Showing papers in "Developmental Neuroscience in 1996"

Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress.

Abstract: The adrenal glucocorticoids and catecholamines comprise a frontline of defense for mammalian species under conditions which threaten homeostasis (conditions commonly referred to as stress). Glucocorticoids represent the end product of the hypothalamic-pituitary-adrenal (HPA) axis and along with the catecholamines serve to mobilize the production and distribution of energy substrates during stress. The increased secretion of pituitary-adrenal hormones in response to stress is stimulated by the release of corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) from neurons in the nucleus paraventricularis. In this way, a neural signal associated with the stressor is transduced into a set of endocrine and sympathetic responses. The development of the HPA response to stressful stimuli is altered by early environmental events. Animals exposed to short periods of infantile stimulation or handling show decreased HPA responsivity to stress, whereas maternal separation, physical trauma and endotoxin administration enhance HPA responsivity to stress. In all cases, these effects persist throughout the life of the animal and are accompanied by increased hypothalamic levels of the mRNAs for CRH and often AVP. The inhibitory regulation of the synthesis for these ACTH releasing factors is achieved, in part, through a negative feedback loop whereby circulating glucocorticoids act at various neural sites to decrease CRH and AVP gene expression. Such inhibitory effects are initiated via an interaction between the adrenal steroid and an intracellular receptor (either the mineralocorticoid or glucocorticoid receptor). We have found that these early environmental manipulations regulate glucocorticoid receptor gene expression in the hippocampus and frontal cortex, regions that have been strongly implicated as sites for negative-feedback regulation of CRH and AVP synthesis. When the differences in glucocorticoid receptor density are transiently reversed, so too are those in HPA responses to stress. Taken together, our findings indicate that the early postnatal environment alters the differentiation of hippocampal neurons. This effect involves an altered rate of glucocorticoid receptor gene expression, resulting in changes in the sensitivity of the system to the inhibitory effects of glucocorticoids on the synthesis of CRH and AVP in hypothalamic neurons. Changes in CRH and AVP levels, in turn, determine the responsivity of the axis to subsequent stressors; increased releasing factor production is associated with increased HPA responses to stress. Thus, the early environment can contribute substantially to the development of stable individual differences in HPA responsivity to stressful stimuli. These data provide examples of early environmental programming of neural systems. One major objective of our research is to understand how such programming occurs within the brain.

Regulation of Neuronal Birth, Migration and Death in the Rat Dentate Gyrus

Abstract: The granule cell population of the rat dentate gyrus forms over an extended period which begins during gestation and continues into adulthood. During the embryonic period, the postnatal period and in adulthood, granule cells proliferate, migrate and degenerate. We have found that granule cell production is dependent on the levels of circulating adrenal steroids and NMDA receptor-mediated excitatory input throughout life. In general, increases in adrenal steroid levels or NMDA receptor activation diminish the rate of cell proliferation whereas decreases in adrenal steroid levels or NMDA receptor activation increase the rate of cell production. This paper describes the regulation of granule cell proliferation, migration and survival by adrenal steroids and excitatory input and presents evidence that these factors may affect dentate gyrus-mediated behaviors.

The Estrogen/Neurotrophin Connection during Neural Development: Is Co-Localization of Estrogen Receptors with the Neurotrophins and Their Receptors Biologically Relevant?

Abstract: Estrogen enhances neurite growth within the developing rodent forebrain. Estrogen receptor mRNA is co-expressed with the mRNA for the neurotrophins and their receptors. Estrogen may act independently by altering growth-related genes directly, but may interact additionally with growth factors (neurotrophins) and their receptors (trks). Estrogen may also act permissively to facilitate neurotrophin actions by genomic cross-talk with neurotrophin regulatory pathways whose nuclear end points may be the same estrogen-responsive genes. Differential and reciprocal regulation of estrogen and nerve growth factor (NGF) receptor gene expression by their ligands suggests that estrogen and the neurotrophins may influence each other's actions by regulating receptor/ligand availability or by reciprocal regulation at the level of gene transcription or signal transduction. Steroid/neurotrophin interactions may stimulate the synthesis of proteins required for neuronal differentiation, survival and maintenance of function.

Immunohistochemical study on superoxide dismutases in spinal cords from autopsied patients with amyotrophic lateral sclerosis.

