Search

We have identified cells in the central nervous system of the marine mollusc Aplysia that react with antibody raised against the small cardioactive peptide B (SCPB). Antisera to this neuropeptide stained a subset of central neurons that include the large identified buccal neurons, B1 and B2. The distribution of SCP-containing neurons was used in a strategy to isolate a cDNA clone encoding the precursor protein for the peptide. RNA from neurons B1 and B2 and from cells that did not stain with SCPB antisera was used to direct the synthesis of radiolabeled cDNA probes. A cDNA clone complimentary to mRNA specifically expressed in the B1 and B2 cells was isolated by differentially screening a buccal cDNA library with these probes. The cloned cDNA segment is 1394 nucleotides in length and contains a 408-base-pair open reading frame. The predicted precursor protein is composed of 136 amino acids and has a characteristic hydrophobic leader sequence. The sizes of the precursor protein with and without this leader sequence agree with in vivo and in vitro labeling studies. The amino acid sequences for SCPB and a related peptide, SCPA, are present and are flanked by known proteolytic processing sites.

A model system that consists of a muscle utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons innervating this muscle, neurons B15 and B16, has been used to study the expression of food-induced arousal in the marine mollusk Aplysia. The ARC muscle receives modulatory input from an extrinsic source, the serotonergic metacerebral cells, which partially accounts for the progressive increase in the strength of biting seen in aroused animals. Another source of modulation may arise from the ARC motor neurons themselves, which synthesize neuropeptides that can potentiate ARC contractions. Neuron B15 synthesizes the two homologous peptides, small cardioactive peptides A and B, whereas neuron B16 synthesizes the structurally unrelated peptide myomodulin. Here we report the purification and sequencing of a neuropeptide termed buccalin and show that it is colocalized with the small cardioactive peptides to neuron B15. Buccalin is also bioactive at the ARC neuromuscular junction but, in contrast to the small cardioactive peptides, when exogenously applied, it decreases rather than increases the size of muscle contractions elicited by firing of the motor neurons. Also unlike the small cardioactive peptides, which exert postsynaptic actions, buccalin seems to act only presynaptically. It has no effect on muscle relaxation rate and decreases motor neuron-elicited excitatory junction potentials in the ARC without affecting contractions produced by direct application of acetylcholine to the muscle. Neuron B15, therefore, appears to contain three modulatory neurotransmitters, two of which may act postsynaptically on the muscle to potentiate the action of the primary neurotransmitter acetylcholine and one of which may act presynaptically on nerve terminals to inhibit acetylcholine release.

In previous studies, we demonstrated that B15, one of the two cholinergic motor neurons of the accessory radula closer muscle of Aplysia, synthesizes two peptides, small cardioactive peptides A and B (SCPA and SCPB), that, when exogenously applied, increase the size and relaxation rate of muscle contractions elicited by motor neuron stimulation. In the present experiments, we obtained evidence that the SCPs are released under physiological conditions. Specifically, we characterized firing patterns of motor neuron B15 during normal behavior, simulated them in vitro, and demonstrated that this type of neuronal activity produces decreases in SCP levels in neuronal processes and terminals. We also obtained evidence that suggests that enough SCP is released under physiological conditions to modulate neuromuscular activity in the accessory radula closer. We demonstrated that physiological activity of neuron B15 produces significant increases in muscle cAMP levels. Furthermore, increases in the size and relaxation rate of muscle contractions can be produced by changes in stimulation parameters that are also likely to maximize effects of released endogenous SCPA and SCPB.

The neuropeptide designated SCPB (small cardioactive peptide B), the sequence of which has recently been determined, was found in the accessory radula closer muscle, a muscle involved in biting movements. The ganglia and nerves that innervate the accessory radula closer muscle also contain SCPB. At nanomolar concentrations, it enhances the contractions of the muscle. The effect of SCPB on the muscle resembles the effect of an identified serotonergic neuron that previously was shown to mediate behavioral effects that reflect a food arousal state in Aplysia. Like serotonin, SCPB enhances contractions by a postsynaptic action, which appears to involve an increase in cAMP levels in the muscle. Our findings suggest that parallel peptidergic and serotonergic pathways may mediate similar aspects of arousal in Aplysia.

