Neuropeptides and degenerin/epithelial Na+ channels: a relationship from mammals to cnidarians.

Ion channels of the degenerin (DEG)/epithelial Na+ channel (ENaC) family serve diverse functions ranging from mechanosensation over Na+ reabsorption to H+ sensing and neurotransmission. However, several diverse DEG/ENaCs interact with neuropeptides; some are directly activated, whereas others are modulated by neuropeptides. Two questions arise: does this interaction have a common structural basis and does it have an ancient origin? Current evidence suggests that RFamide neuropeptides activate the FMRFamide-activated Na+ channels (FaNaCs) of invertebrates via binding to a pocket at the external face of their large extracellular domain. It is likely that RFamides might activate DEG/ENaCs from the freshwater polyp Hydra (the HyNaCs) via binding to a similar pocket, although there is not yet any experimental evidence. In contrast, RFamide neuropeptides modulate acid-sensing ion channels (ASICs) from vertebrates via binding to a central cavity enclosed by ß-sheets of the extracellular domain. Dynorphin opioid peptides, for their part, bind to the acidic pocket of ASICs, which might be evolutionarily related to the peptide binding pocket of FaNaCs, but instead of opening the channels they work as antagonists to stabilize its closed state. Moreover, peptides interacting with DEG/ENaCs from animals of different phyla, although having similar sequences, are evolutionarily unrelated to each other. Collectively, it appears that despite a seemingly similar interaction with similar peptides, the interaction of DEG/ENaCs with neuropeptides has diverse structural bases and many origins.

Neuromodulation Can Be Simple: Myoinhibitory Peptide, Contained in Dedicated Regulatory Pathways, Is the Only Neurally-Mediated Peptide Modulator of Stick Insect Leg Muscle.

In the best studied cases (Aplysia feeding, crustacean stomatogastric system), peptidergic modulation is mediated by large numbers of peptides. Furthermore, in Aplysia, excitatory motor neurons release the peptides, obligatorily coupling target activation and modulator release. Vertebrate nervous systems typically contain about a hundred peptide modulators. These data have created a belief that modulation is, in general, complex. The stick insect leg is a well-studied locomotory model system, and the complete stick insect neuropeptide inventory was recently described. We used multiple techniques to comprehensively examine stick insect leg peptidergic modulation. Single-cell mass spectrometry (MS) and immunohistochemistry showed that myoinhibitory peptide (MIP) is the only neuronal (as opposed to hemolymph-borne) peptide modulator of all leg muscles. Leg muscle excitatory motor neurons contained no neuropeptides. Only the common inhibitor (CI) and dorsal unpaired median (DUM) neuron groups, each neuron of which innervates a group of functionally-related leg muscles, contained MIP. We described MIP transport to, and receptor presence in, one leg muscle, the extensor tibiae (ExtTi). MIP application reduced ExtTi slow fiber force and shortening by about half, increasing the muscle's ability to contract and relax rapidly. These data show neuromodulation does not need to be complex. Excitation and modulation do not need to be obligatorily coupled (Aplysia feeding). Modulation does not need to involve large numbers of peptides, with the attendant possibility of combinatorial explosion (stomatogastric system). Modulation can be simple, mediated by dedicated regulatory neurons, each innervating a single group of functionally-related targets, and all using the same neuropeptide.SIGNIFICANCE STATEMENT Vertebrate and invertebrate nervous systems contain large numbers (around a hundred in human brain) of peptide neurotransmitters. In prior work, neuropeptide modulation has been complex, either obligatorily coupling postsynaptic excitation and modulation, or large numbers of peptides modulating individual neural networks. The complete stick insect neuropeptide inventory was recently described. We comprehensively describe here peptidergic modulation in the stick insect leg. Surprisingly, out of the large number of potential peptide transmitters, only myoinhibitory peptide (MIP) was present in neurons innervating leg muscles. Furthermore, the peptide was present only in dedicated regulatory neurons, not in leg excitatory motor neurons. Peptidergic modulation can thus be simple, neither obligatorily coupling target activation and modulation nor involving so many peptides that combinatorial explosion can occur.

Immunohistochemical and Direct Mass Spectral Analyses of Plautia stali Myoinhibitory Peptides in the Cephalic Ganglia of the Brown-Winged Green Bug Plautia stali.