Abstract: A role mutations in the superoxide dismutase (SOD)-1 gene in the pathogenesis of amyotrophic lateral sclerosis (ALS) has been discussed. To investigate immunohistochemical alterations of SOD in the spinal cord affected with the disease, we examined 3 patients with SOD1 mutation-associated family with ALS, 20 patients with sporadic ALS and 10 control individuals. Lewy body-like hyaline inclusions (LBHIs) were seen in the anterior horn cells of all the familial patients and 10 of the 20 sporadic patients, while skein-like inclusions (SIs) and Bunina bodies (BBs) were present in the 20 sporadic patients but not in the familial patients. The primary antibodies used for immunostaining were rabbit antisera raised against human SOD1 and SOD2. The anti-SOD1 antibody reacted strongly with all LBHIs of each familial patient and with some LBHIs of each sporadic patient. The cytoplasm of morphologically intact and degenerated spinal cord neurons as well as spheroids seen in the cases examined was only weakly stained by the antibody to SOD1 or not at all. The reactive astrocytes displayed weak to moderate staining for SOD1. The anti-SOD2 antibody strongly immunolabeled the reactive astrocytes and microglia. LBHIs of both familial and sporadic ALS were negatively stained for SOD2. Spinal cord neurons and spheroids of each case exhibited no significant SOD2 immunoreactivity. Neither antibodies reacted with SIs nor BBs. These results indicate that SOD1 may be involved in the formative process of LBHIs especially in familial ALS but not always in that of SIs or BBs, and imply that SOD2 may have no connection with any of these ALS-related abnormal structures.

Neurogenesis in Adult Human

Abstract: THIS report describes neurogenesis in the adult human olfactory epithelium in vitro. Olfactory epithelium was collected at autopsy and by biopsy, and grown in serum-free medium. Basic fibroblast growth factor induced the differentiation of bipolar cells which were immunopositive for several neuronal proteins but not glial proteins. [3H]thymidine autoradiography confirmed that these neurones were born in vitro. The results demonstrate that the adult human olfactory epithelium retains the capacity for neurogenesis and neuronal differentiation, at least until the age of 72 years. It is now possible to examine neurones and neurogenesis in biopsies from patients with disorders that may involve a neurodevelopmental or neurodegenerative aetiology such as schizophrenia, bipolar disorder and Alzheimer's disease.

Juvenile Hormone, Behavioral Maturation, and Brain Structure in the Honey Bee

Abstract: Juvenile hormone regulates metamorphosis in insects, and its effects on the nervous system during the larval-pupal transition have been studied primarily in the hawk moth, Manduca sexta. <

Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implications for neuronal/astrocytic trafficking and neurodegeneration.

Abstract: In this study we have examined the susceptibility of the mitochondrial respiratory chain of astrocytes and astrocytes depleted of glutathione to peroxynitrite exposure. Astrocytes, as reported previously by us, appeared resistant to the actions of peroxynitrite. In contrast, depletion (-94%) of astrocytic glutathione rendered the cells susceptible with mitochondrial complexes I and II/III being decreased in activity by 80 and 64%, respectively, after peroxynitrite exposure. Furthermore, cell death, as judged by lactate dehydrogenase release, was significantly increased (+81%) in the glutathione-depleted astrocytes exposed to peroxynitrite. Glutathione depletion alone had no effect on any of the measured parameters. It is concluded that glutathione is an important intracellular defence against peroxynitrite and that when glutathione levels are compromised the mitochondrial respiratory chain is a vulnerable target and cell death ensues. In view of the relative paucity of neuronal glutathione, it is possible that astrocyte-derived peroxynitrite may, in certain pathological conditions, be released and diffuse into neighboring neurones where mitochondrial damage may occur.

