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

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.

The peptide hormone pQDLDHVFLRFamide (crustacean myosuppressin) modulates the Homarus americanus cardiac neuromuscular system at multiple sites.

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(-6) mol 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.

Quantifying the effects of forestry practices on the recovery of upland streams and lochs from acidification.

Trends in long-term chemistry data are presented for 37 acidified upland streams and lochs, located in four areas (A, B, C and D) across Scotland, to provide a comparison between recovery rates of moorland catchments and forest catchments at different stages of the management cycle. For all sites, non-marine sulfate (nm-SO(4)) showed a significant decline in annual median concentrations, the greatest decline being in streams draining felled catchments, which showed a 50% greater decline than catchments with moorland or young, aggrading forests. A similar pattern was found for chloride (Cl) concentrations in Area C, which reflected the reduced interception of sea-salt aerosols following clearfelling. However, high elevation moorland sites in Area D also revealed significant declines in Cl while trends in aggrading forest sites in this area were insignificant. Alkalinity (ALK) and pH increased more at sites where felling had taken place than at moorland or young forest sites while aggrading forest catchments appeared to be most resistant to changes in pH and ALK. Associated with these acid-base changes was a corresponding decline in labile aluminium (Al-L) concentrations. The pattern of nitrate (NO(3)) change was especially affected by the timing of felling in forested catchments. Large negative trends in NO(3) at stream sites were associated with felling during the early part of the study period. This downward trend was further enhanced as NO(3) concentrations fell below pre-felling levels as the second rotation crop became established. Few forest sites showed significant increases in NO(3) due to felling in the latter part of the study period. Most moorland loch sites showed a small but significant increase in NO(3,) probably in response to similar increases in N deposition and/or climatic impacts. Dissolved organic carbon (DOC) increased significantly at both forest and moorland sites, however, the extent of these increasing trends appeared to be positively correlated with absolute DOC concentrations. Despite the complex response of streams and lochs during the various stages of the forest cycle, especially for NO(3), both forest and moorland catchments showed generally similar and rapid responses to reductions in S deposition. Nevertheless, forested sites are still more acid and have higher concentrations of toxic forms of Al than moorland sites. Although the proposed emission reductions in Europe are likely to result in a continuing decline in S and N loadings to catchments, the continuing policy of planting second rotation forests in these acidified catchments may, in the long-term, delay or halt chemical and biological recovery. However, in the short-term, any increase in the uptake of N deposition by aggrading forests should help to counteract the acidifying effects of a small increase in the interception of S and N compounds.

Species-specific modulation of pattern-generating circuits.

Phylogenetic comparison can reveal general principles governing the organization and neuromodulation of neural networks. Suitable models for such an approach are the pyloric and gastric motor networks of the crustacean stomatogastric ganglion (STG). These networks, which have been well studied in several species, are extensively modulated by projection neurons originating in higher-order ganglia. Several of these have been identified in different decapod species, including the paired modulatory proctolin neuron (MPN) in the crab Cancer borealis [Nusbaum & Marder (1989) J. Neurosci., 9,1501-1599; Nusbaum & Marder (1989), J. Neurosci., 9, 1600-1607] and the apparently equivalent neuron pair, called GABA (gamma-aminobutyric acid) neurons 1 and 2 (GN1/2), in the lobster Homarus gammarus [Cournil et al. (1990) J. Neurocytol., 19, 478-493]. The morphologies of MPN and GN1/2 are similar, and both exhibit GABA-immunolabelling. However, unlike MPN, GN1/2 does not contain the peptide transmitter proctolin. Instead, GN1/2, but not MPN, is immunoreactive for the neuropeptides related to cholecystokinin (CCK) and FLRFamide. Nonetheless, GN1/2 excitation of the lobster pyloric rhythm is similar to the proctolin-mediated excitation of the crab pyloric rhythm by MPN. In contrast, GN1/2 and MPN both use GABA but produce opposite effects on the gastric mill rhythm. While MPN stimulation produces a GABA-mediated suppression of the gastric rhythm [Blitz & Nusbaum (1999) J. Neurosci., 19, 6774-6783], GN1/2 activates or enhances gastric rhythmicity. These results highlight the care needed when generalizing neuronal organization and function across related species. Here we show that the 'same' neuron in different species does not contain the same neurotransmitter complement, nor does it exert all of the same effects on its postsynaptic targets. Conversely, a different transmitter phenotype is not necessarily associated with a qualitative change in the way that a modulatory neuron influences target network activity.

GABA and responses to GABA in the stomatogastric ganglion of the crab Cancer borealis.

