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.

The peptide sequence Arg-Glu-Arg, present in the amyloid precursor protein, protects against memory loss caused by A beta and acts as a cognitive enhancer.

Amino acid sequences containing the palindromic tripeptide RER, matching amino acids 328-330 of the amyloid precursor protein APP, when injected intracerebrally prior to or just after training, protect against memory loss induced by amyloid-beta (A beta) in a one-trial passive avoidance task in the young chick. RER also acts as a cognitive enhancer, strengthening memory for a weak version of the task. N-terminal acylation of RER protects it against rapid degradation, and AcRER is effective in restoring memory if administered peripherally. Biotinylated RER binds to chick neuronal perikarya in an APP-displaceable manner via 66 and approximately 110 kDa neuronal cell membrane proteins. We suggest that RER binding is likely to exert effects on memory retention via receptor-mediated events that include activation of second messenger pathways. These findings suggest that RER and its derivatives may offer a novel approach to enhancing the neuroprotective effects of APP and alleviating the effects of memory loss in the early stages of Alzheimer's disease.

Passive avoidance training enhances cell proliferation in 1-day-old chicks.

One-day-old domestic chicks were injected i.p. with bromodeoxyuridine (BrdU) before training on a one-trial passive avoidance task where the aversive experience was a bead coated with a bitter tasting substance, methyl anthranilate (MeA). Animals were tested 24 h later; those avoiding (if MeA-trained) or pecking if water (W)-trained (which they peck appetitively), along with a group of untrained naïve chicks, were used to determine cell proliferation either 24 h or 9 days post BrdU injection. In all three groups, BrdU positive cells were identified sparsely throughout the forebrain but labelling was pronounced around ventricular zone (VZ) surfaces at both 24 h and 9 days post-BrdU-injection. Double immunolabelling with neuronal specific antibodies, to either NeuN, or beta-tubulin III, confirmed that most BrdU labelled cells appeared to be neurons. Unbiased stereological analysis of labelled cells in selected forebrain areas 24 h post BrdU injection showed a significant MeA-training induced increase in labelled cells in both the dorsal VZ surface bordering the intermediate and medial hyperstriatum ventrale (IMHV) and the tuberculum olfactorium (TO). By 9 days post-BrdU-injection, there was a significantly greater number of BrdU labelled cells in MeA-trained birds within the IMHV, lobus parolfactorius (LPO) and TO. These results demonstrate that avoidance training in 1-day-old chicks has a marked effect on cell proliferation, in the LPO and IMHV, regions of the chick previously identified as a key loci of memory formation, and in a second region (TO), which has olfactory functions, but has not been previously investigated in relation to avoidance learning.

APP is required during an early phase of memory formation.

The amyloid beta/A4 protein precursor (APP) has been shown to be implicated in age-associated plastic changes at synapses that might contribute to memory loss in Alzheimer's disease. As APP has previously been reported to have multiple functions during normal development, we have employed a one-trial passive avoidance task in day-old chicks to study its role in the process of memory formation. Administration of anti-APP antibodies, injected 30 min pretraining, prevented memory for a one-trial passive avoidance task in day-old chicks without effects on general behaviour or initial acquisition. Amnesia was apparent by 30 min post-training and lasted for at least 24 h. The same result was obtained by down-regulation of APP expression by APP-antisense, injected 8-12 h pretraining. However, injections of anti-APP antibodies or APP antisense at later post-training time did not cause amnesia for the task. Unlike antibodies and antisense, injection of the APP328-332 pentapeptide, in either orientation, 30 min pretraining, rescued the memory and prevented antisense-induced amnesia. The post-training time within which the antibody- and antisense-induced amnesia, and within which the APP peptides prevent amnesia, correspond to that during which memory formation is vulnerable to disruption of the putative signal transduction functions of APP. These results suggest that: (i) APP is required during an early phase of memory formation, and (ii) the memory enhancing effect of secretory APP is localized within a 5-mer sequence of growth-promoting domain.

Sequence-specific impairment of memory formation by NCAM antisense oligonucleotides.

