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This study aimed to reveal parvalbumin (PVA) expression in cells of the subventricular zone (SVZ), identify the morphological type of these cells, and estimate the effect of acute hypoxia on the size of this cell population in the Wistar rat neonatal period. PVA-expressing cells were revealed immunohistochemically using Abcam rabbit polyclonal antibodies to this calcium-binding protein. Rats were exposed to hypoxia in a special chamber with 7.8% oxygen in a breathing gas mixture. The brain was examined in the neonatal period on postnatal days 5 and 10. It was found that the SVZ, both in control and experimental rats, contains PVA- expressing cells of the same morphological type, which represent migrating immature neuroblasts (type A). In control animals, throughout the entire neonatal period, the number of these cells remains constant and accounts for more than 20% of the total cell population. Perinatal hypoxic exposure leads to increase the number of PVA-expressing cells, as well as the total number of cells, in the SVZ (to 33%). Thus, perinatal hypoxia activates neurogenesis in the SVZ during the neonatal period. In the same period, some neuroblasts express PVA and may reach a certain degree of differentiation of their neurotransmitter phenotype.

Objective. To study the distribution of GAT1 GABA transporter in the Botzinger complex at different time points in early postnatal development in rats in normal conditions and after prenatal serotonin deficiency. Materials and methods. Studies were performed on Wistar laboratory rats. Decreases in endogenous serotonin levels in the embryonic period were produced by inhibition of tryptophan hydroxylase with para-chlorophenylalanine (PCPA). GAT1 transporter protein was detected using an immunohistochemical reaction with primary rabbit anti-GABA transporter 1 polyclonal antibodies (AbCam, UK). Brains were studied on postnatal days 5, 10, and 20. Results. At the early periods of postnatal development, the Botzinger complex of control animals showed oscillations in the GAT1 GABA transporter level. At one week of life, the GAT1 level was high, both in networks of processes and terminals and in synapses. During the second week of life, the GAT1 level decreased, and by the end of the third week it again increased, reaching the initial level. Serotonin deficiency during the prenatal period induced a significant increase in the GAT1 level in the neuropil of the Botzinger complex in the experimental animals at all time points of postnatal development. Conclusions. Prenatal serotonin deficiency led to a significant increase in the level of GAT1 GABA transporter in the early periods of postnatal development, which may lead to changes in GABA transmission and, as a result, disruption of the balance between inhibitory and excitatory effects in the respiratory nucleus.

Objectives. To study the expression of GAT1 (GABA transporter) at the early periods of postnatal development in the ventrolateral part of the solitary tract nucleus in prenatal serotonin deficiency in rats. Materials and methods. The endogenous serotonin level in the embryonic period was decreased by inhibition of tryptophan hydroxylase with para-chlorophenylalanine (PCPA) (Sigma, USA). GAT-1 transporter was detected using primary rabbit polyclonal antibodies (anti-GABA transporter 1, GAT1) (AbCam, UK). Results. From the early neonatal period to the beginning of juvenile age, the neuropil and the processes, terminals, and synaptic structures of the lateral and ventral parts of the solitary tract nucleus of control rats showed a gradual increase in GAT1 expression in experimental animals. The level of expression of GAT1 transporter was significantly greater in the lateral and ventral parts during postnatal weeks 1 and 2 than in controls, though by the end of week 3, i.e., by the beginning of juvenile age, it decreased to become significantly lower than in controls. Conclusions. The respiratory areas of the solitary tract nucleus in control rats in the first three weeks of postnatal development showed a gradual increase in GAT1 expression. Serotonin deficiency during the prenatal period led to impaired expression of GAT1 in the early postnatal period. These changes can lead to changes in GABA transmission, which may in turn be the cause of imbalance between inhibitory and excitatory effects in the respiratory center in the early postnatal period and, thus, may be the basis of the development of respiratory dysfunctions at early age.

Objectives. To study the dynamics of structural changes in elements of the blood-brain barrier of the hippocampus during the early postnatal period and subsequent ontogeny after exposure to perinatal hypoxia and to investigate the potential for pharmacological correction of these changes. Materials and methods. Experiments were performed on Wistar laboratory rats with a model of the encephalopathy of premature human infants using electron microscopy. Results. Perinatal hypoxia was followed by detection of structural damage to capillary walls: there were increases in the size of endotheliocytes, increases in the numbers of cytoplasmic processes, changes in the diameter of capillary lumens, and delays in the formation of the basal membrane. A marked endothelium-protecting effect was seen with the formulation Salifen at all study time points. Salifen was found to decrease the intensity of the reactions of elements of the blood-brain barrier to the harmful actions of perinatal hypoxia. Conclusions. Perinatal hypoxia has marked damaging effects on all elements of the blood-brain barrier of the hippocampus, the induced lesions being detected at both the early stages of postnatal development and in animals reaching adulthood. Use of Salifen immediately after exposure to perinatal hypoxia eliminated much of the structural damage to endotheliocytes and the basal membrane by the juvenile period. This study provides grounds for believing that the use of Salifen in ischemic brain injury in neonates has potential and our series of further preclinical studies will be continued.

