The Journal of Neuroscience, January 1988, 8(l): 197-211 Dishabituation and Sensitization Emerge as Separate Processes During Development in Ap/ysia Catherine H. Rankin and Thomas J. Carew Departments of Psychology and Biology, Yale University, New Haven, Connecticut 06520 Until recently, dishabituation and sensitization have com- inhibitory component of tail shock that produces reflex monly been considered to reflect a unitary process: Sensi- depression. Moreover, there was a clear progression in the tization refers to a general facilitation produced by strong net effects of tail shock during development: reflex depres- or noxious stimuli that enhances subsequent responding; sion was produced in stages 11 and early stage 12, followed dishabituation has been thought to represent a special in- by a transition to reflex facilitation (sensitization) in late stage stance of sensitization in which the facilitation is simply su- 12. Finally, when sensitization emerged in late stage 12, the perimposed on a habituated response level. The unitary pro- process of dishabituation showed a significant increase cess hypothesis was based on the observation that both compared with previous developmental stages. decremented and nondecremented responses are facilitat- Our results permit 2 principal conclusions. First, in contrast ed by a common noxious or strong stimulus. However, this to a unitary process view, dishabituation and sensitization observation does not rule out the possibility that dishabitua- emerge as separate behavioral processes according to very tion and sensitization could reflect separate processes that different developmental timetables in Aplysia. Second, the are activated in parallel by a strong stimulus. Recent cellular magnitude of dishabituation appears to be determined by experiments by Hochner et al. (1986) suggest that this, in the interaction of 3 underlying processes: (1) the dishabit- fact, occurs in the sensory neurons of the gill withdrawal uation process itself; (2) an inhibitory process that com- reflex in Aplysia. A developmental analysis of learning in petes with dishabituation (see also Rankin and Carew, 1987b); the marine mollusc Aplysia permits a direct behavioral test and (3) a facilitatory process (sensitization) that augments of this hypothesis. If dishabituation and sensitization reflect dishabituation (see also Hochner et al., 1986). a unitary process then they should emerge at the same time ontogenetically. On the other hand, if they reflect different processes, then they might emerge according to different The 3 most commonly observed forms of nonassociativeleam- ontogenetic timetables. ing are habituation, dishabituation, and sensitization. Habit- In the present study we examined the temporal emergence uation involves a progressivedecrement in the amplitude of a of dishabituation and sensitization in the defensive siphon behavioral responseproduced by the repeatedelicitation of that withdrawal reflex in 3 stages of juvenile Aplysia: stage 11, response.Dishabituation involves the facilitation of habituated early stage 12, and late stage 12. Animals received one of responsesby the presentation of a strong or noxious stimulus, 2 kinds of training: D&habituation training, in which the effect and sensitization involves that facilitation of nonhabituated re- of strong tail shock on habituated responses was observed, sponsesby a similar presentation of a strong stimulus (Pavlov, and Sensitization training, in which the effect of strong tail 1927; Grether, 1938; for reviews, seeThompson and Spencer, shock on nondecremented responses was observed. We 1966; Groves and Thompson, 1970; Carew and Kandel, 1974). found that, while dishabituation was present in all stages Until recently, it has commonly been assumedthat dishabitua- examined, sensitization did not emerge until several weeks tion and sensitization reflect a unitary process:Sensitization has later, in late stage 12. These results were confirmed and been viewed asa generalfacilitatory process,and dishabituation extended in a group of animals that were tested twice: first has been thought to representa special instanceof sensitization in stage 11, when they showed no sensitization, and again in which the facilitatory processis simply superimposedon a 13 weeks later, in late stage 12, when they then showed habituated responselevel. This general conclusion has come significant sensitization. from studiesexamining a variety of systems,including the EEG Our analysis of nondecremented responses prior to the arousal response(Sharpless and Jasper, 1956) human abdom- emergence of sensitization also revealed an unexpected inal reflexes (Hagbarth and Kugelberg, 1958), the flexion reflex in the cat (Spencer et al., 1966a, b), and the gill and siphon