Mechanisms of Memory Reorganization during Retrieval of Acquired Behavioral Experience in Chicks: the Effects of Protein Synthesis Inhibition in the Brain

According to current concepts, memory can be disrupted by administration of protein synthesis inhibitors over a relatively short time period before and after learning. However, data have been obtained indicating that protein synthesis inhibitors can induce amnesia when given long after learning if administration is performed in reminder conditions, i.e., when the animal is presented with one of the environmental components which previously formed the learning situation. The aim of the present work was to confirm the possibility of inducing memory disruption in chicks at late post–learning stages by administering the protein synthesis inhibitor cycloheximide in association with a reminder procedure. Day–old chicks were trained to perform a standard passive avoidance task. Chicks were given cycloheximide (20 g, intracerebrally) 5 min before the reminder procedure, which was performed 2, 24, or 48 h after training. Testing was conducted 0.5, 1, 3, 24, and 48 h after the reminder. Administration of cycloheximide in association with the reminder procedure induced the development of temporary amnesia, whose duration gradually decreased as the interval between training and reminding increased. These data led to the hypothesis that a memory reactivated by a reminder undergoes a process of reorganization and reconsolidation, which depends on the synthesis of new proteins. The quenching of the ability of protein synthesis inhibition during the reminder to disrupt memory demonstrates the existence of a gradual process resulting in consolidation of memory between 2 and 48 h of learning.     

Effects of protein synthesis inhibitors during reactivation of associative memory in the common snail induces reversible and irreversible amnesia

The effects of protein synthesis inhibitors on the reactivation of an associative skill consisting of refusing a particular food by common snails were studied. Animals were given single injections of a protein synthesis inhibitor (cycloheximide at 0.6 mg/snail or anisomycin at 0.4 mg) 24 h after three days of training, and were then presented with a “reminding” stimulus (the “conditioned reflex” food-banana) and tested for retention of the skill. Observations revealed an impairment of reproduction of the acquired skill 2.5 h after the “reminder, ” with spontaneous restoration at 4.5–5.5 h. Other snails were given single 1.8-mg doses of cycloheximide or three 0.6-mg doses with intervals of 2 h. “Reminders” were presented after each injection. In these conditions, impairment of reproduction of the conditioned reflex also appeared 2.5 h after the first “reminder, ” though amnesia lasted at least 30 days and repeat training of the animals produced only partial recovery of the skill. Thus, we have provided the first demonstration that recovery of a long-term memory “trace” on exposure to relatively low doses of protein synthesis inhibitors produces transient and short-lived amnesia, lasting 2–3 h, while long-term, irreversible amnesia occurrs after longer-lasting or more profound suppression of protein synthesis. These results suggest that the “reminding” process induces reconsolidation of the “ initial” memory, suppression of which by protein synthesis inhibitors leads to “erasure” of the memory “trace” and impairs consolidation on repeat training. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 9, 1058–1068. September, 2006.     

Selective Impairments to Memory Consolidation in Chicks Produced by 5′-Iodo-2′-Deoxyuridine

The aim of the present work was to study the role of DNA synthesis in the formation of different types of memory in neonatal chicks. The nucleotide analogs 5′-iodo-2′-deoxyuridine (IdU) and 5′-bromo-2′-deoxyuridine (BrdU) were used; these are incorporated into DNA, impairing its function, and have amnestic actions in defined models of learning in mice. We studied the effects of 5′-iodo-2′-deoxyuridine of the formation of long-term memory in chicks during training in different models: passive avoidance, imprinting, taste aversion, and spatial learning in a maze. In the taste aversion model, i.p. administration of IdU (10 mg/kg 5 min before or 50 min after training) had an amnestic effect on testing 1–2 days after training. IdU-induced amnesia developed more than 6 h after training, while administration of IdU 2 h after training had no amnestic effect. 5′-Bromo-2′-deoxyuridine also had a similar amnestic action in the taste aversion model. In the passive avoidance, imprinting, and spatial maze learning models, administration of IdU at the same dose before and after training did not induce amnesia. These data lead to the suggestion that DNA synthesis in the brain may play a critical role in the mechanisms of memory consolidation in chicks in types of learning such as taste aversion.     

