Effects of Agonists of NMDA and Serotonin Receptors at Different ...

ISSN 18197124, Neurochemical Journal, 2010, Vol. 4, No. 3, pp. 189–195. © Pleiades Publishing, Ltd., 2010. Original Russian Text © S.V. Solntseva, V.P. Nikitin, 2010, published in Neirokhimiya, 2010, Vol. 27, No. 3, pp. 214–220.

EXPERIMENTAL ARTICLES

Effects of Agonists of NMDA and Serotonin Receptors at Different Stages of Amnesia Caused by Impairment of LongTerm Memory Reconsolidation S. V. Solntseva and V. P. Nikitin1 Anokhin Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, Russia Received March 30, 2010

Abstract—Previously we showed that in snails that were conditioned for food aversion impairment of mem ory reconsolidation MK801, an antagonist of NmethylDaspartate (NMDA) receptors, induced amnesia, at the early stage of which ( 0.05) in animals of both groups and were 25–40 s on the average. Another 3day training session focused on the aversion of the same type of food as in the first training session did not restore the skill. The testing of the snails on the 20th day (3 days after the second learning session) demonstrated that latencies of responses to the conditioning stimulus were 33 ± 11 s and 37 ± 9 s in control groups 2 and 3, respectively. These values were significantly lower (p < 0.0001) than the latency of responses to the condition ing stimulus 20 days after MK801 injection alone (94 ± 15 s). Thus, MK801/reminding induced stable amnesia in trained snails, and the second learning ses sion did not result in acquisition of conditioned taste aversion. Moreover, an additional reminding proce

Effects of NMDA on the Development of Amnesia Induced by MK801/Reminding in Snails with Conditioned Taste Aversion

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The snails of th e first group (n = 8) were placed in the training situation and exposed to reminding stim uli 24 h after MK801 injection (Fig. 1); 3 days later, the snails were injected with NMDA and subjected to the reminding procedure. It should be noted that after NMDA injection the latencies of consummatory responses to reminding stimuli were at the level of responses to differentiation stimuli (20–40 s). Dur ing the second learning session (Fig. 1) 15 days after MK801/reminding, we found that the latencies of responses to conditioning and differentiation stimuli did not differ during the first presentation (30 ± 7 s and 23 ± 5 s, р = 0.75). However, at the end of the first training day, as well as at the first presentation of con ditioning stimulus on the next day, the latencies of the responses exceeded 100 s (Fig. 1), and snails received less electrical stimulation than during the primary training (2.9 ± 0.4 and 7.1 ± 0.9, respectively, p =

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Fig. 2. NMDA injections and reminding by the training situation (without conditioning stimuli) on the 3rd day after amnesia induction. The designations are the same as in Fig. 1.

0.0005). At 20 days after MK801/reminding the latencies of consummatory responses to conditioning stimuli did not differ from those of control animals, which received MK801 injection alone without the reminding procedure (116 ± 4 s and 94 ± 15 s, respec tively, p = 0.9), and were larger than the latencies for the differentiation stimulus (28 ± 7 s, p = 0.00005). The animals of the second group (n = 8) were also injected with NMDA 3 days after MK801/reminding but then only placed in the training situation without the reminding stimuli (reminding by learning, Fig. 2). During the second learning session, which was per formed 15 days after MK801/reminding the latencies of consummatory responses to the first presentation of conditioning and differentiation stimuli were 36 ± 11 and 24 ± 7 s, respectively (p = 0.9). The second training session did not elaborate the skill: the latencies of responses to conditioning and differentiation stimuli measured on the 20th day still showed no difference (40 ± 9 and 24 ± 8 s, respectively, p = 0.8). During their second training session, animals were stimulated with current more than during the first session (11.5 ± 1.0 vs.. 8.2 ± 0.6, respectively, p = 0.003). The snails of the third group (n = 8) received NMDA with following reminding session 12 days after MK801/reminding (Fig. 3). The latencies of responses to conditioning stimulus were 30–35 s. During the second training session (15 days after MK801/reminding) the latencies of consummatory

