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Biomedical subjects

I B Black

Publications and source records attributed to I B Black.

At least 19 recordsLinked to original sources
PubMed1992

Detection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a common, lethal, chromosome X-linked inherited disease. Moderate cognitive impairment is a feature of DMD, but the underlying mechanisms are unknown. DMD is characterized by a defect in a protein, dystrophin, that is located predominantly in muscle but has been detected in brain. We sought to directly localize dystrophin within the complex synaptic structure of the cerebral cortex by focusing on the postsynaptic density (PSD), which appears to be central to synaptic function. We report that a specific anti-dystrophin antibody (anti 6-10) recognizes three distinct proteins in the purified PSD: the 400-kDa dystrophin and two previously unidentified dystrophin-related proteins of 120 and 110 kDa. These proteins exhibited differential regional expression in PSDs from cerebral cortex, cerebellum, and olfactory bulb. In the cortical PSD, the 400-kDa dystrophin was predominant, whereas the 120-kDa protein was the major species in cerebellum and olfactory bulb PSDs. The three proteins were differentially expressed in the PSD during cortical development: the 400-kDa protein exhibited a selective 9-fold increase during postnatal days 7 to 10, suggesting a normal physiological role in synaptic maturation. The PSD from the mdx mouse, a model of human DMD, contained no detectable 400-kDa dystrophin but expressed the two dystrophin-related proteins. Our results indicate that brain dystrophins are localized to the PSD, potentially as three isoforms, and raise the possibility that cognitive abnormalities in DMD are attributable to synaptic dysfunction associated with deficits in brain dystrophin molecules.

Animals↗
PubMed1992

Mesencephalic type I astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture.

We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1992

On the identity of the major postsynaptic density protein.

Increasing evidence suggests that the postsynaptic density (PSD) plays a critical role in synaptic communication and plasticity. The major PSD protein (mPSDp), a calcium/calmodulin-dependent protein kinase, appears to be central to PSD function. The mPSDp has long been considered identical to the alpha subunit of the soluble calmodulin kinase II (alpha-CKII). However, mPSDp and alpha-CKII do differ in solubility and antigenicity, raising the possibility that the two proteins are distinct. To further define the relationship between the two proteins, we purified the mPSDp to homogeneity from adult rat cerebral cortex and compared the proteins. In contrast to alpha-CKII, the purified mPSDp was insoluble in high concentrations of salt, various detergents, chelators of divalent cations, and the strong denaturant guanidine hydrochloride. The pI value of the mPSDp was 6.2, whereas that of alpha-CKII was 6.7-7.2. The purified mPSDp bound calmodulin in the presence of Ca2+ and was autophosphorylated in a Ca2+/calmodulin-dependent manner. Polyclonal antiserum raised against mPSDp (anti-mPSDp) recognized purified mPSDp or mPSDp in synaptic membrane, indicating immunologic specificity among the synaptic proteins. Anti-mPSDp did not recognize alpha-CKII, whereas anti-alpha-CKII antibodies reacted only weakly with mPSDp, suggesting that the proteins are distinct but structurally similar. Moreover, sequence analysis of protease V8-digested polypeptides revealed that there was at least an 8-amino acid sequence, MLKVPNIS, that is not present in alpha-CKII. Finally, HPLC analysis of V8-digested fragments of mPSDp and alpha-CKII in parallel revealed dissimilar peptide patterns. Thus our observations suggest that mPSDp and alpha-CKII are similar but not identical. The unique physicochemical and structural properties of the mPSDp may provide insights into molecular mechanisms mediating synaptic plasticity.

Amino Acid Sequence↗
PubMed1991

Depolarizing stimuli regulate nerve growth factor gene expression in cultured hippocampal neurons.

Although trophic factors and neuronal activity have been implicated in regulating functional synaptic circuits, the relationship of trophic interaction to impulse activity in synaptogenesis remains unclear. Using cultured hippocampus as a model system, we provide direct evidence that depolarization and impulse activity specifically increase nerve growth factor gene expression in neurons. Depolarizing stimuli, such as a high K+ concentration or the Na+ channel agonist veratridine, elicited a 3-fold increase of nerve growth factor mRNA levels in both explant and dissociated cultures. Blockade of depolarization by tetrodotoxin prevented the increase of neuronal nerve growth factor mRNA. Further, nerve growth factor gene expression was stimulated by picrotoxin, a gamma-aminobutyric acid antagonist frequently used to enhance hippocampal neuronal activity. Impulse regulation of trophic gene function may be relevant to developmental synaptogenesis and synaptic strengthening in learning and memory.

