The Feeding Connectome: Convergence of Monosynaptic and Polysynaptic Sensory Paths onto Common Motor Outputs

Little is known about the organization of central circuits by which external and internal sensory inputs act on motor outputs to regulate fundamental behaviors such as feeding. We reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of Drosophila larvae. The input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS, as defined by distribution patterns of their presynaptic sites. The output neurons consist of pharyngeal motor neurons, serotonergic modulatory neurons, and neuroendocrine neurons that target the ring gland, a key endocrine organ. Monosynaptic connections from a set of sensory synaptic compartments cover the motor and endocrine targets in overlapping domains. Polysynaptic routes can be superimposed on top of the monosynaptic connections, resulting in divergent sensory paths that converge on common motor outputs. A completely different set of sensory compartments is connected to the mushroom body calyx of the memory circuits. Our results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths.


Introduction
Motor outputs of a nervous system can be broadly defined into those carried out by the muscles to produce movements and by the neuroendocrine glands for secretion (Shepherd 1987). Both of these behavioral and physiological events are regulated by a network of motor neurons, interneurons and sensory neurons, and a major open question is how one neural path is selected from multiple possible paths to produce a desired motor output (Grillner et al. 2005). Nervous system complexity and tool availability have strongly dictated the type of experimental system and analysis that can be used to address this issue, such as a focus on a particular organism, behavior or type of neuron. In this context, the detailed illustrations of different parts of nervous systems at neuronal level as pioneered by Cajal, to the first complete description of a nervous system wiring diagram at synaptic level for C. elegans, demonstrate the power of systematic neuroanatomical analysis in providing a foundation and guide for studying nervous system function (Ramon y Cajal 1894; White et al. 1986). However, the technical challenges posed by such analysis have limited the type of organisms for which synaptic resolution mapping can be performed at the scale of an entire nervous system (Swanson and Lichtman 2016;Schlegel et al. 2017;Kornfeld and Denk 2018).
Analysis of the neural circuits that mediate food intake in the Drosophila larvae offers numerous advantages in meeting the challenge of neuroanatomical mapping at a whole brain level, and combining it with the ability to perform behavioral and physiological experiments. The muscle system that generates the different movements necessary for transporting food from the pharynx to the esophagus, as well as the endocrine system responsible for secreting various hormones for metabolism and growth, have both been well described (Kühn 1971;Siegmund and Korge 2001;Buch and Pankratz 2009;Schoofs et al. 2010).
These are also complemented by the analysis of feeding behavior in adult flies (Gelperin 1971;Dethier 1976;McKellar 2016). Although there is broad knowledge at the morphological level on the organs underlying larval feeding behavior and physiology, as well as on the nerves innervating them in the periphery (Schoofs et al. 2010(Schoofs et al. , 2014b, the central connectivity of the afferent and efferent neurons within these nerves are largely unknown. At the same time, advances in the EM reconstruction of an entire CNS of a first instar larva (Ohyama et al. 2015;Schlegel et al. 2016;Schneider-Mizell et al. 2016;Berck et al. 2016;Eichler et al. 2017;Gerhard et al. 2017) (summarized in Kornfeld & Denk, 2018 offers an opportunity to elucidate an animals' feeding system on a brain-wide scale and at synaptic resolution. As part of this community effort, we recently performed an integrated analysis of fast synaptic and neuropeptide receptor connections for an identified cluster of 20 interneurons that express the neuropeptide hugin, a homolog of the mammalian neuropeptide neuromedin U, and which regulates food intake behavior (Melcher et al. 2006;Schoofs et al. 2014a;Schlegel et al. 2016). This analysis showed that the class of hugin neurons modulating food intake receives direct synaptic inputs from a specific group of sensory neurons, and in turn, makes mono-synaptic contacts to output neuroendocrine cells. The study not only provided a starting point for a combined approach to studying synaptic and neuropeptidergic circuits (Diao et al. 2017;Williams et al. 2017), but a basis for a comprehensive mapping of the sensory and motor neurons that innervate the major feeding and endocrine organs.
