Involvement of the NO synthase system in stress-mediated brain reactions.

The neurosis-like state developing as a result of chronic stress in animals and accompanied by transient cerebral hypoxia can lead to significant impairments to many brain structures. The effects of the humoral components of the stress reaction on the brain are mediated by both extra- and intracellular signal pathways, among which nitric oxide (NO) is of great importance. We report here immunohistochemical studies of the expression of the constitutive neuronal (nNOS) and inducible (iNOS) isoforms of NO synthase in neurons in the brains of white rats in conditions of chronic stress leading to the development of a neurosis- like state. Chronic stress was found to induce increases in nNOS and iNOS expression in many parts of the brain, predominantly in the neocortex and hippocampus. Administration of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) led to increases in the animals' depression, which was accompanied by reductions in motor and investigative activity assessed using traditional tests. This NOS inhibitor produced a minor increase in the expression of iNOS only. Thus, NO was shown to be involved in mediating the effects of stress with development of a neurosis-like state.

[Participation of NO-synthase system in the stress-mediated reactions of the brain].

Neurosis-like status developing as a result of the exposure of animals to chronic stress, which is associated with a transitory cerebral hypoxia, could cause significant structural and functional alterations in many brain structures. Realization of humoral stress effects on the brain is mediated by both extra- and intracelullar signal molecules, among which nitric oxide (NO) is considered to be one of the most potent ones. Expression of neuronal constitutive (nNOS) and inducible (iNOS) isoforms of NO-synthase was studied by immunohistochemistry in the neurons of albino rat brain after exposure of animals to chronic stress resulting in the development of neurosis-like status. Chronic stress was shown to result in the increased expression of both nNOS and iNOS in many brain areas with the predominance in neocortex and hippocampus. The administration of nonspecific inhibitor of NOS, Nomega-nitro-1-arginine methyl ester hydrochloride (L-NAME) (10 mg/kg) resulted in the aggravated depression of the animals, associated with a decrease of locomotor and exploring activities that were evaluated using the traditional tests. The application of NOS activity inhibitor caused an insignificant rise only in iNOS expression. Thus the results obtained suggest that NO is involved in the realization of stress effects with the development of a neurosis-like status.

Studies of damage to hippocampal neurons in inbred mouse lines in models of epilepsy using kainic acid and pilocarpine.

Identification of the mechanisms of damage to neurons is an important task in contemporary neuroscience and is of enormous importance in medicine. This report compares two models of neuron damage due to hyperexcitation induced by kainic acid and pilocarpine, using two lines of mice, C57BL/6J and FVB/NJ. Neuron damage was more marked in FVB mice, though lethality was greater in C57BL mice. The levels of convulsive activity were not significantly different. Kainic acid had greater tropism for the hippocampus than pilocarpine. Hsp-70 and Egr-1 expression was not significantly different in C57BL and FVB mice. Analysis of the isolated mitochondrial fraction showed that free radical production was different in these mouse lines; this may be one of the reasons for the differential resistance of neurons to hyperexcitation.

Nestin-expressing cells in the human hippocampus.

Nestin, a protein of the intermediate filament family, is typical of undifferentiated neural stem and progenitor cells. The present report describes studies of nestin expression in the hippocampus of patients with epilepsy and identifies five types of nestin-immunopositive cells differing in terms of their morphological phenotype and immunological characteristics. These were cells with the phenotype of radial glial cells, bipolar cells, small dendritic cells, cells of the subependymal zone, and astrocyte-like cells. Two types of cell - radial gliocytes of the dentate fascia and NG2-immunopositive bipolar cells - can be regarded as neural precursors of different levels of commitment.

[Neuronal damage in the hippocampus of inbred mouse strains in the models of epilepsy induced by kainic acid and pilocarpine]. / Issledovanie povrezhdenii neironov hippokampa v inbrednykh liniiakh myshei v modeliakh épilepsii s ispol'zovaniem kainovoi kisloty i pilokarpina.

Elucidation of the mechanisms of neuronal damage is an important task of modem neuroscience and is of paramount importance for medicine. Present work compares two models of excitotoxic neuronal damage induced by kainic acid and pilocarpine, in which inbred C57BL/6J (C57BL) and FVB/NJ (FVB) mice were used. Both models produced higher neuronal damage in FVB although mortality was higher in C57BL. No significant differences between two strains of mice were found in seizures severity. Kainic acid demonstrated greater tropism to hippocampus in comparison with pilocarpine. Hsp-70 and Egr-1 expression was not significantly different in C57BL and FVB. Analysis of the isolated mitochondrial fraction has shown different degree of free radical production in the strains studied, that could be one of the reasons for unequal susceptibility of their neurons to excitotoxic cell death.

[Nestin-expressing cells in the human hippocampus]. / Nestin-ékspressiruiushchie kletki v gippokampe cheloveka.

