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

Steel mutant mice are deficient in hippocampal learning but notlong-term potentiationBENNY 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)

ABSTRACT Mice carrying mutations in either the dom-inant white-spotting (W) or Steel (Sl) loci exhibit deficits inmelanogenesis, gametogenesis, and hematopoiesis. Wencodesthe Kit receptor tyrosine kinase, while SI encodes the Kitligand, Steel factor, and the receptor-ligand pair are contig-uously expressed at anatomical sites expected from the phe-notypes ofWand Sl mice. The c-kit and Steel genes are also bothhighly expressed in the adult murine hippocampus: Steel isexpressed in dentate gyrus neurons whose mossy fiber axonssynapse with the c-kit expressing CA3 pyramidal neurons. Wereport here that SI/Sid mutant mice have a specific deficit inspatial learning. These mutant mice are also deficient inbaseline synaptic transmission between the dentate gyrus andCA3 but show normal long-term potentiation in this pathway.These observations demonstrate a role for Steel factor/Kitsignaling in the adult nervous system and suggest that a severedeficit in hippocampal-dependent learning need not be asso-ciated with reduced hippocampal long-term potentiation.

Lesion analyses in rodents, monkeys, and humnans point to thecritical role of hippocampal formation in a specific kind oflearning and memory variously called declarative, explicit, orconfigural (1-3). Configural learning, defined as the processesthat establish and store a representation of the relations (e.g.,in space) between more than two stimuli, can be differentiatedfrom a hippocampal-independent, simpler type of learningabout the relations between two stimuli variously called pro-cedural, implicit, or simple association learning (4). Modula-tion of synaptic strength has long been postulated as the centralmechanism of learning and memory. In certain parts of thecentral nervous system, a long-term potentiation (LTP) ofsynaptic efficiency is observed. LTP can be induced by a briefhigh-frequency stimulation of afferent fibers, in both the intactanimal and in tissue slices, and in most sites exhibits thespecificity, cooperativity, and associativity predicted by Hebb(5-7). Although these electrophysiological characteristics ofLTP make it a good candidate for the physiological mechanismof configural learning, the evidence remains equivocal (8, 9).The molecular mechanisms that underlie and modulate

synaptic plasticity (as well as configural learning) are poorlyunderstood. We examined the possible involvement of the Kitreceptor tyrosine kinase in hippocampal-dependent learning.Mutations in the murine genes for either the Kit receptor [theW locus (10, 11)] or its ligand Steel factor [the Steel (Sl) locus(12-14)] have deleterious effects on development of the mel-anocyte, germ cell, and hematopoietic lineages (15, 16). Aspredicted from the W/Sl mutant mice phenotypes, c-kit isexpressed in the three cell lineages known to be affected by Wor Sl mutations, while Steel is expressed in cells located in theirmigratory pathways and adjacent to their final destinations(17-20). However, the c-kit and Steel genes are also contigu-ously expressed in sites not known to be affected in the

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mutants, most noticeably in certain structures of the nervoussystem (17, 20). For example, high levels of Steel transcripts arefound in the enthorhinal cortex and dentate gyrus, while c-kitis found in the nearby CA3 and CAl pyramidal neurons andsubiculum in the hippocampus (20).The contiguous high level expression of c-kit and Steel in the

adult murine hippocampus suggests a possible role for theSteel factor/Kit signaling pathway in hippocampal-dependentlearning and memory. To test this possibility, we used theMorris water maze task to examine the spatial learning abilityof Steel mutant mice. The Morris water maze is the mostcommonly used test to examine deficits in configural learningtasks following damage or dysfunction in the hippocampus(21), including its mossy fiber system (22, 23). We show herethat, compared to +/+ control mice, Steel mutant mice havepoorer performance in a configural learning version of theMorris water maze but not in a similar version of the task thatrequires only simple associations. We demonstrate also thatnormal synaptic transmission between dentate gyrus andhippocampal CA3 pyramidal cells is deficient in the mutantmice. No deficit was found in hippocampal LTP. These resultsdemonstrate a role for the Steel factor/Kit receptor tyrosinekinase signaling pathway in the brain. They also argue thatinefficient hippocampal synaptic transmission, without a def-icit in LTP, can cause a specific impairment in configurallearning and memory.

