Non-competitive NMDA antagonist MK-801 prevents memory reconsolidation impairment caused by protein synthesis inhibitors in young chicks.

Introduction: Reactivation of already consolidated memory can initiate its destabilization, making the memory trace labile. Normally, this destabilization is followed by reconsolidation of the memory trace, enriched by newly acquired experience. Disrupting the reconsolidation process, for example, by inhibiting protein synthesis, impairs subsequent memory retrieval, leading to reminder-related amnesia. Previous studies in various species have shown that this impairment can be prevented by using NMDA receptor antagonists, which interfere with memory destabilization. Methods: In the present study we examined this phenomenon using a one-trial passive avoidance learning model in newborn chicks, the hypothesis being that inactivation of the NMDA-mediated transmission during memory reactivation would inhibit the memory trace labilization and thus prevent the reminder-related amnesia. Results: We found that reminder-associated administration of the NMDA receptor antagonist MK-801 or one of the protein synthesis inhibitors (anisomycin, cycloheximide, 2-deoxygalactose) each alone produced amnesia. However, when combined, injection of MK-801 before the reminder prevented amnesia induced by protein synthesis inhibitors. Discussion: We suggest that the observed paradoxical effect implicates the involvement of NMDA receptors in both protein synthesis-independent engram destabilization upon its retrieval and protein synthesismediated engram stabilization after its updating. This puzzling dual role of NMDA receptors in memory destabilization/restabilization requires further investigation.

The power of innate: Behavioural attachment and neural activity in responses to natural and artificial objects in filial imprinting in chicks.

Newly hatched domestic chicks are known to orient preferentially toward naturalistic stimuli, resembling a conspecific. Here, we examined to what extent this behavioral preference can be transcended by an artificial imprinting stimulus in both short-term and long-term tests. We also compared the expression maps of the plasticity-associated c-fos gene in the brains of chicks imprinted to naturalistic (rotating stuffed jungle fowl) and artificial (rotating illuminated red box) stimuli. During training, the approach activity of chicks to a naturalistic object was always higher than that to an artificial object. However, the induction of c-fos mRNA was significantly higher in chicks imprinted to a box than to a fowl, especially in the intermediate medial mesopallium, hyperpallium apicale, arcopallium, and hippocampus. Initially, in the short-term test (10 min after the end of training), chicks had a higher preference for a red box than for a stuffed fowl. However, in the long-term test (24 h after imprinting), the response to an artificial object decreased to the level of preference for a naturalistic object. Our results thus show that despite the artificial object causing a stronger c-fos novelty response and higher behavioral attachment in the short term, this preference was less stable and fades away, being overtaken by a more stable innate predisposition to the naturalistic social object.

Influence of Natural Asymmetric Embryonic Visual Afferentation on the Neuronal Activations in the Caudomedial Mesopallium during the Freezing Response in Altricial Nestlings.

The study was designed to investigate the role of asymmetric prenatal visual stimulation on the activation of caudomedial mesopallium (CMM) neurons in nine-day-old pied flycatcher nestlings during auditory-guided freezing. Four groups of nestlings were studied: groups 1 and 2 included nestlings with normal vision and visually deprived, respectively, that were incubated and hatched in normal light environment; groups 3 and 4, nestlings with normal vision and visually deprived, respectively, that were incubated and hatched in the dark. The eyes of visually deprived nestlings were covered with non-transparent cups 2 h before the experiment. C-Fos expression was studied. It was shown that densities of neurons activated during freezing response differed in right vs. left CMM only in the group of visually deprived nestlings incubated under light. This suggests that the presence or absence of the asymmetric embryonic visual afferentation may result in the development of different strategies of the visual system integration into defense behavior.

Memory reacquisition deficit: Chicks fail to relearn pharmacologically disrupted associative response.

