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C F Dreyfus

Publications and source records attributed to C F Dreyfus.

At least 37 records · Page 2Linked to original sources
PubMed1994

Embryonic sensory development: local expression of neurotrophin-3 and target expression of nerve growth factor.

Development and maintenance of peripheral sensory and sympathetic neurons are regulated by target-derived neurotrophins, including nerve growth factor (NGF). To determine whether trophins are potentially critical prior to and during target innervation, for neuronal survival or axon guidance, in situ hybridization was performed in the rat embryo. We examined the expression of genes encoding NGF, neurotrophin-3 (NT-3), and their putative high-affinity receptors, trk A and trk C, respectively. Trks A and C were detected in dorsal root sensory ganglia (DRG) on embryonic day 12.5 (E12.5), implying early responsiveness to NGF and NT-3. NGF mRNA was expressed in the central spinal cord target and by the peripheral somite, at this early time, which thereby may function as a transient "guidepost" target for sensory fibers. Somitic expression was transient and was undetectable by E17.5. NT-3 was expressed in the DRG itself from E13.5 to 17.5, suggesting local transient actions on sensory neurons. NT-3 was also expressed in the ventral spinal cord at low levels on E13.5. We examined the trigeminal ganglion to determine whether cranial sensory neurons are similarly regulated. Trk A was detected in the trigeminal ganglion, while NGF was expressed in the central myelencephalon target, paralleling observations in the DRG and spinal cord. However, NT-3 and trk C were undetectable, in contrast to DRG, suggesting that the environment or different neural crest lineages govern expression of different trophins and trks. Apparently, multiple trophins regulate sensory neuron development through local as well as transient target mechanisms prior to innervation of definitive targets.

Animals↗
PubMed1994

Multiple astrocyte transcripts encode nigral trophic factors in rat and human.

The recent discovery of glial cell line-derived neurotrophic factor (GDNF) identified a novel trophin that selectively increases survival of substantia nigra dopaminergic neurons, which degenerate in Parkinson's disease. Our previous studies indicated that GDNF RNA can be amplified from cultured rat nigral type 1 astrocytes and from rat striatum in vivo, implying local as well as target trophic support. The current study establishes the regional pattern of GDNF RNA expression in adult human brain. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the highest expression of GDNF mRNA in the human caudate, with low levels in the putamen and no detectable message in the nigra, suggesting that GDNF is a target-derived factor in humans. We also report the isolation of two additional GDNF-related cDNAs, termed astrocyte-derived trophic factors (ATF), which apparently result from differential RNA processing. Sequence analysis of rat ATF-1 revealed a 78-bp deletion corresponding to a loss of 26 amino acids within the prepro region of the predicted GDNF protein. The RNA processing events responsible for ATF-1 formation in rat brain are conserved in humans; we report the isolation of a full-length human ATF-1 homologue. We identified a second alternative transcript, human ATF-2; the transcript encodes a protein which differs in its first 18 amino acids from the predicted mature GDNF and ATF-1 proteins and shares the terminal 115 residues with the other two forms. To begin assessing the biologic significance of multiple transcript expression we characterized the actions of COS-expressed GDNF and ATF-1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Activating Transcription Factor 1↗
PubMed1994

An improved method detects differential NGF and BDNF gene expression in response to depolarization in cultured hippocampal neurons.

Differential regulation of individual neurotrophins by impulse activity potentially allows transformation of instantaneous signalling into diverse, long-lasting neural alterations. To define the temporal profiles of trophin gene expression we examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs in dissociated cell cultures of rat hippocampus using an improved solution hybridization technique. Traditional methods lack the precision and sensitivity to detect small changes during brief intervals and the facility to process large sample numbers simultaneously. This improved method has now allowed us to better define the dynamics of depolarization-induced changes in expression of individual trophin genes. Using elevated K+ as a depolarizing stimulus, NGF mRNA increased 40% after 48 h. In contrast, BDNF message rose almost 4-fold within 3 h and attained a maximal 6-fold increase within 6 h. Similar increases in BDNF mRNA levels were exhibited following treatment of cultures with glutamate, an excitatory neurotransmitter. To document the sensitivity of BDNF mRNA to depolarizing conditions, we examined expression after K+ withdrawal. BDNF message began decreasing within one hour post-depolarization, and returned to basal levels after 6 h. Observations indicate that BDNF and NGF mRNAs are induced differentially in response to impulse activity; BDNF message is acutely responsive to ongoing changes, whereas NGF mRNA responds more slowly and sluggishly. The physiological implications of this differential regulation are discussed.

