A. V. Bannova

Region-Specific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and hippocampus respectively were also in a good agreement with apoptotic proteins expression in these structures. In the cortex, hippocampus and cerebellum DNA fragmentation intensity was proportional to the active caspase-3 level. In contrast to these structures, in the brainstem, the lowest level of this protease was accompanied by the highest intensity of DNA fragmentation among the brain regions studied. The data suggest that cell death normally occurring during early postnatal life could be realized in the developing brainstem via caspase-3-independent pathways in animals that express this protease.

Quantitative Evaluation of DNA Fragmentation

A new method for evaluation of DNA fragmentation in tissue is based on computer-aided densitometric standardization of the sum of optical densities of DNA fragments visualized by electrophoresis (180-2500 b. p.) in comparison with the density of high molecular weight DNA in the same sample. The method allows evaluation of DNA fragmentation, an acknowledged marker of apoptosis, in the fetal rat brain and detection of increased DNA fragmentation during stress and under the effect of glucocorticoids.

Dexamethasone suppresses the locomotor response of neonatal rats to novel environment

Locomotion of animals in the novel environment is determined by two main factors-the intrinsic motor activity and the specific locomotor response to novelty. Glucocorticoids alter neurobehavioral development of mammals and its locomotor manifestations. However, it remains unclear whether the intrinsic and/or the novelty-induced activity are affected by glucocorticoids during early life. Here, the principal component analysis was used to determine the main factors that underlie alterations in locomotion of rat pups treated with dexamethasone. It was shown that neonatal rats exhibited an enhanced locomotion in the novel environment beginning from postnatal day (PD) 5. We found for the first time that this reaction was significantly suppressed by dexamethasone. The effect was specific to the novelty-induced component of behavior, while the intrinsic locomotor activity was not affected by glucocorticoid treatment. The suppression of the behavioral response to novelty was maximal at PD7 and vanquished at PD10-11. In parallel with the hormonal effect on the behavior, dexamethasone upregulated the main cell death executor-active caspase-3 in the prefrontal cortex of 7-day old rats. Thus, dexamethasone-induced alterations in the novelty-related behavior may be the earliest visible signs of the brain damage that could lead to forthcoming depressive state or schizophrenia, emerging as a result of neonatal stress or glucocorticoid treatment.

Bax and Bcl-XL apoptosis protein mRNA in rat brain stem and cortex during ontogeny.

Bax mRNA level in fetal rat brain stem increases by day 40 of life and then decreases, while level of Bcl-XL mRNA reaches the adult value over one month. Bax mRNA level in the cerebral cortex decreases from day 8 to day 90 of life, while Bcl-XL mRNA level does not change. Judging from Bcl-XL/Bax mRNA ratio, cortical cells exhibit higher readiness to apoptosis than brain stem cells.

The interrelationship between BDNF and its precursor and the level of active caspase-3 in the brain regions of neonatal rats

Brain-derived neurotrophic factor (mat-BDNF) promotes neuronal survival, whereas its precursor protein (pro-BDNF) induces cell death, which indicates the opposite but potentially important roles of both forms of this neurotrophin in brain development. We studied the contents of mat- and pro-BDNF and active caspase-3 in the brain regions of 8-day-old rat pups. Taking the level of caspase-3 in the cerebral cortex into account we may conclude that intense apoptosis occurs in this structure; however, the ratio of mat-BDNF to pro-BDNF in this region was the lowest among all structures studied. In contrast, in the brainstem of neonatal rats, where processes of cell proliferation and elimination are completed, the mat-BDNF/pro-BDNF ratio was substantially higher compared to the cortex. The mat-BDNF/pro-BDNF ratio in the cerebellum and hippocampus also supports the inverse relationship between this index and the level of active caspase-3 in different brain regions. Our data suggest that both forms of BDNF are involved in the determination of cell fate in the developing brain.

Effects of short-term exposure to lithium on antiapoptotic Bcl-xL protein expression in cortex and hippocampus of rats after acute stress

The antiapoptotic protein Bcl-xL is involved in development of neurobiological resilience to stress; hence, the possibility of use of psychotropic drugs to increase its expression in brain in response to stress is of considerable interest. Lithium is a neurotropic drug widely used in psychiatry. In work, we studied effects of lithium administration (for 2 or 7 days) on the expression of Bcl-xL mRNA and protein in the hippocampi and cortices of rats subjected to stress that induced depression-like behavior in the animals. In contrast to the brain-derived neurotrophic factor (BDNF), whose expression decreased in the hippocampus in response to acute stress, stress increased the level of Bcl-xL mRNA in the hippocampus, but decreased it in the frontal cortex. Treatment of stressed animals with lithium for 2 or 7 days increased Bcl-xL protein levels 1.5-fold in the hippocampus, but it decreased them in the cortex. Therefore, Bcl-xL expression in the brain can be modulated by both stress and psychotropic drugs, and the effects of these factors are brain region-specific: both stress exposure and lithium administration activated Bcl-xL expression in the hippocampus and suppressed it in the frontal cortex. The activation of Bcl-xL expression in the hippocampus by lithium, demonstrated for the first time in this study, suggests an important role of this protein in the therapeutic effects of lithium in the treatment of stress-induced psychoemotional disorders.

