Reminder effects – reconsolidation or retrieval deficit? Pharmacological dissection with protein synthesis inhibitors following reminder for a passive‐avoidance task in young chicks

It is generally accepted that memory formation involves an irreversible passage via labile phases to the stable form of 'long-term memory' impervious to amnestic agents such as protein synthesis inhibitors. However, recent experiments demonstrate that reactivation of memory by way of a reminder renders it labile to such inhibitors, suggesting that such retrieval is followed by a so-called reconsolidation process similar or identical in its cellular and molecular correlates to that occurring during the initial consolidation. We compared the effects of the protein synthesis inhibitor anisomycin and the glycoprotein synthesis inhibitor 2-deoxygalactose on the temporal dynamics and pharmacological sensitivity of initial consolidation and memory expression following a reminder in a one-trial passive-avoidance task in day-old chicks. This comparison revealed three differences between the action of the inhibitors on newly formed compared with reactivated memory. First, the recall deficit after the reminder was temporary, whilst the amnesia following inhibitor treatment during training was stable. Second, the sensitive period for the effect of anisomycin was shorter in the reminder than in the training situation. Third, the effective dose for either inhibitor for reminder-associated amnesia was several times lower than for amnesia developing after training. Thus though like initial consolidation, memory expression at delayed periods following reminder depends on protein and glycoprotein synthesis, the differences between the temporal and pharmacological dynamics in the two situations point to the distinct character of the molecular processes involved in postreminder effects.     

Changes in phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in processing of short-term and long-term memories after passive avoidance learning

Characteristic autophosphorylation of calcium/ calmodulin-dependent protein kinase II (CaMKII) and its consequences have made this kinase an interesting target in studying the molecular pathway for important neuronal functions including learning and memory formation. In this article, we use immunoprecipitation and immunoblotting methods to detect changes in phosphorylation of CaMKII during memory formation in 1-day-old chicks trained in a single trial passive avoidance task. A 60-kDa protein has been immunoprecipitated from the chick brain with an anti-rabbit CaMKII antibody. This protein shows strong and specific immunoactivities with antibodies against the calmodulin binding site of CaMKII, and the N and C terminals of beta-CaMKII. Commercially available anti-phosphoserine and anti-phosphothreonine antibodies are shown to sensitively detect phosphorylation of purified CaMKII. The basal phosphorylation of CaMKII from the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) regions of the chick brain is shown to be largely right hemisphere-lateralized. When chicks are subjected to a passive avoidance training experience, a specific increase in CaMKII phosphorylation is induced in the IMHV and LPO of the left hemisphere from those chicks whose memory for the training experience is successfully retrieved. While this specific increase in CaMKII phosphorylation is seen in both the left IMHV and left LPO in short-term memory, it is detectable only in the left LPO associated with long-term memory retrieval. The present results provide evidence that in vivo changes in phosphorylation of CaMKII are associated specifically with processing of distinct memory stages, which take place in specific brain regions.     

Cyclin S: a new member of the cyclin family plays a role in long-term memory

Memory is thought to be subserved by structural and functional alteration in synaptic connectivity. But although neuronal plasticity requires gene expression, the identity of the proteins involved is largely unknown. Using the chick 1-day-old passive avoidance learning paradigm and differential display RNA fingerprinting, we identified 13 candidate genes which are upregulated in the intermediate medial hyperstriatum ventrale (IMHV), an area that has been correlated with the initial processing of memory formation. One of the induced genes is a new member of the cyclin family, with high homology to cyclin L (ania-6a). Analysis of the expression pattern of this gene after training revealed two time waves of induction: the first correlated with learning and initial memory process in the IMHV; the second correlated with memory consolidation, first in the IMHV, and then in the lobus paraolefactoris. There is a correlation between methylanthranilate (MeA) concentrations (the malaise substrate in the passive avoidance training procedure), the duration of memory and the expression level of cyclin S. While training chicks on low concentrations of MeA causes short-term memory and low expression level of cyclin S, high concentration of MeA induces long-term memory and high expression level of cyclin S in the IMHV. The role of cyclins in the regulation of neuronal-plasticity-related gene expression was overlooked, and it might serve as a key step in long-term memory formation.     