—We studied the role of vascular endothelial growth factor (VEGF) in post-hypoxic damage of the rat brain and in the effect of saliphene, a GABA derivative that is considered as a potential neuroprotector. The method of quantitative immunohistochemistry was used to evaluate the expression of VEGF in the rat brain (neocortex and hippocampus) at the early and late periods after perinatal hypoxia and the subsequent administration of saliphene. Hypoxia resulted in an increase in the expression of vascular endothelial growth factor in the developing rat brain, and injections of saliphene prevented an increase in VEGF expression, leaving this index at the level of the control values. Thus, the changes the expression of vascular endothelial growth factor (VEGF) in the rat brain after perinatal hypoxia may be considered as a marker of the neuroprotective effect of saliphene.

Objective. to study the dynamics of the expression of mGluR2/3 metabotropic receptors in the ventral and lateral respiratory subnuclei of the solitary tract nucleus (STN) in the early postnatal period in health and after prenatal serotonin deficiency in rats. Materials and methods. Studies were performed on Wistar laboratory rats (n = 19). Tryptophan hydroxylase was inhibited using parachlorophenylalanine. The ventral and lateral respiratory subnuclei of the STN were studied on days 5, 10, and 20. An immunohistochemical method was used to study the distribution of mGluR2 and mGluR3 metabotropic receptors (mGluR2/3). Results. During the first postnatal week, both subnuclei showed high levels of mGluR2/3 expression. During the second week, there was a sharp reduction in mGluR2/3 expression, followed by an increase by the end of the third week. Decreases in serotonin content during the prenatal period affected the intensity of mGluR2/3 expression in both subnuclei of the STN. During the early postnatal period, there was a significant reduction (more than two-fold) in the expression of mGluR2/3 expression at all time pints, this being more marked in the ventral subnucleus. Conclusions. Changes in the level of expression of mGluR2/3 metabotropic glutamate receptors during the early postnatal period occurred in the ventrolateral part of the STN. Serotonin deficiency led to a sharp reduction in the expression of mGluR2/3 receptors in the respiratory subnuclei during the early postnatal period.

Experiments on Wistar rats (n = 18) were performed to study changes in the expression of ionotropic glutamate receptor subunits (GluR2) in the respiratory subnuclei (ventral and lateral) of the solitary tract nucleus (STN). Observations were made during the early postnatal period (days 5, 10, and 20, 5–6 animals per group) in normal conditions and with prenatal decreases in serotonin levels due to inhibition of tryptophan hydroxylase with parachlorophenylalanine. Immunocytochemical detection of GluR2 revealed significant increases in GluR2 expression in the respiratory subnuclei of the STN in the early postnatal period (a 2-fold increase in the lateral subnucleus, 2.6-fold in the ventral). Prenatal serotonin deficiency altered GluR2 expression in the respiratory subnuclei of the STN. There was a significant delay in GluR2 expression in the early period, which increased to control levels by two weeks after birth, though GluR2 expression by juvenile age was lower (twofold) than in controls. Impairments to the glutamatergic receptor networks in the respiratory nuclei may be the basis of respiratory dysfunction.

We investigated structural changes in the Wistar rat hippocampal CA1 field and fascia dentate during the pubertal period (on P60) after perinatal hypoxic exposure as well as the distribution of GAD67-expressing neurons in these structures. It was established that in the granular layer of the fascia dentata and in the CA1 field acute perinatal hypoxia leads to irreversible homotypic abnormalities as expressed in the reduced number of neurons and their rows as well as injury of a considerable portion of cells, which exhibit the signs of chromatolysis and vacuolization of the cytoplasm. Both in experimental and control animals, GAD67-expressing neurons in the fascia dentata are scattered diffusely and share approximately the same size of their populations. In the CA1 field, immunoreactive neurons lie in the lower rows of the pyramidal layer, while neurons in the upper layers exhibit no immunolabeling and have less synaptic structures in experimental animals than in control. We suggest that neurons in the hippocampal structures are involved in the regulation of functions and formation of prenatal pathology.

The dynamics of the formation of the inhibitory neural GABAergic network in the ventral and lateral subnuclei of the solitary tract nucleus (STN) in the early postnatal period were studied in Wistar rats in normal conditions and in prenatal serotonin deficiency (n = 27). GABAergic neurons were detected immunocytochemically using rabbit polyclonal antibodies to GAD-67. The STN subnuclei studied were found to display high levels of GABA expression during the first week of postnatal development, as evidenced by the significant number of GAD-67-immunopositive neurons, of which the number in the ventral subnucleus was almost 1.3 times greater than that in the lateral subnucleus. GABA expression decreased during the second and third weeks, with a simultaneous increase in the density of the network of terminals and GABAcontaining synaptic structures in the neuropil of both subnuclei. Prenatal serotonin deficiency was found to produce sharp decreases in GABA expression in the ventral and lateral subnuclei of the STN during the early postnatal period of development. By the end of the third week, a loose network of GABAergic terminals persisted in the neuropil and the density of synaptic structures increased.

The dynamics of the distribution of GABAergic neurons and neurons expressing different types of GABA receptors (GABA-Aα1 and GABA-B1) in the Botzinger complex during the early postnatal period (the period of functional maturation of the receptor system in mammals) were studied in normal conditions and after prenatal reductions in serotonin content in Wistar rats. Brains from rat pups of two groups were studied on days 5, 9, and 20 after birth: controls (n = 9), born to intact females, and experimental (n = 13), born to mothers treated with parachlorophenylalanine during pregnancy to decrease endogenous serotonin content. GABA-synthesizing neurons and neurons expressing GABA-Aα1 and GABA-B1 receptors were detected immunocytochemically. Maturation of the inhibitory GABAergic network in the Botzinger complex occurred during the early postnatal period (by day 9). GABA production occurred simultaneously with GABA-Aα1 and GABA-B1 expression, though their maturation differed. GABA-Aα1 receptors formed earlier (by day 9) and coincided in time with GABA expression. Maturation of GABA-B1 receptors occurred later – only by week 3. Prenatal serotonin deficit led to a delay in the expression of GABA and GABA-Aα1 receptors in neurons of this nucleus and produced impairment to the formation of a network of terminals and synapses containing GABA and GABA-Aα1 and GABA-B1 receptors.

