Repeated stimulation of feeding mechanoafferents in Aplysia generates responses consistent with the release of food.

How does repeated stimulation of mechanoafferents affect feeding motor neurons? Monosynaptic connections from a mechanoafferent population in the Aplysia buccal ganglia to five motor followers with different functions were examined during repeated stimulus trains. The mechanoafferents produced both fast and slow synaptic outputs, which could be excitatory or inhibitory. In contrast, other Aplysia mechanoafferents produce only fast excitation on their followers. In addition, patterns of synaptic connections were different to the different motor followers. Some followers received both fast excitation and fast inhibition, whereas others received exclusively fast excitation. All followers showed strong decreases in fast postsynaptic potential (PSP) amplitude within a stimulus train. Fast and slow synaptic connections were of net opposite signs in some followers but not in others. For one follower, synaptic contacts were not uniform from all subareas of the mechanoafferent cluster. Differences in properties of the buccal ganglia mechanoafferents and other Aplysia mechanoafferents may arise because the buccal ganglia neurons innervate the interior of the feeding apparatus, rather than an external surface, and connect to motor neurons for muscles with different motor functions. Fast connection patterns suggest that these synapses may be activated when food slips, biasing the musculature to release food. The largest slow inhibitory synaptic PSPs may contribute to a delay in the onset of the next behavior. Additional functions are also possible.

Multiple changes in connectivity between buccal ganglia mechanoafferents and motor neurons with different functions after learning that food is inedible in Aplysia.

Changes caused by learning that a food is inedible in Aplysia were examined for fast and slow synaptic connections from the buccal ganglia S1 cluster of mechanoafferents to five followers, in response to repeated stimulus trains. Learning affected only fast connections. For these, unique patterns of change were present in each follower, indicating that learning differentially affects the different branches of the mechanoafferents to their followers. In some followers, there were increases in either excitatory or inhibitory connections, and in others, there were decreases. Changes in connectivity resulted from changes in the amplitude of excitation or inhibition, or as a result of the number of connections, or of both. Some followers also exhibited changes in either within or between stimulus train plasticity as a result of learning. In one follower, changes differed from the different areas of the S1 cluster. The patterns of changes in connectivity were consistent with the behavioral changes produced by learning, in that they would produce an increase in the bias to reject or to release food, and a decrease in the likelihood to respond to food.

An in vitro analog of learning that food is inedible in Aplysia: decreased responses to a transmitter signaling food after pairing with transmitters signaling failed swallowing.

An in vitro analog of learning that a food is inedible provided insight into mechanisms underlying the learning. Aplysia learn to stop responding to a food when they attempt but fail to swallow it. Pairing a cholinergic agonist with an NO donor or histamine in the Aplysia cerebral ganglion produced significant decreases in fictive feeding in response to the cholinergic agonist alone. Acetylcholine (ACh) is the transmitter of chemoreceptors sensing food touching the lips. Nitric oxide (NO) and histamine (HA) signal failed attempts to swallow food. Reduced responses to the cholinergic agonist after pairing with NO or HA indicate that learning partially arises via a decreased response to ACh in the cerebral ganglion.

Exogenous expression of an allatotropin-related peptide receptor increased the membrane excitability in Aplysia neurons.

Neuropeptides act mostly on a class of G-protein coupled receptors, and play a fundamental role in the functions of neural circuits underlying behaviors. However, physiological functions of some neuropeptide receptors are poorly understood. Here, we used the molluscan model system Aplysia and microinjected the exogenous neuropeptide receptor apATRPR (Aplysia allatotropin-related peptide receptor) with an expression vector (pNEX3) into Aplysia neurons that did not express the receptor endogenously. Physiological experiments demonstrated that apATRPR could mediate the excitability increase induced by its ligand, apATRP (Aplysia allatotropin-related peptide), in the Aplysia neurons that now express the receptor. This study provides a definitive evidence for a physiological function of a neuropeptide receptor in molluscan animals.

Identification of an allatostatin C signaling system in mollusc Aplysia.