Analysis of differential gene expression supports a role for amyloid precursor protein and a protein kinase C substrate (MARCKS) in long-term memory

Previous work has identified the intermediate and medial part of the hyperstriatum ventrale (IMHV) as a region of the chick brain storing information acquired through the learning process of imprinting. We have examined in this brain region changes in expression of candidate genes involved in memory. Chicks were exposed to a rotating red box and the strength of their preference for it, a measure of learning, determined. Brain samples were removed approximately 24 h after training. Candidate genes whose expressions were different in IMHV samples derived from strongly imprinted chicks relative to those from chicks showing little or no learning were identified using subtractive hybridization. The translation products of two candidate genes were investigated further in samples from the left and right IMHV and from two other brain regions not previously implicated in imprinting, the left and right posterior neostriatum. One of the proteins was the amyloid precursor protein (APP), the other was myristoylated alanine rich C kinase substrate (MARCKS). In the left IMHV the levels of the two proteins increased with the strength of learning. The effects in the right IMHV were not significantly different from those in the left. There were no effects of learning in the posterior neostriatum. This is the first study to relate changes in the amounts of MARCKS and APP proteins to the strength of learning in a brain region known to be a memory store and demonstrates that the systematic identification of protein molecules involved in memory formation is possible.     

Neurosilence: profound suppression of neural activity following intracerebral administration of the protein synthesis inhibitor anisomycin

Early in their formation, memories are thought to be labile, requiring a process called consolidation to give them near-permanent stability. Evidence for consolidation as an active and biologically separate mnemonic process has been established through posttraining manipulations of the brain that promote or disrupt subsequent retrieval. Consolidation is thought to be ultimately mediated via protein synthesis since translational inhibitors such as anisomycin disrupt subsequent memory when administered in a critical time window just following initial learning. However, when applied intracerebrally, they may induce additional neural disturbances. Here, we report that intrahippocampal microinfusions of anisomycin in urethane-anesthetized rats at dosages previously used in memory consolidation studies strongly suppressed (and in some cases abolished) spontaneous and evoked local field potentials (and associated extracellular current flow) as well as multiunit activity. These effects were not coupled to the production of pathological electrographic activity nor were they due to cell death. However, the amount of suppression was correlated with the degree of protein synthesis inhibition as measured by autoradiography and was also observed with cycloheximide, another translational inhibitor. Our results suggest that (1) the amnestic effects of protein synthesis inhibitors are confounded by neural silencing and that (2) intact protein synthesis is crucial for neural signaling itself.     

The dynamics of change in striatal activity following updating training

Increases in striatal activity have been suggested to mediate training‐related improvements in working‐memory ability. We investigated the temporal dynamics of changes in task‐related brain activity following training of working memory. Participants in an experimental group and an active control group, trained on easier tasks of a constant difficulty in shorter sessions than the experimental group, were measured before, after about 1 week, and after more than 50 days of training. In the experimental group an initial increase of working‐memory related activity in the functionally defined right striatum and anatomically defined right and left putamen was followed by decreases, resulting in an inverted u‐shape function that relates activity to training over time. Activity increases in the striatum developed slower in the active control group, observed at the second posttest after more than 50 days of training. In the functionally defined left striatum, initial activity increases were maintained after more extensive training and the pattern was similar for the two groups. These results shed new light on the relation between activity in the striatum (especially the putamen) and the effects of working memory training, and illustrate the importance of multiple measurements for interpreting effects of training on regional brain activity. Hum Brain Mapp, 2013. © 2012 Wiley Periodicals, Inc.     

Protein degradation, as with protein synthesis, is required during not only long-term spatial memory consolidation but also reconsolidation

The formation of long-term memory requires protein synthesis, particularly during initial memory consolidation. This process also seems to be dependant upon protein degradation, particularly degradation by the ubiquitin-proteasome system. The aim of this study was to investigate the temporal requirement of protein synthesis and degradation during the initial consolidation of allocentric spatial learning. As memory returns to a labile state during reactivation, we also focus on the role of protein synthesis and degradation during memory reconsolidation of this spatial learning. Male CD1 mice were submitted to massed training in the spatial version of the Morris water maze. At various time intervals after initial acquisition or after a reactivation trial taking place 24 h after acquisition, mice received an injection of either the protein synthesis inhibitor anisomycin or the protein degradation inhibitor lactacystin. This injection was performed into the hippocampal CA3 region, which is specifically implicated in the processing of spatial information. Results show that, in the CA3 hippocampal region, consolidation of an allocentric spatial learning task requires two waves of protein synthesis taking place immediately and 4 h after acquisition, whereas reconsolidation requires only the first wave. However, for protein degradation, both consolidation and reconsolidation require only one wave, taking place immediately after acquisition or reactivation, respectively. These findings suggest that protein degradation is a key step for memory reconsolidation, as for consolidation. Moreover, as protein synthesis-dependent reconsolidation occurred faster than consolidation, reconsolidation did not consist of a simple repetition of the initial consolidation.     

