Stomatogastric Nervous System Research Articles

Neuropeptide Modulation Enables Biphasic Inter-network Coordination via a Dual-Network Neuron.

Linked rhythmic behaviors, such as respiration/locomotion or swallowing/chewing often require coordination for proper function. Despite its prevalence, the cellular mechanisms controlling coordination of the underlying neural networks remain undetermined in most systems. We use the stomatogastric nervous system of the crab Cancer borealis to investigate mechanisms of inter-network coordination, due to its small, well characterized feeding-related networks (gastric mill [chewing, ∼0.1 Hz]; pyloric [filtering food, ∼1 Hz]). Here, we investigate coordination between these networks during the Gly1-SIFamide neuropeptide modulatory state. Gly1-SIFamide activates a unique triphasic gastric mill rhythm in which the typically pyloric-only LPG neuron generates dual pyloric- plus gastric mill-timed oscillations. Additionally, the pyloric rhythm exhibits shorter cycles during gastric mill rhythm-timed LPG bursts, and longer cycles during IC, or IC plus LG gastric mill neuron bursts. Photoinactivation revealed that LPG is necessary to shorten pyloric cycle period, likely through its rectified electrical coupling to pyloric pacemaker neurons. Hyperpolarizing current injections demonstrated that although LG bursting enables IC bursts, only gastric mill rhythm bursts in IC are necessary to prolong the pyloric cycle period. Surprisingly, LPG photoinactivation also eliminated prolonged pyloric cycles, without changing IC firing frequency or gastric mill burst duration, suggesting that pyloric cycles are prolonged via IC synaptic inhibition of LPG, which indirectly slows the pyloric pacemakers via electrical coupling. Thus, the same dual-network neuron directly conveys excitation from its endogenous bursting and indirectly funnels synaptic inhibition to enable one network to alternately decrease and increase the cycle period of a related network.Significance Statement Related rhythmic behaviors frequently exhibit coordination, yet the cellular mechanisms coordinating the underlying neural networks are not determined in most systems. We investigated coordination between two small, well-characterized crustacean feeding-associated networks during a neuropeptide-elicited modulatory state. We find that a dual fast/slow network neuron directly shortens fast network cycles during its slow, intrinsically generated bursts, likely via electrical coupling to fast network pacemakers, despite rectification favoring the opposite direction. Additionally, the fast network is indirectly prolonged during another slow-network phase, via chemical synaptic inhibition that is likely funneled through the same electrical synapse. Thus, a dual-network neuron alternately reinforces and diminishes neuropeptide actions, enabling distinct frequencies of a faster network across different phases of a related slower rhythm.

Neuropeptide Modulation of Bidirectional Internetwork Synapses.

Oscillatory networks underlying rhythmic motor behaviors, and sensory and complex neural processing, are flexible, even in their neuronal composition. Neuromodulatory inputs enable neurons to switch participation between networks, or participate in multiple networks simultaneously. Neuromodulation of internetwork synapses can both recruit and coordinate a switching neuron in a second network. We previously identified an example in which a neuron is recruited into dual-network activity via peptidergic modulation of intrinsic properties. We now ask whether the same neuropeptide also modulates internetwork synapses for internetwork coordination. The crab (Cancer borealis) stomatogastric nervous system contains two well-defined feeding-related networks (pyloric, food filtering, ~1 Hz; gastric mill, food chewing, ~0.1 Hz). The projection neuron MCN5 uses the neuropeptide Gly1-SIFamide to recruit the pyloric-only LPG neuron into dual pyloric plus gastric mill-timed bursting via modulation of LPG's intrinsic properties. Descending input is not required for a coordinated rhythm, thus intra-network synapses between LPG and its second network must underlie coordination among these neurons. However, synapses between LPG and gastric mill neurons have not been documented. Using two-electrode voltage clamp recordings, we found that graded synaptic currents between LPG and gastric mill neurons (LG, IC, DG) were primarily negligible in saline, but were enhanced by Gly1-SIFamide. Further, LPG and gastric mill neurons entrain each other during Gly1-SIFamide application, indicating bidirectional, functional connectivity. Thus, a neuropeptide mediates neuronal switching through parallel actions, modulating intrinsic properties for recruitment into a second network and as shown here, also modulating bidirectional internetwork synapses for coordination.

