Decision letter: Synaptic transmission parallels neuromodulation in a central food-intake circuit

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract NeuromedinU is a potent regulator of food intake and activity in mammals. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner. Here, we use EM-based reconstruction to generate the entire connectome of hugin-producing neurons in the Drosophila larval CNS. We demonstrate that hugin neurons use synaptic transmission in addition to peptidergic neuromodulation and identify acetylcholine as a key transmitter. Hugin neuropeptide and acetylcholine are both necessary for the regulatory effect on feeding. We further show that subtypes of hugin neurons connect chemosensory to endocrine system by combinations of synaptic and peptide-receptor connections. Targets include endocrine neurons producing DH44, a CRH-like peptide, and insulin-like peptides. Homologs of these peptides are likewise downstream of neuromedinU, revealing striking parallels in flies and mammals. We propose that hugin neurons are part of an ancient physiological control system that has been conserved at functional and molecular level. https://doi.org/10.7554/eLife.16799.001 Introduction Multiple studies have demonstrated functional conservation of fundamental hormonal systems for metabolic regulation in mammals and Drosophila. This includes insulin (Ikeya et al., 2002; Rulifson et al., 2002), glucagon (Kim and Rulifson, 2004), and leptin (Rajan and Perrimon, 2012). In addition to these predominantly peripherally released peptides, there is a range of neuropeptides that are employed within the central nervous systems (CNS) of vertebrates and have homologs in invertebrates, e.g. neuropeptide Y (NPY), corticotropin-releasing hormone (CRH) or oxytocin/vasopressin (Nässel and Winther, 2010; Nässel and Wegener, 2011; Grimmelikhuijzen and Hauser, 2012; Mirabeau and Joly, 2013; Jékely, 2013). Among these, neuromedinU (NMU) is known for its profound effects on feeding behavior and activity; NMU inhibits feeding behavior (Howard et al., 2000), promotes physical activity (Novak et al., 2007; Chiu et al., 2016), and is involved in energy homeostasis (Nakazato et al., 2000; Ivanov et al., 2002) and stress response (Hanada et al., 2001; Zeng et al., 2006). Hugin is a member of the pyrokinin/PBAN (pheromone biosynthesis activating neuropeptide) peptide family and a Drosophila homolog of NMU that has recently gained traction due to similar effects on behavior in the fly: increased hugin signaling inhibits food intake and promotes locomotion (Melcher et al., 2006; Schoofs et al., 2014; Bader et al., 2007b). In mammals, distribution of the NMU peptide, NMU-expressing cells and NMU-positive fibers is wide and complex. High levels of NMU have been reported in the arcuate nucleus of the hypothalamus, the pituitary, the medulla oblongata of the brain stem, and the spinal cord (Domin et al., 1987; Ballesta et al., 1988; Howard et al., 2000; Ivanov et al., 2004). The number of neurons involved and their morphology is unknown. In Drosophila, the distribution of hugin is less complex, yet similar: the peptide is produced by neurons in the subesophageal zone that have hugin-positive projections into the ring gland, the pars intercerebralis and ventral nerve cord (Melcher and Pankratz 2005) (Figure 1). While comparisons across large evolutionary distances are generally difficult, these regions of the fly brain were suggested to correspond to aforementioned regions of NMU occurrence based on morphological, genetic and functional similarities (Ghysen, 2003; Hartenstein, 2006). Consequently, NMU/hugin has previously been referred to as a clear example of evolutionary constancy of peptide function (Taghert and Nitabach, 2012). Figure 1 Download asset Open asset Comparison of mammalian neuromedinU and Drosophila hugin. (A) NeuromedinU (NMU) is widely distributed in the rodent CNS. NMU peptide, NMU-expressing cells and NMU-positive fibers are found in several regions of the brain stem, hypothalamus, pituitary and spinal cord (black dots). The number of neurons and their morphology is unknown. (B) In Drosophila, distribution of the homologous neuropeptide hugin is less complex and well known: hugin is expressed by sets of neurons in the subesophageal zone (SEZ) that project into the pars intercerebralis, ring gland and ventral nerve cord. (C, D) Increased NMU and hugin signaling has similar effects: feeding behavior is decreased, whereas physical activity/locomotion is increased. https://doi.org/10.7554/eLife.16799.002 Although functional and morphological aspects of neurons employing either neuropeptide have been extensively studied in the past, knowledge about their connectivity is fragmentary. While large-scale connectomic analyses in vertebrates remain challenging, generation of high-resolution connectomes has recently become feasible in Drosophila (Ohyama et al., 2015; Berck et al., 2016; Fushiki et al., 2016; Schneider-Mizell et al., 2016). We took advantage of this and performed an integrated analysis of synaptic and G-protein-coupled receptor (GPCR)-mediated connectivity of hugin neurons in the CNS of Drosophila. Our data demonstrates that hugin neurons employ small molecule transmitters in addition to the neuropeptide. We identify acetylcholine as a transmitter that is employed by hugin neurons and find that it is required for their effect on feeding behavior. Next, we show that hugin neurons form distinct units, and demonstrate that clusters of neurons employing the same neuropeptide are remarkably different in their synaptic connectivity. One unit of hugin neurons is presynaptic to subsets of median neurosecretory cells (mNSCs) in the protocerebrum. In parallel to the synaptic connectivity, mNSCs also express the G-protein-coupled receptor PK2-R1, a hugin receptor, rendering them likely targets of both fast synaptic transmission and neuromodulatory effects from hugin neurons. These mNSCs produce diuretic hormone 44 (DH44, a CRH-like peptide) and Drosophila insulin-like peptides, both of which have mammalian homologs that are likewise downstream of NMU (Wren et al., 2002; Malendowicz et al., 2012). Endocrine function is essential to ensure homeostasis of the organism and coordinate fundamental behaviors, such as feeding, mating and reproduction, and acts as integrator of external and internal sensory cues (Swanson, 2000). Consequently, connections between sensory and endocrine systems are found across species (Yoon et al., 2005; Tessmar-Raible et al., 2007; Strausfeld 2012; Abitua et al., 2015). We show that hugin neurons receive chemosensory input in the subesophageal zone (SEZ), thereby linking chemosensory and neuroendocrine systems. Results Input and output compartments of hugin neurons The hugin gene is expressed in only 20 neurons in the Drosophila CNS. This population comprises interneurons, which are confined within the CNS, as well as efferent neurons, which leave the CNS. The interneuron type can be subdivided into those projecting to the protocerebrum (hugin-PC, eight neurons) or the ventral nerve cord (hugin-VNC, four neurons). The efferent type can be subdivided into those projecting to the ring gland (hugin-RG, four neurons) or the pharynx (hugin-PH, four neurons) (Figure 2A) (Bader et al., 2007a). Based on these morphological features, we first reconstructed all hugin neurons in an ssTEM volume covering an entire larval CNS and the major neuroendocrine organ, the ring gland (Figure 2B; see Materials and methods for details). We then localized synaptic sites, which could be readily identified as optically dense structures (Prokop and Meinertzhagen, 2006). Comparing neurons of the same class, we found the number as well as the distribution of pre- and postsynaptic sites to be very similar among hugin neurons of the same class (Figure 2C–E, Video 1). Presynaptic sites are generally defined as having small clear core vesicles (SCVs) containing classic small molecule transmitter for fast synaptic transmission close to the active zone (Prokop and Meinertzhagen, 2006). Efferent hugin neurons (hugin-RG and hugin-PH) showed essentially no presynaptic sites (<1 average/neuron) within the CNS and we did not observe any SCVs. For hugin-RG neurons, membrane specializations resembling presynaptic sites were evident at their projection target, the ring gland. These sites did contain close-by DCVs but no SCVs and had in many cases no corresponding postsynaptic sites in adjacent neurons. Instead they bordered haemal space indicating neuroendocrine release (Figure 2—figure supplement 