long-term potentiation - NCBI

Proc. Nati. Acad. Sci. USA Vol. 93, pp. 1808-1813, March 1996 Genetics

Steel mutant mice are deficient in hippocampal learning but not long-term potentiation BENNY MOTRO*t#, J. MARTIN WOJTOWICZt§, ALAN BERNSTEIN*T, AND DEREK VAN DER KooYII *Program in Molecular Biology and Cancer, Sarnuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON Canada

M5G 1X5; and Departments of

§Physiology, 1Molecular and Medical Genetics, and IlAnatomy, University of Toronto, Toronto, ON Canada M5S 1A8

Communicated by Elizabeth Russell, The Jackson Laboratory, Bar Harbor, ME, October 30, 1995 (received for review September 19, 1994)

mutants, most noticeably in certain structures of the nervous system (17, 20). For example, high levels of Steel transcripts are found in the enthorhinal cortex and dentate gyrus, while c-kit is found in the nearby CA3 and CAl pyramidal neurons and subiculum in the hippocampus (20). The contiguous high level expression of c-kit and Steel in the adult murine hippocampus suggests a possible role for the Steel factor/Kit signaling pathway in hippocampal-dependent learning and memory. To test this possibility, we used the Morris water maze task to examine the spatial learning ability of Steel mutant mice. The Morris water maze is the most commonly used test to examine deficits in configural learning tasks following damage or dysfunction in the hippocampus (21), including its mossy fiber system (22, 23). We show here that, compared to +/+ control mice, Steel mutant mice have poorer performance in a configural learning version of the Morris water maze but not in a similar version of the task that requires only simple associations. We demonstrate also that normal synaptic transmission between dentate gyrus and hippocampal CA3 pyramidal cells is deficient in the mutant mice. No deficit was found in hippocampal LTP. These results demonstrate a role for the Steel factor/Kit receptor tyrosine kinase signaling pathway in the brain. They also argue that inefficient hippocampal synaptic transmission, without a deficit in LTP, can cause a specific impairment in configural learning and memory.

ABSTRACT Mice carrying mutations in either the dominant white-spotting (W) or Steel (Sl) loci exhibit deficits in melanogenesis, gametogenesis, and hematopoiesis. W encodes the Kit receptor tyrosine kinase, while SI encodes the Kit ligand, Steel factor, and the receptor-ligand pair are contiguously expressed at anatomical sites expected from the phenotypes of Wand Sl mice. The c-kit and Steel genes are also both highly expressed in the adult murine hippocampus: Steel is expressed in dentate gyrus neurons whose mossy fiber axons synapse with the c-kit expressing CA3 pyramidal neurons. We report here that SI/Sid mutant mice have a specific deficit in spatial learning. These mutant mice are also deficient in baseline synaptic transmission between the dentate gyrus and CA3 but show normal long-term potentiation in this pathway. These observations demonstrate a role for Steel factor/Kit signaling in the adult nervous system and suggest that a severe deficit in hippocampal-dependent learning need not be associated with reduced hippocampal long-term potentiation.

Lesion analyses in rodents, monkeys, and humnans point to the critical role of hippocampal formation in a specific kind of learning and memory variously called declarative, explicit, or configural (1-3). Configural learning, defined as the processes that establish and store a representation of the relations (e.g., in space) between more than two stimuli, can be differentiated from a hippocampal-independent, simpler type of learning about the relations between two stimuli variously called procedural, implicit, or simple association learning (4). Modulation of synaptic strength has long been postulated as the central mechanism of learning and memory. In certain parts of the central nervous system, a long-term potentiation (LTP) of synaptic efficiency is observed. LTP can be induced by a brief high-frequency stimulation of afferent fibers, in both the intact animal and in tissue slices, and in most sites exhibits the specificity, cooperativity, and associativity predicted by Hebb (5-7). Although these electrophysiological characteristics of LTP make it a good candidate for the physiological mechanism of configural learning, the evidence remains equivocal (8, 9). The molecular mechanisms that underlie and modulate synaptic plasticity (as well as configural learning) are poorly understood. We examined the possible involvement of the Kit receptor tyrosine kinase in hippocampal-dependent learning. Mutations in the murine genes for either the Kit receptor [the W locus (10, 11)] or its ligand Steel factor [the Steel (Sl) locus (12-14)] have deleterious effects on development of the melanocyte, germ cell, and hematopoietic lineages (15, 16). As predicted from the W/Sl mutant mice phenotypes, c-kit is expressed in the three cell lineages known to be affected by W or Sl mutations, while Steel is expressed in cells located in their migratory pathways and adjacent to their final destinations (17-20). However, the c-kit and Steel genes are also contiguously expressed in sites not known to be affected in the