MATERIALS AND METHODSMice. Because mice homozygous for null alleles of the SI

locus die prenatally of severe anemia, we used viable com-pound heterozygous (Sl/Sld) mice, which have one null allele(Sl) (12, 14), while the other allele (Sld) produces only thesoluble, 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/Sld4- to 5-month-old male mice (n = 12) and their +/+ controllittermates (n = 10) in the hidden platform version of theMorris water maze. The apparatus was a white plastic circularpool (diameter, 122 cm; wall height, 76 cm). The water wasmade opaque with nontoxic Crayola paint. A circular, plasticplatform (diameter, 5 cm; height, 60 cm) was placed in onequadrant of the pool just below the surface of the water. In thistask, mice were released from one of four randomly chosenstarting points in the circular pool to search for the hiddenescape platform for 60 sec. The mice were allowed to rest for20 sec on the platform after finding it (or after the experi-menter placed the animal on the platform if the platform wasnot found within 60 sec). The mice received five trials a day for9 days.

Abbreviations: LTP, long-term potentiation; PTP, posttetanic poten-tiation; 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.

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A probe test was performed after 5 days of training on thehidden platform task. The platform was removed and the micewere allowed to search for 60 sec. Their paths were videotapedand the times they spent in each of the quadrants and thenumber of times they swam over the former location of theplatform were recorded.

After completion of the hidden platform task, we comparedthe ability of these mice to swim to a visible, above-waterplatform (a simple association version of the task). The micewere given five trials over 1 day in this task and their latenciesto find the platform were recorded.Anatomy. At the completion of the behavioral experiments,

the brains of some of the SlI/Sid and + /+ mice were processedhistologically. After an anesthetic overdose, the mice wereperfused transcardially with 10% formalin, and their brainswere quickly removed and placed in 10% formalin and 30%sucrose. Frontal sections (30 ,um) were cut through the hip-pocampus on a cryostat, mounted on gelled slides, and Nissl-stained with thionin. Other mice were perfused transcardiallywith 1.17% buffered sodium sulfide (pH 7.35) for 3 min,followed by 3% glutaraldehyde in 0.15 M Soerensen's phos-phate buffer (pH 7.35) for 5 min, and then for a further 7 minwith sodium sulfide. Cryostat sections were thawed on slidesand developed in Timm's solution, as described (22), tovisualize the hippocampal mossy fibers.

Electrophysiology. Animals were anaesthetized with halo-thane and decapitated, and the hippocampi were quicklyremoved. Transverse hippocampal slices (400 ,um thick) werecut with a tissue chopper and kept moist and oxygenated atroom temperature for 1-5 hr until use. For electrical record-ings, slices were transferred to a perfusion chamber and viewedwith a dissecting microscope. Synaptic field potentials wereevoked with the use of tungsten electrodes and recorded viaglass pipettes filled with artificial cerebrospinal fluid [(ACSF)124 mM NaCl/3 mM KCl/1.25 mM NaH2PO4/2 mM MgCl2/2mM CaCl2/26 mM NaHCO3/10 mM dextrose]. The ACSF wasequilibrated with 95% oxygen/5% C02, pH 7.4 at 32°C. Theslicing procedure usually yielded 10-15 slices from each ani-mal. However, due to time constraints, only 2 or 3 slices wereused for recordings. To reduce the strong GABAergic inhibi-tion in the dentate gyrus, 10 ,tM bicuculline was added to theperfusate as described (26). To study LTP in the dentate gyrus,both the stimulating and recording electrodes were placed inthe middle molecular layer and the stimulation intensity wasadjusted to produce a current sink only in the middle molecularlayer (and a current source in the outer molecular layer). Thisprocedure selectively activates the medial perforant pathway(27).To study LTP in the mossy fiber CA3 pathway, the stimu-