It is generally assumed that if memory is disrupted by pharmacological inhibitors during its consolidation, it can be later acquired afresh. In our experiments, we trained day-old chicks in a one-trial passive avoidance task and interfered with memory formation using protein synthesis inhibitor anisomycin or NMDA receptor antagonist MK-801. Second training was then given to amnestic animals with either the same conditioning stimulus (retraining) or a new one (novel training). Retraining with the same stimulus failed to produce efficient memory at all the examined between-training and training-to-test intervals, while a new conditioned stimulus was learned successfully. We suggest that this memory reacquisition deficit may result from the failure of associative memory co-allocation mechanisms.

Recovery of Impaired Memory: Expression of c-Fos and Egr-1 Transcription Factors during Restoration of Damaged Engram in the Chick Brain.

Impairment of protein synthesis in the brain during learning prevents memory consolidation and results in amnesia, which until recently has been regarded irreversible. However, in some cases impaired memory could be restored by various "reminder" stimuli. The present study is based on the hypothesis that even in behaviorally profound amnesia, some disintegrated fragments of the engram are preserved in the brain and could be re-integrated into the whole system by specific types of stimuli. The aim of the present study was to test this hypothesis in an experimental model of pharmacologically induced memory impairment in young chicks and to reveal the brain areas involved in this process by mapping of reminder-induced expression of transcriptional factors c-Fos and Egr-1. We show that reminder treatment results in the recovery of memory impaired by protein synthesis inhibition during learning and induces c-Fos and Egr-1 expression in the brain regions involved in learning in this behavioral model. The patterns of c-Fos and Egr-1 induced expression in animals with impaired memory differed from the patterns of animals with unimpaired memory and as well as naïve animals with no memory. Thus, analysis of activity-induced c-Fos and Egr-1 expression revealed the brain regions that were specifically activated by the reminder treatment. At the behavioral level, this treatment led to memory recovery. Altogether, these results suggest that the reminder-induced transcriptional activity in the brain of amnestic animals occurs in regions maintaining the engram fragments that reintegrate to recover the impaired memory.

Prenatal Sensory Stimulation Induces BDNF Gene Expression in the Brain and Potentiates the Development of Species-Specific Predisposition in Newborn Chicks.

We studied the effects of light and non-specific sound stimulation of domestic chick embryos on their filial preference as well as on the expression of two transcriptional factors c-Fos and Egr-1 and neurotrophin BDNF in the embryo brain. Prenatal light stimulation increased preference of the "natural" object, thus producing a priming effect. In the brain of E19 embryos, c-Fos and Egr-1 were expressed at a high basal level and neither light nor sound stimulation affected the number of cells expressing these factors. BDNF mRNA was also present in a number of brain areas of non-stimulated embryos, but light and sound stimulation enhanced the expression of BDNF mRNA in brain structures associated with filial imprinting. These findings suggest that BDNF is probably involved in the effects of prenatal priming on the development of species-specific behavior.

Paradoxical Effect of NMDA Receptor Blockade in Chicks on Learning and Memory in Passive Avoidance Model.

Activity of NMDA receptors is a prerequisite for numerous but not all forms of neuronal plasticity and learning. The present study examined the role of NMDA receptors in standard, weak, and repeated passive avoidance training in young chicks. Injection of MK-801, an antagonist of NMDA receptor, prior to strong training episode impaired subsequent memory recall. Moreover, repeated training did not restore the lost memory. In the double weak training protocol, the impairing effect of MK-801 was observed only when it was injected prior to the second but not to the first training episode. These results suggest that activation of NMDA receptors is not a necessary stage for memory acquisition in the weak training task. In contrast, the mechanisms of strong training depending on activation of NMDA receptors can be probably involved into the second training episode performed against the background of existing NMDA receptor-independent memory about the first training episode.

Pro-Cognitive Effects of Non-Peptide Analogues of Soluble Amyloid Peptide Precursor Fragment sAPP.