MATERIALS AND METHODS Mice. Because mice homozygous for null alleles of the SI locus die prenatally of severe anemia, we used viable compound heterozygous (Sl/Sld) mice, which have one null allele (Sl) (12, 14), while the other allele (Sld) produces only the soluble, but not the membrane-bound, form of Steel factor (24, 25). As controls, we used their wild-type (+/+) brothers. Behavior. We compared the performance of mutant Sl/Sld 4- to 5-month-old male mice (n = 12) and their +/+ control littermates (n = 10) in the hidden platform version of the Morris water maze. The apparatus was a white plastic circular pool (diameter, 122 cm; wall height, 76 cm). The water was made opaque with nontoxic Crayola paint. A circular, plastic platform (diameter, 5 cm; height, 60 cm) was placed in one quadrant of the pool just below the surface of the water. In this task, mice were released from one of four randomly chosen starting points in the circular pool to search for the hidden escape platform for 60 sec. The mice were allowed to rest for 20 sec on the platform after finding it (or after the experimenter placed the animal on the platform if the platform was not found within 60 sec). The mice received five trials a day for 9 days. Abbreviations: LTP, long-term potentiation; PTP, posttetanic potentiation; PKC, protein kinase C. tPresent address: Department of Life Sciences, Bar Ilan University, Ramat-Gan 52 900 Israel. tB.M. and J.M.W. contributed equally to this work. ITo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Genetics: Motro et al. A probe test was performed after 5 days of training on the hidden platform task. The platform was removed and the mice were allowed to search for 60 sec. Their paths were videotaped and the times they spent in each of the quadrants and the number of times they swam over the former location of the platform were recorded. After completion of the hidden platform task, we compared the ability of these mice to swim to a visible, above-water platform (a simple association version of the task). The mice were given five trials over 1 day in this task and their latencies to find the platform were recorded. Anatomy. At the completion of the behavioral experiments, the brains of some of the SlI/Sid and + / + mice were processed histologically. After an anesthetic overdose, the mice were perfused transcardially with 10% formalin, and their brains were quickly removed and placed in 10% formalin and 30% sucrose. Frontal sections (30 ,um) were cut through the hippocampus on a cryostat, mounted on gelled slides, and Nisslstained with thionin. Other mice were perfused transcardially with 1.17% buffered sodium sulfide (pH 7.35) for 3 min, followed by 3% glutaraldehyde in 0.15 M Soerensen's phosphate buffer (pH 7.35) for 5 min, and then for a further 7 min with sodium sulfide. Cryostat sections were thawed on slides and developed in Timm's solution, as described (22), to visualize the hippocampal mossy fibers. Electrophysiology. Animals were anaesthetized with halothane and decapitated, and the hippocampi were quickly removed. Transverse hippocampal slices (400 ,um thick) were cut with a tissue chopper and kept moist and oxygenated at room temperature for 1-5 hr until use. For electrical recordings, slices were transferred to a perfusion chamber and viewed with a dissecting microscope. Synaptic field potentials were evoked with the use of tungsten electrodes and recorded via glass pipettes filled with artificial cerebrospinal fluid [(ACSF) 124 mM NaCl/3 mM KCl/1.25 mM NaH2PO4/2 mM MgCl2/2 mM CaCl2/26 mM NaHCO3/10 mM dextrose]. The ACSF was equilibrated with 95% oxygen/5% C02, pH 7.4 at 32°C. The slicing procedure usually yielded 10-15 slices from each animal. However, due to time constraints, only 2 or 3 slices were used for recordings. To reduce the strong GABAergic inhibition in the dentate gyrus, 10 ,tM bicuculline was added to the perfusate as described (26). To study LTP in the dentate gyrus, both the stimulating and recording electrodes were placed in the middle molecular layer and the stimulation intensity was adjusted to produce a current sink only in the middle molecular layer (and a current source in the outer molecular layer). This procedure selectively activates the medial perforant pathway (27). To study LTP in the mossy fiber CA3 pathway, the stimulating electrode was placed in the dentate gyrus and the recording electrode in a clearly defined, translucent band visible in the CA3 area. Stimulation of the dentate gyrus produced a synaptic current sink in the mossy fiber layer, which contrasted with a biphasic antidromic/collateral response obtained by stimulation of the CAl area (see Fig. 2C). The mossy fiber responses were further distinguished from other synaptic responses in the CA3 region by their pronounced frequency facilitation. These criteria for mossy fiber responses were developed to overcome the difficulties, pointed out by others (28), in identifying intracellular mossy fiber responses. For this reason, we standardized the magnitude of effective stimulating strength by evoking the maximal presynaptic volley, ensuring optimal locations of the stimulating and recording electrodes for evoking the maximal mossy fiber response. To study LTP in the CAl area, the stimulating and recording electrodes were placed in the stratum radiatum to activate collateral/commissural afferents. Stimulus intensity was adjusted to produce a field synaptic response just subthreshold to evoking a population spike.

