1. We tested for functional neural regeneration in the feeding system of Aplysia after bilateral transections or crushes of the cerebral-buccal connectives (CBCs) with the use of behavioral analyses and electrophysiological recordings. 2. Both types of lesion selectively abolished rhythmic consummatory behavior, dramatically increasing bite latency and interbite interval, and decreasing bite magnitude. Appetitive feeding behavior was not affected. 3. About 2 wk after CBC crush, bite latency, bite magnitude, and interbite interval began to recover, as rhythmic biting reappeared; complete recovery of rhythmic biting occurred within 60 days. Rhythmic biting never recovered after transection of the CBCs. 4. The recovery of rhythmic biting was correlated with changes in buccal motor output, which were assessed with the use of in vivo recordings from buccal nerve 4 in freely moving Aplysia. Initially, some bursting in nerve 4 occurred without overt bites; with full recovery of biting, a 1:1 correspondence between bursts in nerve 4 and overt bites returned. 5. CBC lesions caused a functional separation between biting and swallowing; at early times postlesion, subjects displayed apparently normal rhythmic swallowing even though rhythmic biting had been eliminated. However, there was a disruption of the 1:1 correspondence between nerve 4 bursts and swallows, which persisted until consummatory feeding fully recovered. 6. Transection of the CBCs in animals that had fully recovered from a previous CBC crush again abolished rhythmic biting, suggesting that the recovery of consummatory feeding behavior was due to functional neural regeneration of cerebral-buccal connections.

The intrinsic muscles and peripheral nerves in the buccal system of the sea hare Aplysia californica were studied to build a foundation on which to base future investigations of feeding in intact animals. A detailed description of the bilaterally paired intrinsic muscles is given identifying previously unreported muscles. Each of the six buccal nerves (n1-n6) and the cerebrobuccal connective (CBC) have been characterized in several respects. Cell bodies in the buccal ganglion with projections into each of the buccal nerves have been identified via the cobalt backfilling technique. All nerves contain axons of cell bodies in the ipsilateral as well as the contralateral ganglia. For each nerve, there is a consistent pattern in the distribution of cell bodies in the paired ganglia with the number of cell bodies in the contralateral ganglion being less than or equal to the number in the ipsilateral ganglion. Although the total number of backfilled cell bodies varies among the nerves, their size ranges are similar with the majority being small. Nerves 1, 2, 4, 5, and 6 provide motor innervation to the intrinsic buccal muscles in varying degrees with nerve 4 supplying all the intrinsic muscles; nerve 2 supplies only one. The axon composition of each nerve was scrutinized and revealed large numbers of axon profiles, the majority of which were less than 2 microns in diameter. The present study provides a framework for analysis of feeding behavior in Aplysia californica.

Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia. J. Neurophysiol. 78: 2372-2387, 1997. Numerous studies of plasticity in the feeding behavior of Aplysia have shown that substantial plasticity is due to peripheral neuromodulation of the feeding musculature. Extensive previous work focusing on the accessory radula closer (ARC) muscle has led to the realization that a major function of the modulation in that muscle may be to ensure efficient coordination between its contractions and those of its antagonist muscles. For a more complete understanding, therefore, we must study these muscles also. Here we have studied the radula opener muscles I7-I10. Using single isolated muscle fibers under voltage clamp, we have characterized ion currents gated by voltage and by the physiological contraction-inducing neurotransmitter acetylcholine (ACh) and the effects of the physiological modulators serotonin, myomodulins A and B, and FMRFamide. Our results explain significant aspects of the electrophysiological behavior of the whole opener muscles, as well as why the opener and ARC muscles behave similarly in many ways yet differently in some key respects. Opener muscles express four types of K currents: inward rectifier, A-type [IK(A)], delayed rectifier [IK(V)], and Ca2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and a leakage current. ACh activates a positive-reversing cationic current [IACh(cat)] and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ from the ARC in that, in the openers, activation of IK(A) occurs approximately 9 mV more positive and there is much less IACh(Cl). In both muscles, IACh(cat) most likely serves to depolarize the muscle until ICa activates to supply Ca2+ for contraction, but further depolarization and spiking is opposed by coactivation of IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) and IACh(Cl) may well be key factors that prevent spikes in the ARC but often allow them in the opener muscles. As in the ARC, the modulators enhance ICa and so potentiate contractions. They also activate a modulator-specific K current, which causes hyperpolarization and depression of contractions. Finally, in the opener muscles but not in the ARC, the modulators activate a depolarizing cationic current that may help phase-advance the contractions. Each modulator exerts these effects to different degrees and thus has a distinct effect on voltage and contraction size and shape. The overall effect then will depend on the specific combinations of modulators released in different behaviors. By understanding the modulation in the opener muscles, as well as in the ARC, we are now in a position to understand how the behavior of the two muscles is coordinated under a variety of circumstances.

Morphological techniques were used to study regeneration of central neural pathways involved in feeding behavior following bilateral crushes of the cerebral-buccal connectives (CBCs). Electron microscopic analysis revealed that CBC crushes completely transect axons within the nerve core while leaving a remnant of the nerve sheath intact. Changes in the ultrastructure of the CBCs at the crush site were determined for 1, 7, 14, 21, and 50 days postlesion. At 1 day postlesion, the crush site was no longer compressed, and the nerve core had assumed a circular shape. In addition, several small axon profiles were evident, and large areas of tissue debris and prominent microglial cells were observed. Membranous debris and hemocytes were also present in sinuses that appeared in the sheath adjacent to the crush site. From 7 to 50 days postlesion, the core of the nerve at the crush site increased in size due to the addition of small diameter axons. Initially, the sheath surrounding the crush site exhibited hyperplasia and contained a few small bundles of processes, apparently due to newly sprouted axons that had strayed from the nerve core. By 50 days postlesion, the crush site appeared nearly normal; the nerve core was reacquiring the normal radial pattern of axon profiles with some medium-sized axon profiles covered with glial sheath and exhibiting invaginations typical of the intact CBC. However, there was still a distinct lack of large diameter axons. Cobalt backfills across the crush site revealed neurons in the cerebral ganglion by postlesion day 9. Positions of stained cell bodies were consistent with those observed in controls, although the numbers of stained neurons did not recover to control levels even by postlesion day 63. The changes in the crush site and return of cell body staining with time postlesion are correlated with the recovery of consummatory feeding.

We used physiological recordings, intracellular dye injections and immunocytochemistry to further identify and characterize neurons in the buccal ganglia of Aplysia californica expressing Small Cardioactive Peptide-like immunoreactivity (SCP-LI). Neurons were identified based upon soma size and position, input from premotor cells B4 and B5, axonal projections, muscle innervation patterns, and neuromuscular synaptic properties. SCP-LI was observed in several large ventral neurons including B6, B7, B9, B10, and B11, groups of s1 and s2 cluster cells, at least one cell located at a branch point of buccal nerve n2, and the previously characterized neurons B1, B2 and B15. B6, B7, B9, B10 and B11 are motoneurons to intrinsic muscles of the buccal mass, each displaying a unique innervation pattern and neuromuscular plasticity. Combined, these motoneurons innervate all major intrinsic buccal muscles (I1/I3, I2, I4, I5, I6). Correspondingly, SCP-LI processes were observed on all of these muscles. Innervation of multiple nonhomologous buccal muscles by individual motoneurons having extremely plastic neuromuscular synapses, represents a unique form of neuromuscular organization which is prevalent in this system. Our results show numerous SCPergic buccal motoneurons with widespread ganglionic processes and buccal muscle innervation, and support extensive use of SCPs in the control of feeding musculature.