Chloramphenicol-Induced Amnesia for Passive Avoidance Training in the Day-Old Chick

The antibiotic chloramphenicol, an inhibitor of mitochondrial protein synthesis, was used to investigate the time-related changes in protein synthesis following passive avoidance training in the day-old chick (white leghorn–black Australorp). Retention of memory for this simple learning task is known to be prevented by an inhibitor of cytosolic protein synthesis, anisomycin, in a biphasic manner, with the first phase of sensitivity occurring up to 90 min post-training and the second phase between 4 and 5 h post-training (Freeman, Rose, & Scholey, 1995). Birds received bilateral intracranial injections of chloramphenicol (10 μl/hemisphere of a 7.4 mM solution) at various times relative to training and were tested 24 h later. This report shows that at the second phase of anisomycin susceptibility there was a chloramphenicol-sensitive period (5 h post-training) which had an onset time less than 1 h after injection. The effect of chloramphenicol appears not to be due to the mitochondria being energetically compromised since intracranial injections of an uncoupler of mitochondrial oxidative phosphorylation, 2,4-dinitrophenol (0.1 mM), did not disrupt memory formation when injected 5 h after training, even though it did cause amnesia when injected at the earlier time point of 20 min post-training. These results are discussed in the context of what is already known about memory formation in the day-old chick.     

Involvement of Glutamate Receptors, Protein Kinases, and Protein Synthesis in Memory for Visual Discrimination in the Young Chick

Two-day-old chicks were trained to discriminate between edible chick crumbs and arrays of colored beads glued to the floor of their cage. Normal chicks learned this task within a few minutes and retained it for at least 24 h. The role of several biochemical systems known to be required for other forms of early learning in the chick was explored in this task. Antagonists and inhibitors of these systems were used in the doses known to produce amnesia in a related passive avoidance learning model. Drugs were injected intracerebrally just before training, and retention was tested at various times subsequently. The protein synthesis inhibitor anisomycin (240 nmol/chick) was without effect on retention at 30 min posttraining, but chicks were amnestic at 3 and 24 h. The protein kinases inhibitors melittin (1.2 nmol/chick) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine hydrochloride (100 nmol/chick) were without effect on retention at 30 min posttraining but were amnestic by 3 h. While these effects are similar to those found for one-trial passive avoidance training, neither theN-methyl-D-aspartate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (up to 15 nmol/chick) orDL-2-amino-5-phosphonovalerate (1.3 nmol/chick), both of which are amnestic for passive avoidance, nor the non-NMDA-glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3,-dione (4 nmol/chick) were amnestic for the visual discrimination task. By contrast, the metabotropic glutamate receptor blocker (RS)-α-methyl-4-carboxyphenylglycine (300 nmol/chick) injected 5 min pretraining resulted in amnesia at 3 h posttraining. The implications of these findings for the putative “memory consolidation cascade” are discussed.     

Two critical periods of protein and glycoprotein synthesis in memory consolidation for visual categorization learning in chicks

A protein synthesis inhibitor, anisomycin (ANI), and an inhibitor of glycoprotein synthesis, 2-deoxygalactose (2-D-gal), were used to investigate memory consolidation following visual categorization training in 2-day-old chicks. ANI (0.6 micromole/chick) and 2-D-gal (40 micromoles/chick) were injected intracerebrally at different time intervals from 1 hr before to 23 hr after the training. Retention was tested 24 hr post-training. Both ANI and 2-D-gal injections revealed two periods of memory sensitivity to pharmacological intervention. ANI impaired retention when injected from 5 min before to 30 min after the training or from 4 hr to 5 hr post-training, thus demonstrating that consolidation of long-term memory in this task requires two periods of protein synthesis. 2-D-Gal first produced an amnesia when it was injected in the interval from 5 min before to 5 min after the training. Injections made between 5 min and 5 hr post-training were without effect on the retention. The second period of memory impairment by 2-D-gal started at 5 hr post-training and lasted until 21 hr after the training. Administration of 2-D-gal made 23 hr after the training did not influence retention in the test at either 24 hr or 26 hr. These results are consistent with the hypothesis that two waves of protein and glycoprotein synthesis are necessary for the formation of long-term memory. The prolonged duration of performance impairment by 2-D-gal in the present task might reflect an extended memory consolidation period for a categorization form of learning.     

