Patsy S. Dickinson

Neuromodulation of central pattern generators in invertebrates and vertebrates.

Central pattern generators are subject to extensive modulation that generates flexibility in the rhythmic outputs of these neural networks. The effects of neuromodulators interact with one another, and modulatory neurons are themselves often subject to modulation, enabling both higher order control and indirect interactions among central pattern generators. In addition, modulators often directly mediate the interactions between functionally related central pattern generators. In systems such as the vertebrate respiratory central pattern generator, multiple pacemaker types interact to produce rhythmic output. Modulators can then alter the relative contributions of the different pacemakers, leading to substantial changes in motor output and hence to different behaviors. Surprisingly, substantial changes in some aspects of the circuitry of a central pattern generator, such as a several-fold increase in synaptic strength, can sometimes have little effect on the output of the CPG, whereas other changes have profound effects.

Interactions among neural networks for behavior

In recent years, as our understanding of the pattern-generating networks responsible for a variety of behaviors has increased, the interactions of multiple neural networks have been examined in a number of systems. These studies have shown that functionally related pattern generators can interact extensively, and that the extent to which two or more of these networks interact is not fixed, but can be altered by neuromodulators. Furthermore, a number of studies have begun to elucidate the mechanisms responsible for those interactions. In the crustacean stomatogastric system, for example, neurons can switch between different pattern generators, and whole networks can fuse into single patterns. In addition, several networks can be dismantled, and their components used to generate a new network. The mechanisms responsible for these changes are the same as those involved in other circuit re-configurations, namely alterations of both intrinsic membrane properties of component neurons and alterations in the strength of synapses within the networks.

Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags

The development of expressed sequence tags (ESTs) for crustacean cDNA libraries and their deposition in publicly accessible databases has generated a rich resource for peptide discovery in this commercially and ecologically important arthropod subphylum. Here, we have conducted in silico searches of these databases for unannotated ESTs encoding putative neuropeptide precursors using the BLAST program tblastn, and have predicted the mature forms of the peptides encoded by them. The primary strategy used was to query the database with known decapod prepro-hormone sequences or, in some instances, insect precursor protein sequences. For neuropeptides for which no prepro-hormones are known, the peptides themselves were used as queries. For those peptides expected to originate from a common precursor, the individual sequences were combined, with each peptide flanked by a dibasic processing site and, if amidated, a glycine residue. Using these approaches, 13 unannotated ESTs encoding putative neuropeptide precursors were found. For example, using the first strategy, putative Marsupenaeus japonicus prepro-hormones encoding B-type allatostatins, neuropeptide F (NPF), and orcokinins were identified. Similarly, several Homarus americanus ESTs encoding putative orcokinin precursors were found. In addition to the decapod prepro-hormones, ESTs putatively encoding a NPF isoform and a red pigment concentrating hormone-like peptide were identified from the cladoceran Daphnia magna, as was one EST putatively encoding multiple tachykinin-related peptides from the isopod Eurydice pulchra. Using the second strategy, we identified a Carcinus maenas EST encoding HIGSLYRamide, a peptide recently discovered via mass spectrometry from Cancer productus. Using mass spectral methods we confirmed that this peptide is also present in Carcinus maenas. Collectively over 50 novel crustacean peptides were predicted from the identified ESTs, providing a strong foundation for future investigations of the evolution, regulation and function of these and related molecules in this arthropod taxon.

High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders.

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) has become an important method for identifying peptides in neural tissues. The ultra-high-mass resolution and mass accuracy of MALDI-FTMS, in combination with in-cell accumulation techniques, can be used to advantage for the analysis of complex mixtures of peptides directly from tissue fragments or extracts. Given the diversity within the decapods, as well as the large number of extant species readily available for analysis, this group of animals represents an optimal model in which to examine phylogenetic conservation and evolution of neuropeptides and neuropeptide families. Surprisingly, no large comparative studies have previously been undertaken. Here, we have initiated such an investigation, which encompasses 32 species spanning seven decapod infraorders. Two peptides, APSGFLGMRamide and pQDLDHVFLRFamide, were detected in all species. A third peptide, GYRKPPFNGSIFamide, was detected in all species except members of the Astacidean genus Homarus, where a Val(1) variant was present. Our finding that these peptides are ubiquitously (or nearly ubiquitously) conserved in decapod neural tissues not only suggests important conserved functions for them, but also provides an intrinsic calibrant set for future MALDI-FTMS assessments of other peptides in this crustacean order.

