Showing 1 - 10 of 24 Items

Date: 2020-01-01

Creator: Benjamin Harley Wong

Access: Open access

Neuropeptides are important modulators of neural activity, allowing neural networks, such as the central pattern generators (CPGs) that control rhythmic movements, to alter their output and thus generate behavioral flexibility. Isoforms of a neuropeptide family vary in physical structure, allowing potentially distinct functional neuromodulatory effects on CPG systems. While some familial neuropeptide isoforms can differentially affect a system, others in the same family may elicit indistinguishable effects. Here, we examined the effects elicited by members of a novel family of six peptide hormone isoforms (GSEFLamides: I-, M-, AL-, AM-, AV-, and VM-GSEFLamide) on the pyloric filter and gastric mill CPGs in the stomatogastric nervous system (STNS) of the American lobster, Homarus americanus. Recent unpublished work from the Dickinson lab found that five of the six GSEFLamides elicited similar increases in contraction amplitude when perfused through the isolated lobster heart, while one (AVGSEFLamide) had virtually no effect. Using extracellular recordings, we found the pattern of GSEFLamide effects on the STNS gastric mill to be similar to the pattern observed in the lobster cardiac system; the gastric mill circuit was fairly consistently activated by all isoforms except AVGSEFLamide. The intrinsically active pyloric pattern was also significantly enhanced by three out of five peptide isoforms, and nearly significantly enhanced by two more, but was likewise non-responsive to AVGSEFLamide. While the reason AVGSEFLamide had no effect on either pattern is unknown, the similar phenomenon noted in the isolated whole heart potentially indicates that this isoform lacks any function in the lobster.

Date: 2007-07-01

Creator: Patsy S. Dickinson, Jake S. Stevens, Szymon Rus, Henry R. Brennan, Christopher C., Goiney, Christine M. Smith, Lingjun Li, David W. Towle, Andrew E. Christie

Access: Open access

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.

• Restriction End Date: 2027-06-01

Date: 2022-01-01

Creator: Emily Yuan-ann Pan

Access: Access restricted to the Bowdoin Community

Date: 2018-09-01

Creator: Giap H. Vu, Daniel Do, Cindy D. Rivera, Patsy S. Dickinson, Andrew E., Christie, Elizabeth A. Stemmler

Access: Open access

We report on the characterization of the native form of an American lobster, Homarus americanus, β-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to β-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.

• Restriction End Date: 2029-06-01

Date: 2024-01-01

Creator: Olivia Sewon Choi

Access: Access restricted to the Bowdoin Community

Date: 2018-05-01

Creator: Patsy S. Dickinson, Matthew K. Armstrong, Evyn S. Dickinson, Rebecca Fernandez, Alexandra, Miller, Sovannarath Pong, Brian W. Powers, Alixander Pupo-Wiss, Meredith E. Stanhope, Patrick J. Walsh, Teerawat Wiwatpanit, Andrew E. Christie

Access: Open access

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Date: 2009-12-15

Creator: J. S. Stevens, C. R. Cashman, C. M. Smith, K. M. Beale, D. W., Towle, A. E. Christie, P. S. Dickinson

Access: Open access

pQDLDHVFLRFamide is a highly conserved crustacean neuropeptide with a structure that places it within the myosuppressin subfamily of the FMRFamide-like peptides. Despite its apparent ubiquitous conservation in decapod crustaceans, the paracrine and/or endocrine roles played by pQDLDHVFLRFamide remain largely unknown. We have examined the actions of this peptide on the cardiac neuromuscular system of the American lobster Homarus americanus using four preparations: the intact animal, the heart in vitro, the isolated cardiac ganglion (CG), and a stimulated heart muscle preparation. In the intact animal, injection of myosuppressin caused a decrease in heartbeat frequency. Perfusion of the in vitro heart with pQDLDHVFLRFamide elicited a decrease in the frequency and an increase in the amplitude of heart contractions. In the isolated CG, myosuppressin induced a hyperpolarization of the resting membrane potential of cardiac motor neurons and a decrease in the cycle frequency of their bursting. In the stimulated heart muscle preparation, pQDLDHVFLRFamide increased the amplitude of the induced contractions, suggesting that myosuppressin modulates not only the CG, but also peripheral sites. For at least the in vitro heart and the isolated CG, the effects of myosuppressin were dose-dependent (10 -9 to 10-6mol l-1 tested), with threshold concentrations (10-8-10-7 mol l-1) consistent with the peptide serving as a circulating hormone. Although cycle frequency, a parameter directly determined by the CG, consistently decreased when pQDLDHVFLRFamide was applied to all preparation types, the magnitudes of this decrease differed, suggesting the possibility that, because myosuppressin modulates the CG and the periphery, it also alters peripheral feedback to the CG.