responses to conditioning and differentiation stimuli did not differ after the first presentation (25 ± 5 and 29 ± 6 s, р = 0.99). The second training session also did not result in successful skill elaboration: the testing of the skill performed on the 20th day did not reveal any difference between the latencies of consummatory responses to conditioning and differentiation stimuli (41 ± 12 s and 31 ± 8 s, respectively, p = 0.8). In addi tion, the snails of the third group were stimulated with more current during the second training session than during the first one (11.9 ± 0.7 vs.. 8.5 ± 0.4, respec tively, p = 0.018). Thus, injection of NMDA to the snails and the reminding procedure both performed 3 days after amnesia induction did not improve the latencies of responses to the conditioning reminding stimulus. However, the consequent second training session quickly restored the conditioned reflex. In contrast, if NMDA injection and reminding were performed 12 days after amnesia induction, the second training session was unable to restore the skill. Effects of 5HTP on the Development of Amnesia Induced by MK801/Reminding At 3 days after MK801/reminding the snails (n = 8) were injected with 5HTP and subjected to the reminding procedure (Fig. 4). During this procedure, the latencies of responses to the conditioning stimulus were 30–45 s. During the second training session NEUROCHEMICAL JOURNAL

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Fig. 3. The ffects of NMDA and reminding on the 12th day after amnesia induction. The designations are the same as in Fig. 1.

15 days after MK801/reminding, we found that the latencies of consummatory reactions to conditioning and differentiation stimuli did not differ from the first presentation (29 ± 8 s and 26 ± 4 s, р = 0.9). The second training session did not result in the appearance of the conditioned reflex: the final test on the 20th day showed that latencies of responses to conditioning and differentiation stimuli were 49 ± 17 and 28 ± 7 s, respectively (p = 0.8). The snails were stimulated with more electric current during the second training ses sion than during the first one (11.6 ± 0.8 vs. 9.1 ± 0.8, respectively, p = 0.001). Thus, 5HTP injection and reminding on the 3rd day after amnesia induction did not influence the responses induced by reminding conditioning stimuli, and the second training session failed to elaborate the skill in these animals. DISCUSSION In our previous studies [6], we showed that in snails that were conditioned for taste aversion, reminding to a conditioning food stimulus during NMDA antago nist administration resulted in the development of amnesia. At its early stage (less than 10 days) the resto ration of the skill was still possible after a second train ing session, while the late stage of amnesia (≥10 days) was resistant to the secondary training. In the present study, we investigated the influence of NMDA and 5HT receptor agonists, NmethylDaspartate (NMDA) and 5HTP, on the mechanisms of amnesia NEUROCHEMICAL JOURNAL

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induced by the impairment of reconsolidation of a conditioned taste aversion memory by the NMDA antagonist MK801. We found that NMDA/reminding at the early stage of amnesia did not result in memory restoration. Indeed, the latencies of the responses to the reminding conditioning stimuli did not differ from the latencies of responses to differentiation stimuli 3 days after amnesia induction (just after NMDA injec tion), as well as at the first presentation of condition ing stimuli during the second training session 15 days after amnesia induction. However, the second learning session led to rapid restoration of the skill in these ani mals. These results suggest that NMDA receptor acti vation and reminding had no direct effect on memory restoration but transformed the mechanisms of amne sia development in a way such that, instead of the irre versible processes typical of “NMDAdependent” amnesia, we observed the development of processes that were sensitive to consequent learning. NMDA injection and reminding by training situa tion (without the presentation of conditioning stimuli) on the 3rd day after amnesia induction did not influ ence its development: a stable amnesia developed that was insensitive to repetitive training. We also did not find any influence of NMDA agonist in combination with reminding on amnesia progression if presented on the 12th day after its induction (i.e., at the late stage). Thus, for the occurrence of the antiamnestic effect of NMDA, the combination of the agonist and

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Fig. 4. Injection of 5HT metabolic precursor 5hydroxytriptophane (5HTP) and reminding on the 3rd day after amnesia induc tion. The designations are the same as in Fig. 1.

