Small Cardioactive Peptide Research Articles

Convergent effects of neuropeptides on the feeding central pattern generator of Aplysia californica.

These experiments focus on an interneuron (B63) that is part of the feeding central pattern generator (CPG) in Aplysia californica. Previous work has established that B63 is critical for program initiation regardless of the type of evoked activity. B63 receives input from a number of different elements of the feeding circuit. Program initiation occurs reliably when some are activated, but we show that it does not occur reliably with activation of others. When program initiation is reliable, modulatory neuropeptides are released. For example, previous work has established that an ingestive input to the feeding CPG, cerebral buccal interneuron 2 (CBI-2), releases feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP-2). Afferents with processes in the esophageal nerve (EN) that trigger egestive motor programs release small cardioactive peptide (SCP). Previous studies have described divergent cellular and molecular effects of FCAP/CP-2 and SCP on the feeding circuit that specify motor activity. Here, we show that FCAP/CP-2 and SCP additionally increase the B63 excitability. Thus, we show that peptides that have well-characterized divergent effects on the feeding circuit additionally act convergently at the level of a single neuron. Since convergent effects of FCAP/CP-2 and SCP are not necessary for specifying the type of network output, we ask why they might be important. Our data suggest that they have an impact during a task switch, i.e., when there is a switch from egestive to ingestive activity.NEW & NOTEWORTHY The activity of multifunctional central pattern generators (CPGs) is often configured by neuromodulators that exert divergent effects that are necessary to specify motor output. We demonstrate that ingestive and egestive inputs to the feeding CPG in Aplysia act convergently (as well as divergently). We ask why this convergence may be important and suggest that it may be a mechanism for a type of arousal that occurs during task switching.

Comparative Analysis of Neuropeptides in Homologous Interneurons and Prohormone Annotation in Nudipleuran Sea Slugs.

Despite substantial research on neuronal circuits in nudipleuran gastropods, few peptides have been implicated in nudipleuran behavior. In this study, we expanded the understanding of peptides in this clade, using three species with well-studied nervous systems, Hermissenda crassicornis, Melibe leonina, and Pleurobranchaea californica. For each species, we performed sequence homology analysis of de novo transcriptome predictions to identify homologs to 34 of 36 prohormones previously characterized in the gastropods Aplysia californica and Lymnaea stagnalis. We then used single-cell mass spectrometry to characterize peptide profiles in homologous feeding interneurons: the multifunctional ventral white cell (VWC) in P. californica and the small cardioactive peptide B large buccal (SLB) cells in H. crassicornis and M. leonina. The neurons produced overlapping, but not identical, peptide profiles. The H. crassicornis SLB cells expressed peptides from homologs to the FMRFamide (FMRFa), small cardioactive peptide (SCP), LFRFamide (LFRFa), and feeding circuit activating peptides prohormones. The M. leonina SLB cells expressed peptides from homologs to the FMRFa, SCP, LFRFa, and MIP-related peptides prohormones. The VWC, previously shown to express peptides from the FMRFa and QNFLa (a homolog of A. californica pedal peptide 4) prohormones, was shown to also contain SCP peptides. Thus, each neuron expressed peptides from the FMRFa and SCP families, the H. crassicornis and M. leonina SLB cells expressed peptides from the LFRFa family, and each neuron contained peptides from a prohormone not found in the others. These data suggest each neuron performs complex co-transmission, which potentially facilitates a multifunctional role in feeding. Additionally, the unique feeding characteristics of each species may relate, in part, to differences in the peptide profiles of these neurons. These data add chemical insight to enhance our understanding of the neuronal basis of behavior in nudipleurans and other gastropods.

The Distribution and Possible Roles of Small Cardioactive Peptide in the Nudibranch Melibe leonina.