The functional role of NCAM gene expression in memory formation was studied in the one-trial passive avoidance task in day-old chicks by pretraining injections of one of three different 18-mer end-protected oligonucleotides corresponding to positions 190-, 207-, and 332- of the NCAM Ig1 domain. Twenty-four-hour-old chicks were trained by pecking at a bitter-tasting bead and tested for avoidance 30 min, 3, 8, or 24 hr later. Memory retention was significantly reduced only in the group of animals injected with the NCAM antisense corresponding to position 207- (AS-ODN-207), and only if given twice, both immediately after hatching and 12 hr before training. This antisense was without effect on the general behavior of the chicks, training or acquisition, and did not produce observable neurotoxic damage. Under such conditions amnesia was evident by 3 hr after training and lasted until at least 24 hr after training. The two other tested oligonucleotides were without behavioral effect. To control for nonsequence-specific effects of AS-ODN-207, brains from injected and trained animals were processed for Western blotting and probed using anti-NCAM, anti-L1, and anti-actin antibodies. NCAM antisense corresponding to position 207- significantly reduced the level of NCAM, whereas the level of L1 and actin remained unchanged. These results confirm our earlier conclusion that NCAM is necessary for longer term memory retention.

Apolipoprotein E antibodies affect the retention of passive avoidance memory in the chick.

Isoforms of apolipoprotein E (ApoE) have been implicated as risk factors in Alzheimer's disease. We have, therefore, examined the possible role of ApoE in memory formation, using a one-trial passive avoidance task in day-old chicks. Birds were trained on the task and then at various times pre or post-training were injected intracerebrally with anti-ApoE. Immunofluorescence staining demonstrated the presence of the antibody bound to the neuropil, close to the injection site and adjacent to the ventricle, with a residence time in the brain of up to 30 min. Chicks that were injected 30 min pre-training or just post-training with 5 micrograms/hemisphere of the antibody learned the task, but were amnesic when tested at 30 min or at subsequent times up to 24 hr post-training. When tested at 24 hr, birds injected 5.5 hr post-training showed unimpaired retention. Birds injected with 5 micrograms/hemisphere of anti-ApoA-I (which has a brain distribution similar to that of anti-ApoE) at 30 min pretraining showed no amnesia, indicating the specificity of the effect to the ApoE. Possible mechanisms for this effect are discussed.

Neural cell adhesion molecules play a role in rat memory formation in appetitive as well as aversive tasks.

A polyclonal antibody (R1), raised against chick synaptic membrane glycoproteins and recognizing the neural cell adhesion molecule (NCAM) caused amnesia for avoidance tasks when injected into day-old chicks and adult rats 5.5 h post-training. We investigated the effects of R1 antibody on memory formation in a non-aversive task, where stress is minimal: a massed trial odour discrimination task in rats. Preimmune serum or R1 antibody was injected i.c.v. 5.5 h after the last training session. Forty-eight hours after the training session, control rats showed very good retention whereas R1 antibody injection significantly disrupted retention. The results suggest that glycoproteins recognized by R1 in the rat play a specific role in memory formation for appetitive events as well as in memory formation for aversive situations.

Antibody to day-old chick brain glycoprotein produces amnesia in adult rats.

Polyclonal antibody R-1, raised against a chick synaptic membrane glycoprotein fraction whose synthesis is enhanced following training on a passive avoidance task, produces amnesia when injected into chick forebrain 5.5 h posttraining. The amnestic IgG fraction specifically recognizes a low sialylated isoform of NCAM (Mileusnic Rose, Lancashire, & Bullock, 1995). We have now investigated the effects of this antibody on memory formation in adult rats. R-1, preimmune serum, or saline was injected intracerebroventricularly 5.5 h posttraining through bilaterally implanted cannulae. Rats injected with R-1 and tested 48 h later showed a significant amnesia for avoidance compared with the controls. Amnesia was not apparent at 24 h posttraining. R-1 injections were without effect on spontaneous locomotor or exploratory activity in a holeboard test. The results contribute to the argument that the role of cell adhesion molecules in neuronal plasticity is not limited to the developing nervous system, but they play a more general role in the experience-dependent synaptic remodeling underlying long-term memory.