The distribution of GABAergic interneurons as well as terminal and synaptic networks in different layers of the rat sensorimotor neocortex were studied at different stages of the postnatal period under normal conditions and after exposure to perinatal hypoxia. In control animals, the architectonics of the inhibitory network in different layers of the sensorimotor neocortex was shown to display distinctive features at different stages of the postnatal development. At early postnatal stages, a significant portion of neurons in layers II–V are immunopositive for GAD-67, indicative of a high level of GABA expression, however, GABA transmission is extremely weak, thus supporting the presence in the neuropil of only sporadic GABAergic terminals and synapses. By the juvenile age, a dramatic drop in the number of GABAergic neurons and an increase in the density of the network of GABA-immunopositive processes and synaptic structures occur in the neuropil, suggesting a considerable increase in GABA transmission. A higher level of GABA transmission is revealed in layers IV and V, persisting over the prepubertal period. Our results demonstrate that acute perinatal hypoxia affects the state of the inhibitory GABAergic network in the rat sensorimotor neocortex during the postnatal period. GABA expression and transmission were shown to change virtually in all layers.

The long-term effects of perinatal hypoxic exposure followed by the administration of salifen, a GABA derivative, on morphological characteristics of the vascular wall elements in the neocortical microvasculature were studied in rats. Salifen, when applied at therapeutic doses, was established to exert an endothelial protective effect. It prevents such typical pathomorphological consequences of perinatal hypoxia as endothelial hypertrophy, the appearance of multiple intravascular endothelial outgrowths, narrowing of the capillary lumen, malformation of the lamina densa, swelling of perivascular astrocytes, and reactive changes in pericytes. In all neocortical layers, a density of blood vessel distribution across microvasculature as well as their cross-sectional area were found to be approximately the same in adult animals both in control and after hypoxic exposure followed by salifen administration. Changes and rearrangements in the capillary bed after hypoxic exposure and salifen administration were shown to occur at earlier developmental stages, while by the maturity period the structural parameters of microvasculature become stabilized. The endothelial protective effect of salifen promises high clinical efficiency of this preparation and serves as a basis for further studies in this direction.

Time course of formation of the inhibitory receptor network (GABA-Aα1 and GABA-B1) in the respiratory subnuclei (ventral and lateral) of the solitary tract (NTS) during the early postnatal period was studied on laboratory Wistar rats under normal conditions and prenatal serotonin deficiency. It was found that in normal rats the maturation of the inhibitory receptor networks in both NTS subnuclei occurs within the first three postnatal weeks. Some features of their formation were noticed. The dynamics of changes in intensity of GABA-Аα1 expression in the ventral and lateral subnuclei proceeds in a similar way. During the first postnatal week, GABA-Aα1 expression is low. In the neuropil, the network of GABA-Aα1-containing presynaptic terminals and synapses is poorly developed. Within the second week, the number of GABA-Aα1-expressing neurons increases in both subnuclei with a simultaneous rise in the density of the network of terminals and synaptic structures. By the end of the third week, the number of GABA-Aα1-expressing neurons decreases, but the network density continues to increase. GABA-B1 expression in the ventral and lateral subnuclei occurs also simultaneously, although with some distinctions. During the first postnatal week, intensity of GABA-B1 expression is weak. In the neuropil, few GABA-B1-containing terminals form a loose network with sporadic synaptic structures. During the second week, expression of the receptor increases, being particularly considerable in the ventral subnuclei. Simultaneously, the density of the presynaptic terminals increases. By the end of the third week, the number of GABA-B1-expressing neurons in the ventral subnuclei decreases, while in the lateral subnuclei remains almost intact and the network density increases. The data obtained show that prenatal serotonin deficiency leads to malformation and impaired maturation of the GABAergic inhibitory neuronal network in the ventrolateral part of NTS.

Morphological characteristics of the elements of vessel walls in the microvasculature of rat neocortex were studied after perinatal hypoxia and subsequent introduction of salifen, a derivative of GABA, at long-term postnatal periods. It is shown that salifen at a therapeutic dose has a protective effect on the endothelium of blood vessels in the microvasculature of neocortex. It is found that after exposure to hypoxia followed by application of salifen no endothelial hypertrophy, appearance of numerous processes of endotheliocytes in the vascular lumen, and narrowing of the capillary lumen occur. The formation of la- mina densa is almost identical to that in the control. Swelling of perivascular astrocytes and reactive chan- ges of pericytes are also absent. In all layers of the neocortex the density of vessel distribution in the mic- rovasculature as well as their cross-sectional area in adult animals of the control group and after exposure to hypoxia and salifen application were approximately identical. It is shown that changes and restructu- ring of the capillary bed after exposure to hypoxia and application of salifen take place at earlier terms of development whereas by the maturity period stabilization of the structural parameters of the microvas- culature occurs. The protective effect of salifen on the elements of capillary walls determines the clinical effectiveness of its use and serves as a basis for further research in this direction.