Feeding is one of the most universal and important activities that animals engage in. Despite large differences in the morphology of the external feeding organs, the internal gut structures are quite similar across different animals (Campbell 1990); indeed, even within closely related species, there can be large differences in the external organs that detect and gather food, whereas the internal organs that transport food through the alimentary canal are much more similar. Recent studies have also pointed out the functional similarities between the subesophageal zone in insects and the brainstem in vertebrates for regulating feeding behavior (Schoofs et al. 2014a;Yapici et al. 2016;McKellar 2016). In mammals, the different cranial nerves from the medulla innervate distinct muscles and glands of the foregut ( Figure 1A). For example, the VIIth cranial nerve (facial nerve) carries taste sensory information from anterior 2/3 of the tongue, and innervates the salivary glands, and lip and facial muscles. The IXth cranial nerve (glossopharyngeal nerve) receives taste inputs from the posterior 1/3 of the tongue, and innervates the salivary glands and pharynx muscles. The Xth cranial nerve (vagus nerve) receives majority of the sensory inputs from the enteric nervous system of the gut, and innervates pharynx and esophagus muscles. The XIth cranial nerve (spinal accessory nerve) and the XIIth cranial nerve (hypoglossal nerve) are thought to carry strictly motor information which innervate the pharynx and neck muscles, and the tongue muscles (Cordes 2001;Simon et al. 2006). The distinct cranial nerves project onto topographically distinct areas in the medulla of the brainstem ( Figure 1A). We also note that olfactory information is carried by cranial nerve I, a strictly sensory nerve that projects to the olfactory bulb (OB), an area topographically distinct from the brainstem. In addition, there are direct neuronal connections between the brainstem and the hypothalamus, the key neuroendocrine center of vertebrates (D'Agostino et al. 2016;Liu et al. 2017).
Analogously, distinct pharyngeal nerves of the Drosophila larva are connected to the subesophageal zone (SEZ), and also carry sensory and motor information that regulate different parts of the body (Figure 1B). The AN (antennal nerve) carries sensory information from the olfactory, pharyngeal and internal organs, and innervates the pharyngeal muscles for pumping in food. The neurons that innervate the major endocrine center and the enteric nervous system also project through the AN. Note also that the olfactory sensory organs project to the antennal lobe (AL), which abuts the SEZ yet is topographically separate. The MN (maxillary nerve) carries external and pharyngeal sensory information, and innervates the mouth hooks, whose movements are involved In sum, although a large body of knowledge exists on the gross anatomy of the nerves that target the feeding organs in vertebrates and invertebrates, the synaptic pathways within the brain that interconnect the sensory inputs and motor outputs of the individual nerves remain to be elucidated.
In this paper, we have reconstructed all sensory and motor neurons of the three pharyngeal nerves that underlie the feeding motor program of Drosophila larvae. The activity of these nerves has previously been shown to be sufficient for generating the feeding motor pattern in isolated nervous system preparations, and that the central pattern generators (CPGs) for food intake lie in the SEZ (Schoofs et al. 2010;Hückesfeld et al. 2015). We then identified all monosynaptic connections between the sensory and motor neurons, thus providing a full monosynaptic reflex circuit for food intake. We also mapped polysynaptic pathways that are integrated onto the monosynaptic reflex circuits. In addition, we mapped the multisynaptic non-olfactory neuron connections from the sensory neurons to the mushroom body memory circuit (Eichler et al. 2017), and show that these are different from those involved in monosynaptic reflex circuits. Reflex circuits can be seen to represent the simplest synaptic architecture in the nervous system, as formulated by Charles Sherrington (Sherrington 1906). Anatomical reconstructions of monosynaptic and poly-synaptic reflex circuits can also be seen in the works of Cajal (Ramon y Cajal 1894; Swanson 2000). We propose a model of how different mono-and polysynaptic pathways can be traversed from a set of sensory neurons to specific output neurons, which has relevance for understanding the mechanisms of action selection.