Identification of SYWKQCAFNAVSCFamide: a broadly conserved crustacean C-type allatostatin-like peptide with both neuromodulatory and cardioactive properties.

SUMMARY The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif –YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a –WX6Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus– PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-type-like peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species.

Identification, physiological actions, and distribution of VYRKPPFNGSIFamide (Val1)-SIFamide) in the stomatogastric nervous system of the American lobster Homarus americanus.

In this study, the peptide VYRKPPFNGSIFamide (Val1‐SIFamide) was identified in the stomatogastric nervous system (STNS) of the American lobster, Homarus americanus, using matrix‐assisted laser desorption/ionization‐Fourier transform mass spectrometry (MALDI‐FTMS). When bath‐applied to the stomatogastric ganglion (STG), synthetic Val1‐SIFamide activated the pyloric motor pattern, increasing both burst amplitude and duration in the pyloric dilator (PD) neurons. To determine the distribution of this novel SIFamide isoform within the lobster STNS and neuroendocrine organs, a rabbit polyclonal antibody was generated against synthetic Val1‐SIFamide. Whole‐mount immunolabeling with this antibody showed that this peptide is widely distributed within the STNS, including extensive neuropil staining in the STG and commissural ganglia (CoGs) as well as immunopositive somata in the CoGs and the oesophageal ganglion. Labeling was also occasionally seen in the pericardial organ (PO), but not in the sinus gland. When present in the PO, labeling was restricted to fibers‐of‐passage and was never seen in release terminals. Adsorption of the antibody by either Val1‐SIFamide or Gly1‐SIFamide abolished all Val1‐SIFamide staining within the STNS, including the STG neuropil, whereas adsorption by other lobster neuropeptides had no effect on immunolabeling. These data strongly suggest that the staining we report is a true reflection of the distribution of this peptide in the STNS. Collectively, our mass spectrometric, physiological, and anatomical data are consistent with Val1‐SIFamide serving as a locally released neuromodulator in the lobster STG. Thus, our study provides the first direct demonstration of function for an SIFamide isoform in any species. J. Comp. Neurol. 496:406–421, 2006.

Identification and cardiotropic actions of sulfakinin peptides in the American lobster Homarus americanus.

SUMMARY In arthropods, a group of peptides possessing a– Y(SO3H)GHM/LRFamide carboxy-terminal motif have been collectively termed the sulfakinins. Sulfakinin isoforms have been identified from numerous insect species. In contrast, members of this peptide family have thus far been isolated from just two crustaceans, the penaeid shrimp Penaeus monodon and Litopenaeus vannamei. Here, we report the identification of a cDNA encoding prepro-sulfakinin from the American lobster Homarus americanus. Two sulfakinin-like sequences were identified within the open-reading frame of the cDNA. Based on modifications predicted by peptide modeling programs, and on homology to the known isoforms of sulfakinin, particularly those from shrimp, the mature H. americanus sulfakinins were hypothesized to be pEFDEY(SO3H)GHMRFamide (Hoa-SK I) and GGGEY(SO3H)DDY(SO3H)GHLRFamide (Hoa-SK II). Hoa-SK I is identical to one of the previously identified shrimp sulfakinins, while Hoa-SK II is a novel isoform. Exogenous application of either synthetic Hoa-SK I or Hoa-SK II to the isolated lobster heart increased both the frequency and amplitude of spontaneous heart contractions. In preparations in which spontaneous contractions were irregular, both peptides increased the regularity of the heartbeat. Our study provides the first molecular characterization of a sulfakinin-encoding cDNA from a crustacean, as well as the first demonstration of bioactivity for native sulfakinins in this group of arthropods.

Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus.

SUMMARY Over a quarter of a century ago, Mykles described the presence of putative endocrine cells in the midgut epithelium of the crab Cancer magister (Mykles, 1979). In the years that have followed, these cells have been largely ignored and nothing is known about their hormone content or the functions they play in this species. Here, we used a combination of immunohistochemistry and mass spectrometric techniques to investigate these questions. Using immunohistochemistry, we identified both SIFamide- and tachykinin-related peptide (TRP)-like immunopositive cells in the midgut epithelium of C. magister, as well as in that of Cancer borealis and Cancer productus. In each species, the SIFamide-like labeling was restricted to the anterior portion of the midgut, including the paired anterior midgut caeca, whereas the TRP-like immunoreactivity predominated in the posterior midgut and the posterior midgut caecum. Regardless of location, label or species, the morphology of the immunopositive cells matched that of the putative endocrine cells characterized ultrastructurally by Mykles (Mykles, 1979). Matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry identified the peptides responsible for the immunoreactivities as GYRKPPFNGSIFamide (Gly1-SIFamide) and APSGFLGMRamide [Cancer borealis tachykinin-related peptide Ia (CabTRP Ia)], respectively, both of which are known neuropeptides of Cancer species. Although the function of these midgut-derived peptides remains unknown, we found that both Gly1-SIFamide and CabTRP Ia were released when the midgut was exposed to high-potassium saline. In addition, CabTRP Ia was detectable in the hemolymph of crabs that had been held without food for several days, but not in that of fed animals, paralleling results that were attributed to TRP release from midgut endocrine cells in insects. Thus, one function that midgut-derived CabTRP Ia may play in Cancer species is paracrine/hormonal control of feeding-related behavior, as has been postulated for TRPs released from homologous cells in insects.