1A). The configuration of hugin-PH terminals is unknown as their peripheral target was outside of the ssTEM volume. For the interneuron classes (hugin-PC and hugin-VNC), we found SCVs at larger presynaptic sites, indicating that they employ classic neurotransmitter in addition to the hugin peptide (Figure 2—figure supplement 1B,C). Hugin-PC and hugin-VNC neurons’ projections represent mixed synaptic input-output compartments as they both showed pre- as well as postsynaptic sites along their neurites (Figure 2D,E). Figure 2 with 1 supplement see all Download asset Open asset EM reconstruction of hugin neurons and their synaptic sites. (A) Hugin neurons are known to form four morphologically distinct classes: hugin-PC (protocerebrum), hugin-VNC (ventral nerve cord), hugin-RG (ring gland) and hugin-PH (pharynx, asterisks mark nerve exit sites). (B) Reconstruction of all hugin neurons based on serial section electron microscopy (EM) of an entire larval brain. (C–E) Spatial distribution of synaptic sites for all hugin classes. Hugin interneurons (hugin-PC and hugin-VNC) show mixed input/output compartments, and presynaptic sites indicate the existence of a small molecule transmitter in addition to the hugin neuropeptide. In contrast, Hugin-RG and hugin-PH show almost exclusively postsynaptic sites within the CNS. Each dot in D and E represents a single synaptic site. Graphs show distribution along dorsal-ventral and anterior-posterior axis of the CNS. See also Video 1. https://doi.org/10.7554/eLife.16799.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Morphology of hugin-producing neurons. Video shows morphology of hugin-producing neurons as well as distribution of their presynaptic and postsynaptic sites. Hugin interneurons (PC and VNC) have mixed input-output compartments, whereas efferent hugin neurons (PH and RG) show almost exclusively postsynaptic sites within the CNS. Outlines of the CNS including the ring gland are shown in white. https://doi.org/10.7554/eLife.16799.005 All hugin neurons receive inputs within the SEZ [previously called subesophageal ganglion (SOG)], a chemosensory center that also houses the basic neuronal circuits generating feeding behavior (Hückesfeld et al., 2015). However, only the hugin-PC neurons showed considerable numbers of synaptic outputs in the SEZ, consistent with their previously reported effects on feeding (Schoofs et al., 2014; Hückesfeld et al., 2016) (Figure 2E). Acetylcholine is a co-transmitter in hugin neurons The existence of presynaptic sites containing SCVs in addition to large DCVs led to the assumption that hugin-PC and hugin-VNC (possibly also hugin-PH neurons) employ small molecule neurotransmitters parallel to the hugin neuropeptide. To address this, we checked for one of the most abundantly expressed neurotransmitter in the Drosophila nervous system: acetylcholine (ACh) (Yasuyama and Salvaterra, 1999; Salvaterra and Kitamoto, 2001). In the past, immunohistochemical and promoter expression analyses of choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, were successfully used to demonstrate cholinergic transmission (Barnstedt et al., 2016; Miyamoto, 2012; Yapici et al., 2016). We used both, anti-ChAT antibody as well as a ChAT promoter analysis, and investigated co-localization with hugin neurons. In the EM data, we found hugin neurons to have comparatively few SCVs, suggesting only low amounts of small transmitters. In addition, ChAT is preferentially localized in the neuropil and less so in the somas (Sámano et al., 2006). Consistent with this, we found that ChAT immunoreactivity in hugin cell bodies was relatively low and varied strongly between samples. Therefore, we quantified the anti-ChAT signal to show that while ChAT levels were in some cases indiscernible from the background, overall highest levels of ChAT were found in hugin-PC and hugin-VNC/PH neurons (Figure 3A). Note that while hugin-PC and hugin-RG neurons were easily identifiable based on position and morphology, hugin-PH and hugin-VNC neurons usually clustered too tightly to be unambiguously discriminated and were thus treated as a single mixed group. Similar to the immunohistochemical analysis, the ChAT promoter (ChAT-GAL4) drove expression of a fluorescent reporter in all hugin-PC neurons plus a subset of