Brain Research, 585 (1992) 272-274

272

Elsevier Science Publishers B.V.

BRES 25222

Recovery of consummatory feeding behavior after bilateral lesions of the cerebral-buccal connectives in Aplysia californica M a r s h a L. Scott a n d M a r k D. Kirk Dirision of Biological Sciem'es, UnirersiO'of Missouri-Coh~mbia, Lefet're Hall, Columbia, MO 65211 (USA) (Accepted 3 March 1992)

Key words: Behavioral recovery; Regeneration; Feeding; Aplysia; Central nervous system; Mollusc

In the sea hare, Aplysia californica, con,~ummatory feeding behavior is selectively abolished by bilateral crushes of the cerebral-buccal connectives and recovers by postlesion day 13. Recovered biting responses are initially weak and increase in magnitude gradually with time. The lesions do not affect appetitive feeding behavior or unrelated reflexive behaviors. Thus, feeding in Aplysia can be used to examine the neural basis of behavioral recovery after CNS injury.

Understanding recovery of behavioral functions that are lost following damage to the central nervous system is a central issue in neurobiological research, in contrast to higher vertebrates, following a central lesion, lower vertebrates and invertebrates may regain at least partial function with time, although the mechanisms of behavioral recovery are poorly understood t,:,5,7, in Aplysia, consummatory feeding behavior is eliminated with bilateral cuts of the cerebral-buceal connectives (CBCs), and with these lesions, no return of function occurs within 3 weeks t°, However, in the same species escape locomotion is selectively abolished and subsequently recovers with bilateral crushes to the cerebral-pleural connectives, leaving normal Iocomo. tion unaffected 7. Thus, the type of lesion may dictate the potential for functional recovery in molluscs. Evidence from in rive and in vitro studies using a variety of molluscs also suggests that, once injured, neurons sprout processes across the lesion and that some physiological connections are reestablished following a recovery period ~.4,~-s,tt-~3. Transections of the CBCs do not affect the initial appe'~tive phase of feeding behavior, during which the animals locate and contact the food source (seaweed),~-t..This phase begins when the animal encoun-

ters a food-related chemical and/or mechanical stimulus in the environment, which initiates a series of head-waving movements alternated with locomotion until the seaweed is located. The subject then assumes a characteristic head-up feeding posture, and makes head movements to center the food over the lips. This is followed by the consummatory phase of feeding behavior, which consists of a series of bites and swallows that draw the seaweed into the buccal cavity and then into the esophagus'~°1o. Several lines of evidence indicate that the central pattern generator underlying cyclic consummatory feeding movements resides primarily in the buccal ganglia, and command neurons for this behavior are located in the cerebral ganglion 1°'14-''~'21 and communicate with the buccal ganglia via axons that pass through the CBCs. in this report we show that consummatory feeding behavior is initially abolished, but then recovers, following bilateral CBC crushes. Aplysia californica used in our behavioral studies were tested preoperatively for a variety of normal feeding parameters which were introduced previously 17 and are only briefly described here. Appetitive behavior was quantified using LiD Latency (LL)--the time between presentation of a food stimulus (dried sea'

Coreespondcnce: M.D. Kirk, University of Missouri-Columbia, Division of Biological Sciences, 103 Lefevre Hall, Columbia, M e 65211, USA. Fax: (i) (314) 882-0123,

273 weed laver, 1 cm 2) to the rhinophores (chemo- and mechanoreceptive structures) and the appearance of the lips. Consummatory feeding behavior was quantified using Bite Magnitude (BM)--an assessment of the magnitude of the bite according to a defined scale (0-4), or Interbite Interval (IBl)--the time between peak radula protraction of successive bites. The righting reflex, an unrelated behavior used as a measure of non-specific behavioral effects, was quantified as Righting Reflex Latency (RRL)--the time between the release of an animal held upside down over the substrate and its assumption of an upright position. Testing was performed in a separate testing tank (12" x 6" × 8") containing artificial sea water (ASW) from the animals' home tank, which was maintained at 16 :t: I°C. Animals were acclimated to the testing tank for 5 rain before the onset of testing. Data were recorded on audio-tape with a cassette recorder, and intervals were measured from tapes with a stopwatch. After preoperative testing, subjects were anesthetized with isotonic MgC! 2 injections (50% of body weight), then a small dorsal incision was made between the rhinophores over the cerebral ganglion. Both CBCs were located and crushed with No. 5 forceps, and the wound was closed by suturing the skin in layers ")'17. Animals were allowed to recover 24 h, and testing was performed every other day until the animal was sacrificed for morphological studies. One animal was excluded from the study because it exhibited consummatory feeding behavior on postlesion day 1, indicating an incomplete crush. Consummatory feeding was initially abolished by the lesions, and while the rate of recovery was somewhat variable between animals, the first day that all animals showed biting responses was postlesion day 13 (crite-

