Studying the Specific Activity of the Amide Form of HLDF-6 Peptide using the Transgenic Model of Alzheimer's Disease.

The neuroprotective and nootropic activities of the amide form (AF) of the HLDF-6 peptide (TGENHR-NH2) were studied in transgenic mice of the B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line (the animal model of familial Alzheimer's disease (AD)). The study was performed in 4 mouse groups: group 1 (study group): Tg+ mice intranasally injected with the peptide at a dose of 250 µg/kg; group 2 (active control): Tg+ mice intranasally injected with normal saline; group 3 (control 1): Tg- mice; and group 4 (control 2): C57Bl/6 mice. The cognitive functions were evaluated using three tests: the novel object recognition test, the conditioned passive avoidance task, and the Morris water maze. The results testify to the fact that the pharmaceutical substance (PhS) based on the AF of HLDF-6 peptide at a dose of 250 µg/kg administered intranasally efficiently restores the disturbed cognitive functions in transgenic mice. These results are fully consistent with the data obtained in animal models of Alzheimer's disease induced by the injection of the beta-amyloid (ßA) fragment 25-35 into the giant-cell nucleus basalis of Meynert or by co-injection of the ßA fragment 25-35 and ibotenic acid into the hippocampus, and the model of ischemia stroke (chronic bilateral occlusion of carotids, 2VO). According to the overall results, PhS based on AF HLDF-6 was chosen as an object for further investigation; the dose of 250 µg/kg was used as an effective therapeutic dose. Intranasal administration was the route for delivery.

A Hardware-and-Software System for Experimental Studies of the Acoustic Startle Response in Laboratory Rodents.

We developed and tested a novel hardware-and-software system for recording the amplitude of the acoustic startle response in rodents. In our experiments, the baseline indexes of acoustic startle response in laboratory rats and pre-stimulation inhibition under the standard delivery of acoustic stimulation were similar to those evaluated by other investigators on foreign devices. The proposed system is relatively cheap and provides the possibility of performing experiments on freely moving specimens. It should be emphasized that the results of studies can be processed with free-access software.

Delayed effects of NMDA receptor antagonist MK-801 on storage and reconsolidation of spatial memory in rats.

We studied the effects of NMDA receptor antagonist MK-801 on the storage and reconsolidation of spatial memory in Morris water maze in adult rats. MK-801 (50 µg/kg) administered 24 h after the completion of training was shown to improve the resistance of spatial memory to spontaneous extinction, while reminder against the background of MK-801 suppresses its ameliorating effect on memory storage. The detected behavioral effects of MK-801 persisted over 60 days after administration and can be associated with its influence on coupled neurogenesis/apoptosis processes induced during memory trace formation in adult animals.

Neurogenesis enhancer RO 25-6981 facilitates repeated spatial learning in adult rats.

The effects of Ro 25-6981 (selective NMDA receptor blocker) in a dose stimulating neurogenesis on repeated learning, reversal learning, and memory reconsolidation were studied in adult rats in Morris water maze. Ro 25-6981 facilitated repeated learning 13 days after injection, but did not influence reversal learning. The blocker injected directly before reminder did not disturb repeated learning and reversal learning in Morris water maze. These effects of Ro 25-6981 on the dynamics of repeated learning seemed to be due to its effects on neurogenesis processes in adult brain.

[Effects of the neurogenesis stimulator Ro 25-6981 upon formation of spatial skill in adult rats depend on the term of its administration and the animals' ability to learn].

Effect of administration of selective N-methyl-D-aspartate (NMDA) receptor antagonist Ro 25-6981 on learning and memory in a dose which is known to stimulate neoneurogenesis was assessed in adult rats with different abilities to formation of spatial skills in different time periods after the antagonist injection. Wistar male rats were trained to find hidden platform in the Morris water maze for 5 consecutive days. Rats' learning ability for spatial skill formation was evaluated depending on platform speed achievements. In re-training sessions (cues and platform location changed), it was found that all rats received Ro 25-6981 13 days before the re-training demonstrated impaired spatial memory. At the same time the inhibitor injected 29 days before re-training selectively facilitated the formation of spatial skill in animals with initially low learning abilities.

