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Marine Biological Laboratory, Woods Hole, Massachusetts 02543
1 Ohio University, Athens, OH
The nudibranch mollusc Hermissenda crassicornis can be trained through classical Pavlovian conditioning to contract its foot (the conditioned response) when presented with light alone. If, between 5 and 55 min after conditioning, a sensory block (SB) is induced by inverting and re-righting animals, the expression of the conditioned response, when tested 90 min after training, will be inhibited. A similar inhibitory effect also will occur when the SB is given between 70 and 85 min after training (1). However, animals that receive the SB at 60 min post-training will demonstrate recall of the conditioned response when tested at 90 min. Thus, 60 min after training must be a key time in the formation of long-term memory in Hermissenda. To investigate the nature of this inhibitory effect further, animals that had a SB administered at 50, 60, or 65 min after conditioning were chemically fixed, and their central nervous systems were examined immunocytochemically for the protein calexcitin, a known mediator of learning.
Calexcitin is a GTP/Ca2+ binding protein that is present in the visual cells of Hermissenda (2) as well as in other specific but restricted areas of the brain (3). After calexcitin is phosphorylated by protein kinase C, it binds to the ryanodine receptors of the ER, triggering the release of internal Ca2+ stores. Concomitantly, calexcitin blocks the voltage-dependent outward K+ currents (IA; ICa-K). The release of Ca2+ from the ER into the cytosol and the blockade of K+ currents are believed to cause the enhanced long-lasting depolarization (LLD) that has been observed in photoreceptors of Hermissenda and shown to be necessary for learning and memory to occur in this animal (4). The intensity of calexcitin immunostaining has not only been shown to be higher in trained Hermissenda, but also appears to be positively correlated with the strength of the memory (3,5).
Specimens of Hermissenda were purchased from Sea Life Supply (Sand City, California) and adapted to laboratory conditions for 3 days. Previous research had shown that nine training events of classical Pavlovian conditioning are sufficient to initiate long-term memory (LTM) in Hermissenda when tested 90 min later (1). After 10 min of dark adaptation, the animals were conditioned with nine training events (TEs), in which they were exposed to 6 s of bright light (700 lux; conditioned stimulus, CS), paired, after a 2-s delay, with 4 s of horizontal orbital rotation (unconditioned stimulus, US). The inter-trial interval was 1 min. The animals reacted to the US by contracting their foot (unconditioned response; UR). Animals were tested with the light (CS) alone, and a positive learning response was indicated by a contraction of the animal’s foot (i.e., shortening of body length) with the CS alone; no change in length or elongation as in normal locomotory movement (6) meant that no learning had occurred.
For this study, four to six animals per group were given nine TEs as described above. A sensory block was applied to three groups of animals at 50, 60, and 65 min following training; the procedure was to turn the tray containing the animals upside down for 5 s and then to re-right it. As mentioned above, this additional vestibular stimulus had been demonstrated to interfere with the formation or completion of LTM in Hermissenda (1). After testing at 90 min, all animals were immediately removed from the training tray and submerged in 4% paraformaldehyde in Tris-buffered artificial seawater (Tris-ASW; pH 8.0). The animals’ brains were subsequently removed and fixed overnight in 4% paraformaldehyde/0.2% glutaraldehyde in Tris-ASW. The brains were then washed, dehydrated in a series of increasing alcohol concentrations to 95% ethanol, embedded in polyethylene glycol-400-distearate, and sectioned (5 µm). Following de-waxing and rehydration, sections containing photoreceptors were immunolabeled for calexcitin using a polyclonal antibody (25U2; cloned from squid optic lobe and raised in rabbit; 1:1000 dil.) (3,5). A biotinylated horse anti-rabbit secondary antibody and avidin-bound microperoxidase (ABC method, Vector) were applied, followed by staining with the chromogen, 3-amino-9-ethylcarbazole (AEC). The negative control used was normal horse serum (NHS at 2%). NIH Image software was used to measure grayscale intensities from digital images of the original sections. Intensity levels were taken from areas of interest drawn around the cytoplasm of the B-photoreceptors of the eyes. That same perimeter was then transferred to an unstained area of the central neuropile to obtain background levels. The background readings were then subtracted from the photoreceptor values and the difference data analyzed statistically (ANOVA, Student’s t test).
The behavioral data (Fig. 1A) show that animals experiencing SBs at 50 and 65 min did not contract when tested with the CS alone at 90 min; they demonstrated no memory recall. However, when the SB was applied at 60 min and the animals tested at 90 min, these animals exhibited a positive learning response (memory recall); the SB was ineffective. From the previous study (1) and from experiments currently being conducted using transcription and translation inhibitors, this 60-min time point appears to define the establishment of LTM in Hermissenda. Thus, SBs applied before 60 min interfere with LTM formation, while SBs after 65 min interfere with the more permanent cellular changes of consolidated long-term memory (CLTM; work in progress).
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The authors acknowledge Drs. Daniel Alkon and Tom Nelson, Blanchette Rockefeller Institute, Johns Hopkins University, Rockville, Maryland, for their support and for providing Hermissenda and calexcitin antibodies for this study. This work was supported in part by a NSF-REU Site Grant (9912287) to the Marine Biological Laboratory, Woods Hole, Massachusetts.
Literature Cited
This article has been cited by other articles:
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  A. M. Kuzirian, H. T. Epstein, C. J. Gagliardi, T. J. Nelson, M. Sakakibara, C. Taylor, A. B. Scioletti, and D. L. Alkon Bryostatin Enhancement of Memory in Hermissenda Biol. Bull., June 1, 2006; 210(3): 201 - 214. [Abstract] [Full Text] [PDF]
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A. M. Kuzirian, F. M. Child, H. T. Epstein, M. E. Motta, C. E. Oldenburg, and D. L. Alkon Training Alone, Not the Tripeptide RGD, Modulates Calexcitin in Hermissenda Biol. Bull., October 1, 2003; 205(2): 220 - 222. [Full Text] [PDF]
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