Neuropeptides, as pervasive intercellular signaling molecules in the CNS, modulate a variety of behavioral systems in both protostomes and deuterostomes. Allatostatins are neuropeptides in arthropods that inhibit the biosynthesis of juvenile hormones. Based on amino acid sequences, they are divided into three different types in arthropods: allatostatin A, allatostatin B, allatostatin C. Allatostatin C (AstC) was first isolated from Manduca sexta, and it has an important conserved feature of a disulfide bridge formed by two cysteine residues. Moreover, AstC appears to be the ortholog of mammalian somatostatin, and it has functions in common with somatostatin, such as modulating feeding behaviors. The AstC signaling system has been widely studied in arthropods, but minimally studied in molluscs. In this study, we seek to identify the AstC signaling system in the marine mollusc Aplysia californica. We cloned the AstC precursor from the cDNA of Aplysia. We predicted a 15-amino acid peptide with a disulfide bridge, i.e., AstC, using NeuroPred. We then cloned two putative allatostatin C-like receptors and through NCBI Conserved Domain Search we found that they belonged to the G protein-coupled receptor (GPCR) family. In addition, using an inositol monophosphate 1 (IP1) accumulation assay, we showed that Aplysia AstC could activate one of the putative receptors, i.e., the AstC-R, at the lowest EC50, and AstC without the disulfide bridge (AstC') activated AstC-R with the highest EC50. Moreover, four molluscan AstCs with variations of sequences from Aplysia AstC but with the disulfide bridge activated AstC-R at intermediate EC50. In summary, our successful identification of the Aplysia AstC precursor and its receptor (AstC-R) represents the first example in molluscs, and provides an important basis for further studies of the AstC signaling system in Aplysia and other molluscs.

Nitric oxide and l-arginine have mixed effects on mammalian feeding in condition of a high motivation to feed.

Feeding inhibition caused by satiation in rats is partially mediated by the unconventional neurotransmitter nitric oxide (NO). Thus, in satiated rats blocking NO production increases feeding, and treatment with the NO precursor l-arginine or with an NO donor reduces feeding beyond that caused by satiation. Do NO and l-arginine also inhibit feeding when feeding motivation is high? When feeding motivation in satiated animals was hedonically increased by offering a highly attractive food, blocking NO production reduced the quantity eaten, rather than increasing it, indicating that hedonic aspects of food are partially mediated by NO. Increasing NO via an NO donor or l-arginine did not further increase the quantity eaten, indicating a ceiling effect. The NO donor, but not l-arginine, also decreased some motivation-dependent parameters of feeding. When feeding motivation was increased by hunger, quantities of food eaten were unaffected by an NO donor, blocker or precursor, with only the blocker of NO production affecting feeding patterning. We also examined effects on feeding of dissolving l-arginine in drinking water over 3 weeks. Chronic l-arginine administration had different effects during the first and in subsequent weeks, increasing feeding at first, but not later. The data indicate that NO has complex, state dependent effects on both the quantity of food eaten, and on patterns of feeding, probably reflecting different sites and mechanisms of action in the nervous system.

Multiple Local Synaptic Modifications at Specific Sensorimotor Connections after Learning Are Associated with Behavioral Adaptations That Are Components of a Global Response Change.

Learning causes local changes in synaptic connectivity and coordinated, global changes affecting many aspects of behavior. How do local synaptic changes produce global behavioral changes? In the hermaphroditic mollusc Aplysia, after learning that food is inedible, memory is expressed as bias to reject a food and to reduce responses to that food. We now show that memory is also expressed as an increased bias to reject even a nonfood object. The increased bias to rejection is partially explained by changes in synaptic connections from primary mechanoafferents to five follower neurons with well defined roles in producing different feeding behaviors. Previously, these mechanoafferents had been shown to play a role in memory consolidation. Connectivity changes differed for each follower neuron: the probability that cells were connected changed; excitation changed to inhibition and vice versa; and connection amplitude changed. Thus, multiple neural changes at different sites underlie specific aspects of a coordinated behavioral change. Changes in the connectivity between mechanoafferents and their followers cannot account for all of the behavioral changes expressed after learning, indicating that additional synaptic sites are also changed. Access to the circuit controlling feeding can help determine the logic and cellular mechanisms by which multiple local synaptic changes produce an integrated, global change in behavior.SIGNIFICANCE STATEMENT How do local changes in synapses affect global behavior? Studies on invertebrate preparations usually examine synaptic changes at specific neural sites, producing a specific behavioral change. However, memory may be expressed by multiple behavioral changes. We report that a change in behavior after learning in Aplysia is accomplished, in part, by regulating connections between mechanoafferents and their synaptic followers. For some followers, the connection probabilities change; for others, the connection signs are reversed; in others, the connection strength is modified. Thus, learning produces changes in connectivity at multiple sites, via multiple synaptic mechanisms that are consistent with the observed behavioral change.