SynopsisThe neuropeptide small cardioactive peptide (SCP) plays an integrative role in exciting various motor programs involved in feeding and locomotion in a number of gastropod species. In this study, immunohistochemistry, using monoclonal antibodies against SCPB, was used to localize SCPB-like-immunoreactive neurons in the central nervous system, and map their connections to various tissues, in the nudibranch, Melibe leonina. Approximately 28–36 SCPB-like-immunoreactive neurons were identified in the M. leonina brain, as well as one large neuron in each of the buccal ganglia. The neuropil of the pedal ganglia contained the most SCPB-like-immunoreactive varicosities, although only a small portion of these were due to SCPB-like-immunoreactive neurons in the same ganglion. This suggests that much of the SCPB-like immunoreactivity in the neuropil of the pedal ganglia was from neurons in other ganglia that projected through the pedal–pedal connectives or the connectives from the cerebral and pleural ganglia. We also observed extensive SCPB innervation along the length of the esophagus. Therefore, we investigated the impact of SCPB on locomotion in intact animals, as well as peristaltic contractions of the isolated esophagus. Injection of intact animals with SCPB at night led to a significant increase in crawling and swimming, compared to control animals injected with saline. Furthermore, perfusion of isolated brains with SCPB initiated expression of the swim motor program. Application of SCPB to the isolated quiescent esophagus initiated rhythmic peristaltic contractions, and this occurred in preparations both with and without the buccal ganglia being attached. All these data, taken together, suggest that SCPB could be released at night to arouse animals and enhance the expression of both feeding and swimming motor programs in M. leonina.

Evaluation of dicloran phototoxicity using primary cardiomyocyte culture from Crassostrea virginica

Dicloran is a commonly used fungicide throughout the Southern and Western United States. Runoff of dicloran from agriculture systems to nearby waterbodies can accumulate in the organisms that inhabit those areas. Although severe damage of dicloran to ecological systems has not been reported, its toxicity has been modified by photodegradation. The objective of this study is to assess the changes of dicloran toxicities during photo exposure using a reliable in vitro biological model. In the present investigation, the photodegradation of dicloran in vitro showed over 90% of dicloran was degraded within 24h of UV exposure in water. Two major intermediate degradation products, 2-chloro-1,4-benzoquinone (CBQ) and 1,4-benzoquinone (BQ), were detected upon UV exposure of dicloran; however, they were rapidly degraded via photolysis. To estimate the impact of the phototoxicity of dicloran to aquatic organisms, we developed an in vitro cell culture system using the C. virginica cardiomyoctes (CvCMs) which were isolated from heart tissues and formed beating cell clusters. The CvCM clusters were treated with irradiated dicloran or the two intermediate standards, CBQ and BQ, and they showed up to 41% decrease in beating rates compared to control cell clusters. Expression levels of selected genes: def, hsp70, and cam, were upregulated in response to stimulations of UV irradiated dicloran and the two standard intermediates. The four-hour irradiated dicloran also resulted in more significant inhibition in the proliferation and small cardioactive peptide β production of CvCMs than other treatment. Tested solutions of photolyzed dicloran showed elevated toxicities opposed to the standard intermediates, CBQ and BQ, suggesting additive toxicity of these dicloran products or toxicity due to other unidentified degradation products. Results of this study supported our hypothesis that the degradation of dicloran caused by photo irradiation results in an elevated toxicity which can be evaluated by the in vitro CvCM model.

The Gastric Ganglion of Octopus vulgaris: Preliminary Characterization of Gene- and Putative Neurochemical-Complexity, and the Effect of Aggregata octopiana Digestive Tract Infection on Gene Expression.

The gastric ganglion is the largest visceral ganglion in cephalopods. It is connected to the brain and is implicated in regulation of digestive tract functions. Here we have investigated the neurochemical complexity (through in silico gene expression analysis and immunohistochemistry) of the gastric ganglion in Octopus vulgaris and tested whether the expression of a selected number of genes was influenced by the magnitude of digestive tract parasitic infection by Aggregata octopiana. Novel evidence was obtained for putative peptide and non-peptide neurotransmitters in the gastric ganglion: cephalotocin, corticotrophin releasing factor, FMRFamide, gamma amino butyric acid, 5-hydroxytryptamine, molluscan insulin-related peptide 3, peptide PRQFV-amide, and tachykinin–related peptide. Receptors for cholecystokininA and cholecystokininB, and orexin2 were also identified in this context for the first time. We report evidence for acetylcholine, dopamine, noradrenaline, octopamine, small cardioactive peptide related peptide, and receptors for cephalotocin and octopressin, confirming previous publications. The effects of Aggregata observed here extend those previously described by showing effects on the gastric ganglion; in animals with a higher level of infection, genes implicated in inflammation (NFκB, fascin, serpinB10 and the toll-like 3 receptor) increased their relative expression, but TNF-α gene expression was lower as was expression of other genes implicated in oxidative stress (i.e., superoxide dismutase, peroxiredoxin 6, and glutathione peroxidase). Elevated Aggregata levels in the octopuses corresponded to an increase in the expression of the cholecystokininA receptor and the small cardioactive peptide-related peptide. In contrast, we observed decreased relative expression of cephalotocin, dopamine β-hydroxylase, peptide PRQFV-amide, and tachykinin-related peptide genes. A discussion is provided on (i) potential roles of the various molecules in food intake regulation and digestive tract motility control and (ii) the difference in relative gene expression in the gastric ganglion in octopus with relatively high and low parasitic loads and the similarities to changes in the enteric innervation of mammals with digestive tract parasites. Our results provide additional data to the described neurochemical complexity of O. vulgaris gastric ganglion.

