ERK1/2 inhibition increases dopamine release from differentiated PC12 cells.

The release of dopamine (DA) is one of the main steps in the control of neuronal functioning and all CNS. It was demonstrated that many factors such as protein kinases and synaptic proteins are tightly involved in the regulation of DA secretion, but the data are contradictory. Here we analysed an effect of ERK1/2 inhibition on DA secretion from differentiated PC12 cells and evaluated the correlation between the activity of kinases/synaptic proteins and the level of released DA. PC12 cells were differentiated by NGF for 6 days. On the 7th day the cells were incubated for 1, 2 and 4 h with 10µM U0126. Obtained data demonstrated a significant accumulation of DA in the media after 4 h incubation with U0126 that accompanied with upregulation of PKG activity. Analysis of exocytosis proteins demonstrated decreased phosphorylation level of synapsin I and content of SNAP25. Taken together our data proposed an inhibitory role of ERK1/2 in the regulation of catecholamine secretion and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from the cells.

Analysis of ERK1/2 kinases in the inferior colliculus of rats genetically prone to audiogenic seizures during postnatal development.

The activity of ERK1/2 kinases in the quadrigemina inferior colliculus of Krushinsky-Molodkina rats of different age, which are characterized by an increased seizure readiness compared to Wistar rats, was analyzed. An increased (probably genetically determined) activity of these enzymes during the development of epileptiform activity in ontogeny was found, which may be the cause of abnormalities in the neurotransmitter system functioning.

Selective specificity of calcium-binding proteins calbindin and calretinin expression in the magnocellular neurosecretory hypothalamic nuclei of tortoises and turtles.

We have studied the distribution of calcium-binding proteins in the magnocellular neurosecretory nuclei of nonapeptidergic neurosecretory nuclei of the preoptic-hypothalamic complex in a tortoise (Testudo horsfieldi) and a pond turtle (Emys orbicularis) using immunohistochemistry. We have found that different types of cells in the paraventricular and supraoptic nuclei predominantly express calbindin and, to a lesser extent, calretinin, but not parvalbumin. The selective calbindin/calretinin control of the neurohormone secretion in these hypothalamic nuclei is an evolutionary conservative feature typical of reptiles and mammals.

[COMPARATIVE STUDY OF NIGROSTRIATAL SYSTEMS IN WISTAR RATS AND RATS PRONE TO SEIZURES].

In this work we analyzed the levels of functional activity of dopaminergic, GABA-ergic and glutamatergic neurons in the nigrostriatal system of control Wistar rats and Krushinsky-Molodkina (KM) rats prone to audiogenic seizures. In KM rats we have revealed disturbed activity of GABA- and dopaminergic neurons in substania nigra whereas the level of glutamatergic neurotransmission remained unchanged. We have also observed no significant differences in GAD65/67 and phospho-tyrosine hydroxylase contents in the striatum of KM and control Wistar rats. However, a high level of D1 dopamine receptor and a decreased level of D2 receptor found can mediate the upregulation of glutamatergic neurotransmission. Indeed, the expression of vesicular glutamate transporter type 2 (VGlut2) and NR2B subunit of NMDA receptor was increased in the striatum of KM rats. In striatal glutamatergic fibers phosphorylated ERK1/2 kinases have been revealed; at the same time, in KM rats an increased ERK1/2 activity has been detected both in striatum and substantia nigra. This finding correlated with activation of exocytosis rate as evidenced by downregulation of SNAP25 level. Apart from other reasons, the activation of glutamatergic system may be a result of disruption of the inhibitory effect of the dopamine- and GABAergic systems of substantia nigra that innervate striatum. We suppose that the increased activity of striatal glutamatergic neurons of KM rats without an adequate inhibition by GABA- and dopaminergic systems may be one of the reasons of high convulsive susceptibility in KM rats.

[MOLECULAR MECHANISMS OF ERK1/2 KINASES REGULATION IN THE GLUTAMATE- AND GABA-ERGIC NEURONS DURING SEIZURE EXPRESSION IN KRUSHINSKY-MOLODKINA RATS].

The aim of the present study was to analyze a role of the ERK1/2 signaling pathway in the regulation of excitation and inhibitory neurons in the hippocampus and the temporal cortex of Krushinsky-Molodkina rats during seizure development finalizing with ataxia. Analysis was done by Western bloting as well as by immunohistochemistry. The results demonstrated significant up-regulation of ERK1/2 activity in the hippocampus in several seconds after sound stimulation. At the same time increased ERK1/2 activity was correlated with enhanced level of SNARE protein SNAP-25 and activation of synapsin I, the proteins which regulate exocytosis machinery. Decreased level of VGLUT2 associated with activation of ERK1/2 and exocytosis proteins supposed activation of glutamate release in the hippocampus, while in the temporal cortex diminished activity of ERK1/2 and synapsin I associated with VGLUT2 up-regulation assumed inhibition of glutamatergic transmission. Our data let us supposed that decreasing of glutamate release in th& temporal cortex could be a trigger for the inhibition of hippocampal glutamatergic system and the beginning of further ataxia stage. Our data demonstrated correlation between expression and activity of exocytosis proteins and ERK1/2 mainly in the glutamategic neurons of the hippocampus and the temporal cortex that let us proposed significant role of ERK1/2 kinases as a positive regulator of glutamate release and as a result initiation of seizure expression.

Role of the ERK signaling pathway in regulating vasopressin secretion in dehydrated rats.