lating electrode was placed in the dentate gyrus and therecording electrode in a clearly defined, translucent bandvisible in the CA3 area. Stimulation of the dentate gyrusproduced a synaptic current sink in the mossy fiber layer, whichcontrasted with a biphasic antidromic/collateral responseobtained by stimulation of the CAl area (see Fig. 2C). Themossy fiber responses were further distinguished from othersynaptic responses in the CA3 region by their pronouncedfrequency facilitation. These criteria for mossy fiber responseswere developed to overcome the difficulties, pointed out byothers (28), in identifying intracellular mossy fiber responses.For this reason, we standardized the magnitude of effectivestimulating strength by evoking the maximal presynaptic vol-ley, ensuring optimal locations of the stimulating and record-ing 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 activatecollateral/commissural afferents. Stimulus intensity was ad-justed to produce a field synaptic response just subthreshold toevoking a population spike.

Different series of slices were used for the LTP studies ineach of the dentate gyrus areas CA3 and CAl. In all threeareas of the hippocampus, LTP was induced by application oftwo trains of stimuli at 100 Hz, lasting 1 sec each and repeatedat a 5-sec interval. LTP was usually monitored for 20-30 minafter the tetanic stimulation. The magnitude of LTP wasexpressed as a relative change (%) with respect to the averageof responses during the 10-min period preceding the tetanicstimulation. The initial slope of field synaptic responses wastaken 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 runin the hidden platform version of the Morris water maze. Onthe first trials, the mice find the platform by chance and thenlearn and remember the platform location based on distal cues.On the first day of training, both Si/Sld and + /+ mice requiredequivalent times to find the hidden platform (Fig. 1A). On thesubsequent 8 days, both +/+ and Sl/Sld mice showed areduction in the times required to find the hidden platform;however, there were significant differences in both the rate atwhich 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 and9 of the mutant mice compared to their +/+ littermates. Inthis probe test, the hidden platform was removed at the end ofthe 5th day of training and the mice searched for the missingplatform for 60 sec. Spatial learning of the platform locationshould result in a targeted search strategy, as measured by anincrease in the time spent in the quadrant that previouslycontained the platform and the number of times the mouseswims over the previous location of the platform. Wild-typemice spent significantly more time in the quadrant that hadcontained the hidden platform than in any other quadrant (Fig.1B) (F(3,27) = 13.5; P < 0.05) and crossed over its previouslocation significantly more times than they crossed symmetri-cal points in the other three quadrants (F(3,27) = 5.04; P <0.05). In contrast, after an identical 5 days of training, theSi/Sld mice did not show significant preference for the quad-rant previously containing the platform (F(3,33) = 1.29; P >0.05), nor did they swim over its former location more thanover corresponding points in other quadrants (F(3,33) = 0.38; P> 0.05). These results suggest that, after 5 days of training, theSl/Sld mice have not yet learned the platform location and thattheir initial improvement in latency can be attributed tovariables such as adapting to the water and swimming orlearning a nonspatial response strategy, such as swimmingsome minimum distance from the wall of the pool. Withcontinued training, the mice further improved, possibly bylearning to follow a single distal cue rather than learningspatial information (29).The Sl/Sld and +/+ mice were also compared for their

ability to swim to a visible, above-water platform (a simpleassociation version of the Morris task). In this task, configuralcues are not necessary for navigation and successful learningrequires only the association on the animal's behavior with asingle visual cue (the platform). In this control experiment, theSi/Sld animals had the same rate of learning as +/+ mice(F(4,80) = 0.56; P > 0.05) (Fig. 1C), suggesting that the learningimpairment in the mutant Sl/Sld mice is specific for configurallearning and is not the result of differences in motivation,motor, or visual abilities, nor is it a deficit in learning simpleassociations. The specificity of the behavioral deficit of Sl/Sldmice is highly similar to the learning impairments observed inrodents after hippocampal lesions (1, 30, 31).