EM reconstruction of the pharyngeal nerves
We reconstructed all axons within the three pharyngeal nerves into the CNS using a ssTEM volume of an entire larval CNS (Ohyama et al. 2015) (Figure 1C). The sensory projections were those that ended blindly, whereas the motor neurons were those with somata in the CNS. For sensory inputs, a regionalization of the target areas can already be seen, reflecting the fact that the nerves are fusions of different axon bundles that arise during embryonic development (Hartenstein et al. 2017;Kendroud et al. 2017). For example, only the AN has sensory projections that extend into the proto-    (Schlegel et al. 2016). We now identify here all pre-and post-synaptic sites of all the motor neurons of the different pharyngeal nerves ( Figure   2C). This includes a special class of four serotonergic neurons (the Se0 cluster) that project to the entire enteric nervous system (Huser et al. 2012;Schoofs et al. 2014b;Shimada-Niwa and Niwa 2014). These four serotonergic neurons can be further divided into one that projects anteriorly to the pharynx (Se0ph), and three that project posteriorly towards the enteric nervous system (Se0ens) (Figure 2-figure supplement 6).
A schematic summary of the pre-and post-synaptic compartments of the input and output neurons, along with their projection regions, is shown in Figure 2D. Taken together, these data define all sensory input convergence zones and motor output compartments of the three pharyngeal nerves underlying feeding motor program at synaptic resolution.

Axo-dendritic connections from sensory to output motor neurons
Having annotated all central synapses of in-and output neurons, we surprisingly found the most basic element of circuit architecture: direct monosynaptic connections between input and output neurons ( Figure 3A).

Axo-axonic connections between sensory neurons
Unexpectedly we found a high number of synaptic connections between the sensory projections within the CNS. This is in contrast to the well characterized olfactory sensory neurons that project onto the antennal lobe (AL) (Figure 4A). For example, at a threshold of two synapses the AL has none, whereas 50% of the ACa neurons have above threshold inter-sensory connections. The majority of the inter-sensory connections were between neurons of the same synaptic target compartment ( Figure 4B), which underscores the clear-cut boundaries between the sensory compartments; these connections are made both in an hierarchical manner as well as reciprocally, suggesting that sensory information processing is occurring already at an inter-sensory level in the brain (Figure 4-figure supplement 1). Viewed from output synapses of the sensory neurons, the percentage of sensory synapses connecting to other sensory neurons are small relative to total sensory outputs (less than 2% of 73,000 synapses); however viewed from input side of the sensory neurons, a high percentage (e.g., 45% for ACa) of their total synaptic inputs originate from other sensory neurons (Figure 4C,D). We also note that sensory neurons from ACp and VM have inter-sensory connections even between neurons of different nerves (Figure 4-figure supplement 1-2), indicating integration of sensory information from different body regions at the sensory neuron level.

