Radmila Mileusnic

Passive Avoidance Learning Results in Region‐Specific Changes in Concentration of and Incorporation into Colchicine‐Binding Proteins in the Chick Forebrain

Abstract: Incorporation of [14T]leucine into trichloracetic acid‐precipitable material and tubulin‐enriched fractions, and total tubulin levels as determined by colchicine‐binding activity and retention on DE81 filter discs, were measured in various regions of the chick brain following training on a one‐trial passive avoidance task, suppression of pecking at a chromed bead as a consequence of the aversive taste of methylanthranilate. Radioactive pulse time was 0.5 h. The only brain region in which changes were found was the anterior forebrain roof, the same area in which biochemical changes in response to exposure of the birds to an imprinting stimulus have been observed previously. In the anterior forebrain roof the changes observed as a consequence of training were detectable at 0.5 and 24 h after the 10‐s training experience but not 48 h subsequently. One‐half hour after training, there were increases of the order of 20 or 30% in [14T]leucine incorporation into particulate and postmitochondrial TCA‐precipitable material and a tubulin‐enriched fraction purified as above. There were comparable increases in the total amount of colchicine‐binding activity. By 48 h, none of these increases were detectable. Subcellular fractionation of the particulate fraction showed that most of the increase of incorporation into the tubulin‐enriched fraction and in colchicine‐binding activity was present in the soluble content of the synaptosomes; there were no increases in either measure in the synaptic membrane fraction. The possible role of changed levels and turnover of tubulin in the plastic responses of the brain to learning experiences is discussed.

APP is required during an early phase of memory formation

The amyloid β/A4 protein precursor (APP) has been shown to be implicated in age‐associated plastic changes at synapses that might contribute to memory loss in Alzheimers 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.

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.

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

A polyelonal 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.

Characterisation of Antibodies Specific for Chick Brain Neural Cell Adhesion Molecules Which Cause Amnesia for a Passive Avoidance Task

Abstract: 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. As we have previously shown that long‐term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training and the second 5–8 h later, the present results suggest strongly that isoforms of N‐CAM molecules with a low level of sialic acid are involved specifically in the establishment of an enduring memory for the experience of the passive avoidance task in chicks, possibly by stabilising changes in synaptic connectivity that encode the memory.

The peptide sequence Arg-Glu-Arg, present in the amyloid precursor protein, protects against memory loss caused by Aβ 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 (Aβ) 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 ≈ 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 Alzheimers disease.

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.

Amyloid precursor protein: from synaptic plasticity to Alzheimer’s disease

Abstract: The amyloid precursor protein (APP) has been shown to be implicated in age‐associated plastic changes at synapses that might contribute to memory loss in Alzheimers disease. As APP has previously been reported to have multiple functions during normal development, and as human and avian APP share 95% homology in amino acid sequence, 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, raised against human APP, APP‐antisense, and Aβ during pre‐training, prevented memory formation without effects on general behavior or initial acquisition. Amnesia is apparent by 30 min post‐training and lasts for at least 24 hours. Injection of APP‐derived peptides RERMS (APP328‐332) and RER (APP328‐330) homologous to the short stretches of amino acids in the Kang sequence (APP319‐335), rescue the memory in animals rendered amnestic by previous (anti‐APP antibody, antisense, and Aβ pretreatments. The protected form of RER, with a prolonged half‐life (acetylated RER), proved to be effective when injected intracranially and peripherally. The tripeptide RER exerts its biological activity by binding to two neuronal plasma membrane proteins (60 and 110 kDa). The results obtained in this study suggest that RER alleviates memory deficits via receptor‐mediated events, and that short APP‐derived peptides might represent a novel group of therapeutically active molecules for the alleviation of memory deficits in age‐related dementias.

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 β‐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.

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 5μg/ 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 5μg/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.