Psychobiology of memory: Towards a model of memory formation

Biobehavioral Reviews, Vol. 1, pp. 113--136, 1977. Copyright © ANKHO International Inc. All rights o f reproduction in any form reserved. Printed in the U.S.A.

Psychobiology of Memory: Towards a Model of Memory Formation MARIE E. GIBBS 1 AND KIM T. NG

Department o f Psychology, La Trobe University, Bundoora, Victoria, 3083, Australia (Received 4 June 1977) GIBBS, M. E. AND K. T. NG. Psychobiology of memory: towardsa model of memory formation. BIOBEHAV. REV. 1(2) 113-136, 1977. -- Evidence from the use of inhibitory drugs and antagonists to these drugs suggest a three phase model of memory formation, with phases sequentially dependent. Hyperpolarization due to potassium conductance changes following learning are postulated to underlie the formation of a short-term memory phase. Hyperpolarization associated with sodium pump activity appears to be involved in the formation of the succeeding labile phase. Long-term memory formation appears to involve sodium pump associated amino acid uptake occuring during labile phase formation. Protein synthesis is accepted as underlying the formation of long-term memory. Although reference is made to available evidence, in the literature, this review deals in detail with evidence from our laboratories. Short-term memory Labile memory Long-term memory Hyperpolarization Sodium pump activity Protein synthesis Amino acid uptake

THERE is a reasonable, although by no means universal concensus among researchers in the field of memory and learning for the view that memory formation involves two related but distinct stages: a short-term (primary) memory stage and a long-term (secondary) stage. In the field of cognitive psychology, the distinction between the two stages has been based on behavioral data from human subjects and on mathematical and quasi-mathematical postulates [8, 20, 21, 120]. The same distinction has also been suggested on the basis of physiological data and neurological postulates [61,82]. Short-term memory (STM) is said to persist for approximately 30 sec to 1 min and may be extended by a process of rehearsal, while long-term memory (LTM) is relatively permanent. A further stage of sensory (iconic) memory has also been suggested [107], lasting approximately 1 sec under optimum conditions and preceding STM.

Potassium conductance changes

animal subjects, provide findings at least partially consistent with a sequential dual-trace model.

Parallel Dual-Trace Model However, the results are equally, and according to some, more consistent with a parallel dual-trace hypothesis [9,85]. Under this hypothesis, learning simultaneously initiates STM and LTM traces. STM traces develop rapidly but decay while LTM traces develop more slowly and are relatively permanent. With this model, inhibition of the STM processes does not lead to consequential inhibition of LTM processes, and STM traces merely serve the function of providing immediate post-learning recall facilities, since permanent neurobiological coding of information requires time for completion.

Single Trace Dual-Process Model McGaugh and Gold [87] and Gold and McGaugh [56] suggest the alternative interpretation that the timedependence of memory processes may reflect the biological utility of selecting biologically significant information for permanent storage. Gold and McGaugh [56] propose a single-trace dualprocess model of memory in which it is argued that learning produces a single memory trace which develops rapidly and decays rapidly unless decay is arrested by non-memory processes also instigated by the learning experience and related to the significance of the experience. RA resulting from post-learning treatment may reflect direct interference with the memory trace itself or failure to consolidate the trace as a result of interference with the non-memory modulator processes. Thus there is no independent STM

Sequential, Dual-Trace Models Similar models have been generated from research into the physiological and pharmacological bases of memory formation where the focus has been on the biological status of the distinction between STM and LTM. The model proposed by Hebb [61] involves a sequential dual-trace development of memory. Recent experience produces a labile short-term process which sustains the experience and promotes the development of a relatively long-term trace. Studies on retrograde amnesia (RA) induced by postlearning treatment with electroconvulsive shock (ECS), convulsant agents, drugs affecting RNA and protein synthesis, drugs affecting neuro-transmitter systems and neurophysiological processes, and hormones, primarily with 1Formerly Marie E. Watts. 113

114 trace, and what appears to be a STM retention function is "simply a special case in which either the experience produces minimal nonspecific influences or those influences are blocked" [56]. One major modulator process accompanying learning is general central nervous system arousal. Particularly in the case of avoidance based training hormonally mediated specific physiological effects are also indicated, related to the release of adrenocorticotropic hormone (ACTH), vasopressin, peripheral epinephrine and gonadotropic hormones among others. Peripheral norepinephrine, ACTH and vasopressin administered after learning have been shown to facilitate retention of a single trial passive avoidance task in mice, the dose response function being inverted U-shaped [56]. Increasing the intensity of the avoidance stimulus produces disruption of retention with a dose that facilitates retention with low intensity avoidance stimulus. Thus motivational modulation of the memory function is infered to be disruptive at low and high intensities, with an optimum facilitative intensity in between. Following avoidance learning there is a transient loss of the learned avoidance response [68] known as the Kamin effect or Kamin deficit. This has been interpreted by some authors as being due to suppression of the learned reaction through conditioned fear or incubation of anxiety. Kamin effects and Kamin-like effects in passive avoidance tasks (cf. [25]) are interpreted by Gold and McGaugh in terms of the modulating processes. Immediately after learning, weak motivational levels lead to weak initial modulating processes. The development of these modulating processes is slow. Thus the memory trace is not strengthened until later into the post-learning period. A decay of the memory trace prior to modulating processes strengthening it again will produce the temporary performance deficit characteristic of the Kamin effect. Multitrace models would interpret the effects in terms of a transition from one tract to the other (e.g. [25,92] ). While the single trace model appears to enjoy theoretical parsimony when compared with multiple trace models, this advantage may be more ephemereal than real. Motivationally related modulator processes undoubtedly play a role in the ultimate consolidation of memory but their elevation to a central role in explaining existing experimental data demands greater theoretical specificity than is currently provided by their proponents, especially with respect to (1) the nature of such processes, (2)the possible biological mechanisms underlying them, and (3) the relationship between these and corresponding characteristics of the memory processes. Otherwise explanations under the model may entail more assumption than is warranted in a model claiming parsimony. Consider, for example, the finding that ECS administered 8 sec after learning of a single trial passive avoidance task involving footshock produces severe retention deficits 1 hr after learning, while the same intensity ECS administered 20 sec after learning results in similar retention deficits not 1 hr but 6 hr after learning [88]. It is not sufficient to attribute the former to the greater lability of the memory trace immediately after learning [56,87]; it merely constitutes a restatement of that which is to be explained. The significant issue is why and in what sense the trace is more labile, and what the difference is between the trace at 1 hr after learning and at 6 hr after learning. Gold and McGaugh [56] suggest that the reaction deficit at 1 hr after learning is due to disrupted modulator processes initiated by the learning experience. Lability must then imply greater susceptibility of the trace

GIBBS AND NG to modulating influences. What then is the basis for the effect of ECS at 6 hr when administered 20 sec after learning? The McGaugh and Dawson [86] model involving multiple traces would seem to be more viable. Three Phase Models

Halstead and Rucker [59,60] postulate three stages in the formation of memory. On the basis of existing evidence from electrophysiological and molecular-biological research, they suggest: a dynamic stage peaking within a few milliseconds after learning and completely dissipated by 1 sec; an intermediate phase developed completely within the first minute following learning, lasting approximately 60 min, and dissipating by 24 hr; and, a permanent phase, possibly initiated within the first few minutes following learning, and fully developed by 24 hr. The first phase appears to correspond in both time parameters and in characterisation to the sensory memory store of Sperling [107], although it is not clear what is meant by dynamic. The second and third phases correspond to the STM and LTM traces of McGaugh [85] and Barondes [9]. While these two stages are sequential, they are not considered sequentially dependent - a parallel dual-process model, but for the initial dynamic stage. The retention level at any point in time is said to be the sum of the retention level at each phase, and a temporary reduction in retention occurs when the total retention level falls below a given threshold. This mainly occurs in the cross-over between the developmental trace of one phase and the decay trace of the preceding phase. Behaviorally, these temporary reductions in performance yield the Kamin effect [25,92]. The Halstead model has been subsequently extended by Matthies [78, 79, 80], the dynamic stage being equated with "systemic synaptic activity" defining a short-term memory, the intermediate phase with "synaptosomal regulation," and the final phase with some form of "neuronal regulation." Booth [19] arrives at a model of memory formation, similar to that of Halstead and Rucker, but possibly more substantially based on empirical grounds. On the basis of decay functions following electrical (ECS) and chemical disruption and the effects of varying the time of administration of these disruptive agents, he suggests a first phase of memory formation, sustained electrically, susceptible to ECS disruption, and developing within the first 0.1 to 1 sec after learning. Decay of this phase is substantially completed within 1 hr but complete decay may take as long as 24 hr. This electrically mediated phase is followed by two non-electrically mediated phases: a labile phase, inferred from the disruptive effects of anaesthetics, developed completely between 1 min and 1 hr after learning, and maintained from an hour to a few days; and, a permanent, protein synthesis-dependent phase, susceptible to disruption by protein synthesis inhibitors, and developed within 1 hr to 10 or more days following learning. There are clearly significant differences in time parameters between this and the Halstead and Rucker [59,60] model. Squire and Barondes [112] and Squire [111] argue for a short-term phase of memory formation persisting for at least 3 hr after learning. In arriving at the temporal parameters, Booth [19] in particular is defining a range of times suggested by a number of studies. He points out that the variation in temporal parameters from different studies may be due to species or anatomical differences, to the proportion of

PSYCHOBIOLOGY OF MEMORY

115

brain capacity pre-empted for the learning in the species used, or complexity of the processes involved, as defined by the tasks used. He also points to the equally likely, but not exclusive possibility that the variation in temporal parameters from different studies may reflect the confounding of a n u m b e r of additional stages in the memory formation sequence with traditionally inferred ones. Squire [ 111], in arguing for the status of short-term memory as a biological entity distinct from long-term memory, concludes that the "most complete and satisfying" answers to questions about memory are to be found in experimental approaches involving enquiry at both the behavioral and the cellular level. Temporal parameters remain the principal arbiter in the choice of terminology for different phases of memory formation by different theorists. In the light of Booth's [19] observations, temporal parameters may be highly misleading in a research environment where neither experimental task, nor experimental subject, nor experimental paradigm is comparable from one study to another. In this context some sympathy may be felt for Gold and van Buskirk's [57] view that retrograde effects on memory may not provide direct information about temporal properties of memory processes, particularly when, as they point out, RA gradients are affected by the severity of amnesic treatments. On the other hand, as argued earlier, an interpretation of the time-dependent nature of post-learning effects on memory in terms of modulator processes is no more satisfactory.' Undoubtedly all of the factors suggested by Booth [ 19] are relevant. The fact that very few of the studies reported in the literature employ a reasonably full time course of retention and of administration for any given experimental treatment, for any given task, for any given species adds confusion to what is already a confusing picture. In the search for species and behavioral generality, specificity of information may be sacrificed. What is required is a systematic exploration at both the behavioral and t h e cellular level with one species and one task. This will generate an initial "family of curves" of the sort which Gibbs and Mark [50] regard as useful but not sufficient to the problem of resolving the question of retrograde amnesia. This has to be dealt with in conjunction with information regarding the nature of the treatment involved. Generalizations to other species and to other tasks for the same species gain in interpretative value when viewed within the context of a stable experimental and theoretical framework. In this paper we review findings from studies conducted from our laboratories within this experimental philosophy. BASIC EXPERIMENTAL