Repetition priming of motor activity mediated by a central pattern generator: the importance of extrinsic vs. intrinsic program initiators.

Repetition priming is characterized by increased performance as a behavior is repeated. Although this phenomenon is ubiquitous, mediating mechanisms are poorly understood. We address this issue in a model system, the feeding network of Aplysia This network generates both ingestive and egestive motor programs. Previous data suggest a chemical coding model: ingestive and egestive inputs to the feeding central pattern generator (CPG) release different modulators, which act via different second messengers to prime motor activity in different ways. The ingestive input to the CPG (neuron CBI-2) releases the peptides feeding circuit activating peptide and cerebral peptide 2, which produce an ingestive pattern of activity. The egestive input to the CPG (the esophageal nerve) releases the peptide small cardioactive peptide. This model is based on research that focused on a single aspect of motor control (radula opening). Here we ask whether repetition priming is observed if activity is triggered with a neuron within the core CPG itself and demonstrate that it is not. Moreover, previous studies demonstrated that effects of modulatory neurotransmitters that induce repetition priming persist. This suggests that it should be possible to "prime" motor programs triggered from within the CPG by first stimulating extrinsic modulatory inputs. We demonstrate that programs triggered after ingestive input activation are ingestive and programs triggered after egestive input activation are egestive. We ask where this priming occurs and demonstrate modifications within the CPG itself. This arrangement is likely to have important consequences for "task" switching, i.e., the cessation of one type of motor activity and the initiation of another.

Differential peptide expression in the central nervous system of the land snail Theba pisana, between active and aestivated

Hypometabolism is a physiological state of dormancy entered by many animals in times of environmental stress. There are gaps in our understanding of the molecular components used by animals to achieve this metabolic state. The availability of genomic and transcriptome data can be useful to study the process of hypometabolism at the molecular level. In this study, we use the land snail Theba pisana to identify peptides that may be involved in the hypometabolic state known as aestivation. We found a total of 22 neuropeptides in the central nervous system (CNS) that were differentially produced during activity and aestivation based on mass spectral-based neuropeptidome analysis. Of these, 4 were upregulated in active animals and 18 were upregulated in aestivation. A neuropeptide known to regulate muscle contractions in a variety of molluscs, the small cardioactive peptide A (sCAPA), and a peptide of yet unknown function (termed Aestivation Associated Peptide 12) were chosen for further investigation using temporal and spatial expression analysis of the precursor gene and peptide. Both peptides share expression within regions of the CNS cerebral ganglia and suboesophageal ganglia. Relative transcript abundance suggests that regulation of peptide synthesis and secretion is post-transcriptional. In summary, we provide new insights into the molecular basis of the regulation of aestivation in land snails through CNS peptide control.

Specificity of repetition priming: the role of chemical coding.

We investigate stimulus specificity of repetition priming in a tractable model system; the feeding network of Aplysia. Previous studies primarily focused on an aspect of behavior that is altered during ingestive priming, radula opening. Priming of radula opening occurs when two modulatory peptides [feeding circuit activating peptide (FCAP) and cerebral peptide-2 (CP-2)] are released from the cholinergic command-like neuron cerebral buccal interneuron 2. Effects of FCAP/CP-2 on radula opening motor neurons are cAMP mediated. The present experiments sought to determine whether FCAP/CP-2 and cAMP are also involved in the priming of radula opening during an incompatible activity, i.e., during egestive motor programs. Egestive priming is induced when motor programs are triggered by afferents with processes in the esophageal nerve. We demonstrate that egestive priming is not FCAP/CP-2 mediated. Instead, it is induced by an unrelated peptide (small cardioactive peptide), which exerts PKC-mediated effects. Our data, therefore, suggest that different feeding motor programs are primed via actions of different sets of intercellular and intracellular substances. We suggest that this accounts for the stimulus specificity that can be characteristic of repetition priming. Different stimuli activate different central pattern generator inputs. These inputs release different modulators, which induce functionally distinct motor programs.

