possess modulatory neurons that arborize broadly across multiple brain regions. Such modulatory systems are critical for proper sensory, motor, and cognitive processing. How single modulatory neurons integrate into circuits within their target destination remains largely unexplored due to difficulties in both labeling individual cells and imaging across distal parts of the CNS. Here, we take advantage of an identified modulatory neuron in Drosophila that arborizes in multiple olfactory neuropils. We demonstrate that this serotonergic neuron has opposing odor responses in its neurites of the antennal lobe and lateral horn, first and second order olfactory neuropils respectively. Specifically, processes of this neuron in the antennal lobe have responses that are inhibitory and odor-independent, while lateral horn responses are excitatory and odor-specific. The results show that widespread modulatory neurons may not function purely as integrate-and-fire cells, but rather their transmitter release is locally regulated based on neuropil. As nearly all vertebrate and invertebrate neurons are subject to synaptic inputs along their dendro-axonic axis, it is likely that our findings generalize across phylogeny and other broadly-projecting modulatory systems.

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Equestrian sports vary in degree of human-animal bond and affiliation, from catch-ride to perceptions of ownership. The Modern Pentathlon has not garnered mainstream media coverage until the 2020 Olympics in Tokyo. Unfortunately, the rise in conversation and coverage was in response and reaction to poor horsemanship, negative behavior, and aggressive coaching. The events of the 2020 Modern Pentathlon were profound in that they highlighted the extreme juxtaposition of competitions that focus on bonded relationships and catch-riding experiences. The implications are far reaching with increased critique of equestrian sports, transparency about the lack of riding training athletes engaged in prior to competition, and the omission of partnership between horse and rider. The Modern Pentathlon is discussed in the context of the human-equine bond. The standards for equine welfare and wellbeing is considered in regard to the role of the rider in supporting their equine partner in the face of competition, including the implications of emotional distress in reaction to disappointment, and tragedy. Additionally, the role of riders when they are not engaged in the competitive sport (e.g., training and general care of their equine partners). Recommendations for the process and guidelines of the sport are delineated, as well as professional opportunities to strengthen the sport.