PARADIGM

The paradigm adopted for most of the experiments reported here follows that introduced by Cherkin [ 24], and involves a single trial passive avoidance learning task. Provided single trial learning can be achieved, this task has decided advantages in research into memory in general and in the program undertaken here in particular. It is nontime-consuming, easily replicated, and provides an accurate measurement of time of learning [77]. In so far as we are attempting to isolate different phases of memory, if such exist, any paradigm involving multiple trials generates problems with respect to the effects of repeated reinforcement and rehearsal. If it is assumed that a single input can theoretically generate a complete memory sequence, the effect of temporally overlapping sequences arising from

multiple inputs may be to confound temporal characteristics associated with different phases in the different sequences. Even the assumption of strict structural differentiation of the different phases does not overcome this problem. One Trial Passive Avoidance Task Day-old cockerels are pre-trained to peck at a 4 mm chromed bead. For the training trial, the bead is coated with a chemical aversant. Subsequent refusal to peck a visually similar but non-aversive bead is regarded as representing memory for the association of taste with the bead. General Conditions Male, white-Leghorn black-Australorp chickens are obtained from a local hatchery on the morning of hatching. They are used in groups of 20 and housed in pairs in wooden boxes (20 x 25 x 20 cm). Housing in pairs reduces stress as indicated by behavioural indices such as attempting to jump out of the box and distress calls. Chick food is scattered on the floor of the box. Temperature is maintained at 2 0 - 2 9 ° C by the use of red 25 W light globes suspended above each box. Chicks are placed in the boxes (prewarmed if necessary), with the lights switched off, immediately on arrival from the hatchery. Pretraining of the chicks commences 30 min after their placement in the boxes. Pretraining Trials The chicks are pretrained go peck at a small chromed bead (2.5 mm dia.) attached to a straight wire, and dipped in water. The function of pretraining is to increase the probability of all chicks pecking on the training trial. Before presentation of the bead into the cage on each trial, the attention of the chicks is gained by tapping gently with the finger on the front panel of the cage. In most cases the chicks will approach the front of the cage before the bead is presented. The front panel has 2 rows of 5 x .5 cm dia. holes at chick eye level ( 8 - 1 0 cm). Tapping therefore results in both auditory and visual stimulation. These two classes of stimuli could be important procedures for subsequent learning. Chicks are attracted by maternal calls, by repetitive tapping (e.g. 4 per sec) and by visual flicker. Fischer [34] suggests that responses to these stimuli have been of adaptive significance (in the ancestral Jungle Fowl) inkeeping the young chick close to the mother hen in dense foliage. Two pretraining trials are given to accustom the chickens and to encourage them to peck at foreign objects entering their cage. The pretraining trials with a smaller bead are considered necessary as chicks often display fear reactions if the larger 4 mm dia. bead is used on the first pretraining trial. The red lights are switched on immediately after the first pretraining trial. A third pretraining trial is given involving a 10 sec presentation of a 4 mm dia. chromed bead attached to the end of a wire with a right-angled bend 1 cm from the bead. This bead is also dipped in water. The number of pecks and the latency to first peck on this and all subsequent trials are recorded on an Esterline Angus event recorder. Training Trial A similar bead to that used in the third pretraining trial

116 is dipped in the chemical aversant m e t h y l anthranilate and presented for 10 sec on the training (learning) trial. Evidence of distaste is seen in the chicken shaking its head and wiping its beak vigorously on the floor of the cage i m m e d i a t e l y after pecking. Chickens failing to peck within the 10 sec are excluded from later data analyses (at m o s t

10%). Retention Trials Tests for retention are carried out with the same bead as used on the third pretraining trial, the dry bead being presented for 10 sec. Typically a chick refusing to peck the bead on this retention trial will either shake its head or wipe its beak on the floor as it did in training, or it will try to escape from the situation physically or visually by closing its eyes. An a t t e m p t is m a d e to present the bead about 3 to 4 cm from the chicken. Closer presentation may encourage aggressive pecking. R e t e n t i o n tests are carried out at various learning-retention intervals b e t w e e n 5 and 180 min. However, although n o t r e p o r t e d in this review, 180 min results are in most cases c o n f i r m e d with tests at 24 hr.

GIBBS A N D NG ability of a positive response be a well-defined (e.g. ogival) f u n c t i o n of the strength of the m e m o r y trace. The probabilistic nature of the relationship is attributed to the operation of random n o n - m e m o r y factors. Theoretically, an actual retention test for a given animal at any p o i n t in time represents a r a n d o m sample of one trial from a universe of possible trials. That is, theoretically repeated testing of the same animal at the same time for a finite n u m b e r n trials w i t h o u t effects due to the repetition itself would generate an estimate of the probability of a positive response and hence an indication of the strength of the m e m o r y trace. In practice, assuming that identical e x p e r i m e n t a l t r e a t m e n t on n different animals will yield identical m e m o r y traces, the p r o p o r t i o n of animals yielding a positive response may be taken as an estimate of the probability of such a response for any given animal. The n different animals, each tested once, are taken to represent a sample of n trials on a single animal. It is realized that animals and their m e m o r y capacities are not as simple as this justification sounds. Subsequent paradigms to be described, give an individual measure of retention, and provide a basis for validation of the results obtained from the passive avoidance task using binary choice.

Retention Measures

Discrimination Paradigms

As m e n t i o n e d above, a different group of 20 chickens is used for each t r e a t m e n t - r e t e n t i o n testing condition. The n u m b e r of chickens in the group of 20 that avoided pecking at the bead during the 10 sec presentation is used as the index of retention. Over a group of 20 chicks which received identical t r e a t m e n t the p r o p o r t i o n avoiding is taken to represent the probability of any one chicken r e m e m b e r i n g on the r e t e n t i o n test. The concept of a " m e m o r y t r a c e " carries with it considerable surplus meaning b e y o n d the usual operations used to define it within experimental settings. It implies at least some underlying physical representation, whether in terms of activities of neurological structures or in terms of some state of the neurological structures themselves. F u r t h e r m o r e , to the e x t e n t that it is meaningful to speak of the " d e v e l o p m e n t " and " d e c a y " of a m e m o r y trace, the representation is assumed to vary quantitatively on some theoretical dimension of strength. Indeed, Gold and McGaugh's [56] m o d u l a t o r processes are said to strengthen or weaken the trace. A quantitative measure of m e m o r y poses a problem for a single learning trial paradigm involving a binary choice response, as in the experiments discussed here. A positive response (avoidance) at best indicates the presence of some minimal strength of the trace, assuming no n o n - m e m o r y factors contributing to the response. Thus the response alone provides no i n f o r m a t i o n on the strength of the trace for a given animal. The problem of avoidance or pecking on retention trials being due to some n o n - m e m o r y factor rather than presence or absence of m e m o r y is considered in the discrimination control experiments to be m e n t i o n e d shortly. On any occasion of retention testing, it may be assumed that the response is determined in part by the strength of the trace and in part by factors n o t related to m e m o r y . The latter may be systematic or random. In the case of systematic factors, separation of these from m e m o r y depends on experimental control and manipulation. Assuming that such controls are present, the response is a function of m e m o r y and random factors. Let the prob-

Learning in the pretraining trials of the paradigm discussed above involves arousal, orientation and approach in the learning to peck small objects. A l t h o u g h the pecking response to small objects is an innate disposition, the procedures e m p l o y e d here reduce stress and fear, and ensure that the rejection rate on the final learning trial is very low. The chick has in fact to learn what not to peck and so discriminate. This discrimination has been e x t e n d e d to colour discrimination in a similar paradigm [ 4 8 ] , to o v e r c o m e some shortcomings of the use of the passive avoidance task. Since learning and m e m o r y are evidenced by avoidance of pecking, general inhibition of the pecking response as a result of some t r e a t m e n t can be differentiated from inhibition of m e m o r y . By the same token, however, e n h a n c e m e n t of m e m o r y may be c o n f o u n d e d with inhibition of pecking and inhibition of m e m o r y with enh a n c e m e n t of the pecking response. T w o control paradigms using successive discriminations have been divised [48] to deal with these possible sources of c o n f o u n d i n g (Table 1). It may be noted that chicks have been shown to discriminate as well with successive discrimination tasks as with simultaneous discrimination tasks [63]. The first paradigm outlined in Table 1 provides a basis for confirming that an observed high percentage of chicks avoiding the bead during retention tests in a single trial passive avoidance paradigm may be attribut'ed to good m e m o r y for the learned association and not to a perf o r m a n c e effect arising from a generalized inhibition of the peck response by the drug treatment. Chicks are pretrained to peck small (4 m m alia.) red and blue glass beads. On the training trial, only one of these beads (e.g. red) is m a d e aversive. On retention trials chicks are presented b o t h beads in either order. The drug is administered at the same time relative to the training trial as in the single trial passive avoidance paradigm. If the effect of the drug is to enhance m e m o r y , chicks should avoid the aversive red bead and peck at the non-aversive blue bead, If, on the o t h e r hand, the drug induces a generalized inhibition of the peck response, chicks should avoid b o t h colored beads. The

117

PSYCHOBIOLOGY OF MEMORY TABLE 1 DISCRIMINATION PARADIGMS AND I N T E R P R E T A T I O N OF PATTERNS OF RESULTS

Re~Zt

C~8e

High I avoidance

(I) (2)

Pr~gza£n RED

Test

~,a£n

BLUE

drug

~HY HTm~IO~ PnFOF~4ANCE EFF~.Cr g e n e r a l l z a t i o n or inhibition of peck response

E~DN

Inte~19retat£on

RED

BLUE

avoid avoid

peck avoid

ResuZt

CQJ~S@

Lowg avoidance

(3) (4)

Pr~t~ain RED

BLUE

~D t

~ENORY IImIBITION PnFOmUmCE E~CT i n c r e a s e d pecking Intmrpretat4on

Test

~a£n ~

memory (1) per for.,ance (2)

drug

G~ROI~*

RED

L'~IRONE

BLUE

avoid peck

peck peck

peck peck

memory i n h i b i t i o n (3) performance (4)

*methyl anthranilate on bead second paradigm distinguishes the possibility that low percentage avoidance of the bead during retention tests in the single trial passive avoidance paradigm may be attributed to inhibition of memory from the possibility that it may be due to a generalized enhancement of the peck response by the drug. Chicks are pretrained to peck at small (4 mm) red glass, blue glass and chromed beads. Well before the drug treatment, chicks are trained on the color discrimination by making the red bead, say, aversive. The temporal separation between this training trial and the drug treatment is long enough to ensure that the drug does not affect the discrimination. On a second training trial chicks are given an aversive chromed bead in conjunction with the drug treatment. If the drug inhibits memory, chicks should avoid the red bead and peck at the blue and chromed beads. If, on the other hand, the drug induces generalized enhancement of the peck response, chicks should peck at all three beads. The learning paradigms discussed so far are not subject to the difficulties outlined in a recent paper by Gallagher and Peeke [45]. In that paper the authors investigate the ontogeny of the peck response in young chicks and found that the spontaneous peck response, although high at 3 days post hatch, disappears by Day 4. There are basic differences between the task employed by Gallagher and Peeke and that employed here. Chicks are pretrained to peck at the lure, hence spontaneous pecking is not the basis of our learning task. The lure is a bead rather than a lamp; Cherkin, Meinecke and Garman [27] have reported that chicks have a higher peck rate on a 3.2 mm bead than on the lamp. We have found that 7 day old chicks can learn the task we employ and in addition will discriminate between colored beads, i.e, avoid the colored bead which was aversive on the learning trial but continue to peck the non-aversive bead. Ouabain in these chicks produced

essentially the same results, as in day old chickens (unpublished observation). Finally, when chicks are kept in isolation, the memory for the task has different characteristics than when chicks are kept in pairs, particularly with respect to drug treatment (Gibbs and Cherkin, in preparation; De Vaus, Gibbs and Ng, in preparation).

Alternative Learning Tasks To overcome the problem of measures of retention associated with the single trial passive avoidance binary choice paradigm and to provide greater generality of our conclusions two further tasks have also been devised. 1. A versive wheat task. Two-day-old chickens are pretrained twice in pairs to peck at crushed wheat scattered on a white paper floor. This is done in testing boxes identical to the passive avoidance box except that the boxes have a clear perspex front. On a final pretraining trial yellow dyed wheat is presented for 30 sec and the number of pecks recorded. Training consists of placing the chickens on a floor scattered with red dyed wheat which has been made aversive with a quinine-mustard mixture. Chickens are allowed 30 sec in the box and individual pecking activity is recorded. For retention testing chickens are first placed on a floor sprinkled with damp but non-aversive red wheat. After 20 sec the chickens are transferred to a box containing non-aversive yellow wheat. Scores are calculated by comparing the number of pecks on yellow and corrected for color preference by comparison with non-trained chickens. This method gives a whole group score and a score for individual chickens. Retention tests have been given between 1 min and 180 min after the learning trial or at 24 hr. 2. Pebble floor. This task is essentially the same as the visual discrimination task described by Rogers, Drennen and Mark [ 104]. Chickens, 5 - 6 days old, are housed in

118 pairs and given experience of feeding in isolation in a testing cage similar to the experimental cage, each day prior to the experimental day. For these adaptation periods millet grain is scattered on the perspex floor. After a 3 hr food deprivation period on the experimental day, the chickens are trained to discriminate pebbles from grains of chick mash. The grains are scattered on a perspex floor to which pebbles have been glued down. Choices of pebble or grain are scored in blocks of 20 pecks. Characteristically, chickens make more pecks at pebbles than on grain in the first block of 20 choices ( 1 2 - 1 7 errors). Learning is assumed to have been achieved when the chicken makes less than 4 errors (pecks at pebbles) in 20 trials. This training normally takes less than 5 min and usually requires 3 - 4 blocks of 20 pecks. After training, chickens are returned to their home cage and only fed if retention tests are made longer than 3 hr after training. Memory for the discrimination is tested between 10 and 180 min or 24 hr after training by placing the chicken back in the experimental cage and the number of errors recoded in the first 20 choices. A total of 60 choices is recorded for the retention tests; as in the case of amnesia producing drug treatment it is necessary to ensure that the chickens can relearn.

