Changes in Aplysia biting responses during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs) The nervous system of the marine mollusc Aplysia provides an advantageous model system for the study of the neural basis ofbehavior and the modification ofbehavior by learning and motivational states such as arousal. We have been studying a form of arousal that is elicited by food and is characterized by general changes in locomotion (1) and cardiovascular responses (2) as well as by specific alterations in feeding behavior-such as progressive increases in the strength and speed of biting (1,3,4).Previous studies (5-7) have demonstrated that changes in biting during food arousal are partially mediated by the serotonergic metacerebral cells (MCCs). The MCCs exert central actions within the nervous system and peripheral actions on buccal muscles. We have studied the peripheral actions of the MCCs on the accessory radula closer muscle (ARCM), a muscle used in biting, which is innervated by the MCC and two cholinergic buccal motor neurons B15 and B16 (8). The MCC is not a motor neuron; its activity does not produce ARCM contractions. Instead it increases the strength of contractions produced by stimulation of neurons B15 and B16, presumably by increasing cAMP levels in the muscle (9).This modulatory input from the MCCs, however, accounts for only a part of the manifestations of arousal, because animals in which the MCCs have been lesioned still exhibit a progressive buildup ofthe magnitude and frequency ofbiting, although to a lesser degree (7). This suggests that arousal may be produced by more than one modulatory system. In fact, we have demonstrated that nerve fibers and varicosities in the ARCM contain the neuropeptide SCPB and that exogenous application of SCPB exerts modulatory actions similar to those of serotonin and the MCC-i.e., SCPB increases cAMP in the ARCM and enhances contractions produced by motor neuron stimulation without itself producing a contraction (10). Therefore, there may be peptidergic as well as serotonergic modulation of the ARCM. As a first step in understanding the functional significance of this dual modulation, we have now identified sources of peptidergic input to the ARCM. METHODS Extraction of Peptides from Motor Neurons. Identified neurons B15 and B16 (8) were marked by iontophoresis of fast green dye. In some experiments, peptides were radiolabeled in vivo (11). Buccal ganglia were incubated with 0.5 mCi of [35S]methionine (1 Ci = 37 GBq; Amersham) for 24 hr at 18°C in 1 ml of 50% artificial sea water (ASW)/50% sterile (0.2-,um filtered) hemolymph/100 ,l of antibiotics (penicillin and streptomycin each at 50 units per ml)/colchicine (2.5 ,ul of 1 M colchicine dissolved in Me2SO). Colchicine was added to inhibit axonal transport, which raised intrasomatic peptide levels and reduced, or eliminated, labeled peptides that might be present in fibers and terminals near the somata of dissected neurons (12). After 24 hr, ganglia were rinsed and incubated i...

Adipocytes participate in the microenvironment of the bone marrow (BM), but their exact role remains to be determined. It has recently been shown that leptin, a hormone secreted from extramedullary adipocytes, could be involved in hematopoiesis. Therefore we have developed a primary culture system of human BM adipocytes to characterize their differentiation and determine whether leptin is also secreted from these adipocytes. BM cells were cultured with fetal calf and horse sera. In the presence of dexamethasone, cells with vesicles containing lipids appeared within 15 days. They expressed glycerol phosphate dehydrogenase activity and a lipolytic activity in response to isoproterenol, but expressed neither the adrenergic beta3 receptor nor the mitochondrial uncoupling protein UCP1. The addition of insulin alone to the culture media did not promote adipocyte differentiation. Leptin was expressed and secreted at high levels during adipocyte differentiation. Acute exposure of differentiated adipocytes to insulin had little effect on leptin expression whereas forskolin strongly inhibited it. These results show that although human BM adipocytes differ from extramedullary adipose tissues in their sensitivity to different effectors, they are a secondary source of leptin production. They suggest that BM adipocytes could contribute to hematopoiesis via the secretion of leptin in the vicinity of hematopoietic stem cells.