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Different series of slices were used for the LTP studies in each of the dentate gyrus areas CA3 and CAl. In all three areas of the hippocampus, LTP was induced by application of two trains of stimuli at 100 Hz, lasting 1 sec each and repeated at a 5-sec interval. LTP was usually monitored for 20-30 min after the tetanic stimulation. The magnitude of LTP was expressed as a relative change (%) with respect to the average of responses during the 10-min period preceding the tetanic stimulation. The initial slope of field synaptic responses was taken as a measure of synaptic efficacy. RESULTS Si/SI Mice Have a Specific Deficit in Configural Larning. To test for configural learning, SlI/Sid and +/+ mice were run in the hidden platform version of the Morris water maze. On the first trials, the mice find the platform by chance and then learn and remember the platform location based on distal cues. On the first day of training, both Si/Sld and + / + mice required equivalent times to find the hidden platform (Fig. 1A). On the subsequent 8 days, both +/+ and Sl/Sld mice showed a reduction in the times required to find the hidden platform; however, there were significant differences in both the rate at which Sl/Sld mice improved at this task (F(8,60) = 2.22; P < 0.05) and in the higher plateau escape latencies on days 8 and 9 of the mutant mice compared to their +/+ littermates. In this probe test, the hidden platform was removed at the end of the 5th day of training and the mice searched for the missing platform for 60 sec. Spatial learning of the platform location should result in a targeted search strategy, as measured by an increase in the time spent in the quadrant that previously contained the platform and the number of times the mouse swims over the previous location of the platform. Wild-type mice spent significantly more time in the quadrant that had contained the hidden platform than in any other quadrant (Fig. 1B) (F(3,27) = 13.5; P < 0.05) and crossed over its previous location significantly more times than they crossed symmetrical points in the other three quadrants (F(3,27) = 5.04; P < 0.05). In contrast, after an identical 5 days of training, the Si/Sld mice did not show significant preference for the quadrant previously containing the platform (F(3,33) = 1.29; P > 0.05), nor did they swim over its former location more than over corresponding points in other quadrants (F(3,33) = 0.38; P > 0.05). These results suggest that, after 5 days of training, the Sl/Sld mice have not yet learned the platform location and that their initial improvement in latency can be attributed to variables such as adapting to the water and swimming or learning a nonspatial response strategy, such as swimming some minimum distance from the wall of the pool. With continued training, the mice further improved, possibly by learning to follow a single distal cue rather than learning spatial information (29). The Sl/Sld and +/+ mice were also compared for their ability to swim to a visible, above-water platform (a simple association version of the Morris task). In this task, configural cues are not necessary for navigation and successful learning requires only the association on the animal's behavior with a single visual cue (the platform). In this control experiment, the Si/Sld animals had the same rate of learning as +/+ mice (F(4,80) = 0.56; P > 0.05) (Fig. 1C), suggesting that the learning impairment in the mutant Sl/Sld mice is specific for configural learning and is not the result of differences in motivation, motor, or visual abilities, nor is it a deficit in learning simple associations. The specificity of the behavioral deficit of Sl/Sld mice is highly similar to the learning impairments observed in rodents after hippocampal lesions (1, 30, 31). Sl/Sld Mice Have Deficient CA3 Synaptic Transmission but Normal LTP. The observed configural learning deficit in SlI/Sid mice might suggest hippocampal dysfunction. Therefore, we compared normal synaptic transmission and induced persist-