presentation of conditional stimuli have to occur at the early stage of amnesia. Previously, we showed that exposure of snails to methiothepin, a 5HT receptor antagonist, during the elaboration of conditioned taste aversion and just before the reproduction of this skill caused impair ment in the consolidation and reconsolidation of longterm memory and consequent amnesia develop ment [4, 5]. Another conditioning performed 2 weeks later quickly restored the learned skill. On the other hand, in the current study we demonstrated that a 5HT receptor agonist, 5HTP, along with a remind ing procedure 3 days after the induction of “NMDA dependent” amnesia did not affect its development: the second training session did not result in restoration of the conditioned reflex. Taken together, these results suggest that 5HT receptors, which play a great role during consolidation and reconsolidation of memory, seem not to be involved in its storage or restoration, if the memory was damaged. A similar view on the role of 5HT receptors in the mechanism of conditioned taste aversion in snails has been reported by other authors [9]. Thus, on the basis of behavioral manifestations, “5HTdependent” amnesia seems to be similar to “NMDAdependent” amnesia, which is transformed on the 3rd day after its induction by NMDA/reminding combination. In both cases, skill loss was reversible

using subsequent repetitive training. These data sug gest that the molecular alterations that develop during the early phase of amnesia predispose its reversibility during the late phase. However, it is still not clear what type of neurochemical mechanisms are involved in the progression of amnesia at different stages. Earlier, we demonstrated that a possible cause of amnesia may be altered molecular mechanisms of longterm synaptic plasticity in the neurons involved in engram genera tion and storage [3]. We proposed several possible mechanisms for irreversible amnesia: stable suppres sion of transcription of neuroplasticity–responsive genes, epigenetic mechanisms (such as modification of chromatin due to DNA methylation), priontype autotransformations, and other modulations of molecular machinery regulating longterm synaptic plasticity [14, 18, 20]. Another possible cause of irre versible amnesia is the destruction of the morphologi cal substrate of the engram due to neuronal death (e.g., via apoptosis) or the elimination of functionally important interneuronal synapses. The lack of experimental data makes further vali dation of the contribution of each of the above factors impossible but, in any case, any substantial progress in this area requires further fundamental research in the area of the basic molecular events that underlie mem ory reconsolidation and the development of amnesia. NEUROCHEMICAL JOURNAL

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CONCLUSIONS The experimental data obtained on snails that were trained for conditioned taste aversion support our assumption that during impaired memory reconsoli dation different stages of amnesia development and its sensitivity to the action of mnemotropic substances occur. During early phase of amnesia (the 3rd day after its induction), an NMDA agonist and reminding altered the process of amnesia development, which resulted in the rapid restoration of a memory after repeated conditioning. NMDA injections and remind ing during the late phase of amnesia (12 days after its induction) did not influence its development, and repeated conditioning did not restore the skill. A 5HTP injection/reminding that was performed 3 days after amnesia induction also did not influence its develop ment. These data indicate the critical and specific role of glutamate NMDA receptors in the mechanisms of acquisition and storage of a memory of conditioned taste aversion, as well as amnesia development and memory restoration. ACKNOWLEDGEMENTS The study was supported by the Russian Founda tion for Basic Research, project no. 080400833. REFERENCES 1. Dubrovina, N.I., Zinov’ev, D.R., and Zinov’eva, D.V., Byull. Eksperim. Biol. Med, 2007, vol. 144, no. 11, pp. 484–486. 2. D’yakonova, V.E., Usp. Fiziol. Nauk, 2007, vol. 38, no. 3, pp. 3–20. 3. Kozyrev, S.A. and Nikitin, V.P., Rossiiskii fiziol. zhurn. im. I.M. Sechenova, 2009, vol. 95, no. 6, pp. 652–662. 4. Solntseva, S.V. and Nikitin, V.P., Ros. Fiziol. Zhurn. im I.M. Sechenova, , 2007, vol. 93, no. 10, pp. 1101–1111. 5. Solntseva, S.V. and Nikitin, V.P., Rossiiskii fiziol. zhurn. im. I.M. Sechenova, 2008, vol. 94, no. 8, pp. 860–870.

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