We studied pro-cognitive effect of two heterocyclic low-molecular-weight compounds that serve as non-peptide analogues of soluble fragment of amyloid peptide precursor (sAPP). Intracerebroventricular and systemic administration of peptide mimetics P2 and P5 improved weak memory on the model of passive avoidance in chicks and in the object location task in mice. Both compounds were effective if administered close to the moment of training or 4 h after it. The time windows and dose range for the pro-cognitive effects of the mimetics were similar to those observed in previous studies with sAPP peptide fragments.

[Wulst Activation in Pied Flycatcher Nestlings in Feeding Behavior Induced by Patterned Visual Stimulus].

Immunohistochemical detection of c-Fos was used to study the transcriptional activation in two higher visual centers (Wulst area and Entopallium) of 12-day-old pied flycatcher (Ficedula hypoleuca) during the realization of feeding behavior guided by patterned visual stimulus, simulating the species-specific one. Activation was compared in 4 groups of nestlings. Control group was not subjected to any experimental influence. In binocular, right-field (deprivation of the left eye) and left-filed (deprivation of the right eye) groups the vision feeding responses were provoked, reinforced and evaluated. It was shown that the visual afferentation from the right eye was more significant for the organization of early feeding behavior guided by a moving patterned visual stimulus as compared with the afferentation from the left eye. Feeding behavior induced activation of c-Fos expression only in neurons of the higher center of thalamofugal system--Wulst area. The comparison of transcriptional activation in different groups revealed the significant increase of c-Fos induction related with feeding behavior only in the left hemisphere and only in binocular and right-field groups.

Involvement of protein kinase Mζ in the maintenance of long-term memory for taste aversion learning in young chicks.

The effects of an inhibitor of protein kinase Mζ on long-term memory were studied using the model of taste aversion in newborn chicks. Memory was impaired by intracerebral injection of 10 or 20 nmol of ζ-inhibiting peptide 24 h after training. Memory impairment was found 2 h after peptide administration, and repeated examination 24 h after treatment showed no recovery. Memory impairment was not observed 24 h after inhibitor administration if the testing 2 h after treatment was not performed. The results indicate the contribution of protein kinase Mζ in the maintenance of long-term memory in the avian brain. These data confirm the hypothesis of several authors that inhibition of protein kinase Mζ does not abolish memory, but rather interacts with processes of memory retrieval and/or reconsolidation.

[Inhibition of histone deacetylases in the chick brain modulates expression of c-Fos and ZENK transcription factors and facilitates establishment of long-term memory].

The aim of the work was to examine the role of histone acetylation in memory consolidation in newborn chicks. We studied the effects of histone deacetylase inhibitor trichostatin A (TSA) on a "weak" memory for passive avoidance and on expression of two transcription factors c-Fos and ZENK known to play a role in neuronal plasticity in the chick brain. Intraventricular administration of trichostatin A prior to training produced a dose-dependent enhancement of memory when tested 24 hours after the training. It also increased neuronal expression of c-Fos and ZENK proteins: the density of ZENK immunopositive cells increased in the hippocampus and intermediate medial mesopallium and the density of c-Fos immunopositive cells increased in intermediate arcopallium and dorsocaudal nidopallium. Weak passive avoidance training did not produce further enhancement of c-Fos and ZENK expression in any of these brain areas. These data demonstrate possibility of facilitating long-term memory in day-old chicks by a histone deacetylases inhibitor, thus supporting the hypothesis on the role of histone acetylation in long-term memory formation. They also suggest that these effects might be mediated through modulation of transcriptional response in brain areas involved in consolidation of this form of memory.

[Visual input affects the expression of the early genes c-Fos and ZENK in auditory telencephalic centers of pied flycatcher nestlings during the acoustically-guided freezing].

