Testing the NMDA, Long-term Potentiation, andCholinergic Hypotheses of Spatial Learning

DONALD PETER CAIN*

Department of Psychology and Graduate Program in Neuroscience, University of Western Ontario, London,Ontario N6A 5C2, Canada

CAIN, D. P.Testing the NMDA, long-term potentiation, and cholinergic hypotheses of spatial learning. NEUROSCI BIOBEHAV REV22(2), 181–193, 1998.—The problems and issues associated with the use of pharmacological antagonists in studies on learning andmemory are considered in a review of the role ofN-methyl-d-aspartate (NMDA) receptors, NMDA receptor-mediated long-termpotentiation (LTP), and muscarinic receptors in spatial learning in the water maze. The evidence indicates that neither NMDA normuscarinic receptors, nor NMDA receptor-mediated LTP, are required for spatial learning, although they might normally contribute toit. Detailed behavioral analyses have indicated that the water maze task is more complex than generally has been appreciated, and has anumber of dissociable components. Naive rats trained under NMDA or muscarinic antagonism display sensorimotor disturbances thatinterfere with their ability to acquire the task. Rats made familiar with the general requirements of the task can learn the location of ahidden platform readily under NMDA or muscarinic antagonism. The ability of a rat to acquire the water maze task depends on its abilityto apply instinctive behaviors to performance of the task in an adaptive manner. The instinctive behaviors undergo modification as therat learns the general strategies required in the task. The evidence suggests that at least some of the plastic changes involved in acquiringthe task occur in existing neural circuits situated in widespread areas of the brain, including sensory and motor structures in the cortexand elsewhere, and are therefore difficult to distinguish from existing sensorimotor mechanisms. More generally, the findings indicatethe difficulty of inferring the occurrence or nonoccurrence of learning from behavior, and the difficulty of causally linking the action ofparticular receptor populations with the formation of specific memories.q 1998 Elsevier Science Ltd. All rights reserved.

N-Methyl-d-aspartate NMDA Scopolamine Muscarinic receptor Spatial learning Water maze Sensorimotordisturbances Behavior

CERTAIN PERIODS in the history of brain research havebeen dominated by a search for the mechanisms of learningand memory. Karl Lashley, the father of modern behavioralneuroscience, spent much of the period from 1920 to 1950searching for the engram in a long series of learning experi-ments involving rats and monkeys with brain lesions. ForLashley the engram represented the sites of lasting change inthe nervous system—presumably specific synapses—thatwere associated with particular instances of behavioral learn-ing, and were necessary for it to occur. His approach of beha-viorally training rats to discriminate visual stimuli or negotiatemazes set the tone of such research for years. Lashley’s con-tributions are usually, but incompletely, summed up by the‘‘principle of mass action’’, the conclusion that for complextasks such as learning to traverse a maze, the amount learnedand remembered was proportional to the amount of remainingneocortex; the locationof the damagemattered little. Althoughthe common view that Lashley failed in his attempts to locatethe engram for some forms of learning is mistaken (104), theperceived negativity of his findings, and the accumulatingevidence for the localization of function in the neocortex, ledmany researchers to discredit his conclusions.

More recently, behavioral neuroscience has experienceda strong resurgence of interest in the engram. Two develop-ments during the 1970s were probably important in thisresurgence. First, Eric Kandel and his colleagues set outto map the nervous system ofAplysia, a simple invertebrate,and to identify the neurons and events required for simpleforms of behavioral learning (14). Second, Bliss and Lomo(10) discovered long-term potentiation (LTP), a relativelylong-lasting form of neural plasticity that could be artificiallyinduced in the brain of mammals by electrical stimulation.The first development showed that detailed knowledge ofrelevant neural circuitry was beneficial and perhaps evennecessary, and that this knowledge, together with electro-physiological analysis, could identify the nature and locationof the engram in a simple animal. The second developmentprovided a laboratory model of the kind of synaptic plasticitythat could underlie complex forms of learning in mammals(9). LTP appeared to embody the activity of the ‘‘Hebbsynapse’’, a modifiable synapse that Donald Hebb, Lashley’sstudent, proposed in 1949 as a learning mechanism.

The idea that the hippocampus was uniquely importantfor engram formation stemmed from the discovery in the

Neuroscience & Biobehavioral Reviews, Vol. 22, No. 2, pp. 181–193, 1998q 1998 Elsevier Science Ltd. All rights reserved

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1950s that human patients with hippocampal removalsexperienced severe disturbances in learning and memory(87). Later, based on animal research, O’Keefe and Nadel(71) proposed that the hippocampus served as a spatial map.Their hypothesis was supported by the finding that indivi-dual complex spike cells in the hippocampus, called placecells, fired when an animal was in a specific location withinan environment (70). This suggested that place cells codedspatial locations in the environment, giving anatomical andfunctional support to the hypothesis that the hippocampusserved as a spatial map that was crucial for spatial learn-ing—the learning of a location in the environment inrelation to cues in that environment. The fact that damageto the hippocampus or related structures severely disruptedacquisition of tasks that required spatial information wasconsistent with their hypothesis (65,69).

Further impetus for the idea that the hippocampus mightbe important for engram formation came from researchshowing that LTP occurred most readily in the hippo-campus. Although hippocampal LTP has been theoreticallylinked to various kinds of learning, it has been most closelylinked to spatial learning (15,63,64). LTP at most hippo-campal synapses was found to be dependent on activity atN-methyl-d-aspartate (NMDA) receptors (17,34,64), andNMDA receptor antagonists were found to severely disruptacquisition of spatial learning tasks (63,64). This suggestedthat NMDA receptor-mediated LTP in the hippocampusestablished the engram for spatial learning.

This review will evaluate the hypothesis that NMDAreceptor-mediated LTP in the hippocampus is necessaryfor establishing the engram for spatial learning. The numberof laboratory tasks that could potentially involve aspects ofspatial learning for their acquisition is large, so the discus-sion will focus on selected studies involving the water maze.This task requires a rat or mouse to swim in a circular poolof water 1–2 m in diameter until it finds and climbs onto asmall platform hidden just beneath the surface, whichprovides the only refuge. This task is well suited for thestudy of spatial learning because it requires the animal tolearn to swim to a specific location using visual cues in theroom (61,71,96). Normal rats learn to do this quickly, butrats given NMDA antagonists or hippocampal lesions dopoorly on this task (63–65).