Excitatory amino acids stimulate aerobic glycolysis in astrocytes via an activation of the Na+/K+ ATPase

Abstract: Astrocytes appear to be ideally localized to couple neuronal activity to energy metabolism. First, specialized processes, the astrocytic end-feet surround blood vessels, the source of glucose for the brain. Second, other processes ensheath synapses and express receptors and transporters for various neurotransmitters, endowing astrocytes with the capacity to 'sense' synaptic activity. We have previously described the stimulation by L-glutamate of glucose utilization by astrocytes, monitored by 2-deoxyglucose uptake and phosphorylation. Here we have further characterized the pharmacological characteristics and molecular mechanisms of this action, which involve a massive influx of Na+ as a result of the cotransport of the amino acid with Na+, by Na(+)-dependent transporters and a subsequent activation of the Na+/K+ ATPase. To fuel the ATPase, glucose is processed glycolytically thus leading to increased lactate production. Since excitatory amino acids are released during activation by cortical afferents, these data reveal a simple mechanism for coupling neuronal activity to glucose utilization and provide further evidence for the concept of a transient stimulation of aerobic glycolysis during activation occurring preferentially in astrocytes.

Development and regeneration of the nervous system: a role for neurosteroids.

Abstract: Several steroids, termed 'neurosteroids', are synthesized from cholesterol within both the central and peripheral nervous systems. These include pregnenolone and its sulfate ester, progesterone and it

'Hot spots' of creatine kinase localization in brain: cerebellum, hippocampus and choroid plexus.

Abstract: Creatine kinase (CK) isoenzymes, with emphasis on the mitochondrial CK isoenzymes, were characterized and localized in chicken cerebellum. Chicken cerebellar extracts analyzed by two-dimensional gels, using anti-peptide antibodies specific for sarcomeric muscle-type mitochondrial CK (Mib-CK) and revealed the presence of a Mib-CK variant in avian cerebellum. This CK isoform was localized by immunofluorescence staining exclusively in the Purkinje neurons. The co-expression of this Mib-CK together with cytosolic muscle-type MM-CK, as observed in the same Purkinje neurons, may reflect the specific energy requirements associated with highly fluctuating Ca2+ levels (Ca2+ spiking) in these specialized neurons. Ubiquitous brain-type mitochondrial Mia-CK was found together with cytosolic BB-CK mainly in the glomeruli structures of the cerebellar granular layer. BB-CK, but much less so Mia-CK, however, was also very prominent in Bergmann glial cells of the molecular layer. Thus, an isoenzyme-specific differential localization of the two mitochondrial Mi-CK isoenzymes in the chicken cerebellum is demonstrated. Other hot spots of CK localization were the granule and pyramidal cells of the hippocampus in rat. There, a developmental stage-dependent immunofluorescence staining, especially with antibodies against Mia-CK, was noted. Epithelial cells of the choroid plexus were also highly enriched in CK. The possible implications of a CK/PCr circuit at these various cellular locations of the brain are discussed with respect to normal brain physiology and pathology.

New Insights into the Compartmentation of Glutamate and Glutamine in Cultured Rat Brain Astrocytes

Abstract: Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production from U-[14C]glutamate and U-[14C]glutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.

Regulation of Oligodendrocyte Precursor Migration by Extracellular Matrix: Evidence for Substrate-Specific Inhibition of Migration by Tenascin-C

Abstract: In order to analyse the role of the extracellular matrix (ECM) in the migration of oligodendrocyte precursor cells we have used a chemotaxis chamber assay in which the filter separating the wells is coated with different ECM molecules. Two molecules, fibronectin and the laminin family member merosin, promoted migration either alone or in combination with the chemotactic growth factor platelet-derived growth factor. The effects of the ECM molecules and growth factor were additive, and demonstrated that the migrating oligodendrocyte precursors respond both to haptotactic and chemotactic stimuli. A third extracellular molecule, tenascin-C, inhibited migration. This inhibition was substrate-specific; while migration on fibronectin was inhibited there was no effect on merosin-stimulated migration. This specificity confirms that tenascin-C inhibits migration by modulation of individual cell-ECM interactions rather than by a non-specific process of interference with substrate adhesion. An understanding of the role of tenascin-C in central nervous system development will therefore require characterisation of the different colocalising ECM molecules at different developmental stages.