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FIG. 1. Impaired learning of SlI/Sid mice in the hidden but not the visible platform task. (A) Escape latencies (mean ± SEM) to find a hidden platform in control (n = 10) and Si/Sld (n = 12) mice. (B) Time spent in the four different quadrants on a probe test. Control but not Sl/Sld mice spent significantly more time in quadrant 3 (previous platform location) than in the other three quadrants. (C) Latencies to find a visible platform. Following the hidden platform task, the mice were trained to swim to a visible above-water platform. No significant difference was found between the performances of the two groups. ent synaptic enhancement (in the form of LTP) in +/+ and

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mice in in vitro hippocampal slice preparations. The primary synaptic pathways in the hippocampal formation include enthorhinal cortex neurons projecting to the dentate gyrus the (the perforant path), dental gyrus neurons projecting to CA3 pyramidal neurons (the mossy fibers), and CA3 neurons projecting to CAl pyramidal neurons (the Schaffer collaterals). We examined LTP in dendate gyrus neurons after tetanic stimulation of the medial perforant path, which, unlike the lateral path, is believed to carry nonolfactory information into the hippocampus and consequently may be important for spatial learning (32). The high-frequency stimulation of the medial perforant path induced strong posttetanic potentiation (PTP) that decayed rapidly in -5 min. In the subsequent phase of potentiation, the decay was less rapid and the transmission was enhanced by 34% 25-30 min after the tetanus (LTP). Statistically significant LTP (t = 4.82; df = 7; P < 0.05, compared to the pretetanic stimulation baseline) was seen in + /+ mice and was similar in time course and duration to LTP observed by others (27). The magnitude of PTP in the mutant mice was unchanged, but LTP in Sl/Sld mice was slightly reduced. However, an ANOVA on the data after tetanic stimulation revealed only a significant main effect of time

(F(29,551) = 7.1; P < 0.05) but no significant main effect of group (+/+ compared to SlI/Si mice) (F(l,l9) = 2.04; P > 0.15) or significant interaction of time with group (F(29,55l) = 0.16; P > 0.15. The difference between +/+ and Si/Sld slices in LTP was not statistically significant at any time during the course of

potentiation. We next examined synaptic potentiation in the dentate gyrus CA3 pathway (Fig. 2 C and D). Comparisons of PTP and LTP did not reveal any significant differences between control and mutant animals (Fig. 2A). An ANOVA on the data after tetanic stimulation revealed no significant main effect of group (+/+ versus Si/Sid) (F(l,17) = 0.51; P > 0.15) nor any significant interaction of group with time (F(l8,306) = 0.81; P > 0.15). However, the baseline level (before tetanic stimulation) of synaptic transmission was significantly lower by 37% (MannWhitney U test = 424; n = 15; P < 0.05) in the mossy fiber pathway of Sl/Sld compared to control mice (Fig. 2B). In a few additional Si/Sld preparations, the baseline responses in CA3 were too small to be measured at the usual stimulation frequencies between 0.2 and 1.0 Hz, although increasing the frequency above 5 Hz did produce a response. Such higher frequencies tended to cause prolonged potentiations and were not suitable for monitoring of baseline transmission. Thus, it appears that baseline synaptic transmission in the mossy fiber

Genetics: Motro et al. A

Proc. Natl. Acad. Sci. USA 93 (1996) B

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lasting at least 20 min. Data points represent means ± SEM. (B) Comparison of initial slopes of baseline mossy fiber field responses in control and SlI/Sid slices shows a significant difference. (C) Upper traces, typical mossy fiber responses in Sl/Sid slice (S2) facilitated when stimulation frequency was increased from 0.2 to 2 Hz. Asterisk indicates presynaptic volley. Antidromic/collateral responses (Si) did not facilitate, indicating selectivity and independence of the two inputs. Lower traces, LTP was induced in the mossy fiber synapses but not in the collateral input to