Dehydration activates the vasopressinergic system of the hypothalamus. We analyzed the effects of dehydration induced by water deprivation for 3 days on the activities of ERK1/2 and transcription factors, Elk1 and cAMP response element-binding protein (CREB) in vasopressinergic neurons, as well as the distribution and level of the motor protein, kinesin, in the hypothalamo-neurohypophyseal system. We showed that dehydration resulted in enhanced vasopressin (VP) expression and activation of CREB, and increased the activity of the MEK/ERK/Elk1 pathway in magnocellular neurons of the supraoptic nucleus. The activation of VP secretion was associated also with accumulation of phospho-ERK1/2 in the VP-ergic terminals of the posterior lobe of the pituitary. Analysis of the amount and distribution of kinesin and SNAP25, the proteins associated with transport and secretion, demonstrated that dehydration enhanced kinesin content in the perikarya of magnocellular neurons in the supraoptic nucleus and decreased kinesin and SNAP25 levels in the posterior pituitary. ERK1/2 and ERK1/2-dependent transcription factors, Elk1 and CREB, participate in the regulation of dehydration-evoked VP expression. We propose that ERK1/2 and kinesin participate in regulation of anterograde transport of VP dense core vesicles.

Effect of p53 inhibition by pifithrin-alpha on functional activity of vasopressin neurones in rat hypothalamus.

In the present work we investigated the effects of p53 inhibition by pifithrin-alpha (PFT) in vitro and in vivo on functioning vasopressinergic magnocellular neurones of rat hypothalamus. In vivo treatments with PFT were done by intra-hypothalamic microinjections or by intra peritoneal injections. In in vitro experiments hypothalamic slices containing supraoptic nuclei and intact pituitary were incubated with or without PFT. In all experiments we observed accumulation of vasopressin (VP) in the cell perikarya after PFT injections, however expression of VP mRNA was not changed. Analysis of VP content in the posterior pituitary demonstrated that amount of VP was significantly decreased after PFT treatments. Additionally, long-term inhibition of p53 in experiments with intra-hypothalamic injections of PFT resulted in an increased diuresis rate. The obtained results demonstrated that in all experiments PFT treatments inhibited VP anterograde transport from the cells of supraoptic nuclei. Moreover, analysis of MEK/ERK activities revealed that phosphorylation levels of MEK1/2 and ERK1/2 were decreased after PFT treatments. Our findings provide new evidences that p53 could be involved in the control of VP secretion from hypothalamo-hypophyseal system and that this action probably can be mediated by ERK signalling pathway.

Tet-system for the regulation of gene expression during embryonic development.

The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.

Effect of Hypericum extract on the hypothalamic-pituitary-adrenal system in rats.

We studied the effect of Hypericum extract on activity of the hypothalamic-pituitary-adrenal system in rats. In rats exposed to stress after a 30-day daily oral treatment with Hypericum extract, the weight of the adrenals and ACTH concentration were lower than in controls. Hence, treatment with Hypericum extract improved resistance to stress and prevented exhausting of the hypothalamic-pituitary-adrenal system.

Different pathways of neurohormonal hypothalamic control of the adrenal cortex function in young and old rats.

The pathways of hypothalamic control of the adrenal cortex function were studied in the experiments on young and old male Wistar rats. Immunohistochemical, morphometric, densitometric and radioimmunoassay methods were used. It was shown that in young rats under stress vasopressin (VP) is released into the portal circulation and probably in this way stimulates ACTH and enhances secretion of corticosterone in the adrenal cortex. In old rats stress-reaction is delayed and less adequate compared to young animals. It seems likely that in old rats VP is secreted into the general circulation mainly via the posterior pituitary lobe (PP), exerting a direct influence on the adrenal cortex. The conclusion is made that in old rats regulation of the adrenal cortex function is realised via the pathway phylogenetically more ancient than in young animals.

Oxytocinergic neurosecretory system in genetically selected rats differing in emotionality. A morphometric investigation.

The study was carried out on lines of rats selected for high (KHA) and low (KLA) rates of development of an active avoidance conditioned reflex in a shuttle box. The volume of nucleoli of oxytocinergic cells of the paraventricular and supraoptic nuclei of the hypothalamus, as well as the content of oxytocin-immunopositive material in the posterior lobe of the hypophysis, were measured. The volume of the nucleoli in the intact KHA rats was smaller, and the amount of oxytocin-immunopositive material in the posterior lobe of the hypophysis greater, than in the KLA rats. A stressor (unavoidable electrodermal stimulation) exerts an opposite effect on the oxytocinergic system of the hypothalamus in rats of the two lines: the volume of the nucleoli increases in the KHA rats, while the amount of oxytocin-immunopositive material in the posterior lobe of the hypophysis decreases; the changes are opposite in the KLA rats. These results suggests that the reactivity of the oxytocinergic system is closely associated with the specific features of the animal's adaptive behavior, in particular, with the capacity for learning.

The hypothalamo-hypophysial system in acipenseridae. X. Corticoliberin-like immunoreactivity in the hypothalamus and hypophysis of Acipenser ruthenus L.

Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level the distribution of immunoreactive corticoliberin has been studied in the hypothalamus and hypophysis of the sterlet. Corticoliberin-containing cells have been revealed in the nucleus tuberalis and to lesser degree in the nucleus preopticus of the hypothalamus. The majority of labeled cells is located subependymally or between the ependymal cells. Some dendrites of corticoliberin-containing cells can be followed toward the ventricular lumen. Their axons project to the anterior neurohypophysis (proximal neurosecretory contact region) where they terminate in contact with the primary portal capillaries. Single terminals containing immunoreactive corticoliberin are seen in the roots of the posterior neurohypophysis on the border region between the neurohypophysis and the hypophysial pars intermedia, and sometimes in contact with the capillaries of the general circulation. The possible role of corticoliberin in the regulation of the function of glandular cells of both the pars distalis and pars intermedia is discussed.