Identification of NSC in the Drosophila brain.

(A) Schematic drawing of the different types of NSC and their projections to different release sites within the fly. Based on (Nässel et al., 2013). (B) All NSC projections exit the brain via the nervi corpora cardiaca (NCC). Electron micrograph showcasing a cross section of the NCC. Scale bar = 750nm. (C) Cosine similarity matrix of all NSC based on their total inputs. The darker the color, the higher the similarity between neurons. Neurons within the clades are colored based on the schematic in (D). (D) Reconstructions of the 80 NSC within the adult brain connectome. (E) Classification of NSC based on their location and neuropeptide expression. Refer to Table 1 for further details. Abbreviations: SEZ, subesophageal zone; CC, corpus cardiacum; CA, corpus allatum.

Classification of Drosophila neurosecretory cells (NSC) based on their cell body position in the central brain

Quantification of NSC.

(A) On average, there are 16 m-NSCDILP as labelled by DILP3-Gal4 and DILP2 antibody. Note that some preparations contain 18 m-NSCDILP, in agreement with the number determined based on the connectome. (B) MS-T2A-Gal4 drives expression in several neuronal populations across the brain including (C) six m-NSC in pars intercerebralis and (D) the pair of SEZ-NSCCAPA (filled arrowheads). (E) CRZ is expressed in 7 pairs of neurons in adult flies, 4 of which co-express Gr64a (empty arrowheads). These smaller Gr64a-expressing CRZ neurons form dense arborizations in the lateral horn. They project contralaterally but do not send projections via the nervii corpora cardiaca (NCC) and are thus not considered neurosecretory.

Synaptic inputs to NSC.

(A) Postsynaptic sites of different NSC subtypes. Majority of the dendrites are found in the protocerebrum and SEZ. (B) Input to NSC grouped by the neuronal super classes annotated in the FlyWire connectome. Central neurons are the largest group providing inputs to NSC. (C) Proportion of inputs from various neuronal super classes to different types of NSC. (D) Reconstructions of neurons from different super classes which provide major inputs to NSC. Only the top 10 cell types per super class are shown. (E) Number of strong input connections (greater than 50 synapses) to each NSC subtype and the total number of synapses constituting these connections. (F) Reconstructions of neurons that provide major inputs (more than 50 synapses per connection) to SEZ-NSCCAPA, l-NSCDH31, m-NSCDH44 and m-NSCDILP. (G) Proportion of inputs from individual neurons to different NSC subtypes. In total, 76 neurons provide inputs to more than one type of NSC, with m-NSCDH44 receiving inputs from most of these neurons. Out of these 76 neurons, (H) 53 neurons provide inputs to two types of NSC, (I) 22 neurons provide inputs to three types of NSC and (J) 1 neuron provides input to four types of NSC. Reconstructions of corresponding neurons below each schematic. For (E) and (G), bars have been color coded according to the legend in the panel (A).

Sensory inputs to NSC.

(A) Direct and indirect (disynaptic) sensory inputs to NSC. Interneurons mediating connectivity between sensory neurons and NSC are referred to as sensory interneurons. The donuts represent proportion of cells and the number in the donut reflects the total number of neurons in that group. NSC receive very minimal direct sensory inputs. Only gustatory, mechanosensory and unknown sensory inputs provide monosynaptic and disynaptic inputs to NSC. Note that l-NSCITP do not receive any significant synaptic inputs and are thus not represented here. (B) Reconstructions of sensory neurons (separated by class) providing direct inputs to NSC. (C) Reconstructions of sensory neurons providing indirect inputs to NSC. (D) Number of sensory neurons (grouped by sub class) that provide indirect inputs to NSC. (E) Schematic showing the projections of labellar and tarsal gustatory receptor neurons (GRN) from the periphery to the SEZ (adapted from (Freeman and Dahanukar, 2015)). Reconstructions of four tarsal GRN (colored red; classified as ascending neurons on Codex) that provide indirect inputs to six NSC (also shown). Abbreviations: acc. pharyngeal, accessory pharyngeal.

Olfactory inputs to NSC.