An immunocytochemical study method was used to investigate the expression of the serotonin transporter (5-HTT) in the ventrolateral part of the solitary tract nucleus in Wistar rats during the early postnatal period (days 5 and 10) in normal conditions (n = 10) and in conditions of prenatal serotonin deficiency (n = 12). During the early postnatal period, the ventrolateral section of the solitary tract nucleus was found to show transient expression of 5-HTT, most marked in the caudal part of the ventral subnucleus. The numbers of neurons synthesizing 5-HTT in the rostral part of the ventral and lateral subnuclei were small and did not change with age. The caudal part of the ventral subnucleus contained a large number of neurons synthesizing 5-HTT on day 5 of the postnatal period, and this number decreased significantly with age. The caudal part of the lateral subnucleus contained a small number of cells expressing 5-HTT, and this number also decreased with age. 5-HTT expression levels were significantly higher in the caudal areas of both the ventral and lateral subnuclei than in the rostral areas. Prenatal serotonin deficiency decreased the numbers of neurons synthesizing 5-HTT in the nuclei studied here.

Expression of the serotonin transporter protein (5-NTT) in the dorsal raphe nucleus (DNR) during the early postnatal period was investigated in laboratory Wistar rats. Immunocytochemical labeling showed that during the first 3 postnatal weeks the intensity of 5-NTT expression in DNR of control animals changes. During the earliest postnatal stages, most of DNR subnuclear neurons (dorsal, DNR-d; ventral, DNR-v; lateral, DNR-lat) were found to intensely express 5-NTT. 5-NTT localization sites were revealed on the membrane surface of neuronal cell bodies and their processes in neuropil. On P10, the number of 5-NTT expressing neurons and 5-NTT binding sites decreases. At this time, the 5-NTT binding sites were shown to undergo redistribution becoming very few on neuronal cell bodies and dendrites, but rather densely packed in the axonal membrane. The number of 5-NTT expressing neurons and density of 5-NTT localization sites in neuropil gradually increases with age. The reduction in the serotonin level in all DNR regions during prenatal development leads to the reduction both in the number of 5-NTT expressing neurons and 5-NTT localization sites during the early postnatal period. This tendency was shown to persist with age.

The aim of the present work was to study the distribution of GABAergic neurons in structures of the pneumotaxic center (medial subnucleus of the parabrachial complex and the Kolliker–Fuse nucleus) in health and in conditions of serotoninergic system insufficiency during the prenatal period of development in Wistar rats. Endogenous serotonin content was decreased in fetuses by inhibition of tryptophan hydroxylase with parachlorophenylalanine, which was given to females on day 16 of pregnancy. Specimens from the pons from experimental and control (intact) rat pups were investigated at early postnatal (days 5, 10, and 12) and juvenile (day 20) ages. Groups of 5–6 experimental and control rat pups were used at each time point. GABAergic neurons were detected using antibodies to its synthetic enzyme glutamate decarboxylase (GAD67). The results showed that the Kolliker–Fuse nucleus contained a population of GABAergic neurons during the early periods of postnatal development, the numbers of cells in this population persisting to juvenile age. The medial subnucleus of the parabrachial complex contained an insignificant number of GABAergic neurons at the early time points, with a slight increase by juvenile age, though neurons continued to give weak immune reactions. Serotonin deficiency in structures of the pneumotaxic center led to decreases in the number of GABAergic neurons and decreases in the numbers of GABAergic synapses and their accumulations. The decrease in serotonin during the prenatal period could induce impairments of inhibitory afferentation of the nuclei of the pneumotaxic center and changes in local inhibitory GABAergic networks in its nuclei, which could result in impairments to inhibitory processes in the structures of this center.

Experiments were performed on Wistar rats divided into three groups: group 1 (experimental group) consisted of rats subjected to hypoxia and treated with Salifen (15 mg/kg for 14 days); group 2 (controls) consisted of rats subjected to hypoxia but not treated; group 3 consisted of intact animals (each group included 8–10 animals). Light and electron microscopy methods were used to study the effects of Salifen on the structural characteristics of elements of the blood-brain barrier (BBB) of the neocortex in rats after exposure to hypoxia during the early postnatal period – on day 2 of life (a model of premature birth in humans). The results showed that Salifen had positive actions on the state of the microcirculatory bed after perinatal hypoxia, particularly in relation to state of endotheliocytes. Salifen was found to take active part in compensatory-adaptive reactions of the BBB in response to hypoxia; these experiments identified potential for further studies of the protective properties of Salifen.

The dynamics of the distribution of GABAergic neurons and neurons expressing different types of GABA receptors (GABAAα1 and GABAB1) was studied in Bötzingercomplex (BötC) in the early postnatal period (the period of functional maturation of the respiratory system in mammals) in norm and prenatal reduction of serotonin content in Wistar rats. The brain was studied on postnatal Days 5, 9 and 20 in two groups of rat pups: control (n=9), born by intact females, and experimental (n=13), born from mothers that received parachlorophenylalanine, causing the depression of endogenous serotonin level. Imunocytochemical methods were used to detect the neurons producing GABA and expressing GABAAα1 and GABAB1 receptors. It was shown that the maturation of the inhibitory GABAergic network in BötC occurred in the early postnatal period (by Day 9). Simultaneously with GABA, the expression of GABAAα1 and GABAB1 receptors took place, however their maturation has the distinctive features. The formation of GABAAα1 receptors occurred earlier (by Day 9) and coincided in time with the expression of GABA. The maturation of GABAB1 receptors happened later — only by the third week. Prenatal serotonin deficiency caused a delay in the expression of GABA and GABAAα1 receptors by the neurons of BötC, as well as the disruption of the formation of a network of terminals and synapses containing GABA, GABAAα1 and GABAB1 receptors.