Recalling an aversive experience by day-old chicks is not dependent on somatic protein synthesis

Long-term memory is dependent on protein synthesis and inhibiting such synthesis following training results in amnesia for the task. Proteins synthesized during training must be transported to the synapse and disrupting microtubules with Colchicines, and hence, blocking transport, results in transient amnesia. Reactivating memory for a previously learned avoidance triggers a biochemical cascade analogous to that following the initial training and renders the memory labile once more to protein synthesis inhibitors. However, the reminder-induced cascade differs in certain key features from that following training. Here we show that in a one-trial passive avoidance task in chicks, in contrast with initial consolidation following training, memory following a reminder is not impaired by Colchicine. We conclude that recall after a reminder does not require synaptic access to somatically synthesized proteins in this task. Our results support the hypothesis that in the chick, a reminder may instead engage local protein synthesis at the synapse, rather than in the soma.     

Some biochemical and behavioural aspects of the paradoxical sleep window

It has recently been proposed that there is a vulnerable period of time following successful learning when paradoxical sleep (PS) is necessary for learning. This vulnerable time period has been called the PS window. In Experiment 1, the protein synthesis inhibitor anisomycin (ANI) was administered following shuttle avoidance training in the Sprauge-Dawley rat to coincide with the onset of an established PS window. Control groups received either saline or ANI either 3 hours before or 3 hours after the beginning of the PS window. Three hours after the injection, each group was retested. Then animals were immediately sacrificed, and whole brain biochemical analyses were done on levels of acetylcholine (ACh) and activity of acetylcholinesterase (AChE). Only the rats given ANI timed to coincide with the beginning of the PS window showed learning deficits. All ANI-treated groups had less ACh and AChE activity. In Experiment 2, the ACh antagonist scopolamine (SCOP) was injected at the same times as in Experiment 1, and each of these groups had a corresponding saline control group as before. Retesting was done 1 day later; once again, the only group to show learning deficits was the group receiving SCOP timed to coincide with the PS window. Results suggested that the transmitter ACh plays an important role in learning/memory processes at the PS window.     

Lidocaine attenuates anisomycin-induced amnesia and release of norepinephrine in the amygdala

When administered near the time of training, protein synthesis inhibitors such as anisomycin impair later memory. A common interpretation of these findings is that memory consolidation requires new protein synthesis initiated by training. However, recent findings support an alternative interpretation that abnormally large increases in neurotransmitter release after injections of anisomycin may be responsible for producing amnesia. In the present study, a local anesthetic was administered prior to anisomycin injections in an attempt to mitigate neurotransmitter actions and thereby attenuate the resulting amnesia. Rats received lidocaine and anisomycin injections into the amygdala 130 and 120 min, respectively, prior to inhibitory avoidance training. Memory tests 48 h later revealed that lidocaine attenuated anisomycin-induced amnesia. In other rats, in vivo microdialysis was performed at the site of amygdala infusion of lidocaine and anisomycin. As seen previously, anisomycin injections produced large increases in release of norepinephrine in the amygdala. Lidocaine attenuated the anisomycin-induced increase in release of norepinephrine but did not reverse anisomycin inhibition of protein synthesis, as assessed by c-Fos immunohistochemistry. These findings are consistent with past evidence suggesting that anisomycin causes amnesia by initiating abnormal release of neurotransmitters in response to the inhibition of protein synthesis.     