For seasonal adaptation, the brown-winged green bug Plautia stali (Hemiptera: Pentatomidae) enters reproductive diapause by suppressing juvenile hormone biosynthesis. Plautia stali myoinhibitory peptides (Plast-MIPs) are known to have allatostatic effects and to suppress juvenile hormone biosynthesis. We examined Plast-MIP-producing neurons in the brain with immunohistochemistry and Fourier transform ion cyclotron resonance mass spectrometry. Rabbit polyclonal antiserum against Plast-MIP revealed immunoreactive cells in seven regions of the brain, including the posterior antennal lobe, basal optic lobe, dorsal anterior protocerebrum, ventrolateral protocerebrum, pars intercerebralis, posterior protocerebrum, and dorsal posterior region to the calyx of the mushroom body, aside from the gnathal ganglion. Anatomical locations of the immunoreactive cells in the pars intercerebralis and dorsal posterior region to the mushroom body calyx partly overlapped with the cell body location stained by retrograde dye fills from the corpus allatum and corpus cardiacum complex. Direct mass spectrometry revealed the molecular ion peaks corresponding to the predictive mass of Plast-MIPs in the pars intercerebralis and the corpus allatum-corpus cardiacum complex. Plast-MIP immunoreactivity in different cell types suggests that Plast-MIPs have different functions in the cephalic ganglia. Considering the anatomical location of neurons projecting to the corpus allatum-corpus cardiacum and results of mass spectrometry, Plast-MIP immunoreactive cells in the pars intercerebralis may play a role in suppressing juvenile hormone biosynthesis.

Function and Distribution of the Wamide Neuropeptide Superfamily in Metazoans.

The Wamide neuropeptide superfamily is of interest due to its distinctive functions in regulating life cycle transitions, metamorphic hormone signaling, and several aspects of digestive system function, from gut muscle contraction to satiety and fat storage. Due to variation among researchers in naming conventions, a global view of Wamide signaling in animals in terms of conservation or diversification of function is currently lacking. Here, I summarize the phylogenetic distribution of Wamide neuropeptides based on current data and describe recent findings in the areas of Wamide receptors and biological functions. Common trends that emerge across Cnidarians and protostomes are the presence of multiple Wamide receptors within a single organism, and the fact that Wamide signaling likely functions across an extensive variety of biological systems, including visual, circadian, and reproductive systems. Important areas of focus for future research are the further identification of Wamide-receptor pairs, confirmation of the phylogenetic distribution of Wamides through largescale sequencing and mass spectrometry, and assignment of different functions to specific subsets of Wamide-expressing neurons. More extensive study of Wamide signaling throughout larval development in a greater number of phyla is also important in order to understand the role of Wamides in hormonal regulation. Defining the evolution and function of neuropeptide signaling in animal nervous systems will benefit from an increased understanding of Wamide function and signaling mechanisms in a wider variety of organisms, beyond the traditional model systems.

Molecular characterization of ligand selectivity of the sex peptide receptors of Drosophila melanogaster and Aedes aegypti.

Drosophila melanogaster sex peptide receptor (DrmSPR) is a G protein-coupled receptor (GPCR) with 'dual ligand selectivity' towards sex peptide (SP) and myoinhibitory peptides (MIPs), which are only remotely related to one another. SPR is conserved in almost all the sequenced lophotrochozoan and ecdysozoan genomes. SPRs from non-drosophilid taxa, such as those from the mosquitoes Aedes aegypti (AeaSPR), Anopheles gambiae (AngSPR), and the sea slug Aplysia californica (ApcSPR), are highly sensitive to MIP, but not to SP. To understand how Drosophila SPRs evolved their SP sensitivity while maintaining MIP sensitivity, we examined ligand selectivity in a series of chimeric GPCRs that combine domains from the SP-sensitive DrmSPR and the SP-insensitive AeaSPR. We found replacement of Pro 238 (P238) in DrmSPR with the corresponding residue from AeaSPR (L310) reduced its SP sensitivity 2.7 fold without altering its MIP sensitivity. The P238 residue located in the third extracellular loop (ECL3) is conserved in Drosophila SPRs and in SPR from the moth Bombyx mori (BomSPR), which is considerably more sensitive to SP than AeaSPR, AngSPR, or ApcSPR. We found, however, that rather than improving AeaSPR's sensitivity to SP, replacement of L310 in AeaSPR with Pro significantly reduces its MIP sensitivity. Thus, our identification of a single amino acid residue critical for SP sensitivity, but not for MIP sensitivity is an important step in clarifying how DrmSPR evolved the ability to detect SP.

A Neural Circuit Encoding the Experience of Copulation in Female Drosophila.