Protein Synthesis Is Required for Induction of Amnesia Elicited by Disruption of the Reconsolidation of Long-Term Memory

Studies on common snails previously trained to an associative skill consisting of rejecting a defined foodstuff addressed the effects of NMDA glutamate receptor antagonists (MK-801 and APV) and protein synthesis inhibitors (cycloheximide and anisomycin) on long-term memory reconsolidation processes. Injections of each of the study compounds before the reminding procedure 24 h after training were found to lead to impairment of the reproduction of the acquired skill, which lasted at least three weeks. Repeat training of these animals to reject the same foodstuff as used in the initial training did not lead to acquisition of the skill. However, simultaneous injections of a protein synthesis inhibitor and an NMDA receptor antagonist (MK-801 + cycloheximide or APV + anisomycin) did not impair the skill. In subsequent experiments, snails received cycloheximide at different times after exposure to MK-801/reminding. Administration of cycloheximide 3 and 6 h after MK-801/reminding led to the development of incomplete amnesia and repeat training of the animals led to rapid restoration of memory. Administration of cycloheximide 9 h after MK-801/reminding evoked the development of stable amnesia characterized by impairment of skill formation on repeat training. We propose that the mechanisms of amnesia induced by the NMDA glutamate receptor antagonist, by analogy with the mechanisms of other long-term adaptive rearrangements of the brain, depend on translation and can be suppressed by inhibitors of translation. The “time window” for the dependence of amnesia induction processes on the synthesis of protein molecules was 6–9 h after exposure to MK-801/reminding.     

Involvement of protein synthesis in the reconsolidation of memory at different time points after formation of conditioned reflex freezing in mice

The aim of the present work was to study the involvement of protein synthesis in the reconsolidation of memory at different periods of time after training. In mice trained in a conditioned reflex freezing model, memory was reactivated by a reminder combined with administration of the protein synthesis inhibitor cycloheximide. The results showed that suppression of protein synthesis on reactivation of memory 3, 6, and 24 h and 14 and 30 days after training impaired acquired conditioned reflex freezing. These data provide evidence that memories retrieved by a reminder require protein-dependent reorganization at both short (3–6 h) and long (14–30 days) periods after training. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 56, No. 2, pp. 274–281, March–April, 2006.     

Recovery of Memory by the Glutamate NMDA Receptor Agonist D-Cycloserine Depends on the Stage of Development of Amnesia

We have previously observed that in common snails trained to an associative skill consisting of refusing a defined foodstuff, impairment of memory reconsolidation by the NMDA receptor antagonist MK-801 evokes amnesia in which the skill can be recovered by retraining at the early stage (<10 days) while retraining at the late stage does not lead to formation of the skill. In the experiments reported here, amnesia was induced with MK-801 and a reminder 24 h after training of snails to conditioned taste aversion, and the antiamnestic effects of D-cycloserine, a partial agonist of the glycine site of the NMDA receptor, were studied in the early (day 3) and late (day 12) stages. Three days after induction of amnesia, injections of D-cycloserine and a reminder of the conditioned food stimulus led to recovery of the memory; administration of D-cycloserine without a subsequent reminder was ineffective. Injection of D-cycloserine and a reminder 12 days after induction of amnesia had no effect on its development and retraining did not lead to recovery of the memory. Thus, this study is the first to show that the NMDA receptor agonist D-cycloserine influences memory recovery processes only at the early stage of development of amnesia induced by lesioning of reconsolidation processes, while the late stage of amnesia was not sensitive to this agonist.     

