Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na⁺ saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a “borrowed transmitter”. Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron‐evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron‐evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave‐form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 380–392, 2003     

The actions of crustacean cardioactive peptide on adult and developing stomatogastric ganglion motor patterns

The motor patterns produced by the stomatogastric ganglion (STG) are strongly influenced by descending modulatory inputs from anterior ganglia. With these inputs intact, in control saline, the motor patterns produced by the stomatogastric nervous system of embryonic and larval lobsters are slower and less regular than those of adult lobsters. We studied the effects of the hormonal modulator, crustacean cardioactive peptide (CCAP) on the discharge patterns of STG motor patterns in embryos, larvae, and adult Maine lobsters, Homarus americanus, with the anterior inputs present and absent. In adults, CCAP initiated robust pyloric rhythms from STGs isolated from their descending control and modulatory inputs. Likewise, CCAP initiated robust activity in isolated embryonic and larval STGs. Nonetheless, quantitative analyses revealed that the frequency and regularity of the STG motor neuron discharge seen in the presence of CCAP in isolated STGs from embryos were significantly lower than those seen late in larval life and in adults under the same conditions. In contrast, when the descending control and modulatory pathways to the STG were left intact, the embryonic and larval burst frequency seen in the presence of CCAP was increased by CCAP, whereas the burst frequency in adults was decreased by CCAP, so that in CCAP the frequencies at all stages were statistically indistinguishable. These data argue that immature embryonic motor patterns seen in the absence of CCAP are a function of immaturity in both the STG and in the descending and modulatory pathways.     

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val¹]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.     

Glutamatergic motoneurons in the stomatogastric ganglion of the mantis shrimp Squilla oratoria

1. Transmitters of motoneurons in the stomatogastric ganglion (STG) of Squilla were identified by analyzing the excitatory neuromuscular properties of muscles in the posterior cardiac plate (pcp) and pyloric regions. 2. Bath and iontophoretic applications of glutamate produce depolarizations in these muscles. The pharmacological experiments and desensitization of the junctional receptors elucidate the glutamatergic nature of the excitatory junctional potentials (EJPs) evoked in the constrictor and dilator muscles. The reversal potentials for the excitatory junctional current (EJC) and for the glutamate-induced current are almost the same. 3. Some types of dilator muscle show sensitivity to both glutamate and acetylcholine (ACh) exogenously applied. The pharmacological evidence and desensitization of the junctional receptors indicate the glutamatergic nature of neuromuscular junctions in these dually sensitive muscles. The reversal potentials for the EJC and for the ACh-induced current are not identical. 4. Glutamate is a candidate as an excitatory neuro-transmitter at the neuromuscular junctions which the STG motoneurons named PCP, PY, PD, LA and VC make with the identified muscles. Kainic and quisqualic acids which act on glutamate receptors are potent excitants of these muscles. Extrajunctional receptors to ACh are present in two types of the muscle innervated by LA and VC. 5. Neurotransmitters used by the STG motoneurons of stomatopods are compared to those of decapods.     

Phylogenetic,ontogenetic and adult adaptive plasticity of rhythmic neural networks: a common neuromodulatory mechanism?

Neuromodulatory inputs are known to play a major role in the adaptive plasticity of rhythmic neural networks in adult animals. Using the crustacean stomatogastric nervous system, we have investigated the role of modulatory inputs in the development of rhythmic neural networks. We found that the same neuronal population is organised into a single network in the embryo, as opposed to the two networks present in the adult. However, these adult networks pre-exist in the embryo and can be unmasked by specific alterations of the neuromodulatory environment. Similarly, adult networks may switch back to the embryonic phenotype by manipulating neuromodulatory inputs. During development, we found that the early established neuromodulatory population display alteration in expressed neurotransmitter phenotypes, and that although the population of modulatory neurones is established early, with morphology and projection pattern similar to adult ones, their neurotransmitter phenotype may appear gradually. Therefore the abrupt switch from embryonic to adult network expression occurring at metamorphosis may be due to network reconfiguration in response to changes in modulatory input, as found in adult adaptive plasticity. Strikingly, related crustacean species express different motor outputs using the same basic network circuitry, due to species-specific alteration in neuromodulatory substances within homologous projecting neurones. Therefore we propose that alterations within neuromodulatory systems to a given rhythmic neural network displaying the same basic circuitry may account for the generation of different motor outputs throughout development (ontogenetic plasticity), adulthood (adaptive plasticity) and evolution (phylogenetic plasticity).Abbreviations CoG Commissural ganglion - OG Oesophageal ganglion - STG Stomatogastric ganglion - STNS Stomatogastric nervous system     