Advantages of Day Old Chicks The advantages of using day-old chickens and the above task and procedure have been detailed elsewhere [24,77]. Chickens have a strong tendency to peck at small objects, making the task a relatively simple and possibly biologically significant one. Young chicks will peck and ingest many different small objects irrespective of their nutritive value. However, they can learn very rapidly to discriminate in one or two trials [63]. Selection may be based on visual preferences or cues and on immediate gustatory or tactile feedback. Chickens can show very strong reactions to stimuli on the basis of taste. Hogan [63] gives the example of chicks showing clear signs of disgust when they peck at fresh droppings, where their reaction includes those behaviors mentioned above in the avoidance paradigm with methyl anthranilate. Their reaction includes head shaking with the mouth open, bill wiping, and retreat from the offending stimulus. After one such experience, a chick may avoid all further contact with the object. Hogan mentions that "not infrequently a chick makes an intention to peck towards a previously experienced, distasteful object, but does not touch it; it may then retreat one or two steps and make a trill call, shakes its head, or even wipe its bill," suggestive that the visual characteristics of the stimulus are remembered. The aversion to a visual stimulus that has been associated with methyl anthranilate, once learned, is still present 72 hr later [77]. There are many similarities in responsiveness between the chick and vertebrate brain to various neurochemical agents, and this indicates that the basic functions of the chick CNS are regulated by neuronal principles in common with mammals [ 108]. The immature development of the blood-brain barrier in the young post-hatch chick allows effective central administration of certain drugs by subcutaneous injections [ 94,108 ]. This has an added advantage of permitting some multiple drug treatments.

Details of Drug Administration Drugs, generally made up in sterile 154 mM (0.9% w/v) sodium chloride (NaC1), are administered either intra-

GIBBS AND NG cranially or subcutaneously. Intracranial injections are administered freehand into each side of the forebrain in volumes of 10 ul per hemisphere, using a Hamilton repeating dispenser syringe, fitted with a stop to ensure an injection depth of 3 . 0 - 3 . 5 mm. In the experiments reported in this review a volume of 10 ul per hemisphere is used. This differs from the 25 ul per hemisphere used by Mark and Watts [77] and the 2 and 5 ul per hemisphere found to be effective in inducing ouabain amnesia by Gibbs and Cherkin (in preparation). With 2 and 5 ul only injection into a neostriatal site produced reliable amnesia compared with injections into the hyperstriatum, paleostriatum and hippocampus. Diffusion of drug from a neostriatal injection could influence ectostriatal regions which receive visual projections from the optic rectum. In all experiments, spot checks are made to ensure consistency and accuracy of location of placements of the 10 ul injections. It is realized that a potential confounding factor lies in possible differences in diffusion rates of different drugs. Time of administration studies suggest that this is not a significant factor in the results reported in this paper. But autoradiographic investigations are underway to obtain accurate indications of the relative distribution rates of the various drugs used. Subcutaneous injections are given in 0.1 ml (100 ul) volumes into a fold of skin on the ventral side of the rib cage. Table 2 summarizes the main drugs and doses used. EVIDENCE FOR STAGES IN MEMORY FORMATION In this section we present evidence from our laboratories in support of a three-phase, sequentially dependent, model of memory formation: a short-term phase developed within 5 min after learning and decaying after 10 rain; a labile phase developed by 15 to 20 min after learning, with decay initiated after 30 min; a long-term, relatively permanent, phase developed by about 45 min after learning, with no evidence of decay by 24 hr. The model does not rule out a phase of memory formation preceding the short-term phase and susceptible to disruption by electroconvulsive (ECS) or sub-convulsive (SCC) shock [15, 16, 19, 70, 71, 72, 74], with an apparent consolidation period of approximately 30 sec following learning. However, our present paradigm does not permit direct behavioural observation of this phase. In a multiphase sequentially dependent memory system, disruption of any phase prior to its initiation should prevent input of information into that and any succeeding phase from any preceding ones. It should not, however, have any effect on the preceding phase. Hence, the temporal gradient of decay of retention following such a disruption should reflect the decay function of the preceding phase. Conversely, varying the time of administration of a disruptive agent after learning should produce a temporal gradient defining the rise function of the disrupted phase, the earliest time when the phase can be triggered, and the earliest time when it can be disrupted. It will also define the latest time at which the phase can be disrupted [19]. Recovery of memory following disruption should not occur if the disruptive effect of the agent is on memory formation processes. Such recoveries need to be interpreted in terms of disruptive effects on retrieval mechanisms or performance parameters. In a multiphase non-sequentially dependent system, disruption of any phase should have no effect on preceding and succeeding phases. Behaviorally, the distinction between disrupted memory formation and disrupted retrieval

PSYCHOBIOLOGY OF MEMORY

119 TABLE 2

D E T A I L S O F M A J O R D R U G S U S E D IN S T U D I E S R E V I E W E D

D~g

Abl~

Sou.Poe

Vol~ne Inj#oted

Conoontration Injected (n~)

Dose Range Lu3/ohiek

Dose used ~g/chick (raM)

sodium chloride

Nael

BDH

20~1

120 -

140 - 180 (0.7-0.9Zw/v)

164 180

Potasslum chloride

KCI

BDH

20~I

0.1 - I0.0 154

0.149 - 14.9 229.8

2.98 (2 mM) 229.8 (154 raM) 130.6 326.5

154 mM

(140 taM) ( i S 4 mM)

LithluBa c h l o r i d e

Lie1

May & Baker

20~1 50~1

154 15.4 - 154

130.6 32.7 - 326.5

(154 ~ ) (154 m/4)

Copper chloride

Cu + +

May & Baker

50~I

.265 - 1.061~

.0008 - .33

Ouabaln octahydrate

Sigma

20~I 50pl

.027 - .041 Ix 10 -4-. 015

0.4 - 0.6 .0037 - .548

0.4 (.027 raM) .37

Ethacrynlc acid

Merck, S h a r p & Dohme

20~i

.017 - .16S

0.I - 1.0

1.0 (.165 raM)

Cyclohexlmlde

CXM

U p J o h n Co.

20~tl 50~I

3.5 .27 - 21.3

20 3.75 - 300

20 (3.5 raM) 37.5

Anlsomycin

ANI

g i f t from S.H. Barondes

20~I

1.88 - 5.64

I0 - 30

20

• -Amlnolsobutyrate

AIB

Sigma

20~I I00~1

.25 - 250 .25 - 250

.515 - 515 2.5811g-2.5flmg

515 (250 raM) 2.58 uKj (250 raM)

Merck, S h a r p & DoMe

20~I I00~I

300 300

690.8 3.45 mg

6 9 0 . 8 (300 mM) 3.45 mg (300 raM)

L-Prollne

(3.8 raM)

D - A m p h e t a m l n e SO 4

RJ~H

Sigma

I00~i

0.1 - 2.2

4.0 - 80 (0.1 - 2.0mg/kg)

.04 m g

l-Noreptnephrine d-bitartrate

NE

Sigma

I00~I

0.01 - 0.24

0.2 - 4.0 (.005 - 0.1~j/kg)

2 ~

Diphenylhydantoln

DPII

Sigma

100~tl

Ixl0 -7 -l.0mM

2.7xi0 -7- 27.4 (6.9xi0 -8- .69~j/kg)

27.4 ~g (0.1raM) (0.069 mg/kg)

(Na salt)

(l.0mq/kg)

(.05mq/kg)

Note: 50pl was used in the experiments reported by Mark & Watts (1971) and Watts & Mark (1971). 100pl was used in subcutaneous administration, all other administrations were intracranial. is obscured. By the same token, the rise function of a phase cannot be unequivocally established on the basis of behavioral evidence alone, since at any m o m e n t of interruption, memory may be available from either the preceding or the succeeding phase. Some information may be obtained from a comprehensive monitoring of the retention course using a comprehensive range of disruption times, provided the rise and decay functions of the different phases do not overlap excessively in time. In any event, inferences regarding the nature of the memory processes must rely, in such a model, principally on pharmacological, physiological and biochemical data on the postulated actions of the disruptive agents and by correlation of drugs having the same action by different mechanisms. Within a sequentially dependent model, however, complete information of the memory formation sequence, within the limits of sampling of the time dimension, may be obtained from varying (1) the time of administration of the disrupting agent, and (2) the learning-retention interval for a given time of administration of the agent, although validity of inferences will also depend on physiological, pharmacological and biochemical evidence.

Short-term Memory Formations Watts and Mark [77, 121, 122] argue for at least two

stages in the formation of memory on the basis of the delayed effect of protein synthesis inhibitors [3, 11, 28, 29]. They provide evidence to show that the phase present in the absence of protein synthesis, the long-term memory precursor, is susceptible to blockage by inhibition of the sodium pump [122]. Two aspects of their results are significant: no recovery of memory is observed 24 hr after learning following inhibition of sodium pump activity, and more importantly for the present discussion, retention deficits following sodium pump inhibition with drugs administered 5 min before learning consistently occurs at 30 min following learning but not at 10 min. With two of the drugs (lithium chloride (LiC1) and cupric chloride (CuCI~)) a noticeable but not statistically significant reduction in retention levels compared with saline controls is observed at 10 min. On the basis of the evidence provided by Watts and Mark [ 122], Gibbs and Ng [51 ] suggest that another phase of memory may precede the sodium pumpdependent phase, with decay occuring after 10 min following learning. Using the same paradigm as Watts and Mark [122], but with drugs injected in 10 pl volumes instead of the 25 pl volumes of the latter (see Table 2), we demonstrated significant retention deficits for a single trial passive avoidance task in day-old chickens 5 min after learning following treatment 5 min before learning with 20 pl of 154 mM LiC1 and 1 mM KC1 but not with 0.027 mM

120

GIBBS A N D NG

ouabain (0.4 ug/chicken), the sodium p u m p inhibitor. Ouabain-induced amnesia did n o t occur until 15 min following learning irrespective of whether the drug was administered 5 or 15 min before learning. No recovery from the amnesic effects of any of the drugs was evident at 3 hr. F u r t h e r m o r e , it was d e m o n s t r a t e d that while the effect of ouabain was c o u n t e r a c t e d by 100 ul of 0.1 mM diphenylh y d a n t o i n (DPH) administered subcutaneously 5 min after learning, those of LiC1 and KC1 were n o t [ 5 1 , 5 2 ] . DPH, at the dose used, stimulates Na÷/K÷ ATP'ase activity in chicken forebrain total h o m o g e n a t e [ 5 2 ] . We concluded that a phase of m e m o r y formation, i n d e p e n d e n t of sodium p u m p activity, preceded the short-term labile phase of Watts and Mark [ 1 2 2 ] . To distinguish the two phases, the term 'short-term' has been restricted to the new phase and the term 'labile phase' is used for the ouabain-sensitive phase. This terminology is maintained in this paper. We now review new and existing evidence in an a t t e m p t to characterise in more detail the short-term phase. Sodium chloride (20 ul of 154 mM NaC1) administered intracranially 5 rain before learning yields retention levels of 95% and 85% at 5 and 10 min after learning respectively. R e t e n t i o n level drops to 70% at 20 min after learning and is maintained for at least 3 hr (Fig. 1). Gibbs and Barnett [48] have shown that chickens under this t r e a t m e n t discriminate successfully between an aversive red bead (avoid) and a non-aversive blue bead (peck), suggesting that the above administration of NaC1 does not lead to general inhibition of pecking. It may be noted, however, that the retention levels for 154 mM NaC1 are consistently lower than those for 140 mM at any given learning-retention interval. This issue will be taken up later. For the time being it is sufficient to point out that, unless otherwise indicated, all references to NaC1 imply a c o n c e n t r a t i o n of 154 raM.

100 - O LiCt 154 mM V KCL 2raM 80

60

D

0

z_ Q 0

<~

4o-

I

I

I

I

I

I

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5

10 15 20 25 30 180 LEARNING RETENTION INTERVAL(min) FIG. 2. Retention levels over time following intracranial administration of 20 ~1 injections of 2 mM KC1 (dissolved in 154 mM NaCI) or 154 mM LiC1 solutions.

Both 2 mM KC1 and LiC1 are maximally effective in producing amnesia when administered close to the time of learning, b e t w e e n 5 min before and 5 - 1 0 sec after learning for LiC1, and b e t w e e n 5 min before and 2.5 rain after learning for 2 mM KC1 (Fig. 3). In both cases, m e m o r y deficits are slight with injections 5 min after learning, and the short-term m e m o r y process is no longer susceptible to inhibition by these drugs 10 min after learning. 100

-

100 -

~

\

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~

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80

z o

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20

,

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to

~

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~o

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LEARNING RETENTION

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,o

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~2o INTERVAL (rain)

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FIG. 1. Percentage of chicks avoiding on retention tests made between 5 and 180 min after the single learning trial. Five min before learning chicks were given bilateral intracranial injections of 140 or 154 mM NaC1. Each data point represents a separate group of 18-20 chicks. Intracranial injection of 20 ul of 1 or 2 mM KC1 in NaCI 5 min before learning yields a retention level of about 30% 5 min after learning ([51,53] Gibbs, Gibbs and Ng, in preparation). Although LiC1 yields a lesser deficit than that obtained with 2 mM KC1 at 5 min after learning, it reaches levels observed with 2 mM KC1 by 10 min [ 5 1 ] . The retention deficits are maintained up to 180 min.