When Aplysia are initially exposed to food stimuli, their biting responses show progressive increases in speed and strength. The accessory radula closer (ARC) buccal muscles have been used to study this phenomenon, and it has been shown that changes in ARC muscle contraction are partially due to activity of a serotonergic neuron that modulates this muscle, by both a direct action and an action on two ARC motor neurons (B15 and B16). The motor neurons use acetylcholine as their excitatory transmitter, but they also contain bioactive peptides that can potentiate muscle contractions when they are exogenously applied. Motor neuron B15 contains the structurally related small cardioactive peptides A and B, whereas motor neuron B16 contains a different peptide--termed myomodulin. In the present study we determined the full amino acid sequence of myomodulin. Myomodulin is present in the ARC muscle, and exogenous application of the peptide potentiates ARC muscle contractions in a manner similar to the potentiation by small cardioactive peptides A and B. The structure of myomodulin, however, bears little resemblance to the small cardioactive peptides. Thus it appears that ARC muscle contractions may be regulated by at least three distinct classes of neuromodulators: serotonin, the small cardioactive peptides, and myomodulin.

Through the construction of a probability distribution, a test is proposed for the randomness of the occurrence of an epidemiological event in a series of structured ordered sequences. Such sequences may be of persons admitted to hospital in some specified order, individuals seen in a large set of extended screening program, or individuals in a large set of extended genealogies. Modifications to account for the limited length of ascertained sequences are also given. The method will be particularly important in the first-stage evaluation of genealogical data to indicate the existence and degree of familial aggregation of diseases such as cancer and to justify more complex methods of analysis designed to evaluate the specific elements of causation that may be operating. The statistical power of this approach is also derived, considering the anticipated sample sizes in its application, to detect some forms of familial aggregation in a large genealogical data base from Laredo, Texas.

An important criterion for classifying a substance as a neurotransmitter is that it is released in an activity-dependent fashion. We have utilized cell culture of individual neurons of Aplysia to demonstrate the release of the neuropeptides SCPA and SCPB (small cardioactive peptides A and B). Neurons B1 and B2 were isolated from the buccal ganglion of Aplysia and maintained in cell culture. The cells grew new processes, which were immunoreactive to antibodies for the neuropeptide SCPB. These processes contained SCPA and SCPB that were detectable by bioassay on snail heart. The cells synthesized the SCPs from radiolabeled precursors and transported the peptides to their neurites. Single cells released SCPs in a calcium-dependent fashion upon intracellular electrical stimulation. Taken together, these results provide critical evidence that SCPs are neurotransmitters. The results also indicate that the cell culture of individual identified neurons can be used to investigate the release of peptides.

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells.

Admixture between genetically different populations may produce gametic association between gene loci as a function of the genetic difference between parental populations and the admixture rate. This association decays as a function of time since admixture and the recombination rate between the loci. Admixture between genetically long-separated human populations has been frequent in the centuries since the age of exploration and colonization, resulting in numerous hybrid descendant populations today, as in the Americas. This represents a natural experiment for genetic epidemiology and anthropology, in which to use polymorphic marker loci (e.g., restriction fragment length polymorphisms) and disequilibrium to infer a genetic basis for traits of interest. In this paper we show that substantial disequilibrium remains today under widely applicable situations, which can be detected without requiring inordinately close linkage between trait and marker loci. Very disparate parental allele frequencies produce large disequilibrium, but the sample size needed to detect such levels of disequilibrium can be large due to the skewed haplotype frequency distribution in the admixed population. Such situations, however, provide power to differentiate between disequilibrium due just to population mixing from that due to physical linkage of loci--i.e., to help map the genetic locus of the trait. A gradient of admixture levels between the same parental populations may be used to test genetic models by relating admixture to disequilibrium levels.

Changes in Aplysia biting responses during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs). The MCCs potentiate contractions of a muscle utilized in biting, the accessory radula closer (ARCM), when contractions are elicited by stimulation of either of the two cholinergic motor neurons B15 or B16 that innervate the muscle. We have now shown that ARCM contractions may also be potentiated by peptide cotransmitters in the ARCM motor neurons. We found that motor neuron B15 contains small cardioactive peptides A and B (SCPA and SCPB)--i.e., whole B15 neurons were bioactive on the SCP-sensitive Helix heart, as were reverse-phase HPLC fractions of B15 neurons that eluted like synthetic SCPA and SCPB. Furthermore, [35S]methionine-labeled B15 peptides precisely coeluted with synthetic SCPA and SCPB. SCPB-like immunoreactivity was associated with dense-core vesicles in the soma of B15 and in neuritic varicosities and terminals in the ARCM. B16 motor neurons did not contain SCPA or SCPB but contained an unidentified bioactive peptide. RP-HPLC of [35S]methionine-labeled B16s resulted in one major peak of radioactivity that did not coelute with either SCP and which, when subject to Edman degradation, yielded [35S]methionine in positions where there is no methionine in the SCPs. Exogenously applied B16 peptide potentiated ARCM contractions elicited by stimulation of B15 or B16 neurons. Thus, in this system there appear to be two types of modulation; one type arises from the MCCs and is extrinsic to the motor system, whereas the second type arises from the motor neurons themselves and hence is intrinsic.