Mapping peripheral origins of monosynaptic circuits
We next investigated the peripheral origins of the sensory neurons that comprise the different synaptic compartments. This was accomplished by using various sensory receptor Gal4 lines to follow the projections from the sensory organs into the CNS. The mapping was aided by the fact that the pharyngeal projections enter the SEZ in distinct bundles that can be observed in both light and EM microscopic sections (Figure 5A,B). The AN and the MN each have three bundles (these nerves are formed by fusion of several axon bundles during development (Hartenstein et al. 2017), whereas the PaN has just one. The well characterized projections from the external olfactory organ (DOG) to the antennal lobe (AL), for example, use one of the bundles in the AN (Bundle 3 of the AN). Figure   5B illustrates the basic strategy, using two of the gustatory receptors (GRs) to follow the projections from the enteric nervous system into the CNS. This analysis, denoting the receptor line used and their expression in the sensory organs and the axon bundles of each pharyngeal nerve, is summarized in Figure 5C ( Figure 5-figure supplement 1-9 for detailed stainings). These results were then used to determine the peripheral origin (enteric/internal, pharyngeal, external) of the sensory neurons that comprise the 7 synaptic compartments defined earlier (Figure 5C,D). This revealed a wide spectrum in compartment composition. For example, the ACa is derived 100% from the enteric nervous system, while the AVa is 93 % enteric; these are the only two sensory compartments with enteric origin. As a comparison, the antennal lobe (AL) is derived 100% from a single external sensory organ, the dorsal organ. Interestingly, the topographical location of the sensory compartments within the CNS broadly mirrors in a concentric manner the peripheral origins from which they derive: the inner-most enteric organs project to the anterior most region, the pharyngeal sensory organs project to the middle region, while the most external organs project to the outer-most region ( Figure 5E). Recent light microscopy study on the projections of somatosensory neurons onto the adult brain also showed a topographically separate target areas in the brain (Tsubouchi et al. 2017).
In addition, as we progress from the inner to the outer layers, there is a graded contribution of connections having monosynaptic sensory-to-output contacts (highest being between the inner layers).
In other words, the greatest number of monosynaptic connects occur between the enteric system and the neuroendocrine system, followed by the pharyngeal sensory organs to the AN motor neurons, and the least from the external organs.
We point out, for example, that the olfactory projections from the external dorsal organ have no monosynaptic connections whatsoever to any output neurons. In this context, the Se0 serotonergic neurons appear to play a special role, as these have the greatest number of monosynaptic contacts from both the enteric system and the pharyngeal sensory organs.

Multi-synaptic connections to the mushroom body (MB) memory circuits
As a contrast to direct input-to-output connections, we additionally looked at connections to a higher brain center for learning and memory, the mushroom body. To this end, we checked previ- gustatory projections neurons derive from ACp ( Figure 6C, right panel). This is also consistent with the view that the ACp is the primary sensory compartment onto which the external and pharyngeal gustatory sensory organs project (Colomb et al. 2007;Hartenstein et al. 2017).

Integration of polysynaptic connections onto monosynaptic circuits
We then asked how the hugin neuropeptide (Drosophila neuromedin U homolog) circuit, which relays gustatory information to the protocerebrum (Schlegel et al. 2016;Hückesfeld et al. 2016), would be positioned with respect to the monosynaptic reflex and multisynaptic MB memory circuits. Based on our earlier studies on mapping sensory inputs onto hugin protocerebrum neurons (huginPC) (Schlegel et al. 2016;Hückesfeld et al. 2016), we were expecting most inputs from the ACp, which is the primary gustatory sensory compartment (Colomb et al. 2007;Hartenstein et al. 2017). However, most of the huginPC neurons receive inputs from the sensory compartments ACa and AVa, which are the two major monosynaptic compartments that originate from enteric organs.
HuginPC neurons do receive inputs from the external and pharyngeal organs (i.e., through sensory compartment ACp), but to a much smaller degree (Figure 7-figure supplement 1). Thus, unlike the MB circuit that utilizes a completely new set of sensory inputs, the huginPC circuit is associated with a feeding related monosynaptic circuit.  (Figure 7A,B). We then asked, using the same threshold, how many different di-synaptic paths exist and how often a particular interneuron is used for the different possible converging paths ("degree" of convergence). We also calculated the relative synaptic strengths of the connection among the various paths ("ranking index" A potential functional consequence of such circuit architecture can be seen if we now include all the sensory inputs onto the interneurons. As an example, we take Dilp 1L as the common output, and the interneurons H1 (a huginPC neuron) and "S" (not previously described) as two of the polysynaptic paths onto a common output ( Figure 7C).
One consequence of such superimposition is that the type of sensory information that can reach a In sum, we propose that the different path possibilities allow different strength and combination of sensory inputs to be evaluated, which would then determine which synaptic path will dominate to a given output.