hugin-VNC/PH neurons (Figure 3B). Hugin-RG showed weak ChAT signal with either method, consistent with these neurons lacking SCVs in the EM data. Figure 3 Download asset Open asset Acetylcholine (ACh) is a neurotransmitter of hugin neurons. (A,B) Co-localization of the biosynthetic enzyme for ACh, choline acetyltransferase (ChAT), in hugin neurons using a ChAT antibody (A) or a ChAT promoter-GAL4 driving a fluorescent reporter (B). ChAT immunoreactivity was variable but strongest signals were found in hugin-PC and hugin-VNC/PH neurons. Similarly, ChAT-GAL4 consistently drove expression in hugin-PC and subsets of hugin-VNC/PH. Shown are exemplary scans and quantification of ChAT co-localization in the different hugin classes. Note that while hugin-PC and hugin-RG neurons are easily identifiable, hugin-PH and hugin-VNC neurons were usually too close to be unambiguously discriminated and were thus treated as a single mixed group. Each data point in the dot plots represents a single hugin neuron. Horizontal lines mark median. (C,D) ACh is necessary for the effect of hugin neurons’ activation on food intake (C) and pharyngeal pumping (D). Food intake was measured in intact larvae. Pharyngeal pumping was monitored by extracellular recordings of the antennal nerve (AN) and analyzed with respect to the cycle frequency of the motor patterns. Activation of hugin neurons using the thermosensitive cation channel dTrpA1 (HugS3-GAL4 x UAS-dTrpA1) led to a decrease in food intake and pharyngeal pump activity compared to the control (OrgR, OrgR x UAS-dTrpA1). Knockdown of either the hugin neuropeptide or ChAT but not LacZ control (UAS-dTrpA1;HugS3-GAL4 x UAS-RNAi) rescued the effect of hugin neuron activation on food intake as well as on pharyngeal pumping. For details see Materials and methods, and Schoofs et al., (2014). Numbers below box plots give N [C: # larvae; D: # trials (# larvae)]. Mann-Whitney Rank Sum Test (*** = p<0.001; **=p < 0.01). https://doi.org/10.7554/eLife.16799.006 These findings suggested that ACh may be a co-transmitter in hugin neurons. We previously demonstrated that RNAi-induced knockdown of the hugin neuropeptide rescues the phenotype of feeding suppression caused by induced activation of hugin neurons in behavioral and electrophysiological experiments (Schoofs et al., 2014). Here, we present the knockdown of ChAT using an established UAS-ChAT-RNAi line (Plaçais et al., 2013; see Materials and methods). Under unimpeded conditions (i.e., without ChAT knockdown), activation of hugin neurons leads to severe decrease of food intake in intact larvae. This decrease in food intake was rescued by knockdown of ChAT in hugin neurons to a similar degree as the knockdown of the hugin neuropeptide itself (Figure 3C). In addition to a general decrease in food intake, activation of hugin neurons leads to a decrease in cycle frequency of pharyngeal pump motor activity (Schoofs et al., 2014). We used extracellular recordings of the antennal nerve (AN) in isolated CNS for precise monitoring of motor patterns of the pharyngeal pump (Schoofs et al., 2009). As for the food intake, knockdown of ChAT in hugin neurons also rescued the suppressive effect of hugin neuron activation on pharyngeal pumping (Figure 3D). Taken together, these data clearly demonstrate that ACh plays a functional role in hugin neurons. Moreover, this suggests that hugin neuropeptide and ACh have to be employed together in order to regulate feeding behavior. Hugin classes form distinct units that share synaptic partners Reconstruction of hugin neurons and localization of synaptic sites revealed that neurons of the two interneuron classes, hugin-PC and hugin-VNC, were reciprocally connected along their main neurites to ipsilateral neurons of the same class (Figure 4, Figure 2—figure supplement 1E,F). These connections made up a significant fraction of each neuron's total synaptic connections, implying that their activity might be coordinately regulated. Figure 4 Download asset Open asset Hugin neurons synapse reciprocally within-class but not across-class. (A) Connectivity matrix of hugin to hugin connections. Each row indicates number of synaptic connections of given hugin neuron to other hugin neurons. Connections that could not be recapitulated for both hemisegments are grayed