Animals↗
PubMed1994

Muscarinic stimulation promotes cultured Purkinje cell survival: a role for acetylcholine in cerebellar development?

The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6-7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 microM) and pirenzepine (10 nM), but not by methoctramine (25 nM). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.

Acetylcholine↗
PubMed1993

Hippocampal regulation of the survival and morphological development of locus coeruleus neurons in dissociated cell culture.

The influence of target structures on neural development, originally described for the peripheral nervous system, has more recently been investigated in the central nervous system. We sought to determine whether targets regulate the development of the locus coeruleus, with its diffuse and complex projections in marked contrast to the simpler neural circuits of the peripheral nervous systems. Dissociated locus coeruleus cells were grown alone or with the hippocampus in serum-free, chemically defined medium that minimized non-neuronal growth. Coculture with the hippocampus resulted in a significant increase in locus coeruleus tyrosine hydroxylase activity. Elevated tyrosine hydroxylase activity was accompanied by a commensurate increase in the number of tyrosine hydroxylase-immunoreactive cells, suggesting hippocampal enhancement of locus coeruleus survival. Furthermore, when hippocampal cells were added to locus coeruleus dissociates at zero time, or after two days, tyrosine hydroxylase-positive cell number was increased only by hippocampal cells added initially, suggesting that the target does indeed foster survival. The apparent target enhancement of locus coeruleus survival seems to be selective since total protein and total neuron number, estimated with neuron-specific enolase immunocytochemistry, were not affected by the hippocampus. Coculture with the hippocampus also increased the length and complexity of locus coeruleus cell processes. Neither the increase in tyrosine hydroxylase cell number nor the changes in morphology could be attributed to nonspecific effects of the increased cell density in cocultures. Our observations thus suggest that the target hippocampus influences the survival and neurite elaboration of afferent locus coeruleus neurons.

Aging↗
PubMed1993

Differential actions of neurotrophins in the locus coeruleus and basal forebrain.

The neurotrophin gene family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/NT-5, supports the survival of distinct peripheral neurons, however, actions upon central neurons are relatively undefined. In this study we have compared different neurotrophins in the regulation of neuronal survival and function using dissociated embryonic cell cultures from two brain regions, the basal forebrain (BF) and locus coeruleus (LC). In the BF, NGF increased choline acetyl transferase (ChAT) activity, but did not influence cholinergic cell survival. In contrast to NGF, BDNF, NT-3, and the novel neurotrophin, NT-4, all increased ChAT activity and cholinergic cell survival. We also examined embryonic LC neurons in culture. LC neurons are unresponsive to NGF. In contrast, NT-3 and NT-4 elicited significant increases in survival of noradrenergic LC neurons, the first demonstration of trophic effects in this critical brain region. Identification of factors supporting coeruleal and basal forebrain neuronal survival may provide insight into mechanisms mediating degeneration of these disparate structures in clinical disorders.

Animals↗
PubMed1993

Depolarizing influences increase low-affinity NGF receptor gene expression in cultured Purkinje neurons.

Multiple cellular and molecular interactions are required for the differentiation and development of central neurons. For example, neural activity may modulate trophic function. In the developing cerebellum, establishment of functional excitatory synaptic connections coincides with the expression of NGF and its receptors. We have previously shown that excitatory signals and NGF act in concert to regulate the survival and morphological differentiation of cerebellar Purkinje cells in culture. To begin investigating the molecular mechanisms by which trophic interactions and neural activity modulate cerebellar development, we have now studied the role of excitatory signals on the expression of both NGF and the p75 glycoprotein (the low-affinity component of the NGF receptor) by cerebellar cells in culture. We used p75 as a model of potential responsiveness, since it is well characterized and conveniently monitored. Expression of the NGF and p75 mRNA's was studied in either mixed, neuron-enriched, or pure glial cultures. Expression of the NGF gene was localized to proliferating glial cells, while expression of p75 was restricted to developing Purkinje cells. To evaluate whether presynaptic activation may potentially modulate trophic factor receptor expression, the expression of the p75 gene was studied in cultures exposed to excitatory signals. Depolarization of cultures with high potassium, veratridine, or exposure to the excitatory neurotransmitter aspartate, resulted in a two- to threefold increase in the expression of both the p75 protein and messenger RNA. These increases did not require the presence of glia, indicating a direct effect of the excitatory signals on the neuronal population. Moreover, message and receptor increased per neuron. Our study suggests that local glia provide trophic support for Purkinje cell development, and that impulse activity modulates Purkinje cell responsiveness by regulating expression of trophic receptor subunits.