Antisense oligodeoxynucleotides to c-fos are amnestic for passive avoidance in the chick.

Induction of c-fos occurs in the forebrain when chicks are trained on a passive avoidance task. Suppression of c-Fos protein synthesis with antisense oligodeoxynucleotides prevented long-term memory retention when injected 10-11 h before training, but not if injections were made between 3 h pre-and 3 h post-training. c-fos expression is thus necessary for long-term memory retention, presumably because of its control of induction of the signalling pathway that ultimately results in the protein synthesis subserving synaptic remodelling.

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 the N-methyl-D-aspartate receptor antagonists (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (up to 15 nmol/chick) or DL-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)- alpha-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.

Corticosterone facilitates long-term memory formation via enhanced glycoprotein synthesis.

Long-term memory formation for a passive avoidance task in one-day-old chicks requires a late phase of synaptic glycoprotein synthesis (including the neural cell adhesion molecule), commencing 5.5 h post-training. This phase occurred in chicks trained with a strong, but not a weak aversant, which only retained this memory for a few hours (< 10). In addition, previous work has shown that a corticosteroid action through central corticosteroid receptors is also required for long-term passive avoidance memory. Here, we tested the hypothesis that the corticosteroid action on memory formation might be exerted via modulation of the late phase of neural glycoprotein synthesis. One-day-old chicks were used as experimental subjects. Incorporation of the radiolabelled glycoprotein precursor [3H]fucose into synaptic membranes of the chick forebrain was used as an index of glycoprotein fucosylation. Bilateral intracerebral injections of a corticosterone dose (0.5 micrograms/hemisphere) that facilitates long-term retention of weak learning were able to induce the late phase of glycoprotein synthesis in undisturbed chicks. A further experiment examined the effect of antibodies against the neural cell adhesion molecule on the facilitatory action of corticosterone on long-term memory for the weak passive avoidance training. Chicks trained on a weak aversant were injected with corticosterone (0.5 micrograms/hemisphere) 30 min post-training and testing occurred 24 h post-training. Administration of the neural cell adhesion molecule antibodies during the late phase (5.5 h post-training) blocked the facilitatory action of corticosterone on long-term memory. These findings further support the view that corticosteroids have a role in memory consolidation. In addition to previously proposed effects on gene transcription, these data suggest a post-translational glycosylation mechanism for the modulatory effect of corticosteroids on long-term memory formation.

Characterisation of antibodies specific for chick brain neural cell adhesion molecules which cause amnesia for a passive avoidance task.

Antisera were prepared against six postsynaptic density glycoprotein fractions (150-180, 62-80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day-old chicks in a one-trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R-1 and R-6, or their IgGs (IgG-1 and IgG-6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later, IgG-1 and IgG-6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti-N-CAM antibodies. IgG-1 and IgG-6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N-linked mannose and fucose, and a very low amount of polysialic acid and O-linked galactose. Absorption of IgG-6 antibodies with neural cell adhesion molecule (N-CAM) isolated from synaptic plasma membranes derived from day-old chick brain resulted in loss of amnestic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

A role for a chicken homolog of the neural cell adhesion molecule L1 in consolidation of memory for a passive avoidance task in the chick.

Intracranial injection of antibodies directed against the neural cell adhesion molecule L1 resulted in amnesia for passive avoidance training in day-old chicks tested 24 hr subsequently. L1 antibodies were amnesic when administered at one of two time windows: 30 min pretraining and 5.5-8 hr post-training. No amnesia was apparent if injections were made at times before, between, or after these time windows (-2, +1, +3, +4, or +12 hr relative to training). A fragment of the L1 molecule derived from the external fibronectin domains FN1-5 produced amnesia only when injected at the 5.5-hr timepoint, whereas a fragment of the immunoglubin-like domains Ig I-VI produced amnesia only when injected 30 min prior to training. We have shown previously that long-term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training, and the second some 6 hr thereafter. The glycoprotein synthesis inhibitor 2-deoxygalactose results in amnesia if injected at either time, whereas the neural cell adhesion molecule (N-CAM) is specifically involved only in the second wave. The coincidence of the time course of memory disruption resulting from injection of L1 antibodies with that occurring with 2-deoxygalactose supports the hypothesis that establishment of an enduring memory for the experience of passive avoidance training requires two waves of glycoprotein synthesis, each wave being biochemically and functionally discrete. The differential effects of the two L1 fragments suggests that separate mechanisms of synaptic stabilization are involved at the two time points.