Successful and unsuccessful attempts to swallow in a reduced Aplysia preparation regulate feeding responses and produce memory at different neural sites.

Sensory feedback shapes ongoing behavior and may produce learning and memory. Motor responses to edible or inedible food in a reduced Aplysia preparation were examined to test how sensory feedback affects behavior and memory. Feeding patterns were initiated by applying a cholinomimetic onto the cerebral ganglion. Feedback from buccal muscles increased the response variability and response rate. Repeated application of the cholinomimetic caused decreased responses, expressed in part by lengthening protractions. Swallowing strips of "edible" food, which in intact animals induces learning that enhances ingestion, increased the response rate, and shortened the protraction length, reflecting more swallowing. Testing memory by repeating the procedure prevented the decrease in response rate observed with the cholinomimetic alone, and shortened protractions. Training with "inedible" food that in intact animals produces learning expressed by decreased responses caused lengthened protractions. Testing memory by repeating the procedure did not cause decreased responses or lengthened protractions. After training and testing with edible or inedible food, all preparations were exposed to the cholinomimetic alone. Preparations previously trained with edible food displayed memory expressed as decreased protraction length. Preparations previously trained with inedible food showed decreases in many response parameters. Memory for inedible food may arise in part via a postsynaptic decrease in response to acetylcholine released by afferents sensing food. The lack of change in response number, and in the time that responses are maintained during the two training sessions preceding application of the cholinomimetic alone suggests that memory expression may differ from behavioral changes during training.

Nitric oxide and l-arginine regulate feeding in satiated rats.

Nitric Oxide (NO) and its precursor l-arginine were found to inhibit feeding in rats with a low motivation to eat, as they do in Aplysia. In rats that are relatively satiated, treatment with an NO blocker increased feeding, and treatment with an NO donor or with either of 2 doses of l-arginine inhibited feeding. NO and l-arginine modulated several parameters of feeding, such as the total duration of appetitive behaviors, the time spent feeding, the quantity of food eaten and the number of feeding bouts. The inhibitory effect of l-arginine on feeding could not be attributed to changes in locomotion. These data indicate that satiation is partially mediated by increased production of NO. NADPH-Diaphorase histochemical staining, which is specific for tissues actively producing NO, showed significantly greater staining in satiated compared to hungry rats in all 4 hypothalamic nuclei (paraventricular and arcuate nuclei, lateral and ventromedial hypothalamus) that were examined. l-arginine may act as a regulator of feeding by controlling NO production in several hypothalamic nuclei, specifically under condition of a low feeding motivation.

NO is required for memory formation and expression of memory, and for minor behavioral changes during training with inedible food in Aplysia.

A learning experience may lead to changes in behavior during the experience, and also to memory expressed at a later time. Are signals causing changes in behavior during the learning experience related to the formation and expression of memory? We examined this question, using learning that food is inedible in Aplysia Treatment of an isolated buccal ganglia preparation with an NO donor elicited rejection-like motor programs. Rejection initiated by NO production is consistent with aspects of behavioral changes seen while animals learn, and with memory formation. Nonetheless, applying the NO donor during training had only minor effects on behavior during the training, and did not improve memory, indicating that the induction of rejection in the buccal ganglia is unlikely to be the means by which NO during training contributes to memory formation. Block of NO during memory retrieval prevented the expression of memory, as measured by a lack of savings in time to stop responding to food. Applying an NO donor to the cerebral ganglion while eliciting fictive feeding inhibited the expression of feeding activity, indicating that some NO effects on memory consolidation and on expression of memory may be via effects on the cerebral ganglion.

Molecular correlates of separate components of training that contribute to long-term memory formation after learning that food is inedible in Aplysia.