MK-801 Blockade of Fos and Jun Expression Following Passive Avoidance Training in the Chick

Training chicks on a one-trial passive avoidance task results in transient up-regulation of the N -methyl- d -aspartate (NMDA) receptor in the left intermediate medial hyperstriatum ventrale (IMHV) of the forebrain 30 min post-training. Injection of the non-competitive NMDA receptor inhibitor, (+)-5-methyl-10, 11-dihydro-5H-dibenzo- (a.d)-cyclohepten 5, 10-imine maleate (MK-801), around the time of training renders chicks amnesic for the task. Training also results in enhanced expression of the immediate early gene (IEG) c- fos in the IMHV. To determine the relationship between NMDA receptor up-regulation and IEG induction during memory formation we have examined the expression of Fos, Jun and their related proteins 1 h following training in the presence/absence of the putative amnestic agent MK-801. Western blotting of IMHV samples revealed two protein bands with immunoreactivity to the Fos antibody at 47 and 54 kDa. Using an antibody to Jun, two immunoreactive bands were revealed at 39 and 54 kDa. All bands were enhanced in the left IMHV following passive avoidance training. Post-training intraperitoneal injections of MK-801 (75 mM) produced amnesia in ∼50% of the birds when tested 1 h after training. Injection of MK-801 significantly attenuated expression of these proteins in birds rendered amnesic, but not in those that recalled the task. We conclude that NMDA receptor activation precedes immediate early gene expression in the memory formation cascade.     

Striato-telencephalic and striato-tegmental circuits: relevance to learning in domestic chicks

Memory formation for a passive avoidance task in the domestic chick is likely to involve a hyperstriatum ventrale (IMHV)-archistriatum-lobus parolfactorius (LPO) arc. The present study summarises previous findings, relevant to this neural system, and is also supplemented with some recent data from our laboratory. Projections from the IMHV on the archistriatum, as well as from the archistriatum on the LPO, have been characterised using a combination of anterograde pathway tracing (Phaseolus lectin), and post-embedding GABA and glutamate immunocytochemistry. The majority of IMHV efferents have been found to synapse with dendritic spine heads and necks of densely spiny projection neurons of the ventral archistriatum, and the ultrastructure of synapses suggested a potent excitatory input. Similar synaptic connections of the excitatory type were ultrastructurally verified between ventral archistriatal afferent terminals and dendrites or spines of the LPO, suggesting an involvement of the medium sized spiny neurons, which are typical of the striatum. Although some of the IMHV boutons terminating in the archistriatum were immunoreactive to glutamate, this was not observed in the archistriatal-LPO pathway. Tegmental connections of the basal ganglia, in particular LPO, are also likely to play a role in processing of the avoidance response. We have demonstrated reciprocal connections between the LPO and dopaminergic (TH-positive) neurons of the substantia nigra and ventral tegmentum. Dopamine D1 receptors were upregulated bilaterally in the LPO following avoidance learning and this response was not accompanied by significant changes in the level of dopamine or its metabolites (HVA, DOPAC), as revealed by HPLC chromatography of brain samples dissected from the LPO of control and trained chicks. The dopamine receptor-related phosphoprotein DARPP-32 was localised in dendritic elements of the LPO, often forming asymmetric synapses with glutamate immunoreactive axon terminals. The findings are consistent with a scenario in which the striatum acts as a suppressor of natural pecking behaviour. Learned visual association with the target (bead) occurs in the IMHV and is relayed to the basal ganglia via the limbic archistriatum (amygdala equivalent), the latter introducing a motivational element (aversion, fear). Suppression of a brainstem pecking centre is likely to involve activation of the nigrostriatal (tegmentostriatal) dopaminergic circuit.     

Neural cell adhesion molecules, CaM kinase II and long-term memory in the chick

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain is a site of recognition memory for filial imprinting. Previous results have demonstrated learning-related changes in the amounts of the three major isoforms of neural cell adhesion molecule (NCAM) in the left IMHV. The increases were present 24 h after training. The present study enquired whether the increases persisted and were present 48 h after training. The brain regions analysed were the left and right IMHV and the left and right hyperstriatum accessorium (HA), a visual projection area. The alpha-subunit of calcium/calmodulin protein kinase II (CaMKIIalpha) was also assayed. There were significant correlations between a measure of the strength of learning and the amount of NCAM 180 in the right IMHV (r = +0.65; p = 0.012) but not in the left, and in the left HA (r = -0.61; p = 0.02), but not in the right. There were no learning-related changes for CaMKIIalpha. We conclude that in IMHV the effects of imprinting on NCAM 180 are expressed mainly in the left IMHV 24 h after training, but 48 h after training are expressed mainly in the right IMHV.     

