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.     

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.     

Serotonin and NMDA glutamate receptor antagonists selectively impair the reactivation of associative memory in the common snail

Experiments on the common snail were performed to study the influences of serotonin and glutamate receptor antagonists on the processes of reactivation of an associative habit consisting of refusing a particular type of food. Twenty-four hours after training, animals were injected with the non-selective serotonin receptor antagonist methiothepin (0.1 mg/snail) or the NMDA glutamate receptor antagonist MK-801 (0.005 mg/snail), after which they were presented with a "reminder" stimulus (the "conditioned reflex" foodstuff, a banana) and tested for retention of the habit. Three hours after antagonist injections and the "reminding" procedure, snails showed impairments in the reproduction of the acquired habit, which persisted for more than two weeks. Furthermore, animals with amnesia after treatment with methiothepin/reminding showed facilitation of repeated acquisition of the aversive habit to banana. Repeat training of animals which had shown amnesia after MK-801/reminding did not result in acquisition of the habit. It is suggested that serotonin receptors are involved in the mechanisms underlying extraction of the memory trace of the aversive habit to the foodstuff in snails, while NMDA glutamate receptors are involved in memory trace storage processes.     

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.     

Neurochemical Mechanisms of Consolidation of Associative Aversive Training to Food in the Common Snail

The effects of the protein synthesis inhibitor cycloheximide and serotonin and NMDA glutamate receptor antagonists on the processes of consolidation of an associative skill consisting of refusing a particular foodstuff were studied in the common snail. When animals were trained on the background of cycloheximide, the skill was not acquired. Repeat training of “amnestic” snails to refuse the same food without the inhibitor also failed to produce the skill. Training of snails on the background of the nonselective serotonin receptor antagonist methiothepin or the NMDA glutamate receptor antagonist MK-801 (dizocilpine maleate) did not lead to acquisition of the conditioned reflex to food. However, on repeat training, the skill was formed more quickly. The studies included the first observation that using a single type of training, treatments addressing different molecular mechanisms evoke reversible or irreversible impairments to the mechanisms of consolidation of long-term memory. It is suggested that the reversible effect is associated with suppression of the processes of reproduction, while the irreversible effect is linked with impairment to engram storage.     

Neurochemical Mechanisms of Consolidation of Associative Aversive Learning to Food in the Common Snail

Studies of the effects of the protein synthesis inhibitor cycloheximide and serotonin and NMDA glutamate receptor antagonists on the processes of consolidation of the associative skill of rejecting particular foodstuffs were performed in the common snail. The skill was not acquired when animals were trained on the background of cycloheximide. Repeated training of “amnestic” snails to reject the same foodstuff in the absence of the inhibitor also failed to produce learning. Training of snails on the background of the nonselective serotonin receptor antagonist methiothepin or the NMDA glutamate receptor antagonist MK-801 (dizocilpine maleate) did not result in the acquisition of the conditioned reflex to food. However, repeated training led to the rapid formation of the skill. These experiments provide the first evidence that interventions influencing different molecular mechanisms during a single type of training produce either reversible or irreversible impairment of the mechanisms of consolidation of long-term memory. It is suggested that the reversible effect is associated with suppression of reproduction processes, while irreversible impairment was associated with impairments to engram storage. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 8, pp. 860–870, August, 2008.     

Irreversible Amnesia in Rats and Edible Snails under Conditions of Associative Memory Reconsolidation Disturbance Caused by NMDA-Glutamate Receptor Antagonist

The effect of MK-801, an antagonist to NMDA-glutamate receptors, on reconsolidation of olfactory discrimination task in rats and taste discrimination in edible snails was examined. Twenty-four hours after conditioning, the animals received a single systemic injection of MK-801 followed by a reminding conditional stimulus. Disturbances in retrieval of the acquired task were observed 10 days after injection followed by a reminding procedure. Repeated conditioning of these animals did not restore the task. Injection of MK-801 without reminding stimulation had no effect on task retention. Thus, disturbances of NMDA-dependent reconsolidation of the associative memory in animals of different taxonomic groups irreversibly eliminated long-term memory.     