Ontogeny of rhythmic motor networks in the stomatogastric nervous system

We used the lobster Homarus gammarus to study the ontogeny of neural networks involved in rhythmic behaviours. Since in the adult the neural networks belonging to the stomatogastric nervous system and controlling the rhythmic movements of the foregut are well characterised, we have studied them during ontogeny. While this foregut develops slowly throughout embryonic and larval stages, the neuronal population of these motor networks is quantitatively established since the mid-embryonic period. Moreover, in the embryo, this neural population is organised into a single functional network that displays a unique motor output. By contrast, in the adult the same neuronal elements are organised into three neural networks that express independent motor programs. Our results indicate that the multiple adult networks are partitioned progressively from a single embryonic network during development. Accepted: 23 May 1999     

Patterns of appearance of serotonin and proctolin immunoreactivities in the developing nervous system of the American lobster

Serotonin (5-HT) and proctolin, neurohormones widely distributed in the lobster nervous system, have been implicated in a variety of behaviors and also are known to coexist in large pairs of identified neurons in the fifth thoracic (T5) and first abdominal ganglia (A1) of adults (Siwicki, Beltz, and Kravitz, 1987). Earlier studies also have shown that these paired neurons already contain 5-HT in embryos approximately halfway through development, whereas proctolin immunoreactivity does not appear in these cells until near the time of hatching (Beltz and Kravitz, 1987a). In the current studies, the brain and ventral nerve cord have been screened for the appearance of serotonin and proctolin immunoreactivities using immunocytochemical and biochemical methods, in order to determine whether the late appearance of proctolin in the paired T5 and A1 cells is a general feature of development in other neurons as well. In embryos approximately halfway through development, the adult complement of 5-HT-staining cells is already present. In several cases, embryonic serotonin cells are proportionally very large and prominent, suggesting possible developmental roles. In contrast to serotonin, fewer than 10% of the proctolin-staining neurons of juvenile animals are seen in embryos halfway through development. The number of immunoreactive cells gradually increases, but even by the sixth larval stage only half the number of cells that will eventually stain for proctolin are observed. Therefore, the developmental appearance of proctolin in lobster neurons, assayed using immunocytochemical methods, is relatively late and protracted compared to the appearance of serotonin. Quantitative measurements for 5-HT in lobster larvae were performed using high pressure liquid chromatography (HPLC) with dual electrochemical detection and for proctolin using radioimmunoassay. A gradual, probably growth-related increase in the amounts of serotonin and proctolin were seen during larval development. The implications of the biochemical data, in light of the immunocytochemical studies, are discussed.     

NO/cGMP Signaling and the Flexible Organization of Motor Behavior in Crustaceans

The basic elements of the NO/cGMP signaling pathway have beenidentified in the nervous systems of animals from nearly allof the major phyla. In crustaceans, the NO/cGMP pathway is associatedwith certain fundamental neuronal processes, including sensoryintegration and the organization and production of motor behavior.Here I review the evidence for NO synthesis and action in crustaceanneural networks, with an emphasis on the rhythmic motor circuitsof the crab stomatogastric ganglion (STG). In the STG, NO appearsto be released as an orthograde transmitter from descendingprojection neurons. NO's receptor, a cytopasmic isoform of guanylatecyclase (sGC), is expressed in a subset of the cells that participatein the gastric mill and pyloric central pattern generating networks.In spontaneously-active, in vitro preparations of the STG, pharmacologicalinhibitors of the NO/cGMP pathway cause the two rhythmic motorpatterns to collapse into a single conjoint rhythm. Parallelmotor output is restored when the ganglion is returned to normalsaline. Although precise mechanisms have yet to be determined,these data suggest that NO and cGMP play an important role inthe functional organization of STG networks. The STG, as wellas other crustacean models, provides a promising context forstudying the physiological and behavioral aspects of NO-mediatedsignaling in the nervous system.     