Characteristics of retrograde amnesia following reactivation of memory in mice

Amnesia for approach-avoidance learning was induced in mice by injecting the protein synthesis inhibitor anisomycin (ANI) immediately, 1, or 2 hours, but not 3 hours after training. A robust amnesia could be demonstrated if ANI was administered 3 hours after training, immediately following a 60 second exposure to the training apparatus or to a structurally similar environment. The temporal gradient of effectiveness of amnesia production by ANI was significantly steeper following reactivation treatment than it was following initial training. In addition, while amnesia produced by the conventional procedure remained stable for 6 days, the amnesia induced following reactivation treatment spontaneously recovered 4 days after training. These findings are discussed in terms of their relevance to interpretations of retrograde amnesia studies.     

Different temporal profiles of amnesia after intra-hippocampus and intra-amygdala infusions of anisomycin

Systemic or intra-hippocampal administration of the protein synthesis inhibitor anisomycin generally leads to impairments in memory tested 24 hr or more after training but spares memory for a few hours after training. Thus, amnesia does not appear immediately after training but develops with time, findings most often interpreted as evidence for distinct short- and long-term memory processes. However, time courses for the onset of amnesia vary substantially after treatment with protein synthesis inhibitors. Some of the variability across experiments may reflect task-related differences or, perhaps relatedly, may reflect memory processing mediated by different neural systems. In the present experiments, anisomycin was infused into either the hippocampus or the amygdala 20 min before inhibitory avoidance training. Similar to previous findings, intra-hippocampus injections of anisomycin impaired memory tested 48 hr after training yet spared memory tested 4 hr after training. In contrast, intra-amygdala injections of anisomycin impaired memory tested at 0.5, 4, and 48 hr after training, revealing no evidence for spared memory at short times after training. The distinct temporal properties for amnesia following anisomycin injections into the hippocampus or amygdala may reflect different consequences for memory of perturbations of the neural system in which the manipulation is made.     

The roles of information reactivation and nonassociative arousal in recovery from ECS-induced retrograde amnesia

Restoration of memory by pretest reminder treatments following ECS-induced amnesia in principle could be mediated by nonassociative arousal and/or reactivation of information by the reminder stimuli. In the present research, arousal value of the reminder treatment was largely a function of the novelty of the context in which the reminder cues occurred. After producing ECS-induced amnesia in rats for a signaled passive avoidance task, presentation of the signal outside of the training and test context proved to be an effective reminder cue. However, prereminder familiarization with the reminder location, which was sufficient to reduce exploratory behavior suggestive of a decrease in the arousal value of the context, did not influence recovery from amnesia. These data indicate that reminder treatments are capable of reversing experimental amnesia by initiating further processing of previously acquired information even when the treatment does not produce appreciable arousal.     

Reversible and Irreversible Stages in the Development of Amnesia after Disruption of the Reactivation of Associative Memory in Snails

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.     

Genetic mechanisms of the involvement of SMP-69 protein in memory consolidation

Intracerebral administration of antibodies to SMP-69 protein to rats 24 h prior to passive avoidance conditioning impaired memory processes, while the administration 48 h after learning had no such effect. Activation of RNA and protein synthesis was observed 24 h after the administration. It is suggested that the impaired consolidation of memory traces is due to the synthesis of “anticonsolidation” proteins. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 8, pp. 141–143, August, 1999     

Protein synthesis inhibition and amnesia for saccharin aversion memory in rats after intra-cisternal administration of cycloheximide

The series of experiments reported further investigated the time course relationship of the amnesia for saccharin aversion memory in rats and the inhibition of protein synthesis resulting from cycloheximide (CXM). The first experiment showed that intraventricular CXM injected rats which had been trained 7 hrs after injection had learned the contingency under a considerable inhibition of protein synthesis. However, 1 hr after CXM injection protein synthesis inhibition was 86% and yet there was no amnesia observed 24 hr after training. It was also apparent that there were no marked regional differences in the extent of protein synthesis inhibition after intraventricular CXM. Finally, intracisternal injection of CXM 1 hr before training resulted in amnesia 24 hrs after training although this effect was greater when the injection was performed 7 hr before training. The findings (1) are consistent with Day et al's [4] suggestion of maximal protein synthesis inhibition at 1 hr post CXM injection with an approximately linear decline thereafter, and (2) provide no support for the involvement of the brain stem nucleus solitarius in taste aversion learning.     