Sl/Sld Mice Have Deficient CA3 Synaptic Transmission butNormal LTP. The observed configural learning deficit in SlI/Sidmice might suggest hippocampal dysfunction. Therefore, wecompared 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 hiddenplatform 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 micespent 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 betweenthe performances of the two groups.

ent synaptic enhancement (in the form of LTP) in +/+ andSlIS/d mice in in vitro hippocampal slice preparations. Theprimary synaptic pathways in the hippocampal formationinclude enthorhinal cortex neurons projecting to the dentategyrus the (the perforant path), dental gyrus neurons project-ing to CA3 pyramidal neurons (the mossy fibers), and CA3neurons projecting to CAl pyramidal neurons (the Schaffercollaterals).We examined LTP in dendate gyrus neurons after tetanic

stimulation of the medial perforant path, which, unlike thelateral path, is believed to carry nonolfactory information intothe hippocampus and consequently may be important forspatial learning (32). The high-frequency stimulation of themedial perforant path induced strong posttetanic potentiation(PTP) that decayed rapidly in -5 min. In the subsequent phaseof potentiation, the decay was less rapid and the transmissionwas 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 LTPobserved by others (27). The magnitude of PTP in the mutantmice was unchanged, but LTP in Sl/Sld mice was slightlyreduced. However, an ANOVA on the data after tetanicstimulation revealed only a significant main effect of time

(F(29,551) = 7.1; P < 0.05) but no significant main effect ofgroup (+/+ 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 LTPwas not statistically significant at any time during the course ofpotentiation.We next examined synaptic potentiation in the dentate gyrus

CA3 pathway (Fig. 2 C and D). Comparisons of PTP and LTPdid not reveal any significant differences between control andmutant animals (Fig. 2A). An ANOVA on the data aftertetanic stimulation revealed no significant main effect ofgroup(+/+ versus Si/Sid) (F(l,17) = 0.51; P > 0.15) nor anysignificant 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% (Mann-Whitney U test = 424; n = 15; P < 0.05) in the mossy fiberpathway of Sl/Sld compared to control mice (Fig. 2B). In a fewadditional Si/Sld preparations, the baseline responses in CA3were too small to be measured at the usual stimulationfrequencies between 0.2 and 1.0 Hz, although increasing thefrequency above 5 Hz did produce a response. Such higherfrequencies tended to cause prolonged potentiations and werenot suitable for monitoring of baseline transmission. Thus, itappears that baseline synaptic transmission in the mossy fiber

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pathway was suppressed in Sl/Sld animals, sometimes belowdetectable levels.No differences were observed in baseline synaptic trans-

mission, PTP or LTP between Sl/Sld and +/+ mice in theSchaffer collateral pathway from CA3 to CAl. As shown inFig. 3 (which compares the average LTP 20-25 min afterinduction), none of the hippocampal synaptic pathwaysexhibited differences in LTP between Si/Sld and controlmice. Because a 30-min time period was sufficient to detectabnormalities in hippocampal LTP in mice carrying targetedmutations (33-37), we measured LTP for a subsequent10-20 min in several experiments but again no differencesbetween Sl/Sld and control slices were observed. However,these studies do not rule out possible differences at muchlater times during the later phases of LTP that have beendescribed recently (38).Hippocampal Anatomy Is Unchanged in SlI/Si Mice. The

specific reduction in baseline responses (but not LTP) in theCA3 region might be caused by an impairment in the devel-opment of dentate gyrus CA3 connectivity in the Sl/Sldmutants, leading to less efficient transmission. However, Nisslstaining revealed no gross cellular alterations in the hip-pocampus of Si/Sld adult mice and certainly not the in-creased neuronal numbers and undulations described in fyn-/- mutant mice (33). In addition, Timm's staining of themossy fibers did not show detectable changes in granule cellaxons, and MAP2 immunostaining did not reveal differencesin the dendrites of the pyramidal neurons. Electron micro-

