Influence of ethanol on neonatal cerebellum of BDNF gene‐deleted animals: analyses of effects on Purkinje cells,apoptosis‐related proteins,and endogenous antioxidants

The sensitivity of the developing central nervous system (CNS) to the deleterious effects of ethanol has been well documented, with exposure leading to a wide array of CNS abnormalities. Certain CNS regions are susceptible to ethanol during well‐defined critical periods. In the neonatal rodent cerebellum, a profound loss of Purkinje cells is found when ethanol is administered early in the postnatal period [on postnatal days 4 or 5 (P4–5)], while this neuronal population is much less vulnerable to similar ethanol insult slightly later in the postnatal period (P7–9). Prior studies have shown that neurotrophic factors (NTFs) can be altered by ethanol exposure, and both in vitro and in vivo studies have provided evidence that such substances have the potential to protect against ethanol neurotoxicity. In the present study, it was hypothesized that depletion of an NTF shown to be important to cerebellar development would exacerbate ethanol‐related effects within this region, when administration was confined to a normally ethanol‐resistant ontogenetic period. For this study, brain‐derived neurotrophic factor (BDNF) gene‐deleted (“knockout”) and wild‐type mice were exposed to ethanol via vapor inhalation or to control conditions during the normally ethanol‐resistant period (P7 and P8). Two hours after termination of exposure on P8, analyses were made of body weight, crown‐rump length, and brain weight. In subsequent investigations, the number and density of Purkinje cells and the volume of cerebellar lobule I were determined, and the expression of anti‐ and pro‐apoptotic proteins and the activities of endogenous antioxidants were assessed. It was found that the BDNF knockouts were significantly smaller than the wild‐type animals, with smaller brain weights. Purkinje cell number and density was reduced in ethanol‐treated knockout, but not wild‐type animals, and the volume of lobule I was significantly decreased in the gene‐deleted animals compared to wild‐types, but was not further affected by ethanol treatment. The loss of Purkinje cells in the BDNF knockouts was accompanied by decreases in anti‐apoptotic Bcl‐xl and in phosphorylated (and hence inactivated) pro‐apoptotic Bad, and reduced activity of the antioxidant glutathione reductase, while the antioxidant catalase was increased by ethanol treatment in this genotype. In the wild‐type animals, anti‐apoptotic Bcl‐2 was decreased by ethanol treatment, but the pro‐apoptotic c‐Jun N‐terminal kinase (JNK) was markedly diminished by ethanol exposure, while the activity of the protective antioxidant superoxide dismutase (SOD) was significantly enhanced. These results suggest that neurotrophic factors have the capacity to protect against ethanol neurotoxicity, perhaps by regulation of expression of molecules critical to neuronal survival such as elements of the apoptosis cascade and protective antioxidants. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 160–176, 2002     

Cumulative effects of alcohol (ethanol) on the activity of Purkinje cells in the cat cerebellum

The effects of three consecutive injections of 2 ml/kg 30% alcohol (ethanol, i.v.) on the activity of identified cerebellar Purkinje cells were investigated during experiments on adult cats anesthetized by a Nembutal-chloralose mixture. It was found that repeated alcohol injections exert a cumulative effect on the firing rate of these cells. The alcohol-induced rise in Purkinje cell firing rate, produced by excitation of the mossy fibers, was generally accompanied by a reduction in that of cells responding to excitation of climbing fibers. The inhibitory pause occurring after complex discharges in cerebellar Purkinje cells grew shorter with successive alcohol injections.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 74–80, January–February, 1987.     

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.     

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.     

Age-related changes in activity of cerebellum Purkinje cells,shape of the complex spike,and locomotion of Wistar rats under effect of ethanol

The work deals with study of peculiarities of effect of ethanol upon the Purkinje cell activity, shape of the complex spike, and locomotion of rats at different stages of ontogenesis, such as the stage of the morphofunctional maturation of the cerebellar cortex, the mature stage, and in the process of aging. The experiments were carried out on three age groups of Wistar rats: rat pups (2 weeks), adult rats (3–6 months), and senile animals (22–26 months). The administration of ethanol has been established to produce an increase in frequency of simple spikes, a decrease in frequency of complex spikes, a shortening of duration of depression of simple spikes, a decrease in the total duration of the complex spike, the number and frequency of its impulses as well as reduction of the motor activity of animals of all age groups. The change of the majority of the studied parameters occurred by the common temporal scheme. The earliest responding were the rat pups, later-the adult rats, and the last-the animals of the senior group. The stronger effect of ethanol was observed in adult rats. Their differences of all studied parameters, as compared with rat pups and senile animals, were characterized on the whole by the longer period of time and by the higher percent of changes relative to the initial values. Analysis of the obtained results has shown that the most pronounced changes in parameters of the cerebellum Purkinje cell activity and of the complex spike shape corresponded to the more significant decrease in the locomotion level, i.e., were recorded in adult rats. Thus, the work has demonstrated different sensitivity to administration of ethanol in the Wistar rats at different stages of ontogenetic development.     

Influence of gastrin on the expression of cyclooxygenase-2, hepatocyte growth factor and apoptosis-related proteins in gastric epithelial cells.