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 ofa 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 peptidetermed 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.An important issue in behavioral neuroscience is the nature of the modulatory synaptic mechanisms that affect behavior. We have taken advantage of the simplicity of the nervous system of Aplysia to study synaptic modulatory processes and their possible role in motivational states. One such motivational state is arousal, which can be induced by exposing an animal to food. Food arousal in Aplysia is characterized by changes in locomotion, posture, and cardiovascular function (1-3), as well as by progressive increases in the speed and strength of biting as the animal begins to feed (1, 4, 5). Modulation of feeding responses has been studied in the experimentally advantageous muscle the accessory radula closer (ARC), which is innervated by two identified motor neurons, B15 and B16 (6). Contraction of the ARC is modulated in part by activity of a serotonergic neuron, the metacerebral cell (7-9).We have begun to investigate possible additional sources of neuromodulation in the ARC muscle (10, 11). We have shown that both of the ARC motor neurons B15 and B16 are cholinergic (6) but also contain neuropeptides that, when exogenously applied, potentiate muscle contractions elicited by motor neurons. We showed that B15 contains the characterized (12)(13)(14) small cardioactive peptides A and B (SCPA and SCPB) and that B16 contains a peptide that has methionine in positions 2 and 4 of its amino acid sequence and, therefore, is not one of the SCPs. This peptide was preliminarily named myomodulin (11). In this study we obtained the complete primary structure of the B16 peptide myomodulin. METHODS Extraction and Purification. ARC muscle (30 g derived from 1000 Aplysia californica) were heated for 10 min at 100'C...

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 ofbiting 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.We have been studying a motivational state, food-induced arousal, that is manifested in the consummatory biting response of the marine mollusk Aplysia californica as progressive increases in both the speed and strength of biting (1, 2). Work in a model system (3) that consists of one of the muscles utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons that innervate this muscle, buccal motor neurons B15 and B16, has led us to conclude that arousal in the Aplysia biting response is partially mediated by activity of the serotonergic metacerebral cells (4-6).We have also demonstrated that motor neurons B15 and B16 synthesize neuropeptides that have bioactivity at the ARC muscle (7,8). Neuron B15 contains the two structurally homologous (9-11) peptides, small cardioactive peptides A and B (SCPA and SCPB) (7), whereas neuron B16 contains the unrelated peptide myomodulin (8). When exogenously applied, all three peptides produce increases in the size and the relaxation rate of muscle contractions elicited by motor neuron stimulation (7,8,12). Therefore, we have hypothesized that the ARC neuromuscular system is modulated both extrinsically (by means of the metacerebral ...

In previous studies, we demonstrated that B15, one of the two cholinergic motor neurons of the accessory radula closer muscle ofAplysia, 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.In the marine mollusc Aplysia californica, the food arousal state is manifested both as alterations in appetitive feeding behaviors and as increases in the efficiency of consummatory responses (1, 2). Manifestations of arousal in consummatory biting responses are partially produced by modulatory effects of the serotonergic metacerebral cells (MCCs), which have both central and peripheral actions (3)(4)(5). The central actions of the MCCs are exerted on motor neurons utilized in feeding behavior and on the biting central pattern generator. Peripheral effects of the MCCs are exerted directly on the feeding musculature; they increase the size and relaxation rate of muscle contractions elicited by motor neuron stimulation. However, manifestations of arousal in biting responses are not produced exclusively by the MCCs, since effects of arousal can still be partially seen in animals in which the MCCs have been destroyed (5).We have used the accessory radula closer (ARC), an experimentally advantageous muscle, to explore other sources of modulation of the biting musculature. The two cholinergic motor neurons of the ARC synthesize a number of myoactive peptides that are present in fibers and varicosities in the muscle. For example, buccal neuron B15 synthesizes two peptides, small cardioactive peptides A and B (SCPA and SCPB; ref. 6), that, when exogenously applied, exert actions similar to those ofthe MCCs; they increase both the size (7) and relaxation rate (8) of motor neuron-elicited muscle contractions. We have hypothesized that SCPA and SCPB are released as cotransmitters and are at least partially responsible for producing the increase in the efficiency of biting responses that is seen during arousal (6).In the present study, we obtained evidence that the SCPs are released under physiological conditions. Specifically, we recorded B15 synaptic acti...