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pathway was suppressed in Sl/Sld animals, sometimes below detectable levels. No differences were observed in baseline synaptic transmission, PTP or LTP between Sl/Sld and +/+ mice in the Schaffer collateral pathway from CA3 to CAl. As shown in Fig. 3 (which compares the average LTP 20-25 min after induction), none of the hippocampal synaptic pathways exhibited differences in LTP between Si/Sld and control mice. Because a 30-min time period was sufficient to detect abnormalities in hippocampal LTP in mice carrying targeted mutations (33-37), we measured LTP for a subsequent 10-20 min in several experiments but again no differences between Sl/Sld and control slices were observed. However, these studies do not rule out possible differences at much later times during the later phases of LTP that have been described recently (38). Hippocampal Anatomy Is Unchanged in SlI/Si Mice. The specific reduction in baseline responses (but not LTP) in the CA3 region might be caused by an impairment in the development of dentate gyrus CA3 connectivity in the Sl/Sld mutants, leading to less efficient transmission. However, Nissl staining revealed no gross cellular alterations in the hippocampus of Si/Sld adult mice and certainly not the increased neuronal numbers and undulations described in fyn -/- mutant mice (33). In addition, Timm's staining of the mossy fibers did not show detectable changes in granule cell axons, and MAP2 immunostaining did not reveal differences in the dendrites of the pyramidal neurons. Electron micro-

scopic analysis revealed normal giant mossy fiber terminal boutons making synapses in the CA3 region of Sl/Sld mice. Furthermore, we have previously shown that, unlike the

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FIG. 3. LTP magnitude is the same in control and mutant animal slices. LTP is shown as the mean + SEM relative increase of synaptic responses 20-25 min after induction in the Schaffer collateral CA1, mossy fiber CA3, and perforant pathway dentate gyrus projections. The numbers of experiments (slices) from control and mutant animals were 8 and 6 in CA1, 9 and 10 in CA3, and 8 and 14 in the dentate gyrus pathway. In none of the areas were the differences between control and Sl/Sld animals statistically significant.

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cellular deficits in the three major lineages affected in Wand Sl mutant mice, there are no obvious reductions in the numbers of c-kit-expressing cells in the brains of W and Sl mutant mice (20).

DISCUSSION The contiguous expression in the adult mouse of the Steel ligand in dentate gyrus neurons and its c-kit receptor tyrosine kinase in CA3 target neurons (20), which receive the mossy fiber synapses of the dentate gyrus neurons, led us to examine the anatomical, behavioral, and electrophysiological consequences of mutation at the Sl locus on this hippocampal pathway. Although we could see no anatomical deficits in the SlIS/d hippocampus, there was a selective reduction of baseline synaptic transmission at the mossy fiber synapse and a severe deficit in hippocampal-dependent configural learning as assessed in the Morris water maze. No changes in LTP were detected in any hippocampal pathway in the Sl/Sld mice. The Si/Sld animals analyzed in this study can express only the soluble, and not the membrane-bound form, of Steel factor (24, 25). Thus, these data argue that the membrane-bound form of Steel factor is required for proper signaling in the brain, as it is in the hematopoietic, reproductive, and melanocyte lineages (15, 16). It is also possible that the remaining learning and synaptic transmission observed in Sl/Sld mice reflects the residual activity of the soluble steel factor still expressed by the Sld allele. These results broaden the role of the Kit receptor pathway to include a postmitotic signaling function in the adult central nervous system. In humans, heterozygous mutations in the KIT gene have been identified in kindreds with a rare disorder known as piebald trait (39, 40). Such individuals have a dominant defect in melanocyte development, visible as a white hair forelock, and areas of hypopigmentation on the anterior trunk, similar to the dominant white-spotting seen in W and SI mutant mice. It is of interest to note that in several independent kindreds, an association between piebald trait and neurological impairment has been noted (41, 42). These deficiencies can include cerebellar ataxia, mental retardation, impaired motor coordination, and intestinal dysfunction similar to that observed observed in W mutant mice (43). An analysis of synaptic responses and of synaptic plasticity in three regions of the mouse hippocampal formation (CA1, CA3, and the dentate gyrus) revealed only one significant difference between control and Sl/Sld mice-a reduction of baseline responses in the CA3 area. It is not yet clear how the absence of the membrane-bound form of the Steel factor in Sl/Sld animals leads to the observed electrophysiological deficit, but mechanisms can be suggested. For example, dynorphin-related peptides, acting on K receptors, are known to inhibit synaptic transmission at mossy fiber synapses (44). It is believed that dynorphin peptides are produced and released from dentate granule neurons (45). Steel factor ligand in the mossy fibers could have a similar (although in the opposite direction) modulatory role, although no details on the electrophysiological effects of Steel factor at mossy fiber synapses are available. Our results, made with naturally occurring mutations at the Steel locus, demonstrate the importance of phosphorylation reactions in mediating synaptic transmission in the hippocampus and in learning and memory. These data extend observations made with mice created by introducing null mutations by gene targeting in embryo-derived stem cells. In these studies, mice homozygous for null mutations in the fyn tyrosine kinase (33), the a-calcium calmodulin kinase II (34, 35), or the y-isoform of protein kinase C (PKC'y) (36, 37) all exhibited deficits both in learning and memory and in hippocampal LTP. Thus, these data argue that phosphorylation reactions, initiated via receptor tyrosine kinases such as Kit and continued