Inter- and intraspecific plasticity in distribution patterns of immunoreactive compounds in actinotroch larvae of Phoronida (Lophotrochozoa)

Recent molecular data place Phoronida within the protostome superclade Lophotrochozoa, where they have been suggested to form a monophyletic assemblage with Brachiopoda and/or Nemertea. Herein, the anatomy of the nervous system and the structure of the apical organ are described for two larval stages of Phoronis muelleri in order to contribute to the discussion concerning the evolution of lophotrochozoan nervous systems. Specimens were investigated by confocal laser scanning microscopy using antibodies against the serotonin-like immunoreactive (serotonin-lir), the FMRF-amide-like immunoreactive (FMRFamide-lir) and the small cardioactive peptide B-like immunoreactive (small cardioactive peptide B-lir) compounds of the nervous system. Consistent with larvae of other phoronid species, we found a complex apical organ that consists of numerous serotonin-lir flask-shaped cells, additional bi- or multipolar serotonin-lir cells and several FMRFamide-lir perikarya. A detailed comparison between our results and those of a previous study on the same species shows significant differences in the innervation of the preoral lobe, the tentacles and the telotroch. Our work is the first to prove the presence of small cardioactive peptide B in phoronids. In larvae of P. muelleri, it is expressed in neurites along the margin of the preoral hood, in the mesosome, in the tentacles, in the trunk as well as in the apical organ. A positive signal for this peptide is also known from molluscs, annelids and arthropods, indicating that it was also part of the protostomian groundplan. In contrast to a recent study on another phoronid species, Phoronopsis harmeri, we did not find a ventral neurite bundle in the larval stages investigated herein, thus leaving the question open whether this structure was part of the phoronid groundplan or evolved de novo in P. harmeri.

Neurochemical and Neuroanatomical Identification of Central Pattern Generator Neuron Homologues in Nudipleura Molluscs

Certain invertebrate neurons can be identified by their behavioral functions. However, evolutionary divergence can cause some species to not display particular behaviors, thereby making it impossible to use physiological characteristics related to those behaviors for identifying homologous neurons across species. Therefore, to understand the neural basis of species-specific behavior, it is necessary to identify homologues using characteristics that are independent of physiology. In the Nudipleura mollusc Tritonia diomedea, Cerebral Neuron 2 (C2) was first described as being a member of the swim central pattern generator (CPG). Here we demonstrate that neurochemical markers, in conjunction with previously known neuroanatomical characteristics, allow C2 to be uniquely identified without the aid of electrophysiological measures. Specifically, C2 had three characteristics that, taken together, identified the neuron: 1) a white cell on the dorsal surface of the cerebral ganglion, 2) an axon that projected to the contralateral pedal ganglion and through the pedal commissure, and 3) immunoreactivity for the peptides FMRFamide and Small Cardioactive Peptide B. These same anatomical and neurochemical characteristics also uniquely identified the C2 homologue in Pleurobranchaea californica (called A1), which was previously identified by its analogous role in the Pleurobranchaea swim CPG. Furthermore, these characteristics were used to identify C2 homologues in Melibe leonina, Hermissenda crassicornis, and Flabellina iodinea, species that are phylogenetically closer to Tritonia than Pleurobranchaea, but do not display the same swimming behavior as Tritonia or Pleurobranchaea. These identifications will allow future studies comparing and contrasting the physiological properties of C2 across species that can and cannot produce the type of swimming behavior exhibited by Tritonia.