Discussion
We This set of monosynaptic connections can thus be seen to represent an elemental circuit for feeding, since the connections between the input and output neurons cannot be broken down any further.
Vast majority of the sensory inputs comprising this "elemental feeding circuit" derive from the enteric nervous system to target the pharyngeal muscles involved in food intake and neuroendocrine output organs. However, there is a small number of monosynaptic reflex connection that originate from the somatosensory compartment. The output neurons targeted by these somatosensory neurons are motor neurons that control mouth hook movements and head tilting, movements which are involved in both feeding and locomotion. In this context, it is noteworthy that monosynaptic reflex connections are found to a much lesser degree in the larval ventral nerve cord, which generates locomotion (unpublished data from Ohyama et al., 2015). An analogous situation exists in C. elegans, where majority of the monosynaptic reflex circuits are found in the head motor neurons and not in the body (Yan et al. 2017).
One reason could be due to the relative complexity in the response necessary for food intake as compared to locomotion. For example, a decision to finally not to swallow a harmful substance, once in the mouth, may require a more local response, e.g. muscles limited to a very specific region of the pharynx and esophagus, where monosynaptic arc might suffice. By contrast, initiating escape behaviors requires a more global response with respect to the range and coordination of body movements involved.

Monosynaptic connections between the sensory neurons
The inter-sensory connections show a combination of hierarchical and reciprocal connections, which may increase the regulatory capability and could be especially important for monosynaptic circuits. By contrast, very few monosynaptic connections exist between the larval olfactory, chordotonal or nociceptive class IV sensory in the body (Ohyama et al. 2015;Jovanic et al. 2016;Gerhard et al. 2017). Interestingly, there is also a much higher percentage of intersensory connections between olfactory neurons in the adult as compared to the larva, which could function in gain modulation at low signal intensities (Tobin et al. 2017). This might be attributable to adults requiring faster processing of olfactory information during flight navigation (or mating), and/or to minimize metabolic cost (Wilson 2014). Whether such explanation also applies to the differences in intersensory connection between the different types of sensory neurons in the larvae remains to be determined.

Superimposition of polysynaptic pathways onto monosynaptic circuits
We found very few cases where a monosynaptic path between any sensory-output pair is not additionally connected via a polysynaptic path. An interesting question in the context of action selection mechanism is which path a sensory signal uses to reach a specific target neuron. For exam-  (Schoofs et al. 2014a).
The coexistence of polysynaptic and monosynaptic paths could also be relevant for circuit variability and compensation (Leonardo 2005;Marder and Goaillard 2006): destruction of any given path would still enable the circuit to function, but with more restrictions on the precise types of sensory information it can respond to. In certain cases, this may even lead to strengthening of alternate paths as a form of synaptic plasticity. An open issue is how the sensory synaptic compartments might be connected to the feeding central pattern generators (CPGs) which have been demonstrated to exist in the SEZ (Schoofs et al. 2010;Hückesfeld et al. 2015), especially since CPGs are defined as neural circuits that can generate rhythmic motor patterns in the absence of sensory input. However, the modulation of CPG rhythmic activity can be brought about by sensory and neuromodulatory inputs (Marder and Bucher 2001;Marder 2012). A complete circuit reconstruction of the larval SEZ circuit may shed some light on the circuit structure of feeding CPGs. signal and an autonomic secretory response in response to food (Pavlov 1927;Todes 2001). Although a comparable autonomic response has not been analyzed in the larvae, analogous associative behavior based on odor choice response has been well studied (Aceves-Pina and Quinn 1979;Gerber and Stocker 2007;Eichler et al. 2017;Widmann et al. 2018). It is also noteworthy that in the Aplysia, classical conditioning of the gill withdrawal reflex involves monosynaptic connections between a sensory neuron (mechanosensory) and a motor neuron, and neuromodulation by serotonin (Bailey et al. 2000). This constellation has similarities with the elemental feeding circuit consisting of sensory, motor and serotonergic modulatory neurons. For more complex circuits of feeding behavior in the mouse, a memory device for physiological state, such as hunger, has been reported involving synaptic and neuropeptide hormone circuits (Yang et al. 2011). Identification of output neurons of the MB calyx would help address how memory circuits interact with reflex feeding circuits.