withdrawal reflex in Aplysia (Carew et al., 1971; for a general Received Mar. 3, 1987; revised June 15, 1987; accepted June 17, 1987. review, seeGroves and Thompson, 1970). We wish to thank Deda Gillespie for her excellent help with scoring the data, In all of these studies, the basic observation that supported and our colleagues Emilie Marcus, Thomas G. Nolen, Mark Stopfer, Allan Wagner, and William Wright for their helpful criticism of an earlier version of the manu- the unitary processview wasthat both decrementedand nondec- script. We are also very grateful to the Howard Hughes Medical Institute, and to remented responseswere simultaneously facilitated by the pre- Tom Capo, for generously supplying juvenile Aplysia. This work was supported sentation of a single strong or noxious stimulus. The most par- by NIH BSRG Grant 507-RR-075 015 and NSF Grant BNS 83 11300 (to T.J.C.). Correspondence should be addressed to Thomas J. Carew, Department of Psy- simonious explanation of this kind of result was that a noxious chology, P.O. Box 11A Yale Station, New Haven, CT 06520. stimulus initiated a general arousal-like processthat was wide- Copyright 0 1988 Society for Neuroscience 0270-6474/88/010197-15$02.00/O spread in the nervous system, facilitating habituated and non- 198 Rankin and Carew - Development of Sensitization in Aplysia habituated responses alike. Although both reasonable and log- obtained from the Howard Hughes Medical Institute (Woods Hole, ically consistent, this explanation does not rule out the possibility MA). Small animals (2.5-8 mm) were maintained at 15°C in groups of 4-8 on seaweed (Gracilluria) in 50 ml plastic centrifuge tubes filled with that dishabituation and sensitization could reflect separate fa- seawater obtained from Marine Biological Laboratories. Larger animals cilitatory processes that are activated in parallel by a strong (18-25 mm) were kept in a 30 gal aquarium of artificial seawater main- stimulus. In fact, direct cellular evidence for 2 separate facili- tained at 15°C (Instant Ocean). The animals used in this study were tatory processes has recently been provided in Aplysia sensory developmental stages 11 and 12, as described by Kreigstein (1977). neurons by Hochner and colleagues(1986) who suggestedthat However, additional morphological criteria were used to further sub- divide stage 12. Our combined criteria were as follows: Stage 11 animals dishabituation and sensitization in adult Aplysia are produced, were at least 47 d posthatching and 1.5-3.0 mm in length. The rhi- at least in part, by different cellular mechanisms. nophores were seen only as rudimentary buds above the eyes. Early One way to addressthe question of whether dishabituation stage 12 animals were at least 60 d posthatching. They were 5.0-8.0 and sensitization reflect a unitary process behaviorally is to mm in length, had well-developed anterior tentacles and rhinophores, and had white spots distributed over their bodies, with large patches of examine how they emergeduring development. If a single pro- white on the parapodia. The genital groove was visible on the right side cessis involved, both forms of learning should be expressed of the animal stretching from the anterior insertion of the parapodia to simultaneouslywhen that processemerges developmentally. Al- the propodium. Late stage 12 animals were 18-25 mm in length, had ternatively, if more than one processis involved, it might be developed mottled colored patterns like adult animals, and weighed possibleto separatethem ontogenetically. The defensive with- 400-600 mg. Late stage 12 animals looked very much like miniature adults. drawal reflex of the gill and siphon in the marine molluscAplysia Behavioralprocedures. Details of the behavioral procedures have been calijbrnica provides an excellent preparation in which to ex- previously described by Rankin and Carew (1987a). Briefly, a Zeiss amine this question directly. First, adult Aplysia exhibit habit- stereomicroscope (model SV8) fitted with a color video camera (JVC uation, dishabituation, and sensitization of this reflex, and these S-62U) was used to videotape behavioral responses. A timing signal from a video timer (Panasonic Time-Date Generator WJ-8 10) was su- processeshave been extensively analyzed on both behavioral perimposed onto the video image. Image and signal were recorded by and cellular levels (for review, seeHochner et al., 1986; Carew, a videotape recorder (Panasonic NV-8950) which permitted slow-mo- 1987; Hawkins et al., 1987). Second,it has recently been shown tion and stop-frame playback analysis when displayed on a color video to be possibleto analyze the development of different forms of monitor (Sony PMV- 12704). Aplysia Stage II animals were restrained with 3 suction micropipettes