Sequential recruitment of identified I1/I3 motor neurons enhances muscle contraction through modulatory interactions

Event Abstract Back to Event Sequential recruitment of identified I1/I3 motor neurons enhances muscle contraction through modulatory interactions Hui Lu1*, Jeffrey M. McManus1, Miranda J. Cullins1 and Hillel J. Chiel2 1 Case Western Reserve University, Biology, United States 2 Case Western Reserve University, Biology, Neurosciences, Biomedical Engineering, United States Previous studies have shown that motor neurons for a single muscle are recruited in an orderly fashion based on a ‘size principle’ to produce movements with different force and speed (Henneman, 1957; 1965a; 1965b; Mendell, 2005). Although sequential recruitment of motor neurons has been extensively studied in vertebrates during the past 50 years (Zajac and Faden, 1985; Gabriel et al, 2011; Mantilla and Sieck, 2011), it is difficult to study in vivo at the individual neuron level. To explore the behavioral significance and force outputs of motor neuron recruitment sequences, we studied it in the marine mollusk Aplysia californica, in which individual motor neurons can be identified, monitored and controlled. Aplysia use the same feeding apparatus, the buccal mass, to perform multiple feeding behaviors: biting, swallowing and rejection. All feeding behaviors have two phases, protraction and retraction of the food grasper (radula/odontophore). Previous magnetic resonance imaging evidence (Neustadter et al, 2007) and our current data both suggest that the retraction phase is a major difference between biting and swallowing, and is primarily mediated by the contraction of the I1/I3 muscle. We found that buccal nerve 2 (BN2), which contains the axons of multiple I1/I3 motor neurons (e.g., B3, B6, B9, B10, B38, B39 and B43), is activated more intensely during the retraction phase in swallowing than in biting in vivo. In addition, identified I1/I3 motor neurons B3, B6 and B9 are more active in swallowing than in biting. Thus, we hypothesize that the increased retraction force during swallowing is due to the sequential recruitment of the identified I1/I3 motor neurons B6 and B9, followed by B3. We found that BN2 neural activity of the I1/I3 motor neurons during retraction in vivo and in vitro can be divided into four sub-phases (blocks) based on the BN2 unit sizes, relative timing, bursting features, and muscle innervation. The B3, B6 and B9 neurons are activated during the middle blocks of the retraction phase in all three feeding behaviors. More specifically, B6 and B9 fire in both the second and third blocks of retraction, whereas B3 only fires in the third block. The duration and force of retraction are both primarily determined by the second and third blocks containing B3, B6 and B9. In addition, we found that the frequency range (10-15 Hz) of B3 firing in vivo is not high enough to generate large forces. However, the activation of B6 and B9 prior to B3 can enhance the I1/I3 muscle contraction evoked by B3, consistent with the modulatory effects from B9 to B3 (Keating and Lloyd, 1999), which could be caused by the small cardioactive peptide (SCP) released by B6 and B9 (Church and Lloyd, 1991; Weiss et al, 1992). Moreover, we found that repetitive activation of a single motor neuron, B3, B6, or B9, can also increase its own forces. Therefore, the sequential recruitment of B6 and B9, prior to B3, enhances muscle contraction through modulatory interactions both within single responses and from response to response. Acknowledgements This research was funded by NIH Grant NS047073 and NSF Grant DMS1010434.

The presence and location of small cardioactive-like peptides in larvae of Crassostrea virginica

We examined the presence and location of small cardioactive peptide (SCP)-like neuropeptides in both the central and peripheral nervous systems of D-hinge, newly eyed, and pediveliger larvae of Crassostrea virginica. Results indicate that SCP-like substances are present early in development (D-hinge larvae), and that presence in the central and peripheral nervous systems increases as the larva develops toward metamorphic competence (pediveliger larvae). In addition, in newly eyed and pediveliger larvae, SCP-like labeling is found in a varying number of neurons within all central ganglia, with the possible exception of the accessory ganglia. Varicose labeling of axons is also documented within the ganglia, commissures, and connectives, as well as in peripheral nerves. Peripheral tissues innervated by axons exhibiting SCP-like immunoreactivity include the velum, foot, esophagus, mantle, and various musculatures. As indicated by the location of SCP-like labeling in various organs and tissues, it is likely that these neuropeptides modulate muscle contraction or ciliary beating in molluscan larvae such as those of C. virginica.