Irreversible amnesia in rats and edible snails under conditions of associative memory reconsolidation disturbance caused by NMDA-glutamate receptor antagonist.

The effect of MK-801, an antagonist to NMDA-glutamate receptors, on reconsolidation of olfactory discrimination task in rats and taste discrimination in edible snails was examined. Twenty-four hours after conditioning, the animals received a single systemic injection of MK-801 followed by a reminding conditional stimulus. Disturbances in retrieval of the acquired task were observed 10 days after injection followed by a reminding procedure. Repeated conditioning of these animals did not restore the task. Injection of MK-801 without reminding stimulation had no effect on task retention. Thus, disturbances of NMDA-dependent reconsolidation of the associative memory in animals of different taxonomic groups irreversibly eliminated long-term memory.

[Selective involvement of cerebellar neurotransmitter systems in mechanisms of various types of defensive behavior].

The extracellular level of neurotransmitter amino acids and total content of serotonin and its metabolite 5-HIAA were estimated by microdialysis in cerebellar vermis at different stages of habituation of the acoustic startle reaction (ASR) and conditioned freezing in a startle chamber prior to acoustic stimulation. An increase in GABA and serotonin levels was observed 10 min after training. Twenty four hours after training, glycine and glutamate levels increased, whereas serotonin and 5-HIAA contents decreased. Positive correlations were revealed between the GABA and glycine levels during training and characteristics of the freezing behavior during testing. Glutamate level during the test session positively correlated with the index of long-term habituation of the ASR orienting component. NMDA receptor antagonist APV applied on cerebellar vermis before training suppressed, whereas AM PA receptor antagonist CNQX enhanced the long-term habituation of the ASR orienting component. Under the same conditions, GABA receptor antagonists bicuculline and phaclofen suppressed the long-term habituation of the defensive ASR component and formation of the conditioned freezing behavior. Applied before training 5-HT antagonist ritanserin impaired long-term habituation of defensive ASR component and formation of conditioned freezing behavior, but its application 1 h before testing enhanced the habituation of the defensive ASR component without any effect on conditioned fear. The results testify to the involvement of glutamatergic cerebellar system activity predominantly in long-term habituation of orienting ASR component and participation of GABA interneurons and serotoninergic projections to cerebellum in the mechanisms of the long-term habituation of the defensive ASR component and formation of conditioned freezing behavior.

[Neuroprotective effect of choline succinate in rats with experimental chronic cerebral ischemia evaluated by cognitive ability tests].

The neuroprotective effect of choline succinate was studied in rats with chronic cerebral ischemia induced by ligation of both common carotid arteries. Two approaches were used to evaluate the neuroprotective effect: cognitive ability tests (passive avoidance test and Morris water maze test) and determination of the brain content of N-acetylaspartate, a marker of functional neurons, by 1H NMR spectroscopy in vivo. Choline succinate administration significantly improved memory and learning in ischemic rats and prevented the ischemia-induced decrease in the cerebral level of N-acetylaspartate. Thus, choline succinate demonstrated a neuroprotective effect in conditions of ischemic brain injury.

Corrective effect of flavonoid-containing preparation Extralife on the development of Parkinson's syndrome.

Oral administration of flavonoid-containing preparation Extralife (daily dose 40 mg/kg) to animals with modeled Parkinson's syndrome considerably improved their survival and the main diagnostic and nosological parameters characterizing the state of locomotor functions. The preparation decreased animal mortality, rigidity, disturbances in dynamic muscular work and coordination of movements, and reduced oligokinesia. Experimental data confirm the involvement of mitochondrial enzyme complex I dysfunction in the pathogenesis of the disease and showed the possibility of by-passing this site of the respiratory chain with Extralife.

Hexapeptides HLDF-6 and PEDF-6 restore memory in rats after chronic intracerebroventricular treatment with beta-amyloid peptide Abeta(25-35).

Effects of homologous peptides HLDF-6 and PEDF-6 on behavior of animals with experimental Alzheimer's disease induced by chronic intracerebroventricular administration of beta-amyloid peptide Abeta(25-35) were studied in the zoosocial recognition test and Morris water maze. Peptides HLDF-6 and PEDF-6 possessed neuroprotective activity and counteracted the toxic effect of Abeta(25-35). Peptides HLDF-6 and PEDF-6 mainly improved long-term memory and working memory, respectively.

