Prenatal Hypoxia Impairs Olfactory Function in Postnatal Ontogeny in Rats.

Analysis of the age-related dynamics of olfactory behavior in the odor preference and food search testsshowed that all male Wistar rats, regardless of age, preferred valerian essential oil, whose components have the properties of pheromones in rodents, when given a selection of eight essential oils; young rats displayed better food-seeking results than adult and old animals. Acute prenatal hypoxia (PH) on E14 (7% O2 for 3 h) led to impairment of the valerian odor preference at all ages studied and to decreased productivity of food searches. Neurodegenerative processes were seen in the piriform cortex after PH, with reductions in the number of neurons and increases in glial elements. We have previously observed these changes in the entorhinal cortex and hippocampus, but not in the olfactory bulbs. This suggests that PH-induced decreases in olfactory function in rats may result from impairments to the formation of the central elements of the analyzer during the first months of postnatal ontogeny.

Regulation of Neprilysin Activity and Cognitive Functions in Rats After Prenatal Hypoxia.

The amyloid-degrading enzyme neprilysin (NEP) is one of the therapeutic targets in prevention and treatment of Alzheimer's disease (AD). As we have shown previously NEP expression in rat parietal cortex (Cx) and hippocampus (Hip) decreases with age and is also significantly reduced after prenatal hypoxia. Following the paradigms for enhancement of NEP expression and activity developed in cell culture, we analysed the efficacy of various compounds able to upregulate NEP using our model of prenatal hypoxia in rats. In addition to the previous data demonstrating that valproic acid can upregulate NEP expression both in neuroblastoma cells and in rat Cx and Hip we have further confirmed that caspase inhibitors can also restore NEP expression in rat Cx reduced after prenatal hypoxia. Here we also report that administration of a green tea catechin epigallocatechin-3-gallate (EGCG) to adult rats subjected to prenatal hypoxia increased NEP activity in blood plasma, Cx and Hip as well as improved memory performance in the 8-arm maze and novel object recognition tests. Moreover, EGCG administration led to an increased number of dendritic spines in the hippocampal CA1 area which correlated with memory enhancement. The data obtained allowed us to conclude that the decrease in the activity of the amyloid-degrading enzyme NEP, as well as a reduction in the number of labile interneuronal contacts in the hippocampus, contribute to early cognitive deficits caused by prenatal hypoxia and that there are therapeutic avenues to restore these deficits via NEP activation which could also be used for designing preventive strategies in AD.

Delayed effect of prenatal exposure to hypoxia on the susceptibility of rats to electric seizures.

We studied the delayed effects of prenatal exposure to hypoxia on the susceptibility of rats to seizures. The later was estimated using graded electroshock. The experiments were performed in two groups of 1.5-year-old male Wistar rats. The experimental group consisted of the animals that were exposed to hypoxia on day 14 of prenatal development, and the control group consisted of the animals that developed under the normal conditions. In the rats subjected to prenatal hypoxia, seizure episodes induced by weak currents in the range of 10-40 mA and their average duration were more pronounced as compared to the control animals.

[ROLE OF CASPASE-3 IN REGULATION OF THE CONTENT OF THE AMYLOID-DEGRADING NEUROPEPTIDASE NEPRILYSIN IN THE CORTEX OF RATS AFTER HYPOXIA].

Analysis of the effect of a caspase-3 inhibitor on the content of the amyloid-degrading neuropeptidase neprilysin (NEP) in the cortex of rats subjected to prenatal hypoxia (7% O2, 3 h) on the 14-th day of the embryonic development (E14) was performed. It was found that rats subjected to prenatal hypoxia on days 20-30 after birth have an increased content and activity of caspase-3 with reduced levels of NEP and of the C-terminal fragment of the amyloid precursor protein (AICD) regulating NEP expression. In hypoxic animals 3 days after a single injection of a caspase inhibitor (i. v., Ac-DEVD-CHO, P20) the content of AICD and NEP was found to be increased up to the levels observed in control rats. The data obtained suggest that the increase of caspase-3 enzyme activity could affect NEP expression via proteolytic degradation of its transcription factor AICD. These data for the first time demonstrate the role of caspases in AICD-dependent regulation of NEP production in the brain of mammals under hypoxic conditions.

