L-arginine via nitric oxide is an inhibitory feedback modulator of Aplysia feeding.

An increase in L-arginine hemolymph concentration acts as a postingestion signal inhibiting Aplysia feeding. At physiological concentrations (a 10-µM increase over background), the inhibitory effect of L-arginine is too weak to block feeding in hungry animals. However, a 10-µM increase in L-arginine concentration acts along with another inhibitory stimulus, the sustained presence of food odor, to inhibit feeding after a period of access to food. A physiological concentration of L-arginine also blocked the excitatory effect of a stimulus enhancing feeding, pheromones secreted by mating conspecifics. High concentrations of L-arginine (2.5 mM) alone also inhibited ad libitum feeding. L-arginine is the substrate from which nitric oxide synthase (NOS) produces nitric oxide (NO). Both an NO donor and a 10-µM increase in L-arginine inhibited biting in response to a weak food stimulus. Treatment with NOS inhibitors initiated food-finding and biting in the absence of food, indicating that food initiates feeding against a background of tonic nitrergic inhibition. Increased feeding in response to blocking NOS is accompanied by firing of the metacerebral (MCC) neuron, a monitor of food arousal. The excitatory effect on the MCC of blocking NOS is indirect. The data suggest that L-arginine acts by amplifying NO synthesis, which acts as a background stimulus inhibiting feeding. Background modulation of neural activity and behavior by NO may also be present in other systems, but such modulation may be difficult to identify because its effects are evident only in the context of additional stimuli modulating behavior.

Comparative neuroethology of feeding control in molluscs.

Over the last 30 years, many laboratories have examined, in parallel, the feeding behaviour of gastropod molluscs and the properties of the nervous system that give rise to this behaviour. Equal attention to both behavioural and neurobiological issues has provided deep insight into the functioning of the nervous system in generating and controlling behaviour. The conclusions derived from studies on gastropod feeding are generally consistent with those from other systems, but often provide more detailed information on the behavioural function of a particular property of the nervous system. A review of the literature on gastropod feeding illustrates a number of important messages. (i) Many of the herbivorous gastropods display similarities in behaviour that are reflected in corresponding similarities in neural anatomy, pharmacology and physiology. By contrast, the same aspects of the behaviour of different carnivorous species are quite variable, possibly because of their specialised prey-capture techniques. Nonetheless, some aspects of the neural control of feeding are preserved. (ii) Feeding in all species is flexible, with the behaviour and the physiology adapting to changes in the current environment and internal state and as a result of past experience. Flexibility arises via processes that may take place at many neural sites, and much of the modulation underlying behavioural flexibility is understood at a systems and at a cellular level. (iii) Neurones seem to have specific functions that are consistent with their endogenous properties and their synaptic connections, suggesting that individual neurones code specific pieces of information (i.e. they are 'grandmother cells'). However, the properties of a neurone can be extremely complex and can be understood only in the context of the complete neural circuit and the behaviour that it controls. In systems that are orders of magnitude more complex, it would be impossible to understand the functional properties of an individual neurone, even if it also coded specific information. (iv) Systems such as gastropod feeding may provide a model for understanding the functional properties of more complex systems.

Serotonergic and peptidergic modulation of the buccal mass protractor muscle (I2) in aplysia.

Plasticity of Aplysia feeding has largely been measured by noting changes in radula protraction. On the basis of previous work, it has been suggested that peripheral modulation may contribute to behavioral plasticity. However, peripheral plasticity has not been demonstrated in the neuromuscular systems that participate in radula protraction. Therefore in this study we investigated whether contractions of a major radula protraction muscle (I2) are subject to modulation. We demonstrate, first, that an increase in the firing frequency of the cholinergic I2 motoneurons will increase the amplitude of the resulting muscle contraction but will not modulate its relaxation rate. We show, second, that neuronal processes on the I2 muscle are immunoreactive to myomodulin (MM), RFamide, and serotonin (5-HT), but not to small cardioactive peptide (SCP) or buccalin. The I2 motoneurons B31, B32, B61, and B62 are not immunoreactive to RFamide, 5-HT, SCP, or buccalin. However, all four cells are MM immunoreactive and are capable of synthesizing MMa. Third, we show that the bioactivity of the different modulators is somewhat different; while the MMs (i.e., MMa and MMb) and 5-HT increase I2 muscle relaxation rate, and potentiate muscle contraction amplitude, MMa, at high concentrations, depresses muscle contractions. Fourth, our data suggest that cAMP at least partially mediates effects of modulators on contraction amplitude and relaxation rate.