(A) Schematic showcasing the flow of olfactory information from olfactory receptor neurons (ORN) in the antenna to the higher-order brain centers (e.g. mushroom bodies and lateral horn) via the antennal lobe (adapted from (Zhao and McBride, 2020)). (B) Number of neurons (grouped by different categories) that comprise the shortest pathway from ORN to NSC. ORN have been grouped based on their behavioral significance (based on (Zheng et al., 2022)). Antennal lobe associated neurons (AL*) include projection neurons (ALPN) and local interneurons (ALLN). IN represent interneurons that link AL* neurons and NSC. (C) Numbers of each ORN type that provide indirect inputs to NSC. (D) Number of synapses formed by these ORN. Note that the ORN which detect aversive odors followed by those that detect food odors provide the strongest indirect inputs to NSC. (E) Reconstructions of top ten ORN types. (F) Number of AL* in the pathway. v2LN30 is the only ALLN whereas the rest are ALPN. (G) Reconstructions of top four ALPN types and (H) top three NSC types that are part of this pathway. Bars in (C) and (D) and neurons in (E) and (G) have been colored based on their behavioral significance. (I) Pheromonal and egg-laying associated olfactory information is relayed to m-NSCDILP. (J) ORN belonging to all five behavioral categories provide inputs to l-NSCDH31. (K) SEZ-NSCCAPA primarily receive aversive olfactory inputs. For I-K, the numbers within the circles indicate the number of neurons or the name of that neuron. Arrows have been weighted based on the number of synapses and colored based on the neurotransmitter mediating those connections (see legend). Abbreviations: LN, local interneuron; uni. PN, uniglomerular projection neuron; multi. PN, multiglomerular projection neuron; KC, Kenyon cell; LHON, lateral horn output neuron.

Synaptic output from NSC.

(A) Presynaptic sites of different NSC subtypes. (B) Output from NSC grouped by the neuronal super classes annotated in the FlyWire connectome. Central neurons receive inputs from l-NSCunknown and descending neurons receive inputs from l-NSCCRZ. (C) Proportion of outputs from different types of NSC to various neuronal super classes. (D) Reconstructions of l-NSCunknown and all their postsynaptic partners. (E) Reconstructions of l-NSCCRZ and all their postsynaptic partners (descending neurons). The descending neurons primarily innervate the wing tectulum. (F) Weighted connections between l-NSCCRZ and DNg27 descending neurons which innervate the wing tectulum and could thus regulate flight. (G) Individual postsynaptic partners of l-NSCunknown and l-NSCCRZ sorted based on the number of synapses and colored based on their neurotransmitter identity.

NSC interconnectivity and endocrine output.

(A) Identification of single-cell transcriptomes representing different NSC subsets in the adult brain (Davie et al., 2018). All NSC express genes required for neuropeptide processing and release (amon, svr, Pal2, Phm and Cadps) and were identified primarily based on the neuropeptides that they express. (B) Dot plot showing expression of receptors in NSC. Expression of only those receptors whose corresponding neuropeptides are expressed in NSC are shown. (C) Connectivity diagram (weighted based on neuropeptide and receptor expression) showing putative paracrine connectivity between different types of NSC. Note that short neuropeptide F (sNPF) and myosuppressin (DMS) are expressed in two different NSC subtypes. Ion transport peptide and CAPA pathways are not included because their receptors were not detected in these transcriptomes. Leucokinin was excluded because its expression levels were below the threshold used here. Dot plot showing the expression of neuropeptide receptors in (D) adipokinetic hormone cells of the corpus cardiacum and (E) all the tissues in adults. “General” in panel E includes cell types that are found across multiple tissues including sensory neuron, visceral muscle and hemocytes amongst others. See Figure 7 Supplement 3 for all the different cell types that are part of this cluster.

Differences between m-NSCDH44 and m-NSCDMS.

