I. A. Zhuravin

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.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.

Epigenetic and pharmacological regulation of the amyloid-degrading enzyme neprilysin results in modulation of cognitive functions in mammals

145 The present work reports an experimental testing of a hypothesis that epigenetic and pharmacological reg ulation of the activity of amyloid degrading enzyme neprilysin (NEP) results in changes in the plasticity of the nervous system and affects cognitive functions. It was found that prenatal hypoxia or prolonged admin istration to rats of a neprilysin inhibitor phosphorami don led to a decrease in the number of labile spines in the cortical regions of the brain and to deterioration of their short term working memory tested in a radial maze. On the contrary, injections to rats with reduced NEP activity (after prenatal hypoxia) of an inhibitor of histone deacetylase, sodium valproate, led to an enhanced NEP activity, increased number of labile spines and improved memory. The data obtained allowed us to conclude that the decrease in the activity of amyloid degrading enzymes, in particular of NEP, is accompanied by a reduced number of labile intra neuronal contacts which might be one of the reasons of cognitive decline caused by pathological develop ment or ageing. This decline can be compensated by regulating expression and activity of amyloid degrad ing enzymes.

[Changes in the activity of amyloid-degrading metallopeptidases leads to disruption of memory in rats].

In old male Wistar rats (older than 12 months), or adult males (3–4 months) subjected to prenatal hypoxia (7% O2, 3 h, E14), a disruption of short-term memory was observed. Prenatal hypoxia also led to a decrease in the brain cortex of the levels of expression of the metallopeptidases neprilysin (NEP) and endothelinconverting enzyme (ECE-1) which regulate some neuropeptides and are the main amyloid–beta (Αβ)-degrading enzymes. Moreover we have demonstrated a significant decrease (by 2.7 times) of NEP activity in the sensorimotor cortex of old rats and of adult rats subjected to prenatal hypoxia (by 1.7 times). To confirm possible involvement of these enzymes in memory we have performed an analysis of the effect of microinjections of phosphoramidon – an inhibitor of NEP and ECE-1, and thiorphan – an inhibitor of NEP – into the rat sensorimotor cortex. Using a two-level radial maze test, disruption of short-term memory was observed 60 and 120 min after i.c. injections of phosphoramidon (10–2 M) and 30 and 60 min after i.c. injections of thiorphan (10–2 M). Thus, involvement of NEP and ECE-1 in short-term memory observed in this study allows us to suggest that one of the main factors in disruption of cognitive functions after prenatal hypoxia or in the process of ageing could be a decrease in the level of expression and activity of metallopeptidases participating in metabolism of Αβ and other neuropeptides.

Effect of hypoxia on cholinesterase activity in rat sensorimotor cortex

This study reports the dynamics of changes in postnatal ontogenesis of the activity of soluble and membrane-bound forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the sensorimotor cortex of rats as well as the character of their changes after prenatal hypoxia (E14, 7% O2, 3 hours) or acute hypoxia in adult animals (4 months, 7% O2, 3 hours). In normally developing rats the activity of the membrane-bound form of AChE in the sensorimotor cortex gradually increased up to the end of the first month after birth and remained at this high level during all further postnatal ontogenesis while the activity of the soluble form of the enzyme reached its maximum value on the 10th day after birth and decreased significantly by the end of the first month. In animals subjected to prenatal hypoxia the activity both of the soluble and membrane bound forms of AChE during the first two weeks after birth was 20–25% lower compared to controls but increased by the end of the first month and even exceeded the control values and remained increased up to old age (1.5 years). The activity of both forms of BChE in rat sensorimotor cortex at all stages of postnatal ontogenesis was significantly lower than of AChE although the dynamics of their changes was similar to AChE. Prenatal hypoxia led to a decrease in the activity of the membrane-bound form of BChE compared to controls practically at all studied stages of development but was higher at the end of the first month after birth. At the same time, the activity of the soluble form of BChE was decreased only on the 20th day of development compared to the control but increased starting from the end of the first month of life and further. Acute hypoxia in adult rats also led to a decrease in the activity of both forms of AChE and BChE in the sensorimotor cortex but the dynamics of these changes was different. Thus, insufficient oxygen supply to the nervous tissue at different stages of ontogenesis has a significant effect on the activity and ratio of various forms of cholinesterases possessing either growth factor or mediator properties which might lead to the changes in brain development and formation of behavioural reactions including learning and memory as well as increase the risk of development of the sporadic form of Alzheimer’s disease (AD)—one of the most common neurodegenerative diseases of advanced age. This study widening our understanding of the properties of brain cholinesterases under normal and pathological conditions might be useful for developing new approaches towards prevention and treatment of AD.