Identification of a calcitonin-like diuretic hormone that functions as an intrinsic modulator of the American lobster, Homarus americanus, cardiac neuromuscular system.

SUMMARY In insects, a family of peptides with sequence homology to the vertebrate calcitonins has been implicated in the control of diuresis, a process that includes mixing of the hemolymph. Here, we show that a member of the insect calcitonin-like diuretic hormone (CLDH) family is present in the American lobster, Homarus americanus, serving, at least in part, as a powerful modulator of cardiac output. Specifically, during an ongoing EST project, a transcript encoding a putative H. americanus CLDH precursor was identified; a full-length cDNA was subsequently cloned. In silico analyses of the deduced prepro-hormone predicted the mature structure of the encoded CLDH to be GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH), which is identical to a known Tribolium castaneum peptide. RT-PCR tissue profiling suggests that Homam-CLDH is broadly distributed within the lobster nervous system, including the cardiac ganglion (CG), which controls the movement of the neurogenic heart. RT-PCR analysis conducted on pacemaker neuron- and motor neuron-specific cDNAs suggests that the motor neurons are the source of the CLDH message in the CG. Perfusion of Homam-CLDH through the isolated lobster heart produced dose-dependent increases in both contraction frequency and amplitude and a dose-dependent decrease in contraction duration, with threshold concentrations for all parameters in the range 10–11 to 10–10 mol l–1 or less, among the lowest for any peptide on this system. This report is the first documentation of a decapod CLDH, the first demonstration of CLDH bioactivity outside the Insecta, and the first detection of an intrinsic neuropeptide transcript in the crustacean CG.

Molecular and mass spectral identification of the broadly conserved decapod crustacean neuropeptide pQIRYHQCYFNPISCF: The first PISCF-allatostatin (Manduca sexta- or C-type allatostatin) from a non-insect

The PISCF-allatostatins (Manduca sexta- or C-type allatostatins) are a family of pentadecapeptides characterized by a pyroglutamine blocked N-terminus, an unamidated-PISCF C-terminus, and a disulfide bridge between two internal Cys residues. Several isoforms of PISCF-AST are known, all from holometabolous insects. Using a combination of transcriptomics and mass spectrometry, we have identified the first PISCF-type peptides from a non-insect species. In silico analysis of crustacean ESTs identified several Litopenaeus vannamei (infraorder Penaeidea) transcripts encoding putative PISCF-AST precursors. Translation of these ESTs, with subsequent prediction of their putative post-translational processing, revealed the existence of as many as three PISCF-type peptides, including pQIRYHQCYFNPISCF (disulfide bridging between Cys(7) and Cys(14)). Although none of the predicted isoforms was detected by mass spectrometry in L. vannamei, MALDI-FTMS mass profiling identified an m/z signal corresponding to pQIRYHQCYFNPISCF (disulfide bridge present) in neural tissue from 28 other decapods, which included members of six infraorders (Stenopodidea, Astacidea, Thalassinidea, Achelata, Anomura and Brachyura). Further characterization of the peptide using SORI-CID and chemical derivatization/enzymatic digestion supported the theorized structure. In both the crab Cancer borealis and the lobster Homarus americanus, MALDI-based tissue surveys suggest that pQIRYHQCYFNPISCF is broadly distributed in the nervous system; it was also detected in the posterior midgut caecum. Collectively, our data show that members of the PISCF-AST family are not restricted to the holometabolous insects, but instead may be broadly conserved within the Pancrustacea. Moreover, our data suggest that one highly conserved PISCF-type peptide, pQIRYHQCYFN-PISCF, is present in decapod crustaceans, functioning as a brain-gut paracrine/hormone.