Female behavior changes profoundly after mating. In Drosophila, the mechanisms underlying the long-term changes led by seminal products have been extensively studied. However, the effect of the sensory component of copulation on the female's internal state and behavior remains elusive. We pursued this question by dissociating the effect of coital sensory inputs from those of male ejaculate. We found that the sensory inputs of copulation cause a reduction of post-coital receptivity in females, referred to as the "copulation effect." We identified three layers of a neural circuit underlying this phenomenon. Abdominal neurons expressing the mechanosensory channel Piezo convey the signal of copulation to female-specific ascending neurons, LSANs, in the ventral nerve cord. LSANs relay this information to neurons expressing myoinhibitory peptides in the brain. We hereby provide a neural mechanism by which the experience of copulation facilitates females encoding their mating status, thus adjusting behavior to optimize reproduction.

Identification of allatostatins in the brown-winged green bug Plautia stali.

Juvenile hormone (JH) biosynthesis is inhibited under short-day conditions in the brown-winged green bug Plautia stali. We investigated allatostatic molecules in the brain of P. stali. Methanol brain extracts strongly inhibited JH biosynthesis. The allatostatic activities of the brain extracts were heat stable but gently suppressed by trypsin treatment, indicating that the allatostatic molecules were peptides. Grybi-MIP1, found in Gryllus bimaculatus as an allatostatic molecule, inhibited JH biosynthesis in P. stali. In contrast, peptides such as Dippu-AST2, 8, and 9, found in Diploptera punctata, did not affect JH biosynthesis in P. stali. We found a cDNA sequence encoding a peptide precursor of myoinhibitory peptides (MIPs), which we named Plast-MIP. Three synthetic peptides, AWKDLSKAW-NH2 (Plast-MIP1), GWSDLQSAGW-NH2 (Plast-MIP5), and AADWGSFRGSW-NH2 (Plast-MIP8), deduced from the precursor sequence, showed clear inhibition of JH biosynthesis in P. stali. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and tandem mass spectrometry showed that Plast-MIP8 resides in the brain. Expression of the Plast-MIP mRNA precursor was detected in the brain of insects reared under short- and long-day conditions. These results suggest that Plast-MIP is an allatostatic molecule and that MIPs are synthesized irrespective of photoperiod. To our knowledge, this is the first study to identify Plast-MIP as a functional allatostatin in hemipteran insects.

Functions of corazonin and histamine in light entrainment of the circadian pacemaker in the Madeira cockroach, Rhyparobia maderae.

The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi- and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light-dependent delays or advances to the clock. Injections of neuroactive substances combined with running-wheel assays suggested that GABA, pigment-dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light-like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple-label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin-immunoreactive (-ir) terminals overlapped with projections of putatively light-sensitive interneurons from the ipsi- and contralateral compound eye. Thus, we hypothesize that the corazonin-ir medial neuron integrates ipsi- and contralateral light information as part of the phase-advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250-1272, 2017. © 2016 Wiley Periodicals, Inc.

The degradome and the evolution of Drosophila sex peptide as a ligand for the MIP receptor.

The male sex peptide (SP) of Drosophila melanogaster has wide ranging effects on females, including rejection of courting males, increased egg production, changes to the feeding habit, increased synthesis of antimicrobial peptides and elevated locomotor activity during day-time. The peptide activates receptors in sensory neurons of the female reproductive tract and can also traverse into the hemolymph and reach the central nervous system. The SP receptor involved in rejection and egg-laying responses has been shown to be identical to the receptor for the evolutionary conserved myoinhibitory peptides (MIPs) that function as neuropeptides in both males and females. Intriguingly, MIPs cannot substitute for SP when either expressed in the male accessory glands or injected into virgin females. MIPs are linear peptides with an amidated C-terminus which protects them from cleavage by carboxypeptidases, but leaves them exposed to potential attack from aminopeptidase and endopeptidase activities. In contrast, the SP region responsible for eliciting the post-mating response is cyclic and has several hydroxyproline residues N-terminal to the disulfide bridge which is expected to protect the biological activity of SP from peptidases of the male accessory gland and seminal fluid. We now present in vitro data showing that SP is metabolically stable, whereas MIPs are much more susceptible to degradation by peptidases of the male accessory gland and the hemolymph of virgin female D. melanogaster. SP has evolved relatively recently as a MIP receptor ligand that is particularly well adapted to surviving in the hostile degradome of the male accessory gland and seminal fluid.

Diverse in- and output polarities and high complexity of local synaptic and non-synaptic signaling within a chemically defined class of peptidergic Drosophila neurons.

Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators.