Substrates for Neuronal Cotransmission With Neuropeptides and Small Molecule Neurotransmitters in Drosophila

Abstract

It has been known for more than 40 years that individual neurons can produce more than one neurotransmitter and that neuropeptides often are colocalized with small molecule neurotransmitters (SMNs). Over the years much progress has been made in understanding the functional consequences of cotransmission in the nervous system of mammals. There are also some excellent invertebrate models that have revealed roles of coexpressed neuropeptides and SMNs in increasing complexity, flexibility, and dynamics in neuronal signaling. However, for the fly Drosophila there are surprisingly few functional studies on cotransmission, although there is ample evidence for colocalization of neuroactive compounds in neurons of the CNS, based both on traditional techniques and novel single cell transcriptome analysis. With the hope to trigger interest in initiating cotransmission studies, this review summarizes what is known about Drosophila neurons and neuronal circuits where different neuropeptides and SMNs are colocalized. Coexistence of neuroactive substances has been recorded in different neuron types such as neuroendocrine cells, interneurons, sensory cells and motor neurons. Some of the circuits highlighted here are well established in the analysis of learning and memory, circadian clock networks regulating rhythmic activity and sleep, as well as neurons and neuroendocrine cells regulating olfaction, nociception, feeding, metabolic homeostasis, diuretic functions, reproduction, and developmental processes. One emerging trait is the broad role of short neuropeptide F in cotransmission and presynaptic facilitation in a number of different neuronal circuits. This review also discusses the functional relevance of coexisting peptides in the intestine. Based on recent single cell transcriptomics data, it is likely that the neuronal systems discussed in this review are just a fraction of the total set of circuits where cotransmission occurs in Drosophila. Thus, a systematic search for colocalized neuroactive compounds in further neurons in anatomically defined circuits is of interest for the near future.

Keywords: circadian clock; fly brain; mushroom bodies; neuromodulation; neurosecretory cells; olfactory system; presynaptic facilitation; short neuropeptide F.

FIGURE 1

Outline of Drosophila brain centers and some definitions. (A) The adult Drosophila brain with some of the centers/regions discussed in this review. The glomerular antennal lobes (AL) are located anteriorly. Projection neurons from the ALs supply axons to the calyx (Ca) of the mushroom bodies (MBs) and the lateral horn (LH). The MBs have three major lobes, α, β, and γ lobes. Neurosecretory cells are located in the pars intercerebralis (PI) and pars lateralis (PL), as well as in the subesophageal ganglion (SEG). Bilateral pacemaker centers of the circadian clock are located near the optic lobe and are formed by the LNv clock neurons and others not shown. (B) Some definitions of terms used in this review for signaling with neuroactive substances. So-called classical- or SMNs are sometimes referred to as fast neurotransmitters. The four listed ones are discussed in this review. Monoamines or biogenic amines can be subdivided into catecholamines, indolamines and phenolamines, and the four amines discussed here are listed. Neuropeptides/peptide hormones are very diverse and some larger ones are termed protein hormones. Commonly peptides/proteins of this kind act as cotransmitters, modulators or hormones over a slower and more long lasting temporal scale than the neurotransmitters. The neurotransmitters often activate ion channels leading to rapid changes in membrane potential.

