Neuroscience and Behavioral Physiology, Vol. 40, No. 6, 2010
Reversible and Irreversible Stages in the Development of Amnesia after Disruption of the Reactivation of Associative Memory in Snails S. V. Solntseva and V. P. Nikitin
UDC 612.822.3+612.821.6
Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 59, No. 3, pp. 344–352, May–June, 2009. Original article submitted July 14, 1008. Accepted October 20, 2008. Our previous studies on common snails have demonstrated that inhibition of NMDA glutamate receptors during reactivation of a skill consisting of refusal of a defined foodstuff leads to impairment of long-term memory. We report here our studies of the dynamics of the development of amnesia. Snails were trained to refuse a defined foodstuff and were injected 24 h later with the NMDA glutamate receptor antagonist MK-801, and were then presented with the conditioned food stimulus (a reminder). Testing on days 1 and 3 after exposure to MK-801 and the reminder showed gradual decreases in the number of refusals of the conditioned food stimulus. Repeat training of the animals to refuse the same foodstuff performed during these periods led to restoration of the skill seen after the initial training. The number of refusals by snails of the conditioned food stimulus 10 days after MK-801 and the reminder decreased to a minimal level. Repeat training at this time did not lead to the formation of a conditioned reflex to food. Thus, we have provided the first demonstration that impairment of the reactivation of long-term memory induces two stages in the development of amnesia. The first, reversible, stage, which lasted less than 10 days, was characterized by the potential for long-term memory to be restored by repeat training of the snails. The second, irreversible, stage developed 10 days after induction of amnesia and was characterized by disruption of the ability of long-term memory to be restored. These results may have practical value in terms of understanding the mechanisms of acute memory loss due to trauma and neurological diseases. KEY WORDS: learning, long-term memory, reconsolidation, amnesia, NMDA glutamate receptors, acute memory loss, common snail.
Studies in recent decades have generated significant progress in understanding the neurophysiological and molecular mechanisms of the formation and stabilization of long-term memory. “Reversed” mnestic processes, i.e., memory loss, forgetting, and amnesia, despite their undoubted theoretical, social and medical importance, remain virtually unstudied. This situation changed quite recently with the discovery of the processes of reactivation and reconsolidation of long-term memory. In particular, presentation of one of the components of the training situa-
tion to animals with acquired behavioral skills was found to “transform” memory into a labile state, in which it can be enhanced or impaired by different physical or chemical treatments [4, 13, 15, 19, 21, 24]. The procedure consisting of presentation of a training component was termed “reminding;” without reminding, reactivation of the memory did not occur. Disruption of memory reactivation by protein and RNA synthesis inhibitors, antagonists of a number of neurotransmitter receptors, including NMDA (N-methyl-D-aspartate) glutamate receptors, blockers of intracellular protein kinases and transcription factors, etc., produces transient or stable amnesia [6, 8, 9, 15, 16, 19, 24]. The sensitivity of memory to these treatments persists with a relatively narrow “time window” lasting a few hours after presentation of reminder
Anokhin Research Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow; e-mail:
[email protected].
679 0097-0549/10/4006-0679 ©2010 Springer Science+Business Media, Inc.
680 stimuli. It is important to note the specificity of the mechanisms of memory reactivation and amnesia – memory is lost only for the stimuli used as reminders. Evidence has been reported suggesting that memory loss after induction of amnesia is not instantaneous, but develops over a period of time, depending on the type of treatment used and the type of learning. For example, the onset time of retrograde amnesia induced by protein synthesis inhibitors varied significantly from 1 to 48 h in rats using both different and identical types of training [5, 9, 17, 22]. Memory loss at essentially the same times could be reversed using a series of experimental manipulations: presentation of the animals with the conditioned or reinforcing stimuli, repeated testing of memory, placing the animal in the training context, or exposure to a variety of psychostimulator substances. It should, however, be noted that most studies have assessed memory only at particular time points after impairment; systematic studies of the dynamics and characteristics of the development of amnesia have not been undertaken, and the cellular and molecular mechanisms of the induction and persistence of amnesia have not been investigated. Our previous studies addressed the mechanisms of reactivation of the associative skill of refusal of particular foodstuffs in the common snail [1–3]. Reactivation of longterm memory “traces” by presentation of the conditioned food stimulus during exposure to protein synthesis inhibitors or NMDA receptor antagonists led to stable impairments to the reproduction of the skill – repeat training two or more weeks after the initial training did not lead to recovery of the skill. We suggested that memory might be sensitive to recovery-promoting treatments, particularly repeat treating, at the earlier time points after the induction of amnesia. The present study used a repeat training procedure to study the possibility of restoring long-term memory for refusal of a defined foodstuff in common snails at different time points after impairment to memory reactivation in conditions of exposure to the NMDA glutamate receptor antagonist MK-801.