Alterations in network robustness upon simultaneous temperature and pH perturbations.

Nervous systems have evolved to function consistently in the face of the normal environmental fluctuations experienced by animals. The stomatogastric nervous system (STNS) of the crab, Cancer borealis, produces a motor output that has been studied for its remarkable robustness in response to single global perturbations. Changes in environments, however, are often complex and multifactorial. Therefore, we studied the robustness of the pyloric network in the stomatogastric ganglion (STG) in response to simultaneous perturbations of temperature and pH. We compared the effects of elevated temperatures on the pyloric rhythm at control, acid, or base pHs. In each pH recordings were made at 11°C, and then the temperature was increased until the rhythms became disorganized ("crashed"). Pyloric burst frequencies and phase relationships showed minor differences between pH groups until reaching close to the crash temperatures. However, the temperatures at which the rhythms were disrupted were lower in the two extreme pH conditions. This indicates that one environmental stress can make an animal less resilient to a second stressor.NEW & NOTEWORTHY Resilience to environmental fluctuations is important for all animals. It is common that animals encounter multiple stressful events at the same time, the cumulative impacts of which are largely unknown. This study examines the effects of temperature and pH on the nervous system of crabs that live in the fluctuating environments of the Northern Atlantic Ocean. The ranges of tolerance to one perturbation, temperature, are reduced under the influence of a second, pH.

Switching neuron contributions to second network activity.

Network flexibility is important for adaptable behaviors. This includes neuronal switching, where neurons alter their network participation, including changing from single- to dual-network activity. Understanding the implications of neuronal switching requires determining how a switching neuron interacts with each of its networks. Here, we tested 1) whether "home" and second networks, operating via divergent rhythm generation mechanisms, regulate a switching neuron and 2) if a switching neuron, recruited via modulation of intrinsic properties, contributes to rhythm or pattern generation in a new network. Small, well-characterized feeding-related networks (pyloric, ∼1 Hz; gastric mill, ∼0.1 Hz) and identified modulatory inputs make the isolated crab (Cancer borealis) stomatogastric nervous system (STNS) a useful model to study neuronal switching. In particular, the neuropeptide Gly1-SIFamide switches the lateral posterior gastric (LPG) neuron (2 copies) from pyloric-only to dual-frequency pyloric/gastric mill (fast/slow) activity via modulation of LPG-intrinsic properties. Using current injections to manipulate neuronal activity, we found that gastric mill, but not pyloric, network neurons regulated the intrinsically generated LPG slow bursting. Conversely, selective elimination of LPG from both networks using photoinactivation revealed that LPG regulated gastric mill neuron-firing frequencies but was not necessary for gastric mill rhythm generation or coordination. However, LPG alone was sufficient to produce a distinct pattern of network coordination. Thus, modulated intrinsic properties underlying dual-network participation may constrain which networks can regulate switching neuron activity. Furthermore, recruitment via intrinsic properties may occur in modulatory states where it is important for the switching neuron to actively contribute to network output.NEW & NOTEWORTHY We used small, well-characterized networks to investigate interactions between rhythmic networks and neurons that switch their network participation. For a neuron switching into dual-network activity, only the second network regulated its activity in that network. In addition, the switching neuron was sufficient but not necessary to coordinate second network neurons and regulated their activity levels. Thus, regulation of switching neurons may be selective, and a switching neuron is not necessarily simply a follower in additional networks.

Oscillatory network spontaneously recovers both activity and robustness after prolonged removal of neuromodulators.