FIGURE 2

Sets of neuroendocrine cells in the Drosophila brain that express colocalized neuropeptides and peptide hormones. (A) Neurosecretory cells and interneurons of the adult brain. The insulin producing cells (IPCs) co-express DILP1, 2, 3, and 5. Some of the IPCs (cross hatched) also produce drosulfakinins (DSK1 and 2). The IPCs are regulated by DLP neurons that produce short neuropeptide F (sNPF), corazonin (CRZ), and proctolin (Proct) (Kapan et al., 2012). These DLPs are likely to also release CRZ into the circulation via axons passing through the NCC nerves to peripheral release sites in the CC, anterior aorta and intestine (Kubrak et al., 2016). Another set of lateral neurosecretory cells (LNCs), designated ipc-1 and ipc-2a, express the peptides sNPF, tachykinin (TK) and ion transport peptide (ITP) (Kahsai et al., 2010). These neurons, like the DLPs, are parts of the LNC clusters and have axon terminations in peripheral sites overlapping those of the IPCs and the DLPs. A pair of large interneurons in the brain (ICLI) were shown to co-express AstA, MIP and natalisin Diesner et al. (2018). Further abbreviations: MT, medially projecting axon tract; PLT, posterior lateral axon tract; MB, median bundle. This figure is slightly altered and updated from Nässel et al. (2013). (B) Neuroendocrine cells in one hemisphere of the larval brain (dorsal view). The cells shown (numbered 1–43) are Dimmed positive cells, most of which have neuropeptides assigned to them. The bona fide MNCs include cells numbered 19–25, 28–30, and 31–32, the other adjacent neurons are peptidergic interneurons. Note several cases of colocalized peptides, some of which so far have not been observed in the adult brain: NPF/DH31, DMS/ITP, dTK/AstB, DSK/AstC, and sNPF/DMS (acronyms as in text). Left image from Park et al. (2008) with permission (PLOS, Open access).

FIGURE 3

Neuroendocrine cells colocalizing neuropeptides and neurotransmitters in the CNS of adult and larval Drosophila. (A) Neuroendocrine cells colocalizing leucokinin and diuretic hormone 44 in the adult CNS. In the brain there are 4 neurons expressing leucokinin (LK), 6 neurons producing diuretic hormone 44 (DH44) and 8 ipc-1 neurons producing ion transport peptide (ITP; shown because they express Lk-Gal4), tachykinin (TK) and sNPF. In the abdominal ganglion at least 8 of the 22 ABLKs coexpress LK and DH44. Note that in adults, but not larvae the brain MNCs coexpress DH44 and DILP2 (Ohhara et al., 2018). This figure is slightly altered from Zandawala et al. (2018). (B) In third instar larvae all ABLKs coexpress LK and DH44. In younger larvae the ipc-1 neurons, in addition to ITP and sNPF, express weak LK (de Haro et al., 2010; Kahsai et al., 2010). One subset of midline neurons expressing DILP7, the DP1 neurons, coexpress sNPF and acetylcholine (Ach) (Nässel et al., 2008). (C) In adult flies the posterior DILP7 expressing neurons in abdominal ganglia consist of neurons derived from the embryo (pDILP7 in B) and adult-specific ones that are generated later. There is a sex-dimorphism in that females have a larger number of adult DILP7 neurons that also produce glutamate, whereas in males the adult DILP7 neurons produce additional serotonin (5-HT) (Castellanos et al., 2013). (D) In each neuromere of the larval ventral nerve cord (VNC) there is a pair of midline motor neurons, aCC and RP2 that signal with glutamate. The RP2 neurons also produce proctolin from Luo et al. (2017).

FIGURE 4

Neurons and neuropeptides that control ecdysis motor activity in Drosophila. (A) Schematic depiction of peptidergic neurons in the larval CNS that express the ecdysis triggering hormone receptor ETHR-A. These neurons respond to ETH in a sequence and trigger ecdysis behavior as shown in (B). The color-coding depicts the expression of various neuropeptides in the neurons (note that some colocalize 2–3 peptides). ETH is released from peritracheal Inka cells (not shown). SN, subesophageal neuromeres 1–3; ETH, ecdysis triggering hormone; FMRFa, FMRFamide; EH, eclosion hormone; CCAP, crustacean cardioactive peptide; MIP myoinhibitory peptide; burs, bursicon. Redrawn from Kim et al. (2006), Nässel and Winther (2010).