0 I -15

I --10

i -5

] 0

TIMEOF INJECTION (rain)

I "+5

I +10

FIG. 3. The time of intracranial injection of 20 ~1 of KC1 (2mM in NaC1) or LiC1 (154 mM) is varied around the learning trial (0 min) and retention measured as percentage of chicks avoiding the bead on testing at 180 rain after learning. These data are compared with 154 mM NaC1 controls. Preliminary findings have suggested two further candidates for inhibition of m e m o r y at this STM stage: glutamate and isotonic (154 mM) KC1. With both of these agents chicks show p o o r retention 5 min after learning. Both agents show a decreasing effectiveness when injected 2.5 min after learning, with no effect at 5 min after

PSYCHOBIOLOGY OF MEMORY learning. The results are identical to those observed with LiC1. Of importance to any conclusion drawn from our studies with LiC1 and KC1 is the observation that the strong amnesic effects of KC1 decrease rapidly with increasing and decreasing concentrations from 1 and 2 mM [53] Gibbs, Gibbs and Ng, in preparation). In particular, 0 and 7 mM KC1 in 154 mM NaC1 administered 5 min before learning produce normal retention at all learning-retention intervals, while performance is markedly impaired only after 15 min following learning with 3 and 4 mM KC1 in 154 mM NaC1. The possibility arises that 1 or 2 mM KC1 may be affecting a phase of memory formation n o t affected by concentrations of 3 or 4 mM KC1. The precise nature of the effects and a possible mechanism underlying them will be discussed later. There is also an effect produced by the concentration of NaC1 used. 154 mM NaC1 produces a small but consistent deficit when compared with 140 mM NaC1, particularly from 15 min onwards following learning (see Fig. 1). However, investigations with different concentrations of KC1 using these two concentrations of NaC1 as vehicles suggest that the effects of KC1 and NaC1 are independent and additive ([53], Gibbs, Gibbs and Ng, in preparation). The optimal concentration of 7 mM KC1 and 140 mM NaC1 for memory retention correspond to normal CSF levels of Na~ and K~ in 6-week-old chickens [6]. Finally, while retention levels following administration of 140 mM NaC1 remain high at all learning-retention intervals, the retention function shows two temporary performance deficits, one at 15 min and the other at between 60 and 90 min following learning (see Fig. 1). These are reminiscent of the Kamin effects cited by Halstead and Rucker [ 59,60] and Cherkin [25 ] as evidence for the crossing over of retention functions for two different phases of memory formation. The evidence reviewed to date is consistent with the postulation of an early phase of memory formation preceding the ouabain-sensitive labile phase of Watts and Mark [122]. This short-term phase (STM) has a rise function occuring less than 5 min after learning and a decay initiated by 15 min following learning. It is susceptible to inhibition by 1 or 2 mM KC1, glutamate and isotonic KC1 but not by the sodium pump inhibitor ouabain or concentration of KC1 between 3 and 7 mM. Results from the pebble floor task show memory retention to be considerably reduced when 5 - 6 day old chickens are tested 10 min after learning with injection of 2 mM KC1 5 min before learning, even thotigh the chickens learn the discrimination in the same number of choices as do saline-treated chickens. Preliminary results from the aversive wheat task show memory to be reduced to approximately 20% at 10 min after learning with both 2 and 154 mM KC1 administration 5 min before learning, whereas saline injected chickens in this task yield approximately 60% at this time. These findings clearly confirm those obtained with the single trial passive avoidance task. Benowitz and Sperry [16], using an identical experimental paradigm, task, and species of animal, report retention deficits with LiC1 24 hr after learning, but not 20 min after learning. They interpret these results as indicating a phase of memory formation distinguishable from Watts and Mark's [122] ouabain-sensitive phase, a conclusion similar to ours. However, on the basis of reported evidence [72, 73, 74] that long-term memory consolidation is

121 disruptible by electroshock (ES) treatment when applied within 30 sec after learning, they equate Watts and Mark's ouabain-sensitive phase with an ES-sensitive long-term memory precursor. This conclusion appears unwarranted on the grounds that the ES effects are maximal by 10 min after learning, and substantial after 5 min [73]. We report evidence later that, unlike ES, ouabain is still partially effective in inhibiting retention when administered as late as 5 min after learning, and the retention deficits are not observed until after 10 min after learning. A more consistent interpretation of the data suggests the possibility of an electroshock-sensitive phase of memory processing preceding the LiCl-sensitive phase, with at least the transfer of information from the early to the LiCl-sensitive phase being disruptible by electroshock [32]. The differences in finding with LiC1 between our study and that of Benowitz and Sperry may be attributed in part to a major procedural difference between the two studies. In our study chickens were housed in pairs, while in the other study they were housed in isolation. Flexner and Flexner [35] and Gold and McGaugh [56] have argued for the role of motivational factors in memory processing. The effects of electroconvulsive shock on memory processing have been shown to be affected by isolation [1, 23, 93]. Similarly, Golub, Varn and McCluer [58] have demonstrated that the inhibitory effects of cycloheximide on memory for a passive avoidance task in mice can be cycloheximide on memory for a passive avoidance task in mice can be counteracted by individual housing of the animals. In view of studies showing that arousal-producing treatments can alter the effects of amnesic treatments [ 12, 46, 47, 81], it is possible that isolation may alter the effects of LiC1 in the Benowitz and Sperry study through enhanced arousal (stress). It has been shown that the onset of ouabain-induced amnesia is delayed when chickens are trained and tested under isolation conditions (Gibbs and Cherkin, in preparation). Similar findings have also been observed in our laboratories with respect to KC1, ouabain, and CXM-induced amnesias. We extend the tentative suggestion that isolation (possibly through concomitant stress-related release of hormones like ACTH and vasopressin; cf. [35] may extend an electroshock-sensitive phase of memory formation preceding the LiCl-sensitive phase, thus giving the impression of absence of early amnesia with LiC1 claimed by Benowitz and Sperry. The effect of stress may be directly on the memory trace or on modulator processes of the sort suggested by Gold and McGaugh [56]. While the evidence reviewed above suggests strongly a short-term memory phase which may be demarcated from the labile phase of memory formation postulated by Watts and Mark [122], the strength of the inference depends in part on evidence delineating the decay function of this phase from the rise function of the next. The amnesic effects arising from inhibition of this phase of memory formation persist for at least 24 hr after learning and this may imply that the subsequent phases are dependent on this phase or that the treatments also affect the succeeding phases or both. The resolution of this issue also awaits information on the succeeding phases~ and information regarding the possible mechanisms underlying the different phases. Labile Memory Formation Watts and Mark [ 122] provide evidence in support of a

122

GIBBS A N D NG

phase of m e m o r y f o r m a t i o n susceptible to inhibition by sodium p u m p inhibitors, and distinguishable from a protein synthesis-dependent long-term phase in two respects. Ouabain shows a greater dose dependence than CXM at retention intervals from 30 to 180 min. Increasing the c o n c e n t r a t i o n of the sodium p u m p inhibitor produces an increasingly rapid decay of m e m o r y but increasing the c o n c e n t r a t i o n of CXM did not. This suggests that ouabain interferes with a phase of m e m o r y storage of m e m o r y that is n o t susceptible to inhibition of protein synthesis. There is also a difference in effective time of injection after learning. Ouabain has no effect administered 10 min after learning, a time when CXM is still m a x i m a l l y effective. A n o t h e r sodium p u m p inhibitor, ethacrynic acid, has been reported to have the same inhibitory effect on m e m o r y f o r m a t i o n [ 105]. In none of these studies are retention tests carried out between 10 and 30 min after learning or prior to 10 min. We now review m o r e c o m p l e t e evidence from our laboratories. With retention measured at 180 min after learning, intracranial injections of ouabain (20 pl of 0.027 mM, 0.4 pg/chicken) between 15 min before and 2.5 min after learning yield substantial reductions in retention level (Fig. 4) when c o m p a r e d to NaC1 controls (see Fig. 1). Similar

100 -

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60(.~

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FIG. 5. Time course of memory decay following intracranial administration of 0.4 pg/chick ouabain at times between 30 rain before and 10 min after learning. Retention of the memory was followed between 5 and 30 min (upper graph) and between 30 and 180 min (lower graph).

+10

100

FIG. 4. Time of intracranial injection of 0.4 pg ouabain or 1.0/~g ethacrynic acid relative to the learning trial (0 rain). Retention was measured at 180 rain after learning. results are obtained with ethacrynic acid. Administration of either drug 5 rain after learning results in a lesser deficit, and by 10 min after learning retention is normal. These results suggest a phase of m e m o r y with a rise f u n c t i o n initiated soon after learning and reaching m a x i m u m levels 10 min following learning. It contrasts with the short-term phase susceptible to inhibition by LiC1, 2 mM and isotonic KC1 and glutamate which have a rise function occurring within 5 min of learning. F r o m the evidence reported earlier, this labile phase of m e m o r y appears to be also susceptible to inhibition by KCI in concentrations of 2.5 to 5 mM. The inhibitory effects of ouabain and ethacrynic acid are not evident until 15 min after learning (Figs. 5 and 6). Relative to NaC1 controls (Fig. 1), with all times of administration of ouabain, retention levels are normal 5 and 10 min after learning (Fig. 5). When the dose of ouabain is raised to 0.6 pg/~l, m e m o r y is still unimpaired 10 min following learning, but the extent of the deficit is greater at

I

30 60 90 120 LEARNING RETENTION INTERVAL (rain)

-

80

•

ETHACRYNIC ACID

6O z a ~> 40

20

1

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[

180

FIG. 6. Retention course for memory following intracranial injection of 1.0 #g/chick ethacrynic acid given 5 min before learning. 30 min than with the 0.4 ug/pl dose normally used (Table 3). Ethacrynic acid administered 5 min before learning also yields normal retention levels 5 and 10 min after learning (Fig. 6), and similar results have been observed with 3 or 4

PSYCHOBIOLOGY OF MEMORY

123 TABLE 3 COMPARISON OF RETENTION LEVELS OF 0.4/~g AND 0.6 #g OUABAIN

Time of I~ection - 5 m ln

- 1 5 min

Concentration

Time of ~est $0 rain 180 rain

20 rain

0.4

88.4

43.8

16.5

0.6

79.0

30.0

10.5

0.4

84.3

50.0

15.0

0.6

79.0

25.0

5.3

mM KCI (Gibbs, Gibbs and Ng, in preparation). A pronounced deficit, especially in the case of ethacrynic acid, occurs at 15 min after learning and this is maintained up to 180 min. Gibbs and Barnett [48] have also shown that chickens administered ouabain discriminate successfully between an aversive red bead and a non-aversive blue bead when tested 10 min after learning. The capacity to discriminate is noticeably reduced by 30 min after learning. The above evidence confirms Watts and Mark's [122] suggestion of a labile phase of memory formation preceding a long-term protein synthesis-dependent phase, and implies a distinct difference between the mechanisms underlying the short-term phase and the ouabain-sensitive labile phase. It may be argued that the separation between the phases may be an artifact arising from different distribution rates of the drugs used. Thus 1 or 2 mM KC1 may diffuse further and faster than ouabain, and differences in temporal parameters arising from the use of these drugs may result from differences in the extent or the nature of brain structures reached. If this is the case, however, it is difficult to explain why (1) ouabain administered as early as 15 min before learning should yield the same effects as when administered as late as 5 min after learning; in both cases retention deficits are not evident until 15 min after learning (Fig. 4), and (2) KC1 is only effective when administered between 5 min before and 2.5 min after learning (Fig. 3), while ouabain is still effective when administered 5 min after learning; one may have expected the reverse to be the case if the above argument holds. The fact that ouabain and ethacrynic acid exert similar effects when injected both before and after learning rules out learning deficits. Moreover, since under these times of administration no retention deficits are observed at 5 and 10 min after learning but only after 15 min following learning, the action of these drugs cannot be attributed, at least solely, to effects on retrieval mechanisms. Further evidence in support of Watts and Mark's [ 122] contention that ouabain exerts its effects on sodium pump activity and that this mechanism underlies the labile phase of memory formation will be discussed later. For the moment, what needs emphasizing is that the mechanisms underlying the formation of labile memory cannot be the mechanism underlying the subsequent maintenance since neither ouabain nor ethacrynic acid is effective in inhibiting this phase of memory when applied after 10 min following learning (see Fig. 4), at a time when the phase is still maximally active (see Fig. 8); also Watts and Mark [ 122] ). Clearly the same conclusion needs to be drawn about the mechanisms underlying short-term memory formation and

its subsequent maintenance. Again isotonic KC1, LiC1, and glutamate are not very effective when administered after 2.5 min following learning, when the evidence suggests that the short-term phase is still active up to 10 min after learning. The time of action of ouabain is not a significant factor since it is effective as measured by retention at the same time when administered 15 min before learning. The conclusion appears inescapable that these drugs are acting on memory processes during the period when memory is being formed. The effect of ouabain (20 t~l of 0.027 mM) in the pebble floor and aversive wheat tasks is essentially the same as in the passive avoidance and discriminated passive avoidance tasks [48]. In the aversive wheat task, when ouabain is injected 5 min before the learning trial, retention is found to be high at 5 and 10 min after learning and then to decay when tests are carried out from 15 min or more after learning. In the pebble floor task, with ouabain (20 ~1 of 0.027 mM) given 10 min before learning, there is good retention at 10 min after learning but this had decayed by 30 min after learning. The chicks show acquisition rates very similar to chicks pretreated with saline. This latter result is in contrast to that shown by Rogers et al. [105] who find that performance is impaired during and up to 30 min after learning with ouabain (50 ~1 of 0.4 ug) injected 10 min before learning but long-term memory is unimpaired. It may be significant that Rogers et al. administered the same amount of ouabain but in a 50 ul volume compared to 20 ul used here.

