The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine ... more The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.
The transplantation of fetal neurons has gained notoriety in recent years for its perceived poten... more The transplantation of fetal neurons has gained notoriety in recent years for its perceived potential to reverse neurological deficits caused by loss of one or another neuronal population. The present paper describes a neural grafting approach employed by our laboratory to gain more insight into the drug-induced neurobehavioral teratogenicity. Mice were exposed prenatally to phenobarbital by feeding the barbiturate to the pregnant dam on gestation days 9-18. Heroin exposure was accomplished by injecting dams during the same gestational period. At maturity, the drug-exposed offspring displayed profound deficits in specific behavioral tasks, suggesting alterations in the septohippocampal cholinergic pathway. Biochemically, we observed increased presynaptic activity in the pathway, which was not accompanied by a corresponding reduction in postsynaptic activity. Rather, there was a general hyperactivation along the different postsynaptic phases. In contrast, we noted a desensitization of protein kinase C activity in response to the exposure of a cholinergic agonist to the drug-exposed offspring. Subsequent transplantation of embryonic cholinergic cells from normal mice to the impaired hippocampus reversed the behavioral deficits, whereas sham-operated controls exhibited no improvement. Concomitantly, all the biochemical alterations studied, both presynaptic and postsynaptic, were either partially or completely reversed following grafting.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 4, 2009
Zn(2+) is coreleased with glutamate from mossy fiber terminals and can influence synaptic functio... more Zn(2+) is coreleased with glutamate from mossy fiber terminals and can influence synaptic function. Here, we demonstrate that synaptically released Zn(2+) activates a selective postsynaptic Zn(2+)-sensing receptor (ZnR) in the CA3 region of the hippocampus. ZnR activation induced intracellular release of Ca(2+), as well as phosphorylation of extracellular-regulated kinase and Ca(2+)/calmodulin kinase II. Blockade of synaptic transmission by tetrodotoxin or CdCl inhibited the ZnR-mediated Ca(2+) rises. The responses mediated by ZnR were largely attenuated by the extracellular Zn(2+) chelator, CaEDTA, and in slices from mice lacking vesicular Zn(2+), suggesting that synaptically released Zn(2+) triggers the metabotropic activity. Knockdown of the expression of the orphan G-protein-coupled receptor 39 (GPR39) attenuated ZnR activity in a neuronal cell line. Importantly, we observed widespread GPR39 labeling in CA3 neurons, suggesting a role for this receptor in mediating ZnR signaling ...
The transplantation of fetal neurons has gained notoriety in recent years for its perceived poten... more The transplantation of fetal neurons has gained notoriety in recent years for its perceived potential to reverse neurological deficits caused by loss of one or another neuronal population. The present paper describes a neural grafting approach employed by our laboratory to gain more insight into the drug-induced neurobehavioral teratogenicity. Mice were exposed prenatally to phenobarbital by feeding the barbiturate to the pregnant dam on gestation days 9-18. Heroin exposure was accomplished by injecting dams during the same gestational period. At maturity, the drug-exposed offspring displayed profound deficits in specific behavioral tasks, suggesting alterations in the septohippocampal cholinergic pathway. Biochemically, we observed increased presynaptic activity in the pathway, which was not accompanied by a corresponding reduction in postsynaptic activity. Rather, there was a general hyperactivation along the different postsynaptic phases. In contrast, we noted a desensitization of protein kinase C activity in response to the exposure of a cholinergic agonist to the drug-exposed offspring. Subsequent transplantation of embryonic cholinergic cells from normal mice to the impaired hippocampus reversed the behavioral deficits, whereas sham-operated controls exhibited no improvement. Concomitantly, all the biochemical alterations studied, both presynaptic and postsynaptic, were either partially or completely reversed following grafting.
Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates ... more Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates a number of critical developmental processes in both vertebrates and invertebrates, including angiogenesis, vasculogenesis and hematopoiesis. We employed an organotypic rat explant model (produced from embryonic day 17 fetuses) to assess the effects of vascular endothelial growth factor on brain microvasculature in general and the ventral midbrain specifically. Immunohistochemistry using antisera to rat endothelial cell antigen and laminin demonstrated a robust, dose-dependent effect of vascular endothelial growth factor, resulting in increased vessel neogenesis, branching and lumen size by three days in vitro. This effect was blocked by addition of an anti-vascular endothelial growth factor antibody. At higher doses of vascular endothelial growth factor, the effect was attenuated, though a statistically significant increase in both astrocyte, and neuronal density was observed using antisera to glial and neuronal markers. Tyrosine hydroxylase-immunoreactive (i.e. dopaminergic) neurons, particularly, exhibited increased survival in response to vascular endothelial growth factor application. Vascular endothelial growth factor had a mitogenic effect on endothelial cells and astrocytes, but not dopaminergic neurons, as demonstrated by the addition of [3H]thymidine to the cultures 2 h after the cultures were established. Similarly, results of a radioreceptor assay indicated that specific vascular endothelial growth factor binding sites were present on blood vessels and astrocytes, and were up-regulated by exposure to vascular endothelial growth factor. We conclude that, in explants of the ventral mesencephalon, exogenously applied vascular endothelial growth factor is mitogenic for endothelial cells and astrocytes, and promotes growth/survival of neurons in general and dopaminergic neurons in particular.
Zinc maintains a diverse array of functions in the mammalian central nervous system as a key comp... more Zinc maintains a diverse array of functions in the mammalian central nervous system as a key component of numerous enzymes, via its role in the activation of transcription factors, and as a neuroregulator, modulating neuronal receptors such as N-methyl-D-aspartate and gamma-aminobutyric acid. Zinc has a dark side, however, with massive influx of Zn(2+) to neurons considered to be a key factor in neuronal death secondary to ischemia and seizure. Several different putative zinc transporters, ZnT-1-4, have recently been identified and characterized. Among them, ZnT-1 has been suggested to play a key role in reducing cellular Zn(2+) toxicity. In the present study, we describe the regional and cellular distribution of ZnT-1 in the adult mouse brain using an antibody raised against the C-terminal domain of mouse ZnT-1. The distribution of ZnT-1 was compared to that of chelatable Zn(2+), visualized by means of neoTimm histochemistry or N-(6-methoxy-8-quinolyl)-p-toluene-sulfonamide (TSQ) histofluorescence. Extracts from various brain regions specifically stained a 60-kDa peptide corresponding to the expected molecular weight of ZnT-1. The expression of ZnT-1 was highest in the cerebral cortex and cerebellum, moderate in the hippocampus, hypothalamus, and olfactory bulb, and lowest in the striatum and septum. In brain sections, ZnT-1-immunoreactive neurons, in particular principle neurons, in the somatosensory cortex, hippocampus, and olfactory bulb, were closely related to synaptic Zn(2+). Robust ZnT-1 immunoreactivity was also observed in cerebellar Purkinje cells. Although the function of the protein in these cells is unclear, in the forebrain, ZnT-1 is strikingly present in cells and regions where significant Zn(2+) homeostasis is required. This finding suggests a protective role for neuronal ZnT-1 in the context of both normal and pathophysiological activity.
Despite the basic differences in their underlying biological targets, prenatal exposure to heroin... more Despite the basic differences in their underlying biological targets, prenatal exposure to heroin or phenobarbital produces similar syndromes of neurobehavioral deficits, involving defects in septohippocampal cholinergic innervation-related behaviors. At the cellular level, these deficits are associated with cholinergic hyperactivity, characterized by increased concentrations of muscarinic receptors and enhanced second messenger activity linked to the receptors. In the present study, we determined whether the cellular changes are mechanistically linked to altered behavior, using two different approaches: neural grafting and correlations between behavior and biochemistry within the same individual animals. Mice were exposed transplacentally to phenobarbital or heroin on gestation days 9-18 and, as adults, received fetal cholinergic grafts or were sham-operated. Prenatal drug exposure resulted in deficits in behavioral performance tested in the eight-arm radial maze, accompanied by increases in hippocampal M(1)-muscarinic receptor expression and muscarinic receptor-mediated IP formation. Neural grafting reversed both the behavioral deficits and the muscarinic hyperactivity. In the drug-exposed offspring, there was a significant correlation between maze performance and carbachol-induced inositol phosphate (IP) formation. These studies indicate that deficits of cholinergic function underlie the neurobehavioral deficits seen in the hippocampus of animals exposed prenatally to heroin or phenobarbital, and consequently that the observed cholinergic hyperactivity is an unsuccessful attempt to compensate for the loss of cholinergic function. The fact that the damage can be reversed by neural grafting opens up novel approaches to the restoration of brain function after prenatal insults.
The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine ... more The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei. We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway. Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively.