METHODS Experiments were performed on common snails (Helix lucorum) which were kept in “home” boxes and fed with raw carrot for at least two weeks prior to the experiments and during the intervals between experimental procedures. Animals were deprived of food for three days before training and testing for retention of the skill. The procedure for training to refusing a defined foodstuff was as described previously [2, 7]. Snails were fixed by the shell to a bar such that the animal could move relatively “freely” across a plastic sphere floating in water. The conditioned food stimulus was banana and the differential stimulus was cooked carrot.
Solntseva and Nikitin Pieces of food (2–3 g) were placed 0.5 cm from the animal’s oral cavity. The reinforcing stimulus was a direct electric current of amplitude 1.2 mA lasting 300 msec. The current was passed through the food and the snail’s body at the moment of the first consumatory feeding reactions (foodscraping radula movements). The latent periods of the onset of food eating were recorded using a webcam and an IBM computer. Tests were terminated if the animal did not start to eat the food within 120 sec. Combined presentations of the food and electrical current were made every 15–20 min. The conditioned stimulus was presented 14–16 times and the differential stimulus 8–10 times. Three training sessions were performed, one every day for three days. The latent periods of consumatory reactions on training day 3 in response to the last presentation of the conditioned stimulus was generally more than 100 sec. Snails received single injections of MK-801 into the body cavity through the skin of the mid part of the foot using a syringe 24 h after training and the reminder procedure was performed 15–20 min later: the animals were placed in the training apparatus (on plastic spheres) and were presented with a new stimulus (banana) three times with intervals of 10–15 min without reinforcement with the current. One hour after presentation of the first reminder stimulus, the snails were removed from the spheres and were placed in their “home” boxes. The first series of experiments addressed changes in the latent periods of consumatory reactions and the numbers of refusals of the conditioned food stimulus 1, 3, 10, and 15 days after MK-801 injections and reminding. Control animals received MK-801, after which the snails were placed in the training apparatus for 1 h; reminder stimuli were not presented and the skill was tested after 1, 3, 10, and 15 days. Each snail was tested at no more than two time points. The skill was tested by placing the snail in the training apparatus for 30 min, where they were presented with the conditioned and differential food stimuli with intervals of 10–15 min; latent periods of consumatory reactions were measured for 120 sec. The test was terminated if the animal started to eat the food within this time period. The reinforcement stimulus was not presented during testing. In the second series of experiments, three groups of animals received repeat training to the banana refusal skill, i.e., the food stimulus used in initial training, at 1, 3, and 10 days after MK-801 injections and reminding. Repeat training was performed over 1–3 days. Training to the skill was terminated if the latent periods of consumatory reactions reached 100 sec or more on the training day and these same latent periods were recorded on the next day in response to the first presentation of the conditioned stimulus. Control animals were injected with MK-801 without the subsequent reminding procedure. Retention of the skill in experimental and control snails was tested 15 days after MK-801 injections. The specific NMDA glutamate receptor antagonist MK-801 ((+)-MK-801 hydrochloride maleate (dizocilpine
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Fig. 1. Dynamics of feeding reactions to the conditioned food stimulus after administration of MK-801 and the reminder procedure in common snails trained to refuse a defined foodstuff. Left: the vertical axis shows latent periods of consumatory reactions to presentation of the conditioned food stimuli, sec. Right: the vertical axis shows the number of refusals of the conditioned food stimulus, % (logarithmic scale, used for clearer presentation of the data). On both plots, squares show animals given MK-801 and presented with reminder stimuli; circles show snails injected with MK-801 but not presented with reminder stimuli. Horizontal axes: 1 shows feeding reactions to the last presentation of the conditioned stimuli on training; 0 and the arrow show 24 h of training, the moment of administration of MK-801 and presentation of reminder stimuli; 1, 3, 10, and 15 show days after MK-801/reminders. *p < 0.05 compared with the preceding test day; single-factor analysis of variance ANOVA and a posteriori analysis using the Tukey HSD for unequal N.