Robustness of neuronal activity is a property necessary for a neuronal network to withstand perturbations, which may otherwise disrupt or destroy the system. The robustness of complex systems has been shown to depend on a number of features of the system, including morphology and heterogeneity of the activity of the component neurons, size of the networks, synaptic connectivity, and neuromodulation. The activity of small networks, such as the pyloric network of the crustacean stomatogastric nervous system, appears to be robust despite some of the factors not being consistent with the expected properties of complex systems, e.g., small size and homogeneity of the synaptic connections. The activity of the pyloric network has been shown to be stable and robust in a neuromodulatory state-dependent manner. When neuromodulatory inputs are severed, activity is initially disrupted, losing both stability and robustness. Over the long term, however, stable activity homeostatically recovers without the restoration of neuromodulatory input. The question we address in this study is whether robustness can also be restored as the network reorganizes itself to compensate for the loss of neuromodulatory input and recovers the lost activity. Here, we use temperature changes as a perturbation to probe the robustness of the network's activity. We develop a simple metric of robustness, i.e., the variances of the network phase relationships, and show that robustness is indeed restored simultaneously along with its stable network activity, indicating that, whatever the reorganization of the network entails, it is deep enough also to restore this important property.

Thermal acclimation and habitat-dependent differences in temperature robustness of a crustacean motor circuit.

At the cellular level, acute temperature changes alter ionic conductances, ion channel kinetics, and the activity of entire neuronal circuits. This can result in severe consequences for neural function, animal behavior and survival. In poikilothermic animals, and particularly in aquatic species whose core temperature equals the surrounding water temperature, neurons experience rather rapid and wide-ranging temperature fluctuations. Recent work on pattern generating neural circuits in the crustacean stomatogastric nervous system have demonstrated that neuronal circuits can exhibit an intrinsic robustness to temperature fluctuations. However, considering the increased warming of the oceans and recurring heatwaves due to climate change, the question arises whether this intrinsic robustness can acclimate to changing environmental conditions, and whether it differs between species and ocean habitats. We address these questions using the pyloric pattern generating circuits in the stomatogastric nervous system of two crab species, Hemigrapsus sanguineus and Carcinus maenas that have seen a worldwide expansion in recent decades. Consistent with their history as invasive species, we find that pyloric activity showed a broad temperature robustness (>30°C). Moreover, the temperature-robust range was dependent on habitat temperature in both species. Warm-acclimating animals shifted the critical temperature at which circuit activity breaks down to higher temperatures. This came at the cost of robustness against cold stimuli in H. sanguineus, but not in C. maenas. Comparing the temperature responses of C. maenas from a cold latitude (the North Sea) to those from a warm latitude (Spain) demonstrated that similar shifts in robustness occurred in natural environments. Our results thus demonstrate that neuronal temperature robustness correlates with, and responds to, environmental temperature conditions, potentially preparing animals for changing ecological conditions and shifting habitats.

Multiscale head anatomy of Megaphragma (Hymenoptera: Trichogrammatidae)

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps.

A Feeding-Related Mechanoreceptor Identified in the Crab Cancer borealis Shares Similarities and Differences with Homologs in Other Crustaceans.

AbstractSensory feedback plays an essential role in shaping rhythmic animal movements. In the crustacean stomatogastric nervous system, which is responsible for grinding and filtering food particles in the animal's foregut, a number of mechanoreceptors whose activity affects motor output have been characterized. The hepatopancreas duct receptor neurons, which are located in the pyloric region of the foregut that is responsible for filtering, are among the less well understood groups of stomatogastric mechanoreceptors. Although they were first described decades ago in a number of decapod species, many questions remain about their role in shaping the movements produced by the stomatogastric nervous system. Here we provide the first anatomical and physiological evidence that there are also hepatopancreas duct receptors in the crab Cancer borealis, and we demonstrate that hepatopancreas duct receptor spiking produced by mechanical stimulation modifies the properties of an ongoing pyloric motor program.

Multiple intrinsic membrane properties are modulated in a switch from single- to dual-network activity.