The effect of biogenic monoamine antagonists on the development of preimplantation mouse embryos cultured in vitro

The effect of serotonin and adrenaline antagonists was tested on the early embryos of mice of three lines. All the substances tested produced an arrest or inhibition of cleavage division and the appearance of anomalies. Serotonin introduced in the incubation medium was effective against some serotoninolytics. We were unable to test the protective effect of adrenaline, as in the concentrations used it has its own effect on the development. From the data obtained, a conclusion is made of the existence in early mouse embryos of the structures sensitive to serotonin and adrenaline antagonists. The assumptions is made from the previously obtained data on the presence of biogenic monoamines in early mouse embryos, of functional activity of prospective mediators of the nervous system at the earliest stages of embryonic development of mammals.     

The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages

The locus elav (ella-vee) of Drosophila melanogaster, which is necessary for the proper development of the embryonic and adult nervous systems, has been characterized both genetically and molecularly. This locus has been shown to be transcribed exclusively within, and ubiquitously throughout, the developing nervous system during Hours 6 to 12 of embryogenesis. We present in situ RNA localization data which demonstrate that elav is expressed in the central nervous system as well as the peripheral nervous system of embryos, larvae, pupae, and adults. We also demonstrate that elav is not transcribed in embryonic or larval neuroblasts (the neuronal progenitor cells), or in at least one type of glial cell. These data provide evidence that the requirement for elav function is not limited to the 6- to 12-hr embryonic nervous system and the adult eye and developing optic lobe, but that its function is required for the development and continued maintenance of all neurons of the organism.     

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT₁ receptors, antagonists of 5-HT₂, 5-HT₃ or 5-HT₄ receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.     

Developmental modulation of a glial cell-associated glycoprotein, 5B12, in an insect, Acheta domesticus

The expression of an insect (Acheta domesticus) adult glial cell-specific antigen, 5B12 undergoes major changes during development. The 5B12 antigen is detected as early as 20-25% of embryonic development, when immunoreactivity is distributed throughout the periphery, present at the luminal surface of epithelial cells which compose developing limb buds, sensory appendages, and the body cavity. The antigen is also localized on the cell surface of neural elements within commissural tracts in the embryonic CNS. 5B12 is secreted extracellularly in the periphery, where it is associated with the embryonic basal lamina in developing cercal sensory appendages. Luminal surface expression is transient, and disappears by 95% of embryonic development. As development proceeds, 5B12 distribution becomes more restricted, so that in the adult the antigen is predominantly associated with specific glial elements within the nervous system where it occurs as a specialized component of the extracellular matrix. The 5B12 antigen is also associated with discrete central and peripheral fiber tracts. Antigen 5B12 is present in whole embryos and in the adult CNS as a Mr 185-kDa glycoprotein. Distinct carbohydrate moieties with chondroitin sulfate-like properties are situated on the 5B12 epitope. Thus the glia-associated 5B12 macromolecule has the characteristics of a small proteoglycan. Based upon features of its distribution, pattern of spatiotemporal expression, and biochemical properties, it is speculated that 5B12 participates in events related sequentially to the development and the function of the insect nervous system.     