The transplantation of fetal neurons has gained notoriety in recent years for its perceived poten... more The transplantation of fetal neurons has gained notoriety in recent years for its perceived potential to reverse neurological deficits caused by loss of one or another neuronal population. The present paper describes a neural grafting approach employed by our laboratory to gain more insight into the drug-induced neurobehavioral teratogenicity. Mice were exposed prenatally to phenobarbital by feeding the barbiturate to the pregnant dam on gestation days 9-18. Heroin exposure was accomplished by injecting dams during the same gestational period. At maturity, the drug-exposed offspring displayed profound deficits in specific behavioral tasks, suggesting alterations in the septohippocampal cholinergic pathway. Biochemically, we observed increased presynaptic activity in the pathway, which was not accompanied by a corresponding reduction in postsynaptic activity. Rather, there was a general hyperactivation along the different postsynaptic phases. In contrast, we noted a desensitization of protein kinase C activity in response to the exposure of a cholinergic agonist to the drug-exposed offspring. Subsequent transplantation of embryonic cholinergic cells from normal mice to the impaired hippocampus reversed the behavioral deficits, whereas sham-operated controls exhibited no improvement. Concomitantly, all the biochemical alterations studied, both presynaptic and postsynaptic, were either partially or completely reversed following grafting.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 4, 2009
Zn(2+) is coreleased with glutamate from mossy fiber terminals and can influence synaptic functio... more Zn(2+) is coreleased with glutamate from mossy fiber terminals and can influence synaptic function. Here, we demonstrate that synaptically released Zn(2+) activates a selective postsynaptic Zn(2+)-sensing receptor (ZnR) in the CA3 region of the hippocampus. ZnR activation induced intracellular release of Ca(2+), as well as phosphorylation of extracellular-regulated kinase and Ca(2+)/calmodulin kinase II. Blockade of synaptic transmission by tetrodotoxin or CdCl inhibited the ZnR-mediated Ca(2+) rises. The responses mediated by ZnR were largely attenuated by the extracellular Zn(2+) chelator, CaEDTA, and in slices from mice lacking vesicular Zn(2+), suggesting that synaptically released Zn(2+) triggers the metabotropic activity. Knockdown of the expression of the orphan G-protein-coupled receptor 39 (GPR39) attenuated ZnR activity in a neuronal cell line. Importantly, we observed widespread GPR39 labeling in CA3 neurons, suggesting a role for this receptor in mediating ZnR signaling ...
The transplantation of fetal neurons has gained notoriety in recent years for its perceived poten... more The transplantation of fetal neurons has gained notoriety in recent years for its perceived potential to reverse neurological deficits caused by loss of one or another neuronal population. The present paper describes a neural grafting approach employed by our laboratory to gain more insight into the drug-induced neurobehavioral teratogenicity. Mice were exposed prenatally to phenobarbital by feeding the barbiturate to the pregnant dam on gestation days 9-18. Heroin exposure was accomplished by injecting dams during the same gestational period. At maturity, the drug-exposed offspring displayed profound deficits in specific behavioral tasks, suggesting alterations in the septohippocampal cholinergic pathway. Biochemically, we observed increased presynaptic activity in the pathway, which was not accompanied by a corresponding reduction in postsynaptic activity. Rather, there was a general hyperactivation along the different postsynaptic phases. In contrast, we noted a desensitization of protein kinase C activity in response to the exposure of a cholinergic agonist to the drug-exposed offspring. Subsequent transplantation of embryonic cholinergic cells from normal mice to the impaired hippocampus reversed the behavioral deficits, whereas sham-operated controls exhibited no improvement. Concomitantly, all the biochemical alterations studied, both presynaptic and postsynaptic, were either partially or completely reversed following grafting.
Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates ... more Vascular endothelial growth factor is a highly conserved, heparin-binding protein which mediates a number of critical developmental processes in both vertebrates and invertebrates, including angiogenesis, vasculogenesis and hematopoiesis. We employed an organotypic rat explant model (produced from embryonic day 17 fetuses) to assess the effects of vascular endothelial growth factor on brain microvasculature in general and the ventral midbrain specifically. Immunohistochemistry using antisera to rat endothelial cell antigen and laminin demonstrated a robust, dose-dependent effect of vascular endothelial growth factor, resulting in increased vessel neogenesis, branching and lumen size by three days in vitro. This effect was blocked by addition of an anti-vascular endothelial growth factor antibody. At higher doses of vascular endothelial growth factor, the effect was attenuated, though a statistically significant increase in both astrocyte, and neuronal density was observed using antisera to glial and neuronal markers. Tyrosine hydroxylase-immunoreactive (i.e. dopaminergic) neurons, particularly, exhibited increased survival in response to vascular endothelial growth factor application. Vascular endothelial growth factor had a mitogenic effect on endothelial cells and astrocytes, but not dopaminergic neurons, as demonstrated by the addition of [3H]thymidine to the cultures 2 h after the cultures were established. Similarly, results of a radioreceptor assay indicated that specific vascular endothelial growth factor binding sites were present on blood vessels and astrocytes, and were up-regulated by exposure to vascular endothelial growth factor. We conclude that, in explants of the ventral mesencephalon, exogenously applied vascular endothelial growth factor is mitogenic for endothelial cells and astrocytes, and promotes growth/survival of neurons in general and dopaminergic neurons in particular.
Zinc maintains a diverse array of functions in the mammalian central nervous system as a key comp... more Zinc maintains a diverse array of functions in the mammalian central nervous system as a key component of numerous enzymes, via its role in the activation of transcription factors, and as a neuroregulator, modulating neuronal receptors such as N-methyl-D-aspartate and gamma-aminobutyric acid. Zinc has a dark side, however, with massive influx of Zn(2+) to neurons considered to be a key factor in neuronal death secondary to ischemia and seizure. Several different putative zinc transporters, ZnT-1-4, have recently been identified and characterized. Among them, ZnT-1 has been suggested to play a key role in reducing cellular Zn(2+) toxicity. In the present study, we describe the regional and cellular distribution of ZnT-1 in the adult mouse brain using an antibody raised against the C-terminal domain of mouse ZnT-1. The distribution of ZnT-1 was compared to that of chelatable Zn(2+), visualized by means of neoTimm histochemistry or N-(6-methoxy-8-quinolyl)-p-toluene-sulfonamide (TSQ) histofluorescence. Extracts from various brain regions specifically stained a 60-kDa peptide corresponding to the expected molecular weight of ZnT-1. The expression of ZnT-1 was highest in the cerebral cortex and cerebellum, moderate in the hippocampus, hypothalamus, and olfactory bulb, and lowest in the striatum and septum. In brain sections, ZnT-1-immunoreactive neurons, in particular principle neurons, in the somatosensory cortex, hippocampus, and olfactory bulb, were closely related to synaptic Zn(2+). Robust ZnT-1 immunoreactivity was also observed in cerebellar Purkinje cells. Although the function of the protein in these cells is unclear, in the forebrain, ZnT-1 is strikingly present in cells and regions where significant Zn(2+) homeostasis is required. This finding suggests a protective role for neuronal ZnT-1 in the context of both normal and pathophysiological activity.
Despite the basic differences in their underlying biological targets, prenatal exposure to heroin... more Despite the basic differences in their underlying biological targets, prenatal exposure to heroin or phenobarbital produces similar syndromes of neurobehavioral deficits, involving defects in septohippocampal cholinergic innervation-related behaviors. At the cellular level, these deficits are associated with cholinergic hyperactivity, characterized by increased concentrations of muscarinic receptors and enhanced second messenger activity linked to the receptors. In the present study, we determined whether the cellular changes are mechanistically linked to altered behavior, using two different approaches: neural grafting and correlations between behavior and biochemistry within the same individual animals. Mice were exposed transplacentally to phenobarbital or heroin on gestation days 9-18 and, as adults, received fetal cholinergic grafts or were sham-operated. Prenatal drug exposure resulted in deficits in behavioral performance tested in the eight-arm radial maze, accompanied by increases in hippocampal M(1)-muscarinic receptor expression and muscarinic receptor-mediated IP formation. Neural grafting reversed both the behavioral deficits and the muscarinic hyperactivity. In the drug-exposed offspring, there was a significant correlation between maze performance and carbachol-induced inositol phosphate (IP) formation. These studies indicate that deficits of cholinergic function underlie the neurobehavioral deficits seen in the hippocampus of animals exposed prenatally to heroin or phenobarbital, and consequently that the observed cholinergic hyperactivity is an unsuccessful attempt to compensate for the loss of cholinergic function. The fact that the damage can be reversed by neural grafting opens up novel approaches to the restoration of brain function after prenatal insults.
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Papers by William Silverman