Neural network flexibility includes changes in neuronal participation between networks, such as the switching of neurons between single- and dual-network activity. We previously identified a neuron that is recruited to burst in time with an additional network via modulation of its intrinsic membrane properties, instead of being recruited synaptically into the second network. However, the modulated intrinsic properties were not determined. Here, we use small networks in the Jonah crab (Cancer borealis) stomatogastric nervous system (STNS) to examine modulation of intrinsic properties underlying neuropeptide (Gly1-SIFamide)-elicited neuronal switching. The lateral posterior gastric neuron (LPG) switches from exclusive participation in the fast pyloric (∼1 Hz) network, due to electrical coupling, to dual-network activity that includes periodic escapes from the fast rhythm via intrinsically generated oscillations at the slower gastric mill network frequency (∼0.1 Hz). We isolated LPG from both networks by pharmacology and hyperpolarizing current injection. Gly1-SIFamide increased LPG intrinsic excitability and rebound from inhibition and decreased spike frequency adaptation, which can all contribute to intrinsic bursting. Using ion substitution and channel blockers, we found that a hyperpolarization-activated current, a persistent sodium current, and calcium or calcium-related current(s) appear to be primary contributors to Gly1-SIFamide-elicited LPG intrinsic bursting. However, this intrinsic bursting was more sensitive to blocking currents when LPG received rhythmic electrical coupling input from the fast network than in the isolated condition. Overall, a switch from single- to dual-network activity can involve modulation of multiple intrinsic properties, while synaptic input from a second network can shape the contributions of these properties.NEW & NOTEWORTHY Neuropeptide-elicited intrinsic bursting was recently determined to switch a neuron from single- to dual-network participation. Here we identified multiple intrinsic properties modulated in the dual-network state and candidate ion channels underlying the intrinsic bursting. Bursting at the second network frequency was more sensitive to blocking currents in the dual-network state than when neurons were synaptically isolated from their home network. Thus, synaptic input can shape the contributions of modulated intrinsic properties underlying dual-network activity.

Modulatory capacity correlates with dietary diversity in three species of decapod crustaceans

The central nervous systems of decapod crustaceans contains numerous amines, amino acids, and peptides that modulate the central pattern generating (CPG) circuits found within its stomatogastric nervous system (STNS) and cardiac ganglion (CG); over 30 modulators have been identified in one species of crab (C. borealis). CPGs in the STNS are responsible for internally chewing (Gastric Mill CPG) and filtering food (Pyloric CPG) into the midgut, while the CG drives the heartbeat. Neuromodulation allows these CPGs to flexibly produce a variety of output patterns; however, the variety of modulators exceeds the variety of induced output, leading us to ask why so many modulators are present. Although modulatory capacity, a measure of the extent to which a neuronal circuit can be modulated, may be solely an inherited trait, we hypothesize a functional correlation: decapods that need more behavioral flexibility utilize a broader range of modulators than those with more stereotyped behaviors. We thus predicted that decapods with broad diets would have a larger modulatory capacity in the STNS, but not the CG, than those with limited diets. To test this, we compared responses of the STNS and CG CPGs in three species of majoid crabs (Pugettia producta, Libinia emarginata, and Chionoecetes opilio) to the same neuromodulators. L. emarginata and C. opilio both have broad diets, while P. producta primarily eats kelp. With one exception, each species’ CG exhibited similar responses to all modulators. The STNS of crabs with broader diets responded similarly to all modulators, whereas the P. producta STNS showed limited responses to the same set of neuromodulators. CG responses did not follow this trend, as all crabs showed varied CG response to modulation. Modulators: Cancer borealisTachykinin Related Peptide (CabTRP), Crustacean Cardioactive Peptide (CCAP), Red Pigment Concentrating Hormone (RPCH), Myosuppressin (Myo), Oxotremorine (Oxo), Dopamine (DA), Proctolin (Proc).

Suppression of a Novel Vitellogenesis-Inhibiting Hormone Significantly Increases Ovarian Vitellogenesis in the Black Tiger Shrimp, Penaeus monodon.