The anatomy and physiology of the stomatogastric nervous system ofSquilla

Summary The stomatogastric nervous system of a mantis shrimp,Squilla oratoria, is described. The motor nerves of the stomatogastric ganglion (STG) and their innervation of muscles of the posterior cardiac plate (pcp) and pyloric systems are detailed.The STG contains more than 25 neurons. It sends out one pair of major output nerves. The pcp-pyloric cycle recorded from the motor axons in this nerve consists of rhythmic bursts of several units which fire with a characteristic phase relationship to each other. The rhythm is intrinsic to the STG itself, but it is modifiable.Recordings from the peripheral nerves reveal that identifiable cardiac plate, pyloric dilator and pyloric neurons control sequential contractions of the pcp and pyloric muscles to constrict or dilate a number of their attached ossicles.Several modulatory input fibres in the stomatogastric nerve, activated via stimulation of the superior or inferior oesophageal nerve (son, ion), prime or trigger the cyclic motor outputs. The son inputs induce distinct effects on the cardiac and pcp-pyloric pattern generators, while the ion inputs, via the oesophageal ganglion, excite only the pcp-pyloric generator.On the basis of anatomical and physiological observations, the possible functions of motor neurons involved in the pcp-pyloric cycle are described with reference to opening of the pcp and pyloric channels.This stomatogastric nervous system inSquilla is compared to that in decapods which has been well analyzed.Abbreviations CG commissural ganglion - ion inferior oesophageal nerve - lvn lateral ventricular nerve - OG oesophageal ganglion - pep posterior cardiac plate - son superior oesophageal nerve - STG stomatogastric ganglion - stn stomatogastric nerve - ivn inferior ventricular nerve     

A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster

Individuals expressing recessive mutations in the Deformed (Dfd) locus of Drosophila melanogaster were examined for embryonic and adult defects. Mutant embryos were examined in both scanning electron microscope and light microscope preparations. The adult Dfd recessive mutant phenotype was assessed in somatic clones and in survivors homozygous for hypomorphic alleles of the gene. The time of Dfd+ action was determined by studying a temperature conditional allele. Dfd+ is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. Shift studies utilizing a temperature conditional allele have shown that the gene product is necessary during at least two periods of development, during embryonic segmentation and head involution and during the late larval and pupal stages. From these studies we conclude that Dfd is a homeotic gene necessary for proper specification of both the embryonic and the adult head.     

Neuropilar Projections of the Anterior Gastric Receptor Neuron in the Stomatogastric Ganglion of the Jonah Crab,Cancer Borealis

Sensory neurons provide important feedback to pattern-generating motor systems. In the crustacean stomatogastric nervous system (STNS), feedback from the anterior gastric receptor (AGR), a muscle receptor neuron, shapes the activity of motor circuits in the stomatogastric ganglion (STG) via polysynaptic pathways involving anterior ganglia. The AGR soma is located in the dorsal ventricular nerve posterior to the STG and it has been thought that its axon passes through the STG without making contacts. Using high-resolution confocal microscopy with dye-filled neurons, we show here that AGR from the crab Cancer borealis also has local projections within the STG and that these projections form candidate contact sites with STG motor neurons or with descending input fibers from other ganglia. We develop and exploit a new masking method that allows us to potentially separate presynaptic and postsynaptic staining of synaptic markers. The AGR processes in the STG show diversity in shape, number of branches and branching structure. The number of AGR projections in the STG ranges from one to three simple to multiply branched processes. The projections come in close contact with gastric motor neurons and descending neurons and may also be electrically coupled to other neurons of the STNS. Thus, in addition to well described long-loop pathways, it is possible that AGR is involved in integration and pattern regulation directly in the STG.     

Localization of a SALMFamide Neuropeptide in the Larval Nervous System of the Sand Dollar Dendraster excentricus

Using immunocytochemical methods we describe the localization of serotonin and the SALMFamide peptide, S1 (GFNSALMFamide), during embryonic and larval development of the echinoid Dendraster excentricus. Anti-SI immunoreactivity first appears in the apical ganglion in late gastrulae at the same time as anti-serotonin immunoreactivity. Initially, anti-S1 immunoreactivity is restricted to fibres of the neuropile, but in later feeding stages, cell bodies are also immunoreactive. Anti-S1 immunoreactivity appears as 2–4 cells in the oral ganglion of early prism stage larvae, whereas anti-serotonin immunoreactivity does not occur in the oral ganglion until the 8-arm stage. Anti-S1 immunoreactivity also occurs in diffuse fibres in the oesophagus and in a single fibre encircling the pyloric sphincter of the gut. A reticular network associated with the apical surface of the epithelial cells of the vestibule of the adult rudiment was anti-S1 immunoreactive. In double-labelling experiments, anti-serotonin and anti-S1 immunoreactivity co-localize in the neuropile of the apical ganglion. The distribution of S1, in association with putative sensory cells in the apical and oral ganglia and with muscles of the oesophagus and gut, suggests S1 may have diverse functions in the larval nervous system. The distribution of anti-S1 immunoreactivity in echinoid embryos and larvae supports the proposal that SALMFamide-like peptides are widely shared in echinoderms and potentially have a fundamental role in neural function.     

