Paternal alcohol exposure: developmental and behavioral effects on the offspring of rats.

The effect of paternal alcohol exposure on neurochemical and behavioral parameters was investigated using as a model system glial cells derived from newborn rat brain and cultured for 4 weeks. The total brain neurochemical parameters from rats born to mothers sired by an alcohol treated father were also investigated. Enzymatic markers of nerve cell development (enolase isoenzymes and glutamine synthetase) and the defense system (superoxide dismutase) against free radicals formed during alcohol degradation were measured in order to evaluate nerve cell damage. Behavioral locomotor tests (open-field, novelty-seeking, light/dark) were carried out to show long-lasting effects of paternal alcoholization on the offspring. Behavioral and developmental alterations were found until 1 year of age in the offspring and a significant growth retardation was observed in the males. Our results suggest that paternal alcohol exposure produces developmental and behavioral effects in the offspring. The consequence of either alcohol withdrawal during stage one spermatogenesis, or maternal diet supplementation with manganese during pregnancy were investigated. It was observed that some of the effects of paternal alcohol exposure on the offspring may be reversed by these treatments.

An improved method for the preparation of rat cerebellar glomeruli.

Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransmitter receptors.

Factors involved in expression of neuron-specific and non-neuronal enolase activity in developing chick brain and in primary cultures of chick neurons.

The effect of various factors affecting non-neuronal enolase (NNE) and neuron-specific enolase (NSE) was investigated in developing brain of two different chick strains, in primary cultures of pure neurons and of mixed cultures of neuronal and glial cells. NNE and NSE activities reached their maximum at an earlier stage of brain development in the fast growing Hybro strain than in the Leghorn strain. In pure neurons cultured during 6 days, NNE was stimulated by hydrocortisone in presence or in absence of serum. Dibutyryl cyclic AMP (diBcAMP) stimulated NNE only in serum-free medium. NSE activity was increased by glial cell-conditioned medium in presence of serum and by removal of serum from the medium. Hydrocortisone and diBcAMP had no effect on NSE. In mixed cultures of neurons and glial cells both enolase activities were raised in absence of serum. Hydrocortisone and diBcAMP had no effect. Steroid hormones, insulin and serum albumin also modify both enolase activities in pure neurons and in mixed cultures of neurons and glial cells. Our results suggest that NNE and NSE are regulated separately by various factors involved in nerve cell maturation.

Neuron-specific and non-neuronal enolase in developing chick brain and primary cultures of chick neurons.

Developmental changes of the neuron-specific enolase (NSE) and the non-neuronal enolase (NNE) were determined in pure neuronal, or mixed cultures of neuronal and glial cells, prepared from brains of 8-day-old chick embryos, as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain during the development of the nervous tissue in the embryo and during the post-hatching period. In the whole brain, the total enolase activity increases progressively until the adult level, the NSE appears only 4 days before hatching. The total enolase activity of pure neurons increases until 8 days of culture but the NSE never appears during this period. Mixed cultures of neurons and glial cells present an increase of total activity until 14 days of culture and neuron-specific form of the enzyme can be observed after 6 days of culture. Our results suggest that NSE appears only in the functional brain and is expressed in cultivated neurons only when glial cells are present simultaneously.

Comparative investigations of glutaminase development in cerebral cortex of chick embryo and in primary cultures of neurons and glial cells.

Glutaminase activity was determined in pure cultures of neurons, glial cells and in mixed cultures obtained from chick embryo brain. The development of this enzyme was observed periodically over time and compared to its evolution in corresponding cerebral hemispheres during embryonic and postnatal development. The specific activity of brain glutaminase increased between the twelfth and sixteenth day of embryogenesis. A similar increase was observed in cultures of neuroblasts during the corresponding period of time, although the activity in culture was about one-third lower than in vivo. In contrast to neurons, there was no significant increase of glutaminase activity in glial cells before the fifteenth day of culture. The enzyme level in glial cells between the thirteenth and fifteenth days of culture was approximately 25% of that in 7- and 8-day-old neurons. The different development of glutaminase activity in neurons and glial cells was demonstrated in both pure and mixed cultures. The results support the hypothesis that there is a glutamine shunt from glial cells to neurons.