Long-term Memory Formation Mark and Watts [77] show that CXM in 25 ~1 x 2 volumes produces amnesia 24 hr after learning when administered between 30 min before and 10 min after learning. The results of injection 5 min before learning [ 121,122] show the amnesic effect to occur as early as 60 min after learning. The effect is found not to vary with doses up to 300 ~g/50 t~l [ 1221. The data in Figs. 7 and 8 from our laboratories confirm the above findings, with a more complete time course of administration and time course of retention. Both anisomycin (20 ul of 3.8 mM or 20 ug/20 ~tl) and CXM (20 ~tl of 3.5 mM or 20 ug/20 ~1) are successful in inducing retention deficits measured at 180 min after learning when administered between 30 min before and 10 min after learning. Similar results have been reported with anisomycin by Bull, Ferrera and Orrego [22] using essentially the same experimental paradigm and 1 - 2 day old chickens. In their behavioural study 21 ~g/chicken of intracranial

124

GIBBS A N D NG

10C

L~ CXM • ANISOMVCIN

80-Z :E

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40--

20--

I

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-45

-30

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TIME OF

I

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,'1.45

m g / k g subcutaneously administered anisomycin yields 91% inhibition in the first 2 hr and 75% in the subsequent 2 hr. However, we have found 68% inhibition of leucine inc o r p o r a t i o n into chicken forebrain protein at 30 min following intracranial administration of 20 ug/chicken. Biill e t al. [22] do not report the behavioural effects of 21 ug/chicken intracranial administration of anisomycin, the effects of variation in times of administration, or retention levels earlier than 24 hr after learning. Our results suggest a phase of m e m o r y f o r m a t i o n susceptible to inhibition by protein synthesis inhibitors, with a rise f u n c t i o n occurring within 45 min of learning and c o m p l e t e l y developed by 60 min. The temporal gradient of retention under various times of administration of CXM is shown in Fig. 8 and under anisomycin administered 5 min before learning in Fig. 9. No r e t e n t i o n deficit is observed up to 30 min after learning,

FIG. 7. Time of intracranial injection of CXM (20 txg/chick) or anisomycin (20 /~g/chick) relative to the learning trial (0 min). Retention was measured at 180 min after learning.

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LEARNING RETENTION INTERVAL(rain) FIG. 8. Time course of memory decay following intracranial administration of CXM (20 ug/chick) at times between 45 min before and 30 min after learning. Retention of the memory was followed between 5 and 30 min (upper graph) and between 30 and 180 min (lower graph).

anisomycin is augmented by 53 mg/kg administered subcutaneously on the grounds that intracranial administration of 21 ug/chicken produces only 30% inhibition of brain protein synthesis at 2 hr, while augmentation with 53

I 5

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I I I __ [ 60 90 120 180 LEARNING RETENTION INTERVAL(min) FIG. 9. Time course of retention following intracranial administration of 20/~g of anisomycin 5 min before the learning trial. in contrast with the effects of ouabain (cf. Fig. 5). R e t e n t i o n deficits are clearly evident by 60 min for times of administration which produce amnesia at 3 and 24 hr. The time course of effects of CXM administered 5 min before learning has been c o n f i r m e d in discrimination experiments by Gibbs and Barnett [ 4 8 ] . Assuming that administration of the drugs 5 rain before learning is the most effective in inducing amnesia, the corresponding temporal gradient of retention defines the decay function for the labile, ouabain-sensitive phase. Decay is virtually complete 90 min after learning. The sodium p u m p inhibitors, ouabain and ethacrynic acid, are ineffective in inducing amnesia when administered 10 min or later after learning and the amnesic effects are observed as early as 15 min following learning. On the other hand, the protein synthesis inhibitors are still partially effective when administered as late as 20 min after learning with the amnesic effects occurring only after 30 min following learning. There are grounds, therefore, for arguing that the two groups of drugs are affecting two different phases in the m e m o r y f o r m a t i o n sequence. The results cannot be attributed to differential diffusion rates of the two groups of drugs for the same reasons as brought out in the discussion on the distinction between short-term and labile m e m o r y . As with all previous phases, it would appear that the effect of the protein synthesis inhibitors are restricted to

PSYCHOBIOLOGY OF MEMORY the memory formation stage of the phase affected. Once formed, memory during this phase is no longer susceptible to disruption. Evidence from Mark and Watts [77] suggests that no spontaneous or test-induced recovery from the effects of CXM occurs within 72 hr after learning. To this extent, the inhibitory effects are relatively permanent and are consistent with an interpretation in terms of formation inhibition rather than retrieval inhibition. Comparable results have been found with anisomycin for the aversive wheat and pebble floor task. Results obtained with CXM are somewhat more equivocal. In the pebble floor task, CXM has been found to have no effect on retention tested 30 min after learning but memory is severely impaired when the chickens are tested for retention at 24 hr [ 105]. We have confirmed that memory is good at 10 and 30 min after learning, but decaying by 120 min with partial retention at 60 min after learning. For the aversive wheat task we have found essentially the same decay time course with the protein synthesis inhibitor anisomycin as in the passive avoidance task. However, so far we have been unable to reliably demonstrate a memory loss with CXM, it appears that CXM has a deleterious side effect on performance in this task. Co n clusio n

The evidence reviewed so far suggest a model of memory formation for a single-trial passive avoidance task in day-old chickens consistent with that proposed by Gibbs and Ng [51]. A short-term memory phase, susceptible to disruption by drugs like LiC1, glutamate, and specific low (1 or 2 mM) or isotonic concentrations of KC1, develops within the first 5 min after learning, is fully developed by 10 min, and decays thereafter with almost complete decay by 30 min. This is followed by a labile phase, susceptible to disruption by the sodium pump inhibitors ouabain and ethacrynic acid and selected low ( 3 - 5 mM) concentrations of KC1. It develops within the first 15 min following learning and decays after 30 rain, with complete decay occurring by 90 min. A long-term phase develops within the first 45 min after learning and appears to be relatively permanent. It is susceptible to inhibition by protein synthesis inhibitors like CXM, puromycin and anisomycin. The evidence is neutral with respect to the issue of sequential dependence of the phases, in so far as it is not clear whether drugs affecting earlier phases may also affect the later phases, either in terms of formation, maintenance, or retrieval. What is clear is that the effects of the drugs are on the formation stage of the relevant phase, suggesting that the mechanisms underlying formation may not be the mechanisms underlying maintenance. The evidence is consistent with a sequential retrieval system which, under abnormal conditions of drug treatment, at least, is partly dependent on temporal parameters. A threshold concept along [59,60] is not adequate to explain the non-zero retention levels observed during inhibition of a given phase by disrupting agents. If one accepts the retention function as indicating the amount of memory left in the disrupted phase, it is necessary to assume a strict temporal criterion for retrieval, unless one argues for a variable threshold concept with monitoring by feedback mechanisms triggered by disruption. If, on the other hand, one argues that the retention function obtained with disruption of a given phase represents the decay function of the preceding phase, it must be assumed that the disruption is complete, an assumption not susceptible

125 to confirmation or denial with behavioural data. An explanation involving both temporal and threshold criteria would fit the data. It is suggested that retrieval at any point in time is from that phase of memory which is normally active, subject to a threshold test. If the threshold criterion is not met, then memory is sought from the preceding phase, but not subject to threshold tests. In a sequentially dependent system, such an arrangement of retrieval mechanisms would be optimally efficient since disruption is an abnormal occurrence. In a sequential model where the phases are not dependent, the failure to meet the threshold criterion may entail a search among all existing phases. In a parallel model, where all phases develop in parallel and independently, decay functions should be virtually absent early after learning under either a temporal or a threshold basis for retrieval. The answer to this issue depends upon precise knowledge of the possible actions of the disrupting agents and the mechanisms underlying the different phases. FURTHER

EVIDENCE FOR PROCESSES UNDERLYING MEMORY FORMATION

The known pharmacological actions of drugs which induce amnesia provide information about the possible physiological and biochemical processes which may be involved in memory f o r m a t i o n . However, any inference that is drawn must be qualified by the fact that a given drug at a given dose may have more than one pharmacological action; that different doses of the same drug may have different pharmacological actions; that different pharmacological actions of the same or different drugs may yield the same behavioral outcome as a result of patterns of relationships, known or u n k n o w n , between underlying physiological and biochemical mechanisms. Nonetheless, known common actions of a number of drugs having a common behavioral effect may generate useful hypotheses. The specificity of such inferences may be augmented by challenging the drugs with other drugs whose actions are antagonistic. Antagonistic and agnostic drugs may provide a means for verifying such hypotheses when used judiciously. S h o r t - t e r m M e m o r y Processes

No effective counteractive agent to the effects of 1 and 2 mM KC1 and LiC1 on the postulated short-term memory phase has been isolated. On the other hand, the negative findings from challenges instituted to date may provide positive inferences about what processes are n o t likely to be involved in this phase of memory formation. We reported that 0.1 mM diphenylhydantoin (DPH) succeeded in overcoming ouabain- and CXM-induced amnesia but not amnesia induced by 1 mM KC1 or LiC1 [52]. We have since confirmed these findings with 1 and 2 mM KC1 (Gibbs, Gibbs and Ng, in preparation) as well as LiC1. Retention levels with DPH administered 5 min after learning to chickens pretreated with one of the above drugs 5 min before learning are unchanged from amnesic levels measured between 5 and 30 min, and 180 min after learning (Figs. 10 and 11). Sodium pump dependent hyperpolarization and Na÷/K÷ ATP'ase activity in neurones depend on critical levels of extracellular K ions and intracellular Na ions and are inhibited at low K levels [84]. It is possible therefore that administration of low concentrations of KC1 may inhibit Na÷/K+ ATP'ase activity in the chicken brain. DPH (0.1 mM) has been shown to stimulate Na÷/K÷ ATP'ase activity

126

GIBBS A N D NG

100 Z

I o

~ Z

80 o

60

o

o

OSALINE

0

~ 4o 20

0 v

OUABAIN

LiCt 1S4mM KCt 2mM

I o

I 15

I 30

30

60

90

120

10o

• ANISOMYCIN

z o

80

o

60

Z

~

4o

20

Labile Memory Processes

AIB CXH

I 0

in the c h i c k e n in vitro [52] a n d m i g h t be e x p e c t e d , t h e r e f o r e , to o v e r c o m e the i n h i b i t o r y effects of KC1 o n m e m o r y . T h e fact t h a t DPH does n o t o v e r c o m e KC1i n d u c e d amnesia a p p e a r s to rule o u t i n v o l v e m e n t of s o d i u m p u m p activity in the s h o r t - t e r m p h a s e of m e m o r y form a t i o n . KC1, at these c o n c e n t r a t i o n s m a y still i n h i b i t the labile phase of m e m o r y f o r m a t i o n via i n h i b i t i o n o f Na÷/K + ATP'ase activity. If this is the case, t h e n the fact t h a t DPH does n o t o v e r c o m e 1 m M K C l - i n d u c e d a m n e s i a at r e t e n t i o n intervals o f 10 m i n or longer (Fig. 11) s u p p o r t s o u r c o n t e n t i o n t h a t the labile phase is d e p e n d e n t o n the short-term phase for formation. Since d - a m p h e t a m i n e s u l p h a t e (1 m g / k g ) does n o t o v e r c o m e a m n e s i a i n d u c e d b y 1 m M KCI [54] b u t does o v e r c o m e o u a b a i n - and C X M - i n d u c e d a m n e s i a [46] it is u n l i k e l y t h a t c a t e c h o l a m i n e s are involved in the s h o r t - t e r m phase of m e m o r y f o r m a t i o n . As seen in Figs. 12 and 13, d-amphetamine sulphate administered 5-10 sec a f t e r l e a r n i n g to c h i c k e n s p r e t r e a t e d w i t h 1 mM KC1 5 rain before l e a r n i n g p r o d u c e s n o r e c o v e r y from amnesia, with r e t e n t i o n m e a s u r e d b e t w e e n 5 and 30 m i n a f t e r learning, and at 180 rain. T h e i n v o l v e m e n t of n o r a d r e n e r g i c p a t h ways will be e x p l o r e d again w h e n we c o n s i d e r the labile and l o n g - t e r m phases.