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during recovery from bilateral crushes to the CBCs (n = 4) using a defined scale. Numbers above data points indicate the number of animals responding on that day. BM = 0 indicates that a minimum bite criterion (at least two unequivocal bites during 2 min of continuous lip stimulation) was not met. BM = 1 indicates minimal mouth opening, radula not visible; BM = 2 indicates radula visible but not protracted beyond the plane of the jaws; BM = 3 indicates radula protraction beyond the plane of the jaws; BM = 4 indicates maximum radula protraction showing muscle insertion sites along the sides of the radula,

rion ffi at least two unequivocal bites within a 2-min time period). Due to the sacrifice of select animals at intervals for morphological studies, the entire initial group of 15 subjects was not maintained throughout the experiment. Therefore, behavioral analysis was performed on a subgroup of 4 animals that were tested through at least postlesion day 19. In this latter group, the first consummatory responses (using the interbite interval measurement) were observed on day 9, with all animals responding by day 13 (Fig. 1). Mean preoperative bite magnitude (BM) was initially between 3 (the radula protracts beyond the plane of the jaws) and 4 (maxirr~al radula protraction; muscle insertion sites on radula are visible). No biting (i.e. BM = 0) was observed until day 5, when one animal made minimum criterion with two bites (BM - 1; minimal jaw opening, radula not visible; Fig. 2). By day 13 mean BM was approximately 2 (jaws open, radula is visible but not protracted beyond the plane of the jaws). Bite magnitudes on day 19 were not significantly different from preoperative BM measurements (paired t-test, P > 0.05). BM showed a steady increase toward preoperative levels with time, revealing that recovery is not an all-or-none event, but requires further maturation as regeneration progresses. The behavioral deficit in BM was a specific result of the CBC lesion, as all sham-operated animals ( n - 3) showed normal feeding re-

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POSTLESION DAY Fig. 3. Mean latenci¢~ for lip exposure following seaweed stimulus (Lip Latency. an :~ :~¢titive feeding behavior) and righting reflex (RRL, a behavior umelated to feeding) measured preoperatively and during recovery from bilateral crushes to the CBCs (n = 4). Asterisks above data points indicate that no data was obtained for RRL from one animal on that day due to technical difficulties.

sponses by day 3. In addition, as shown in Fig. 3, appetitive feeding behavior and the righting reflex are not affected by bilateral CBC crushes; both of these latter behaviors occur on day 1 with latencies near preoperative levels or even shorter. Ultrathin sections taken through the crush site and examined with the electron microscope reveal camplete disruption of the axon core of the nerve and disorganization of the nerve sheath immediately after a crush lesion (Scott, Govind and Kirk, unpublished observations); thus, nerve crush is effective in severing axons in the CBCs. By postlesion day 17, the crushed nerve shows signs of regeneration with many intact axons bridgi~tg the crush site (unpublished observations). We are currently investigating these changes in more detail. Cobalt backfills of the CBCs:" suggest that elongation of sprouts from axotomized neurons into the target ganglia (cerebral and buccal) parallels the tinte course of behavioral recovery (unpublished observations). The return of soma staining parallels the time course of behavioral recovery, supporting the hypothesis that regenerated connections contribute to behavioral recovery. A contribution to behavioral recovery by regeneration of cerebral neurons seems likely, as bilateral CBC cuts do not permit return of consummatory feeding (at least over a 3-week postlesion observation periodt°). Preliminary electrophysioiogical experiments indicate that some cerebral to buccal synaptic connections are reestablished by postlesion day 31 (unpublished observations), including buccal connections of command-like