Amnesia induced by 2-deoxygalactose in the day-old chick: lateralization of effects in two different one-trial learning tasks

2-Deoxy-D-galactose, an inhibitor of brain glycoprotein fucosylation, was injected intracranially (10 mumole dose in 10 microliters) into either the left or the right forebrain hemisphere of day-old chicks (Gallus domesticus). Bilateral injection of this dose of 2-deoxy-D-galactose is known to induce amnesia for several learning tasks including one-trial passive avoidance and sickness-induced learning. When a tritiated form of the drug was injected into one forebrain hemisphere only, a significantly large proportion of the dose remained in that hemisphere. Chicks were trained in two different one-trial learning tasks. The first was a passive avoidance task in which the chicks were allowed to peck at a green training stimulus (a small light-emitting diode, LED) coated in the bitter liquid, methylanthranilate, giving rise to a strong disgust response and consequent avoidance of the green stimulus. In the second paradigm the chicks were allowed to peck at a similarly colored dry stimulus but, 30 min later, were injected intraperitoneally with lithium chloride (0.1 ml of 1 M solution), causing a sickness-induced aversion for the green LED. 2-Deoxy-D-galactose caused amnesia for the passive avoidance task when injected before training into the right hemisphere but not the left. However, unilateral injection of the drug before training on the sickness-induced learning task did not cause amnesia. The results indicate that fucosylation of brain glycoproteins is required in the right hemisphere for learning the passive avoidance task but that memory for sickness-induced learning can be retained by either hemisphere.     

Retrieval does not induce reconsolidation of inhibitory avoidance memory

It has been suggested that retrieval during a nonreinforced test induces reconsolidation instead of extinction of the mnemonic trace. Reconsolidation would preserve the original memory from the labilization induced by its nonreinforced recall through a hitherto uncharacterized mechanism requiring protein synthesis. Given the importance that such a process would have in terms of maintaining, as part of the animal behavioral repertoire, a learned response that has been devalued by experience, we analyzed its existence for the memory associated with a one-trial, step-down inhibitory avoidance task (IA), a memory whose consolidation and extinction require protein synthesis in the CA1 region of the dorsal hippocampus (CA1) and involve the participation of the basolateral amygdala (BLA) and entorhinal cortex (ENT). Rats were trained in IA, and 24 h later they were submitted either to a pure reactivation session (retrieval without stepping down), which was unable by itself to initiate extinction of the avoidance response, or to a second training session. Fifteen minutes before or 3 h after either the reactivation or the retraining sessions, animals were infused with the protein synthesis inhibitor anisomycin (ANI) into CA1, BLA, or ENT. Contrary to the prediction of the reconsolidation hypothesis, none of these treatments affected subsequent memory retention. Because reconsolidation is regarded to be a direct consequence of retrieval, one would expect that, when given before a retention test or a pure reactivation session, enhancers of memory expression should permanently improve retention and, therefore, facilitate retrieval both in that and in subsequent sessions. Using two well-known retrieval enhancers, noradrenaline and adrenocorticotropin(1-24), we could not find any evidence suggestive of reconsolidation. Hence, our results indicate that there is no retrieval-induced, protein synthesis-dependent process that would cause reconsolidation of IA memory.     

Long-term memory formation in chicks is blocked by 2-deoxygalactose, a fucose analog

When day-old chicks are trained on a passive avoidance task there is enhanced synthesis of glycoproteins. Bilateral intracerebral injections of 20 mumole of 2-deoxygalactose (2-D-gal), administered just before and just after training on the task, produce amnesia for the avoidance. Amnesia develops slowly over the first hour and persists for at least 24 h subsequently. If 2-D-gal injections are administered 4 h prior to the training or delayed for 3 h after training, no amnesia occurs. Apart from a brief initial suppression of pecking following injection there are no effects of 2-D-gal on other observed behaviors of the birds. Within the first hour this dose of 2-D-gal inhibits [3H]fucose incorporation into acid-insoluble material by 26% (or 68%, calculated relative to free pool fucose). The amnestic effect of 2-D-gal is not shown by galactose, glucose, fucose, or 2-D-glucose. Injecting 40 mumole of galactose simultaneously with the 2-D-gal abolishes the 2-D-gal-induced amnesia; 40 mumole of fucose, however, does not abolish the amnesia. The utility of 2-D-gal as an agent for analyzing the role of glycoproteins in memory formation is discussed.     