Bacopa monniera alleviates Nω-nitro-l-arginine-induced but not MK-801-induced amnesia: A mouse Morris water maze study

N-methyl-d-aspartate (NMDA) receptor and nitric oxide syntheses are the emerging target sites for development of novel drug molecules because their modulation affects the long term potentiation (LTP) process. NMDA receptor antagonists and nitric oxide synthase inhibitors induce amnesia in animals and therefore have been employed for evaluation of efficacy of several novel antiamnesic agents. Bacopa monniera Linn (syn. Brahmi) is commonly used in the ancient Indian medical system for improvement of memory deficit. We have earlier described the involvement of GABAergic and cholinergic system to account for the antiamnesic effects of B. monniera on diazepam- and scopolamine-induced amnesia. In extension to our previous study this study was designed to investigate the downstream mechanism of B. monniera by evaluation of its effect on MK-801 (an NMDA receptor antagonist) and N_ω-nitro-l-arginine (l-NNA) (a nitric oxide inhibitor) induced memory deficit. We used a Morris water maze scale and compared the degree of reversal of amnesia induced by the two agents. Male Swiss albino mice were subjected to a Rota-rod muscle incoordination test followed by water maze tasks. Our data revealed that l-NNA and MK-801 produced anterograde and retrograde amnesia and B. monniera significantly attenuated the l-NNA-induced anterograde amnesia, partially reversing l-NNA-induced retrograde amnesia. On the other hand, B. monniera neither attenuated the MK-801-induced anterograde amnesia nor improved retrograde amnesia caused by it.     

N-methyl-D-aspartate receptor antagonist MK-801 impairs learning but not memory fixation or expression of classical fear conditioning in goldfish (Carassius auratus).

The amnestic effects of the noncompetitive antagonist MK-801 on visually mediated, classic fear conditioning in goldfish (Carassius auratus) was examined in 5 experiments. MK-801 was administered 30 min before the training session on Day 1 to look for anterograde amnestic effects, immediately after training to look for retrograde amnestic effects, and before the training or test session, or both, to look for state-dependence effects. The results showed that MK-801 produced anterograde amnesia at doses that did not produce retrograde amnesia or state dependency and did not impair the expression of conditioned or unconditioned branchial suppression responses (BSRs) to the conditioned stimulus. The results indicate that MK-801 disrupts the mechanism of learning of the conditioned stimulus-unconditioned stimulus relation. Evidence is also presented that the learning processes that are disrupted by MK-801 occur during the initial stage of BSR conditioning.     

NMDA receptors are involved in Ginkgo extract-induced facilitation on memory retention of passive avoidance learning in rats

Herbal therapies are commonly used to enhance memory and learning. Ginkgo biloba has shown to be one of the most popular herbs that is used to treat amnesia and retard age related memory deficits. Although, there have been several reports on the memory enhancing effects of Ginkgo, involvement of glutamatergic system that plays pivotal role in learning and memory has not been precisely assessed so far. The current study intended to investigate the effect of Ginkgo intake on amnesia while NMDA (N-methyl D-aspartic acid) receptors blocked by the administration of MK-801. The study used passive avoidance (PA) task to investigate the effect of chronic administration of Ginkgo extract (40 and 90 mg/kg; oral) on the memory span in male Wistar rats, suffering from MK-801-induced forgetfulness (0.06 and 0.1 mg/kg; i.p.). The results indicate that Ginkgo was able to remove MK-801-induced forgetfulness, indicating that Ginkgo can affect memory retention but not effect on passive avoidance acquisition, using pathways other than glutamatergic system as well. The results might indicate that Ginkgo extract can be effective in removing forgetfulness caused by inhibiting NMDA receptors from performing their activities.     