Special focus is currently laid on the early postnatal period when an organism adapts actively to new living conditions and therefore becomes particularly vulnerable to adverse environmental factors. Among these adaptive processes, one of the most important is the development of homeostatic and, specifically, barrier mechanisms. The aim of this study was to explore the reaction of the hematoencephalic or blood–brain barrier (BBB) structural elements to perinatal normobaric hypoxia (the human incomplete pregnancy model). Using light and electron microscopies, it was demonstrated that after hypoxic exposure all the capillary wall components in the neocortex display structural changes which may underlie microcirculatory disorders and increased BBB permeability. Differentiation of the capillary wall basal membrane was found to occur during the early perinatal period, and the formation of its laminae (laminae rara et densa) serves a differentiation criterion. Hypoxic exposure was found not only to delay the basal membrane formation, but to induce a vesicular type of its degeneration.

In this work an expression of serotonin transport protein (5-HTT) was studied in the dorsal raphe nucleus (DRN)--in its dorsal, ventral and lateral subnuclei in Wistar rats (n = 15) during the early postnatal period. Histological methods and immunocytochemical staining demonstrating 5-HTT were used in the investigation. It was shown that at postnatal Day 5 major part of neurons of the subnuclei studied intensively expressed 5-HTT. However, by Day 10, the level of its expression decreased dramatically, but with age (by Day 20) the expression was increased once again. This was manifested by the augmentation of size of 5-HTT-positive neuronal population, the increase in the density of plexus network of their processes. The detected changes of 5-HTT expression indicate the varying degrees of functional activity of serotonin in the DRN in the early postnatal period.

The responses of forming synapses in the rat neocortex to the actions of hypoxia in the early period of neonatal life (day 2) were studied. Immunocytochemical studies were used to detect synaptophysin and these results, along with electron microscopic studies, addressed the sensorimotor cortex in rat pups at 3, 5, and 10 days of postnatal development (using groups of 6–10 individuals) in an experimental group and a control group (intact animals). Immunocytochemical studies of control animal showed significant differences in the quantitative distribution of synaptophysin-positive structures in different layers of the neocortex during the early neonatal period of development (day 5). Perinatal hypoxia decreased the optical density of the immunocytochemical reaction product more than twofold, and this was accompanied by reductions in the density of synaptophysin-positive granules in all layers of the neocortex. In addition, electron-dense terminals, providing evidence of degenerative processes, were seen. The neuropil of the neocortex showed a sharp decrease in the number of growth cones, small processes, and forming synapses, along with a significant increase in the electron density of synaptic elements, especially postsynaptic membranes and densities. In experimental animals, increases in the numbers of growth cones and forming synaptic structures were seen only by postnatal day 10. Thus, the consequences of hypoxia during the early neonatal period, inducing impairments to synaptogenesis, persisted throughout the study period.

Experiments were performed on the offspring of female Wistar rats (n = 8) given parachlorophenylalanine on day 16 of pregnancy to decrease endogenous serotonin levels in the offspring. Histological, immunocytochemical, and morphometric methods were used in rat pups on days 10 and 14 of postnatal development (5–6 individuals at each time point) for studies of the dorsal and ventral subnuclei of the lateral part of the parabrachial complex. The results showed that serotonin deficiency led to changes in the structure of these subnuclei. The population of large, multipolar neurons in the ventral subnucleus was found to be more sensitive to decreased serotonin levels. Experimental animals showed decreases in the body sizes of these neurons by a factor of 1.6 compared with controls, with virtually no change in the number of cells in this population. The morphological characteristics of small neurons corresponded to those in controls, though there was a reduction in the number of cells, which may explain the rarefaction of neurons in this nucleus. In the dorsal subnucleus of experimental animals, neurons of both of the populations in this structure underwent more marked changes. The sizes of both large and smaller neuron cell bodies decreased significantly, by factors of 2.0 and 1.8 respectively; the cytoplasmic volumes of these cells decreased compared with controls; the numbers of cells in both populations remained largely unaltered. Reactions detecting glial fibrillary acidic protein showed increases in the numbers of the astrocyte glial population in the early postnatal period in the experimental animals.

Morphological studies were performed on the serotoninergic neurons forming the nucleus raphe obscurus (NRO) of the medulla oblongata in rats at the early periods (5, 10, 12, and 14 days) of the postnatal period in normal animals and in animals undergoing prenatal development with serotonin deficiency. The NRO was found to contain three subpopulations of serotonin-synthesizing neurons (large, medium, and small) with different sensitivities to serotonin levels during the developmental period. The results showed that serotoninergic system deficiency during the prenatal period leads to changes in the structural organization of the NRO, with slowing of the rate of formation of the nucleus and differentiation of serotonin-synthesizing neurons, along with a 1.6-fold reduction in the total number of these cells. The sizes of all types of serotoninergic neurons underwent significant changes. In control animals, large neurons were 1.8 times larger than those in experimental animals, while intermediate cells were 1.4 times larger and small cells were 1.5 times larger. Decreases in neuron size were combined with changes in the nucleus:cytoplasm ratio. Cytoplasm volume decreased significantly, as did detectable chromatophilic material. Clear astrocyte reactions, which could subsequently lead to gliosis, developed synchronously with these changes.