Adenosine-amino acid interactions in the chick brain: a role in passive avoidance learning

The present work describes interactions between adenosine and the amino acids glutamate and GABA in slices of intermediate medial hyperstriatum ventrale (IMHV), an area of the chick brain known to be involved in learning and memory events associated with a one-trial passive avoidance task. In slices derived from the IMHV of untrained chicks, the A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 10 microM) specifically inhibited glutamate release. Conversely, cyclopentyltheophylline (CPT; 100 microM an A(1) antagonist) increased glutamate release from the slices and blocked the CHA-induced inhibition of glutamate. The A(2) receptor agonist 2-p-(2-carboxylethyl)-phenylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) selectively increased glutamate release when applied at 5 microM while it selectively inhibited GABA release at a lower concentration (10 nM). The addition of NMDA to the medium, resulted in increased adenosine release equivalent to that found following stimulation with 50 mM KCl. Both the NMDA and the KCl-induced increases were eliminated by addition of D-2-amino-5 phosphopentanoic acid (D-AP5), an NMDA-receptor antagonist. Slices prepared from the IMHV of chicks following successful training on the task showed enhanced adenosine release 30 min, 1, 3 and 6.5 h after training compared to chicks trained to peck a water-coated bead. The results show that changes in adenosine release from the IMHV accompany memory formation in the chick. We suggest that adenosine-amino acid transmitter interactions potentially via the activation of NMDA receptors, a necessary step in long-term memory formation for the task, may modulate the formation of memory for the one-trial passive avoidance task.     

The effects of protein synthesis inhibition on structural changes associated with learning in the chick

The effect of the protein synthesis inhibitor anisomycin on the structural changes associated with passive avoidance learning in the chick was investigated. Chicks were trained when they were 24 h old by allowing them to peck at a shiny bead coated with either water or the aversive-tasting substance methylanthranilate (MeA). Chicks which peck the MeA-coated bead will on subsequent testing avoid pecking a similar, but water-coated bead. Behavioural testing was carried out 12 h after training and immediately afterwards the chicks were killed and their brains prepared for electron microscopy. A specific region of the forebrain, the intermediate and medial part of the hyperstriatum ventrale (IMHV) was investigated. When the IMHV of the MeA trained chicks was compared with that of water-trained controls structural changes of the synapse were detected. These changes involved a significant increase in the mean length of the postsynaptic density (LPSD) of symmetrical synapses in the left IMHV. Chicks injected with 0.8 mg of anisomycin 30 min before training with a MeA-coated bead showed aversion for the shiny bead when tested 12 h later. Electron microscopic analysis of the IMHV from these amnestic chicks showed no evidence for the change in LPSD demonstrated in the water-injected controls. These results are discussed in relation to the nature of the memory trace induced by training on a passive avoidance task.     

Mechanisms of memory stabilization: are consolidation and reconsolidation similar or distinct processes?

Consolidation of new memories depends on a crucial phase of protein synthesis. It is widely held that, once consolidated, memories are stable and resilient to disruption. However, established memories become labile when recalled and require another phase of protein synthesis to be maintained. Therefore, it has been proposed that when a memory is reactivated it must undergo additional consolidation (reconsolidation) to persist. To determine whether reconsolidation recapitulates consolidation, in the past few years several groups have investigated whether the same molecules and pathways mediate the formation of a memory and its maintenance after reactivation. At first glance, the results appear conflicting: although both processes appear to engage the same molecules and mechanisms, brain areas involved in consolidation after initial training are not required for reconsolidation. In addition, the formation of a memory and its maintenance after reactivation seem to have distinctive temporal molecular requirements. This review concludes with a working model that could explain the apparent controversy of memory vulnerability after reactivation.     