Control of reflexes
Feeding behavior manifests itself from the most primitive instincts of lower animals, to deep psychological and social aspects in humans. It encompasses cogitating on the finest aspects of food taste and the memories evoked by the experience, to sudden reflex reactions upon unexpectedly biting down on a hard seed or shell. Both of these extremes are mediated, to a large degree, by a common set of feeding organs, but the way these organs become utilized can vary greatly. The architecture of the feeding circuit described here allows the various types of sensory inputs to converge on a limited number of output responses.
The monosynaptic pathways would be used when fastest response is needed. The presence of polysynaptic paths would enable slower and finer control of these reflex events by allowing different sensory inputs, strengths or modalities to act on the monosynaptic circuit. This can be placed in the context in the control of emotions and survival circuits (LeDoux 2012), or by cortex regulation of basic physiological or autonomic processes (Dum et al. 2016). In a striking example, pupil dilation, a reflex response, has been used as an indicator of cognitive activity (Hess and Polt 1964;Kahneman and Beatty 1966;Larsen and Waters 2018). Here, a major function of having more complex circuit modules on top of monosynaptic circuits may be to allow a finer regulation of feeding reflexes, and perhaps of other reflexes or instinctive behaviors.
As an outlook, our analysis provides an architectural framework of how a feeding circuit is organized in the CNS. The circuit is divided into two main axes that connect the input to the output systems: the sensory-neurosecretory cell axis and the sensory-motor neuron axis (Swanson 2011

Neuronal reconstruction
All reconstructions were based on an ssTEM (serial section transmission electron microscope) data set of a complete nervous system of a 6-h-old [iso] CantonS G1 x w1118 larva as described in (Ohyama et al. 2015). Using a modified version of the web-based software CATMAID (Saalfeld et al. 2009) we manually reconstructed neurons' skeletons and annotated synapses following the methods described in (Ohyama et al. 2015) and . Sensory and motor neurons were identified by reconstructing all axons in the antennal nerve, maxillary nerve and the prothoracic accessory nerve. Further, neurons with their soma in the brain and projections through one of the three pharyngeal nerves have been identified as motor neurons and serotonergic output neurons. All annotated synapses represent fast, chemical synapses equivalent to previously described typical criteria: thick black active zones, pre-and postsynaptic membrane specializations (Prokop and Meinertzhagen 2006).

Morphology similarity score
To neuron morphologies (Figure ), we used a morphology similarity score decribed by Kohl et al. 2013. Briefly, reconstructions of neurons are con- .;< where n is the number of points in the query neuron, d_ij is the distance between point i in the query neuron and its nearest neighbor, point j, in the target neuron and q_i and t_j are the tangent vectors at these points. σ determines how close in space points must be to be considered similar. For our calculations, we used σ of 2 um. Similarity score algorithm was implemented in a Blender plugin (https://github.com/schlegelp/CATMAIDto-Blender).

Synapse similarity score
To calculate similarity of synapse placement between two neurons, we calculated the synapse similarity score (Figure 2; Figure 2-

Clustering
Clusters for dendrograms were created based on the mean distance between elements of each cluster using the average linkage clustering method.

Percentage of synaptic connections
Percentage of synaptic connections was calculated by counting the number of synapses that constitute between neuron A and a given set of pre-or postsynaptic partners divided by the total number of either incoming our outgoing synaptic connections of neuron A. For presynaptic sites, each postsynaptic neurite counted as a single synaptic connection.

Ranking index
Ranking index was calculated by counting the number of synapses that constitute between neuron A and a given target neuron B divided by the highest number of synapses among all incoming synaptic connections of target neuron B.