We studied reactions of neural elements from different neocortex regions (sensorimotor, visual, auditory) to acute normobaric hypoxia on the model of human incomplete pregnancy (the 2-day-old rat pups) and revealed similar and unidirectional reorganization in all these regions. The chosen parameters of hypoxia induced the earliest detectable changes as fast as in a day since exposure: a decrease in cell body size and cytoplasmic volume, intensification of apoptotic cell death. By the end of the neonatal period (day 5), several ultrastructural changes were observed by indicating deceleration of processes of nerve cell differentiation: arrest of complication of smooth and rough endoplasmic reticulum and Golgi apparatus, the reduced number of free ribosomes and polysomes in cytoplasm as well as of axonal and dendritic growth cones in neuropil, delayed and disordered myelination of nerve fibers. All these morphofunctional abnormalities may be the structural basis for development of neonatal encephalopathies.

The dynamics of reactive changes in the population density of microgliocytes and the reversibility of their phenotypic forms were studied in the brains of neonatal rats at different time points after acute 1-h exposure to normobaric hypoxia in a barochamber on postnatal day 2. Different parts of the neocortex (frontal, motor, somatosensory, and visual) and segments of the hippocampus (fields CA1, CA3, and CA4 and the dentate fascia) were studied at 1 and 3 h and 1 and 5 days after hypoxia. Microgliocytes were detected immunocytochemically using polyclonal antibodies to Iba-1 antigen. The results showed that the microglial reactions to acute hypoxia in the neocortex and hippocampus in neonates developed simultaneously and synchronously with increases in cell death. Increases in the population density of ameboid microgliocytes in the brain areas of interest occurred within 1 h and resulted from migration of these cells from the subventricular zone and areas adjacent to the major vessels, where they virtually disappeared. The numbers of ameboid microgliocytes in these areas recovered quite quickly (within 3 h). The population density of microgliocytes, especially those of the ameboid form, increased sharply with increases in cell death and lasted to five days.

Synaptogenesis in the dorsal raphe nucleus (DRN) of the medulla oblongata in Wistar rats was studied at the end of the prenatal (days 19 and 20) and during the early postnatal (days 5 and 20) periods (8–10 animals at each time point); the role of serotonin in forming synaptic contacts at this period of development was also assessed. In control animals (intact), the DRN-d, DRN-v, and DRN-l showed the onset of neuropil formation on day 19 of prenatal development; synaptic contacts appeared on processes at this time, first being detected on the surfaces of neuron bodies on day 20. The distribution density of synaptophysinpositive granules both on processes in the neuropil and on neuron bodies showed a sharp and significant increase by day 5 of postnatal development. Subsequent increases, to day 20, were minor. Serotonin deficiency during the second half of the period of intrauterine development, induced by administration of p-chlorophenylalanine to female animals on day 16 of pregnancy, led to significant delay in synaptogenesis in the DRN in the offspring during both the pre- and early postnatal periods. As postnatal age increased, increases in the distribution density of synaptic contacts were different in different parts of the DRN: contact density was similar to controls in the DRN-d, while it remained low in the DRN-v and DRN-l. These results provide evidence for the involvement of serotonin in synaptogenesis in the DRN.

The distribution of GABAergic neurons in different areas of the neocortex (frontal, sensorimotor, and visual) was studied in Wistar rats at different time points in the postnatal period of development after exposure to perinatal hypoxia. These neurons were detected using antibodies to GAD-67, a marker for GABAergic neurons. Perinatal hypoxia was found to lead to significant decreases in the numbers of GAD-67-expressing neurons in both the upper and the deep layers of the cortex at juvenile age (at day 20 of postnatal development), persisting to the pubertal period (day 40). The number of GAD-67-synthesizing neurons in each of the neocortical layers in experimental animals on day 40 were half those in controls. The sharp decrease in the number of GABAergic neurons in the neocortex is probably due to the harmful action of acute perinatal hypoxia on precursor cell migration from the subventricular zone or on the synthesis of the factors controlling these migration processes and the maturation of GABAergic neurons, and thus decreasing the late expression of GAD-67.

The establishment of the motor nucleus of the trigeminal nerve (MNTN) was studied during the early postnatal period in 21 female Wistar rats given the serotonin synthesis inhibitor parachlorophenylalanine on day 16 of intrauterine development (before the formation of the serotoninergic system). These experiments showed that serotonin deficiency during the prenatal period in rats led to changes in the structural organization of the MNTN. During the early stages of postnatal development, there was a delay in the development of the neuropil, with decreases in neuron body sizes and, in some cells, partial loss of chromatophilic substance, along with the presence of dead cells with signs of hyperchromatosis in all parts of the MNTN and especially its ventrolateral part. Neuron death then slowed, though some cells had morphological abnormalities. With increasing postnatal age (by day 20), there was a reduction in the number of motoneurons, evidently due to a gradual increase in cell death. Motoneuron degeneration in the areas of the MNTN studied was accompanied by astrocyte gliosis.