Glutamine synthetase and energy metabolism enzymes in cultivated chick neurons and astrocytes: modulation by serum and hydrocortisone.

Primary cultures of astroglial cells and of neurons obtained from chick embryos were grown in culture medium with and without serum added. The expression of glutamine synthetase (GS) in the cultured nerve cells was investigated immunocytochemically and biochemically. The cellular localization of GS in cerebellar tissue sections and in cerebral cortex of chick embryos was investigated by immunohistochemical staining. In tissue sections the enzyme is only present in astrocytes and their processes; neurons and their structures do not express the enzyme. In contrast, in pure neuronal primary cultures, a high level of GS was detected by biochemical and immunochemical methods. Thus, our results clearly indicate the presence of GS in pure neuronal cell cultures and its absence in this type of cells in vivo. Removal of serum from the culture medium enhanced GS levels in primary astrocyte cultures, but was without effect on GS activity in neurons. Addition of calf serum to the culture medium induces a two-fold increase of cellular lactate dehydrogenase (LDH) activity in neurons by increasing specifically the M subunit containing isoenzymes. The sensitivity of chick astroglial cells and neurons toward the GS inducing effect of hydrocortisone and modulation of its effect by serum was also investigated. Differences in the sensitivity of the two types of nerve cells in culture toward the GS inducing effect of hydrocortisone, and the effect of serum could be demonstrated.

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: a double labeling immunohistochemical study.

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP.

Alcohol exposure before pregnancy: biochemical and behavioral effects on the offspring of rats.

The effect of maternal alcohol exposure before mating was investigated in the offspring over a period of 6 months concerning some specific aspects of energy metabolism in the brain and the liver. The following biochemical parameters were analyzed: superoxide dismutase (involved in elimination of free radicals produced during ethanol oxidation), enolase isoenzymes (markers of nerve cell maturation), and alcohol and aldehyde dehydrogenase (the main alcohol degradating enzymes). These enzymatic activities were measured at their subcellular level. In these animals never directly exposed to alcohol, superoxide dismutase activity was decreased mainly in the liver cytosol. Only the nonneuronal form of enolase activity was modified. Alcohol dehydrogenase was decreased in the liver as well as in the brain. Aldehyde dehydrogenase was also decreased in the liver and in the brain, mainly in the mitochondria. Behavioral observations showed decreased emotional reactivity as well as an increase in locomotor activity. Our results suggest that long-lasting biochemical and behavioral effects of alcohol may occur in the offspring starting at the earliest stage of development.

Evaluation of a laparoscopic grasper with force feedback.

BACKGROUND: The clinical implementation of laparoscopic tools with haptic feedback is far from becoming a reality, partly because current tools with force feedback are not configured like conventional surgical instruments. Hence, our goal was to incorporate force sensing into existing laparoscopic tools for use in robot-assisted surgery. METHODS: We developed a laparoscopic grasper with force-feedback capability that would help surgeons to differentiate tissue stiffness based on the measured tissue deformation. RESULTS: Surgeons and nonsurgeons alike were able to qualitatively characterize different tissue samples with a high degree of accuracy. The observed force vs deformation characteristic for various tissue samples confirmed the ability of our apparatus to characterize tissues of varying stiffness. CONCLUSION: This innovative laparoscopic grasper with force-feedback capability enables accurate characterization of tissue deformation and is a promising tool for minimally invasive surgery.

[Neuronal and astrocytic plasticity: metabolic aspects]. / Plasticité neuronale et astrocytaire: aspects métaboliques.