I I I I I 15 30 30 60 90 TIME OF DPH INJECTION (MIN)

I 120

FIG. 10. Percentage of chickens avoiding on 180 min retention test. Upper graph: saline (154 mM) or ouabain (0.4 pg) were administered intracranially 5 min before training and 100 pl DPH (10-4M) administered subcutaneously between 5 and 120 rain after training. LiC1 (154 mM) or KC1 (2 mM) were given 5 min before training and DPH 5 min after training. Lower graph: Chickens were pretreated with CXM (20 pg) intracranially or AIB (250 mM) subcutaneously 5 min before training and given subcutaneous injections of DPH from 5 min before in the case of AIB or 5 - 1 0 sec to 120 min after training. Chicks pretreated with intracranial anisomycin (20 pg) were given DPH 5 min after training.

Labile Memory Processes A c o n c e n t r a t i o n of 0.1 m M DPH has b e e n s h o w n to be effective in c o u n t e r a c t i n g o u a b a i n - i n d u c e d a m n e s i a [ 5 2 ] . The e f f e c t o f DPH varies w i t h the time of a d m i n i s t r a t i o n . With r e t e n t i o n m e a s u r e d 180 rain a f t e r learning, i n j e c t i o n s 100

SALINE -AMPH÷ 10 min CXH- AHPH +10 rain

80 \\

40 --

KC[- AHPH+0 rain

20

l 16

I00

I

I

..

I

J

I

20 30 60 90 120 LEARNING RETENTION INTERVAL (mln)

_.

I 180

FIG. 12. Effect on retention between 5 and 180 min of amphetamine (1.0 mg/kg) administered subcutaneously either 5 - 1 0 sec or 10 min after learning. Chickens were pretreated with saline (154 mM), ouabain (0.4 /~g), CXM (20 pg) or KCI (1 mM) 5 min prior to training.

6C

c)

~e ZO - -

I T

10

i

I

I

15 20 25 LEARNING DPH (10"4H)

I

_.

I

I

I

30 60 90 120 RETENTION INTERVAL (mini

-_

-'-'~'

KCt 1 mM

~

LiC[ 154mM

I 180

FIG. 11. Effect on retention between 10 and 180 min of DPH (10-* M) administered subcutaneously 5 min after learning. Chickens were given intracranial injections of saline (154 mM), ouabain (0.4 pg), CXM (20 pg), LiC1 (154 mM) or KC1 (1 mM) 5 min prior to training.

of DPH up to 10 m i n following l e a r n i n g yield levels of r e t e n t i o n in o u a b a i n - p r e t r e a t e d c h i c k e n s similar to t h o s e o b t a i n e d w i t h s a l i n e - p r e t r e a t m e n t (Fig. 10). B e y o n d 10 m i n , a d m i n i s t r a t i o n of DPH p r o d u c e s r e t e n t i o n levels c o m p a r a b l e t o t h o s e o b t a i n e d at c o r r e s p o n d i n g learningr e t e n t i o n intervals using o u a b a i n a l o n e (Figs. 10 a n d 5). R e c o v e r y f r o m o u a b a i n - i n d u c e d a m n e s i a occurs d u r i n g t h e labile p h a s e a n d is s u s t a i n e d for u p to 180 m i n following l e a r n i n g (Fig. 11). We have rejected earlier suggestions t h a t p o s t - t e t a n i c p o t e n t i a t i o n s m a y be i m p l i c a t e d in the labile p h a s e of m e m o r y f o r m a t i o n , a l t h o u g h I z q u i e r d o a n d Nasello [ 6 4 , 6 5 ] have r e p o r t e d t h a t DPH depresses h i p p o c a m p a l

PSYCHOBIOLOGY OF MEMORY

127

facilitation and post-tetanic potentiation of evoked responses in rats and also depresses the acquisition of a conditioned avoidance response (but not retention in rats which acquired the response). However, it should be pointed out that their concentration of DPH is much higher (80 mg/kg) than that used in these experiments (0.07 mg/kg for a facilitating dose). We attribute the effect of DPH to its stimulation of Na*/K÷ ATP'ase activity and suggest that this reactivates the processes underlying the formation of labile memory. Provided sufficient information remains in the preceding short-term store, labile memory formation will be reinstituted. Alternatively, reactivation of the labile memory formation processes may serve to preserve the level of information available in the labile store for an extended time by reactivating the existing labile trace. Our argument earlier that the same mechanisms appear not to sustain both the formation and the maintenance of labile memory does not preclude the above possibility. Ouabain-induced amnesia is also overcome successfully by both amphetamine and norepinephrine [54]. Damphetamine sulphate (1 mg/kg) administered subcutaneously up to 5 min after learning yields normal retention levels at 180 min after learning in ouabainpretreated chickens (Fig. 13). Administration of amphetamine 10 min or later after learning has no effect. 10(~

SALINE z ~"

6C OUABAIN r-i KCI. 1ram V

4O

labile phase by inhibiting the amino acid uptake in the first 10 min. This would be consistent with the observation that retention levels show further decline from 30 min to 180 min in ouabain treated chickens (Fig. 5). That this is not the basis of its action on the labile phase will be demonstrated in the next section. One common pharmacological action in chicken forebrain of DPH, amphetamine and norepinephrine is the stimulation of Na÷/K+ ATP'ase activity [52,67]. Since none of these agents counteracts the effects on short-term memory of 1 mM KC1, but successfully overcomes ouabaininduced amnesia during the postulated labile phase, it may be argued that the latter phase is dependent on Na÷/K÷ ATP'ase activity. This conclusion is further strengthened by our observation that 0.1 mM DPH is effective in overcoming amnesia induced by 3 mM KC1 (unpublished observation), this amnesia occurring during the labile phase and not during the short-term memory phase. The resulting retention levels at 10, 30 and 180 min after learning being 90, 80 and 80% respectively. The fact that neither amphetamine nor NE is effective in overcoming ouabain-induced amnesia if injected 10 rain or later after learning is consistent with the results from DPH and with the earlier argument that the labile phase develops within 15 min or so following learning, while short-term memory is still maximally active. The results are also consistent with the argument that sodium pump activity is important for the formation of labile memory but not for its subsequent maintenance. We suggest that DPH, NE or amphetamine causes recovery from ouabain-induced amnesia by either (1) overcoming the inhibitory effects of ouabain on Na+/K+ ATP'ase activity in time for normal labile memory formation to take place, or (2) reactivating the labile memory formation processes to capitalize on whatever is left in short-term memory at the time of effective action of the counteracting agent.

Long-term Memory Processes

o

"~ 2o

CXH

I 0

I

I

I

15 30 30 TIHE OF AHPH['TAHINE

I

I

I

60

90

120

INJECTION (min)

FIG. 13. Percentage of chickens avoiding at 180 min after learning when pretreated with saline (154 mM), ouabain (0.4 ~g), KC1 (1 mM) or CXM (20 t~g) 5 min before learning and given subcutaneous injections of amphetamine (1.0 mg/kg) at various times between 5-10 sec and 120 min after learning. Amphetamine administered immediately after learning yields a normal retention function up to 30 min following learning, but this is followed by a rapid decline (30 to 90 min) and recovery thereafter (90 to 180 min) (Fig. 12). Saline-pretreated controls treated with amphetamine show the same temporary performance deficit. We have reported data [54] showing that the deficit is related to the time of administration of the drug and is a performance deficit which may indicate interference with retrieval systems. 1-norepinephrine bitartrate (50 ug/kg) yields results comparable to those obtained with amphetamine (Table 4). In addition to inhibition of Na+/K+ ATP'ase activity, ouabain has been shown to associatively inhibit ~4 C-leucine uptake into chicken forebrain and in particular into synaptosomal fractions [49], and to inhibit NE reuptake [18]. It is likely that ouabain does in fact inhibit protein synthesis-dependent memory processes that follow the

Cycloheximide-induced amnesia has been shown to be overcome by DPH [52], amphetamine [46] and norepinephrine [54]. Noradrenergic agonists have also been effective [47]. However, unlike ouabain-induced amnesia, CXM-induced amnesia may be counteracted by DPH and amphetamine administered as late as 30 min after learning (Figs. 10 and 13), although NE is slightly less effective at 30 min (Table 4). If these counteractive agents are administered 60 min or more after learning they are less effective. The DPH-induced recovery is observed as early as 60 min following learning (Fig. 11). With amphetamine and norepinephrine, recovery at 60 min is less pronounced, and is followed by a marked decline in retention levels with complete recovery not achieved until 180 min after learning (Fig. 12 and Table 4). There is a suggestion of a similar dip in performance with DPH, although it is not as marked. To our knowledge, DPH has not been implicated in ribosomal protein synthesis, and so its ability to overcome th eeffect of CXM must be attributed to its action on some other process. Possibly DPH extends the active life of the labile phase through stimulation of Na+/K÷ ATP'ase activity until the processes of protein synthesis recover from CXM inhibition [52]. If this is the case, one might expect that the time of administration of DPH to CXM-pretreated chicks will follow the decay function of the labile phase, that is, DPH maintains the level of memory exhibited at the

128

GIBBS AND NG TABLE 4

EFFECT

OF

ADMINISTRATION

OF

50

/.tg/kg

stimulate the sodium pump would be expected to overcome both effects of ouabain. Gibbs, Robertson and Hambley [55] have demonstrated that the non-metabolizable amino acid a-aminoisobutyrate (AIB), which is transported across cell membranes but not incorporated into proteins [90], reduces '4C-leucine uptake in vivo into chicken forebrain without affecting incorporation into protein. This evidence is consistent with the possibility that AIB interferes with protein synthesis by competing with amino acids for transport (Robertson, Gibbs and Ng, in preparation). If the above is the case, then AIB should be expected to interfere with long-term memory formation. Behavioral evidence is consistent with this view ([ 55 ] Robertson, e t al, in preparation). AIB administered 5 min before learning results in retention deficits 60 min and later following learning, with normal retention levels up to 30 min (Fig. 14). The retention function parallels that obtained with CXM (Fig. 8). Of greater significance is the time of

NOR-

EPINEPHRINE ON OUABAIN- AND CXM-INDUCED AMNESIA Peegpeatment

Ouabaln

T~ne o f Admin£atl, ation

+

+

CX2d

0 mln

~ a~o£cl£~d at 180ra£n

Lear~ainCd retention £nter~aZ

90.0

10 mln

80.0

20 min

84.2

30 min

55.0

60 min

a-doiding

40.0

90 min

60.0

120 m l n

85.0

180

mln

180

mln

15.0

60 mln

42.1

I0 mln

+

I0 mln

68.0

+

30 mln

52.6

+

60 min

40.0

+

90 mln

31.6

+ 120 min

15.4

lOO

-

80 -

-

\

60-

time it is injected. The evidence is clearly consistent with this expectation (Figs. 8 and 11). A similar mode of action is postulated to explain the effects of NE and amphetamine, as NE has b e e n ' s h o w n to stimulate Na*/K*" ATP'ase activity in chicken forebrain total homogenate [47, 52, 67]. Gibbs [47] has shown that the a and ~ noradrenergic blockers, piperoxane and propranolol, prevent the counteractive action of amphetamine on CXM-induced amnesia, while the a and ~ noradrenergic agonists, methoxamine and isoprenaline, mimic the actions of amphetamine and NE. However, only the ~ noradrenergic agonists and antagonists have any effect on Na÷/K÷ ATP'ase activity in chicken brain [67]. It is significant, however, that neither piperoxane nor propranolol prevents DPH counteraction of CXM-induced amnesia [54]. It would appear that it is not NE release p e r se but the resulting stimulation of Na÷/K* ATP'ase activity that forms the basis for the counteractive effects of NE and amphetamine. There may be, however, a more direct link between protein synthesis and Na÷/K÷ ATP'ase activity in memory formation. Ouabain, after in vivo administration results in the absence of l*C-leucine incorporation into chicken forebrain protein ]49]. Gibbs et al. [49] suggest that the observed inhibition of leucine incorporation into protein by ouabain is indirect through reduction in membrane transport of amino acids. The relevance of Na and K concentrations to both protein synthesis and Na÷/K÷ ATP'ase activity has been amply demonstrated [7,83]. It is possible therefore that amino acid transport necessary for protein synthesis associated with long-term memory formation may be associated with sodium pump activity [49,122]. If this is so, then the effect of sodium pump inhibitors like ouabain on memory formation is twofold: inhibition of labile memory formation through inhibition of Na÷/K÷ ATP'ase activity upon which memory formation is directly dependent, and indirect inhibition of long-term memory formation by preventing the sodium pump dependent uptake of necessary amino acids. Drugs like DPH which

z~

\

-

•

AIB 250mH

\

°

~

40-

20 -

~1~ "-.~ I

I

I

10

20

30

--I 60

I

I

90

120

--

I 180

LEARNING RETENTION INTERVAL (rnin)