In addition to a hypothesized role for NMDA receptor-mediated hippocampal LTP in spatial learning, there is evi-dence that other neurochemical mechanisms are relevant.For example, muscarinic receptor antagonists produceacquisition deficits that are comparable to those producedby NMDA antagonists (96). Therefore studies involvingmuscarinic antagonists also will be discussed.

LEARNED AND INSTINCTIVE BEHAVIOR IN THE WATER MAZE

Spatial navigation plays a crucial role in many importantbehaviors in the rodent. Foraging for food, reproductive andparental behavior, escaping predation to a known safe loca-tion, and returning to the nest site depend on the ability tonavigate accurately in space. The fact that normal rats canacquire the water maze task in just a few trials suggests thattheir nervous system was shaped during evolution toproduce, among other things, accurate spatial navigation.As the basic behaviors involved in performing the task areinstinctive (movements of the limbs, the guiding of

movement by visual and other sensory input, climbingonto and standing on the platform), there must be pre-existing neural circuits that the rat brings to the water mazetask for its successful acquisition (51,100,102,104,117), inaddition to engrams that might be formed by neuroplasticprocesses during training.

Successful acquisition of the water maze task requires ananimal to navigate efficiently from any starting point in thepool to a specific location containing the hidden platform.Whishaw and Mittleman (115) showed that normal ratsmade use of a number of different spatial navigationstrategies in acquiring the water maze task, and that thesestrategies were considerably more complex than had beenthought. They suggested that ‘‘some deficits in spatialnavigation that follow brain damage may be as easilyattributed to the loss of search strategies as to the loss of ahypothetical subsystem of memory’’ (p. 428). This generalapproach led to an extensive series of experiments, theconclusion of which was that muscarinic antagonists disrupta sensorimotor subsystem important for the selection ofbehavioral strategies required to solve the water mazetask, and do not prevent spatial learning per se(109,110,117,118). The question whether experimentaltreatments affect the selection of behavioral strategies foracquiring the water maze task, or even basic sensoryprocessing or motor control mechanisms, is important forunderstanding drug and lesion effects.

The relevance of this question is that all conclusionsabout the neural mechanisms of learning, conceived of aschanges in conduction through specific neural circuits, areinferences from observable behavior. For inferences aboutspatial learning to be valid, the behavioral changes cannothave resulted from the disruptive effects of an experimentaltreatment on the mechanisms involved in producing theinstinctive behaviors required to perform the task. Under-standing the effects of treatments on instinctive behaviors,as well as on the engram, is vital for arriving at a useful andcomplete understanding of learning and memory (102,104).

The difficulty of knowing whether experimental treat-ments have affected pre-existing neural circuits that produceinstinctive behavior, or the engram, or both, is a very diffi-cult problem for even the simplest forms of learning. Afurther complication is the fact that instinctive behaviorsthemselves can be modified by experience. There aremany examples of this from a large variety of species. Forexample, instinctive behavior in insects, birds, and mam-mals can depend directly on the animal’s prior experiencewith the stimuli that release or evoke it (1,32,86,104).Further, the nature and extent of modifications by experi-ence can depend on the type of instinctive behavior that ismeasured (80). Some basic human sensorimotor behaviorsare well known to be to modifiable (38). All of this suggeststhat both learned and instinctive behaviors involve both pre-existing and acquired circuits (104). Said another way,‘‘ …there is no evidence to justify the a priori assumptionthat the control of learned and the control of instinctivebehavior are quite separate processes.’’ ((37), p. 169). Forthe water maze task this means that the instinctive behaviorsrequired in the task might well be modified by experience inthe pool; indeed, this modification might be a prerequisitefor successful acquisition of the task. This topic will be dealtwith below in a discussion of the nonspatial pretrainingeffect.

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WATER MAZE PERFORMANCE AND SENSORIMOTORDISTURBANCES

The intertwined nature of learned and instinctivebehaviors, and of acquired and pre-existing neural circuits,raises the question of whether it is possible to devise anexperimental approach to separate the effects of experimen-tal treatments on the engram for spatial learning from effectson instinctive behaviors required to perform the task. If, assuggested in the previous paragraph, the modification of

instinctive behaviors is required for successful acquisitionof the water maze task, then this modification might itselfinvolve establishment of an engram related to the instinctivebehaviors. This would further complicate the task of separ-ating the effects of experimental treatments on various com-ponents of behavior that contribute to the overallperformance of the task. However, a beginning in dealingwith this question can be made by analyzing a broad batteryof behaviors that includes both sensorimotor function andinstinctive behaviors required in the task, in addition to

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FIG. 1. Hidden and visible platform search time (top), hidden platform quadrant dwell time during the probe trial (middle), and percent direct swims to thehidden platform (bottom) for rats given NPC17742, a competitive NMDA receptor antagonist (left) or scopolamine, a muscarinic receptor antagonist (right).Contr, controls; NPC, NPC17742; Platf, platform; Pretr, nonspatially pretrained; Rand, random; SCO, scopolamine. Adapted from Ref. (83).

TESTING THE NMDA, LONG-TERM POTENTIATION, 183

behavioral measures intended to quantify the particular kindof learning that one is interested in. This approach is usefulbecause it can reveal treatment-induced disturbances inbehavior that have the potential to affect the behavioralmeasures of learning, and thus the inferences made fromthem. Further approaches to this general problem will bediscussed in the next section.

Measures for quantifying spatial learning in the watermaze have included hidden platform search time (elapsedtime from release into the water to climbing onto the plat-form (60,61)), platform quadrant dwell time (time spentswimming in the quadrant of the pool that formerly con-tained the hidden platform during training trials (93,96)),and swim accuracy as indicated by heading error (96) or thedirectness of the swim path to the hidden platform ((118);Fig. 1). Since these measures depend on the instinctivebehaviors described earlier, they can be affected by treat-ments that disrupt relevant sensorimotor mechanisms.