Inhibition of monoADP-ribosylation prevents long-term memory consolidation of a single-trial passive avoidance task in the day-old chick

The cytosolic posttranslational protein-modifying mechanism of monoADP-ribosylation has been implicated in long-term potentiation, a synaptic model of memory formation. The current study investigated the effect of inhibiting mono(ADP-ribosyl) transferase on memory for the passive avoidance task in day-old chicks (white Leghorn-black Australorp). Various doses of novobiocin or menadione sodium bisulfite were administered intracranially at different times before or after training. Control chicks were administered saline at matched times. Novobiocin (650 microM) or menadione sodium bisulfite (250 microM) administered between 5.0 min pretraining and 2.5 min posttraining was found to cause a persistent loss of retention from 120 min posttraining. These data provide the first demonstration that monoADP-ribosylation is required for the maintenance of long-term memory. Furthermore, the temporal characteristics of the memory loss caused by monoADP-ribosylation inhibition appears to exclude this mechanism as a downstream effect of the well-established nitric oxide activity previously shown to occur within 40 min of passive avoidance training.     

The timing of multiple retrieval events can alter GluR1 phosphorylation and the requirement for protein synthesis in fear memory reconsolidation

Numerous studies have indicated that maintaining a fear memory after retrieval requires de novo protein synthesis. However, no study to date has examined how the temporal dynamics of repeated retrieval events affect this protein synthesis requirement. The present study varied the timing of a second retrieval of an established auditory fear memory and followed this second retrieval with infusions of the protein synthesis inhibitor anisomycin (ANI) into the basolateral amygdala. Results indicated that the memory-impairing effects of ANI were not observed when the second retrieval occurred soon after the first (within 1 h), and that the inhibitor gradually regained effectiveness as the retrieval episodes were spaced further apart. Additionally, if the second of the closely timed retrievals was omitted prior to ANI infusions, long-term memory deficits were observed, suggesting that the altered effectiveness of ANI was due specifically to the second retrieval event. Further experiments revealed that the second retrieval was not associated with a change in Zif268 protein expression but did produce a rapid and persistent dephosphorylation of GluR1 receptors at Ser845, an AMPAR trafficking site known to regulate the stability of GluR2 lacking AMPARs, which have been shown to be important in memory updating. This suggests that the precise timing of multiple CS presentations during the reconsolidation window may affect the destabilization state of the memory trace.     

Long-term memory for instrumental responses does not undergo protein synthesis-dependent reconsolidation upon retrieval

Recent evidence indicates that certain forms of memory, upon recall, may return to a labile state requiring the synthesis of new proteins in order to preserve or reconsolidate the original memory trace. While the initial consolidation of "instrumental memories" has been shown to require de novo protein synthesis in the nucleus accumbens, it is not known whether memories of this type undergo protein synthesis-dependent reconsolidation. Here we show that low doses of the protein synthesis inhibitor anisomycin (ANI; 5 or 20 mg/kg) administered systemically in rats immediately after recall of a lever-pressing task potently impaired performance on the following daily test sessions. We determined that the nature of this impairment was attributable to conditioned taste aversion (CTA) to the sugar reinforcer used in the task rather than to mnemonic or motoric impairments. However, by substituting a novel flavored reinforcer (chocolate pellets) prior to the administration of doses of ANI (150 or 210 mg/kg) previously shown to cause amnesia, a strong CTA to chocolate was induced sparing any aversion to sugar. Importantly, when sugar was reintroduced on the following session, we found that memory for the task was not significantly affected by ANI. Thus, these data suggest that memory for a well-learned instrumental response does not require protein synthesis-dependent reconsolidation as a means of long-term maintenance.     

Long-term memory formation in the chick requires mobilization of ryanodine-sensitive intracellular calcium stores

Training chicks (Gallus domesticus) on a one-trial passive avoidance task results in transient and time-dependent enhanced increases in N-methyl-d-aspartate- or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated intracellular calcium concentration in synaptoneurosomes isolated from a specific forebrain region, the intermediate medial hyperstriatum ventrale. This increase could result from either calcium entry from the extracellular medium or from mobilization of intracellular calcium stores. We have therefore examined the effects of dantrolene, an inhibitor of calcium release from the intracellular ryanodine-sensitive store, on these processes. Dantrolene, 50 nmol per hemisphere injected intracerebrally 30 min pre- or 30 min posttraining, blocked longer term memory for the passive avoidance task, whereas memory for the task was unaffected when dantrolene was injected at earlier or later times. Preincubation of synaptoneurosomes, isolated from the intermediate hyperstriatum ventrale 10 min after training, with 100 nM dantrolene abolished the enhanced training-induced increase in intracellular calcium concentration elicited by 0.5 mM N-methyl-d-aspartate. By contrast, the training-induced enhancement of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-stimulated increase in intracellular calcium concentration in synaptoneurosomes prepared 6 h posttraining was unaffected by preincubation with dantrolene, which was not amnestic at this time. Calcium release from ryanodine-sensitive intracellular stores may thus be a necessary stage in the early phase of the molecular cascade leading to the synaptic modulation required for long-term memory storage.     