Nestin is a protein that belongs to a family of intermediate filament proteins which are typical for undifferentiated neural stem and progenitor cells. In this work nestin expression was studied in the hippocampus obtained from patients with epilepsy. Immunohistochemical investigation demonstrated five types of nestin-positive cells, differing in morphological and immunological phenotype. These included cell with a radial glia phenotype, bipolar cells, small dendritic cells, subependymal and astrocyte-like cells. Two types of these cells: radial glia of dentate gyrus and bipolar NG2+ cells can be considered as neural progenitor cells possessing different degrees of commitment.

Mouse strain differences in kainic acid sensitivity, seizure behavior, mortality, and hippocampal pathology.

Genetic influences contribute to susceptibility to seizures and to excitotoxic injury, but it is unclear if/how these susceptibilities are linked. This study assessed the impact of genetic background on mouse strain seizure susceptibility, seizure phenotype, mortality, and hippocampal histopathology. A subcutaneous (s.c.) kainic acid multiple injection protocol was developed. Five mouse strains were tested: a and b) C57BL/6J and 129/SvJ, strains commonly used in gene targeting experiments; c) C3HeB/FeJ, a strain with reported sensitivity to the convulsant effects of kainic acid (KA); d) 129/SvEms, a strain reportedly susceptible to hippocampal excitotoxic cell death; and e) a mixed genetic background strain (129/SvJXC57BL/6J) from which targeted gene deletion experiments have been carried out. Histopathological features were examined at early (7-10 day), delayed (2-4 month), and late (6-13 month) time points.Mouse background strains can be genetically segregated based on excitotoxin sensitivity, seizure phenotype, mortality, and hippocampal histopathology. When injected with KA, C3HeB/FeJ and C57BL/6J strains were resistant to cell death and synaptic reorganization despite severe behavioral seizures, while 129/SvEms mice developed marked pyramidal cell loss and mossy fiber sprouting despite limited seizure activity. The mixed background 129/SvJXC57BL/6J group exhibited features of both parental strains. In the mouse strains tested, the duration or severity of seizure activity was not predictive of subsequent hippocampal pyramidal cell death and/or synaptic reorganization. Unlike rats, mice exhibiting prolonged high-grade KA-induced seizure activity did not develop subsequent spontaneous behavioral seizures.

[Neurogenesis in the adult mammalian brain]. / Neirogenez v golovnom mozgu zrelykh mlekopitaiushchikh.

The concept of the CNS cell composition stability has recently undergone significant changes. It was earlier believed that neurogenesis in the mammalian CNS took place only during embryonic and early postnatal development. New approaches make it possible to obtain new results overriding the dogma that neurogenesis is impossible in the adult brain. The present review summarizes the information about the neural stem cell. It has been demonstrated that new neurons are constantly formed in adult mammals, including man. In two brain zones, subventricular zone and denate gyrus, neurogenesis appears proceed throughout the entire life of mammals, including man. The newly arising neurons are essential for some important processes, such as memory and learning. Stem cells were found in the subependymal and/or ependymal layer. They express nestin, and have a low mitotic activity. During embryogenesis, the stem cell divides asymmetrically: one daughter cell resides as the stem cell in the ependymal layer and another migrates to the subventricular zone. There it gives rise very fast to a pool of dividing precursors, from which neural and glial cells differentiate and migrate to the sites of final localization. The epidermal and fibroblast growth factors act as mitogens for the neural stem cell. The neural stem cell gives rise to the cells of all germ layers in vitro and has a wide potential for differentiation in the adult organism. Hence, it can be used as a source of various cell types of the nervous tissue necessary for cellular transplantation therapy.

[Neural stem cells in the brain]. / Stvolovaia nervnaia kletka mozga.

Stem cells in the central nervous system were usually considered as relevant for evaluation only in embryonic time. Recent advances in molecular cloning and immunological identification of the different cell types prove the presence of neurogenesis of the new neurons in adult mammals brains. New neurons are born in two areas of the mammal and human brain--sybventricular zone and subgranular zone of dentate gyrus. New born granular neurons of dentate gyrus have a great importance for memory and learning. New neurons originate from precursors which in culture and in situ could also transform into astrocytes and oligodendrocytes, thus fulfill criteria of neural stem cells. In culture, mitotic activity of these stem sells depends on fibroblast growth factor 2 and epidermal growth factor. Depletion of cultural medium of these factors and addition of serum, other growth factors (Platelet-derived growth factor and ciliary neurotrophic factor) leads to generation of neurons and astrocytes. Isolation and clonal analysis of stem cells is based on immunological markers such as nestin, beta-tubulin III, some types of membrane glicoproteids. Identification and visualization of stem cells in brain revealed two populations of cells which have properties of stem cells. In embryonic time, radial glia cells could give origin to neurons, in mature brain cells expressing glial fibrillar acidic protein typical marker of astrocytes fulfill criteria for stem cells. Neural stem cells could transform not only into mature neurons and glial cells but also into blood cells, thus revealing broad spectrum of progenitors from different embryonic tissues. Further progress in this field of neurobiology could give prosperity in the cell therapy of many brain diseases.