Cellular correlates of stages of memory formation in the chick following passive avoidance training

The process of memory formation has been investigated using the model of one-trial passive avoidance training in the one-day old domestic chick. We have unraveled a biochemically coherent cascade of processes which, beginning with transient ion and neurotransmitter flux, and by way of a sequence of interacting pre- and post-synaptic intracellular signalling steps, results in gene activation and the synthesis of cell adhesion molecules which appear to be the effective agents in the structural processes involved in remodelling of synaptic and neuronal circuits. Further, in a related series of experiments we have shown that these biochemical and morphological changes are accompanied by significant changes in the neurophysiological status of the neurons on the IMHV and LPO, in particular in terms of their engagement in bouts of high-frequency firing. However, much remains to be clarified, particularly the meaning of the time-dependent shifts in the location of the trace, and the ways in which these molecular and cellular events translate into changes in behavior in the animal.     

Adenosine–amino acid interactions in the chick brain: a role in passive avoidance learning

The present work describes interactions between adenosine and the amino acids glutamate and GABA in slices of intermediate medial hyperstriatum ventrale (IMHV), an area of the chick brain known to be involved in learning and memory events associated with a one-trial passive avoidance task. In slices derived from the IMHV of untrained chicks, the A₁ receptor agonist N⁶-cyclohexyladenosine (CHA; 10 μM) specifically inhibited glutamate release. Conversely, cyclopentyltheophylline (CPT; 100 μM an A₁ antagonist) increased glutamate release from the slices and blocked the CHA-induced inhibition of glutamate. The A₂ receptor agonist 2-p-(2-carboxylethyl)-phenylamino-5′-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) selectively increased glutamate release when applied at 5 μM while it selectively inhibited GABA release at a lower concentration (10 nM). The addition of NMDA to the medium, resulted in increased adenosine release equivalent to that found following stimulation with 50 mM KCl. Both the NMDA and the KCl-induced increases were eliminated by addition of -2-amino-5 phosphopentanoic acid ( -AP5), an NMDA-receptor antagonist. Slices prepared from the IMHV of chicks following successful training on the task showed enhanced adenosine release 30 min, 1, 3 and 6.5 h after training compared to chicks trained to peck a water-coated bead. The results show that changes in adenosine release from the IMHV accompany memory formation in the chick. We suggest that adenosine–amino acid transmitter interactions potentially via the activation of NMDA receptors, a necessary step in long-term memory formation for the task, may modulate the formation of memory for the one-trial passive avoidance task.     

Remembering that things have changed: a review of the cellular mechanisms of memory re-consolidation in the day-old chick

It has been one of the unshakeable orthodoxies of memory research that memory is initially laid down in a labile form for a short period following the experience and that over time the memory is “fixed” or “consolidated” into the physical structure of the brain. Over the last decade a large body of data has gathered which demonstrates that a “consolidated” memory can be returned to a labile state following retrieval of material from the store, which can then be re-consolidated, incorporating the newly acquired information into the representation of the world. The process of re-consolidation thus provides a sensible means for the crucial process of memory updating to occur. The paper focuses on pharmaco-behavioural experiments undertaken in our laboratories as well as in those of other groups which use the day-old chick as subject and the passive avoidance learning (PAL) task to examine the behavioural and metabolic parameters of re-consolidation. The data indicate that the consolidation and the re-consolidation processes are similar but not identical physiological processes. The re-processing of the memory following a re-consolidation involves each of the glutamatergic, adrenergic and dopaminergic neurotransmitter systems as well as re-activation of protein synthesis associated with the respective traces. In the chick model system, the ability to undertake re-consolidation is transient, and is observed only for a maximum of 24–48 h following the initial training event. Controversy persists as to whether the re-consolidated memory represents a new memory or whether it is a modification of the original memory processing.     

Quantitative Autoradiographic Demonstration of Changes in Binding to NMDA-sensitive [3H]Glutamate and [3H]MK801, but not [3H]AMPA Receptors in Chick Forebrain 30 min After Passive Avoidance Training

Day-old domestic chicks ( Gallus domesticus ) were trained on a one-trial passive avoidance task in which the aversive stimulus was an unpleasant tasting substance, methyl anthranilate. Thirty minutes later, localization of N -methyl- d -aspartic acid (NMDA)-sensitive [³H]glutamate receptor binding sites, [³H]MK801 and [³H]AMPA binding sites in 17 regions of the forebrain of methylanthranilate-trained and control (water-trained) chicks was determined using quantitative receptor autoradiography. Significant differences in binding to both MK801- and NMDA-sensitive glutamate receptors, but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, were found in three regions of the forebrain of trained compared to control chicks; two of these regions have been implicated from previous lesion, biochemical and morphological studies as playing a key role in the process of memory formation and storage following passive avoidance training. For NMDA-sensitive [³H]glutamate receptors, significant elevations in binding were observed in two regions, the left intermediate and medial hyperstriatum ventrale (IMHV) (39%) and the lobus parolfactorius (LPO) (34%), at 30 min post-training, but a decrease (44%) occurred in binding to the lateral neostriatum. Significant increases in binding to MK801 receptors were observed in the left IMHV (19%) and right IMHV (13%), and left LPO (22%) at 30 min post-training, though there was a decrease in the right LPO (15%). These findings, coupled with those described in a previous paper from our group (Burchuladze and Rose, Eur. J. Neurosci. , 4 , 533–538, 1992), demonstrate that a glutamate receptor subtype is involved in learning and memory formation in the chick.     