The multifunctional neural circuits in the crustacean stomatogastric ganglion (STG) are influenced by many small-molecule transmitters and neuropeptides that are co-localized in identified projection neurons to the STG. We describe the pattern of gamma-aminobutyric acid (GABA) immunoreactivity in the stomatogastric nervous system of the crab Cancer borealis and demonstrate biochemically the presence of authentic GABA in C. borealis. No STG somata show GABA immunoreactivity but, within the stomatogastric nervous system, GABA immunoreactivity co-localizes with several neuropeptides in two identified projection neurons, the modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1). To determine which actions of these neurons are evoked by GABA, it is necessary to determine the physiological actions of GABA on STG neurons. We therefore characterized the response of each type of STG neuron to focally applied GABA. All STG neurons responded to GABA. In some neurons, GABA evoked a picrotoxin-sensitive depolarizing, excitatory response with a reversal potential of approximately -40 mV. This response was also activated by muscimol. In many STG neurons, GABA evoked inhibitory responses with both K(+)- and Cl(-)-dependent components. Muscimol and beta-guanidinopropionic acid weakly activated the inhibitory responses, but many other drugs, including bicuculline and phaclofen, that act on vertebrate GABA receptors were not effective. In summary, GABA is found in projection neurons to the crab STG and can evoke both excitatory and inhibitory actions on STG neurons.

Different proctolin neurons elicit distinct motor patterns from a multifunctional neuronal network.

Distinct motor patterns are selected from a multifunctional neuronal network by activation of different modulatory projection neurons. Subsets of these projection neurons can contain the same neuromodulator(s), yet little is known about the relative influence of such neurons on network activity. We have addressed this issue in the stomatogastric nervous system of the crab Cancer borealis. Within this system, there is a neuronal network in the stomatogastric ganglion (STG) that produces many versions of the pyloric and gastric mill rhythms. These different rhythms result from activation of different projection neurons that innervate the STG from neighboring ganglia and modulate STG network activity. Three pairs of these projection neurons contain the neuropeptide proctolin. These include the previously identified modulatory proctolin neuron and modulatory commissural neuron 1 (MCN1) and the newly identified modulatory commissural neuron 7 (MCN7). We document here that each of these neurons contains a unique complement of cotransmitters and that each of these neurons elicits a distinct version of the pyloric motor pattern. Moreover, only one of them (MCN1) also elicits a gastric mill rhythm. The MCN7-elicited pyloric rhythm includes a pivotal switch by one STG network neuron from playing a minor to a major role in motor pattern generation. Therefore, modulatory neurons that share a peptide transmitter can elicit distinct motor patterns from a common target network.

Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis.

Immunocytochemical and biochemical studies have indicated the presence of many neuroactive substances in the stomatogastric nervous system (STNS) of the crab Cancer borealis. In electrophysiological studies, many of these substances modulate the motor output of neural networks contained within this system. Previous work in the STNS suggested the presence of neuropeptides related to the invertebrate tachykinin-related peptide (TRP) family. Here we isolate and characterize two novel peptides from the C. borealis nervous system that show strong amino acid sequence identity to the invertebrate TRPs. The central nervous systems of 160 crabs were extracted in an acidified solvent, after which four reversed-phase HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay and a radioimmunoassay (RIA) employing an antiserum to locustatachykinin I (Lom TK I) were used to monitor all collected fractions. The amino acid sequences of the isolated peptides were determined by Edman degradation. Mass spectrometry and chemical synthesis confirmed the sequences to be APSGFLGMR-NH2 and SGFLGMR-NH2. APSGFLGMR-NH2 is approximately 20-fold more abundant in the crab central nervous system than is SGFLGMR-NH2. We have named these peptides Cancer borealis tachykinin-related peptide Ia and Ib (CabTRP Ia and Ib), respectively. Both peptides are myoactive in the cockroach hindgut muscle contraction bioassay, with CabTRP Ia being approximately 500 times more potent than CabTRP Ib. RIA performed on HPLC-separated C. borealis stomatogastric ganglion (STG) extract revealed that CabTRP Ia is the only detectable TRP-like moiety in this ganglion. Incubation of synthetic CabTRP Ia with the isolated STG excited the pyloric motor pattern. These effects were suppressed by the broad-spectrum tachykinin receptor antagonist Spantide I. Spantide I had no effect on the actions of the unrelated endogenous peptide proctolin in the STG. There was no consistent influence of CabTRP Ib on the pyloric rhythm. Given its amino acid sequence and minimal biological activity in the crab, CabTRP Ib may be a breakdown product of CabTRP Ia.

Organization of the stomatogastric neuropil of the crab, Cancer borealis, as revealed by modulator immunocytochemistry.