It is well known that brain function is critically dependent upon energy metabolism and that the brain has a relatively high metabolic rate. Experiments using intact brain preparations do not provide information about metabolism in the different cell types that constitute brain tissue. Progress in primary culture techniques has facilitated biochemical investigations and analysis of the metabolic pathways prevailing in specific cerebral cell types. We found that, in the presence of pyruvate or succinate as the substrate, oxygen consumption by neurons grown in culture was always higher than that by glial cells. The relatively low values of hexokinase, malate dehydrogenase and glutamate dehydrogenase activities observed in glial cells and, in contrast, the high levels of lactate dehydrogenase and enolase activities may be the result of a less aerobic metabolism prevailing in this type of brain cell, compared to neurons. On the other hand, the predominance of the aerobic, lactate dehydrogenase, isoenzymatic form in neurons can be associated with a more aerobic metabolism in this type of cell. In the case of severe hypoxia, we observed that astrocytes were the most damaged cells. An increased lactate dehydrogenase level with a modification of its isoenzymatic profile and a decreased glutamine synthetase activity under hypoxic conditions indicated severe derangement of important biochemical functions within the astrocytes. By antagonizing some of these changes, almitrine and raubasine (both present in Duxil) seem to exert some protective effect. One may consider that, among the different cell types present in brain tissue, astroglial cells may represent a target particularly sensitive to hypoxia-induced injury.

Modifications in energy metabolism during the development of chick glial cells and neurons in culture.

Developmental changes in lactate dehydrogenase (LDH), enolase, hexokinase (HK), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) activities were measured in cultures of pure neurons and glial cells prepared from brains of chick embryos (8 day-old for neurons, 14 day-old for glial cells) as a function of cellular development with time in culture. The modifications observed in culture were compared to those measured in brain extracts during the development of the nervous tissue in the chick embryo and during the post-hatching period. A significant increase of MDH, GDH, LDH, and enolase activities are observed in neurons between 3 and 6 days of culture, whereas simultaneously a decrease of HK values occurs. In the embryonic brain between 11 and 14 days of incubation, which would correspond for the neuronal cultures to day 3 through 6, modifications of MDH, GDH, HK, and enolase levels are similar to those observed in neurons in culture. Only the increase of LDH activity is less pronounced in vivo than in cultivated cells. The evolution of the tested enzymatic activities in the brain of the chick during the period between 7 days before and 10 days after hatching is quite similar to that observed in cultivated glial cells (prepared from 14 day-old embryos) between 6 and 18 days of culture. All tested activities increased in comparable proportions. The modifications of the enzymatic profile indicate that some maturation phenomena affecting energy metabolism of neuronal and glial elements in culture, are quite similar to those occuring in the total nervous tissue. A relationship between the development of the energy metabolism of the brain and differentiation processes affecting neuroblasts and the glial-forming cells is discussed.

Distribution of mitochondrial manganese superoxide dismutase among rat glial cells in culture.

Enzymatic antioxidant defense systems, like superoxide dismutase (SOD), may protect neuronal and glial cells from reactive oxygen species (ROS) damage. Beside the cytosolic constitutive CuZn SOD, mitochondrial manganese SOD (Mn SOD) represents a ROS inducible enzyme which should allow the adaptation of brain cells to variation in ROS concentrations resulting from their oxidative metabolism. Using immunocytochemistry, the distribution of Mn SOD among the various representatives of the rat brain glial population (astroglia and microglia in primary culture as well as oligodendroglia in secondary culture) has been examined. Among astroglial cells, only a population of flat polygonal-shaped astrocytes, highly immunostained for glial fibrillary acid protein (GFAP) express Mn SOD immunoreactivity. Microglial cells defined by their shape and OX-42 immunoreactivity also express an intense Mn SOD signal. Exposure of the primary culture to reactive oxygen species generated by a xanthine/xanthine oxidase mixture (X/XO) accentuates the Mn SOD signal in astroglial and microglial cells. On the contrary, oligodendroglial cells grown in secondary culture in a serum-free chemically defined or a serum-containing medium and well characterized by their 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) immunoreactivity never express any immunostaining for Mn SOD, even in response to an extracellular reactive oxygen species generating source like X/XO. Likewise, a population of A2B5-positive glial cells which may represent bipotential O-2A progenitor precursors does not express Mn SOD immunostaining. These results point out that in addition to the well known ability of microglial and astroglial cells to secrete ROS, they also express a high mitochondrial oxygen superoxide decomposition potential. On the contrary, the absence of any observable Mn SOD signal in precursors and in more differentiated oligodendroglial cells could be related to their great sensitivity to ROS damage and could therefore play an important role in the development of various dysmyelinating disorders.

Differential effect of pentobarbital on chick neurons and astrocytes grown in culture.