maleate), Sigma, USA) was diluted in physiological saline (0.5 ml/animal). The antagonist dose was 0.005 mg/snail, corresponding to about 0.25 mg/kg. At this dose, MK-801 effectively influences the processes of consolidation and reconsolidation of the food refusal skill in snails [2]. A “blind” method was used, in which injection of snails with solutions and testing of the skill were performed by different investigators. Data were averaged and standard errors of the mean were calculated. The latent periods of consumatory reactions in response to presentation of the conditioned and differential food stimuli were compared, as were the latent periods to the conditioned stimulus in animals given MK801 and reminders, with values in snails given MK-801 without reminding procedure. Significant differences were identified by single-factor analysis of variance (ANOVA) and a posteriori analysis using the Tukey test for groups of different size.
RESULTS Dynamics of changes in reactions to the conditioned food stimulus after injections of MK-801 and the reminding procedure. By the end of the third training day, the latent period of consumatory reactions of snails (n = 56) to presentation of the conditioned food stimulus was 113 ± 4 sec and the number of refusals of the conditioned food stimulus in
these snails was 88 ± 4% (taking the total number of presentations of the conditioned stimulus as 100%; Fig. 1). Animals were injected with MK-801 24 h after training and were presented with the conditioned food stimuli as reminders. At 1, 3, 10, and 15 days after MK-801/reminding, there were significant reductions in the latent periods of consumatory reactions (F5,42 = 424.6, p < 0.000001) to 43 ± 6 sec (n = 43), 39 ± 3 sec (n = 28), 35 ± 4 sec (n = 31), and 30 ± 3 sec (n = 44) respectively and in the numbers of refusals of the conditioned food stimulus (F6,49 = 46.63, p < 0.000001) to 23 ± 5%, 12 ± 2.7%, 3.1 ± 0.8%, and 2.5 ± 0.7%, respectively. Subsequent a posteriori analysis using the Tukey HSD for unequal N revealed significant differences in the numbers of food refusals between pairs of tests performed on days 0 and 1 (p < 0.000001), 1 and 3 (p = 0.014), and 3 and 10 (p = 0.0106). In control snails given MK-801 but not presented with the reminder stimuli, latent periods of consumatory reactions were greater than 100 sec at all test time points, while the proportions of animals refusing the conditioned food stimulus were 85–92% (Fig. 1). Thus, exposure to MK-801/reminders resulted in significant decreases in the latent periods of consumatory reactions and refusal of the conditioned food stimulus. One of the measures used, i.e., the number of refusals of the conditioned stimulus, demonstrated that changes in feeding behavior from day 1 to day 10 after impairment of memory reactivation were gradual.
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Fig. 2. Repeat acquisition of the defined foodstuff refusal skill 24 h after exposure to MK-801/reminders. Experimental stages are shown at top. Black squares show snails’ responses to presentation of the conditioned food stimulus (banana); white squares show snails’ responses to presentation of the differential stimulus (cooked carrot). The vertical axis shows latent periods of consumatory reactions to presentation of food stimuli, sec. On the horizontal axis: the arrow shows the moment of MK-801 injection; numbers show days after injection of MK-801/reminding. *p < 0.05 compared with the differential stimulus.