Molecular properties of lysine dendrimers and their interactions with Aß-peptides and neuronal cells.

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer's, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aß-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aß-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aß. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs.

Ontogenetic characteristics of behavior in rats subjected to hypoxia on day 14 or day 18 of embryogenesis.

Physiological development, motor activity, and cognitive functions were studied in rats subjected to acute normobaric hypoxic hypoxia (3 h at an O2 concentration of 7%) at different stages of embryogenesis (days E14 or E18). Prenatal hypoxia was found to lead to delays in physiological development and the establishment of motor behavior during the first month of postnatal ontogenesis. These changes were more marked in rats subjected to hypoxia on day 14 of intrauterine development and disappeared with age. In adult rats, regardless of the timing of exposure to hypoxia (E14 or E18), learning ability was degraded and long-term and short-term memory were impaired. These results suggest that exposure to the pathogenic factor during the main period of neuroblast generation and migration (E14) was significant both for physiological development and the establishment of motor behavior in the animals and for the execution of the cognitive functions of the brain, while exposure during the period at which maturation and differentiation processes dominate in the brain (E18) was more significant in relation to the execution of cognitive functions.

Effects of an inhibitor of alpha-secretase, which metabolizes the amyloid peptide precursor, on memory formation in rats.

Intracortical administration of 10(-4) M batimastat, a specific inhibitor of alpha-secretase (a metalloproteinase which cleaves the amyloid peptide precursor), decreased the number of correct runs in a single-level eight-arm maze to 92.78 +/- 1.03% compared with baseline (p < 0.01) within 60 min. However, injection of batimastat into the cerebral cortex of animals during the early postnatal period (days 5 and 7 of life) led to impaired orientation in the simple single-level maze when these adults reached adulthood (90.92 +/- 2.21% correct runs, p < 0.001) as compared with controls. The data obtained here provide evidence for the important role of alpha-secretase in memory processes. The possible role of alpha-secretase in memory processes and the pathogenesis of Alzheimer's disease is discussed.

Formation of the structural and ultrastructural organization of the striatum in early postnatal ontogenesis of rats in altered conditions of embryonic development.

Light (Nissl and Golgi methods) and electron microscopy methods were used to study the formation of the structure of the striatum during the first two weeks after birth in rats subjected to acute hypoxia at different times of embryogenesis. The dynamics of the physiological development of the same population of rats were studied in parallel. Hypoxia at day 13.5 of embryogenesis was found to lead to delayed neurogenesis (delayed establishment of elements of the neuropil and differentiation of cells) and abnormalities in the structure of the striatum (degeneration, particularly chromatolysis, of neurons and the appearance of glial nodes). Morphometric analysis demonstrated a decrease in the total number of cells in the striatum; small changes in large neurons were seen. Hypoxia at day 18.5 of embryogenesis produced no significant changes. Structural abnormalities were accompanied by changes in the process of the animals' physiological development. The data obtained here show that changes in the conditions of embryogenesis (hypoxia) during the period of the most intense proliferation of neuroblasts in the forebrain lead to impairment of the process of formation of striatal nervous tissue and the body as a whole in the period of early postnatal ontogenesis.

Postnatal physiological development of rats after acute prenatal hypoxia.

The aim of the present work was to identify the characteristics of the physiological development of the brain and the formation of behavior in rats subjected to hypoxia on day 13.5 of embryogenesis. These animals showed delayed development and changes in nerve tissue structure in the sensorimotor cortex, along with disturbances to the processes forming normal movement responses during the first month after birth. These changes were partially compensated with age, though adult animals subjected to acute prenatal hypoxia were less able to learn new complex manipulatory movements. Alterations in nerve tissue structure and changes in the neuronal composition of the sensorimotor cortex correlated with the times of appearance of behavioral impairments at different stages of ontogenesis. Thus, changes in the conditions in which the body is formed during a defined period of embryogenesis lead to abnormalities in the process of ontogenetic development and the ability to learn new movements.

Striatal level of regulation of learned forepaw movements in rats.

