N. L. Tumanova

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.

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.

Structural changes in the neocortex nervous tissue in rat ontogenesis after hypoxia at various terms of embryogenesis

The performed study has shown that in rats submitted to hypoxia (3 h, 7% O2) at the 14th day of embryogenesis (E14) as compared with control animals, density of distribution of cells in the brain cortex decreased for the first month of postnatal ontogenesis (maximally by 40.8% by P20). In dying neurons, swelling of the cell body, lyses of or ganoids, and disturbance of the cytoplasm membrane intactness were observed. Two waves of neuronal death by the mechanism of capsize-dependent apoptosis were revealed; the first involved large pyramidal neurons of the layer V (P10–20), the second-small pyramidal and non-pyramidal neurons of the layers II–III (P20–30). In neurosis of molecular layer, a decrease of the mean amount of labile synaptopodin-positive dendrite spines was observed, as compared with control. In rats exposed to hypoxia at E18, no changes of cell composition and structure of the nervous tissue were found in the studied brain cortex areas. Thus, formation of the cortex nervous tissue in postnatal ontogenesis of rats submitted to hypoxia at the period of neuroblast proliferation-migration is accompanied not only by a change of the cell composition of various cortex layers in early ontogenesis, but also by a decrease of the number of the synaptopodin-positive spines in the molecular layer, the decrease being preserved in adult animals.

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.

Role of caspase-3 in regulation of the amyloid-degrading neuropeptidase neprilysin level in the rat cortex after hypoxia

Analysis of the effect of a caspase-3 inhibitor on the level of amyloid-degrading neuropeptidase neprilysin (NEP) in the cortex of rats exposed to prenatal hypoxia (7% О2, 3 h) on day 14 of embryonic development (E14) was performed. It was found that rats exposed to prenatal hypoxia on days 20–30 after birth had an increased level and activity of caspase-3 with reduced levels of NEP and the C-terminal fragment of the amyloid precursor protein intracellular domain (AICD) which regulates NEP expression. In hypoxic animals 3 days after a single intraventricular injection of a caspase inhibitor (Ac-DEVD-CHO) on P20, the AICD and NEP levels were found to be increased up to the levels observed in control rats. The results suggest that the increase in caspase-3 enzyme activity could affect NEP expression via proteolytic degradation of its transcription factor AICD. These findings represent the first demonstration of the role of caspases in AICD-dependent regulation of NEP production in the brain of mammals under hypoxic conditions.

Formation of striatum structural and ultrastructural organization in rat postnatal ontogenesis at changes of conditions of their embryonic development

Using light microscopy (Nissl and Golgi techniques), electron microscopy and immunohistochemistry, formation of structure of the brain striatum dorsolateral part from birth to three month of age was studied in rats submitted to acute hypoxia at the period of embryogenesis. Hypoxia at the 13.5th day of pregnancy (E 13.5) was found to lead to a delay of neuronogenesis for the first two weeks of postnatal development as compared with control animals, and the majority of large neurons for this period were degenerated by the type of chromatolysis with swelling of the cell body and processes and lysis of cytoplasmic organelles. By the end of the third week, shrunken hyperchromic or pycnomorphic neurons with the electron-dense cytoplasm and enlarged tubules of endoplasmic reticulum and Golgi complex were also observed. An increased number of swollen processes of glial cells was found in neuropil around the degenerating neurons. By the 30th day as well as in adult animals, destruction of mitochondrial apparatus, an increased number of lysosomes, and blade-shaped nuclei, which are characteristics of the apoptotic cell death, were observed. This is also confirmed by an increased expression of proapoptotic protein (p53) and its co-localization with caspase-3 in a part of neurons. Morphometric analysis showed a decrease of the cell distribution density in striatum and a change of ratio of different cell types in hypoxia-exposed rats as compared with control group. The most pronounced decrease (42.3% at the 5th day, 14.2% at the 10th day, p < 0.01) of the number of large neurons (larger than 80 μm2) was revealed at early stages of postnatal ontogenesis. After 3 postnatal weeks, the number of middle-sized neurons (30–95 μm2) decreased (by 11.8–19.2% as compared with control, p < 0.05). The obtained data have shown that changes of embryogenesis conditions (hypoxia) at the period of the most intensive proliferation of telencephalon neuroblasts lead to impairment of the process of striatal nervous tissue formation. This might be the cause of delay of development and disturbances of behavior and learning, which are observed in rats exposed to prenatal hypoxia.