Effects of ligands of α2-adrenoceptors on mRNA level of apoptotic proteins in developing rat brain

The effects of antagonist of α2-adrenoceptors yohimbine and their agonist clonidine on Bax and Bcl-XL mRNA levels in neonatal rat brain were studied. Yohimbine decreased Bax mRNA level in the cerebellum, increased the ratio between Bcl-XL and Bax mRNA levels in the cerebellum, cortex, and hippocampus, and increased Bcl-XL mRNA level in the cortex and hippocampus of 6-day-old-rat pups 24 h after injection. Administration of clonidine 20 min after yohimbine administration abolished its effect on Bcl-XL mRNA level in the hippocampus. The data obtained indicate that the blockade of α2-adrenoceptors induces antiapoptotic changes in the developing rat brain, some of which can be abolished after coadministration with the agonist of these receptors.

Negative regulation of caspase-3 expression in the neonatal cerebral cortex by α2A-adrenoceptors

Antisense oligonucleotide to α2A-adrenoceptors increased the levels of mRNA (reverse polymerase chain reaction) and protein for a key executioner protease of apoptosis caspase-3 (immunoblotting assay) in the cerebral cortex of newborn rats. The relationship between the observed effect and low expression of α2-adrenoceptors was confirmed by the possibility of correcting this phenomenon by clonidine (stimulator of α2-adrenoceptors).

Anoxia ameliorates the dexamethasone-induced neurobehavioral alterations in the neonatal male rat pups

&NA; Glucocorticoids and hypoxia are two essential factors affecting the brain development during labor and delivery. In addition to the neurobehavioral alterations induced separately by these factors, glucocorticoids can attenuate the deleterious consequences of severe hypoxia‐ischemia on the brain development, acting as a neuroprotective agent in combination with hypoxia. The role of hypoxia in the combined action with corticosteroids is less clear. Severe hypoxia‐ischemia results in the massive activation of caspase‐3, masking any other effects of hypoxia on the neonatal brain exposed to glucocorticoids. As a result, the effects of mild hypoxia on the developing brain pretreated with glucocorticoids remain unclear. To analyze this problem, 2‐day‐old male rats were treated with dexamethasone (DEX) before the subsequent exposure to mild 10‐min anoxia or normoxia. The treatment with only DEX resulted in the delay in the development of the negative geotaxis reaction and in the decrease in locomotor activity of the neonatal male pups. The mild anoxic event attenuated these DEX‐induced neurobehavioral alterations. The treatment with DEX, but not the mild anoxic exposure alone, resulted in the delayed upregulation of active caspase‐3 in the prefrontal cortex and in the brainstem of the male pups. This glucocorticoid‐induced upregulation of active caspase‐3 was prevented by the anoxic event. The present findings evidence that mild anoxia is capable of ameliorating the glucocorticoid‐induced neurodevelopmental alterations in the neonatal rats if the artificial or the naturally occurring increase in the levels of glucocorticoids occurred just before the episode of hypoxia. HighlightsDexamethasone delays development of the negative geotaxis reaction in the rat pups.Dexamethasone decreases locomotor activity of the rat pups in the novel environment.Mild anoxia attenuates early neurobehavioral alterations induced by dexamethasone.Dexamethasone but not anoxia upregulates active caspase‐3 in the cortex and brainstem.Dexamethasone‐induced upregulation of active caspase‐3 is ameliorated by anoxia.

Estimation of an area between the baseline and the effect curve parameter for lactate levels in the hippocampi of neonatal rats during anesthesia

Naturally occurring caspase-3-dependent cell death is a widespread event in the immature nervous system. Prolonged exposure to anesthetics promotes activation of caspase-3 in the developing hippocampus. In addition, anesthetics can upregulate the levels of metabolite lactate in the adult brain. The long-lasting increase in lactate levels may affect viability of brain cells. However, it remains unknown if anesthetic-induced activation of caspase-3 is accompanied by an increase in lactate levels in the immature brain. We investigated expression of apoptotic proteins by immunoblot and estimated an area between the baseline and the effect curve (ABEC) parameter for lactate levels by high-resolution magnetic resonance spectroscopy in the hippocampi of 2-day-old Wistar rats after treatment with anesthetic urethane. Both 1.5 and 2.5 g/kg of urethane resulted in a dose-dependent increase in the levels of active caspase-3 in the hippocampi in 4 h after injection. This anesthetic-induced increase in the levels of active caspase-3 was preceded by a prolonged dose-dependent rise in lactate levels. The dose-dependent increase in lactate levels was not associated with the urethane-induced changes in respiratory rate in the treated rat pups. Present results evidence that the prolonged dose-dependent elevation in lactate levels in the developing brain can be induced even by urethane, which was suggested to be suitable for various physiopharmacological studies previously. The observed sequence of events after treatment with urethane suggests the possible role of lactate as a neurodamaging agent in the immature brain in case of the sustaining rise in the levels of this metabolite during prolonged anesthesia.