Separate effects of a classical conditioning procedure on respiratory pumping, swimming, and inking in Aplysia fasciata.

We examined whether swimming and inking, two defensive responses in Aplysia fasciata, are facilitated by a classical conditioning procedure that has been shown to facilitate a third defensive response, respiratory pumping. Training consisted of pairing a head shock (UCS) with a modified seawater (85%, 120%, or pH 7.0 seawater--CSs). Animals were tested by re-exposing them to the same altered seawater 1 hr after the training. For all three altered seawaters, only respiratory pumping is specifically increased by conditioning. Swimming is sensitized by shock, and inking is unaffected by training, indicating that the conditioning procedure is likely to affect a neural site that differentially controls respiratory pumping. Additional observations also indicate that the three defensive responses are differentially regulated. First, different noxious stimuli preferentially elicit different defensive responses. Second, the three defensive responses are differentially affected by shock. Inking is elicited only immediately following shock, whereas swimming and respiratory pumping are facilitated for a period of time following the shock. Third, swimming and respiratory pumping are differentially affected by noxious stimuli that are delivered in open versus closed environments. These data confirm that neural pathways exist that allow Aplysia to modulate separately each of the three defensive behaviors that were examined.

Social isolation blocks the expression of memory after training that a food is inedible in Aplysia fasciata.

Isolating a sexually mature Aplysia fasciata for either 1 or 24 hr immediately after training that a food is inedible blocks the subsequent expression of memory measured 24 hr later. Isolation that is delayed for 1 hr after training, but not for 12 hr after training, is also effective in blocking memory. Isolation affects memory because of a specific effect caused by the absence of pheromones secreted by conspecifics rather than by a nonspecific change in the chemical environment, because transferring animals to a novel environment (120% seawater) that contains a conspecific does not affect memory. Isolation also does not affect memory in sexually immature Aplysia, even though immature animals are able to sense one another's presence. Isolation may affect memory because social (and sexual) isolation is a form of stress in mature A. fasciata, and stress after training affects retention in many animals.

Multiple memory processes following training that a food is inedible in Aplysia.

In many organisms, memory after training can be separated into a number of processes. We now report that separable memory processes are also initiated by a training procedure affecting Aplysia feeding behavior, a model system for examining the neural mechanisms underlying the regulation of a complex behavior. Four distinct memory process were identified: (1) a very short-term memory that declines within 15 min, (2) a short-term memory that persists for 0.5-1.0 hr, (3) an intermediate-term memory, observed 4 hr after training, and (4) a long-term memory that is seen only after a 12- to 24-hr delay. The four memory processes can be distinguished by the different training procedures that are required to elicit them. A single 5-min training session is sufficient to elicit the very short-term memory. However, a longer training session that continues until the animal stops responding to food is needed to elicit short-term memory. Intermediate-term memory is observed only after a spaced training procedure (three 5-min training sessions separated by 30-min intervals). A single 5-min training session that does not cause either short-term or intermediate-term memory is sufficient to induce long-term memory, indicating that short- and long-term memory are independent, parallel processes. Short- and long-term memory can also be separated by the effects of a post-training experience. Long-term, but not short-term, memory can be attenuated by cooling animals immediately after training. Cooling before the training does not affect either the training or the subsequent short- or long-term memory.

Different roles of neurons B63 and B34 that are active during the protraction phase of buccal motor programs in Aplysia californica.