The present study analyzed expression of transcriptional factors c-Fos and ZENK in 9-day-old pied flycatcher nestlings' (Ficedula hypoleuca) telencephalic auditory centers (field L, caudomedial nidopallium and caudomedial mesopallium) involved in the acoustically-guided defense behavior. Species-typical alarm call was presented to the young in three groups: 1--intact group (sighted control), 2--nestlings visually deprived just before the experiment for a short time (unsighted control) 3--nestlings visually deprived right after hatching (experimental deprivation). Induction of c-Fos as well as ZENK in nestlings from the experimental deprivation group was decreased in both hemispheres as compared with intact group. In the group of unsighted control, only the decrease of c-Fos induction was observed exclusively in the right hemisphere. These findings suggest that limitation of visual input changes the population of neurons involved into the acoustically-guided behavior, the effect being dependant from the duration of deprivation.

Involvement of Wulst neurons in hiding reaction of pied flycatcher nestlings with different visual afferentation.

We studied transcriptional activity in the higher avian center visual system (Wulst area) in acoustically guided defensive behavior in visually deprived and non-deprived nestlings to evaluate the effects of visual afferentation on functional involvement of visual structures in acoustically guided defensive behavior. Exclusion of visual afferentation from already formed defensive behavior did not significantly change immunoreactivity of Wulst neurons, which attests to substantial contribution of other, non-visual, activating influences. Limitation of visual afferentation during the formation of defensive behavior decreased immunoreactivity of Wulst neurons. Dendritic sprouting in Wulst neurons of visually deprived nestlings unable to promote the formation of complex interneuronic interactions.

Glutamate receptor modulator dimebon stimulates consolidation and reconsolidation of weak memory in chicks.

Effects of glutamate receptor modulator dimebon on memory consolidation and reconsolidation were investigated in passive avoidance paradigm in newborn chicks. Systemic administration of 0.1 mg/kg dimebon 5 min before or 4 h after "weak" training resulted in formation of long-term memory. Dimebon administration in combination with memory reactivation 24 h after "weak" training recovered the memory decayed by the time of reminder and ensured its subsequent long-term maintenance over 24 h. Thus, we showed the possibility for dimebon-induced recovery of the memory that decayed and had no manifestations in behavior. Dimebon administration potentiated early and late stages of memory consolidation in learning as well as in memory reconsolidation following its reactivation.

Effects of systemic administration of histone deacetylase inhibitor on memory formation and immediate early gene expression in chick brain.

We studied the effects of histone deacetylase inhibitor that stimulates transcriptional activity via histone hyperacetylation on memory formation. Sodium butyrate and sodium valproate enhanced memory in chicks following "weak" training with memory transfer into long-term state. Quantitative analysis of c-Fos and ZENK transcriptional factor gene expression in six structures of chick brain revealed induction of these genes in the structures involved in this type of learning. Sodium valproate administration did not increase this induction, but even reduced it. These findings suggest that sodium butyrate and sodium valproate exert cognitive stimulating action in the "weak" memory formation paradigm, and that this effect is not mediated via enhanced expression of transcriptional factors, which are traditionally considered as "molecular switcher" for memory transfer into long-term state.

[Activation of tectofugal visual system of the pied flycatcher nestlings at early developmental stages of feeding behavior guided by diffuse photosensitivity].

Immunohistochemical detection of c-Fos in serial frontal sections of the brain of Pied flycatcher nestlings was used to map the sensory structures involved in early forms of feeding behavior. The c-Fos content was quantitatively analyzed in the higher structures of thalamofugal (Wulst area) and tectofugal (entopallium) visual pathways during visually-guided feeding behavior in 6-day-old nestlings at the stage of diffuse photosensitivity. Induction of c-Fos was not observed in the Wulst area which is known to be involved in the feeding integration in adult bifoveal birds. Induction of c-Fos was detected in the ventral area of entopallium containing neurons that are, according to literature, sensitive to a luminosity change. In the entopallium of 6-day-old nestlings, asymmetry in the evoked c-Fos expression was observed, probably reflecting the asymmetry of the visual projections originating in the embryonic period.

Selective impairments to memory consolidation in chicks produced by 5'-iodo-2'-deoxyuridine.