We used antibodies to a number of neuromodulatory substances, including serotonin, FLRF amide, red pigment-concentrating hormone, substance P, proctolin and cholecystokinin, to investigate the distribution of molecules similar to these substances in the stomatogastric ganglion of the crab, Cancer borealis. No immunoreactivity was seen in the region of the cell bodies that surrounds the neuropil and little was found in the core of the neuropil (where the primary neurites of the intrinsic neurons occupy most of the space). Instead, modulator immunolabel was densely packed in the more peripheral portion of the neuropil that surrounded the core. Within this peripheral neuropil, profiles appeared quite uniformly distributed. Double-labeling showed that there were limited differences in distribution between the labels examined in our study. The only immunolabeled structures that showed a distinct differential distribution within the stomatogastric neuropil were a population of >/=10 microm varicosities that arose from a pair of input fibers that we termed the large varicosity fibers. These varicosities were immunolabelled by antisera for three different peptides. Taken collectively, these data shows that there is a stereotyped distribution of modulator immunoreactivity within the crab stomatogastric neuropil. However, this segregation is more rudimentary than that reported for the intrinsic stomatogastric neurons.

Matrix of neuromodulators in neurosecretory structures of the crab Cancer borealis.

The crustacean stomatogastric ganglion, which is situated in the ophthalmic artery, can be modulated by both intrinsically released molecules and hormones. In the crab Cancer borealis, over a dozen neuroactive compounds have been identified in the input axons that project into the stomatogastric neuropil. However, little is known about the modulator content of the two major neurohemal organs, the sinus glands and the pericardial organs, in this crab. We now report the results of a series of immunocytochemical experiments designed to identify putative neurohormones in these tissues. We find that the majority of modulators present in the input axons of the stomatogastric ganglion are also present in at least one of the neurohemal organs. Specifically, allatostatin-like, buccalin-like, cholecystokinin-like, FLRFamide-like, GABA-like, locustatachykinin-like, myomodulin-like, proctolin-like, red pigment concentrating hormone-like and serotonin-like immunoreactivities are all present in both the stomatogastric neuropil and at least one of the neurohemal organs. Thus, these substances are likely to serve a dual role as both local and hormonal modulators of the stomatogastric network. Two other substances, beta-pigment dispersing hormone and crustacean cardioactive peptide, are not present in the stomatogastric neuropil, but beta-pigment dispersing hormone immunoreactivity is present in the sinus glands and crustacean cardioactive peptide immunoreactivity is present in the pericardial organs. It is likely that crustacean cardioactive peptide exerts its influence on the stomatogastric neural circuit via hormonal pathways. Double-labeling experiments show that the patterns of modulator co-localization present in the stomatogastric neuropil are different from those in the neurosecretory organs, suggesting that few rules of colocalization hold across these tissues.

Distribution and effects of tachykinin-like peptides in the stomatogastric nervous system of the crab, Cancer borealis.

The rhythmically active pyloric and gastric mill motor patterns in the stomatogastric ganglion of the crab, Cancer borealis, are influenced by modulatory projection neurons whose somata are located primarily in the other ganglia of the stomatogastric nervous system. One of these projection neurons exhibits substance P-like immunolabeling. However, bath application of substance P does not influence these motor patterns. To determine whether a different peptide is responsible for the substance P-like immunolabeling, we studied the presence and physiological effects of the locustatachykinins and the leucokinins, two families of tachykinin-like peptides originally identified in insect nervous systems. Locustatachykinin-like immunolabeling has the same distribution in the stomatogastric nervous system as substance P-like immunolabeling and colocalizes with it in the majority of immunopositive structures. Preincubation of locustatachykinin antibody with substance P, and preincubation of substance P antibody with locustatachykinin, blocks subsequent immunolabeling in the stomatogastric nervous system. In contrast, we found no leucokinin-like immunolabeling in this system. Bath application to the stomatogastric ganglion of individual locustatachykinins or leucokinins excited the pyloric rhythm in a state-dependent manner. Each peptide family had distinct effects on the pyloric rhythm. Thus, both of these tachykinin-like peptide families are likely related to native neuropeptides that influence the pyloric rhythm. Furthermore, a member of the locustatachykinin family is likely to be the source of the previously identified substance P-like immunoreactivity in the stomatogastric nervous system.

Immunocytochemical localization of multiple cholecystokinin-like peptides in the stomatogastric nervous system of the crab Cancer borealis.

Three anti-cholecystokinin antibodies were used to label the stomatogastric nervous system of the crab Cancer borealis. Labeled tissues were examined as whole mounts using laser scanning confocal microscopy. Although each of the anti-cholecystokinin antibodies labeled a variety of structures within the stomatogastric nervous system (including somata, fibers and neuropil), the pattern of labeling produced by each antibody was distinct. These results indicate that there is a family of cholecystokinin-like molecules that are differentially distributed among a subpopulation of the neurons in the stomatogastric nervous system of Cancer borealis.

Functional organization of cotransmission systems: lessons from small nervous systems.

Small invertebrate nervous systems allow one to ask a series of questions concerning the functional roles of cotransmitters. This review outlines some of the implications of cotransmission for target selectivity in complex neuropils. We suggest the possibility that a unique constellation of cotransmitters in individual identified modulatory neurons allows a specificity of action even when peptides may act over an extended distance, and when individual modulatory substances may be released from several modulatory neurons.