The role of the striatal adenylyl cyclase (AC) and cholinergic systems in the learning and expression of new forepaw movements (reaching with prolonged pushing on a fixed piston) was studied in male Wistar rats. Motor learning processes, prenatal hypoxia, and cholinergic drugs changed the properties of the AC system in the striatum. After learning, the striatal basal AC activity was decreased compared to untrained control rats. In addition, the AC activity was more decreased in animals with a good ability to learn compared to poor learners (up to 31 % and 51 %, correspondingly; p<0.01). Rats subjected to prenatal hypoxia (13-14th days of embryogenesis) had a lower ability to learn the new movements requiring tactile control and the striatal AC activity in these rats was 1.8 times higher (p<0.001) than controls. In vitro application of the cholinergic agonist carbachol (CARB) 10-5 M (corresponding to approximately 0.3 microg), as well as the antagonist scopolomine (SCOP) 10(-5) M (approximately 0.3 microg) decreased AC activity in the synaptosomal fraction of the striatum. In vivo injections of CARB (0.3-3 microg/1microl) or SCOP (0.3-3 microg/1microl) into the ventral striatum (nucleus accumbens) modified the newly learned sensorimotor skill. After CARB injections the rats performed slower movements with more prolonged pushing. After SCOP the rats could not retain the learned pushing movement. These in vivo and in vitro data suggest that the cholinergic mediator system of the striatum is involved in learning sensory-controlled forepaw movements as well as the regulation of new motor skills by modulating the AC signal transduction process in the striatum. The data confirmed that modification of the striatal AC system resulted in the modulation of reaching behavior and better expression of the learned reflex.

The role of the cholinergic system of the sensorimotor cortex of the rat brain in controlling different types of movement.

The role of the cholinergic system of the sensorimotor cortex of the Wistar rat brain in controlling various types of movements was assessed by studying the effects of microinjections of carbachol and scopolamine into the representation area of the forelimb on the performance of two types of fore-limb food-procuring movements--with and without pressure on an obstacle--as well as on the animals' locomotion. These studies showed that administration of the cholinergic agonist carbachol (0.03-3 microg) leads to slowing of both types of procuring movements and acceleration of locomotor activity in an open field. Injections of the cholinergic antagonist scopolamine (0.3-3 microg) into the same area accelerated procuring movements, while the animals' locomotor activity remained unaltered. These data indicate that the cholinergic system of the sensorimotor cortex has different regulatory influences on movement activity (locomotion) and the performance of learned movements requiring forelimb muscle tone to be maintained for different periods of time (the usual rapid movements used for extracting food from a narrow horizontal tube versus slow movements with additional tactile and tonic components).

Role of the cholinergic systems of the dorsal and ventral striatum of the rat brain in controlling learned movements.

Comparative studies were performed of the effects of injections of a cholinergic agonist (carbachol) and antagonist (scopolamine) into the ventral and dorsal striatum on the performance of a learned movement involving prolonged maintenance of extension of the forelimb in rats. Doses of carbachol (0.03-3.00 micrograms) into the ventral striatum were accompanied by increases in the numbers of movements with prolonged maintenance of extension with application of pressure against an obstacle, with a simultaneous decrease in the percentage of rapid nonreinforced movements (by an average of 18.8%). Injections into the dorsal striatum disrupted slow movements which were not reinforced during training, on a background of stable performance of the learned reflex. Doses of scopolamine (0.3-3.0 micrograms) into both the dorsal and ventral parts of the striatum produced increases (by 22.7 +/- 8.2% and 68.9 +/- 14.3%) in the numbers of rapid nonreinforced movements typical of the repertoire of untrained animals. These data led to the suggestion that the cholinergic system of the ventral striatum is involved in the maintenance of forelimb muscle tone in rats during the performance of movements in which pressure is applied to an obstacle. The cholinergic system of the dorsal striatum does not have this property, but plays a significant role in the process of learning new sensory-controlled movements.

Activity of adenylate cyclase and 5'-nucleotidase in the sensorimotor and limbic structures of the brain in rats after manipulatory training.