The aim of the present work was to study the role of DNA synthesis in the formation of different types of memory in neonatal chicks. The nucleotide analogs 5'-iodo-2'-deoxyuridine (IdU) and 5'-bromo-2'-deoxyuridine (BrdU) were used; these are incorporated into DNA, impairing its function, and have amnestic actions in defined models of learning in mice. We studied the effects of 5'-iodo-2'-deoxyuridine of the formation of long-term memory in chicks during training in different models: passive avoidance, imprinting, taste aversion, and spatial learning in a maze. In the taste aversion model, i.p. administration of IdU (10 mg/kg 5 min before or 50 min after training) had an amnestic effect on testing 1-2 days after training. IdU-induced amnesia developed more than 6 h after training, while administration of IdU 2 h after training had no amnestic effect. 5'-Bromo-2'-deoxyuridine also had a similar amnestic action in the taste aversion model. In the passive avoidance, imprinting, and spatial maze learning models, administration of IdU at the same dose before and after training did not induce amnesia. These data lead to the suggestion that DNA synthesis in the brain may play a critical role in the mechanisms of memory consolidation in chicks in types of learning such as taste aversion.

[Selective disruption of memory consolidation in chicks produced by 5'-iodo-2'-deoxyuridine].

We tested the hypothesis that DNA synthesis is involved in molecular mechanisms of memory consolidation. Nucleotide analogs 5'-iodo- and 5'-bromo-2'-deoxyuridine impair DNA functions being incorporated into elongated DNA chain and cause amnesia in a number of training models in mice. We studied possible amnestic effects of 5'-iodo-2'-deoxyuridine (IdU) in different training models in newborn chicks--in passive avoidance, taste aversion, imprinting and spatial learning in a maze. In the taste aversion model injection of IdU (10 mg/kg 5 min before or 50 min after training) produced amnesia at test 1-2 days after training, at the same time it had no effect on memory retention in test 6 h after training. IdU injection 2 h after training produced no amnesia. Similar amnestic effect in taste aversion model was found for 5'-bromo-2'-deoxyuridine (BrdU). In models of imprinting, passive avoidance and spatial learning IdU injection before or after training had no effect on memory retention. These data presuppose that brain DNA synthesis might play a critical role in mechanisms of memory consolidation in taste aversion learning in chicks.

Chicken synucleins: cloning and expression in the developing embryo.

Synucleins comprise a family of small intracellular proteins that have recently attracted considerable attention because of their involvement in human diseases. Mutations of alpha-synuclein has been found in several families with hereditary early-onset Parkinson's disease and accumulation of this protein in characteristic cytoplasmic inclusions is a pathohistological hallmark of several neurodegenerative diseases that have been recently classified as 'alpha;-synucleinopathies' (reviewed in Brain Res. Bull. 50 (1999) 465; J. Neurosci. Res. 58 (1999) 120; Philos. Trans. R. Soc. Lond. Biol. Sci. 354 (1999) 1101; Brain Pathol. 9 (1999) 733). Aggregates of beta-synuclein and persyn (gamma-synuclein) also have been found in dystrophic neurites associated with Parkinson's and other neurodegenerative diseases (Proc. Natl. Acad. Sci. USA 96 (1999) 13450; and our unpublished observations). Moreover, persyn has been implicated in malignization of breast tumours (Cancer Res. 57 (1997) 759; Cancer Res. 59 (1999) 742; Hum. Mol. Genet. 7 (1998) 1417). All synucleins have distinct, although overlapping, patterns of expression in the embryonic, postnatal and adult mammalian nervous systems, suggesting important, although still not clear, biological functions in neuronal developing. Chicken embryo is a unique object for developmental studies that allows in vivo manipulations not always possible for mammalian embryos. Studies of synucleins expression in this model system could shed light on their functions in the developing nervous system. We cloned three chicken synucleins from the embryonic neural cDNA libraries and studied their expression in normal chicken embryonic tissues by Northern and in situ hybridization with specific probes. Our results demonstrate that primary structures and expression patterns of synucleins are similar in birds and mammals, suggesting that conserved function of synucleins is important for embryonic development of vertebrates.

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.