(A) Retrograde trans-synaptic labelling of m-NSCDH44. m-NSCDH44 are labelled in green and their presynaptic partners are labelled in magenta. Note the ectopic expression in mushroom body which is also visible in the controls. In silico retrograde tracing of (B) m-NSCDH44 and (C) m-NSCDMS. Both of these NSC subtypes receive majority of their inputs from neurons in the SEZ which have similar location and morphology. However, m-NSCDMS also receive inputs from a group of central neurons (marked with an arrow) that are not visible in (A) and (B). (D) Reconstruction of myosuppressin (DMS) descending neurons. Electron micrographs showing a cross section of (E) DMS descending neuron, (F) m-NSCDH44 and (G) m-NSCDMS cell soma. Both types of DMS-expressing cells have darker dense core vesicles (marked by red arrows) compared to those found in m-NSCDH44.

Morphological characteristics of NSC.

(A) cable length, (B) surface area, (C) cell volume and (D) nuclei volume of different NSC subtypes. (E) Principal component analysis of these four features reveals that the NSC of a given subtype generally cluster together. Note the high variability for l-NSCCRZ, l-NSCDH31, m-NSCDH44 and m-NSCDILP populations, suggesting that they comprise of morphologically heterogenous subpopulations.

Postsynaptic sites of NSC.

Reconstructions of different NSC subtypes along with their postsynaptic sites. l-NSCITP are an exception and have very few postsynaptic sites.

Inputs to NSC subtypes.

(A) Individual presynaptic partners of different NSC sorted based on the number of synapses. Presynaptic neurons are colored based on the super class they belong to. Only the top 20 neurons are shown. SEZ-NSCCAPA and l-NSCCRZ receive strong sensory inputs whereas l-NSCDH31, m-NSCDH44 and m-NSCunknown mostly receive inputs from central neurons. (B) Number of presynaptic neurons providing inputs to different types of NSC.

Neurotransmitters providing inputs to NSC subtypes.

(A) Individual presynaptic partners of different NSC sorted based on the number of synapses and colored based on their neurotransmitter identity. l-NSCDH31 and m-NSCDH44 receive strong glutamatergic inputs. (B) Input to NSC grouped by the neurotransmitters. Out of the three fast-acting neurotransmitters, GABA provides the least inputs.

Presynaptic sites of NSC.

Reconstructions of different NSC subtypes along with their presynaptic sites. l-NSCCRZ have several presynaptic sites in the SEZ.

Synaptic output from NSC based on a low synaptic threshold.

(A) Proportion of outputs from different types of NSC to various neuronal super classes when the threshold for a significant connection is lowered to 2 synapses. (B) Output from NSC grouped by the neuronal super classes annotated in the FlyWire connectome. (C) Reconstructions of neurons receiving inputs from NSC. Cells belonging to top four super classes are shown. Note that most of the output from NSC is to partial fragments and non-neuronal cells (undefined), as well as central neurons.

Paracrine interconnectivity between NSC.

NSCs subtypes targeted by (A) myosuppressin (DMS), (B) Hugin, (C) corazonin (CRZ), (D) short neuropeptide F (sNPF), diuretic hormone 31 (DH31), (F) tachykinin (TK), (G) diuretic hormone 44 (DH44) and (H) insulin-like peptides (DILPs). Ion transport peptide and CAPA pathways are not included because their receptors were not detected in these transcriptomes. (I) Dot plot showing the neuropeptides expressed in each NSC type following thresholding. The expression has been scaled and was used to generate the connectivity diagrams in Figure 7C and Figure 7 Supplement 1A-H.

Expression of receptors for hormones released from brain NSC.

t-SNE plots showing expression of hormone receptors across single-cell transcriptomes from all Drosophila tissues (Li et al., 2022). Note that some receptors such as InR and sNPF-R are broadly expressed whereas others such as CapaR and PK2-R1 are sparsely expressed.

Expression of hormone receptors in peripheral tissues.

Dot plots showing expression of hormone receptors in different tissues at single-cell resolution. Expression of only those receptors whose corresponding neuropeptides are expressed in brain NSC are shown.

Expression of hormone receptors in the gut and reproductive tissues.

Dot plots showing expression of hormone receptors in the gut and reproductive tissues at single-cell resolution. Expression of only those receptors whose corresponding neuropeptides are expressed in brain NSC are shown.

Fly strains used in this study.

Antibodies used for immunohistochemistry in this study.