Steroid Receptors and Nervous System Metamorphosis in Insects

Abstract: During CNS metamorphosis, the ecdysteroids evoke diverse neuronal responses including remodeling, maturation, and programmed death. These responses are matched by the equally diverse expression of the

Progressive remodeling of the oligodendrocyte process arbor during myelinogenesis

Abstract: Myelin sheaths develop in the central nervous system (CNS) as elaborations of the processes of oligodendrocytes. Although many details of the spiral wrapping of oligodendrocyte processes around axons

A role for astrocytes in glucose delivery to neurons

Abstract: The present paper examines the possible role of astrocytes in the delivery of glycogen-derived glucose for neuronal metabolism. Such a process would require astrocytic expression of glucose-6-phosphatase. The degree and significance of brain expression of glucose-6-phosphatase (EC 3.1.3.9) has been a subject of controversy. Published immunohistochemical data are consistent with expression of glucose-6-phosphatase by astrocytes, both in vivo and in vitro. In this paper additional confirmation of the expression of glucose-6-phosphatase mRNA in rat brain is presented. Although cultured astrocytes demonstrate glucose-6-phosphatase activity in vitro under assay conditions, there is very limited in vitro evidence that this activity confers a glucose-export capacity on astrocytes. Under most conditions in vitro, lactate export predominates, however this may relate to aspects of the in vitro phenotype. Data relating to astrocytic glucose and lactate export are considered in the context of hypotheses of trafficking by astrocytes of substrates for neuronal metabolism, hypotheses that imply and require compartmentation of these substances, in contrast with current formulations of glucose transport into and within brain that imply no glucose compartmentation. Microdialysis studies of the properties of the brain extracellular fluid (ECF) glucose pool in the freely moving rat were performed seeking evidence of glucose compartmentation. Results of these studies do imply compartmentalisation of brain glucose, and are consistent with a model envisaging the majority of glucose reaching the neuron via the astrocytic intracellular space and the ECF. In addition, such studies provide evidence that rises in ECF glucose concentration are not the direct result of local recruitment of cerebral blood flow, but suggest the influence of intermediate, astrocyte-based mechanisms. Astrocytic glucose-6-phosphatase may permit astrocytes to modulate the trans-astrocytic flux of glucose to adjacent neurons in response to signals reflecting increased neuronal demand.

Expression of the rat brain creatine transporter in situ and in transfected HeLa cells.

Abstract: Using degenerate oligonucleotide probes encoding conserved regions of the gamma-aminobutyric acid/norepinephrine transporter gene family, we have cloned a rat brain cDNA encoding a creatine transporter (rCREAT). rCREAT encodes a highly hydrophobic, 635-amino-acid protein possessing 12 potential transmembrane domains and canonical sites for N-linked glycosylation and protein phosphorylation. Transfection of rCREAT cDNA into mammalian cells results in the expression of [14C]creatine uptake, which is blocked by low micromolar concentrations of recognized creatine uptake inhibitors. Two rCREAT mRNAs are expressed in the rat brain, retina, kidney and heart. Whole-brain rCREAT mRNAs demonstrate a marked postnatal rise to steady-state adult levels. In situ hybridization studies indicate a widespread, differential rCREAT mRNA expression in adult rat brain, with high expression noted over myelinated fiber tracts, cerebellar granule cells, hippocampal pyramidal cells, brainstem nuclei and endothelial cells of the choroid plexus. These studies will allow the development of new molecular probes useful for defining the creatine transporter's cellular expression pattern, function in ATP homeostasis and association with disorders of cellular energy metabolism.

Approaches to Studies on Neuronal/Glial Relationships by 13C-MRS Analysis

Abstract: The use of different 13C-labelled precursors alone or in combination ([1-13C]glucose, [2-13C]glucose, [1-13C]acetate, [2-13C]acetate and [1,2-13C2]acetate) to study neuronal/glial metabolic relationships by MRS is discussed. Glutamine and citrate resonances represent glial metabolism if a combination of [1-13C]glucose + [2-13C]acetate is used, but only for short time periods. A combination of [2-13C]glucose + [2-13C]acetate will label –COO– groups from glucose and – CH2 groups from acetate, respectively, which distinguish well in theory. However, this approach is severely limited by the long T1s of –COO– groups and low S/N. Contributions of the anaplerotic pathway can be assessed using [2-13C]glucose, but again can be limited by the long T1s of –COO– groups. Labelling of glycerol-3-phosphate (believed to be produced in glia) from [1-13C]glucose is difficult to see under normal conditions but has proved useful in, e.g., hypoxia. We believe the most promising approach is the use of [1-13C] glucose with [1,2-I3C] acetate, by analysis of the multiplets (‘isotopomers’) of the amino acid resonances.