BACKGROUND: Several studies have shown a link between gastrin and gastric cancer, both in humans and animals, especially infected with Helicobacter pylori (H. pylori). However, the exact role of hypergastrinemia in gastric carcinogenesis remains still undetermined. The aim of the present study was to evaluate the interaction between gastrin, cyclooxygenase-2 (COX-2), hepatocyte growth factor (HGF) and apoptosis-related proteins (Bax, Bcl-2, caspase-3, survivin) in cultured gastric epithelial cancer cells. MATERIAL AND METHODS: In the present study, gastric cultured cancer cells (KATO III cells) were exposed to increasing concentrations of gastrin (1-1000 nM). Cells incubated with culture medium alone, without added gastrin, served as controls. Using RT-PCR and Western blot, we examined the mRNA and protein expression for COX-2, HGF and apoptosis-related proteins (Bax, Bcl-2, caspase-3 and survivin). In addition, the gene expression of gastrin and gastrin receptor (CCK-2) as well as the release of gastrin in culture medium in the unstimulated cells were examined by RT-PCR and RIA, respectively. The apoptosis rate in cells was measured by flow cytometric analysis. RESULTS: The present study shows that the gastric cultured epithelial cells exhibit the expression of gastrin and CCK-2 receptors and release of gastrin into the culture medium. The epithelial gastric cancer cells incubated with gastrin showed a concentration-dependent increase of COX-2 and HGF expression. Although no significant changes in apoptosis rate were observed, the exposure of these cells was associated with a dose-dependent increase in the expression of antiapoptotic proteins Bcl-2 and survivin. CONCLUSIONS: This study demonstrates that 1) gastrin stimulates the gene and protein expression of COX-2 and HGF in human cultured gastric cancer cells and 2) gastrin shows antiapoptotic activity through the upregulation of Bcl-2 and survivin.     

Autoradiographic research on the dark and light Purkinje cells in the cerebellum of rats

Intensity of the 3H-sodium acetate and 3H-leucine incorporation into dark and light Purkinje cells of the rat cerebellum was studied. The intensity of incorporation into light Purkinje cells was found to be 1.5 times higher than into the dark ones.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in mature- and immature-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV–VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC in the form of simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, on average, from 1 to rarely 11 active PC are recorded in the single microelectrode track. At the one-week age, the highest number of active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals it again exceeds, although slightly, the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in precocial- and altricial-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV-VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC represented by simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, from 1 to rarely 11 active PC are recorded, on average, in the single microelectrode track. At the one-week age, the highest number active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of the cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals exceeds again, although slightly of the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

Co-cultures of inferior olive and cerebellum: Electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca²⁺-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Co-cultures of inferior olive and cerebellum: electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons.

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca(2+)-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.     

Comparison of the effect of gamma-aminobutyric acid and its derivative baclofen on the activity of Purkinje cells of frog cerebellum in vitro

The inhibitory effect of gamma-aminobutyric acid (GABA) and its synthetic derivative baclofen were compared in frog cerebellum in vitro. Baclofen inhibited synaptic transmission from parallel fibres to the Purkinje cells in EC50 concentrations approximately 200-fold lower than for GABA. In addition to its inhibitory effect, GABA induced temporary facilitation of responses in the dendrite zone by a mechanism dependent on the presence of a normal Cl- concentration; the inhibitory phase was only partly sensitive to reduction of the Cl- concentration in the medium and to the administration of picrotoxin. The action of baclofen, which was unaffected by these treatments, requires an intact catecholamine and serotonin pool, since it is ineffective in reserpine-treated animals. Both substances also influence the excitability of parallel fibres. In solutions with a high Mg2+ and a low Ca2+ concentrations GABA inhibits the spontaneous activity of Purkinje cells by acting on the postsynaptic membrane of the soma and the primary dendrites. The effect of baclofen is evidently the outcome of inhibition of transmitter release from presynaptic endings.     

Effects of hypoxia, anoxia, and endogenous ethanol on thermoregulation in goldfish, Carassius auratus

Effects of hypoxia, anoxia, and endogenous ethanol (EtOH) on selected temperature (T(sel)) and activity in goldfish were evaluated. Blood and brain EtOH concentrations ([EtOH]) and brain oxygen partial pressure (PO(2)) were quantified at crucial ambient oxygen pressures. Below a threshold value near 31 Torr, T(sel) decreased as a function of environmental PO(2). T(sel) of 15 degrees C-acclimated fish was approximately 10 degrees C at the onset of anoxia and changed little over 2 h. Activity showed a similar response pattern. Brain [EtOH] was significantly elevated above control levels after 1 h anoxia. In normoxic water, T(sel) remained different in previously anoxic and normoxic control fish for approximately 20 min. Blood [EtOH] of previously anoxic fish remained significantly elevated ([EtOH] >4.0 micromol/g blood), and activity was significantly depressed at 20 min. Brain PO(2) reached normal levels in <3 min. We conclude that [EtOH] (brain or blood) and brain PO(2) are not proximal causes of either behavioral anapyrexia (hypothermia) or inactivity in goldfish exposed to oxygen-depleted environments.     

Effect of Denzimol, a new anticonvulsant drug, on rat cerebellum. Morphological and histochemical patterns of Purkinje cell layer

The effects of Denzimol, a new anticonvulsant drug, were analyzed in the Purkinje cell layer of rat cerebellum. No modifications in the general histochemical and ultrastructural patterns were induced by chronic administration of the drug. A slight increase in the storage of lipopigments in the cytoplasm of Purkinje neurons was found. No effects on secondary fluorescence (indicative of changes in biogenic amines) were found while an increase in specific SSADH activity, a degradative enzyme in GABA metabolism was present. Some observed histological and ultrastructural alterations (presence of "balloned" Purkinje neurons showing, in particular, enlarged cisternae of the endoplasmic reticulum) may be a stress effect probably due to the gastric tube insertion by which the drug was administered.