Animals↗
PubMed1991

Role of voltage-sensitive calcium channels in mitogenic stimulation of neuroblasts.

The present study examines the role of Ca2+ in the regulation of sympathetic neuroblast mitosis. Employing a fully defined neuroblast culture system, we previously found that insulin growth factors (IGFs), depolarization and vasoactive intestinal peptide (VIP) regulated precursor mitosis. We now report that Ca2+ entry via voltage-sensitive channels was required for depolarization-stimulated mitogenesis. Ca2+ channel blockade with nitrendipine completely inhibited the increase in [3H]thymidine incorporation elicited by depolarizing stimuli including 30 mM KCl and the Na+ channel agonist veratridine. However, Ca2+ channel activity was not involved in the stimulation of DNA synthesis by IGFs or VIP. Thus, neuroblast mitosis may be regulated by multiple intracellular as well as extracellular signals.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Sexually dimorphic expression of the NGF receptor gene in the developing rat brain.

To define relations between trophic molecules and known sexually dimorphic traits in brain, we examined possible sex differences in nerve growth factor (NGF) and NGF receptor (NGF-R) gene expression in the rat cholinergic basal forebrain (BF)-hippocampal system. Hippocampal NGF mRNA levels did not differ between sexes; in contrast, BF NGF-R mRNA levels were greater in neonatal females than males, paralleling the known dimorphic development of cholinergic enzyme activity. Cerebellar NGF-R mRNA levels were also dimorphic in the neonate, suggesting that sex-specific influences may regulate trophic receptor gene expression in diverse brain systems.

Animals↗
PubMed1991

Local support cells promote survival of substantia nigra dopaminergic neurons in culture.

Recent studies suggest that brain neurons require extracellular signals for continued survival during maturity as well as development. However, factors underlying the survival of specific populations of central neurons remain to be defined. To examine the regulation of neuronal survival, we have studied the substantia nigra (SN) dopaminergic (DA) system, in dissociated cell culture. DA neuron number was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme. Initially, mixed cultures were grown at low, medium, and high densities in serum-containing media. After 7 days, the number of neuron-specific enolase (NSE)-positive cells, a measure of total neuron number, was proportional to cell plating density. In contrast, high density culture elicited a marked, disproportionate increase in TH-immunopositive cells, suggesting that high density conditions selectively enhanced the DA subpopulation. To define the role of cellular interactions in the selective increase in DA cells, virtually pure neuron cultures were compared to support cell-neuron cocultures, in fully defined medium. In support cell-neuron cocultures, SN support cells evoked a four-fold increase in TH cells, while NSE number did not differ from controls. Moreover, local support cells elicited a greater increase in TH cell number than support cells derived from other brain regions. To determine whether increased TH cell number reflected enhanced survival, or possibly expression of TH by new populations, we monitored the time course of this effect. TH cell number remained constant after 3 days in cocultures, while declining fourfold in controls. In parallel studies, support cells were added to SN dissociates at zero time or after 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Regulation of NGF gene expression in CNS glia by cell-cell contact.

Nerve growth factor (NGF) gene expression in central nervous system (CNS) glia appears to be associated with active glial growth. To study the underlying molecular mechanisms, we examined the effects of a number of growth-related factors on NGF mRNA expression in glial cultures. Our results suggest that glial membrane interaction, as a consequence of growth, actively inhibits NGF gene expression in CNS glia.

Animals↗
PubMed1991

NGF and excitatory neurotransmitters regulate survival and morphogenesis of cultured cerebellar Purkinje cells.

The development of cerebellar Purkinje cells is subject to regulation by multiple epigenetic signals. To define mechanisms by which trophic and presynaptic stimulation may potentially regulate Purkinje cell ontogeny, we studied the effects of NGF and excitatory transmitters on Purkinje cell survival and morphological maturation in dissociated cell culture. Purkinje cells were identified by expression of vitamin D-dependent calcium-binding protein and by their characteristic morphology. NGF receptors were selectively localized to Purkinje cells by both ligand and monoclonal antibody binding, suggesting responsivity to the trophic agent. Simultaneous exposure to depolarizing agents and NGF specifically enhanced Purkinje cell survival in culture. NGF, in combination with either high potassium or veratridine markedly increased survival of Purkinje cells. Furthermore, NGF together with the excitatory neurotransmitters aspartate or glutamate promoted a 2-fold increase in survival. In addition, NGF increased Purkinje cell size and promoted neurite elaboration. These effects required simultaneous exposure to NGF and either aspartate, glutamate, or pharmacologic depolarizing agents. Effects on survival or neurite elaboration were not evoked by exposure to trophic factors or transmitters alone. Our results suggest a novel mechanism for regulation of development, in which trophic factor and afferent stimulation interact to promote survival and morphogenesis of developing Purkinje cells.

