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.

Feeding behavior in Aplysia fasciata and A. oculifera is modified by pairing the behavior with reinforcing consequences. Successful and unsuccessful attempts to transfer food from the buccal cavity to the crop act as positive and negative reinforcers, respectively. A number of changes in feeding behavior occur as a result of pairing of feeding with the negative reinforcer: feeding responses become less effective in leading to the entry of food into the buccal cavity; when food does enter the buccal cavity, it exits sooner; swallowing responses after food entry are less likely to occur; Aplysia eventually cease responding to food. Pairing successful transfer of food into the crop with feeding behavior produces opposite effects. Behavioral change is specific to pairing, as shown by lack of change when reinforcement is explicitly unpaired with feeding behavior. Behavioral change is specific to foods with a particular taste and texture; generalization to alternate foods was not observed. In spite of cessation of feeding, animals remain aroused, as shown by low response latency to alternate foods. Memory of response change persists for at least 48 hr.

Two patterns of neural activity were identified in excised buccal ganglia of Aplysia californica. Both are expressed in many cells, and each can be expressed independently. Using cells B4 and B5 as monitors of the activity patterns, we searched the buccal ganglia for cells initiating the patterns. Two electrically coupled cells, B31 and B32, can initiate what we termed pattern 2. The cells are active before pattern 2 is expressed. Stimuli initiating pattern 2 excite B31/B32. Depolarizing B31/B32 induces the pattern, while hyperpolarizing them can prevent its expression. The cells have unusual features. Their somata do not sustain conventional action potentials, and depolarization causes a regenerative response. B33 differs from B31/B32 in that its soma sustains conventional action potentials but otherwise has similar features. B34 also seems to be inexcitable but has weaker synaptic input than B31/B32 and appears unable to induce pattern 2. B35 and B36 have prominent regenerative capabilities. B35 is also able to initiate pattern 2. B37 is presynaptic to B31/B32 and can initiate pattern 2 via its effects on them. The newly identified cells provide a starting point for investigating factors that initiate and control different patterns of neural activity in the buccal ganglia. Since the buccal ganglia are involved in generating feeding behavior, further studies on the newly identified cells may provide insights into the neural control of feeding behavior, and provide a neural substrate for studying modulation of the feeding patterns by associative learning.

PolyADP-ribose-polymerase 1 is activated in neurons that mediate several forms of long-term memory in Aplysia. Because polyADP-ribosylation of nuclear proteins is a response to DNA damage in virtually all eukaryotic cells, it is surprising that activation of the polymerase occurs during learning and is required for long-term memory. We suggest that fast and transient decondensation of chromatin structure by polyADP-ribosylation enables the transcription needed to form long-term memory without strand breaks in DNA.

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.

Nitric oxide (NO) signaling was inhibited via N(omega)-nitro-L-arginine methyl ester (L-NAME) during and after training Aplysia that a food is inedible. Treating animals with L-NAME 10 min before the start of training blocked the formation of three separable memory processes: (1) short-term, (2) intermediate-term, and (3) long-term memory. The treatment also attenuated, but did not block, a fourth memory process, very short-term memory. L-NAME had little or no effect on feeding behavior per se or on most aspects of the animals' behavior while they were being trained, indicating that the substance did not cause a pervasive modulation or poisoning of many aspects of feeding and other behaviors. Application of L-NAME within 1 min after the training had no effect on short- or long-term memory, indicating that NO signaling was not needed during memory consolidation. Treating animals with the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazdine-1-oxy-3-oxide before training also blocked long-term memory. Memory was not blocked by D-NAME, or by the simultaneous treatment with L-NAME and the NO donor S-nitroso-N-acetyl-penicillamine, confirming that the effect of L-NAME is attributable to its effect as a competitive inhibitor of L-arginine for NO synthase in the production of NO rather than to possible effects at other sites. These data indicate that NO signaling during training plays a critical role in the formation of multiple memory processes.

Understanding modulation of memory, as well as the mechanisms underlying memory formation, has become a key issue in neuroscience research. Previously, we found that the formation of long-term, but not short-term, memory for a nonassociative form of learning, sensitization, was modulated by the circadian clock in the diurnal Aplysia californica. To define the scope of circadian modulation of memory, we examined an associative operant learning paradigm, learning that food is inedible (LFI). Significantly greater long-term memory of LFI occurred when A. californica were trained and tested during the subjective day, compared with animals trained and tested in the subjective night. In contrast, animals displayed similar levels of short-term memory for LFI when trained in either the subjective day or night. Circadian modulation of long-term memory for LFI was dependent on the time of training, rather than the time of testing. To broaden our investigation of circadian modulation of memory, we extended our studies to a nocturnal species, Aplysia fasciata. Contrary to the significant memory observed during the day with the diurnal A. californica, A. fasciata showed no long-term memory for LFI when trained during the day. However, A. fasciata demonstrated significant long-term memory when trained and tested during the night. Thus, the circadian clock modulates memory formation in phase with the animals' activity period. The results from our studies of circadian modulation of long-term sensitization and LFI suggest that circadian modulation of memory formation may be a general phenomenon with potentially widespread implications for many types of long-term learning.biological rhythms ͉ circadian clock ͉ long-term memory T he ability to remember an experience allows an organism to use information gained in planning its response to future events. A variety of factors can modulate the formation and recall of memory. In fact, very few of the events occurring in the environment are remembered. Health, age, motivation, previous experience, stress, and many other factors may modulate learning and memory. Modulation of memory formation can affect how input information is processed, how memories are stored, the length of time that memories last, or even the mechanisms by which memories are recalled. Understanding the modulation of memory will provide insight into the cellular and molecular processes underlying memory formation per se. The circadian clock allows animals to predict when important events occur in the environment and to anticipate changes in the environment based on time of day. Given the enormous and widespread impact that the circadian clock has on an animal's physiology and behavior, we investigated modulation of memory formation by the circadian clock.The time of day can impact learning by becoming part of the context in which the learning occurs (time-stamping), or the time of day can affect the amount of memory that is formed or recalled. Time-stamping has been demonstrated in invertebrates (1) and in mammals (2-...

To gain insight into the function of NO transmission during learning we tested whether blocking NO synthesis affects aspects of feeding that are expressed both in a nonlearning context and during learning. Inhibiting NO synthesis with L-NAME and blocking guanylyl cyclase with methylene blue decreased the efficacy of ad libitum feeding. D-NAME had no effect. L-NAME also decreased rejection responses frequency, but did not affect rejection amplitude. The effect of L-NAME was explained by a decreased signaling that efforts to swallow are not successful, leading to a decreased rejection rate, and a decreased ability to reposition and subsequently consume food in ad libitum feeding. Signaling that animals have made an effort to swallow is a critical component of learning that food is inedible. Stimulation of the lips with food alone did not produce memory, but stimulation combined with the NO donor SNAP did produce memory. Exogenous NO at a concentration causing memory also excited a key neuron responding to NO, the MCC. Block of the cGMP secondmessenger cascade during training by methylene blue also blocked memory formation after learning. Our data indicate that memory arises from the contingency of three events during learning that food is inedible. One of the events is efforts to swallow, which are signaled by NO by cGMP.