The experiments reported here showed that acute hypoxia during the perinatal period of development leads to structural changes in the motor and visual areas of the neocortex, detectable by postnatal day 20 as impairments to the structural organization of the layers of the neocortex. We report the first data showing that different hippocampal fields respond differently to hypoxia during the postnatal period and present evidence for the existence of long-term sequelae of perinatal hypoxia in the structure of the hippocampus. Acute hypoxia was followed by cell death in all fields and thinning of the pyramidal neuron layers. The greatest levels of cell death occurred in fields CA4 and CA3. As postnatal age increased, cell death remained significant in field CA4, decreased in field CA3, and was not seen in field CA1, though granule neuron death in the dentate fascia increased with increasing age. In addition, there were decreases in the sizes of pyramidal neuron cell bodies in all hippocampal fields. All hippocampal fields showed activation of astrocyte reactions, more marked in field CA4, where gliosis persisted to prepubertal age. Hypoxia in the early postnatal period could have effects on synaptogenesis, particularly on the formation of giant synapses in the dentate fascia. Studies of the functional characteristics of the nervous system in such animals showed that hypoxia could induce significant impairments in behavioral reactions. Rats of the experimental group showed impairments to the inhibitory functions of the cerebral cortex, increased anxiety, and disturbances to spatial learning and working memory.

The expression of the serotonin transport membrane protein (5-NTT) in the dorsal raphe nucleus (DNR) was investigated in laboratory Wistar rats during the early postnatal period. The results of the immunocytochemical study using primary antibodies--anti-Serotonin transporter antibody (AbCam, UK)--showed that during the first 3 postnatal weeks the intensity of 5-NTT expression in DNR of control animals changes. At the earliest postnatal times the main part of subnuclear neurons (dorsal, ventral and lateral ones) of the dorsal raphe nucleus (DNR-d, DNR-v, DNR-lat) was shown to intensely express 5-NTT. Sites of 5-NTT localization are found on the membrane surface of neuron bodies and processes in neuropile. The reduction in the number of neurons expressing 5-NTT and of its binding sites was observed on P10. At this time a redistribution of 5-NTT localization sites occurs: they are very few on neuron bodies and dendrites but are located rather densely on the plasma membrane of axons. The number of neurons expressing 5-NTT gradually increases with age and in neuropile the density of 5-NTT localization sites rises. It is shown that during the prenatal development the reduction of serotonin level in all parts of the DNR leads to a reduction in both the number of neurons expressing 5-NTT and sites of its localization in the early postnatal period, this trend continuing with age.

The work was performed on Wistar rats, which were dividedinto 3 groups: 1st group--experimental rats subjected to hypoxia and treated with salifen (15 mg/kg for 14 days), 2nd group--control rats exposed to hypoxia only without treatment, and 3rdgroup--intact animals (8-10 animals in each group). Using themethods of light and electron microscopy, the effect of salifen onthe structural characteristics of the elements of the blood-brainbarrier (BBB) in the neocortex was studied in rats after exposureto hypoxia in the early postnatal period--on postnatal Day 2(model of human preterm pregnancy). The results showed thatsalifen had a positive effect on the state of the microvasculatureafter perinatal hypoxia, in particular, on the state of endothelialcells. Its active participation in the compensatory-adaptive reactions of the BBB in response to hypoxia exposure was detected,and the prospects of further studies of the protective properties ofsalifen are emphasized.

The expression of serotonin transporter (5-HTT) was examined using the immunocytochemical method in the ventrolateral part of the nucleus of the solitary tract in Wistar rats in the early postnatal period (Days 5 and 10) in norm (n=10) and after prenatal serotonin deficiency (n=12). Temporary expression of the 5-HTT was demonstrated during the early postnatal period in the ventrolateral division of the nucleus of the solitary tract, that was more pronounced in the most caudal part of the ventral submucleus. It was shown that in the rostral part of ventral and lateral subnuclei, the number of neurons synthesizing 5-HTT was low and remained unchanged with age. In the caudal region of the ventral subnucleus, a large number of neurons synthesizing 5-HTT was found on Day 5 of postnatal period, that was significantly reduced with age. In the caudal region of the lateral subnucleus, a small number of neurons expressing 5-HTT was detected, that also decreased with age. It was found that in the caudal region of both ventral and lateral subnuclei, the level of 5-HTT expression was significantly higher than in the rostral region. Prenatal deficiency of serotonin resulted in a reduction of the number of neurons synthesizing 5-NTT in all the subnuclei investigated.

For the last time a particular interest of investigators has been attracted to the period of early newborn state when active process of adaptation of the organism to new life conditions occur defining its increased sensitivity to the effect of unfavorable environmental factors. An important place among these processes belongs to formation of homeostasis mechanisms and, primarily, the barrier mechanisms. The purpose of the present study was to investigate reactions of the hematoencephalic barrier (HEB) to action of perinatal normobaric hypoxia (a model of incomplete human pregnancy). Using light and electron microscopy, our investigation showed that after action of hypoxia all wall elements of neocortex capillaries revealed structural alterations which may cause impairment of microcirculation and increased permeability of HEB. It is established that differentiation of the basal membrane of a capillary wall takes place during the early perinatal period and the indicator of its differentiation is the formation of its plates - laminae rara et densa. After action of hypoxia, besides a delay of formation of the basal membrane, a vesicular type of its degeneration occurs. Key words: perinatal hypoxia, hematoencephalic barrier, capillary, endothelial cells, basal membrane.