A practical way of proceeding is by focusing on sensor-imotor function both in the performance of the task itself,and in other sensorimotor tasks conducted outside the spe-cific learning task. Data from the latter source can provideindependent confirmation of sensorimotor disturbances thatmight have affected mechanisms for instinctive behaviorsrequired in the task. The more similar the outside sensor-imotor tasks are to behaviors actually required for perfor-mance of the task, the more confident one can be that theinformation they provide is directly relevant to interpretingthe effects of an experimental treatment.

The earliest use of a test of sensorimotor function in thewater maze was the visible platform task (60,61). Use of aplatform that protruded above the surface of the waterallowed for the evaluation of basic swimming ability, theability of a rat to guide its swimming using visual cues, andthe ability to climb onto and remain on the platform. Animpairment in the visible platform task was taken asevidence that the experimental treatment affected eithersensorimotor abilities other than spatial learning per se, orthe motivation to escape the water (61). Unfortunately, notall water maze studies have made use of this importantcontrol. One of the first to do so reported that hippocampallesions produced a small but reliable increase in visibleplatform search time, in addition to a much larger increasein hidden platform search time (65).

The visible platform task was used in a study withMK801, a noncompetitive antagonist of NMDA receptors,where it was found that MK801 increased visible platformsearch time only at a dose (0.08 mg/kg) greater than thatrequired to increase search time in the hidden platform task(0.05 mg/kg) (79). The first studies to use the visible plat-form task with competitive NMDA and non-NMDA excitatoryamino acid receptor antagonists reported that the antagonistsincreased visible platform search time at the same doses thatincreased hidden platform search time ((11,82,83); Fig. 1),raising the possibility that these treatments caused thehidden platform task deficits by an action on sensorimotormechanisms rather than on spatial learning per se.

Use of the visible platform task with muscarinic antago-nists has produced mixed results. Whishaw found that highdoses of atropine sulfate, a muscarinic antagonist, some-times increased search time early in testing (118), but thatit generally had little or no effect on performance of thevisible platform task (109,118). However, other researchers

found that atropine sulphate or scopolamine, anothermuscarinic antagonist, reliably increased visible platformsearch time ((31,72,83,102); Fig. 1). A possible explanationfor the different results is the nature of the pool and plat-form, which determine the difficulty of the task. The moresalient the visible platform is against the pool wall or theless difficult the task, the less effect muscarinic antagonistsappear to have on the visible platform task (72,117). Thissuggests the possible involvement of sensory deficits in theeffect of muscarinic antagonists on the visible platform task,a topic to be discussed below.

Whishaw’s work appears to provide an explanation forsome of these findings. He was among the first to emphasizethe complexity of the water maze task, which requires a ratto learn how to cope with the task by selecting an appro-priate strategy before it can learn the spatial location of thehidden platform (109). This approach to understanding howthe rat acquires this task is essentially the same one thatLashley (51) and Krechevsky (47) developed many yearsago (117). The rat must learn a number of things before itcan solve the task: to swim; to swim away from the wall;that the platform provides the only refuge; and to use theplatform by climbing onto and remaining on it. Only thencan it engage a strategy to learn the spatial location of thehidden platform in relation to cues in the room.

However, rats trained under a muscarinic antagonist donot appear to learn these strategies for maze acquisition asreadily as normal rats do. Instead, they persist in swimmingaround the perimeter of the pool, close to the wall(72,109,118). Whishaw and Tomie (118) were the first toquantify swimming in the periphery of the pool by analyz-ing the distance swum in the outer 50% of the pool duringposttraining probe trials. This study used a visible platformduring training, which reduced the rats’ need to use a spatialstrategy. Rats trained under atropine sulfate swam morethan 80% of the total distance in the outer portion of thepool, a significantly greater distance than controls. The ratsalso displayed fewer search behaviors (orienting move-ments, head elevations, tight circles, pauses), and ignorednovel cues placed around the pool. These results wereconsistent with the suggestion that atropine impaired theuse of a sensorimotor subsystem for spatial navigation, orimpaired vision, or both. Paylor and Rudy (72) obtainedsimilar results, and further suggested that the long swimtimes that resulted from excessive swimming in the periph-ery of the pool would be expected to fatigue the animals,resulting in further increases in platform search time.

In our experiments with NMDA and muscarinic antago-nists we measured the time swum in the periphery of thepool throughout hidden platform training using a computer-based automatic tracking system. The platform was never inthe periphery, and therefore could not be encountered by arat that spent all of its time swimming there. Rats given anNMDA or muscarinic antagonist spent 70–85% of the timeswimming in the periphery during training, a significantlygreater amount than controls (11,82,83). They also spent asignificantly greater proportion of the time swimming in theperiphery during the visible platform task. As might beexpected, measures of the percent of time swum in theperiphery correlated positively with both hidden and visibleplatform search time, and negatively with platform quadrantdwell time obtained during the probe trial (11,83). Theseresults extended the findings of Whishaw and Tomie (118)

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by showing that even when trained in the hidden platformtask, rats given an NMDA or muscarinic antagonist failed todevelop strategies that were appropriate for the task. Some-times their failure was especially striking, a point illustratedby rats that persisted in swimming around the perimeter ofthe pool throughout every training trial, never once encoun-tering the hidden platform (82).

The exact cause of this peculiar periphery swimmingbehavior is not known. It is possible that periphery swim-ming reflects a specific inability to form a spatial map.Alternatively, the fact that NMDA and muscarinic antago-nists, anxiolytic benzodiazepines (Cain, unpublished obser-vations), hippocampal, thalamic, or neocortical lesions(91,114) (Cain and Vanderwolf, unpublished observations),and both hypo- and hyperthermia (75) all increase swim-ming in the periphery could mean that thigmotaxic swim-ming is a general response to the compromised brainfunction that these treatments cause.