Repeat training of snails one day after disruption of reactivation of the food refusal skill. On repeat training of the first group of snails (n = 12) 24 h after MK-801 and the reminder procedure, the latent periods of consumatory reactions to the first presentation of banana were not significantly different from those to the differential stimulus (27 ± 5 and 18 ± 4 sec, respectively, p = 0.999). However, by the end of the first training day and the first presentation of the conditioned food stimulus on the second day, the latent periods of conditioned food reactions were greater than 100 sec (Fig. 2) and the snails received significantly fewer combinations of food stimuli and electric shocks than on initial training (1.9 ± 0.5 and 9 ± 1.1, respectively, p = 0.000025). There was no difference between the latent periods of consumatory reactions to the conditioned stimulus in experimental and control snails 15 days after MK-801/reminders (114 ± 6 and 109 ± 8 sec, respectively, p = 0.999), while latent periods were greater than that on presentation of the differential stimulus (22 ± 6 sec, p = 0.000037). Thus, repeat training to the banana refusal skill 24 h after exposure to MK-801/reminders led to rapid recovery of the conditioned reflex and required 4.7 times fewer combinations of the conditioned and reinforcing stimuli than on initial training. Repeat training of snails three days after disruption of reactivation of the food refusal skill. On repeat training
of snails (n = 15) three days after MK-801/reminders (Fig. 3), the latent period of consumatory reactions on the first presentation of the conditioned food stimulus was 31 ± 9 sec, which was no different from that to the differential stimulus (23 ± 4 sec, p = 0.999). At the end of the second day of training to the skill and at the first presentation of the conditioned stimulus on training day 3, latent periods reached more than 100 sec and the number of combinations of the food and electric current stimuli was smaller than that during initial training (5.1 ± 0.5 and 8.9 ± 0.6, respectively, p = 0.000345). There was no difference between the latent periods of consumatory reactions to presentation of the conditioned stimulus 15 days after initial training in experimental and control animals (99 ± 11 and 109 ± 8 sec, respectively, p = 0.999), while these latent periods were significantly longer than that on presentation of the differential stimulus (23 ± 3 sec, p = 0.000177). Thus, repeat training three days after MK-801/reminders led to recovery of the aversive feeding skill, the number of combinations of the conditioned and reinforcing stimuli being 1.7 times smaller than that during initial training. Repeat training of snails 10 days after disruption of the reactivation of the food refusal skill. On repeat training of snails (n = 14) 10 days after exposure to MK-801 and reminders (Fig. 4), the latent periods of consumatory reactions to the first presentation of banana and the differential
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Fig. 3. Repeat training three days after exposure to MK-801/reminders. For further details see caption to Fig. 2.
stimulus were 29 ± 7 and 21 ± 3 sec, respectively (p = 0.999). Increases in the latent periods of feeding reactions to the conditioned stimulus were seen on each of the three repeat training days. However, on each subsequent training day, latent periods decreased to the level of responses to the differential stimulus (Fig. 4). Furthermore, the snails received more combinations of the food and electric shock stimuli than during initial training (11.1 ± 0.7 and 8.4 ± 0.5, respectively, p = 0.0018). The latent periods of consumatory reactions to the conditioned stimulus 15 days after initial training were significantly smaller in experimental animals than in controls (51 ± 15 and 109 ± 8 sec, respectively, p = 0.000254) and were not different from the latent period to presentation of the differential stimulus (31 ± 7 sec, p = 0.840). Thus, repeat training 10 days after exposure to MK-801/ reminders did not lead to acquisition of the aversive feeding skill, and the snails received 1.3 times more combinations of the conditioned and reinforcing stimuli then during initial training.
DISCUSSION Our previous studies showed that disruption of the reactivation of the memory of refusing a defined foodstuff by the NMDA glutamate receptor antagonist MK-801 produced amnesia lasting more than two weeks. The studies reported here showed that the development of amnesia is a process which develops over time, consisting of two stages. The first stage lasted less than 10 days and was characterized by a gradual decrease in the number of refusals of the conditioned food stimulus, with the possibility that the
memory could be recovered on repeat training to refuse the same foodstuff as used in the initial training. It should be noted that the possibility of restoring the memory decreased gradually with the passage of time from the moment of induction of amnesia. Thus, restoration of the skill 24 h after disruption required a total of 1.9 presentation of combinations of the banana and electric shock stimuli (4.7 times fewer than during initial training). At the same time, when retraining was performed three days after disruption, memory reactivation required 5.1 combinations of stimuli (1.7 times fewer than during initial training). The second stage of amnesia was characterized by loss of the animals’ ability to show recovery of the memory on repeat training. In particular, repeat training to the food refusal skill 10 days after induction of amnesia showed impairment to the formation of the conditioned reflex despite the fact that the number of combinations of the food and electric shock stimuli was 1.3 times greater than that during initial training. What are the possible mechanisms of amnesia developing after disruption of the reactivation of long-term memory? In the literature we found only one suggestion relating to this problem: that amnesia may be based on cellular and molecular events “repeating” the neurophysiological and molecular processes occurring on memory formation in reverse order [14, 24]. The widely accepted view is that the main mechanism of long-term memory consolidation consists of morphofunctional changes dependent on protein synthesis, leading to the construction and stabilization of new synaptic connections over a period of several days after training [10, 11, 18, 25, 26]. It follows from these data that the following hypothetical sequence of molecular-cellular processes occurring in
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Fig. 4. Repeat training 10 days after exposure to MK-801/reminders. For further details see caption to Fig. 2.