The study has shown that acute hypoxia in newborn rat pups can lead to disturbances in processes of formation of brain structures, behavior reactions, and learning in the subsequent ontogenesis. The single normobaric hypoxia at the 2nd day of life causes retardation of such integrative parameter or general development and growth as body mass at the period of feeding. In such animals, essential disturbances of the sensorimotor development were revealed in forms of delay of reflex reactions of turning on a plane, negative geotaxis, and avoidance of edge. Also detected was action of hypoxia on hanging on a rope by using front legs (a symptom of muscle weakness). Morphological study has shown all studied functional zones of neocortex (motor, sensomotor, auditory, visual) to response stereotypically to the early postnatal action of hypoxia. The death of nerve cells is predominant in the II–III associative layers, the sizes and number of pyramidal neurons are sharply decreased. Different hippocampus fields maturing after death in mammals show a characteristic response to hypoxia. In individual hippocampus fields there was detected a different degree of death of neurons, with predominance in the CA3 and CA4 fields. A possibility of modeling the perinatal encephalopathy with minimal brain dysfunctions in children is discussed.

In this work an expression of serotonin transport protein (5-HTT) was studied in the dorsal raphe nucleus (DRN)--in its dorsal, ventral and lateral subnuclei in Wistar rats (n = 15) during the early postnatal period. Histological methods and immunocytochemical staining demonstrating 5-HTT were used in the investigation. It was shown that at postnatal Day 5 major part of neurons of the subnuclei studied intensively expressed 5-HTT. However, by Day 10, the level of its expression decreased dramatically, but with age (by Day 20) the expression was increased once again. This was manifested by the augmentation of size of 5-HTT-positive neuronal population, the increase in the density of plexus network of their processes. The detected changes of 5-HTT expression indicate the varying degrees of functional activity of serotonin in the DRN in the early postnatal period.

The aim of the present work was to study the long-term consequences of acute normobaric hypoxia during the perinatal period on the establishment of the hippocampal formation in rats. The experiments showed that hypoxia on day 2 of postnatal development led to significant damage to the structure of the hippocampal field, which showed different levels of sensitivity to the harmful factor. On day 20, all fields showed cell death and depletion of the pyramidal cell layers. The most marked neuron death occurred in fields CA4 and CA3. On day 30, a significant level of neuron death persisted in field CA4, though it decreased in field CA3 and was not seen in field CA1; neuron death in the granule layer of the dentate fascia increased. In addition, there were decreases in the sizes of pyramidal neuron bodies in all hippocampal fields. All hippocampal fields also showed activation of astrocyte reactions, which was more marked in field CA4, where gliosis persisted to prepubertal age (30 days).

The role of serotonin in the development of the nucleus raphe pallidus (NRP) and the dynamics of changes in serotonin-synthesizing neurons in this nucleus were studied at different points in the postnatal period in Wistar rats in normal conditions and in animals whose intrauterine development proceeded on the background of serotonin deficiency. The NRP was found to include two populations of serotoninergic neurons, differing in terms of their morphological characteristics. At the initial stages of postnatal development (day 5), only large neurons synthesized serotonin, while the synthetic activity of small neurons appeared later, by day 10. In normal animals, increasing age was associated with increases in the size and number of large neurons, which may be evidence for a continuing process of differentiation and/or an increase in the functional loading. The size and number of small neurons showed virtually no change with age. Serotonin deficit during prenatal development led to abnormalities in the structural organization of the NRP. As compared with controls, there were reductions in the sizes and numbers of serotonin-synthesizing neurons of both populations, with no change in size with age. Some neurons developed degenerative changes, reducing the number of cells. The damage seen would alter the serotoninergic innervation of the medullary nuclei responsible for cardiorespiratory regulation, and would thus impair the activity of the cardiovascular and respiratory systems.

Changes of expression of contractile proteins (smooth muscle cell α-actin and myosin) and of type IV collagen in villous stroma of human placenta were studied at the diagnosed placental insufficiency (PI) in III trimester of pregnancy. The study revealed pronounced disturbances of expression of contractile proteins and type IV collagen at PI. It is shown that in perivascular sheaths of vessels of stem and intermediate villi there is present a much greater amount of cells expressing smooth muscle actin and myosin. These cells are arranged by the denser concentric layers and more compactly than in norm and fill the intervascular space inside the villi. The width of perivascular sheaths of vessels is higher, while vascular lumens are lower than in norm. In terminal villi the capillary walls are thickened and the number of pericytes immunopositive against the smooth muscle cell α-actin and myosin as well as type IV collagen is increased. The change of synthesis of the cytoskeletal contractile proteins and type IV collagen is shown to lead to structural disturbances of villi of different types and of perivascular areas and vessels, which doubtlessly indicates their participation in pathogenesis of placental dysfunction and of disturbance of placental hemodynamics.

Study of born placentas with chronic functional failure established in the III pregnancy trimester has revealed several characteristic structural alterations of placental villi and of its vascular bed elements. There has been shown a decrease of the number of terminal villi and an increase of their sizes (approximately 3 times) as compared with norm, a change of transformation of cytotrophoblast into syncytiotrophoblast, thickening of vascular endothelium, a decrease of the number of capillaries-sinusoids, and a decrease of the number of syncytiocapillary membranes, which leads to deterioration of conditions of the maternal-fetal diffuse exchange. It has been established that in placental failure, expression of the vascular endothelial growth factor (VEGF) by cytotrophoblast cells, syncytiotrophoblast, and Kashchenko-Hoffbauer cells is enhanced as compared with norm, which can be considered an adaptive reaction to a decrease of intensity of placental blood circulation.

It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing at the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.