If a rat does not encounter the hidden platform on a giventrial it will not learn anything about the platform’s locationon that trial, perhaps leading to a biased measure of itsspatial learning ability. This is dealt with by placing therat by hand on the platform after any trial in which it failedto find the platform during the swim. When we attempted todo this with rats given NMDA or muscarinic antagonists we

found that most rats quickly jumped off the platform andcontinued swimming (11,83). This behavior seemed to havea strongly ‘‘driven’’ quality; in many cases the rats con-tinued to make continuous paddling motions with theirlimbs as they were being picked up and transported to theplatform and after they were placed onto it. These observa-tions seemed consistent with the well-known tendency forthese treatments to cause behavioral hyperactivity(27,33,107,116,120). When we measured spontaneousactivity in rats given NMDA or muscarinic antagoniststhey were markedly hyperactive (11,83). The explanationseemed to be that the drug-induced hyperactivity caused theanimals to produce continuous swimming movements,which carried them off the platform and back into thewater. This is an example of how independent measurestaken outside the water maze can help clarify observationsmade during maze training.

A few researchers have noted that rats given an NMDA ormuscarinic antagonist displayed unusual and abnormalbehaviors during training in the pool. Some rats had diffi-culty swimming and interacting adaptively with the hiddenplatform (111), or fell off the platform after climbing on(63). A striking example was the tendency to swim com-pletely over the platform and off the other side in onecontinuous motion as soon as it was encountered (Fig. 2).

FIG. 2. Videotape frames of rats swimming in a pool after being given an NMDA receptor antagonist. The frames proceed from left to right. The top threeframes illustrate a deflection; the hidden platform is darkened on the images for clearer visibility. The bottom three frames illustrate a swimover; the hiddenplatform is indicated by white dots on the images for clearer visibility.

TESTING THE NMDA, LONG-TERM POTENTIATION, 185

These ‘‘swimovers’’ had been described but not measuredand related to maze acquisition (20,62,63,111). Swimoversprobably resulted from the same hyperactivity that producedthe jumping-off behavior described above.

We also observed and measured ‘‘deflections’’, whichoccurred when rats given an NMDA or muscarinic antago-nist bumped into the hidden platform, deflected off it, andcontinued swimming without attempting to climb on(Fig. 2). We found that swimovers and deflections occurredfar more frequently in the drugged rats than in controls. Insome drugged groups more than half of all contacts with thehidden platform resulted in a swimover or deflection(11,83). The incidence of swimovers and deflections corre-lated negatively with measures of learning in both thehidden and visible platform tasks, and positively withother measures of sensorimotor disturbance (11,83). Insome cases the correlations were very high. For rats givenan NMDA antagonist the correlation between the percent ofcontacts with the platform that were deflections or swi-movers, and summed platform search time wasþ0.99 (83).

The overall picture that emerged was that rats that hadpoor acquisition measures in the maze task exhibited moresensorimotor disturbances, and rats that acquired the taskwell had few if any sensorimotor disturbances. When theperformance of the same rats was evaluated under the samedrug treatment in sensorimotor tasks conducted outside thepool, rats that exhibited sensorimotor disturbances duringtraining in the pool also exhibited sensorimotor disturbanceson tasks conducted outside the pool (11,83). This indicatedthat the sensorimotor disturbances were not unique to thetraining conditions in the pool.

The major source of information for the formation of aspatial map in the water maze task is vision (61,71,96). BothNMDA and muscarinic antagonists have been reported todisrupt visual discrimination performance (7,8,16,25,50,56,66,77,98,99), suggesting that these agents might affectwater maze acquisition by producing disturbances in vision.Morris evaluated this possibility for an NMDA antagonistby training rats to discriminate between stable and sinkingplatforms in the maze pool on the basis of their visualappearance, but found that an NMDA antagonist had noeffect on the rate of learning this discrimination (63,64). Inan experiment similar to Morris’, NPC17742 did not affectthe rate of learning to discriminate between stable andsinking platforms during a 9-day training phase (13). How-ever, this NMDA antagonist reduced the ultimate level ofchoice accuracy in the post-learning phase of testing fromdays 10 through 18, after the learning curves had reachedasymptote. The reduction was small, and the animals’choice accuracy remained well above chance (. 80%correct), which is consistent with the good performance ofrats already familiar with the general strategies required inthe task. In the Morris study rats carried osmotic minipumpsfor delivery of the antagonist, which limited testing time to10 days. Therefore Morris’ rats had not been tested duringthe asymptotic phase.

We also examined the effect of NPC17742 on visual dis-crimination in a Y-maze and found that the antagonistreduced the accuracy of well-established discrimination ofvertical vs. horizontal stripes, but had no effect on a blackvs. white discrimination (13). This suggested that NMDAreceptors were not required for simple discriminationsbased on differences in total luminous flux, but that they

were required for discriminations based on pattern differ-ences when luminous flux was held constant.

These results suggested that NMDA activity was requiredfor optimal performance of the visible platform and patterndiscrimination tasks. The rats’ above-chance visual discri-mination performance under an NMDA antagonist probablyallowed them to acquire the water maze task effectivelyafter nonspatial pretraining (see below). The visual discri-mination deficit might have hampered the maze perfor-mance of naive rats given the same treatment. Similarly,muscarinic antagonists disrupted visual discrimination(7,8,16,25,50,56,77), and in tests conducted in a watermaze pool, muscarinic antagonists impaired visual discri-mination performance and decreased visual orienting andsearch behaviors (31,72,118). Although this review is notdirectly concerned with spatial learning in the radial armmaze, results from visual tests of the kind discussed abovewould appear to be relevant to this task. The findingssuggest that radial arm maze deficits obtained withNMDA or muscarinic antagonists need to be considered inthe context of any visual processing deficit the antagonistscause.