Contribution of Astroglia to Functionally Activated Energy Metabolism

Abstract: Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [14C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K+] ([K+]o), or opening of Na+ channels with veratridine stimulates metabolism in neutral tissues, and this increase is blocked by ouabain, a specific inhibitor of Na+,K(+)-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [14C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to stimulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K+], intracellular [Na+], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K+]o stimulated [14C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 microM) stimulated [14C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 microM tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels. The Na+ ionophore monensin (10 microM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 microM) stimulated [14C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na+ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na(+)-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na+ entry into the cells.

Proteolipid Protein: Is It More than Just a Structural Component of Myelin?

Abstract: Proteolipid protein (PLP) is the most abundant protein of central nervous system (CNS) myelin. Because of its predicted topography, PLP has been assumed to function as a structural component of myelin, providing stability and maintaining the compact lamellar structure. However, developmental studies have shown that the PLP gene is active long before myelination begins. This and other evidence from various PLP mutants and transgenic models has fueled speculation that PLP or other products of the gene have additional, nonstructural roles both within and outside the CNS. PLP is structurally related to a family of ion channel proteins which includes the connexins, synaptophysins and various neurotransmitter receptors, and there is some experimental evidence which supports a role for PLP in ion gating. Other provocative ideas are that the PLP gene may influence autocrine signaling within oligodendrocytes or that PLP mRNAs have a function apart from protein coding.

Astrocyte Glucose Metabolism under Normal and Pathological Conditions in vitro

Abstract: Astrocytes in primary culture produce lactate. The net production of lactate from glucose requires that the carbon flux through glycolysis exceed the carbon flux to CO2. This study investigates the control and function of this 'excess' glycolysis in astrocyte cultures. Blockade of glycolysis was found to have minimal effects on astrocyte ATP and function if other substrates for oxidative metabolism were available. In contrast, selective blockade of oxidative metabolism reduced adenosine triphosphate (ATP) levels and slowed glutamate uptake despite a marked increase in glycolytic rate. Acidosis suppressed both glucose utilization and lactate production but had minimal effects on ATP levels. Acidosis in combination with blockade of oxidative metabolism blunted the increase in glycolytic rate and accentuated ATP depletion relative to oxidative blockade alone. These studies suggest that glycolysis in astrocyte cultures is regulated by factors other than energy demand, and that the capacity of glycolysis to support astrocyte metabolism during hypoxia is markedly pH dependent.

Mass isotopomer study of glutamine oxidation and synthesis in primary culture of astrocytes

Abstract: The metabolism of [1, 2-13C2] acetate via the tricarboxylic acid (TCA) cycle leads to the formation of a number of key mass isotopomers of glutamate. The distribution of these isotopomers which is a function of pyruvate carboxylase, pyruvate dehydrogenase and pyruvate recycling was used to determine the relative anaplerotic flux and glutamine oxidation of astrocytes in culture under different substrate conditions. Combinatory analysis of mass isotopomers formed from the condensation of labeled oxaloacetate with labeled acetyl-CoA was used to determine precursor enrichment and fractional glutamine synthesis. When glucose or glutamine was supplied in the medium, the effective anaplerotic flux (Y') was about 1.5 times that of the TCA cycle flux. Under substrate-limiting conditions, Y' and glutamine synthesis was significantly reduced. A unique feature of the use of [1, 2-13C2] acetate in this study is the formation of singly labeled isotopomer of glutamine in the C4 or C5 position when glutamine is irreversibly loss in net oxidation. We observed very little [4-13C] or [5-13C] glutamine either because of the lack of pyruvate recycling or the lack of pyruvate dehydrogenase activity. The lack of 13C recycling to the C4 and C5 position of glutamine suggests that less than 10% of the glutamine is oxidized into astrocytes for energy production. Therefore, glutamine is not a major energy substrate for astrocytes in culture.

Sexual Differentiation of the Brain in Songbirds

Abstract: The brain regions that control song in zebra finches are much larger in males, who sing, than in females, who do not. Two major theories have been proposed to explain sexual differentiation of the neu

Elevation of Amino Acids in the Interstitial Space of the Rat Brain following Infusion of Large Neutral Amino and Keto Acids by Microdialysis: Leucine Infusion

Abstract: A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Sodium channel expression: a dynamic process in neurons and non-neuronal cells.