Discriminative taste aversion learning: a learning task for older chickens

The study of learning and memory using the chicken model has relied on three learning paradigms, passive avoidance learning, imprinting and the pebble floor task. Passive avoidance learning and imprinting have been used predominantly in very young chickens and cannot be used to access learning and memory in older chickens. We have established a new behavioural learning paradigm, Discriminative Taste Aversion Learning (DTAL), that can be used with both young and older animals. The task requires chickens to discriminate between food crumbs dyed either red or yellow with one colour being associated with the aversive tasting substance, methylanthranilate. Learning can be tested at various times after the training session by presenting chickens with the coloured food crumbs without an aversive taste. Both chickens tested at 5 and 15 days post-hatch learned to avoid the aversive crumbs. Furthermore, the protein synthesis inhibitor anisomycin (30 mM; 10 microl per hemisphere) injected into the intermediate medial hyperstriatum ventrale 15 min pre-training or 45 min post-training blocked long-term memory for the DTAL task when tested 24 h later. Memory for the task was unaffected by anisomycin injection 120 min post-training or in control animals injected with saline at similar times. The timing of the cellular processes of protein synthesis needed for consolidation of the DTAL appears to be similar to those described for the other behavioural paradigms in young chickens.     

On the role of hippocampal protein synthesis in the consolidation and reconsolidation of object recognition memory

Upon retrieval, consolidated memories are again rendered vulnerable to the action of metabolic blockers, notably protein synthesis inhibitors. This has led to the hypothesis that memories are reconsolidated at the time of retrieval, and that this depends on protein synthesis. Ample evidence indicates that the hippocampus plays a key role both in the consolidation and reconsolidation of different memories. Despite this fact, at present there are no studies about the consequences of hippocampal protein synthesis inhibition in the storage and post-retrieval persistence of object recognition memory. Here we report that infusion of the protein synthesis inhibitor anisomycin in the dorsal CA1 region immediately or 180 min but not 360 min after training impairs consolidation of long-term object recognition memory without affecting short-term memory, exploratory behavior, anxiety state, or hippocampal functionality. When given into CA1 after memory reactivation in the presence of familiar objects, ANI did not affect further retention. However, when administered into CA1 immediately after exposing animals to a novel and a familiar object, ANI impaired memory of both of them. The amnesic effect of ANI was long-lasting, did not happen after exposure to two novel objects, following exploration of the context alone, or in the absence of specific stimuli, suggesting that it was not reversible but was contingent on the reactivation of the consolidated trace in the presence of a salient, behaviorally relevant novel cue. Our results indicate that hippocampal protein synthesis is required during a limited post-training time window for consolidation of object recognition memory and show that the hippocampus is engaged during reconsolidation of this type of memory, maybe accruing new information into the original trace.     

Effects of d-amphetamine and strychnine on cycloheximide- and diethyldithiocarbamate-induced amnesia in mice.

Groups of C57BL/6J mice were administred cycloheximide (CYC) 30 min before or immediately after training on a passive avoidance task and tested 72 hr later. Some CYC-pretreated groups were given strychnine or d-amphetamine (d-amp) immediately after training and others were given d-amp 1 hr after training. Other groups were given diethyldithiocarbamate (DDC) at various times before or after training. Some DDC-pretreated groups were gived-amp or strychnine as described above for CYC groups. Immediate posttraining administration of 5 mg/kg d-amp, but not strychnine, prevented amnesia in CYC-pretreated mice. The DDC induced an apparent amnesia when administered from 30 min before training to 3 hr after training. Posttraining administration of d-amp or strychnine did not prevent DDC-induced amnesia. These results are discussed in relation to previous suggestions that CYC- and DDC-induced amnesia may be the result of a functional impairment of catecholamine neurotransmitter systems by these drugs.     

Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat

Long-term habituation to a novel environment is one of the most elementary forms of nonassociative learning. Here we studied the effect of pre- or posttraining intrahippocampal administration of drugs acting on specific molecular targets on the retention of habituation to a 5-min exposure to an open field measured 24 h later. We also determined whether the exposure to a novel environment resulted in the activation of the same intracellular signaling cascades previously shown to be activated during hippocampal-dependent associative learning. The immediate posttraining bilateral infusion of CNQX (1 μg/side), an AMPA/kainate glutamate receptor antagonist, or of muscimol (0.03 μg/side), a GABA_A receptor agonist, into the CA1 region of the dorsal hippocampus impaired long-term memory of habituation. The NMDA receptor antagonist AP5 (5 μg/side) impaired habituation when infused 15 min before, but not when infused immediately after, the 5-min training session. In addition, KN-62 (3.6 ng/side), an inhibitor of calcium calmodulin-dependent protein kinase II (CaMKII), was amnesic when infused 15 min before or immediately and 3 h after training. In contrast, the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, and the protein synthesis inhibitor anisomycin, at doses that fully block memory formation of inhibitory avoidance learning, did not affect habituation to a novel environment. The detection of spatial novelty is associated with a sequential activation of PKA, ERKs (p44 and p42 MAPKs) and CaMKII and the phosphorylation of c-AMP responsive element-binding protein (CREB) in the hippocampus. These findings suggest that memory formation of spatial habituation depends on the functional integrity of NMDA and AMPA/kainate receptors and CaMKII activity in the CA1 region of the hippocampus and that the detection of spatial novelty is accompanied by the activation of at least three different hippocampal protein kinase signaling cascades.     

Post-training, but not post-reactivation, administration of amphetamine and anisomycin modulates Pavlovian conditioned approach

The psychostimulant, amphetamine (AMPH), and the protein synthesis inhibitor, anisomycin (ANI), have been shown to modulate the consolidation and reconsolidation of several types of learning. To determine whether Pavlovian conditioned approach (PCA) is modulated in a similar manner, we examined the effects of post-training and post-reactivation administration of both AMPH and ANI on memory for PCA. Male Long-Evans rats received PCA training sessions during which presentations of a CS+ were followed by sucrose delivery. AMPH (1 mg/kg, s.c.) injected immediately but not 6h after the first training session enhanced PCA behavior. ANI (150 mg/kg, s.c.) injected immediately but not 3h after the first training session impaired PCA behavior. This impairment was not due to the development of a conditioned taste aversion. To examine whether PCA can also be modulated by post-reactivation administration of AMPH and ANI, rats were given an injection of AMPH, ANI, or vehicle immediately after a memory reactivation session. Upon testing, the behavior of both the AMPH- and the ANI-treated rats was unaffected. This result remained consistent when the experiment was repeated with changes to various behavioral parameters (i.e., amount of training, length of memory reactivation). These findings indicate that AMPH and ANI act during the post-training but not the post-reactivation period to enhance and impair, respectively, the learning of PCA. This suggests that the consolidation of PCA can be modulated in a manner comparable to other types of learned associations, but once learned, the memory appears to be relatively robust and stable.     

Amnesic effect of cycloheximide in the mouse mediated by adrenocortical hormones.

The involvement of adrenocortical hormones in the amnesic effect of cycloheximide was examined in mice. Subcutaneous injection of cycloheximide shortly before a training trial of a passive avoidance task resulted in an amnesia of the avoidance response. However, amnesia was absent in the adrenalectomized animals in which cerebral protein synthesis was suppressed by cycloheximide. Injection of corticosteroids antagonized the amnesic effect, most effecively if the steroids were given immediately after training. The influence of the hormonal treatments upon the amnesic effect was not ascribable to a change in general activity level. The amnesic effect of subcutaneously injected cycloheximide appears to be mediated by hormonal deficiency, and not related to suppression of the cerebral protein synthesis.