Taken together these studies suggested that the problemof sensorimotor disturbances is an important one in thisresearch. Some researchers have suggested that poor acqui-sition scores in rats given NMDA or muscarinic antagonistscould have resulted from the sensorimotor disturbances(41,58,72), and have pointed out that animals neverthelesslearned a good deal about the task in spite of the drugtreatment (41). Certainly the effect of deflections, swimov-ers, and excessive swimming in the periphery would be tolengthen search times and reduce the information availableto rats about the location of the hidden platform compared torats that swam away from the maze wall and found thehidden platform readily, and climbed onto and remained onthe platform whenever it was encountered. Sensorimotordisturbances could be expected to produce poor mazeacquisition scores in rats even if the drug treatments hadno direct effect on engram formation. However, this doesnot exclude the possibility that the drug treatments alsomight have disrupted neural mechanisms of engram forma-tion for spatial learning. In other words, the drug treatmentsmight have had parallel effects on both sensorimotormechanisms required in the task and on neural mechanismsof engram formation. Evidence of sensorimotor distur-bances due to an experimental treatment can provideimportant information about treatment effects on neuralmechanisms for instinctive behavior, but cannot by itselflead to definitive conclusions about drug effects on theengram.

How might NMDA or muscarinic antagonists affectinstinctive behavior? NMDA receptors are widely distribu-ted in the central nervous system, including virtually all ofthe neocortex and many subcortical sensory and motor areasincluding spinal cord (57,59). In animals NMDA antago-nists can cause hyperactivity, stereotypy, ataxia, musclerelaxation, catalepsy, and other behavioral abnormalities(11,19,27,33,36,42,83,101,107,111,120). They can alsodisrupt normal behavior-related hippocampal electricalrhythms in the rat (53), and cause agitation, confusion,poor concentration, paranoia, ataxia, delusions and halluci-nations in humans (30,97). Many sensory and motormechanisms depend on NMDA receptors for normal

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function (35). NMDA antagonists can markedly alter motorsystem function by altering dopamine release in the basalganglia and hippocampus (29,119), and can interfere withthe processing of visual and somatosensory input (81,88).Disruption of these mechanisms by NMDA antagonistscould contribute to the sensorimotor disturbances found inwater maze studies.

Muscarinic receptors also are distributed widely in thecentral nervous system (26), and muscarinic antagonistscause a variety of behavioral alterations that can affect arat’s ability to perform the behaviors required in the watermaze task (31,72,102,109,117,120). These findings areconsistent with the fact that acetylcholine-sensitive neuronswith muscarinic receptors are frequently found in primarysensory areas of neocortex (48,49), and muscarinic antago-nists block a major component of cortical activation (103).

In sum, both NMDA and muscarinic antagonists causesimilar sensorimotor disturbances in instinctive behaviorsrequired for performance of the water maze task. The natureof the disturbances suggests that they might have contribu-ted to the poor acquisition scores reported for animals giventhese agents. However, the existence of sensorimotor dis-turbances does not exclude possible effects of the antago-nists on mechanisms of engram formation.

MODIFICATION OF INSTINCTIVE BEHAVIOR BY PRETRAINING

An advantage of the water maze task is its proceduralsimplicity and the speed with which normal rats acquirethe task. However, as Whishaw and Mittleman (115) haveemphasized, the water maze task is more complex than itappears, and the spatial navigation strategies used by rats aremore numerous and complex than had been thought. The ratmust learn a number of things before it can deal effectivelywith the task, the most important of which are to swim awayfrom the wall and to climb onto and remain on the platformas soon as it is encountered. Only then can the rat effectivelylearn the location of the hidden platform in relation to cuesin the environment.

In terms of the earlier discussion, learning these generalstrategies involves changes in instinctive behavior. A naiverat normally spends the first few trials swimming close tothe pool wall, scratching at it and trying to find a way out.This behavior has the obvious goal of getting the rat out ofthe water, but it may also reflect the general tendency of ratsto locomote thigmotaxically toward the perimeter for pro-tection whenever they are in a large open space (6). Arequirement for successful acquisition of the water maze isthat the rat learns to curtail its instinctive tendency to swimthigmotaxically, and that it learns to search the open areas ofthe pool away from the wall. As indicated earlier, rats givena variety of drug treatments or brain lesions appear to havegreat difficulty learning to swim away from the wall, whichprevents them from coming into contact with the hiddenplatform. When they do contact the platform, there is a goodchance they will swim over it or deflect off.

Whishaw and Kolb (114) first pretrained decorticate ratsin a visible platform version of the task to reduce theirstrong tendency to swim thigmotaxically. In subsequenttraining in a hidden platform version of the task, thepretrained rats searched the inner areas of the pool thatcontained the hidden platform, but could not learn to swimdirectly to the platform.

Morris (63) and Whishaw (110) were the first to reportthat prior experience with the general task requirementsgreatly improved acquisition of the task when rats subse-quently were trained under either NMDA or muscarinicantagonism. In the Morris study the prior experience, whichwas given in the absence of any drug treatment, was termednonspatial pretraining because black curtains around thepool occluded the room cues, and the hidden platform wasmoved to a new location after every trial. During thepretraining the rats learned to swim away from the walland to climb onto and remain on the platform when it wasencountered (110).

In the Whishaw study (110) rats were first trained in thestandard hidden platform task in one pool in the absence ofdrug treatment, then given drug treatment and trained in thehidden platform task in a second pool in another room with anew set of cues. The results were the same in the twostudies: rats that were familiar with the general task strate-gies had much shorter search latencies when trained underNMDA or muscarinic antagonism than naive rats did.Morris (63) measured instances of falling off the platformand found that falling off the platform was almost elimi-nated in the nonspatially pretrained rats, suggesting that thepretraining reduced the incidence of sensorimotor distur-bances caused by drug treatment. However, in both studiesthe experienced rats had slightly but significantly longersearch times compared to controls.