Training Aplysia with inedible food for a period that is too brief to produce long-term memory becomes effective in producing memory when training is paired with a nitric oxide (NO) donor. Lip stimulation for the same period of time paired with an NO donor is ineffective. Using qPCR, we examined molecular correlates of brief training versus lip stimulation, of treatment with an NO donor versus saline, and of the combined stimuli producing long-term memory. Changes were examined in mRNA expression of Aplysia homologs of C/EBP, CREB1, CREB1α, CREB1ß, and CREB2, in both the buccal and cerebral ganglia controlling feeding. Both the brief training and the NO donor increased expression of C/EBP, CREB1, CREB1α, and CREB1ß, but not CREB2 in the buccal ganglia. For CREB1α, there was a significant interaction between the effects of the brief training and of the NO donor. In addition, the NO donor, but not brief training, increased expression of all of the genes in the cerebral ganglion. These findings show that the components of learning that alone do not produce memory produce molecular changes in different ganglia. Thus, long-term memory is likely to arise by both additive and interactive increases in gene expression.

New learning while consolidating memory during sleep is actively blocked by a protein synthesis dependent process.

Brief experiences while a memory is consolidated may capture the consolidation, perhaps producing a maladaptive memory, or may interrupt the consolidation. Since consolidation occurs during sleep, even fleeting experiences when animals are awakened may produce maladaptive long-term memory, or may interrupt consolidation. In a learning paradigm affecting Aplysia feeding, when animals were trained after being awakened from sleep, interactions between new experiences and consolidation were prevented by blocking long-term memory arising from the new experiences. Inhibiting protein synthesis eliminated the block and allowed even a brief, generally ineffective training to produce long-term memory. Memory formation depended on consolidative proteins already expressed before training. After effective training, long term memory required subsequent transcription and translation. Memory formation during the sleep phase was correlated with increased CREB1 transcription, but not CREB2 transcription. Increased C/EBP transcription was a correlate of both effective and ineffective training and of treatments not producing memory.

Variables controlling entry into and exit from the steady-state, one of two modes of feeding in Aplysia.

BACKGROUND: Aplysia feeding is a model system for examining the neural mechanisms by which changes in motivational state control behavior. When food is intermittently present, Aplysia eat large meals controlled by a balance between food stimuli exciting feeding and gut stimuli inhibiting feeding. However, when food is continuously present animals are in a state in which feeding is relatively inhibited and animals eat little. We examined which stimuli provided by food and feeding initiate steady-state inhibition of feeding, and which stimuli maintain the inhibition. RESULTS: Multiple stimuli were found to control entry into the steady-state inhibition, and its maintenance. The major variable governing entry into the steady-state is fill of the gut with bulk provided by food, but this stimulus cannot alone cause entry into the steady-state. Food odor and nutritional stimuli such as increased hemolymph glucose and L-arginine concentrations also contribute to inhibition of feeding leading to entry into the steady-state. Although food odor can alone cause some inhibition of feeding, it does not amplify the effect of gut fill. By contrast, neither increased hemolymph glucose nor L-arginine alone inhibits feeding in hungry animals, but both amplify the inhibitory effects of food odor, and increased glucose also amplifies the effect of gut fill. The major variable maintaining the steady-state is the continued presence of food odor, which can alone maintain the steady-state for 48-72 hrs. Neither increased glucose nor L-arginine can alone preserve the steady-state, although they partially preserve it. Glucose and arginine partially extend the effect of food odor after 72 hrs. CONCLUSIONS: These findings show that control of Aplysia feeding is more complex than was previously thought, in that multiple inhibitory factors interact in its control.

Localization of molecular correlates of memory consolidation to buccal ganglia mechanoafferent neurons after learning that food is inedible in Aplysia.