The buccal ganglion of Aplysia contains a central pattern generator (CPG) that organizes sequences of radula protraction and retraction during food ingestion and egestion. Neurons B63 and B34 have access to, or are elements of, the CPG. Both neurons are depolarized along with B31/B32 during the protraction phase of buccal motor programs. Both cells excite the contralateral B31/B32 neurons and inhibit B64 and other neurons active during the retraction phase. B63 and B34 also both have an axon exiting the buccal ganglia via the contralateral cerebrobuccal connective. Despite their similarities, B63 and B34 differ in a number of properties, which reflects their different functions. B63 fires during both ingestion and egestion-like buccal motor programs, whereas B34 fires only during egestion-like programs. The bilateral B63 neurons, along with the bilateral B31 and B32 neurons, act as a single functional unit. Sufficient depolarization of any of these neurons activates them all and initiates a buccal motor program. B63 is electrically coupled to both the ipsilateral and the contralateral B31/B32 neurons but monosynaptically excites the contralateral neurons with a mixed electrical and chemical excitatory postsynaptic potential (EPSP). Positive feedback caused by electrical and chemical EPSPs between B63 and B31/B32 contributes to the sustained depolarization in B31/B32 and the firing of B63 during the protraction phase of a buccal motor program. B34 is excited during the protraction phase of all buccal motor programs, but, unlike B63, it does not always reach firing threshold. The neuron fires in response to current injection only after it is depolarized for 1-2 s or after preceding buccal motor programs in which it is depolarized. Firing of B34 produces facilitating EPSPs in the contralateral B31/B32 and B63 neurons and can initiate a buccal motor program. Firing in B34 is strongly correlated with firing in the B61/B62 motor neurons, which innervate the muscle (I2) responsible for much of protraction. B34 monosynaptically excites these motor neurons. B34 firing is also correlated with firing in motor neuron B8 during the protraction phase of a buccal motor program. B8 innervates the I4 radula closer muscle, which in egestion movements is active during protraction and in ingestion movements is active during retraction. B34 has a mixed, but predominantly excitatory, effect on B8 via a slow conductance-decrease EPSP. Thus firing in B34 leads to amplification of radula protraction that is coupled with radula closing, a pattern characteristic of egestion.

The rhinophores sense pheromones regulating multiple behaviors in Aplysia fasciata.

Pheromones released during mating and egg laying in Aplysia facilitate various aspects of behavior. We now show that the chemosensory rhinophores sense these pheromones. Ablating the rhinophores causes a significant decrease in the time spent mating. In addition, the lesion blocks the increases of feeding in response to pheromones released by egg cordons and by mating conspecifics. Respiratory pumping is significantly increased in response to egg cordons, mating conspecifics and egg laying hormone (ELH). The increase in response to egg cordons is blocked by ablating the rhinophores, but not by lesioning the osphradium, a second chemosensory organ.

Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica.

Applying the non-hydrolyzable cholinergic agonist carbachol (CCh) to the cerebral ganglion of Aplysia elicits sustained, regular bursts of activity in the buccal ganglia resembling those seen during biting. The threshold for bursting is approximately 10(-4) M. Bursting begins after a 2 to 5 min delay. The burst frequency increases over the first 5 bursts, reaching a plateau value of approximately 3 per minute. Bursting is maintained for over 10 min. Some of the effects of CCh may be attributed to its ability to depolarize and fire CBI-2, a command-like neuron in the cerebral ganglion that initiates biting. CBI-2 is also depolarized by ACh, and by stimulating peripheral sensory nerves. Excitation of CBI-2 caused by carbachol is partially blocked by the muscarinic antagonist atropine. We examined whether CCh-induced bursting is modified in ganglia taken from Aplysia that previously experienced treatments inhibiting feeding, such as satiation, head shock contingent or non-contingent with food, and training animals with an inedible food. No treatment consistently and repeatedly affected the latency, the peak burst period, the length of time that bursting was maintained, or the threshold CCh concentration for eliciting bursting. However, there was a decrease in the rate of the build-up of the buccal ganglion program in previously satiated animals.

Activity patterns of the B31/B32 pattern initiators innervating the I2 muscle of the buccal mass during normal feeding movements in Aplysia californica.