Expression of adenylyl cyclase type IX and calcineurin in synapses of the central nervous system.

Distribution of type IX adenylyl cyclase and protein phosphatase calcineurin in the brain and in cultured hippocampal neurons from albino rat was immunohistochemically studied. Both enzymes were detected simultaneously in all synaptic structures of most cerebral neurons except for presynaptic sites, where calcium-inhibited type IX adenylyl cyclase was absent.

Characterisation of human adenylyl cyclase IX reveals inhibition by Ca(2+)/Calcineurin and differential mRNA plyadenylation.

The functional diversity of adenylyl cyclases provides for different modes of cyclic AMP signalling in mammals. This study reports the cloning and functional characterisation of a cDNA encoding human adenylyl cyclase IX (ACIX). The data show that human ACIX is a Ca(2+)/calcineurin-inhibited adenylyl cyclase prominently expressed in vital organs, including brain, heart, and pancreas. ACIX mRNA was detected in several brain regions, including neocortex, hippocampus, striatum, and cerebellum. By in situ hybridisation, ACIX mRNA was localised to pyramidal and granule cells of the hippocampus, indicating that it is expressed predominantly in nerve cells. Further analysis of ACIX mRNA expression revealed two major forms of ACIX mRNA that arose through tissue-specific differential mRNA polyadenylation. Taken together, the data show that (a) human ACIX is under inhibitory control by Ca(2+) through calcineurin, (b) ACIX may be involved in higher brain functions, and (c) post-transcriptional regulation of ACIX gene expression is a species-specific control mechanism that may enhance the versatility of cyclic AMP signalling in humans.

Neuropeptide Y-immunoreactive intracardiac neurones, granule-containing cells and nerves associated with ganglia and blood vessels in rat and guinea-pig heart.

Intrinsic neuropeptide Y-containing neurones in rat and guinea-pig hearts were studied at the ultrastructural level by the pre-embedding peroxidase-antiperoxidase immunocytochemical technique. Intracardiac neuronal cell bodies were often weakly or moderately immunostained, and the labelling was usually pronounced in the Golgi complex, multivesicular bodies, some cisterns of granular endoplasmic reticulum and large granular vesicles. Neuropeptide Y-immunoreactive nerve fibres were also observed in association with intracardiac neurones. A subpopulation of neuropeptide Y-immunoreactive granule-containing cells in the rat heart are described for the first time and were very heavily labelled; other granule-containing cells were non-immunoreactive, but were contacted by neuropeptide Y-containing nerves. Preterminal regions of nerve fibres that were located in nerve bundles were only weakly neuropeptide Y-immunoreactive, in contrast to the heavy labelling observed in varicosities that contained many synaptic vesicles. Many neuropeptide Y-immunoreactive nerve fibres were associated with the coronary vasculature and were particularly prominent in the walls of small arteries and arterioles where labelled nerve varicosities were present close to the smooth muscle cells. Immunoreactive nerves were also seen in the myocardium, usually near to capillaries. In axonal varicosities, the central core of large granular vesicles was immunolabelled, and electron-dense immunoreactive material outlined the membranes of small and large clear vesicles. The significance of neuropeptide Y-immunoreactive intracardiac neurones and granule-containing cells and the origin of associated labelled nerve fibres in the heart are discussed.

[Age-related changes in the autonomic ganglia]. / Vozrastnye izmeneniia vegetativnykh gangliev.

Ultrastructure of vegetative ganglions (neck-thoracic, intracardial and intestinal) of Wistar and SHR rats, 26-28 months of age was studied electron-microscopically. The most pronounced changes were found in the neck-thoracic ganglions where, apart from lipofuscin deposits, lamellar bodies were frequently found. Redistribution of neuromediators in the neuron body and an increase of the neuroactive substances release into the intercellular space of the ganglion occur with age. The data on possible postsynaptic influence on the presynapse by means of neuromediators are presented. Nissl bodies hypertrophy was observed in the neck-thoracic and intracardial ganglions of old SHR rats.

[Interneuronal interrelationships in autonomic ganglia (the ultrastructural aspects)]. / Mezhneironnye vzaimootnosheniia v vegetativnykh gangliiakh (ul'trastrukturnye aspekty).

Ultrastructural and immunocytochemical results of studies on interneuronal relationship in vegetative ganglia were represented. Age-related changes of synapses (appearance of synaptic type vesicles and smooth endoplasmic reticulum cisterns in postsynaptic terminals), peculiarities of interneuronal links in neurons containing neuropeptide Y and NO-synthase and structural manifestations of non-synaptic interneuronal links were shown. Modern views on interneuronal communications were discussed.