Training paradigms affecting Aplysia withdrawal reflexes cause changes in gene expression leading to long-term memory formation in primary mechanoafferents that initiate withdrawal. Similar mechanoafferents are also found in the buccal ganglia that control feeding behavior, raising the possibility that these mechanoafferents are a locus of memory formation after a training paradigm affecting feeding. Buccal ganglia mechanoafferent neurons expressed increases in mRNA expression for the transcription factor ApC/EBP, and for the growth factor sensorin-A, within the first 2 h after training with an inedible food. No increases in expression were detected in the rest of the buccal ganglia. Increased ApC/EBP expression was not elicited by food and feeding responses not causing long-term memory. Increased ApC/EBP expression was directly related to a measure of the efficacy of training in causing long-term memory, suggesting that ApC/EBP expression is necessary for the expression of aspects of long-term memory. In behaving animals, memory is expressed as a decrease in the likelihood to respond to food, and a decrease in the amplitude of protraction, the first phase of consummatory feeding behaviors. To determine how changes in the properties of mechanoafferents could cause learned changes in feeding behavior, synaptic contacts were mapped from the mechanoafferents to the B31/B32 neurons, which have a key role in initiating consummatory behaviors and also control protractions. Many mechanoafferents monosynaptically and polysynaptically connect with B31/B32. Monosynaptic connections were complex combinations of fast and slow excitation and/or inhibition. Changes in the response of B31/B32 to stimuli sensed by the mechanoafferent could underlie aspects of long-term memory expression.

Nitric oxide as a regulator of behavior: new ideas from Aplysia feeding.

Nitric oxide (NO) regulates Aplysia feeding by novel mechanisms, suggesting new roles for NO in controlling the behavior of higher animals. In Aplysia, (1) NO helps maintain arousal when produced by neurons responding to attempts to swallow food; (2) NO biases the motor system to reject and reposition food that resists swallowing; (3) if mechanically resistant food is not successfully swallowed, NO mediates the formation and expression of memories of food inedibility; (4) NO production at rest inhibits feeding, countering the effects of food stimuli exciting feeding. At a cellular level, NO-dependent channels contribute to the resting potential of neurons controlling food finding and food consumption. Increases in L-arginine after animals eat act as a post-feeding inhibitory signal, presumably by modulating NO production at rest. NO also signals non-feeding behaviors that are associated with feeding inhibition. Thus, depending on context, NO may enhance or inhibit feeding behavior. The different functions of NO may reflect the evolution of NO signaling from a response to tissue damage that was then elaborated and used for additional functions. These results suggest that in higher animals (1) elicited and background transmitter release may have similar effects; (2) NO may be produced by neurons without firing, influencing adjacent neurons; (3) background NO production may contribute to a neuron's resting potential; (4) circulating factors affecting background NO production may regulate spatially separated neurons; (5) L-arginine can be used to regulate neural activity; (6) L-arginine may be an effective post-ingestion metabolic signal to regulate feeding.

Autaptic muscarinic self-excitation and nitrergic self-inhibition in neurons initiating Aplysia feeding are revealed when the neurons are cultured in isolation.

Properties of a neuron may arise via endogenous mechanisms, or via interactions with other neurons. Culturing a neuron in isolation is a useful tool to distinguish between endogenous and circuit-derived properties. We identified two remarkable functional features of pattern initiator neurons B31/B32 in Aplysia when these neurons were cultured in isolation. These features were also present in situ, but were less prominent, and would have been missed had they not been observed first in the isolated cultured neurons. The properties are likely to be present in neurons of higher animals, but have not yet been observed. One feature was autaptic muscarinic self-excitation that contributes to the neuron's plateau potential, by which it initiates behavior. The other feature was the release of nitric oxide (NO) in the absence of spiking, which causes self-inhibition at rest. The nitrergic modulation of B31/B32 is likely to contribute to the control of feeding by dietary changes in the concentration of L: -arginine, the precursor from which NO is synthesized.

Neurons controlling Aplysia feeding inhibit themselves by continuous NO production.