[Neuroprotective effect of the hexapeptide HLDF-6 on neurons of rat hippocampus on the model of Alzheimer's disease in vivo and in vitro].

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer's disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by administration of the peptide corresponding to the 25-35 sequence of beta-amyloid peptide (25-35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the orientation-investigation activity of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of neurons of the hippocampus and cerebellum of rats under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer's disease and in the medium of the culture of hippocampus neurons in the presence of the Abeta(25-35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.

Different mechanisms of protective and differentiative activities of homological peptides TGENHR and TQVEHR.

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.

Dynamics of intracellular dopamine contents in the rat brain during the formation of conditioned contextual fear and extinction of an acoustic startle reaction.

Extracellular dopamine contents in the caudate nucleus, nucleus accumbens, and prefrontal cortex of the rat brain were measured during two sessions of extinction of an acoustic startle reaction--each consisting of ten sound stimuli, the two sessions separated by 24 h--with simultaneous recording of freezing behavior. The results demonstrated a decrease in extracellular dopamine levels in the caudate nucleus and an increase in the nucleus accumbens during both sessions of extinction, with return to initial immediately after sessions ended. During the second session, the amplitude of startle responses and the magnitude of changes in dopamine levels in both structures were significantly smaller than during the first session. Between the sessions, dopamine levels in the caudate nucleus remained constant, while those in the nucleus accumbens decreased. The prefrontal cortex showed increases in dopamine levels during both sessions of extinction, as well as between the two sessions. The amplitude of the startle reaction was found to correlate with dopamine levels in the prefrontal cortex after the end of the corresponding extinction session and with the dopamine level before the start of the second session. The freezing time before the start of sound stimulation in the second session, this being a measure of conditioned fear, correlated with the dopamine level in the caudate nucleus on the training day and with the dopamine level in the nucleus accumbens before the start of the second session. The role of the dopaminergic system in the mechanisms forming and realizing the various components of defensive behavior are discussed.

Heterochronous involvement of neurotrophic factors in the neurochemical organization of learning and memory processes in adult organisms.

Studies were performed on the involvement of neurotrophic factors in the neurochemical mechanisms of the integrative functions of the brain. The effects of various intrahippocampal doses of antibodies to neurotrophic factors--protein S100 and lectin CSL--were studied on the formation, retention, and reproduction of a habituated acoustic startle response and conditioned fear in adult rats. S100b contents in the hippocampus, hypothalamus, frontal cortex, and cerebellar hemispheres and vermis, and in the basal nuclei were measured in rat brains 0.5, 1, 2, 4, 6, 8, 24, and 48 h after long-term habituation to the startle response. Antibodies to neurotrophic factors had selective and dose-dependent effects on the different memory and learning processes underlying these types of behavior. Changes in S100b in brain structures were seen, which were specific in terms of quantitative levels and dynamics, after acquisition of the behavioral habit. The results obtained here, along with previously reported data on the effects of antibodies to S100b and CSL given into the cerebellum, are discussed as experimental support for the hypothesis of the heterochronous neurochemical organization of integrative brain activity.

S100B protein in pro- and antiapoptotic doses produces different effects on defensive behavior in adult rats.

We studied the effects of S100b protein in doses stimulating (500 and 50 ng) or inhibiting (5 ng) apoptosis in nerve cells on acquisition, retention, and retrieval of extinction of the acoustic startle response and conditioned fear in adult rats. After application to the vermis of the cerebellum S100b protein in doses of 500 and 50 ng impaired, while in a dose of 5 ng facilitated acquisition of both forms of defensive behavior. Different behavioral effects of S100b protein are probably related to its pro- and antiapoptotic effects on cerebellar cells relevant to the studied forms of behavior. Our results suggest that regulators of apoptosis are involved in the mechanisms of learning and memory.

[Dynamics of the level of extracellular dopamine in the rat brain during formation of conditioned fear and acoustic startle habituation]. / Dinamika soderzhaniia vnekletochnogo dofamina v mozge krys pri formirovanii uslovnogo obstanovochnogo strakha i ugasheniia akusticheskoi reakstii vzdragivaniia.