Adenylate cyclase (AC) and 5'-nucleotidase (NT) activities were measured in the limbic (amygdala, hippocampus) and sensorimotor (cortex, striatum) structures of the brain in three groups of rats: untrained rats and rats which were good and poor learners in training to perform movements involving pushing against an obstruction. After training, AC activity decreased in all structures studied. Activity decreased in the cortex and striatum to a greater extent in good learners, and in the amygdala in poor learners. NT activity decreased in all brain structures apart from the striatum, to a greater extent in rats which were less able to learn to produce movements involving prolonged pushing. The striatum was the only structure in which increases in NT activity occurred, from the lowest initial level in the control group. 1.0 +/- 0.04 microgram P(i)/mg protein/min, to 1.3 +/- 0.1 micrograms P(i)/mg protein/min in poor learners and to 2.0 +/- 0.1 micrograms P(i)/mg protein/min in good learners. Interhemisphere asymmetries in AC activity in the cortex and hippocampus were seen, along with an interhemisphere difference in NT activity in the amygdala. Thus, the activity of enzymes involved in adenine and cAMP biosynthesis changed in different ways in the limbic and sensorimotor structures of the brain, depending on the ability of rats to learn. The increase in NT activity after training of rats, which was limited to the striatum, may reflect a special role for the purinergic system in these structures in mediating sensation-regulated movements.

Sensory and motoric components of various forms of learned movement with change in the activity of the cholinergic system of the neostriatum.

The participation of the cholinergic system of the neostriatum in the regulation of sensorily-monitored movements and the differentiation of sensory signals were investigated in 12 Wistar rats, 27 Sprague-Dawley rats, and 6 mongrel dogs, using the following models: 1) the maintenance of learned extension of the forelimb for a specified time (rats); 2) prolonged conditioned reflex flexion of the hind limb (dogs); and 3) active avoidance in a T-maze (rats). It was demonstrated that the injection of carbacholine (0.03 microgram) into the dorsolateral division of the caudate nucleus of the rats does not bring about significant changes in the performance of movements associated with the maintenance of tonus of the forelimb, whereas the injection of carbacholine (0.05-0.1 microgram) into the same division of the caudate nucleus of the dogs improves the realization of movement associated with the maintenance of tonus of the hind limbs. The injection of a blocker of the cholinergic system (scopolamine in the rats and atropine in the dogs) in both the first and the second instance disturbed the performance of the movement by the animals. Bilateral microinjections of carbacholine (0.03 microgram) into the neostriatum of the rats significantly improves the development of a differentiated active avoidance conditioned reflex in a T-maze on the first three days of testing. The differentiation of acoustic signals by dogs also significantly improved against the background of the injection of carbacholine (0.05-0.1 microgram) into the caudate nucleus. Thus, the data obtained in the various behavioral models and different animals suggest that the cholinergic system of the neostriatum participates in the regulation of both motor and sensory mechanisms in connection with the realization of learned movement.

Role of the striatal cholinergic system in the regulation of learned manipulation in rats.

The experiments were performed on adult Wistar male rats trained to push with the forepaw on a fixed piston inside a narrow tube. It was found that after localized intracerebral injection of a cholinergic antagonist into the dorso-lateral (but not medial) neostriatum (i.e., the caudato-putamen) the behavioral performance requiring brief innate movements remained unchanged, but the performance requiring a prolonged pushing movement (> 50 msec) became disrupted. Micoinjection of carbacholine (0.03-3 mu g/l microliters) did not affect the performance of the acquired movements, whereas scopolamine (3 mu g/l microliters) led to the significant decrease in pushing time. We conclude that changes in the state of the dorso-lateral neostriatal cholinergic system result only in disturbances of the sensory-controlled component of a complex instrumental movement.

Neurochemical characteristics of the rat neostriatum and motor cortex after the development of a unilateral manipulatory reflex.

Indicators of the activity of acetylcholinesterase (ACE), 5'-nucleotidase (NT), adenylate cyclase (AC) in the sensorimotor cortex and the neostriatum (NS) of the right and left cerebral hemispheres of control rats and rats trained to perform a food-procuring movement by pressing against an obstacle with the forelimb. An identical level of the averaged bilateral values of the activity of NT and AC in both of the structures in question and an increased ACE activity in the NS were found in the control animals. After the development of a manipulatory skill, the activity of AC decreased in the cortex and the NS in the presence of unchanged ACE activity, while NT activity decreased in the cortex and increased in the NS. The bilateral values of the activity of the enzymes differed significantly in well and poorly trained rats. At the same time, the activity of the enzymes was similar in character in the dominant and subdominant hemispheres for each group of animals. Overall the neurochemical changes obtained can be regarded as specific correlates of the developed unilateral manipulatory reactions that are characteristic for the structures in question of both cerebral hemispheres.