1. B31 and B32 are pattern-initiator neurons in the buccal ganglia of Aplysia. Along with the B61/B62 neurons, B31/B32 are also motor neurons that innervate the 12 buccal muscle via the I2 nerve. This research was aimed at determining the physiological functions of the B31/B32 and B61/B62 neurons, and of the I2 muscle. 2. Stimulating the I2 muscle in the radula rest position produces radula protraction. In addition, in behaving animals lesioning either the muscle or the I2 nerve greatly reduces radula protraction. 3. During buccal motor programs in reduced preparations, B31/B32 and B61/62 fire preceding activity in neuron B4, whose firing indicates the onset of radula retraction. In addition, during both ingestion-like and rejection-like patterns the activity in the I2 nerve is correlated with protraction. 4. B31/B32 fire at frequencies of 15-25 Hz. Neither B31/B32 nor B61/B62 elicit facilitating end-junction potentials (EJPs) and electromyograms (EMGs) in the I2 muscle. EMGs from B31/B32 are smaller than those from B61/B62. B31/B32 and B61/B62 innervate all areas of the muscle approximately uniformly. 5. In behaving animals, EMGs consistent with B31/B32 activity are seen in the I2 muscle during the protraction phase of biting, swallowing, and rejection movements. In addition, the I2 muscle receives inputs that cannot be attributed to either the B31/B32 or B61/B62 neurons, either because the potentials are too large, firing frequencies are too low, or a prominent facilitation is seen. Such potentials are associated with lip movements, and also with radula retraction. 6. EMGs were recorded from the I2 muscle during feeding behavior after a lesion of the I2 nerve. Animals that had severe deficits in protraction showed no activity consistent with B31/B32 or B61/B62, but did show activity during retraction. 7. Our data indicate that the I2 muscle and the B31/B32 motor neurons are essential constituents contributing to protraction movements. Activity in these neurons is associated with radula protraction, which occurs as a component of a number of different feeding movements. The I2 muscle may also contribute to retraction, via activation by other motor neurons.

B64, a newly identified central pattern generator element producing a phase switch from protraction to retraction in buccal motor programs of Aplysia californica.

1. Buccal motor programs in Aplysia are characterized by two phases of activity, which represent protraction and retraction of the radula in intact animals. The shift from protraction to retraction is caused by synaptic activity inhibiting neurons that are active during protraction and exciting neurons that are active during retraction. 2. B64, a newly identified neuron present bilaterally in the buccal ganglia, is partially responsible for the phase shift. Stimulating a single B64 causes bilateral inhibition of neurons B31/B32 and other neurons active during protraction and cause bilateral excitation of neurons B4/B5 and other neurons active during retraction. B64 is active throughout retraction. The amplitude and waveforms of the synaptic potentials caused by firing B64 are similar, but not identical, to those seen during retraction. 3. Some of the effects of B64 on B31/B32 and on B4/B5 are monosynaptic, as shown by their maintained presence in high divalent cation seawater, which blocks polysynaptic activity. 4. A brief depolarization of B64 leads to a long-lasting depolarization and firing. The ability of B64 to respond in this way is at least partially caused by an endogenous plateau potential, as this property is still seen after synaptic transmission is blocked. 5. Hyperpolarization of B64 bilaterally and preventing the somata from firing unmasks a large excitatory postsynaptic potential in B64. This procedure does not block the shift from protraction to retraction, indicating that spiking in the B64 somata is not necessary for the phase shift. 6. The firing pattern and synaptic connections of B64 are consistent with the hypothesis that the neuron is part of a central pattern generator underlying buccal motor programs. B64 is monosynaptically inhibited by neurons that are active along with B31/B32, which are responsible for producing the protraction phase of a buccal motor program. During the later portion of the protraction phase B64 is excited. In addition, firing B64 can phase advance and phase delay buccal motor programs. 7. Regulating the firing of B64 can regulate the expression of buccal motor programs. Stimulation of B64 at frequencies of 0.5-1.0 Hz leads to complete inhibition of buccal motor programs, whereas steady-state depolarization of B64 can lead to repetitive bursts of activity.

Compartmentalization of pattern-initiation and motor functions in the B31 and B32 neurons of the buccal ganglia of Aplysia californica.