[Ultrastructure of the sympathetic ganglia in hypoxia]. / Ul'trastruktura simpaticheskikh gangliev pri gipoksii.

Short-term hypoxia produces ultrastructural alterations in ganglions of rats in the form of dilatation of some membrane neuron compartments and sinuous plasmatic membrane. More long-term hypoxia produces structural changes of membrane cell components. An increase of lysosome number and lipofuscin granules alternations in chronic hypoxia (1 year) are similar to the age changes and are characterized by intensive accumulation of lipofuscin and Nissl bodies hypertrophy.

[Nitric oxide synthase in neuroepithelial brain tumors]. / Sintaza okisi azota v neiroépitelial'nykh opukholiakh golovnogo mozga.

Immunohistochemical investigation of NO-synthase in brain astrocytic tumors revealed intense reaction in many tumor cells as well as direct correlation in the intensity of reaction and the degree of tumor anaplasia. Grade I astrocytomas did not show immunoreactivity in contrast to high anaplastic tumors where many cells had positive reaction with a different degree of intensity. Positive immunoreaction was shown in many giant cells. Small cell glioblastomas and oligodendrogliomas were immunonegative. There was a direct correlation between NO-synthase expression and glial fibrillar acidic protein.

Nitric oxide synthase-containing neurones and nerve fibres within cardiac ganglia of rat and guinea-pig: an electron-microscopic immunocytochemical study.

The nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity of intracardiac neurones in the rat and guinea-pig was studied at the ultrastructural level. While some nitric oxide synthase-containing intracardiac neurones were very heavily labelled, with electron-dense immunoprecipitate distributed throughout the neuronal cell bodies and their processes, most of the labelled neurones exhibited a lighter and more patchy distribution of nitric oxide synthase-immunoreactive material. Synapses made by nitric oxide synthase-negative nerve fibres with labelled intracardiac neurones were seen. Conversely, many nitric oxide synthase-containing nerve fibres that made synaptic contacts with unlabelled intracardiac neurones were also observed. Some small granule-containing cells were nitric oxide synthase-immunoreactive and were associated with unlabelled nerve terminals, while non-immunoreactive small granule-containing cells that were innervated by nitric oxide synthase-immunoreactive nerves were also seen. Small patches of osmiophilic electron-dense material were observed in the cytoplasm of NADPH-diaphorase-positive intracardiac neurones. This is the first description of the ultrastructural distribution of nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity in a subpopulation of intracardiac neurones of rat and guinea-pig heart and provides further evidence in support of a role for nitric oxide in the local control of the heart by intrinsic neurones.

[The structure of the stellate sympathetic ganglia under long-term hypoxia]. / Stroenie zvezdchatykh simpaticheskikh gangliev v usloviiakh dlitel'noi gipoksii.

Changes of mitochondrial ultrastructure indicating not only degenerative processes but also the presence of organelle adaptive reactions were demonstrated in sympathetic neurons. Appearance of osmiophilic inclusions in mitochondrial matrix relates to most typical organelle alterations in ageing. Number of smooth endoplasmic reticulum vesicles equal in size to synaptic vesicles of nerve cells processes grows higher. Such vesicles are usually located near postsynaptic active zones and areas with neuronal plasmolemma free from glia. Their outward resemblance does not allow to claim that neuromediators and neuromodulators may turn up in these vesicles, although dendrites contain and may release certain biologically active substances. Basic difference between 24 and 30-months old rats lies in earlier degenerations of nerve cells in old animals. Forming of dark granules may be also associated with conditions of fixation. Small granular (SG) cells lacked typical lipofuscine granules, while their ultrastructure changed insignificantly. SG cells capacity to divide mitotically in functional maturity is likely to be one of the possible explanations of such difference.

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves associated with coronary blood vessels of rat and guinea-pig.

Ultrastructural investigation of nitric oxide synthase-immunoreactive nerves closely associated with blood vessels in rat and guinea-pig hearts revealed many labelled nerve fibres in the walls of the main branches of the coronary arteries, and in arterioles, capillaries and post-capillary venules. The number of nitric oxide synthase-containing nerve fibres associated with different vessels, even those of the same calibre, varied. Terminal regions of nitric oxide synthase-immunoreactive fibres were observed in the endocardium and myocardium. Nitric oxide synthase-labelled fibres displayed electron-dense immunoproduct in both varicose and intervaricose regions. Immunoreactive axonal varicosities contained both small and large synaptic vesicles. The characteristics of the nitric oxide synthase-immunoreactive nerve fibres observed in the heart and the possibility that these fibres represent the processes of intracardiac neurones and/or sensory neurones of extrinsic origin are discussed.