Primary cultures of neurons and astrocytes prepared from brains of 8-day-old and 15-day-old chick embryos. respectively, were grown for periods between 3 and 23 days. Cellular oxygen consumption was measured at various times in the presence of either pyruvate or succinate as substrate. Neuronal oxygen consumption was significantly higher than glial respiration, irrespective of the substrate employed. Dose-response curves for the effect of pentobarbital on respiratory activity of each cell type were constructed with the two substrates. In the presence of succinate neuronal respiration was more sensitive to pentobarbital than that of glial cells with a shift in the dose-effect curve by at least one order of magnitude. In the presence of pyruvate, glial cell respiration was inhibited at pentobarbital concentrations more than ten times lower than those effective in neurons. It is concluded that the differential sensitivity to pentobarbital between neurons and glia is due to differences in their respective energy metabolism.

Effect of maternal alcohol consumption on nerve cell development in the offspring.

The effect of prenatal alcohol exposure on nerve cell development was investigated in neurons and glial cells cultured from fetal rat brain. Neurons were grown for one week from two week-old cortical brain cells and glial cells were cultured during four weeks from new born cortical brain cells. Two situations were examined: maternal alcohol treatment before and during pregnancy and alcohol exposure only until the beginning of pregnancy. In both situations we found a delayed nerve cell maturation assessed by biochemical markers like enolase and glutamine synthetase. Alterations of lactate dehydrogenase activity and reduced superoxide dismutase activity, involved in free radicals elimination were also observed. "In vitro" addition of alcohol to the culture medium produced an additional effect showing a lower response in the prenatal alcohol exposed brain cells than in the controls. The results confirm our previous "in vivo" experiments showing long lasting effects of maternal alcohol exposure in the offspring.

Trypsinization of chick glial cells before seeding: effects on energy metabolism enzymes and glutamine synthetase.

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine.

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells nor in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.

The effect of ethanol on HSP70 in cultured rat glial cells and in brain areas of rat pups exposed to ethanol in utero.

Prenatal exposure to alcohol is associated with a cluster of symptoms called Fetal Alcohol Syndrome with a characteristic pattern of neuroanatomy and biochemical changes. In recent years it has been shown, that stress exposed cells rapidly increase transcription and translation of heat shock protein genes resulting in an increased appearance of these proteins. It has also been found that heat shock proteins, especially the HSP70 family play a role as molecular chaperons maintaining the native conformation of proteins and participating in protein transport in particular cellular compartments. The aim of this study was to determine the effect of chronic maternal alcohol consumption on HSP70 content in the different regions of the brain of the newborn rats as well as to examine in vitro the effect of ethanol on HSP70 content in cultured glial cells. Chronic maternal ethanol consumption resulted in increased HSP70 in the following regions of developing brain: hippocampus, cerebellum, olfactory bulbs, frontal cortex and septum. Moreover, ethanol applied in vitro, increased HSP70 content in primary astroglial cultures and astrocytes but not in oligodendrocyte cultures. The above described changes may be important in brain maturation and may play a role in Fetal Alcohol Syndrome.

Development of glial cells in primary cultures: energy metabolism and lactate dehydrogenase isoenzymes.

Primary cultures of glial cells prepared from brains of newborn rats were grown for periods of 1-5 weeks. After a proliferative phase of between 2 and 3 weeks, the cultures were maintained in stationary phase, during which a significant increase of oxygen consumption and of the activities of lactate dehydrogenase, succinate dehydrogenase, and mitochondrial glycerolphosphate dehydrogenase could be observed. Furthermore, qualitative changes in the lactate dehydrogenase isoenzyme pattern were found with time, characterized by a shift toward an enhanced synthesis of H subunits. A similar development was found in comparing the LDH isoenzyme pattern in the brain of 15-day-old rat embryo with those of newborn and adult rat brains. It is suggested that some aspects of maturation of glial cells in culture are comparable to those occurring in whole brain in vivo, namely a shift towards an enhanced aerobic metabolism.

Activity and isoenzyme pattern of lactate dehydrogenase in neurons and astroblasts cultured from brains of chick embryos.

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8-day-old and 15-day-old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH-M4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH-H4, and there was a progressive appearance of all other isoenzymes over an 8-day period. These results support the hypothesis of a different energy metabolism in neurons and glia.