• Embargo End Date: 2027-05-19

Date: 2022-01-01

Creator: Usira Ahmed Ali

Access: Embargoed

Date: 2020-01-01

Creator: Emily R Oleisky

Access: Access restricted to the Bowdoin Community

Date: 2019-01-01

Creator: Louis Mendez

Access: Open access

Central pattern generators (CPGs) are neural networks that generate rhythmic motor patterns to allow organisms to perform stereotypical tasks, such as breathing, scratching, flying, and walking. The American lobster, Homarus americanus, is a simple model system whose CPGs are functionally analogous to those in vertebrate models and model complex rhythmic behaviors. CPGs in many Crustacea, including the American lobster, have been studied because of their ability to maintain biological function after isolation in physiologically relevant conditions. The cardiac ganglion (CG) is a CPG consisting of five larger motor neurons and four smaller pacemaker neurons that innervate the cardiac neuromuscular system and generate electrical bursts that drive patterned behaviors. Neuromodulators, such as neuropeptides, are known to modulate neural output in the CPGs of the American lobster. Currently, neuromodulators affecting the cardiac ganglia are thought to be mainly expressed and secreted outside of the cardiac ganglia, acting as extrinsic neuromodulators. However, there is current evidence to support the idea that neuromodulators can be intrinsically expressed within the cardiac ganglion of the American lobster. Preliminary studies using transcriptomic techniques on genomic and transcriptomic information indicate that neuropeptides are likely expressed within the cardiac ganglion. However, little research has been done to determine whether these neuropeptides are expressed in the cardiac ganglion of the American lobster. Therefore, the purpose of this study is to combine bioinformatics and mass spectrometric techniques to determine whether select neuropeptides are present in the cardiac ganglion within the cardiac neuromuscular system of the American lobster, Homarus americanus. Our data mining techniques using protein query sequences obtained from previously annotated brain and eyestalk transcriptomes resulted in the identification of 22 putative neuropeptides preprohormones from 17 neuropeptide families and 20 putative neuropeptide receptors from 17 neuropeptide receptor families in the CG transcriptome. Additionally, 9 putative neuropeptide receptors from 7 neuropeptide receptor families were detected in the cardiac muscle transcriptome. Of the 17 neuropeptide families detected, receptors for 9 of these neuropeptide families were detected in the CG transcriptome. Receptors for 6 of the neuropeptide families were also present in the cardiac muscle transcriptome. Interestingly, receptors for 6 of neuropeptide families detected were not found in either the CG or cardiac muscle transcriptomes, and receptors for 4 neuropeptide families that weren’t detected in the CG transcriptome were found in the cardiac muscle transcriptome. Therefore, our research suggests that neuropeptides are able to modulate CPG activity extrinsically, either though hormonal or local delivery, or intrinsically. Additionally, neuropeptides were extracted from the stomatogastric ganglion and the commissural ganglion using a scaled-down neuropeptide extraction protocol to estimate the number of tissues required to obtain sufficiently strong mass spectrometry signals. Pooled samples with two commissural ganglia and single samples of a commissural ganglion and a stomatogastric ganglion displayed little signal and an increase in larger peptides and impurities relative to single-tissue samples. Therefore, further optimization of the scaled-down neuropeptide extraction protocol must be done prior to analysis of a cardiac ganglion in the American lobster.