out. Numbers in colored boxes give % of incoming (x-axis) and outgoing (y-axis) synaptic connections of the respective hugin neuron. Hugin to hugin contacts are made between hugin interneurons of the same class, not between classes (see schematic). Note that efferent hugin neurons, hugin-RG and hugin-PH, do not have presynaptic sites. (B) Distribution of hugin-hugin synapses. Synaptic contacts between hugin-PC or hugin-VNC neurons are made along their main neurites. Only neurons of one hemisegment are shown. https://doi.org/10.7554/eLife.16799.007 We therefore further explored the different classes within the population of hugin-producing neurons, asking whether hugin classes establish functional units or whether they are independently wired. To this end, we reconstructed 177 pre- and postsynaptic partners of hugin neurons (Figure 5A, see Materials and methods for details). First, we found that neurons of the same hugin class were connected to the same pre- and postsynaptic partners. Furthermore, most synaptic partners were connected exclusively to neurons of a single hugin class (Figure 5B). Second, pre- and postsynaptic partners of each hugin class resided in different parts of the CNS (Figure 5C; Video 2). For hugin-RG and hugin-PH, the vast majority of synapses were made with interneurons, 93 ± 4% and 97 ± 3%, respectively. This percentage was lower for hugin-PC (66 ± 6%) and hugin-VNC (81 ± 2%). To our knowledge, none of these interneuron partners have been previously described, making it difficult to speculate on their functions at this point. Non-interneuron partners will be described in the following sections. In summary, these findings show that neurons of each hugin class form complex microcircuits that are largely separate from one another. Figure 5 with 1 supplement see all Download asset Open asset Each hugin class is part of a distinct microcircuit, weakly or not at all connected to those of the other classes. (A) Synaptic partners of hugin neurons were reconstructed. Pre- and postsynaptic partners of a single hugin-PC neuron are shown as example. (B) Comparison of hugin neurons’ connectivity as measured by connectivity similarity score. High similarity score indicates a large fraction of shared synaptic partners connected by similar numbers of synapses. Neurons are ordered by dendrogram of similarity score of pre- (x-axis) and postsynaptic (y-axis) partners. Matrix shows combined pre- and postsynaptic similarity score. Self-self comparisons were omitted (asterisks). Hugin classes connect to unique sets of pre- and postsynaptic partners. Neurons of each hugin class have the same synaptic partners, and there is little to no overlap with other classes (see schematic). (C) Reconstructed pre- and postsynaptic partners by hugin class. Neurons are color-coded based on total number of synapses to given hugin class [minimum = 1; hugin-PC = hugin-VNC = hugin-RG = hugin-PH = Hugin-RG and hugin-PH neurons do not have postsynaptic partners within the CNS. See also Video 2 and neuron Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Each class of hugin neurons to unique sets of synaptic partners. Video shows all reconstructed presynaptic and postsynaptic partners of hugin neurons (see Figure Neurons are colored by total number of synapses given hugin class. Each hugin class distinct microcircuits with little to no overlap with those of other classes. Hugin neurons receive chemosensory synaptic input Hugin neurons have a significant number of their incoming synapses ± within the This of the CNS is to the and is a chemosensory center that input from sensory (Ghysen, In addition, subsets of hugin neurons were recently shown to be to (Hückesfeld et al., 2016). We therefore for sensory inputs to hugin neurons and found a total of neurons that made synaptic contacts hugin neurons (Figure major a morphologically of neurons projecting one of the pharyngeal antennal a the CNS not We that the reconstructed presynaptic partners of hugin neurons different parts of the we to these neurons by the similarity in distribution of their synaptic sites, synapse similarity score. Figure with 1 supplement see all Download asset Open asset Each class of hugin neurons inputs from distinct subsets of sensory neurons. (A) inputs to hugin neurons the CNS the antennal