intracellularly by cytoplasmic kinases such as fyn, PKC, and a-calcium calmodulin kinase II are central to the postdevelopmental functioning of the central nervous system. However, in contrast to other studies, our results with Sl/Sld mutants suggest that hippocampal LTP and hippocampal-dependent learning can be dissociated. Three lines of evidence support an LTP mechanism underlying hippocampal-dependent learning. First, the saturation of LTP apparently attenuated hippocampal-dependent learning, but these data can no longer be replicated (46-49). These failures to replicate have been attributed to failures to achieve saturation (50). Indeed, maximal electroconvulsive shock, which saturated hippocampal LTP, did attenuate spatial learning in the Morris maze (50), although maximal electroconvulsive shock undoubtedly also produces changes in many brain processes. Second, intracerebral administration of anN-methylD-aspartate (NMDA) receptor antagonist impaired both configural learning in the Morris maze and LTP (51). However, a high concentration of the antagonist in this study completely blocked LTP but left some configural learning intact, as measured by a probe test where treated animals swam significantly more in the quadrant previously containing the escape platform. Furthermore, baseline synaptic transmission could have been reduced by the antagonist since synaptic responses in the dentate gyrus have a substantial NMDA-dependent component at the resting membrane potential (52). Third, targeted mutations of both thefyn (33) and a-calcium-calmodulin kinase II (34, 35) genes produced configural (spatial) learning deficiencies accompanied by defective LTP. Interestingly, both of these targeted mutations showed "collateral" damage, which could have confounded the results. For example, the a-calcium-calmodulin kinase II mutant showed reduced posttetanic potentiation, paired-pulse facilitation, and long-term depression (34,35), in addition to reduced LTP. The fyn mutant had structural abnormalities and increased cell numbers in the hippocampus (23). Moreover, two other lines of mutant mice, the Sl/Sld and PKCy mutants, serve to double dissociate hippocampal LTP from hippocampal-dependent learning and memory. In the PKCy mutants, hippocampaldependent learning was observed in the absence of conventionally elicited LTP (36, 37), and, in the present report on Sl/Sld mutants, conventionally elicited LTP was observed in the absence of hippocampal-dependent learning. Thus, the PKC'y mutants show that hippocampal (CA1) LTP is not necessary for hippocampal-dependent learning and the Sl/Sld mutants show that hippocampal LTP is not sufficient for hippocampal-dependent learning. We thank Shirly Vesely, Sabrina Wang, and Adora Pridgar for technical assistance; Toshi Hattori for help with electron microscopic analysis; Darlene Skinner for help in data analysis; and Kit Carrothers, Pat Bryan, Beblan Soorae, and Linda Houston for help in preparation of the manuscript. B.M. was a fellow of the Leukemia Research Fund. This work was supported by Medical Research Council grants to J.M.W. and A.B., a National Institutes of Health (USA) grant to A.B., and a National Sciences and Engineering Research Council grant to D.v.d.K. A.B. is an International Research Scholar of the Howard Hughes Medical Institute and D.v.d.K. is a Medical Research Council Senior Scientist. 1. Sutherland, R. J. & McDonald, R. W. (1990) Behav. Brain Res. 37, 57-79. 2. Eichenbaum, H., Otto, T. & Cohen, N. J. (1992) Behav. Neural Biol. 57, 2-36. 3. Squire, L. R. (1992) Psychol. Rev. 99, 195-231. 4. Sutherland, R. J. & Rudy, J. W. (1989) Psychobiology 17, 129144. 5. Hebb, D. 0. (1949) The Organization of Behavior (Wiley, New

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