Memory formation in the chick depends on membrane-bound protein kinase C

The role of protein kinase C (PKC) in the formation of memory for a one-trial passive avoidance task in 1-day-old chicks has been studied, following earlier observations that training on this task results in transient and lateralised changes in the phosphorylation state of presynaptic B-50 protein, a PKC substrate. In accord with hypotheses that the activity of PKC is regulated by translocation from cytosol to membrane, a significant increase was found in the fraction of the alpha/beta forms of the enzyme, assayed immunologically, present in a synaptic-membrane-bound, Triton-extractable form in the left intermediate medial hyperstriatum ventrale (IMHV) of chicks 30 min after training on the passive avoidance task. Two inhibitors of PKC, melittin (10 microliters, 120 microM) and H7 (10 microliters, 10 mM), if injected intracerebrally 10 min prior to or 10 min after training, were without effect on the general behaviour of the chicks or their training. However, these injections of the inhibitors produced amnesia in birds tested 3 h later. This effect was lateralised; only left hemisphere injections of the inhibitors produced amnesia. A possible state-dependency interpretation of these results was ruled out. The results are discussed in the context of hypotheses as to the regulatory role of PKC in neural plasticity and memory formation.     

Training Chicks on a Passive Avoidance Task Modulates Glutamate-stimulated Inositol Phosphate Accumulation

The effects of glutamate, N -methyl-d-aspartate (NMDA), (+)-5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten 5,10-imine maleate (MK801), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and quisqualate on the accumulation of inositol phosphates (IP) from the breakdown of phosphoinositides in vitro have been studied in tissue prisms derived from a region of the chick forebrain, the intermediate medial hyperstriatum ventrale (IMHV). In prisms from the left IMHV, glutamate stimulated IP accumulation by 10–20%, AMPA by 55% and quisqualate by 650%. These effects were more marked in the right IMHV, where AMPA stimulated IP accumulation by 157% and quisqualate by 920%. MK801 and NMDA had no significant effect on IP accumulation in either hemisphere. The left IMHV is known to be the site of a biochemical cascade resulting in synaptic remodelling following training day-old chicks on a one-trial passive avoidance task. The effect of such training was to reduce glutamate-stimulated accumulation of IP by 27% ( P < 0.05) in prisms taken 30 min after training. There was no effect on prisms taken at 5 or 180 min after training, and no effect at any time in the right IMHV. MK801, injected intraperitoneally before training at a concentration known to produce amnesia for the passive avoidance task, abolished the training-induced decrease without itself affecting IP accumulation. Taken in conjunction with pharmacological and autoradiographic evidence, these results indicate that memory formation for the passive avoidance task involves the activation of NMDA receptor channels, but not quisqualate or AMPA receptors, in the left IMHV of the chick 30 min after training.     

Tyrosine hydroxylase inhibition by cycloheximide and anisomycin is not responsible for their amnesic effect

It has been reported that the administration of cycloheximide, a protein synthesis inhibitor, depresses brain tyrosine hydroxylase activity as measuredin vitro. This finding raised the possibility that the amnesic effect of this drug could be due to reduction of norepinephrine synthesis rather than to inhibition of protein synthesis required for long-term memory. We have found that (1) amnesic doses of cycloheximide and anisomycin, two protein synthesis inhibitors, produced measurable depression of tyrosine hydroxylase activity although much of the depression may be due to isotope dilution rather than true inhibition, and (2)α-methyl-p-tyrosine a competitive inhibitor of tyrosine hydroxylase, in doses which depressed tyrosine hydroxylase activity as much as or more than either cycloheximide or anisomycin, did not affect memory. Therefore the effect of protein synthesis inhibitors on brain tyrosine hydroxylase activity is not sufficient to explain their amnesic effect.