nerve and (asterisks). Neurons are color-coded based on total number of synapses to hugin neurons. (B) neurons clustered based on synapse similarity score. This score is from the overlap of synaptic sites between two neurons. See also Video (C) of sensory inputs hugin neurons. The antennal nerve sensory from the ganglion and pharyngeal from of the peripheral nervous system of antennal nerve chemosensory in the subesophageal zone receptor neurons in the antennal receptor neurons from different sensory distinct parts of the SEZ on et al., Connectivity matrix of sensory neurons hugin. neurons are ordered by dendrogram of synapse similarity score and to corresponding of and Each row of the matrix shows the number of synaptic connections a single hugin neuron. Numbers in boxes along give percentage of synaptic input each hugin neuron as one neuron of two synapse was See for further based on synapse similarity score in different each of them covering distinct parts of the SEZ (Figure Video see Materials and methods for details). To address the of the of identified sensory we compared our data with of larval sensory neurons. is well established that internal and external sensory of the peripheral nervous This includes neurons) and a range of sensory neurons function is still unknown et al., 2007; et al., and To our knowledge, no sensory neurons with projections into the SEZ such as the one presynaptic to hugin have been However, the majority of neurons hugin neurons from the antennal This pharyngeal nerve the of receptor neurons from internal pharyngeal as well as those of receptor neurons and other from the external sensory (Figure et al., 2007; and can be unambiguously identified as they target of the antennal and but no such sensory neurons were found to input hugin neurons (Figure supplement 1). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg of chemosensory neurons distinct of the subesophageal zone Video shows morphology and presynaptic sites of sensory inputs to hugin neurons. Neurons are clustered based on a synapse similarity score (see Figure Each represents a presynaptic site. with the number of connected neuronal for that The likewise target regions of the SEZ but this is not as well as the antennal et al., 2007; and The antennal nerve neurons that the hugin cells show the morphology of this population of 2007; et al., We thus compared our clustered with previously defined compartments of the SEZ 2 and which the SEZ, likely correspond to two described as the target of from internal pharyngeal only (Figure The were either not previously described or difficult to unambiguously with known Our into is also at the of their connectivity to hugin sensory neurons of 1 have synaptic connections to both hugin-PC and hugin-VNC neurons. the previously described pharyngeal are almost exclusively connected to hugin-PC neurons. sensory neurons few synapses hugin-RG neurons. the neurons, is presynaptic to hugin-VNC (Figure The efferent type hugin neurons, hugin-PH and show little to no sensory In contrast, the interneuron type hugin neurons, hugin-PC and hugin-VNC, receive a significant fraction of their incoming synaptic connections to from sensory neurons. we found two of four of hugin neurons to receive synaptic input from a large but population of sensory neurons, many of which are from external and internal sensory Hugin-PC neurons were recently shown to be by but not or (Hückesfeld et al., 2016). Our data strongly suggests that this activation is at based on connections to Moreover, the among the population of sensory neurons suggests that hugin-PC neurons do not function as but an for example between or external and internal sensory synaptic and peptide-receptor to the neuroendocrine system NMU has been well studied in the of its effect on the We therefore for similar among the downstream targets of hugin neurons. The of hugin-PC neurons their neurites from the SEZ to the the pars neurosecretory cells (mNSCs) in this the major neuroendocrine center in the CNS, homologous to the mammalian hypothalamus, and target the neuroendocrine of Drosophila, the ring gland 2006). different of mNSCs produce distinct neuropeptides in a 3 mNSCs produce diuretic hormone 44 2 mNSCs produce and mNSCs produce Drosophila insulin-like peptides thus called