A new species of Lepidodasys (Gastrotricha, Macrodasyida) from Panama with a description of its peptidergic nervous system using CLSM, anti-FMRFamide and anti-SCP B

A new species of Lepidodasys (Gastrotricha: Macrodasyida: Lepidodasyidae) is described from sublittoral sediments in the Bocas del Toro archipelago in Panama and represents the first species of Lepidodasyidae described from the Caribbean. The new species possesses keeled scales that form a crossed-helical pattern across its dorsal and lateral surfaces and ventral scales that form a herringbone pattern between and lateral to the ciliary columns. A bilateral pair of three ventral adhesive tubes at the posterior end further differentiates this new species from its seven congeners. A confocal laser scanning microscope examination of the nervous system using antibodies to small cardioactive peptide B (SCP B) and FMRFamides reveals a dumbbell-shape cerebral ganglion, paired pharyngeal neurites and paired posterior nerve cords. Expression patterns of immunoreactivity to both classes of neuropeptides show a high degree of similarity. Only within the lateral somata of the cerebral ganglion and a single median pharyngeal neurite is there a difference in immunoreactivity to FMRFamide (positive) compared to SCP B (negative). Results from this investigation reveal that neuropeptides, among other neuronal markers, might provide phylogenetically informative characters in macrodasyidan gastrotrichs, especially regarding the topology of the cerebral ganglion.

Composite Modulatory Feedforward Loop Contributes to the Establishment of a Network State

Feedforward loops (FFLs) are one of many network motifs identified in a variety of complex networks, but their functional role in neural networks is not well understood. We provide evidence that combinatorial actions of multiple modulators may be organized as FFLs to promote a specific network state in the Aplysia feeding motor network. The Aplysia feeding central pattern generator (CPG) receives two distinct inputs-a higher-order interneuron cerebral-buccal interneuron-2 (CBI-2) and the esophageal nerve (EN)-that promote ingestive and egestive motor programs, respectively. EN stimulation elicits a persistent egestive network state, which enables the network to temporarily express egestive programs following a switch of input from the EN to CBI-2. Previous work showed that a modulatory CPG element, B65, is specifically activated by the EN and participates in establishing the egestive state by enhancing activity of egestion-promoting B20 interneurons while suppressing activity and synaptic outputs of ingestion-promoting B40 interneurons. Here a peptidergic contribution is mediated by small cardioactive peptide (SCP). Immunostaining and mass spectrometry show that SCP is present in the EN and is released on EN stimulation. Importantly, SCP directly enhances activity and synaptic outputs of B20 and suppresses activity and synaptic outputs of B40. Moreover, SCP promotes B65 activity. Thus the direct and indirect (through B65) pathways to B20 and B40 from SCPergic neurons constitute two FFLs with one functioning to promote egestive output and the other to suppress ingestive output. This composite FFL consisting of the two combined FFLs appears to be an effective means to co-regulate activity of two competing elements that do not inhibit each other, thereby contributing to establish specific network states.

Identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, Eisenia fetida

AbstractPeriviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR( L/I)amide, SPLPR( L/I)amide, SFVR( L/I)amide, AFVR( L/I)amide, and SPAFVR( L/I)amide, were identified in the central nervous system with the common‐XR( L/I)amide C‐terminal sequence. The exact anatomical position of 13 labeled XR( I/L)amide expressing neuron groups and numerous peptide‐containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole‐mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine‐fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister‐groups of animals such as annelids, mollusks, and insects. J. Comp. Neurol. 514:415–432, 2009. © 2009 Wiley‐Liss, Inc.

Identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, Eisenia fetida

AbstractPeriviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR(L/I)amide, SPLPR(L/I)amide, SFVR(L/I)amide, AFVR(L/I)amide, and SPAFVR(L/I)amide, were identified in the central nervous system with the common −XR(L/I)amide C‐terminal sequence. The exact anatomical position of 13 labeled XR(I/L)amide expressing neuron groups and numerous peptide‐containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole‐mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine‐fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister‐groups of animals such as annelids, mollusks, and insects. J. Comp. Neurol. 514:415–432, 2009. © 2009 Wiley‐Liss, Inc.

Identification of novel neuropeptides in the ventral nerve cord ganglia and their targets in an annelid worm, Eisenia fetida

Periviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR(L/I)amide, SPLPR(L/I)amide, SFVR(L/I)amide, AFVR(L/I)amide, and SPAFVR(L/I)amide, were identified in the central nervous system with the common -XR(L/I)amide C-terminal sequence. The exact anatomical position of 13 labeled XR(I/L)amide expressing neuron groups and numerous peptide-containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole-mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine-fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister-groups of animals such as annelids, mollusks, and insects.