Animals↗
PubMed1991

NGF gene expression in actively growing brain glia.

Previous work suggested that brain NGF acts locally on cells adjacent to sites of synthesis, in addition to any putative actions on distant, projecting perikarya. To define the basis of local action, we used a sensitive nuclease protection assay to identify cells expressing the NGF gene in vivo and in vitro. In addition to neurons, glia from a variety of developing brain areas synthesized NGF mRNA, suggesting that CNS glia exhibit a generalized capacity to express the gene. Expression was associated with active glial growth. Stimulation of growth with serum increased NGF message 2-fold in culture. Moreover, rapidly growing, low-density glial cultures exhibited 8-fold higher levels of NGF mRNA than quiescent, confluent cultures. The optic nerve, which contains all 3 major types of glia, expressed the message in vivo during neonatal development. In contrast, expression was barely detectable in the adult optic nerve. Transection, which induces glial proliferation, elicited de novo appearance of NGF mRNA in the adult nerve. Our observations suggest that active glial growth is associated with expression of the NGF gene and raise the possibility that actively growing glia in the developing or injured brain regulate neuronal growth through the elaboration of NGF.

Animals↗
PubMed1990

Vasoactive intestinal peptide regulation of neuroblast mitosis and survival: role of cAMP.

The role of adenosine 3':5'-cyclic monophosphate (cAMP) in the regulation of neuroblast mitosis and survival by vasoactive intestinal peptide (VIP) was examined. VIP increased the cAMP content of cultured rat sympathetic neuroblasts. Further, cAMP stimulated DNA synthesis and survival in neuroblast cultures, replicating the effects of VIP. Thus, the VIP-cAMP signaling pathway may be involved in the regulation of neuronal development.

Animals↗
PubMed1990

Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts.

Although acute, millisecond-to-millisecond actions of neurotransmitters are well documented, diverse longer-term effects have been discovered only recently. Emerging evidence indicates that these signals regulate a variety of neuronal processes, from phenotypic expression to neurite outgrowth. Here we show that a single putative transmitter, vasoactive intestinal peptide, can exert multiple, long-term effects simultaneously: it stimulates mitosis, promotes neurite outgrowth and enhances survival of sympathetic neuron precursors in culture. As the peptide seems to be a normal presynaptic transmitter in the sympathetic system, synaptic transmission may exert hitherto unexpected effects.

Animals↗
PubMed1990

Regulation of neurotransmitter expression by a membrane-derived factor.

Cell-cell contact appears to play a critical role in the expression of transmitter traits in developing neurons. We have previously shown that cell membrane contact induces the de novo appearance of choline acetyltransferase (CAT) in virtually pure cultures of dissociated sympathetic neurons. A membrane-associated CAT-inducing factor has been extracted and purified 5000-fold. This factor exerts differential effects on transmitter traits in cultured sympathetic neurons. After 3 days in vitro, neurons exposed to the factor contained 40-fold higher levels of the neuropeptide substance P than controls. Somatostatin exhibited a similar dramatic elevation. In contrast, the factor had no effect on leucine-enkephalin. Further, the specific activity of tyrosine hydroxylase was reduced to 5% of control activity in treated cultures. These effects occurred in the absence of any increases in cell number. Thus, it appears that cell contact via membrane-associated factors may exert differential effects on phenotypic expression.

Animals↗
PubMed1990

Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation.