FIG. 2. Low-frequency transmission_ but not synaptic facilitation is altered in_ mossy fibers of Sl/Sld mice. (A) Summary

graph shows normal time course ofLTP in_11 mossy fibers in Si/Sid and control slices.

- ~~~~~~Notea large transient increase of synapticresponses (posttetanic potentiation) fol-

lowing the tetanus and a sustained LTPlasting at least 20 min. Data points repre-

Control si/st sent means ± SEM. (B) Comparison ofinitial slopes of baseline mossy fiber fieldresponses in control and SlI/Sid slicesshows a significant difference. (C) Uppertraces, typical mossy fiber responses inSl/Sid slice (S2) facilitated when stimula-tion frequency was increased from 0.2 to 2Hz. Asterisk indicates presynaptic volley.Antidromic/collateral responses (Si) didnot facilitate, indicating selectivity andindependence of the two inputs. Lowertraces, LTP was induced in the mossy fibersynapses but not in the collateral input tothe CA3 neurons. In a subsequent exper-

iment, Si produced LTP in collateral syn-\ 2 apses (data not shown). (D) Outline of a

[g \ hippocampal slice indicating CA1, CA3,and dentate gyrus (dg) areas. One stimu-lating electrode (Si) was placed in CAl

and the other (S2) was placed in dg; therecording electrode was placed in themossy fiber region of CA3. Responseswere measured at a stimulating frequencyof 0.2-1.0 Hz. In all cases, 12 responseswere averaged before a measurement.

scopic analysis revealed normal giant mossy fiber terminalboutons 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 animalslices. LTP is shown as the mean + SEM relative increase of synapticresponses 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 animalswere 8 and 6 in CA1, 9 and 10 in CA3, and 8 and 14 in the dentate gyruspathway. In none of the areas were the differences between controland Sl/Sld animals statistically significant.

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

DISCUSSIONThe contiguous expression in the adult mouse of the Steelligand in dentate gyrus neurons and its c-kit receptor tyrosinekinase in CA3 target neurons (20), which receive the mossyfiber synapses of the dentate gyrus neurons, led us to examinethe anatomical, behavioral, and electrophysiological conse-quences of mutation at the Sl locus on this hippocampalpathway. Although we could see no anatomical deficits in theSlIS/d hippocampus, there was a selective reduction of baselinesynaptic transmission at the mossy fiber synapse and a severedeficit in hippocampal-dependent configural learning as as-sessed in the Morris water maze. No changes in LTP weredetected 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-boundform of Steel factor is required for proper signaling in thebrain, as it is in the hematopoietic, reproductive, and mela-nocyte lineages (15, 16). It is also possible that the remaininglearning and synaptic transmission observed in Sl/Sld micereflects the residual activity of the soluble steel factor stillexpressed by the Sld allele. These results broaden the role ofthe Kit receptor pathway to include a postmitotic signalingfunction in the adult central nervous system.

In humans, heterozygous mutations in the KIT gene havebeen identified in kindreds with a rare disorder known aspiebald trait (39, 40). Such individuals have a dominant defectin melanocyte development, visible as a white hair forelock,and areas of hypopigmentation on the anterior trunk, similarto 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 impair-ment has been noted (41, 42). These deficiencies can includecerebellar ataxia, mental retardation, impaired motor coordi-nation, and intestinal dysfunction similar to that observedobserved 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 significantdifference between control and Sl/Sld mice-a reduction ofbaseline responses in the CA3 area. It is not yet clear how theabsence of the membrane-bound form of the Steel factor inSl/Sld animals leads to the observed electrophysiological def-icit, but mechanisms can be suggested. For example, dynor-phin-related peptides, acting on K receptors, are known toinhibit synaptic transmission at mossy fiber synapses (44). It isbelieved that dynorphin peptides are produced and releasedfrom dentate granule neurons (45). Steel factor ligand in themossy fibers could have a similar (although in the oppositedirection) modulatory role, although no details on the elec-trophysiological effects of Steel factor at mossy fiber synapsesare available.Our results, made with naturally occurring mutations at the