In this study, a novel Crustacean Hyperglycemic Hormone-type II gene (CHH-type II) was identified and biologically characterized in a shrimp, Penaeus monodon. Based on its structure and function, this gene was named P. monodon vitellogenesis-inhibiting hormone (PemVIH). The complete cDNA sequence of PemVIH consisted of 1,022 nt with an open reading frame (ORF) of 339 nt encoding a polypeptide of 112 amino acids. It was classified as a member of the CHH-type II family based on conserved cysteine residues, a characteristically positioned glycine residue, and the absence of CHH precursor-related peptide (CPRP) domain. The deduced mature PemVIH shared the highest sequence similarities with giant river prawn sinus gland peptide A. Unlike P. monodon gonad-inhibiting hormone (PemGIH), PemVIH was expressed only in the brain and ventral nerve cord, but not the eyestalks. Whole mount immunofluorescence using a newly generated PemVIH antiserum detected positive signals in neuronal cluster 9/11 and 17 of the brain, commissural ganglion (CoG), and neuronal clusters of ventral nerve cord. The presence of PemVIH-positive neurons in CoG, a part of stomatogastric nervous system, suggested a potential mechanism for crosstalk between nutritional and reproductive signaling. The role of PemVIH in vitellogenesis was evaluated using RNA interference technique. Temporal knockdown of PemVIH in female subadults resulted in a 3-fold increase in ovarian vitellogenin expression, suggesting an inhibitory role of PemVIH in vitellogenesis. This study provided novel insight into the control of vitellogenesis and additional strategies for improving ovarian maturation in P. monodon without the current harmful practice of eyestalk ablation.

Neuronal networks: Degeneracy unleashed

Degeneracy in neuronal networks is emerging as a parallel to accepted degeneracy in neuronal and synaptic properties. A new study leverages detailed understanding of neuronal networks and their modulation in the crustacean stomatogastric nervous system to show that network degeneracy is conditional.

Feeding state-dependent modulation of feeding-related motor patterns.

Studies elucidating modulation of microcircuit activity in isolated nervous systems have revealed numerous insights regarding neural circuit flexibility, but this approach limits the link between experimental results and behavioral context. To bridge this gap, we studied feeding behavior-linked modulation of microcircuit activity in the isolated stomatogastric nervous system (STNS) of male Cancer borealis crabs. Specifically, we removed hemolymph from a crab that was unfed for ≥24 h ("unfed" hemolymph) or fed 15 min to 2 h before hemolymph removal ("fed" hemolymph). After feeding, the first significant foregut emptying occurred >1 h later and complete emptying required ≥6 h. We applied the unfed or fed hemolymph to the stomatogastric ganglion (STG) in an isolated STNS preparation from a separate, unfed crab to determine its influence on the VCN (ventral cardiac neuron)-triggered gastric mill (chewing) and pyloric (filtering of chewed food) rhythms. Unfed hemolymph had little influence on these rhythms, but fed hemolymph from each examined time-point (15 min, 1 h, or 2 h after feeding) slowed one or both rhythms without weakening circuit neuron activity. There were also distinct parameter changes associated with each time-point. One change unique to the 1-h time-point (i.e., reduced activity of one circuit neuron during the transition from the gastric mill retraction to protraction phase) suggested that the fed hemolymph also enhanced the influence of a projection neuron that innervates the STG from a ganglion isolated from the applied hemolymph. Hemolymph thus provides a feeding state-dependent modulation of the two feeding-related motor patterns in the C. borealis STG.NEW & NOTEWORTHY Little is known about behavior-linked modulation of microcircuit activity. We show that the VCN-triggered gastric mill (chewing) and pyloric (food filtering) rhythms in the isolated crab Cancer borealis stomatogastric nervous system were changed by applying hemolymph from recently fed but not unfed crabs. This included some distinct parameter changes during each examined post-fed hemolymph time-point. These results suggest the presence of feeding-related changes in circulating hormones that regulate consummatory microcircuit activity.

Neuronal Switching between Single- and Dual-Network Activity via Modulation of Intrinsic Membrane Properties.