Morphofunctional changes in field CA1 of the rat hippocampus after pentylenetetrazole and lithium-pilocarpine induced seizures

Animal models of seizures and epilepsy are very diverse and instrumental for elucidating the mechanisms that underlie convulsive states and epileptogenesis. A single injection of pentylenetetrazole (PTZ) induces seizures, however, does not raise the risk of further development of epilepsy. Pilocarpine, immediately after injection, evokes epileptical state and, following a latent period, results in the development of spontaneous seizures, i.e. the drug triggers epileptogenesis. Assuming that in the PTZ model morphofunctional changes are mainly transient, while changes in the lithium-pilocarpine (PC) model may indicate the brain epileptization, we set ourselves the task of comparing morphological and functional characteristics of the hippocampal field CA1 in control and two experimental animal groups in 24 h after injection of the convulsants. We revealed the changes specific to the PC model and indicating neurodegeneration: a decrease in the cell spacing density, a diminution in the number of the viable NeuN-expressing neurons, an increased activity of the proapoptotic protease caspase-3. A characteristic feature of the PTZ model was the appearance of hyperchromic neurons with normal viability. In both models, the expression of the excitatory amino acid carrier EAAT1 increased by about 40% as compared to control. These morphofucntional correlates of reversible changes in the nervous tissue caused by seizures, as well as the early disorders leading to long-term brain epileptization can be used as indicators allowing assessment of a therapeutic potential of novel anticonvulsive drugs.

Adenylate cyclase system of the rat striatum: regulatory properties and the effects of gangliosides.

The activity and regulatory properties of the adenylate cyclase system of the rat striatum were studied. Agents such as Gpp(NH)p, forskolin, and NaF were found to show classicalin vitro stimulation of adenylate cyclase activity in the striatum membrane fraction. Dosage of rats with a preparation containing gangliosides (30 mg/kg, 6 i.p. injections) led to prolonged reductions in basal striatal adenylate cyclase activity. Two weeks after ganglioside administration, enzyme activity was reduced by 48% and remained lower than in controls (by 18%) even a month after injections.In vitro studies of the effects of gangliosides on adenylate cyclase activity in whole brain and striantal synaptosomes from rats showed that gangliosides have modulating actions on the adenylate cyclase system of the striatum, mainly due to changes in the catalytic function of the enzyme itself, and have no significant effect on the GTP-binding center of G-protein.

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.

Early morphological and functional changes in the GABAergic system of hippocampus in the rat lithium-pilocarpine model of epilepsy

We studied early alterations in the GABAergic system of the rat hippocampus in the lithium–pilocarpine model of epilepsy. Twenty-four hours after the pilocarpine treatment, a decrease in the number of calretinin-positive interneurons was observed in the CA1 field of the hippocampus, whereas the number of parvalbumin-positive interneurons remained unchanged. The decreased levels of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD67) and the membrane GABA transporter GAT1 were revealed using Western blot analysis. These data indicate an altered excitation/inhibition balance in the hippocampus with excitation dominance.

The Ability of NMDA-Type Glutamate Receptor Blockers to Prevent the Development of Pentylenetetrazole Kindling and Morphological Changes to Pyramidal Neurons in the Mouse Hippocampus

Experiments on mice addressed the link between convulsive syndrome and morphological changes in hippocampal neurons occurring on development of pentylenetetrazole (PTZ) kindling. Kindling was induced by i.p. PTZ (35 mg/kg) three times a week for one month. By the end of this period, 70% of the mice responded to administration of PTZ with severe clonic or clonic-tonic seizures. Hippocampal sections (stratum pyramidale, field CA1, Nissl staining) from convulsive mice showed large numbers of altered cells (24.7 ± 2.1%). Most of these were pyramidal neurons. These hyperchromic neurons had reduced body sizes, loss of turgor, wrinkling of the cell body, and deformation of dendritic processes. These dark-type changes were present in 2.3 ± 2.1% of neurons in the hippocampus of intact mice and mice resistant to the convulsogenic effect of PTZ (30% of the population). Immunohistochemical studies demonstrated normal expression of NeuN (Fox3) protein in all hippocampal cells, including dark hyperchromic neurons. This is evidence that neurons did not die en masse and were relatively viable. Prophylactic s.c. administration of NMDA receptor blockers (0.5 mg/kg memantine, 0.1 mg/kg IEM-1921, or 1 mg/kg IEM-1958) decreased the proportion of mice developing PTZ kindling from 70% to 40%. The proportion of altered neurons in the 60% of mice given NMDA blockers and not developing PTZ kindling or convulsions in the presence of blockers was 0.1 ± 0.06%, which was the same as in intact mice. Conversely, the hippocampus of mice demonstrating convulsions despite simultaneous administration of NMDA blockers showed 24.0 ± 5.6% hyperchromic neurons. These results provide evidence that pathologically altered neurons appeared after convulsive seizures in animals after PTZ kindling and that blockade of NMDA glutamate receptors could weaken both the development of convulsive syndrome and the concomitant morphological changes to hippocampal neurons.

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.