In more detailed dose-response experiments Morris andcolleagues found that nonspatially pretrained rats couldlearn the location of the hidden platform when trainedunder NMDA antagonism, as indicated by platform quad-rant dwell data (3,4,20). When we examined the effects ofNMDA or muscarinic antagonists in nonspatially pretrainedrats we found that the pretrained rats had few if anysensorimotor disturbances, and acquired the task as rapidlyas controls, as indicated by three different measures ofacquisition (11–13,82,83). Naive rats given the same drugtreatments displayed numerous sensorimotor disturbancesand obtained very poor maze acquisition scores which, asdescribed above, correlated strongly with the sensorimotordisturbances. The fact that a small elevation in platformsearch time remained in Morris’ nonspatially pretrained ratstrained under NMDA antagonism might be explained by thelarge pool and small hidden platform used in that experi-ment, which could be expected to make the maze task moredifficult than tasks that used a smaller pool and a largerhidden platform (54). With an especially difficult maze task,a drug treatment that interfered with sensorimotor mechan-isms could be expected to impair behavioral performance ofthe task to some extent, by increasing thigmotaxic swim-ming for example. This could lead to increased search timewithout necessarily preventing the rat from learning wherethe platform was located.

Supporting this interpretation about task difficulty, workby Schallert has shown that an easier version of the task thatprevented the learning of inappropriate strategies was readilyacquired by naive rats given either atropine or hippocampallesions (21,84). Rats were given a treatment and first trainedusing a very large hidden platform that nearly filled the pool.Each day thereafter the hidden platform was made smaller,and it effectively shrunk into one quadrant. Quadrant dwellmeasures showed that the rats had learned the location of thehidden platform using a spatial learning strategy.

TESTING THE NMDA, LONG-TERM POTENTIATION, 187

Taken together, these findings indicate that neitherNMDA nor muscarinic activity are required for a rat to beable to learn the location of a hidden platform, confirmingthe conclusion initially arrived at by Keith and Rudy (41).They also suggest that separation of the phase of learningwhen the rats learn the general strategies required in the taskfrom the phase when they learn the location of the hiddenplatform is a useful approach to understanding the complexnature of this apparently simple task. Nonspatial pretrainingappears to involve learning that modifies instinctive beha-viors in ways that allow rats to perform adaptively in thetask when challenged with treatments that normally inter-fere with those instinctive behaviors in naive rats. Oneindication of the learning that rats undergo during nonspatialpretraining is the decrease in platform search time byapproximately two-thirds from day 1 to day 4 of nonspatialpretraining (11,20,63,83).

Although the exact neural basis for the striking effectsthat result from pretraining is not known, the effects ofpretraining have been known since at least 1959, whenHerz showed that prior experience with a pole climbingtask eliminated the debilitating effects of muscarinicantagonists on climbing behavior (39). Similarly, Steinbergshowed that prior experience with the test apparatus elimi-nated the effects of combined administration of ampheta-mine and barbiturate (90). There have been other similardemonstrations of the protective effects of prior experienceon instinctive behaviors that are normally disrupted byscopolamine (22) or brain lesions (86).

It appears that the pretraining experience must be with thespecific apparatus that the animals subsequently will betested with under drug treatment. Nonspatial pretraining inthe maze pool had no effect on the motor ataxia that wascaused by NMDA or muscarinic antagonists, as measuredby the rat’s ability to walk on a narrow wooden beam(11,83). Similarly, rats that were nonspatially pretrained ina large circular pool were markedly impaired in a visibleplatform task in a small rectangular glass aquarium whengiven scopolamine (Caldji and Vanderwolf, unpublishedobservations). The poor performance of nonspatially pre-trained rats under these conditions confirms that the drugswere having the expected effects on the nervous system andon behaviors tested in tasks other than those on which therats were pretrained.

It is not known which components of the nonspatial pre-training experience are important for the pretraining effectin the water maze task. An ongoing experiment designed tofractionate the components of the nonspatial pretrainingexperience by giving different groups of rats only a partof the total nonspatial pretraining experience (swimmingin the pool with no hidden platform present; climbingfrom the water onto the platform; being allowed to sit onthe platform; etc.) suggests that the different parts of thenonspatial pretraining experience are equally beneficial,but that no single part of the experience is as beneficial asthe whole nonspatial pretraining experience (Hoh and Cain,unpublished observations).

NMDA RECEPTOR-MEDIATED LTP AND SPATIAL LEARNING

A number of the experiments discussed aboveincorporated tests of the NMDA/LTP hypothesis of spatiallearning into the design. The hypothesis that NMDA

receptor-mediated LTP underlies spatial learning emergedfrom experiments that showed that the same dose of achronically administered NMDA antagonist that blockedLTP in the dentate gyrus also impaired acquisition of thewater maze task (63,64).

In the earlier experiment (64), which used naive rats, theNMDA antagonist produced a marked elevation of hiddenplatform search time. In the latter experiment (63), whichused nonspatially pretrained rats, the same dose of antago-nist produced a smaller but statistically significant elevationin hidden platform search time. Subsequent work from thesame laboratory indicated that the dose used in theseexperiments was higher than required to block LTP, andthat if a smaller dose was used LTP continued to be blocked,but nonspatially pretrained rats nevertheless learned thelocation of the hidden platform (4,20).

We evaluated the role of NMDA receptor-mediated LTPin spatial learning by using two different competitiveNMDA antagonists (NPC17742 and CGS19755) in differ-ent groups of rats, each in a dose sufficient to completelyblock LTP in the dentate gyrus. The same rats tested forLTP were then nonspatially pretrained and subsequentlytrained in the water maze task after being given the samedose of NMDA antagonist that had been found to blockLTP. As expected from earlier work (11,83), the animalsacquired the maze task as rapidly as controls, as indicated bythree different measures of maze acquisition (11,12,82).This is consistent with the recent finding thatfyn geneknockout mice, in which hippocampal LTP is blunted orabsent, can nevertheless acquire the water maze task (40).

As originally proposed, the NMDA/LTP hypothesisfocused on LTP induced in the dentate gyrus of the hippo-campus. The question arises whether similar findings wouldbe obtained with LTP induced elsewhere in the hippocam-pus, or outside the hippocampus. Other synapses in the hip-pocampus and in the visual neocortex slice preparation alsosupport NMDA receptor-mediated LTP (2,34), and it ispossible that LTP at these sites is important for spatiallearning. The concentration of AP5 required to block LTP inthe CA1 region of the hippocampus (34) and in the visualneocortex slice (2) might be less than that required to blockLTP in the dentate gyrus (64). This suggests that a systemi-cally administered NMDA antagonist that blocks LTP in thedentate gyrus might also block LTP in CA1 and the visualneocortex.