Oscillatory networks underlie rhythmic behaviors (e.g., walking, chewing) and complex behaviors (e.g., memory formation, decision-making). Flexibility of oscillatory networks includes neurons switching between single- and dual-network participation, even generating oscillations at two distinct frequencies. Modulation of synaptic strength can underlie this neuronal switching. Here we ask whether switching into dual-frequency oscillations can also result from modulation of intrinsic neuronal properties. The isolated stomatogastric nervous system of male Cancer borealis crabs contains two well-characterized rhythmic feeding-related networks (pyloric, ∼1 Hz; gastric mill, ∼0.1 Hz). The identified modulatory projection neuron MCN5 causes the pyloric-only lateral posterior gastric (LPG) neuron to switch to dual pyloric/gastric mill bursting. Bath applying the MCN5 neuropeptide transmitter Gly1-SIFamide only partly mimics the LPG switch to dual activity because of continued LP neuron inhibition of LPG. Here, we find that MCN5 uses a cotransmitter, glutamate, to inhibit LP, unlike Gly1-SIFamide excitation of LP. Thus, we modeled the MCN5-elicited LPG switching with Gly1-SIFamide application and LP photoinactivation. Using hyperpolarization of pyloric pacemaker neurons and gastric mill network neurons, we found that LPG pyloric-timed oscillations require rhythmic electrical synaptic input. However, LPG gastric mill-timed oscillations do not require any pyloric/gastric mill synaptic input and are voltage-dependent. Thus, we identify modulation of intrinsic properties as an additional mechanism for switching a neuron into dual-frequency activity. Instead of synaptic modulation switching a neuron into a second network as a passive follower, modulation of intrinsic properties could enable a switching neuron to become an active contributor to rhythm generation in the second network.SIGNIFICANCE STATEMENT Neuromodulation of oscillatory networks can enable network neurons to switch from single- to dual-network participation, even when two networks oscillate at distinct frequencies. We used small, well-characterized networks to determine whether modulation of synaptic strength, an identified mechanism for switching, is necessary for dual-network recruitment. We demonstrate that rhythmic electrical synaptic input is required for continued linkage with a "home" network, whereas modulation of intrinsic properties enables a neuron to generate oscillations at a second frequency. Neuromodulator-induced switches in neuronal participation between networks occur in motor, cognitive, and sensory networks. Our study highlights the importance of considering intrinsic properties as a pivotal target for enabling parallel participation of a neuron in two oscillatory networks.

Dual modulatory effects on feedback from a proprioceptor in the crustacean stomatogastric nervous system.

Neuromodulatory actions that change the properties of proprioceptors or the muscle movements to which they respond necessarily affect the feedback provided to the central network. Here we further characterize the responses of the gastropyloric receptor 1 (GPR1) and gastropyloric receptor 2 (GPR2) neurons in the stomatogastric nervous system of the crab Cancer borealis to movements and contractions of muscles, and we report how neuromodulation modifies those responses. We observed that the GPR1 response to contractions of the gastric mill 4 muscle (gm4) was absent, or nearly so, when the neuron was quiescent but robust when it was spontaneously active. We also found that the effects of four neuromodulatory substances (GABA, serotonin, proctolin, and TNRNFLRFamide) on the GPR1 response to muscle stretch were similar to those previously reported for GPR2. Finally, we showed that an excitatory action on gm4 due to proctolin combined with an inhibitory action on GPR2 due to GABA can allow for larger muscle contractions without increased proprioceptive feedback.NEW & NOTEWORTHY We report that the combination of GABA and the peptide proctolin increases contraction of a stomatogastric muscle while decreasing the corresponding response of the proprioceptor that reports on it. These results suggest a general mechanism by which muscle movements can be modified while sensory feedback is conserved, one that may be particularly well suited for providing flexibility to central pattern generator networks.