1 Arshavskii, Y.I., Gel'land, I.M., Orlovskii, G.N., Pavlova, G.A., Panchin, Y.V. and Popova, L.B., Regeneration of pedal ganglion neurons in the sea butterfly Clione li,nacina, Neirofiziologiya, 17 (1985) 449-455. 2 Baylor, D.A. and Nichoils, J.G., Patterns of regeneration between individual nerve cells in the central nervous system of the leech, Nature, 232 (1971) 268-270. 3 Bulloch, A.G.M. and Kater, S.B., Neurite outgrowth and selection of new electrical connections by adult Helisoma neurons, 1. Neurophysioi., 48 (1982) 569-583. 4 Bulloch. A.G.M. and Kater, S.B., Selection of a novel connection by adult molluscan neurons, Science, 212 (1981) 79-81. 5 Cohen, A., Mackler, S.A., and Seizer, M.E. Behavioral recovery following spinal transection: functional regeneration in the lamprey CNS, Trends NeuroscL, I 1 (1988) 227-231. 6 Croll, R.P. and Baker, M.W., Axonal regeneration and sprouting following injury to the cerebral--buccal connective in the snail Achatina fulica. J. Comp. Nearol., 300 (1990) 273-286. 7 Fredman, S.M., Recovery of escape locomotion following a CNS lesion in Aplysia, Behat'. Neural Biol., 49 (1988) 261-279. 8 Fredman, S.M. and Nutz, P.G., Regeneration of identified neurons and their synaptic connections in the central nervous system of Aplysia. Am. ZooL, 28 (1988) 1099-1108. 9 Kupfermann, l,, Feeding behavior in Aplysia: a simple system for the study of motivation, Behav. Biol., l0 (1974) 1-26. 10 Kupfermann, l., Dissociation of the appetitive and consummatory phases vf feeding behavior in Aplysia: a lesion study, Behav. Biol., l{l (1974) 89-9% i 1 Murphy, A.D., Barker, D.L., Loring, J.F. and Kater, S,B., Sprouting and functional regeneration of an identified serotonergie neuron fi~llowingaxotomy, J. Nc, robiol., 16 (1985) 137-151. 12 Murphy, A.D. and Kater, S.B,, Differential discrimination of appropriate pathways by regenerating identified neurons in Hdi. sonta, Z Comp, NetoY~l,, 19(l (1980)395-403, 13 Murphy, A.D. and Kater, S.B,, Specific rein,ervation of a target organ by a pair of identified molluscan neurons, Brain Re,~., 156 (I 97,';) 322-328. 14 Ros~,, S.C., Miller, M.W., Weiss, K.R. and Kupfermann, I., Control of huccal motor programs m Aplys'ia by identified neurons in the cerebral ganelion, So(', Neurt~ci. Abstr,, 13 (1987) 1061. 15 Rosen, S,C,, Miller, M,W,, Weiss, K.R. and Kupfermann, i., Activity of CBI.2 of Ai,lysia elicits biting-like responses, Soc. Neurosci, Ab,vtr., 14 (1988) 608, 16 Rosen, S.C., Teyke, T,, Miller, M,W,, Weiss, K,R, and Kupfermann, i,, Identification and characterization of cerebral-to-buccal interncurons implicated in the control of motor programs associated with feeding in Aplysia, J, Nt,~rosci,, II (1991) 3630-3655, I? Rosen, S,C,, Weiss, K,R,, Goldstein, R,S, and Kupfermann, l,, The role of a modulatory n~uron in feeding and satiation in Aplysia: effects of lesioning of the serotonergic metacerebral cells, J. Ne, rosci,, 9 (1989) 1562-1578, 18 Rosen, S,C,, Weiss, K,R,, Cohen, LL, and Kupfermann, I,, Interganglionic cerebal buccal mechanoafferents of Aplysia: receptive fields and synaptic connections to different classes of neurons involved in feeding behavior, J. Neurophysiol., 48 (1982) 271-288. 19 Rosen, S.C,, Weiss. K,R, and Kupfermann, I., Command of feeding in Aplvsia: implications of a network of identified interganglionic i'aterneurons in t|le buccal and cerebral ganglia, Neu. rosci, Abstr,, 17 (1991~ 1490, 20 Scott, M,L,, Govind, C.K, and Kirk, M,D,, Neuromuscular organization of the buccal system in Aplysia cali~mzica, J. Comp. Ne, rol,, 312 (1991) 207-222. 21 Susswein, AJ. and Byrne, J.H., Identification and characterization of neurons initiating patterned neural activity in the buccal ganglia of Aplysia, J, Neurosci., 8 (1988)2049-2061.

Recovery of consummatory feeding behavior after bilateral lesions of the cerebral-buccal connectives in Aplysia californica.

In the sea hare, Aplysia californica, consummatory feeding behavior is selectively abolished by bilateral crushes of the cerebral-buccal connectives a...
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