Taken together, these results suggest that NMDA recep-tor-mediated LTP in the hippocampus and visual neocortexmight not be required for spatial learning in the water maze,but they do not exclude the possibility that this form of LTPcontributes in a non-essential way to spatial learning. If thisis true, there must be an alternative mechanism for estab-lishing the engram for the location of the hidden platform.

Recently the suggestion was made that an alternativenavigation mechanism based on inertial (dead reckoning)navigation could help account for spatial learning, andthat the recalibration of such a mechanism in the secondroom of a two-room water maze task might not requirehippocampal LTP (5). The suggestion was based on pre-liminary data indicating that such a mechanism might beavailable to rats in the water maze task (108). If true, thiscould explain why NMDA antagonists do not prevent rapidlearning of hidden platform location in the second room (3).In a test of this possibility we trained rats in a stable hidden

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platform task, with black curtains around the pool toeliminate visual cues. Different groups searched for a plat-form made from acrylic plastic and weighted by a brick, or asimilar platform containing a strong magnet. Both groupswere trained over many days, but neither group gave anyevidence of preferential swimming in the platform quadrantduring probe trials, and platform quadrant dwell time was atchance (Cain, Beiko and Boon, unpublished observations).When the curtains were removed and the rats given furthertraining they all achieved short search times, and signifi-cantly increased their dwell time in the platform quadrantduring the probe trial, indicating that they were capable ofacquiring the task when allowed to use visual cues. Thissuggested that when visual cues were unavailable in thewater maze task the rats could not make use of inertialnavigation or magnetic field information to solve it.

To date, the focus of research on LTP and its relation tolearning has been on the hippocampus. Karl Lashley, speak-ing of the role of the primary visual neocortex and learning,concluded that engrams for sensory discrimination taskswere established in the sensory neocortex, and suggested‘‘that the same cells which bear the memory traces arealso excited and play a part in every other visual reactionof the animal’’ (52). The possibility that engram formationfor other forms of learning could occur in the cortex hasbeen widely discussed (89), but to date there have been nostudies of the possible relation between neocortical LTP andlearning. There is considerable evidence that damage to avariety of cortical areas produces acquisition deficits in thewater maze that are comparable to those produced byhippocampal damage (23,43–46,85,92,93,95,114), andthat some learning deficits formerly attributed exclusivelyto the hippocampus are now known to result from damage tocortex (28,55). It seems that it would be fruitful to explorethe possibility that neocortical LTP might be related tospatial learning.

LTP has been observed in the neocortical slice prepara-tion (2), but its induction is more difficult than in thehippocampus, and in an extensive series of experiments inawake, behaving rats it was not possible to induce LTP inthe neocortex (74). More recently it has proven possible toinduce LTP in the neocortex, but this required manystimulations each day for up to 15 days (73). Thus LTPinduction in the neocortex of awake, behaving rats requiresmuch more stimulation than in the hippocampus. Theneuropharmacological mechanism of this form of neocor-tical LTP is not known.

SEARCHING FOR THE ENGRAM

Study of the involvement of muscarinic and serotonergicmechanisms in behavior has shown that the effect of com-bined antagonism of both receptor types was generallymuch greater than the effect of antagonism of either systemalone, even if antagonism of one system produced no deficit(68,76,78,102,106). Combined antagonism of muscarinicand serotonergic mechanisms produced a severe deficit thatmay be analogous to ‘‘global dementia’’ in the rat, whichprobably resulted from removal of the two major systemsinvolved in activation of the cerebral cortex (102). Theseresults were consistent with the idea that the cerebral cortexis involved in the organization of the instinctive behaviorsrequired in tasks such as the water maze (52,102,105). The

fact that combined muscarinic and serotonergic antagonismoften produced a much greater behavioral deficit thanantagonism of either system alone suggested that the lossof one system was compensated for, at least in part, by theother (106).

However, to date no water maze study has examined theeffect of combined antagonism of two receptor types innonspatially pretrained rats. In doing these experiments,we found that nonspatial pretraining did not protect ratsfrom the effects of combined muscarinic and serotonergicantagonism in the water maze task, in spite of the fact thatnonspatially pretrained rats acquired the task readily ifeither antagonist was given by itself (Beiko and Cain,unpublished observations). The same results were obtainedwith combined antagonism of muscarinic and NMDAreceptors (Cain, unpublished observations). In contrast tothe studies described in the previous paragraph, selectivedamage of both hippocampal cholinergic and hippocampalserotonergic afferents produced only a mild impairment inthe water maze task in naive rats (67). Taken together, thedata suggest that there are multiple systems for productionof the instinctive behaviors required in the water maze task,and that a major part of these systems resides outside thehippocampus. Further, it appears that hippocampal damagedoes not prevent the formation of spatial maps, but insteadinterferes with the use of that information to guide locomo-tion through the environment (24,84,91,94,112,113).

The question of whether a specific type of synapse is asite of engram formation for spatial learning is difficult toanswer definitively. In light of the emerging evidence for theinvolvement of widespread circuitry involving a number ofdifferent receptor types in the various components of watermaze acquisition, this becomes an important question. How-ever, the very requirement that both pre-existing neural cir-cuits for instinctive behavior, as well as an engram for thespecific location of the hidden platform, are required foracquisition of the water maze makes it impossible to iden-tify the specific sites of change that constitute the engramfrom experiments with neurotransmitter antagonists inintact, behaving animals. To illustrate the nature of the pro-blem, the following possibilities are consistent with what isknown about the role of muscarinic and NMDA receptors inthe water maze task.

(1) NMDA and muscarinic receptors function in neuralcircuits involved in the processing of visual and other sen-sory information important for the task. Antagonism ofeither receptor population alone causes a partial sensoryimpairment, but enough sensory processing capabilityremains to allow nonspatially pretrained rats to acquirethe maze task using a spatial learning strategy (Fig. 1).When both receptor populations are antagonized, a greatersensory impairment likely is produced. This leaves insuffi-cient sensory processing capability to allow use of a spatiallearning strategy, and the rats fall back on a taxon strategy,such as swimming away from the wall at a distance thatwould allow interception with the hidden platform. Thisprobably requires less sensory processing because itdepends on larger, closer cues (e.g., the maze wall).