the snail nervous system on disruption of memory reactivation can be proposed. As demonstrated by our previous studies, the induction of amnesia depends on the synthesis of protein molecules and the activity of NMDA glutamate receptors [2, 3]. By analogy with consolidation processes, the further development of amnesia may be linked with molecular and cellular events leading to “reversion” of these morphofunctional changes and elimination of synapses. Elimination of synapses increases with increases in time from the moment at which amnesia appears, though morphological rearrangements and amnesia are relatively easily reversible by repeat training during the first few days. By 10 days, destructive changes are evidently completed by the formation of a qualitatively new morphofunctional state of nerve cells, which is characterized by disruption of the mechanisms of consolidation of this skill on repeat training. It is important to emphasize that the molecular-cellular changes underlying amnesia are system-specific, i.e., are characteristic only for those nerve cells and synaptic connections involved in the system for a given behavioral act. Thus, we have previously shown that snails with irreversible amnesia following disruption of reactivation of the memory for a defined foodstuff were able to learn the associative skill of refusing a different foodstuff [2, 3]. However, it would not be correct to take the view that the processes of amnesia and memory formation are identical and differ only in terms of a reversible sequence of development. Moreover, the processes of amnesia appear to be significantly different from the mechanisms of memory formation. This is evidenced in particular by the fact that completion of the “deconsolidation” process would be expected to be followed by “deletion” of the memory trace,
which does not exclude the possibility of forming the same skill on repeat training. However, as noted above, amnestic processes ended with the nervous system in a state in which acquisition of the skill of refusing the same foodstuff as used in initial training was disrupted. It is currently difficult to define the processes responsible for the stable amnesia arising 10 days after its induction. It follows from the above hypotheses that one cause of amnesia may be “disruption” of the morphological “carriers” of engrams due to neuron death or elimination of functionally vital synaptic connections between nerve cells. Without completely excluding this possibility, we will present data contradicting it. Our experiments showed that on repeat training 10, 11, and 12 days after induction of amnesia, the latent periods of consumatory reactions increased significantly on each training day, though they returned to the initial level on the next day. This fact is evidence for retention of the short-term memory and, consequently, the structural basis of its formation. On the other hand, studies of simple forms of learning in mollusks and long-term potentiation in mammals have demonstrated that the molecular mechanisms of short-term and long-term memory are located in the same nerve cells, though they are significantly different [11, 12]. These data suggest that synaptic connections between neurons are not completely eliminated during the process of development of amnesia in snails: same “basal” proportion of synapses persist and provide the basis for forming short-term and long-term memory, and for the process of reproduction [23, 27]. Another cause for disruption of memory consolidation on repeat training is disruption of the molecular processes of long-term synaptic plasticity in the neurons involved in
Reversible and Irreversible Stages in the Development of Amnesia the processes of engram formation and retention. Possible mechanisms of stable amnesia in this situation are: stable repression of the transcription of genes controlling the plastic properties of neurons, epigenetic mechanisms such as modification of chromatin on DNA methylation, prion-like autotransformations, and other types of modulation of chemical reactions maintaining “protein turnover” and involved in the long-term regulation of synaptic plasticity [11, 13, 20]. Because of the lack of experimental data, the exact role of any of these processes cannot be determined, though resolution of this problem in any case requires widening of the search for the molecular mechanisms of memory reconsolidation and the development of amnesia from signal pathways to structural changes in cells. It is important to note that the two different stages in the development of amnesia identified here may be a general biological phenomenon characteristic of at least some types of memory in different animal species and not restricted to the learning model used here. The existence of a stage at which amnesia is sensitive to modulatory treatments must be taken into consideration when testing memory retention and the possibility of its recovery at different time points after induction of amnesia. Our results should be considered during the clinical analysis of the mechanisms of amnesia, particularly that arising in conditions of “acute” memory loss of various causes [19, 24]. The tactics for correcting impaired memory by therapeutic procedures or using pharmacological agents may depend on the stage of amnesia and the involvement of different molecular and cellular mechanisms.
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CONCLUSIONS 14.
In snails with the acquired associative skill of refusing a defined foodstuff, administration of the NMDA glutamate receptor antagonist MK-801 and presentation of conditions serving as a reminder of the conditioned food stimulus induced disruption to long-term memory consisting of two stages. The first stage lasted less than 10 days and was characterized by gradual decreases in the number of refusals of the conditioned food stimulus and the potential for memory recovery on repeat training to refuse the same foodstuff as used in the initial training. The second stage of amnesia was characterized by disruption of the animals’ ability to recover memory on repeat training. This study was supported by the Russian Foundation for Basic Research (Project No. 08-04-00833).
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