FIGURE 5

Several neuropeptides are colocalized in different types of clock neurons in Drosophila. (A) The different types of clock neurons in one brain hemisphere. There are lateral ventral neurons (s-LNv and l-LNv), lateral dorsal neurons (LNd), dorsal neurons (DN1–DN3), and a set of lateral posterior neurons (LPN). aMe, accessory medulla. This panel is originally from Helfrich-Förster et al. (2007), but slightly modified in Johard et al. (2009). (B) Expression of Tim-Gal4-driven GFP (green) and immunolabeling for ion transport peptide (ITP; magenta). From Johard et al. (2009). (C) Neuropeptides in LNd and LNv neurons are colocalized in different patterns. The figure is compiled from data in Johard et al. (2009) and Schlichting et al. (2016). Note that the s-LNvs also produce the amino acid transmitter glycine (Frenkel et al., 2017) and l- LNvs the cytokine Upd1 (Beshel et al., 2017). (D) The clock output generates daily activity with peaks in the morning (M) and evening (E) and low activity at night and mid-day. This activity is generated by clock neurons that act as morning (M) and evening (E1–E3) oscillators. These express different combinations of neuropeptides and some interactions between LNvs and other neurons are known to be by means of pigment-dispersing factor (PDF) shown in red arrows. The roles of other peptides are less known so far (but, see Figure 6). This figure is compiled in part from data in (Schlichting et al., 2016). (E) This table shows additional neuropeptides in clock neurons indicated by singe cell transcriptomics. Note that the cell types listed were assayed as groups and there is no information on peptides in subtypes of neurons in these groups. Thus it is not known to what extent the neuropeptide transcripts are coexpressed in specific neurons; this is especially prominent for the relatively large group of DN1 neurons, which may be heterogeneous. Data mined from Abruzzi et al. (2017).

FIGURE 6

A scheme depicting PDF-, sNPF-, and light-mediated interactions that orchestrate sequential Ca²⁺ activity phases in different pacemaker groups. Each pacemaker group is represented by one neuron. The position of cells in the day-night circle (yellow–gray) indicates the peak phase of Ca²⁺. Both PDF and sNPF signals inhibit the target neurons and suppress these from being active when the sender neurons (s-LNv for PDF; s-LNV and LNd for sNPF) are active. Light cycles act together with PDF to delay Ca²⁺ phases in LNds. This figure was redrawn from Liang et al. (2017).

FIGURE 7

Networks of peptide cotransmission in the clock circuits of Drosophila. (A) Neuronal circuit that regulates locomotor activity proposed in King et al. (2017). It was suggested that s-LN_vs signal to DN1s that in turn act on DH44-producing MNCs. These signal with DH44 to hugin cells in the SEG via the DH44-R1. Some of the hugin cells have axons that terminate in the VNC where they contact glutamatergic neurons in motor centers that generate locomotion. The two arrows indicate areas where interactions between MNCs and hugin cells can occur. It is not known which peptide/neurotransmitter of the s-LNvs and DN1s that signal in this pathway. For s-LNvs it could be PDF, sNPF or glycine and for DN1s there are several candidate peptides (see Figure 5E). Note that the MNCs also produce DILP2 (Ohhara et al., 2018). (B) The colocalized neuropeptides and other signaling molecules in the LN_vs may target different constellations of neurons within and outside the clock circuitry; these are represented by “other clock neurons” and “other neurons,” respectively. The “other neurons” are effectors downstream to the clock circuits that regulate, e.g., rhythmic activity in behavior and physiology (RA), or neuronal systems that influence other behaviors such as reproduction, foraging and feeding (and indirectly metabolism), or produce systemic responses via hormone release (OA). In the simplest model, shown in (B), all the target neurons for the large and small LN_vs express receptors for all of the signal molecules released. Thus, targets of s-LN_vs within and outside the clock circuit would all express receptors (R) for PDF, sNPF and glycine, and targets of l-LN_vs have receptors for PDF, NPF and Upd1 (unpaired-1). Each of the neurons would thus receive multiple complementary signals. (C) Hypothetically the target neurons of the LN_vs could be more diverse and express only subsets of the relevant receptors in different combinations. In this extreme example each target neuron type expresses only one receptor type and the downstream circuits could generate neuropeptide and signal substance-specific activity. Here, activity in, e.g., s-LN_vs would simultaneously generate a more diverse set of actions within and outside the network. These could include some actions that are targeted only to neurons outside the bona fide clock network (e.g., the Upd1 or sNPF actions in this example). The outputs, RA¹ and OA¹, could be more diversified.