Intrinsic sources of tachykinin-related peptide in the thoracic ganglion mass of the crab, Cancer borealis

Neuropeptides comprise the largest class of neural and neuroendocrine signaling molecules. Vertebrate tachykinins (TKs) and the structurally-related invertebrate tachykinin-related peptides (TRPs) together form the largest neuropeptide superfamily, with a number of conserved neural and neuroendocrine functions across species. Arthropods, including crustaceans, have provided many insights into neuropeptide signaling and function. Crustacean tachykinin-related peptide occurs in endocrine organs and cells and in two of the major crustacean CNS components, the supraoesophageal ganglion (“brain”) and the stomatogastric nervous system. However, little is known about TRP sources in the remaining major CNS component, the thoracic ganglion mass (TGM). To gain further insight into the function of this peptide, we aimed to identify intrinsic TRP sources in the TGM of the Jonah crab, Cancer borealis. We first adapted a clearing protocol to improve TRP immunoreactivity specifically in the TGM, which is a dense, fused mass of multiple ganglia in short-bodied crustaceans such as Cancer species of crabs. We verified that the clearing protocol avoided distortion of cell body morphology yet increased visibility of TRP immunoreactivity. Using confocal microscopy, we found TRP-immunoreactive (TRP-IR) axon tracts running the length of the TGM, TRP-IR neuropil in all ganglia, and approximately 110 TRP-IR somata distributed throughout the TGM, within and between ganglia. These somata likely represent both neural and neuroendocrine sources of TRP. Thus, there are many potential intrinsic sources of TRP in the TGM that are positioned to regulate behaviors such as food intake, locomotion, respiration, and reproduction.

Role of peptide hormones in insect gut physiology.

Nutrient uptake and digestion are essential for optimal growth and development. In insects, these processes are regulated by the gut-brain axis, which is a neurohumoral communication system for maintaining gut homeostasis. The insect gut is a complex organ consisting of three distinct structures, denominated foregut, midgut and hindgut, each with their specific specializations. These specializations are tightly regulated by the interplay of several neuropeptides: a versatile group of signalling molecules involved in a multitude of processes including gut physiology. Neuropeptides take part in the regulation of gut processes ranging from digestive enzyme release to muscle activity and satiety. Some neuropeptide mimetics are a promising strategy for ecological pest management. This review focuses on a selection of neuropeptides that are well-known for their role in gut physiology, and neuropeptides for which the mode of action is yet to be unravelled.

Within-family plasticity of nervous system architecture in Syllidae (Annelida, Errantia)

BackgroundThe ground pattern underlying the nervous system of the last common ancestor in annelids was long thought to be settled, consisting of a dorsal brain, circumoesophageal connectives and a subepithelial, ladder-like ventral nerve cord with segmental ganglia connected by paired connectives. With the advent of immunocytochemical stainings and confocal laser scanning microscopy, it becomes evident that its architecture is extremely diverse, which makes the reconstruction of a ground pattern in annelida challenging. Whereas the nervous systems of many different families has already been described, only very few studies looked at the diversity of nervous systems within such clades to give a closer estimate on how plastic the annelid nervous system really is. So far, little is known on syllid nervous system architecture, one of the largest and most diverse groups of marine annelids.ResultsThe position of the brain, the circumoesophageal connectives, the stomatogastric nervous system, the longitudinal nerves that traverse each segment and the innervation of appendages are relatively uniform within the clade. Both the number of connectives within the ventral nerve cord and the number of segmental nerves, which in earlier studies were used to infer phylogenetic relationships and to reconstruct an annelid ground pattern, are highly diverse and differ between genera or even within a given genus. Differences in the distribution of somata of the brain, the nuchal innervation and its associated cell bodies were found between Syllinae and Exogoninae and may be subfamily-specific.ConclusionsThe nervous system morphology of syllids very likely depends on the taxon-specific ecological requirements. Thus, it is not surprising that in a clade, which occupies such diverse niches as the Annelida, we find similar patterns in phylogenetically widely separated species in similar niches and a high degree of modularity within a family. Only standardized protocols and staining methods can lead to comparable results, but so far different approaches have been taken to describe annelid nervous systems, making homologization of certain structures difficult. This study provides the first thorough description of the nervous system in the family Syllidae, allowing more detailed comparisons between annelid families in the future.