The content of extracellular dopamine (DA) was estimated in dorsal striatum (DS), nucleus accumbens (Nac), and prefrontal cortex (Pfc) of rat brain during two sessions of acoustic startle habituation consisting of 10 trials each with 24-hour intersession and 20-s intertrial intervals. Startle amplitude and freezing behavior were recorded. A decrease in the content of extracellular DA in the DS and an increase in the DA content in Nac were observed during both sessions of habituation with return to a baseline level immediately after termination of the sessions. During the second session, the startle amplitude and change in the DA content in both structures were much lower than during the first session. During the first session, the DA level in DS remained unchanged but dropped in Nac. In the Pfc the DA level increased during both habituation sessions and in the period between the session. Time of freezing prior to acoustic stimulation in the second sessions (that is known to be an indication of conditioned fear) correlated with the DA level in DS on the day of training and with the DA level in Nac just before the beginning of the second session. The role of the dopaminergic system in formation and retrieval of different components of defensive behavior is discussed.

[Heterochronic effects of neurotrophic factors in neurochemical organization of learning and memory in the adult organism]. / Geterokhronia uchastiia neirotroficheskikh faktorov v neirokhimicheskoi organizatsii protsessov obucheniia i pamiati v zrelom organizme.

Following elaboration of long-term habituation to a startle-response, antibodies to neurotrophic factor protein S100b exerted selective and dose-dependent influence on different learning processes and memory of learned behavioural patterns in adult rats. S100b increased at all stages of behavioural skill development in hippocampus, hypothalamus, frontal cortex, cerebellar hemispheres and vermis, basal ganglia.

The involvement of neurotrophic factors in the central mechanisms of behavior in adult animals.

In the light of the similarity of the neurochemical mechanisms of behavior during ontogenesis and in adult organisms, studies were performed on the effects of antibodies to neurotrophic factors--S-100b protein, CSL lectin, and R1 lectin-on the acquisition, retention, and reproduction of habituated acoustic startle reactions and freezing behavior in adult rats. Antibodies against neurotrophic factors were found to have selective, dose-dependent effects on the types of behavior investigated. Antibodies to S-100b, CSL, and R1, at a dose of 5 microg, induced derangements in the processes of consolidation and/or retention of behavioral habits. At a dose of 2 microg, antibodies to S-100b selectively blocked the retention of long-term habituation of the startle reaction, while a dose of 0.5 microg had no such effect. Antibodies to neurotrophic factors had no effect on reproduction of acquired defensive habits.

[Involvement of neurotrophic factors in central mechanisms of behavior in adult animals]. / Uchastie neirotroficheskikh faktorov v tsentral'nykh mekhanizmakh povedeniia u vzroslykh zhivotnykh.

The effect of antibodies against some neurotrophic factors (S-100b protein, CSL, etc.) on elaboration, storage, and retrieval of acoustic startle habituation and concomitant freezing behaviour, was found to be dose-dependent in adult rats. Thus, the 5-mcg dose impaired the consolidation and/or storage process, whereas 2 mcg only impaired the storage of long-term acoustic startle habituation, and 0.5 mcg exerted no effect at all. The habits retrieval displayed no dependence on the antibodies effect.

The selective effects of a monoclonal antibody against neural growth-related protein A3G7 on central mechanisms of several types of defensive behavior in adult rats.

Studies were carried out into the effects of monoclonal antibodies to protein A3G7, which is associated with the differentiation of cerebellar and hippocampal neurons, on the development, retention, and reproduction of a habituated acoustic startle response and freezing behavior in rats. Application of monoclonal antibody (50 ng) to the vermis of the cerebellum selectively disrupted the retention of long-term habituation of the acoustic startle response, while a higher dose (5 micrograms) inhibited both retention of long-term habituation of the acoustic startle response and conditioned freezing behavior. Monoclonal antibody (10 micrograms) disrupted the development and retention of short-term and long-term extinction of the acoustic startle response, as well as conditioned freezing behavior. These data provide experimental support for the systemogenesis hypothesis of behavioral acts and for the concept that there is a single molecular basis for development and learning.