Although neuron generation is precisely regulated during ontogeny, little is known about underlying mechanisms. In addition, relationships between precursor proliferation and the apparent sequence of developmental processes, including cell migration, neurite elaboration, transmitter expression and synaptogenesis remain unknown. To address these issues, we used a fully defined neuronal cell culture system derived from embryonic rat sympathetic ganglia (DiCicco-Bloom, E., and I. B. Black. 1988. Proc. Natl. Acad. Sci. USA. 85:4066-4070) in which precursors enter the mitotic cycle. We now find that, in addition to synthesizing DNA, neuroblasts also underwent division in culture, allowing analysis of developmental relationships and mitotic regulation. Our observations indicate that mitotic neuroblasts expressed a wide array of neuron-specific characteristics including extension of neuritic processes with growth cones, elaboration of neurotransmitter enzyme, synthesis and transport of transmitter vesicles and organization of transmitter release sites. These data suggest that neuroblasts in the cell cycle may simultaneously differentiate. Consequently, the apparent sequence of ontogenetic processes is not an immutable, intrinsic neuronal program. How, then, are diverse developmental events coordinated? Our observations indicate that neuroblast mitosis is regulated by a small number of epigenetic factors, including insulin and EGF. Since these signals also influence other processes in developing neurons, epigenetic regulation normally may synchronize diverse ontogenetic events.

Animals↗
PubMed1989

Developmental regulation of tyrosine hydroxylase in the mediobasal hypothalamus.

In the adult rodent the mediobasal hypothalamus (MBH) interacts extensively with the pituitary gland to regulate a variety of endocrine functions. The dopaminergic (DA) neurons of the mature MBH are influenced by numerous transmitters and hormones, however, little is known about developmental regulation of this system. Ontogeny of DA neurons was examined in vivo and in explant culture by monitoring tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The influence of the depolarizing agent, veratridine, was examined to determine whether development of TH in the MBH is regulated by depolarizing signals as in other CA neurons. Veratridine elicited a significant increase in TH activity in cultures of MBH. Adult MBH neurons are influenced by hormones such as estradiol. We investigated the possible role of estradiol in regulating the ontogeny of MBH DA neurons in culture. We developed a steroid-depleted culture medium to rigorously define the effects of steroids on the developing system. This enabled us to determine that estradiol does not appear to influence TH during embryonic development, though estrogen receptors are present at this stage. These results were confirmed in vivo by injecting neonates with moxestrol, a synthetic estrogen which is not sequestered by alpha-fetoprotein. This treatment did not elicit any change in TH. Our observations suggest that although estrogen regulates TH in the adult MBH, this hormone does not play a role in developmental regulation of TH in this brain region. In contrast, however, depolarizing signals appear to be a widespread mechanism for regulation of TH in numerous neuronal populations.

Animals↗
PubMed1989

Depolarization and insulin-like growth factor-I (IGF-I) differentially regulate the mitotic cycle in cultured rat sympathetic neuroblasts.

Although neuroblast generation is highly reproducible temporospatially, the underlying regulation is undefined. Employing a fully defined sympathetic neuroblast culture system, we previously found that insulin growth factors regulated the mitotic cycle. We now report that depolarizing stimuli, including elevated K+ and veratridine, also increased the proportion of mitotic neuroblasts in vitro. Moreover, Na+ channel blockade prevented effects of depolarization, but not that of insulin-like growth factor I, suggesting that these influences stimulate mitosis by different membrane transduction mechanisms.

Animals↗
PubMed1989

Regulation of beta-adrenergic receptor expression in rat liver.

To begin defining the factors regulating neurotransmitter receptor expression, we examined beta-adrenergic receptors in rat liver in vivo and in primary liver cultures under defined hormonal conditions. beta-receptors described a remarkable developmental profile in vivo, increasing fivefold between embryonic days 16 and 20, and decreasing tenfold by early adulthood. The developmental decrease reflected reduced receptor number without a change in receptor properties. The ontogenetic decrease appeared to be specific for beta-receptors; alpha-receptors developed in a hyperbolic fashion, reaching high plateau values by the third postnatal week. Adult rat liver cells plated into culture re-expressed high beta-receptor levels, exhibiting a 4-8-fold increase. A similar pattern of expression of the beta-receptors, having similar pharmacological properties, was observed in primary liver cultures maintained in serum-free medium, in a serum-supplemented medium or in several variations of a serum-free, hormonally defined medium designed for primary liver cultures. Thus, the degree of expression of the beta-receptors was not found affected by various hormones, by serum, or by any medium condition. By contrast, the degree of expression of the beta-receptors was markedly sensitive to cell density. High expression of the beta-receptors was observed at low cell densities (1-3 x 10(6) cells/150 mm dish), and low expression or no expression was observed in confluent cultures (10-20 x 10(6) cells/150 mm dish). Our experiments suggest that beta-receptor expression does not follow an immutable program, but may be regulated by density-dependent cell-cell interactions.

Age Factors↗