Regulation of pyloric rhythm by I(A) and I(h) in crayfish stomatogastric ganglion

The stomatogastric ganglion (STG) of shellfish includes 30 neurons and produces pyloric rhythms. It is the common model to study central pattern generator (CPG). Regulation of pyloric rhythms not only is related to the property of single neurons in STG but also depends on the connections and property of the whole neuronal network. It has been found that transient potassium current (I(A)) and hyperpolarization-activated cation current (I(h)) exist in certain types of neurons of STG. However, roles played by these two currents in maintaining and regulating the pyloric rhythms are unknown. In the present study, in vitro electrophysiological recordings were performed on crayfish STG to examine the role played by I(A) and I(h) in regulation of pyloric rhythm. 4AP (2 mmol/L), a specific inhibitor of I(A), caused a decrease in pyloric cycle (P < 0.01), an increase in PD (pyloric dilator) ratio, a decrease in PY (pyloric) ratio (P < 0.01) and delay of phases of LP and PY firing. ZD7288 (100 μmol/L), a specific inhibitor of I(h), caused a decrease in pyloric cycle (P < 0.01), an increase in PD ratio (P < 0.01), an increase in LP (lateral pyloric) ratio (P < 0.01), a decrease in PY ratio (P < 0.01) and delay of phases of LP and PY firing. These results indicate that I(A) and I(h) play important roles in regulating pyloric rhythms in crayfish STG.     

All-or-none control of the bursting properties of the pacemaker neurons of the labster pyloric pattern generator

In Crustacea the central pattern generator for the pyloric motor rhythm (filtration to the midgut) is known to be located within the stomatogastric ganglion (STG); its cycling activity is known to be organized by three endogenous burster neurons acting as pacemakers and driving 11 follower neurons. In Homarus, recordings from the isolated stomatogastric nervous system (Fig. 1) indicate that (1) the pyloric output can be generated only when the STG is afferented (i.e., connected to the more rostral oesophageal and commissural ganglia) (Fig. 2) and (2) the deafferntation of the STG results in a complete loss of the bursting properties of the pacemaker neurons (Fig. 4). Manipulation of the STG inputs responsible for unmasking the properties of the pacemakers strongly suggests that (1) they are not phasic inputs (Fig. 5) and (2) they are long-term acting inputs (Fig. 6). These results provide evidence for a neural all-or-none control of the bursting properties of the pacemaker neurons of a motor pattern generator.     

Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)

Serotonin, or 5-hydroxytryptamine (5-HT), plays critical roles as a neurotransmitter and neuromodulator that control or modulate many behaviors in insects, such as feeding. Neurons immunoreactive (IR) to 5-HT were detected in the central nervous system (CNS) of the larval and adult stages of the stable fly, Stomoxys calcitrans, using an immunohistological technique. The location and pattern of the 5-HT IR neurons are described and compared for these two different developmental stages. Anatomical features of the fly feeding system were analyzed in third instar larvae and adult flies using a combination of histological and immunohistological techniques. In third instar larvae, the cibarial dilator muscles were observed within the cibarial pump skeleton and innervated by 5-HT IR neurons in nerves arising from the brain. There were four pairs of nerves arising from the frontal surface of the larval brain that innervate the cibarial pump muscles, pharynx, and muscles controlling the mouth hooks. A strong serotoninergic innervation of the anterior stomatogastric system was observed, which suggests 5-HT may play a role in the coordination of different phases of food ingestion by larvae. Similarly, many 5-HT IR neurons were found in both the brain and the thoracico-abdominal ganglia in the adult, some of which innervate the cibarial pump dilator muscles and the stomatogastric muscles. This is tnhe first report describing neuromuscular structures of the stable fly feeding system. The results reported here suggest 5-HT may play a critical role in feeding behaviors of stable fly larvae and adults.