FIG. 14. Time course of retention after intracranial injection of AIB (20/~1 of 250 mM) 5 min before learning. 100 •

AIB 250rnPI

80

~-

6O

4O

2O

~e

I -10

I -5

1

I

I

I

0 +5 +10 TIHE OF INJECTION (rain)

._

I "*,30

FIG. 15. Time of intracraniai injection of AIB (20 t~l of 250 mM) before or after learning. Retention was measured 180 min after learning. administration function for AIB (Fig. 15). The effect on retention at 180 min following learning, when AIB is administered at different times after learning, closely parallels data obtained with ouabain and ethacrynic acid (cf. Fig. 4), and not that obtained with the protein synthesis inhibitors CXM and anisomycin (cf. Fig. 7). It

PSYCHOBIOLOGY OF MEMORY would appear that the effect of AIB on long-term m e m o r y formation is occurring at a time when labile memory formation is taking place, and not at a time when long-term memory formation postulated to be dependent on protein synthesis is occurring. It seems reasonable to infer that the amino acids necessary for protein synthesis associated with long-term m e m o r y formation are taken up within the first 10 m i n following learning through sodium pump activity associated with the formation of the labile phase. If the action of AIB is as postulated above, then stimulation of Na÷/K÷ ATP'ase activity in the presence of AIB (that is, temporally close to the administration of AIB) should not be effective in overcoming the effects of AIB on long-term memory. The behavioral data (Fig. 10) show that DPH does not counteract the effects of AIB when administered 5 min before and 5 min after learning but does overcome the effects of AIB when administered between 10 and 30 min after learning. The results may imply that stimulation of the Na÷/K÷ ATP'ase activity by DPH (1) leads to progressive recovery from AIB-induced amnesia as AIB becomes replaced by relevant amino acids, (2) ultimately allows relevant amino acids to be transported so that subsequent l o n g - t e r m memory-related protein synthesis can take place, and (3) is effective in overcoming AIB-induced amnesia so long as labile memory is still intact at the time of administration and AIB is no longer competing for transport sites. Similar results have been obtained in our laboratories with L-proline (Gibbs, Ng and Richdale, in preparation), and confirm the findings reported by Van Harreveld and Fifkova [ 119 ] and Cherkin, Eckhardt, and Gerbrandt [ 26 ]. We suggest, however, that the amnesic effect of L-proline arises from disturbances in amino acid balance during the time when uptake of amino acids for subsequent memoryspecific protein synthesis is taking place, an interpretation different from that of the above authors. They attribute the effect of proline to the blocking of release of glutamate, thus leading to inhibition of short-term memory. The inference to short-term memory, however, is based on time of administration of the drug [26] and on retention first tested at 45 min after learning [ 119]. We draw attention to our observation that 4 mM glutamate inhibits short-term memory. Con clusion The results from challenges instituted against the effects of amnesia-inducing agents confirm the existence of at least three phases in the formation of memory for a single trial passive avoidance task in day old chickens, involving different underlying physiological and biochemical mechanisms. Na÷/K÷ ATP'ase appears to be clearly implicated in the formation of labile memory, a process which takes place during the first 10 min or so following learning while the short-term memory phase is still maximally active. The inhibitory effects of drugs like ouabain, ethacrynic acid, and KC1 (2.5 to 5 mM), whose action on labile m e m o r y is postulated to be through inhibition of Na÷/K÷ ATP'ase activity, may be counteracted by drugs which stimulate such activity, either directly (DPH or NE) or indirectly through release of norepinephrine (amphetamine). The fact that DPH does not overcome the effects of 1 or 2 mM KC1 on short-term memory supports the contention that this phase of memory formation is distinguishable from the succeeding labile phase, and that Na÷/K÷ ATP'ase activity is not the substantive basis for it. Furthermore, if it

129 is assumed that 1 or 2 mM KCI may also inhibit labile memory formation via inhibition of Na+/K÷ ATP'ase activity, then the fact that DPH does not produce retention recovery in 1 or 2 mM KCl-pretreated chickens at retention intervals longer than 10 min but does so in 3 mM KCl-pretreated chickens lends support to the idea that the labile phase is dependent on an active short-term phase for formation of labile memory. This argument is strengthened by the fact that not only is the labile phase susceptible to inhibition only within 10 min or so after learning but that any attempt to counteract this inhibition must be instituted within the same time period. The recovery function under different times of administration of the counteractive agents corresponds closely to the retention function observed under inhibition of labile memory. The differences in time of administration effects of DPH on ouabain and CXM based amnesia also highlight the distinction between labile and long-term memory. Recovery from ouabain-induced amnesia can be effected only if the counteractive agent is applied at a time when short-term memory is active, while recovery from CXM-induced amnesia depends on administration of the counteractive agent during the active life of labile memory. T.he latter fact also supports the contention that long-term m e m o r y formation is dependent on an active labile phase. The effect of DPH or NE on CXM-induced amnesia appears to be through stimulation of Na÷/K÷ ATP'ase activity preserving whatever is left of labile memory until protein synthesis recovers from CXM inhibition. Finally, the findings from the use of non-metabolizable amino acid AIB suggests an intimate link between the Na÷/K÷ ATP'ase processes underlying labile memory formation and protein synthesis processes underlying longterm memory formation. It would seem that amino acids required for long term memory-related protein synthesis are taken up at the same time as labile memory is being formed, and both processes are controlled by Na÷/K÷ ATP'ase activity. While DPH also overcomes AIB-induced amnesia, it does so by progressive replacement of AIB with essential amino acids through stimulation of Na÷/K÷ ATP'ase activity. The time of administration effects of DPH on AIB-induced amnesia also suggest that the counteractive action of DPH is, in this instance, dependent on the presence of active labile memory. The formation of long-term memory takes much longer than the time required for uptake of relevant amino acids and occurs at a functionally different period in the entire memory formation sequence. Thus CXM is effective in inducing amnesia when administered up to 30 min after learning, while AIB is effective when administered only up to 10 rain following learning. TOWARDS A MO DEL OF MEMORY FO RMATION A comprehensive and consistent interpretation of the evidence from our laboratories and related evidence from other laboratories implies a model of memory formation along the lines suggested by Gibbs and Ng [51]: consolidation of information into permanent memory involves at least three phases distinguishable from each other in terms of temporal parameters and putative underlying mechanisms. A fourth phase occurring immediately after learning is indicated in the literature on the effects of electroshock on memory, but we have no direct evidence from our laboratories. A diagramatic representation of the possible development and decay of memory traces as-

130

GIBBS AND NG

......

~ss~,L~ E ~ c T ~ .

iiii ',/ 10 ~5

\ 3O

60

1~

Th~,~E AFTER LEARNING (MIN)

FIG. 16. Development and decay of memory for shnple tasks in day-old chicks through short-term, labile and long-term phase. The short-term phase is possibly preceded by an electroshock-sensitive phase. sociated with each of the postulated phases is given in Fig. 16. To the extent that the temporal parameters are estimated from a large series of experiments conducted over a relatively long period of time and to the extent that the basic source of inference is behavioral data, the temporal characteristics depicted must of necessity be treated with reservation. Furthermore, while there is evidence that the general features of the model may apply to other tasks and possibly other species, the model as it stands is confined to simple learning paradigms in young chickens. Finally, the model deals primarily with the formation of memory, while the equally complex and important aspects of maintenance memory and retrieval are less clear. It is postulated that a short-term memory trace is developed within 5 min after the learning experience, is maximally active up to about 10 min following learning, and follows a negatively accelerated decay function asymptotic after 60 rain following learning. It is suggested that the STM trace is held by a phase of hyperpolarization of the neuronal membranes following afferent input (Gibbs, Gibbs & Ng, in preparation). A prolonged hyperpolarization in certain invertebrate and mammalian neurones which lasts for seconds or minutes can occur following trains of impulses generated by electrical stimulation in the normal physiological range or by natural stimulation (for reviews see [69,118]). There seems to be two independent processes that can produce prolonged afterhyperpolarizations; they can be caused by an increase in K conductance or by the activity of an electrogenic sodium pump. Experimental evidence obtained with leech ganglia suggests that the conductance increase occurs first and later on the sodium pump contributes more to the period of hyperpolarization [66]. The relative contributions of the conductance change and the electrogenic pump to the afterhyperpolarization vary with the frequency and duration of the stimulation as well as with the type of cell in the leech. Although in some cells afterhyperpolarization appears to be due predominantly to either the K conductance changes or to the activity of an electrogenic sodium pump, there are many sensory cells in which both mechi~nisms contribute to the hyperpolarization [66 ]. We propose that hyperpolarization due to K conductance changes underlies the formation of the STM trace following neuronal activity consequent on sensory input from the learning experience. On the other hand, the

hyperpolarization resulting from the activity of the neuronal sodium pump is associated with the formation of labile memory. Inactivation of the neuronal sodium pump by agents like ouabain and ethacrynic acid will prevent the formation of labile memory, but an activity-induced increase in neuronal membrane permeability to K ions can still lead to the early phase of hyperpolarization and the formation of the STM trace. In the experiments on memory formation in the chicken, it is suggested that injection of 1 or 2 mM KC1 inhibits the sodium pump generated hyperpolarization as the sodium pump is inhibited by lowered extracellular K. This would be consistent with the data of McDougal and Osborn [84]. As well as this inhibitory action, these levels of K must also prevent the phase of hyperpolarization due to increased K permeability. Thus we suggest that at this particular extracellular K level sensory input will not lead to a phase of hyperpolarization by either mechanism and STM traces will not form. The question arises as to why further reduction of the extracellular potassium allows both the STM and labile memory phases to reappear so that behaviorally there is little difference between 0 or 7 mM KC1 injection. At present we cannot satisfactorily answer this question but draw attention to the role of glia in controlling the extracellular K levels in the brain (Gibbs, Gibbs and Ng, in preparation). One suggestion is that at very low (0 to 0.5 raM) K levels, the glial cells are hyperpolarized and cease to carry out their normal physiological function of removal of neuronally released K. This might allow discrete accumulation of K around neurones that have recently been active and allow reactivation of the neuronal sodium pump leading to labile memory formation. It should be appreciated that we are suggesting that for KC1 injections greater than 2 mM and rising up to 7 mM KC1 there will be gradual reactivation of the neuronal sodium pump and that at these K levels the glial sodium pump will function normally [62]. While the above explanation is highly speculative it is consistent with our experimental findings showing that STM can be interferred with by replacing NaC1 with LiC1, with evidence that LiC1 can abolish post-tetanic hyperpolarization [30, 84, 101], and with the fact that 1 or 2 mM KC1 appears to also affect labile memory formation. Furthermore, preliminary eividence from our laboratories suggests that a concentration of 10 mM calcium prolongs the duration of STM in the presence of ouabain without overcoming the effect of ouabain on labile memory formation. This is consistent with the observation by Jansen and Nicholls [66] that raising the extracellular calcium level to 10 mM increases both the magnitude and the duration of this type of hyperpolarization. Pertinent to our hypothesis that conductance changes following sensory input is associated with the formation of STM while pump activity is associated with the formation of labile memory is the report in Kuffler and Nicholls ([69] p. 368) that hyperpolarization arising from both these mechanisms following sensory input can, in the leech, extend over a period as long as 15 rain. If similar time parameters operate within the chicken, then a physiologically viable basis exists for our postulate that labile memory is formed within the first 10 min or so following learning while the STM trace is still maximally active. What is unclear, however, is the nature of the mechanisms underlying the maintenance of STM and labile traces, and