Serotonergic and SCPb-like innervation of the atrial complex in Gyratrix hermaphroditus (Platyhelminthes, Kalyptorhynchia) revealed with CLSM

The platyhelminth reproductive system is a complex series of canals, glands, and sclerotic components that figure prominently in our understanding of reproductive physiology, taxonomy, and evolution of the Platyhelminthes. Yet, there is limited information on its innervation, especially for free-living species of Rhabdocoela, the most speciose and ecologically diverse assemblage. Here, innervation of the reproductive system in the common marine kalyptorhynch, Gyratrix hermaphroditus, is studied using confocal laser scanning microscopy (CLSM), fluorescent phalloidin, and antibodies to serotonin (5HT) and small cardioactive peptide b (SCPb). Results show that portions of the female atrial system (uterus, female gonopore) are innervated by peptidergic (SCPb) neurons that may function to control muscles involved in egg movement. In contrast, portions of the male atrial system (male atrium, male gonopore) are innervated by both peptidergic and serotonergic neurons. These neurons form a complex series of hoops around the musculature of the male atrium that houses prostate stylet type II. It is hypothesized that 5HT is the primary myoexcitatory neurotransmitter, and that it acts either synergistically with SCPb to trigger muscle contractions of the male atrium and protract the copulatory stylet, or that SCPb plays an inhibitory role during contraction of the male atrium. A comparison of the distribution of peptidergic and serotonergic neurons in G. hermaphroditus with other free-living species and parasitic rhabdocoels (Neodermata) reveals similarities in innervation of comparable (though probably not homologous) reproductive organs. These results suggest that the atrial systems of phylogenetically diverse species may share a common neuronal physiology despite their structural differences and potential independent evolutionary origins within the Platyhelminthes.

Immunostaining of peripheral nerves and other tissues in whole mount preparations from hatchling cephalopods

Newly hatched paralarvae (“hatchlings”) or late-stage embryos of Loligo opalescens were dissected and pieces of tissue removed for immunostaining as flat whole mounts. The general layout of the peripheral nervous system in the mantle and gills was investigated using antisera for tubulin and FMRFamide. Primary sensory neurons are densely distributed in the outer mantle epidermis and show strong FMRFamide immunoreactivity. Their axons form a plexus in the underlying dermis, but do not appear to innervate the chromatophore muscles, which are well visualized with anti-tubulin. Some cross the muscle layer and enter the stellate ganglia via the stellar nerves. The stellate ganglion neuropil contains a rich FMRFamide-immunoreactive mass of axons. It is suggested that these axons originate in large part from sensory neurons in the skin and that the known modulatory effects of FMRFamide-related peptides on motor output of the stellate ganglion may be a reflection of this sensory input in normal life. FMRFamide-immunoreactive primary sensory neurons are also abundant in the gills, but unlike those in the mantle, these cells lack cilia or other external projections. Anti-tubulin staining reveals a network of interstitial cells in the mantle dermis. Such networks may have been mistaken for nerve nets in older accounts. Additional results with Octopus vulgaris hatchlings and immunostaining for serotonin (5HT), small cardioactive peptide (SCP), and gonadotropin releasing hormone (GnRH) are briefly reported.

Topology of the nervous system of Notommata copeus (Rotifera: Monogononta) revealed with anti‐FMRFamide, ‐SCPb, and ‐serotonin (5‐HT) immunohistochemistry

Abstract. The nervous system of the benthic freshwater rotifer, Notommata copeus, was examined using antibody probes, epifluorescence and confocal laser scanning microscopy, and digital imaging to highlight similarities with other monogonont rotifers. Immunoreactivity to anti‐FMRFamide (Phe–Met–Arg–Phe–NH2), ‐SCPb (small cardioactive peptide b), and ‐serotonin (5‐HT, 5‐hydroxytryptamine) was present in the central, peripheral, and stomatogastric nervous system. Specifically, anti‐FMRFamide and ‐SCPb staining was abundant in perikarya and neurites of the cerebral ganglion, ventrolateral nerve cords, and mastax. In addition, a single loop‐like neurite was present in between the nerve cords at the posterior end of the body. Serotonergic neurites were also abundant, and highlighted several cerebral pathways that included connections to the nerve cords and possibly the mastax. Novel neural pathways were also present in the posterior trunk region, where serotonergic neurites innervated the foot and lateral body wall. The results presented herein also highlight the utility of 3D visualization software to gain further insights into the organization and architecture of the rotifer cerebral ganglion.