Influence of isolation stress and inhibited protein biosynthesis on learning and memory in goldfish

Goldfish, Carassius auratus auratus L. (Pisces, Cyprinidae), were trained by different kinds of training procedures under the influence of cycloheximide or puromycin, two inhibitors of the protein biosynthesis. After active avoidance training in a shuttle box an apparent amnesia was found only when the fish were exposed to a one day lasting isolation stress prior to training. If the animals were accustomed to isolation over a period of 20 days the inhibitors did not affect memory formation. After learning by positive reinforcement (food rewarded color discrimination) in groups under stress-free conditions, neither learning nor memory formation were impaired in spite of the presence of cycloheximide. It is suggested that the amnestic effect of the inhibitors is caused by isolation treatment. Lack of the additional stress, however, leads to memory formation.     

Ca2+/calmodulin protein kinase II and memory: learning-related changes in a localized region of the domestic chick brain

The role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the recognition memory of visual imprinting was investigated. Domestic chicks were exposed to a training stimulus and learning strength measured. Trained chicks, together with untrained chicks, were killed either 1 h or 24 h after training. The intermediate and medial hyperstriatum ventrale/mesopallium (IMHV/IMM), a forebrain memory storage site, was removed together with a control brain region, the posterior pole of the neostriatum/nidopallium (PPN). Amounts of membrane total αCaMKII (tCaMKII) and Thr286-autophosphorylated αCaMKII (apCAMKII) were measured. For the IMHV/IMM 1 h group, apCaMKII amount and apCAMKII/tCaMKII increased as chicks learned. The magnitude of the molecular changes were positively correlated with learning strength. No learning-related effects were observed in PPN, or in either region at 24 h. These results suggest that CaMKII is involved in the formation of memory but not in its maintenance.     

Development of learning and memory in mice after brief paradoxical sleep deprivation.

The effects of three hour paradoxical sleep deprivation (3 hr PSD) via the water tank procedure to produce retrograde amnesia of active avoidance and inhibitory avoidance learning was examined in mice. Results indicated no memory impairment in experimentally treated groups. An attempt was then made to induce amnesia by administering ECS immediately after 3 hr PSD thereby increasing the susceptibility of the memory trace to disruption. This procedure, however, also results in good retention. We conclude that the paradoxical sleep (PS) phase immediately after aversively motivated training is not essential for subsequent development of learning and memory. These results do not, however, detract from previously reported protracted PSD effects on memory storage processes.     

Temporal effects of dehydroepiandrosterone sulfate on memory formation in day-old chicks

Dehydroepiandrosterone sulfate (DHEAS) has been shown to enhance memory retention in different animal models and in various learning paradigms. In the present study, we investigated the effect of peripherally administered DHEAS on the acquisition, consolidation and retention of memory using a weak version of the one-trial passive avoidance task in day-old chicks. Intraperitoneally administered DHEAS (20 mg/kg) either 30 min before or 30 min and 4.5 h after training on the weakly aversive stimulus, enhanced recall at 24 h following training, suggesting a potentiation of not only the acquisition but also the early and late phases of memory consolidation. In contrast, when DHEAS was administered at 30 min prior to the 24 h retention test there was no memory enhancement, indicating a lack of effect on memory retrieval. Memory recall was unaltered when DHEAS was administered at 30 min before training in a control group trained on a strongly aversive stimulus, confirming memory-specific effects. Interestingly, the memory enhancement appeared to be sex-specific as male chicks showed higher recall than females. These findings provide further evidence that DHEAS enhances memory and may be involved in the temporal cascade of long-term memory formation.     