FIGURE 8

Coexpression and neuromodulation in the olfactory system. (A) Neuromodulation in the Drosophila antennal lobe. The antennal lobe (AL) is shown highly schematically with only two glomeruli (dashed outlines). Inputs to the glomeruli are from OSNs in the antenna and labial palps. The OSNs synapse on PN that relay signals to higher brain centers (MBs and lateral horn). The OSNs and PNs are modulated by local neurons (LNs), which form intrinsic modulatory circuits and by extrinsic neurons that utilize several neurotransmitters/neuromodulators. The LNs are either GABAergic, cholinergic (Ach), or in some cases glutamatergic. The former two types are known to colocalize the neuropeptides tachykinin (TK), allatostatin-A (AstA) or myoinhibitory peptide (MIP) (Carlsson et al., 2010), whereas it is not known whether glutamatergic ones colocalize any peptide. The extrinsic neurons utilize SIFamide, dromyosuppressin (DMS), IPNamide (from precursor NPLP1), octopamine or serotonin (5-HT) (Dacks et al., 2006; Sinakevitch and Strausfeld, 2006; Roy et al., 2007; Carlsson et al., 2010). It is not known whether any of these extrinsic neurons colocalize other neurotransmitters/neuropeptides. Additionally, a subpopulation of the OSNs coexpress Ach and sNPF (Nässel et al., 2008; Carlsson et al., 2010) and in females some OSNs with Ir-type receptors coexpress MIP (Hussain et al., 2016a). Recent reports from single cell transcriptomics suggest that some PNs may express sNPF and others TK in addition to Ach (Croset et al., 2017). The general outline of this figure is redrawn from (Lizbinski and Dacks, 2017). (B) The OSNs are regulated presynaptically by insulin and sNPF. This figure, based on a paper by Root et al. (2011), shows an OSN synapsing on a PN in the antennal lobe of a fed Drosophila fly (top) and a hungry one (bottom). The synapse is located within a glomerulus. In the fed fly, the level of circulating insulin is high and the activated insulin receptor (dInR) on the OSN inhibits transcription of the sNPF receptor, sNPFR1. Thus, there is low expression of the sNPFR1 presynaptically on the OSN axon termination and signal transfer with acetylcholine at the synapse is weak. As a result, food search/finding is low. In the hungry (starved) fly, insulin levels are low and the transcription of sNPFR1 in the OSNs is activated. Consequently, presynaptic sNPFR1 expression increases and released sNPF activates the presynapse leading to enhanced release of acetylcholine and thus increased signaling in the synapse: food search/finding increases. This is an example of an autocrine presynaptic regulation. The figure is slightly altered from Nässel (2012), which was compiled from Root et al. (2011).

FIGURE 9

Neuropeptides and peptide hormones in the Drosophila intestine. (A) In the midgut enteroendocrine cells (EECs) produce neuropeptides/peptide hormones in a region-specific manner. Four midgut regions are shown here [based on (Veenstra and Ida, 2014)]. Peptides produced are shown in blue text, and those that are colocalized in EECs in the different regions are shown in red or black text (NPF and tachykinin, AstC and orcokinin B, DH31 and tachykinin). DH31 is calcitonin-like diuretic hormone 31. There are also EECs producing tachykinin in the anterior hindgut. This figure is compiled from Veenstra et al. (2008); Veenstra and Ida (2014). (B) EECs in the posterior midgut and anterior hindgut produce tachykinin (TK) as demonstrated by Tk-Gal4 driven GFP (green) and antiserum to TK (magenta). From Liu et al. (2016b). (C) Schematic depiction of an EEC located between enterocytes (EC). The EECs may sense nutrients in the gut lumen and release peptide either locally in a paracrine fashion or into the circulation outside the muscle layer (M). The other cells shown are stem cells (SC) and enteroblasts (EB) that are responsible for renewal of the gut epithelium. This figure was redrawn from a figure in Lemaitre and Miguel-Aliaga (2013).