Selective Effects of an NMDA Glutamate Receptor Antagonist on the Sensory Input from Chemoreceptors in the Snail's Head during Acquisition of Nociceptive Sensitization

Experiments on semi-intact preparations from common snails were used to study the characteristics of the actions of MK-801, an antagonist of NMDA glutamate receptors, on the plasticity of various sensory inputs to defensive behavior command neurons LPl1 and RPl1 during acquisition of nociceptive sensitization. Application of sensitizing stimuli to the head or foot of control snails led to depression of neuron responses to tactile and chemical sensory stimulation during the short-term stage and marked facilitation of these responses during the long-term stage of sensitization. Application of sensitizing stimuli to the snail's head during administration of MK-801 led to marked depression of responses to chemical stimulation of the head in both the short-term and long-term stages of sensitization. In addition, blockade of NMDA receptors during application of sensitizing stimuli to the foot or head had no effect on changes in neuron responses elicited by chemical stimulation of the snail's foot and by tactile stimulation of the foot or head. It is suggested that NMDA-like glutamate receptors are selectively involved in the mechanism of induction of plasticity of synaptic inputs to command neurons LPl1 and RPl1, excited by chemical sensory stimulation of the head – a skin receptor zone specific for these neurons.     

NMDA-Dependent Increases in Extracellular Arginine Levels in the Nucleus Accumbens Are Mediated by Activation of No Synthase

Intracerebral microdialysis studies with HPLC in Sprague–Dawley rats showed that administration of N-methyl-D-aspartate (NMDA, 10, 100, and 1000 μM), an NMDA glutamate receptor agonist, into the nucleus accumbens by dialysis infusion was found to induce dose-dependent increases in extracellular arginine (the substrate of NO synthase) levels in this structure. These increases were prevented by administration of the NMDA glutamate receptor antagonist MK-801 (50 μM) into the nucleus accumbens and were significantly reduced on the background of administration of the NO synthase inhibitor N-nitroarginine (500 μM) into this structure. These data show that the extracellular arginine level in the nucleus accumbens is controlled by NMDA glutamate receptors, whole involvement is partially mediated by activation of NO synthase in this area of the brain.     

Neurochemical,behavioral and architectural changes after chronic inactivation of NMDA receptors in mice

Schizophrenia is a psychotic illness characterized by problems in perception, learning, and memory. Post-mortem clinical data revealed abnormalities in neuronal organization, reduced soma and dendritic tree size. In rodents, reduction of glutamatergic neurotransmission by NMDA receptor antagonists mimics symptoms of schizophrenia. However, the dosage, treatment and species used in previous studies have not been consistent, leading to a lack of correlation between the findings reported in low-dose, long-term treatment models and the results in acute or chronic high dose administration. Thus, the present study investigates whether long-term, low-dose blockade of NMDA receptors with MK-801 in the early postnatal period results in molecular, cellular, morphological and behavioral changes in the mouse, alterations that have been singly described by using different drugs and dosages in either mice or rats. We found that early postnatal administration of 0.1 mg/kg MK-801 for 15 days altered protein translation, synapse formation, hippocampus-dependent learning and neuronal development, resembling findings reported in schizophrenia. These results suggest that there are strong parallels between this animal model and schizophrenia, which validates it as an animal model for this condition and lends further strength of the NMDA receptor hypofunction as a useful model for the study of psychosis.     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) impairs late reconsolidation of passive avoidance learning in the day-old chick

Both NMDA and non-NMDA receptors participate in the consolidation of passive avoidance learning (PAL) in the day-old chick. NMDA antagonists have also been implicated in reconsolidation processes following reminder-trials. In this study, we examined the effect of administering 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, on reconsolidation following memory reactivation. New HampshirexWhite leghorn cockerels were trained using a modified version of the PAL task. When CNQX was administered 20min following a reminder trial, a retention deficit was detected at 90min, but this had resolved by 24h following the reminder. The parameters of the reconsolidation deficit were similar to those induced by CNQX injections post-training with the exception of their transience. This finding suggests that the action of non-NMDA receptors may perform a similar role in both consolidation and reconsolidation processes.     