Disturbances in Formation of the New and Old Cortex at Changes of Conditions of Embryonic Development

Using a model of acute hypoxia during pregnancy of rats, changes in the development of old (hippocampus) and new (sensorimotor) cortex associated with disturbance of neuronogenesis have been revealed in the studied brain structures at the period of action of a pathological factor. It was found that in rats submitted to hypoxia at the 13–14th days of embryogenesis, the number of degenerating neurons (including the pyramidal ones) at various levels of chromatolysis increased since the 5th day after birth; the increase was present for the entire first month of postnatal development. In the cortex of rat pups submitted to prenatal hypoxia there were observed deformation of neuronal bodies, vacuoles in the cytoplasm, shrinkage of apical dendrites of pyramidal neurons and delayed development of the structure (time of the appearance of spikes, formation of structural elements and the size of the cells) of the nervous tissue of the brain of the rat pups exposed to prenatal hypoxia. The columnar structure of the cortex was disturbed. In hippocampus, the process of degeneration of neurons started by 2–3 days later than in the cortex; by two weeks of postnatal development a massive degeneration and death of a part of neurons were also revealed. The morphometrical analysis showed a decrease in the number of neurons and their total area in the sensorimotor cortex (the layer V) and an increase in the number of glial elements at the 10–17th days after birth. In the hippocampus a decrease in the area occupied by neurons and in their size was detected in adult animals. The adult rats submitted to prenatal hypoxia were found to have disturbances of memory and learning. A correlation was shown between the disturbances of the conditions of embryonic development and the changes in the ability of learning and storage of new skills in the offspring.

Role of caspase-3 in development of neuronal plasticity and memory

Caspases are known to play an important role in apoptosis and their increased activity in pre- and postsynaptic terminals leads to proteolysis of synapse-associated proteins, resulting in disruption of synaptic functions. Moreover, caspases degrade the C-terminal fragment of amyloid-precursor protein intracellular domain (AICD) which regulates expression of a variety of genes including an amyloid-degrading enzyme neprilysin (NEP). As such, inhibition of caspases is considered as a tool for prevention and compensation of various synaptic pathologies leading to cognitive deficit and Alzheimer’s disease pathogenesis. In this study we have evaluated the role of prenatal hypoxia on the activity of caspases and neuronal network characteristics in rat brain and the effect of caspase inhibitors on these parameters. We have found that the brain of rats subjected to prenatal hypoxia (Е14, О2 7%, 3 h) is characterised by an increased number of caspase-3-positive neurones and higher activity of this enzyme in the neocortex and hippocampus in the period of intensive synaptogenesis (Р20-30) compared to controls. Subsequently, in later life these animals had a reduced number of synaptopodin-positive dendritic spines and reduced activity of NEP accompanied by disruption of cognitive functions. Single i.v. injection of caspase-3 inhibitors (Ac-DEVD-CHO) to hypoxic rats on Р18-23 led to a decrease in caspase-3 activity and increased NEP expression. In these animals we have also observed restoration of synaptopodin levels and distribution of the labile synaptic spines in the neocortex and hippocampus which were accompanied by improved memory. The effects of inhibitors on memory was observed within one month after administration but not detected 2.5 months later. These data testify to the involvement of caspase-3 in normal brain development and indicate an important role of this enzyme in neuronal plasticity and regulation of cognitive functions. Supported by RFBR (13-04-00388), ARUK.

Study of distribution of the spine apparatus protein synaptopodin in cortical brain parts of rats exposed to hypoxia at different periods of embryogenesis

A comparative study of the nervous tissue and distribution of the spine apparatus protein synaptopodin was performed in all layers of the brain sensorimotor cortex and hippocampal CAl area in control rats and in the rats exposed to hypoxia at E14 and E18. It was found that beginning from the 20th day of postnatal development, a statistically significant decrease of the mean number of labile synaptopodin-positive spines in the stratum radiatum moleculare of the hippocampal area CAl was observed in rats exposed to hypoxia both at E14 and E18. The decrease of the number of labile spines in the sensorimotor brain cortex was revealed only in the I layer beginning from the 20th day after birth in the rats exposed to hypoxia at E14. Maximal differences in the studied brain areas were observed in adult rats exposed to hypoxia at E14 in the neocortex—a decrease by 23 ± 10%, in hippocampus—by 24 ± 8%, respectively. However, no increased degeneration of neurons was detected in adult animals. It is suggested that disturbances in cognitive functions and in the capability for learning observed in rats after prenatal hypoxia can be due to a decrease of the amount of the labile synaptopodin-positive spines, which leads to a change of the structural-functional properties of neuronal networks and to a decrease of their plasticity.