1. The B31 and B32 cells in the buccal ganglia of Aplysia californica have unusual electrophysiological features. The somata of these strongly coupled cells do not sustain conventional action potentials. Brief depolarization of the soma produces a complex, sustained regenerative slow depolarization that is followed by a hyperpolarization. This activity in B31/B32 is correlated with a patterned burst of activity expressed in many of the neurons of the buccal ganglia. 2. Intracellular fills of B31/B32 showed that they have many neurites adjacent to the soma, as well as peripheral axons leaving the buccal ganglia via the radular nerve and innervating the Intrinsic-2 (I2) muscle of the buccal mass. Varicosities of B31/B32 axons are seen within the muscle. Backfills from I2 filled two adjacent B31/B32 cells as well as two newly identified neurons: B61 and B62. 3. Intracellular recording from the B31/B32 axons shows that they sustain conventional action potentials. These are recorded in the soma as approximately 10-mV fast depolarizations. Failed spikes in B31/B32, and conventional spikes in B61/B62, are correlated one for one with end-junction potentials (EJPs) in the I2 muscle. The EJPs are present even when the ganglia and muscles are bathed in high-divalent cations seawater. Thus B31/B32 and B61/B62 are motor neurons to the I2 muscle. 4. To determine whether the ability of B31/B32 to initiate patterned bursts is mediated by spikes in the axon or by slow potentials in the soma, the B31/B32 axon was stimulated directly while recording from the B31/B32 soma. Patterned bursts were never seen in the absence of slow potentials in the soma. Thus the ability of B31/B32 to initiate patterned bursts is localized to the soma and adjacent neurites. Slow potentials influence and cause spiking in adjacent neurons even in the absence of axon spikes. 5. These data show that the B31/B32 cells serve two functions that are compartmentalized in different regions of the cell and are mediated via different electrical signaling mechanisms. The B31/B32 somata utilize slow, sustained potentials as part of a network initiating patterned activity in the buccal ganglia. The B31/B32 axons utilize conventional action potentials, and act as motor neurons to the I2 muscle.

Learned changes in the rate of respiratory pumping in Aplysia fasciata in response to increases and decreases in seawater concentration.

In Aplysia fasciata, the sea hare, shock paired with moderate increases or decreases in the seawater concentration leads to pairing-specific increases in the respiratory pump rate in response to the same solutions an hour later. A common neural circuit underlies learned changes to increased and decreased seawater concentration, as shown by complete generalization of learning between these stimuli. Different neural circuitry controls learning after pairing a shock with pH 7 seawater, as shown by a lack of generalization of learning to this stimulus. Preexposure to strong changes in the seawater leads to sensitization of respiratory pumping. The hypothesis was tested that associative learning and sensitization arise from activation of common pathways. However, patterns of generalization of sensitization elicited by preexposure to altered seawaters differ from those produced by associative learning.

Modulation of respiratory pump rate by reproductive behaviors in freely behaving pairs of Aplysia fasciata.

Respiratory pumping in Aplysia is a spontaneously occurring behavior whose neural circuitry has been explored, but whose natural functions are incompletely understood. Respiratory pump rate was examined in freely behaving pairs of Aplysia fasciata, to determine whether it is modified by the occurrence of mating and other behaviors. The background rate of respiratory pumping was approximately 2/hour. This rate was maintained while animals were immobile, moving in place, crawling, or feeding. The rate was increased to over 8/hour during courtship and to approximately 4/hour during female mating and was reduced to approximately 1/hour during male-mating. These data suggest that respiratory pumping has a reproductive function, perhaps in dispersal of pheromones that are released during female-mating and courtship. Respiratory pumping never occurred while animals were swimming, suggesting that respiratory pumping and swimming may be mutually incompatible behaviors. Respiratory pumping was less common by night than by day.

Separate neural pathways respond to different noxious stimuli affecting respiratory pump frequency in Aplysia fasciata.

Neural circuits responsible for both conditioned and unconditioned respiratory pumping to three stimuli modulating respiratory pumping were examined. The stimuli used were: (i) reduction of pH; (ii) increase and (iii) decrease in seawater concentration. Ablation of the osphradium, but not of the rhinophores, abolished responses to all 3 stimuli. Cutting the pleural-abdominal connectives led to a decrease in responses to lowered pH, but did not affect responses to changes in seawater concentration. Further lesions showed that integrity of the cerebral-pleural ganglion is needed for animals to respond to a decrease in pH. Thus, neural circuitry entirely within the abdominal ganglion and the periphery innervated by the ganglion is sufficient for mediating responses to changes in seawater concentration, while the cerebral ganglion is needed to respond to lowered pH. Different transmitter mechanisms are also used by pathways responding to changes in seawater concentration and to decreased pH: 5,7-dihydroxytryptamine in concentrations which cause depletion of serotonin blocked the response to lowered pH, but not to altered seawater concentrations.