Steel locus, demonstrate the importance of phosphorylationreactions in mediating synaptic transmission in the hippocam-pus and in learning and memory. These data extend observa-tions made with mice created by introducing null mutations bygene 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 they-isoform of protein kinase C (PKC'y) (36, 37) all exhibiteddeficits both in learning and memory and in hippocampal LTP.Thus, these data argue that phosphorylation reactions, initi-ated via receptor tyrosine kinases such as Kit and continued

intracellularly by cytoplasmic kinases such as fyn, PKC, anda-calcium calmodulin kinase II are central to the postdevel-opmental functioning of the central nervous system. However,in contrast to other studies, our results with Sl/Sld mutantssuggest that hippocampal LTP and hippocampal-dependentlearning can be dissociated.Three lines of evidence support an LTP mechanism under-

lying hippocampal-dependent learning. First, the saturation ofLTP apparently attenuated hippocampal-dependent learning,but these data can no longer be replicated (46-49). Thesefailures to replicate have been attributed to failures to achievesaturation (50). Indeed, maximal electroconvulsive shock,which saturated hippocampal LTP, did attenuate spatial learn-ing in the Morris maze (50), although maximal electroconvul-sive shock undoubtedly also produces changes in many brainprocesses. Second, intracerebral administration ofanN-methyl-D-aspartate (NMDA) receptor antagonist impaired both con-figural learning in the Morris maze and LTP (51). However, ahigh concentration of the antagonist in this study completelyblocked LTP but left some configural learning intact, asmeasured by a probe test where treated animals swam signif-icantly more in the quadrant previously containing the escapeplatform. Furthermore, baseline synaptic transmission couldhave been reduced by the antagonist since synaptic responsesin the dentate gyrus have a substantial NMDA-dependentcomponent at the resting membrane potential (52). Third,targeted mutations of both thefyn (33) and a-calcium-calmod-ulin kinase II (34, 35) genes produced configural (spatial)learning deficiencies accompanied by defective LTP. Interest-ingly, both of these targeted mutations showed "collateral"damage, which could have confounded the results. For exam-ple, the a-calcium-calmodulin kinase II mutant showed re-duced posttetanic potentiation, paired-pulse facilitation, andlong-term depression (34,35), in addition to reduced LTP. Thefyn mutant had structural abnormalities and increased cellnumbers in the hippocampus (23). Moreover, two other linesof mutant mice, the Sl/Sld and PKCy mutants, serve to doubledissociate hippocampal LTP from hippocampal-dependentlearning and memory. In the PKCy mutants, hippocampal-dependent learning was observed in the absence of conven-tionally elicited LTP (36, 37), and, in the present report onSl/Sld mutants, conventionally elicited LTP was observed inthe absence of hippocampal-dependent learning. Thus, thePKC'y mutants show that hippocampal (CA1) LTP is notnecessary for hippocampal-dependent learning and the Sl/Sldmutants show that hippocampal LTP is not sufficient forhippocampal-dependent learning.

We thank Shirly Vesely, Sabrina Wang, and Adora Pridgar fortechnical assistance; Toshi Hattori for help with electron microscopicanalysis; Darlene Skinner for help in data analysis; and Kit Carrothers,Pat Bryan, Beblan Soorae, and Linda Houston for help in preparationof the manuscript. B.M. was a fellow of the Leukemia Research Fund.This work was supported by Medical Research Council grants toJ.M.W. and A.B., a National Institutes of Health (USA) grant to A.B.,and a National Sciences and Engineering Research Council grant toD.v.d.K. A.B. is an International Research Scholar of the HowardHughes Medical Institute and D.v.d.K. is a Medical Research CouncilSenior Scientist.

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