(2) NMDA and muscarinic receptors, together withremaining brain mechanisms, are directly involved in learn-ing the general behavioral strategies required in the mazetask; that is, they are involved in the learning that occursduring nonspatial pretraining. Both receptor populations are

TESTING THE NMDA, LONG-TERM POTENTIATION, 189

required for rapid learning of the strategies, as antagonismof either one alone prevents naive rats from learning thestrategies within a single session (11,83). NMDA ormuscarinic synapses might be modified during this learningprocess (Fig. 3A,B), or they might be unmodified but

associated with other neurons whose synapses are modified(Fig. 3C–F).

(3) NMDA or muscarinic receptors are directly involved,together with remaining brain mechanisms, in learning theexact spatial location of the hidden platform. For rats thatare already familiar with the general behavioral strategiesrequired in the maze task, only one of these receptor popu-lations is required. NMDA or muscarinic synapses might bemodified during this learning process (Fig. 3A,B), or theymight be unmodified but associated with other neuronswhose synapses are modified (Fig. 3C–F).

The available data do not allow a decision about which ofthese possibilities holds, or which type of neural circuit orsynapse undergoes modification during training on the task.There is no reason to believe that these possibilities areexhaustive or mutually exclusive, and it seems likely thatall three possibilities might hold. This would not be surpris-ing, given the widespread anatomical distribution of NMDAand muscarinic receptors and the profound disruptions thatNMDA and muscarinic antagonists cause in hippocampaland neocortical electrographic activity and instinctivebehavior (11,53,83,102,103).

Neurotransmitter antagonist or lesion effects by them-selves do not allow conclusions about which specificsynapses form the engram for a particular form of learning.Evidence from neuroanatomy or functional electro-physiology, and preferably both, is needed in addition. Forexample, a series of experiments that obtained behavioralevidence from use of a specific neurotransmitter antagonistor brain lesion, together with neuroanatomical evidenceof plastic change in the brain structure of interest, andelectrophysiological evidence of a functional change inthe structure, could be highly informative. The choice ofbehavioral task is also important. The specific behavioralstrategies required in the task should be well understood,and ideally the task should be amenable to fractionation bypretraining or some similar approach that would allow aclearer understanding of the neural mechanisms of thevarious components of task performance.

CONCLUSIONS

The ability of an animal to acquire the water maze taskdepends on its being able to apply instinctive behaviors toperformance of the task in an adaptive manner, as well as itsability to learn the specific information required by theparticular task conditions. The instinctive behaviorsundergo modification as the rat learns the general strategiesrequired in the task. Thus, learning the water maze taskdepends on activity in pre-existing neural circuits that gen-erate behaviors appropriate for the task, as well as plasticchanges in those neural circuits, and the storage of newinformation about the spatial location of the hidden plat-form. The sites of engram formation for learning thelocation of a hidden platform in the water maze are notknown. Recent experiments have shown that neitherNMDA nor muscarinic receptors are required, althougheither may normally contribute to this form of learning.The available evidence suggests that at least some of theplastic changes involved in learning the general strategiesrequired in the task occur in existing neural circuits situatedin widespread areas of the brain, perhaps including sensory

FIG. 3. Hypothetical neural circuits that might be involved in learning thegeneral behavioral strategies required in the water maze task or the spatiallocation of the hidden platform. Circles represent cell bodies and dendrites;lines represent axons. Filled terminals and apposed darkened regions on thecell body represent synapses that are modifiable in the course of learning(plastic). Unfilled terminals represent synapses that are effective but unmo-difiable. Ach, acetylcholine-releasing neuron; Glu, glutamate-releasingneuron; musc, muscarinic receptors; NMDA,N-methyl-d-aspartate recep-tors; dep, depolarizing (excitatory) synapse; unknown, synapse for whichthe neurotransmitter and receptor type are unknown; T, target neuron. Thehypothetical circuits are conceptual representations only, and do not neces-sarily represent synapses proposed to be required for learning. However,(B) is similar to the NMDA coincidence detector that is hypothesized tounderlie some forms of learning (9,18). The circuits are intended to providea simplified representation of which synapses might undergo plasticmodification to support learning. Activation of muscarinic and NMDAreceptors by acetylcholine or glutamate, respectively, often has excitatoryeffects (35,48,49). (A) Circuit in which a synapse containing muscarinicreceptors undergoes modification during behavioral training. (B) Circuit inwhich a synapse containing NMDA receptors undergoes modificationduring behavioral training. Should this occur when another synapseprovides coincident depolarization (‘‘dep’’), this would resemble a coin-cidence detector (18). (C,D) Circuits in which a synapse with unknownneurotransmitter and receptors undergoes modification during behavioraltraining. In A–D scopolamine or NPC, as appropriate, will block transmis-sion, producing behavioral impairments. In (E) and (F) muscarinic orNMDA receptors do not form modifiable synapses themselves but doplay an essential role in a Hebb-synapse learning mechanism (37).Simultaneous activation of neurons 1 and 2 leads to increased transmissionat the modifiable synapse as a result of coincident firing of neuron 1 and thetarget neuron. In (E) and (F) scopolamine or NPC, respectively, willinterfere with transmission between neuron 2 and the target neuron, thuspreventing the neuroplastic modifications that underlie learning. Adaptedfrom Fig. 8 in Ref. (102).

190 CAIN

and motor structures in the neocortex and elsewhere, mak-ing them difficult to distinguish from existing sensorimotormechanisms. The findings indicate the difficulty of inferringthe occurrence or non-occurrence of learning from behavior,and the difficulty of causally linking the action of particularreceptor populations with the formation of specificmemories.

ACKNOWLEDGEMENTS

Supported by a grant from NSERC. Those familiar withCase Vanderwolf’s career will recognize his many contri-butions to the content of this essay. It is a pleasure toacknowledge these contributions and to have attended thefestschrift marking his 60th year.

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