Animals↗
PubMed1993

Regional and cell-specific expression of GDNF in rat brain.

The survival of ventral mesencephalic substantia nigra (SN) dopamine neurons, which degenerate in Parkinson's disease, is enhanced by glial cells in vitro. The recent isolation of glial cell line-derived growth factor (GDNF), a molecule with apparently selective effects on dopamine (DA) neurons in vitro, raises the question of whether this factor is found in normal brain cells. In this study, the polymerase chain reaction (PCR) was employed to determine the regional distribution and cellular localization of GDNF in the rat central nervous system. GDNF was expressed by SN and basal forebrain Type 1 (T1) astrocytes, with trace transcript levels present in cortical T1 astrocytes. Neuronal cultures of embryonic SN also expressed GDNF. Regionally, postnatal striatum contained the highest GDNF mRNA levels in vivo under the PCR conditions employed. Our data suggest a role for GDNF in both local and target-derived support of DA neurons, as well as potential involvement in the support of other neuronal populations in vivo.

Animals↗
PubMed1993

NGF increases brain astrocyte number in culture.

Nerve growth factor (NGF) promotes survival and maintenance of peripheral and central neurons. In previous studies, we found that low-affinity NGF binding sites were associated with flat nonneuronal cells dissociated and cultured from the embryonic septum. These cells were also labeled with a monoclonal antibody to the NGF receptor, 192 IgG, suggesting that low-affinity NGF receptors are associated with a nonneuronal population. To define the potential effect of NGF on these nonneuronal cells, rat Embryonic Day 17 (E17) septa were dissociated and cultured in fully defined medium in the presence of NGF. Glial fibrillary acidic protein (GFAP) was used as a marker for the astrocyte population. NGF elicited a dramatic ninefold increase in the number of GFAP-positive cells after 7 days. NGF similarly increased astrocyte number in cultures grown from E18 hippocampi and E16 substantia nigra, suggesting that NGF acts on glia from multiple brain regions. To begin defining the mechanism of NGF action on astrocytes, we examined [3H]thymidine incorporation, which increased significantly, but by only 50%, upon exposure to the trophic factor. We tentatively conclude that NGF increases astrocyte number in culture by stimulating mitosis as well as enhancing survival or differentiation.

Animals↗
PubMed1993

Purkinje cell survival is differentially regulated by metabotropic and ionotropic excitatory amino acid receptors.

We previously reported that trophic factors and neurotransmitters in concert regulate survival of cultured cerebellar Purkinje cells. In particular, excitatory amino acid (EAA) transmitters and NGF increased survival, whereas neither alone was effective. In the present studies, we sought to identify molecular mechanisms through which EAAs participate in the survival-promoting interaction. Initially, we characterized the potential role of ionotropic EAA receptors by exposing cultures to the antagonists MK-801, D-2-amino-5-phosphonovaleric acid, and 6,7-dinitroquinoxalinedione. Each increased cell number, suggesting that endogenous ionotropic activity decreased survival. To determine whether metabotropic EAA receptor stimulation modulates survival, the metabotropic agonist ACPD ([1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid; 1 microM) was tested. ACPD alone had no effect on survival. However, simultaneous exposure to ACPD and NGF significantly increased Purkinje number. Moreover, this increase in survival was blocked by L-AP3 [L(+)-2-amino-3-phosphonopropionic acid; 1 microM], a putative antagonist of certain metabotropic responses. L-AP3 also reduced cell number in the absence of exogenous EAA. Thus, endogenous metabotropic stimulation is normally necessary for survival. In sum, these studies reveal a novel mechanism whereby an excitatory neurotransmitter shapes neural development by simultaneous trophic and regressive actions that are, respectively, mediated by metabotropic and ionotropic EAA receptors.

2-Amino-5-phosphonovalerate↗
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↗
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

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↗
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↗