Learning that food is inedible in freely behaving Aplysia californica.

Freely behaving Aplysia californica can learn that food is inedible. Animals were given access to seaweed tied into canvas and attached to a force transducer. Animals repeatedly found the stimulus, attempted to ingest it, and failed. The force transducer provided an objective record of the number of attempts made by the animal to ingest the stimulus, the length of each attempt, and its intensity (i.e., peak force exerted). Within 2.5 hr, animals showed significant declines in these 3 measures of response to the stimulus. When exposed to the same stimulus the next day, animals showed more rapid declines in responsiveness, which indicate a retention of learning. Training appeared to be specific: Responses to the seaweed Laurencia of animals previously trained on the seaweed Ulva do not differ from the responses of naive animals to Laurencia.

Presence of conspecifics facilitates learning that food is inedible in Aplysia fasciata.

The absence of a conspecific, but not of food, interfered with learning and memory of a feeding task in Aplysia fasciata. Interference was shown by a shortened training session and by lack of savings on retraining. The shortened training is not responsible for the lack of savings because brief training in the presence of a conspecific led to savings on retraining. Animals trained in the absence of a conspecific and then tested in its presence did not show signs of having learned, which indicates that the absence of a conspecific interfered with the ability to learn, rather than with the expression of memory. Absence of a conspecific also inhibited other aspects of feeding behavior, such as the latency to respond to food and the length of time that animals respond to food, which indicate that interference with learning was apparently caused by inhibition of feeding behavior, rather than by block of the mechanisms underlying learning.

Common regulation of feeding and mating in Aplysia fasciata: pheromones released by mating and by egg cordons increase feeding behavior.

We examined whether pheromones released by reproductive behaviors (mating and egg-laying) affect feeding behavior. A preliminary experiment demonstrated that the quantity of food eaten can be used to measure the effects of pheromones on feeding. Using this measure, we then showed that Aplysia that were prevented from mating, but that were in the same aquarium as mating conspecifics, eat more food than do Aplysia in a medium lacking mating animals. Mating and feeding were not temporally correlated, indicating that pheromones released by mating probably do not initiate feeding, but rather modulate feeding after it has begun. Aplysia that were in the same aquarium as freshly deposited egg cordons also ate more than did animals in a medium lacking eggs.

Sequencing of behaviors in Aplysia fasciata: integration of feeding, reproduction, and locomotion.

To begin studying the neural basis of higher-order decision-making in Aplysia fasciata, we examined individual bouts of behavior in various conditions of access to food and/or mates. We then determined rules governing transitions between bouts. The data indicate that a single intermediate condition, moving in place, may be centrally related to transitions between behaviors. In all conditions, over 85% of all transitions between behaviors were via moving in place. Moving in place tended to precede and follow other categories of activity (crawling, swimming, immobile), and feeding. Also, moving in place apparently represents a fixed proportion of all bouts of behavior. In each condition, moving in place represented approximately 40% of all bouts, while the number of bouts of other behaviors varied markedly. After a bout of moving in place there was a strong tendency for the animal to return to the behavior performed before moving in place. Additional preferred sequences of behavior were also seen. Courtship tended to precede mating, and crawling preceded swimming.

Variables affecting long-term memory of learning that a food is inedible in Aplysia.

Long-term memory of learning that a food is inedible was studied in Aplysia. Seven days after a single training session, animals retained significant memory, as measured by a number of parameters. A 2nd experiment demonstrated savings 3 weeks after 2 training sessions. Long-term memory was also found after training procedures were altered to resemble those more likely to occur in nature, such as training for only 10 min or training with ad-lib access to inedible food, with no experimenter intervention. The effects were determined of bilaterally sectioning the esophageal nerves that innervate the gut. Denervation of the gut blocked the ability to learn that food is inedible but did not affect memory after the task had already been learned.