Halothane anesthesia causes state-dependent retrieval failure in mice.

Effects of exposure to halothane on memory processing was studied using single-trial inhibitory avoidance learning to measure retention. Mice were anesthetized with halothane either before training, immediately after training, or both before training and before testing. Results showed that memory was not impaired by posttraining halothane exposure, indicating that the anesthetic does not cause retrograde amnesia. Mice trained after recovery from halothane showed a robust memory loss 24 h later. This deficit could be alleviated by reexposure to the anesthetic before the retention test. Mice given multiple training trials following recovery from the anesthetic showed a normal rate of learning when compared with controls, but deficient retention. This indicates that the performance deficit was the result of impaired retention (anterograde amnesia) rather than disrupted acquisition. Anterograde amnesia occurred when training was delayed up to 2 h after recovery from anesthesia. These findings indicate that the memory impairment following halothane anesthesia is the result of a state-dependent retrieval failure.     

Effects of protein synthesis inhibition on memory for active avoidance training.

Inhibition of brain protein synthesis by anisomycin and acetoxycycloheximide was studied in mice for its biochemical and behavioral effects. By employing both drugs in low doses in a series of injections, we were able to maintain inhibition of protein synthesis of 80% or greater for up to 14 hr without causing detectable permanent physiological impairment. The drugs were employed as amnestic agents in mice trained to avoid footshock in a T-maze. As the duration of inhibition increased, the percentage of mice classed as amnesic increased. This amnesia could be reduced by increasing (a) the rate of acquisition, or (b) the number of training trials. Anisomycin was shown to cause a significant degree of amnesia for the escape component as well as the avoidance component of the learning. A single injection of anisomycin given 15 min prior to training did not cause significant changes in the acquisition or retention of avoidance conditioning, when comparison was made with saline-injected controls. Only additional injections given after training to prolong inhibition caused amnesia. Thus, those injections critical in obtaining amnesia were given at a time at which interference with acquisition could not have occurred, so the results bear clearly on memory processes.     

Memory loss caused by beta-amyloid protein is rescued by a beta(3)-adrenoceptor agonist

Accumulation of the neurotoxic beta-amyloid protein (Abeta) in the brain is a key step in the pathogenesis of Alzheimer's disease (AD). Although transgenic mouse models of AD have been developed, there is a clear need for a validated animal model of Abeta-induced amnesia which can be used for toxicity testing and drug development. Intracranial injections of Abeta(1-42) impaired memory in a single trial discriminative avoidance learning task in chicks. Memory inhibition was closely associated with the state of aggregation of the Abeta peptide, and a scrambled-sequence of Abeta(1-42) peptide failed to impair memory. Abeta had little effect on labile (short-term and intermediate) memory, but blocked consolidation of memory into long-term storage mimicking the type of anterograde amnesia that occurs in early AD. Since noradrenaline exerts a modulatory influence on labile memory in the chick, we examined the effects of two beta-adrenoceptor (AR) agonists on Abeta-induced amnesia. A beta(3)-AR agonist (CL316243), but not a beta(2)-AR agonist, rescued Abeta-induced memory loss, suggesting the need for further studies on the role of beta(3)-ARs in AD.     

Recovery from hypoxia and ECS-induced amnesia after a single exposure to training environment

A single exposure to the training environment for 3 min was sufficient to produce recovery from hypoxia-induced amnesia when tested 30 min later. Recovery from ECS-induced amnesia was evidenced 5 min after exposure to the training environment. The results make a state dependent learning explanation for memory loss after ECS or hypoxia unfeasible, since the recovery is seen immediately after the memory loss is evidenced. The author proposes that amnestic treatments prevent the integration of the training event into existing functional memory systems and consequently the training has no significance for the animal. Reexposure to the training environment precipitates a continuation of these integrative processes and the memory becomes organized in such a way that it has a behavioral consequence (avoidance) on the subsequent trial.