PSYCHOBIOLOGY OF MEMORY the specific processes involved. In the case of the STM trace, at least, movement of calcium ions may be involved in maintenance if hyperpolarization is the underlying process. The hyperpolarization may be expected to act on neuronal properties such as threshold of activation, impulse conduction at branch points, efficiency of transmitter release or potassium mediated neuronal interactions between neighboring cells [ 66 ]. Physiologically, prolonged hyperpolarization will increase the period of reduced impulse traffic at its origin at the receptor level, and the subsequent period of impairment of communication within the ganglion and between ganglia (cf. [69] ). Functionally, from the point of view of memory formation, it will serve to (1) reduce further input of information for an extended period and for that period preserve existing information for immediate retrieval prior to the formation of the succeeding phase, and (2) mark previous input for an extended period possibly to permit translation of information into the next phase, It is interesting to note here that Mark [75,76] makes the suggestion that learning occurs by suppressing transmissions from synapses that are not used. This may be a means of modifying connections to make a meaningful pattern of impulse activity in the context of densely interconnected cortical cells. If convergent activity is random, then each synapse will suppress others and the relative efficacy of all remains unchanged. If, on the other hand, one terminal is used a lot, it will suppress others non-randomly so that a particular behavioral pattern is associated with a particular sensory pattern. Following Mark's argument, hyperpolarization may both affect memory pathways directly and define patterns of impulse activity specific to the learning experience. We suggest that the same sodium pump activity responsible for the hyperpolarization associated with the formation of labile memory is responsible for the uptake of amino acids necessary for subsequent protein synthesis specific to long-term memory formation. The latter takes place during the time when the labile trace is maximally active, and it is possible that the same assembly of cells is involved. Whether the same cells are involved in STM and labile memory is a moot point. In terms of the above suggestion, the existence of cells in the leech CNS which show conductance changes and sodium pump induced hyperpolarization is encouraging, although there appears to be no physiological demand that the same cells be involved in the different memory phases for these to be sequentially dependent. The issue of whether protein synthesis underlies the formation of long-term memory has been extensively researched and is the subject of a number of extensive reviews [2, 9, 10, 13, 14, 50, 111, 112]. While there is general agreement that a number of structurally unrelated antibiotics including puromycin [4, 36, 39, 89, 109, 110], anisomycin [22, 42, 43, 44, 115, 116], and cycloheximide [33, 41, 95, 96, 113, 114, 116, 117] induce amnesia for a variety of learning tasks for a variety of species of animals, there is controversy regarding the basis of the amnesic effects. Although it is generally accepted that all these drugs are potent inhibitors of cerebral protein synthesis, there is disagreement as to whether this inhibition is responsible for their amnesic action [116, 117, 122], or whether the drugs exert their amnesic effects through disruption of brain catecholamine synthesis [14, 40, 95, 96]. Furthermore, there is a dispute concerning whether

131 the effect of the drugs, by whatever mechanism, is on the formation of long-term memory or on retrieval from long-term memory [ 14]. The argument for the role of the noradrenergic system in long-term memory processing as an alternative to protein synthesis is based on a number of experimental findings. Protein synthesis inhibitors like anisomycin and cycloheximide have been shown to also inhibit catecholamine synthesis [ 17,40]. Inhibitors of catecholamine synthesis like d i e t h y l d i t h i o c a r b a m a t e (DDC), alpha-methylpara-tyrosine (AMPT), and reserpine produce amnesic effects similar to those observed with glutarimides [5, 31, 91, 95, 100]. Furthermore, stimulators of catecholamine synthesis, uptake or accumulation and release, such as metaraminol, amphetamine, and norepinephrine [106], tranylcypromine [103], caffeine [37] and MAO inhibitors such as pargyline and catron [95] have been shown to successfully counteract antibiotic-induced amnesia. Finally, it has been suggested that the amnesic effect of the glutarimides in particular, either show spontaneous recovery [38, 102, 106, 113] or recovery under behavioral reminders [97, 98, 99]. Logically, the fact that inhibitors of catecholamine synthesis produce amnesic effects similar to those produced by the glutarimides is itself not a significant counter to the protein synthesis inhibition hypothesis unless it can be demonstrated that (1) these agents do not themselves inhibit protein synthesis or (2) they do not indirectly disrupt protein synthesis by affecting processes necessary for protein synthesis, or (3) they do not affect a stage in memory processing prior to or after the stage affected by protein synthesis inhibition. In this context, it is pertinent that preliminary evidence from our laboratories (Carter, Gibbs & Ng, in preparation) show that while the retention course following administration of diethyldithiocarbmate prior to learning must closely follow that obtained with CXM, the time of administration course after learning corresponds not to that obtained with CXM but to that observed with AIB and ouabain. The possibility arises that the effect of DDC is associated with interference with the uptake of amino acids necessary for protein synthesis specific to long-term memory formation, this effect actually occurring at the time when these amino acids are being taken up (within 10 min or so following learning). It is significant that Bloom [17] shows that while amphetamine overcomes CXM-induced amnesia, it does not counteract CXM inhibition of protein synthesis. On the other hand, amphetamine is reported not to counteract DDC-induced amnesia, and to both prolong and enhance DDC-induced protein synthesis inhibition. This evidence supports the conclusion that the effects of DDC and CXM on long-term memory are based on quite distinct actions, although in terms of the model proposed here the net effect of both may be to block consolidation. In view of the above we conclude that the hypothesis that protein synthesis underlies the process of formation of long-term memory represents a comprehensive and consistent interpretation of the evidence. In terms of memory formation, at least part of the role of the catecholamines appears to be associated with their effects on Na÷/K÷ ATP'ase activity. However, they appear to be possibly involved in retrieval mechanisms associated with long-term memory. We have cited preliminary evidence to show that both amphetamine and norepinephrine produce a marked but temporary performance deficit during the period after

132

GIBBS AND NG

learning when the long-term phase is active, and that this deficit is dependent solely upon the time of administration of the drugs. It is possible that this effect is due to marked increases in norepinephrine accumulation. If this hypothesis is correct, drugs like pargyline should yield the same effect. Preliminary observations in our laboratories (Gibbs and Ng, in preparation) appear to support this expectation• We propose, on the basis of our data, a three phase model of memory formation involving: (1) a short-term phase dependent on an early phase of hyperpolarization induced by changes in the membrane permeability to K÷ ions following neural impulse activity - susceptible to inhibition by specific concentrations of KC1 and LiC1 and this is followed by (2) a labile phase resulting from a second phase of hyperpolarization induced by neural sodium pump activity, inhibited by Na÷/K÷ ATP'ase activity inhibitors like ouabain and ethacrynic acid, and by treatments which alter the cellular ionic environment with respect to relative Na÷ and K" ion concentrations; and this is followed by (3) a long-term phase dependent on protein synthesis - inhibited by protein synthesis inhibitors like cycloheximide and anisomycin, and by agents which inhibit the uptake of necessary amino acids. Figure 17 provides a schematic representation of the suggested flow of events following sensory input under the above model. We postulate further a three phase retrieval process defined, under normal conditions, by the time parameters governing the formation and maintenance of each phase of memory processing. Under conditions of inhibition of a given memory phase, retrieval will revert to the preceding phase subject to threshold criteria. The mechanisms for the retrieval process are not understood but the possibility exists that, at least in the long-term phase, noradrenergic systems are involved. Finally, we suggest that the mechanisms underlying the maintenance of memory within any given phase may not be the same mechanisms underlying its formation, although

the physiological processes defining the memory trace may be. At present the nature of the maintenance mechanisms is not known. SUMMARY Attempts to formulate models of memory formation from existing data on the psychobiology of memory suffer from difficulties associated with the use, by different investigators, of different species of animals and of different experimental paradigms. This is compounded by absence of systematic exploration of dose-response functions associated with the use of different drug treatments and temporal parameters associated with administration of treatments and learning-retention intervals. Systematic exploration at both the cellular and the behavioral level with one species of animals and one basic experimental paradigm provides a model of memory formation with a stable theoretical and data base from which generalizations to other species and to other tasks may be sought. We review findings from our laboratories on the processes involved in and the stages underlying the formation of memory for a single trial passive avoidance task in young domestic chicks, with limited generalizations to more complex tasks. Chicks are trained to avoid pecking at a metal lure by associating the lure with an aversive taste. Memory for the association is challenged by a range of pharmacological agents, selected on the basis of known or postulated pharmacological, physiological, or biochemical actions and available evidence or hypotheses about possible processes underlying memory formation• Inferences about the possible processes underlying memory formation and the stages involved arising from these primary treatments are subject to further confirmation by the use of known antagonists to the primary drugs or of other pharmacological agents with known antagonistic actions.

STAGESOF MEMORY FORMATION

I %

'PROCESSES UNDERLYING STAGES OF MEMORY FORMATION

HYPERPOLARIZATION

T

T I HYPERPOLARIZATION

SYNTHESIS

c. .

~[K+CONDUCTACTIVITY

I

ANCE CHANGE

)'i Na+-K+PUMP ACTVT IY

~IAMINO ACID ~1

UPTAKE

Ouabain sensitive

FIG. 17. Postulated sequence of events in the formation of memory, indicating points for sensitivity to inhibitory agents.

I

1

PSYCHOBIOLOGY OF MEMORY

133

On the basis of our findings we postulate a three-phase model of memory formation: a short-term memory STM) phase, a labile memory phase, and a long-term memory (LTM) phase. Existing evidence from studies using electroshock treatments suggest the possibility of an electroshock-sensitive phase preceding our STM phase. We provide no data pertaining directly to this phase. STM appears to be developed fully within 5 min of learning and to decay after 10 min following learning. Formation of STM may be inhibited by specific low doses of potassium chloride (KC1; 1 or 2 mM), isotonic KC1, 154 mM lithium chloride (LiC1), and 4 mM glutamate. On the other hand, doses of KC1 lower than 1 mM or higher than 2.5 mM have no inhibitory effect on STM. It is suggested that this phase of memory formation involves hyperpolarization associated with changes in K÷ conductance following neural activity. Labile memory develops fully by 15 min after learning and decays after 30 min. Its formation is inhibited by the sodium pump inhibitors ouabain and ethacrynic acid, and by 2.5 to 5 mM KC1. While drugs inhibiting STM formation are no longer effective if administered later than 2.5 min after learning, and exert their effects as early as 5 min after learning, the labile memory formation inhibitors are still effective when administered as late as 5 min after learning and do not exert their effects till after 10 min following learning. Furthermore, diphenylhydantoin (DPH), amphetamine and norepinephrine, which directly or indirectly stimulate sodium pump activity, overcome the inhibitory effects of ouabain, ethacrynic acid and 3 mM KC1 on labile memory formation but not that of 1 or 2 mM KC1 on STM formation. These observations together support the contention of two distinct but sequentially dependent phases of memory formation. The labile memory phase is postulated to involve a phase of hyperpolarization associated with sodium pump activity. Since the same drugs do not inhibit memory when administered after the labile memory is fully developed, it is concluded that the processes underlying maintenance differ from those underlying formation. LTM formation is dependent on an intact labile memory, and is completed after 30 min following learning with no evidence of decay of memory by 24 hr. It is inhibited by protein synthesis inhibitors such as cyclo-

heximde and anisomycin, when administered as late as 20 min after learning. The inhibitory effects are not evident, however, until after 30 min following learning. While the formation of LTM appears to involve protein synthesis, the inhibitory effects of CXM and anisomycin can be counteracted by the sodium pump stimulator DPH and by amphetamine and norepinephrine. It is argued that the action of these counteractive drugs is through prolongation of the effective life of the labile memory until after the effects of the inhibitory drugs have dissipated. LTM formation is also inhibited by the non-metabolized amino acid a-aminoisobutyrate (AIB). Of significance is the fact that the time-of-administration effect of AIB parallels closely that observed with ouabain and ethacrynic acid, but the amnesic effect does not appear until after 30 min following learning, the retention function being almost identical to those observed with CXM and anisomycin. Preliminary investigations suggest a similar pattern of results with the amino acid L-proline. These findings are interpreted as providing evidence of a direct biochemical link between protein synthesis specific to LTM formation and sodium pump activity associated with labile memory formation. It is postulated that amino acids necessary for LTM-related protein synthesis are taken up by sodium pump activity associated with labile memory formation and occurs within 10 min of learning. AIB competes with the amino acids for uptake. This interpretation is supported by the finding that DPH overcomes AIB-induced amnesia when administered between 10 and 30 min after learning, while labile memory is active, and not before 10 min and after 30 min following learning. The proposed model appears to have generality with respect to somewhat more complex learning tasks in the young chick, both with respect to the stages involved and the time parameters. Confirmatory evidence has been obtained in preliminary investigations using more complex appetitive and aversive visual discrimination tasks. The model is also interpretatively consistent with existing evidence in the literature. ACKNOWLEDGEMENT We wish to acknowledge Jenny Barnett for successfully developing the aversive wheat task and for assistance in the laboratory.

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