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.     

The amplitude of N2pc reflects the physical disparity between target item and distracters

The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist (+)MK-801 is widely used in animal research (over 3000 publications), however its extracellular brain concentration has never been reported. Here, we show using in vivo microdialysis that systemic injection of (+)MK-801 at doses of 0.05, 0.1 or 0.2 mg/kg resulted in peak brain ECF concentration of 6, 14 or 34 nM, respectively. Moreover, (+)MK-801 resulted in a dose-dependent learning impairment in the Morris water maze as well as hyperactivity in the open field. These data demonstrate for the first time that (+)MK-801 at doses producing behavioural alterations expected from NMDA receptor blockade reaches extracellular brain concentrations corresponding to the affinity at NMDA receptors.     

Behavioral Analysis of the Consequences of Chronic Blockade of NMDA-Type Glutamate Receptors in the Early Postnatal Period in Rats

Considering data on the possible glutamatergic nature of the pathogenesis of schizophrenia, we attempted to model cognitive derangements in animals by chronic blockade of NMDA glutamate receptors. Wistar rats received daily s.c. injections of the non-competitive NMDA glutamate receptor antagonist MK-801 (0.05 mg/kg) from days 7 to day 49 of postnatal life. One day after the antagonist injections given on days 27 and 28 of life, animals of the experimental group showed decreased levels of spontaneous movement and orientational-investigative activity as compared with controls, where there was no change in the elevated locomotor reaction produced in response to the direct action of MK-801. These animals showed decreases in the level of anxiety (on day 40 of life) and derangement in spatial learning with food reinforcement (days 50–54 of life). It is suggested that early neonatal blockade of NMDA glutamate receptors leads to the development in animals of disturbances to situational perception and assessment of incoming sensory information.     

Comparison of the spinal anti-nociceptive effects of ES-242-1 and MK-801, two different NMDA antagonists, in rats

The purpose of this study was to determine whether ES-242-1, a novel N-methyl-D-aspartate (NMDA) receptor antagonist of microbial origin, has anti-nociception at the spinal level and to evaluate how its anti-nociceptive effect differs from that of MK-801, a non-competitive NMDA receptor antagonist. Agents were injected intrathecally (0.1, 1.0 and 10 microg) through a previously implanted PE tube in rats. Formalin (2%, 100 microl) was injected subcutaneously into the left hindpaw 15 min after each antagonist administration. Licking time as a nociceptive behavior was measured in three stages after formalin-injection, such as early phase (0-9 min), late first phase (10-29 min) and late second phase (30-60 min). In the early phase, the largest dose of ES-242-1 significantly decreased total licking time, although MK-801 did not show any significant reduction. With the treatment of 1.0 and 10 microg MK-801, total licking time in both late first and second phases was significantly suppressed, although the smallest dose (0.1 microg) of ES-242-1 showed a significant reduction in the late second phase. These results indicate that ES-242-1 is highly effective against tonic pain, such as inflammatory pain.     

The role of NMDA receptor binding sites in ethanol place conditioning

Little is known about the specific role of glutamate, in particular its actions at N-methyl-D-aspartate (NMDA) receptors, in ethanol reward. Pretreatment with channel blockers MK-801 and ketamine, NMDA NR2B receptor subunit antagonists ifenprodil and CP-101,606, and the glycine(B) partial agonist (+)-HA-966 did not alter acquisition of ethanol-induced conditioned place preference (CPP) in mice. However, pretreatment with the competitive antagonist CGP-37849 attenuated acquisition of ethanol-induced CPP. Follow-up experiments indicated that CGP-37849 also blocked acquisition of ethanol-induced and lithium chloride-induced conditioned place aversion but did not produce rewarding or aversive effects on its own. These results suggest that the NMDA receptor glutamate binding site is important for ethanol place conditioning. Moreover, these results suggest CGP-37849 modulates ethanol place conditioning by impairing the ability to learn these tasks.