Abstract: Although voltage-gated sodium channels have been most carefully studied in neurons, these channels are also present in nonexcitable cells within the nervous system and outside the nervous system. The mRNAs and protein for rat brain type sodium channels are expressed in both non-neuronal nervous system cells (glial cells) and in some cell types outside the nervous system. In most of these cell types, the expression of sodium channels is dynamic, with the levels and proportions of various sodium channel subtypes changing during development, and in response to injury and upon exposure to neurotrophins. It is likely that, in the near future, we will understand the roles played by sodium channels in each of these cell types, and the regulatory mechanisms that control expression of these channels.

Cell culture approaches to understanding the actions of steroid hormones on the insect nervous system.

Abstract: During metamorphosis of the hawkmoth, Manduca sexta, ecdysteroids regulate the dendritic remodeling and programmed death of identified motoneurons. These changes contribute to the dramatic reorganization of behavior that accompanies metamorphosis. As a step toward elucidating cellular and molecular mechanisms by which ecdysteroids affect neuronal phenotype, we have investigated the responses of Manduca motoneurons to ecdysteroids in vitro. Following dendritic regression at the end of larval life, thoracic leg motoneurons placed in culture respond to ecdysteroids by an increase in branching complexity, similar to events in vivo. Growth cone structure is affected markedly by ecdysteroids. At pupation, a rise in ecdysteroids triggers the segment-specific death of proleg motoneurons: the same segmental pattern of death is observed when motoneurons from different segments are removed from the nervous system and exposed to ecdysteroids in vitro. These studies provide strong evidence that Manduca motoneurons are direct targets of steroid action and set the stage for further studies of the specific mechanisms involved.

Modulation of Oxygen-Radical-Scavenging Enzymes by Oxidative Stress in Primary Cultures of Rat Astroglial Cells

Abstract: Cytosolic and mitochondrial alterations induced by exposure of rat astroglial primary cultures to reactive oxygen species (ROS) generated by a xanthine/ xanthine oxidase (X/XO) mixture or by lipopolysaccharide (LPS) have been investigated biochemically and irnmunochemically. In the presence of ROS generated by X/XO, a significant decrease in Cu,Zn superoxide dismutase (Cu,Zn-SOD) and in glutamine synthetase (GS) activity was observed whereas mitochondrial Mn-SOD activity and enzyme protein levels were significantly enhanced. Similar effects on GS, Cu,Zn- and Mn-SOD activities were observed by glucose/glucose oxidase treatment of the cells. Addition of LPS to the cell growth medium also specifically induces Mn-SOD synthesis but was without effect on Cu,Zn-SOD. It is suggested that in all these tested situations, hydrogen peroxide could represent a specific inducer of the observed phenomenon and it may therefore be considered as an intracellular messenger involved in the regulation of some aspects of astroglial oxidative metabolism, particularly the defence against ROS.

Development and Regeneration of the Nervous System: A Role for Neurosteroids (Paart 2 of 2)

Abstract: Several steroids, termed 'neurosteroids', are synthesized from cholesterol within both the central and peripheral nervous systems. These include pregnenolone and its sulfate ester, progesterone and it

Expression of EGFR-, p75NGFR-, and PSTAIR (cdc2)-Like Immunoreactivity by Proliferating Cells in the Adult Rat Hippocampal Formation and Forebrain

Abstract: Immunocytochemical and autoradiographic techniques were used to examine proliferating cells in the adult rat brain along with the expression of specific growth factor receptors and cell cycle proteins

Morphological organization of oligodendrocyte processes during development in culture and in vivo.

Abstract: In order to meet the requirements for myelin biogenesis, the processes of oligodendrocytes must differentiate into specialized cellular compartments. For translation of myelin basic protein mRNA to oc

Respective Roles of Glucose and Ketone Bodies as Substrates for Cerebral Energy Metabolism in the Suckling Rat

Abstract: The postnatal evolution of local cerebral metabolic rates for glucose (LCM-Rglcs) and of regional rates of cerebral uptake of β-hydroxybutyrate (βHB) were studied in the suckling rat between postnatal