BACKGROUND: Neural activity can be affected by nitric oxide (NO) produced by spiking neurons. Can neural activity also be affected by NO produced in neurons in the absence of spiking? METHODOLOGY/PRINCIPAL FINDINGS: Applying an NO scavenger to quiescent Aplysia buccal ganglia initiated fictive feeding, indicating that NO production at rest inhibits feeding. The inhibition is in part via effects on neurons B31/B32, neurons initiating food consumption. Applying NO scavengers or nitric oxide synthase (NOS) blockers to B31/B32 neurons cultured in isolation caused inactive neurons to depolarize and fire, indicating that B31/B32 produce NO tonically without action potentials, and tonic NO production contributes to the B31/B32 resting potentials. Guanylyl cyclase blockers also caused depolarization and firing, indicating that the cGMP second messenger cascade, presumably activated by the tonic presence of NO, contributes to the B31/B32 resting potential. Blocking NO while voltage-clamping revealed an inward leak current, indicating that NO prevents this current from depolarizing the neuron. Blocking nitrergic transmission had no effect on a number of other cultured, isolated neurons. However, treatment with NO blockers did excite cerebral ganglion neuron C-PR, a command-like neuron initiating food-finding behavior, both in situ, and when the neuron was cultured in isolation, indicating that this neuron also inhibits itself by producing NO at rest. CONCLUSION/SIGNIFICANCE: Self-inhibitory, tonic NO production is a novel mechanism for the modulation of neural activity. Localization of this mechanism to critical neurons in different ganglia controlling different aspects of a behavior provides a mechanism by which a humeral signal affecting background NO production, such as the NO precursor L-arginine, could control multiple aspects of the behavior.

A brief retraining regulates the persistence and lability of a long-term memory.

An experience extending the persistence of a memory after training Aplysia californica with inedible food also allows a consolidated memory to become sensitive to consolidation blockers. Long-term (24 h) memory is initiated by 5 min of training and is dependent on protein synthesis during the first few hours after training. By contrast, a more persistent (48 h) memory is dependent on a longer training session and on a later round of protein synthesis. When presented 24 h after training, a 3-min training that produces no memory alone can cause a memory that would have persisted for only 24 h to persist for 48 h. After a 48 h memory has been consolidated, 3 min of training also makes the memory sensitive to a protein-synthesis inhibitor. These findings suggest that a function of allowing a consolidated memory to become sensitive to blockers of protein synthesis may be to allow the memory to become more persistent.

Nitric oxide and histamine signal attempts to swallow: A component of learning that food is inedible in Aplysia.

Memory that food is inedible in Aplysia arises from training requiring three contingent events. Nitric oxide (NO) and histamine are released by a neuron responding to one of these events, attempts to swallow food. Since NO release during training is necessary for subsequent memory and NO substitutes for attempts to swallow, it was suggested that NO functions during training as a signal of attempts to swallow. However, it has been shown that NO may also be released in other contexts affecting feeding, raising the possibility that its role in learning is unrelated to signaling attempts to swallow. We confirmed that NO during learning signals attempts to swallow, by showing that a variety of behavioral effects on feeding of blocking or adding NO do not affect learning and memory that a food is inedible. In addition, histamine had effects similar to NO on learning that food is inedible, as expected if the transmitters are released together when animals attempt to swallow. Blocking histamine during training blocked long-term memory, and exogenous histamine substituted for attempts to swallow. NO also substituted for histamine during training. Histamine at concentrations relevant to learning activates neuron metacerebral cell (MCC). However, MCC activity is not a good monitor of attempts to swallow during training, since the neuron responds equally well to other stimuli. These findings support and extend the hypothesis that NO and histamine signal efforts to swallow during learning, acting on targets other than the MCC that specifically respond to attempts to swallow.

Autaptic excitation elicits persistent activity and a plateau potential in a neuron of known behavioral function.

Synaptic connections from a neuron onto itself (autapses) are not uncommon, but their contributions to information processing and behavior are not fully understood. Positive feedback mediated by autapses could in principle give rise to persistent activity, a property of some neurons in which a brief stimulus causes a long-lasting response. We have identified an autapse that underlies a plateau potential causing persistent activity in the B31/B32 neurons of Aplysia. The persistent activity is essential to the ability of these neurons to initiate and maintain components of feeding behavior. Persistent activity in B31/B32 arises from a voltage-dependent muscarinic autapse and from pharmacologically identical network-based positive feedback. Depolarization via the autapse begins later than network-driven excitation, and the effect of the autapse is therefore overshadowed by the earlier network-based depolarization. In B31/B32 neurons isolated in culture only the autapse is present, and the autapse functionally replaces the missing network-based feedback. Properties of B31/B32 provide insight into a possible general function of autapses. Autapses might function along with synapses from presynaptic neurons as components of feedback loops.