Molecular cloning of crustacean hyperglycemic hormone (CHH) family members (CHH, molt-inhibiting hormone and mandibular organ-inhibiting hormone) and their expression levels in the Jonah crab, Cancer borealis

The crustacean hyperglycemic hormone (CHH) neuropeptide family has multiple functions in the regulation of hemolymph glucose levels, molting, ion, and water balance and reproduction. In crab species, three neuroendocrine tissues: the eyestalk ganglia (medulla terminalis X-organ and -sinus gland = ES), the pericardial organ (PO), and guts synthesize a tissue-specific isoforms of CHH neuropeptides. Recently the presence of the mandibular organ-inhibiting hormone (MOIH) was reported in the stomatogastric nervous system (STNS) that regulates the rhythmic muscle movements in esophagus, cardiac sac, gastric and pyloric ports of the foregut. In this study, we aimed to determine the presence of a tissue-specific CHH isoform in the Jonah crab, Cancer borealis using PCR with degenerate primers and 5′, 3′ rapid amplification of cDNA ends (RACE) in the ES. PO, and STNS. The analysis of CHH sequences shows that C. borealis has one type of CHH isoform, unlike other crab species. We also isolated the cDNA sequence of molt-inhibiting hormone (MIH) in the ES and MOIH in the ES and STNS. The presence of CHH, MOIH and MIH in the sinus gland of adult females and males is confirmed by using a dot-blot assay with the putative peaks collected from RP-HPLC and anti-Cancer sera for CHH, MIH, and MOIH. The present of crustacean female sex hormone (CFSH) in the sinus gland of adult females was examined with a dot-blot assay with anti-Callinectes CFSH serum. Levels of CHH, MOIH, and MIH in the sinus gland and their expressions in the eyestalk ganglia are estimated in the adult males, where CHH is the predominant form among these neuropeptides.

Tachykinins: Neuropeptides That Are Ancient, Diverse, Widespread and Functionally Pleiotropic.

Tachykinins (TKs) are ancient neuropeptides present throughout the bilaterians and are, with some exceptions, characterized by a conserved FX1GX2Ramide carboxy terminus among protostomes and FXGLMamide in deuterostomes. The best-known TK is the vertebrate substance P, which in mammals, together with other TKs, has been implicated in health and disease with important roles in pain, inflammation, cancer, depressive disorder, immune system, gut function, hematopoiesis, sensory processing, and hormone regulation. The invertebrate TKs are also known to have multiple functions in the central nervous system and intestine and these have been investigated in more detail in the fly Drosophila and some other arthropods. Here, we review the protostome and deuterostome organization and evolution of TK precursors, peptides and their receptors, as well as their functions, which appear to be partly conserved across Bilateria. We also outline the distribution of TKs in the brains of representative organisms. In Drosophila, recent studies have revealed roles of TKs in early olfactory processing, neuromodulation in circuits controlling locomotion and food search, nociception, aggression, metabolic stress, and hormone release. TK signaling also regulates lipid metabolism in the Drosophila intestine. In crustaceans, TK is an important neuromodulator in rhythm-generating motor circuits in the stomatogastric nervous system and a presynaptic modulator of photoreceptor cells. Several additional functional roles of invertebrate TKs can be inferred from their distribution in various brain circuits. In addition, there are a few interesting cases where invertebrate TKs are injected into prey animals as vasodilators from salivary glands or paralyzing agents from venom glands. In these cases, the peptides are produced in the glands of the predator with sequences mimicking the prey TKs. Lastly, the TK-signaling system appears to have duplicated in Panarthropoda (comprising arthropods, onychophores, and tardigrades) to give rise to a novel type of peptides, natalisins, with a distinct receptor. The distribution and functions of natalisins are distinct from the TKs. In general, it appears that TKs are widely distributed and act in circuits at short range as neuromodulators or cotransmitters.