Effects of early experience on c-fos gene expression in the chick forebrain.

The expression of c-fos mRNA was studied by blot and in situ hybridisation in the forebrains of young chicks subjected to different types of experience-dependent stimulation. Exposure to one hour of activity in a rich visual environment produced a significant increase in forebrain c-fos mRNA in 1-day or 2-day-old chicks; this increase was most marked in cerebellum and medial forebrain--especially intermediate medial hyperstriatum ventrale (IMHV) regions. Levels of c-fos mRNA were not elevated in birds which had been accommodated to the enriched environment for 2 days, suggesting that the novelty of presented stimuli play a critical role in the c-fos induction. Training chicks on a one-trial passive avoidance task was followed by 2- to 2.5-fold increases in c-fos mRNA in both left and right IMHV and lobus parolfactorius (LPO) 30 min after the learning experience. Occlusion of one eye during training resulted in an asymmetrical expression of c-fos in the hemisphere contralateral to the open eye. Therefore c-fos is induced in the chick forebrain during learning and its activation cannot be attributed to 'stress', arousal or intensity of sensory stimulation alone. These observations support the hypothesis that c-fos expression in the brain is related to the acquisition of new experience.

The effect of electroconvulsive shock on brain tubulin during development and aging.

Effect of electroconvulsive shock on rat brain tubulin content was studied during maturation and aging. The results show that electroconvulsive shock had no effect on soluble tubulin in different brain structures of young animals (22 days) while the same treatment produced a marked decline in adult (95 days) and aged (490-511 days) animals. The same treatment produced inhibition of 3H-leucine incorporation into tubulin and decrease of 3H-colchicine binding in the proteins of synaptosomes isolated from the centricephalic structures of all the ages examined. Tubulin biosynthesis by free polysomes was not diminished to the extent which could explain the decrease of tubulin level found in the soluble or synaptosomal fraction. Thus, our results suggest that changes in soluble tubulin content in response to electroconvulsive shock could be a reflection of changes in equilibrium: tubulin dimers--microtubules--membrane-bound tubulin.

Effects of cortisol treatment on protein synthesis in septum and hippocampus of rat brain.

The effects of cortisol on protein synthesis in distinct brain regions were studied. Hormone administration produced increases in tubulin and actin content in the soluble fraction isolated from septum and hippocampus. Therefore, the poly(A)RNAs were isolated from the same brain regions and their translational capacity was tested in a heterologous cell-free system. Poly(A)RNAs from cortisol-treated animals, as compared to control animals, had a stimulatory effect on 35S-methionine incorporation into tubulin and actin. Thus, our results suggest that cortisol treatment increases the amount of mRNAs coding for tubulin and actin. More importantly, this study indicates that the molecular mechanism of glucocorticoid action in the brain involves modulation of protein synthesis.

Effect of calcium ionophore A23187 on the sensitivity of early sea urchin embryos to cytotoxic neuropharmacological drugs.

The ability of cytotoxic neurochemicals (indole and amphetamine derivatives) to block first cleavage division in the embryos of the sea urchin Arbacia lixula abruptly increases when the embryos are incubated in calcium-free seawater and decreases when the external Ca concentration is raised up to 46.4 mM. Sensitivity of the embryos to these drugs decreases also in the presence of the Ca-ionophore A23187. It is suggested that Ca ions are involved in the realization of physiological effects of "prenervous" neurotransmitters whose presence in early sea urchin embryos was shown by us earlier.