Comparison of transmitter release properties of embryonic sympathetic neurons growing in vivo and in vitro

Perivascular sympathetic nerves are important determinants of vascular function that are likely to contribute to vascular complications associated with hyperglycemia and diabetes. The present study tested the hypothesis that glucose modulates perivascular sympathetic nerves by studying the effects of 7 days of hyperglycemia on norepinephrine (NE) synthesis [tyrosine hydroxylase (TH)], release, and uptake. Direct and vascular-dependent effects were studied in vitro in neuronal and neurovascular cultures. Effects were also studied in vivo in rats made hyperglycemic (blood glucose >296 mg/dl) with streptozotocin (50 mg/kg). In neuronal cultures, TH and NE uptake measured in neurons grown in high glucose (HG; 25 mM) were less than that in neurons grown in low glucose (LG; 5 mM) ( P < 0.05; n = 4 and 6, respectively). In neurovascular cultures, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release from neurovascular cultures grown in HG (1.8 ± 0.2%; n = 5) was greater than that from cultures grown in LG (0.37 ± 0.28%; n = 5; P < 0.05; unpaired t-test). In vivo, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release in hyperglycemic animals (9.4 + 1.1%; n = 6) was greater than that in control animals (5.39 + 1.1%; n = 6; P < 0.05; unpaired t-test). These data identify a novel vascular-dependent effect of elevated glucose on postganglionic sympathetic neurons that is likely to affect the function of perivascular sympathetic nerves and thereby affect vascular function.

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Neurotrophins affect the pattern of DRG neurite growth in a bioassay that presents a choice of CNS and PNS substrates

Development ◽

10.1242/dev.121.5.1301 ◽

1995 ◽

Vol 121 (5) ◽

pp. 1301-1309 ◽

Cited By ~ 1

Author(s):

R. Tuttle ◽

W.D. Matthew

Keyword(s):

Spinal Cord ◽

Central Nervous System ◽

Nervous System ◽

Sciatic Nerve ◽

Sympathetic Neurons ◽

Neurite Growth ◽

Drg Neurons ◽

Substrate Preferences

Neurons can be categorized in terms of where their axons project: within the central nervous system, within the peripheral nervous system, or through both central and peripheral environments. Examples of these categories are cerebellar neurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons, respectively. When explants containing one type of neuron were placed between cryosections of neonatal or adult sciatic nerve and neonatal spinal cord, the neurites exhibited a strong preference for the substrates that they would normally encounter in vivo: cerebellar neurites generally extended only on spinal cord, sympathetic neurites on sciatic nerve, and DRG neurites on both. Neurite growth from DRG neurons has been shown to be stimulated by neurotrophins. To determine whether neurotrophins might also affect the substrate preferences of neurites, DRG were placed between cryosections of neonatal spinal cord and adult sciatic nerve and cultured for 36 to 48 hours in the presence of various neurotrophins. While DRG cultured in NGF-containing media exhibited neurite growth over both spinal cord and sciatic nerve substrates, in the absence of neurotrophins DRG neurites were found almost exclusively on the CNS cryosection. To determine whether these neurotrophin-dependent neurite patterns resulted from the selective survival of subpopulations of DRG neurons with distinct neurite growth characteristics, a type of rescue experiment was performed: DRG cultured in neurotrophin-free medium were fed with NGF-containing medium after 36 hours in vitro and neurite growth examined 24 hours later; most DRG exhibited extensive neurite growth on both peripheral and central nervous system substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

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Neural regulation of intestinal smooth muscle growth in vitro

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.3.g511 ◽

2000 ◽

Vol 279 (3) ◽

pp. G511-G519 ◽

Cited By ~ 16

Author(s):

M. G. Blennerhassett ◽

S. Lourenssen

Keyword(s):

Smooth Muscle ◽

Sympathetic Neurons ◽

Muscle Growth ◽

Scorpion Venom ◽

Enteric Neurons ◽

Intestinal Smooth Muscle ◽

Early Event ◽

Thymidine Uptake

The loss of intrinsic neurons is an early event in inflammation of the rat intestine that precedes the growth of intestinal smooth muscle cells (ISMC). To study this relationship, we cocultured ISMC and myenteric plexus neurons from the rat small intestine and examined the effect of scorpion venom, a selective neurotoxin, on ISMC growth. By 5 days after neuronal ablation, ISMC number increased to 141 ± 13% ( n = 6) and the uptake of [3H]thymidine in response to mitogenic stimulation was nearly doubled. Atropine caused a dose-dependent increase in [3H]thymidine uptake in cocultures, suggesting the involvement of neural stimulation of cholinergic receptors in regulation of ISMC growth. In contrast, coculture of ISMC with sympathetic neurons increased [3H]thymidine uptake by 45–80%, which was sensitive to propranolol (30 μM) and was lost when the neurons were separated from ISMC by a permeable filter. Western blotting showed that coculture with myenteric neurons increased α-smooth muscle-specific actin nearly threefold to a level close to ISMC in vivo. Therefore, factors derived from enteric neurons maintain the phenotype of ISMC through suppression of the growth response, whereas catecholamines released by neurons extrinsic to the intestine may stimulate their growth. Thus inflammation-induced damage to intestinal innervation may initiate or modulate ISMC hyperplasia.

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Effects of Buthus martensii Karsch scorpion venom on the release of noradrenaline from in vitro and in vivo rat preparations

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-121 ◽

1994 ◽

Vol 72 (8) ◽

pp. 855-861 ◽

Cited By ~ 7

Author(s):

Sylvain Foucart ◽

Rui Wang ◽

Pierre Moreau ◽

Rémi Sauvé ◽

Jacques de Champlain ◽

...

Keyword(s):

Intracellular Calcium ◽

Noradrenaline Release ◽

Sympathetic Neurons ◽

Scorpion Venom ◽

Dependent Manner ◽

Release Of Noradrenaline ◽

Adult Rat ◽

Isolated Atria

The aim of this study was to test the neuronal effects of the Chinese Buthus martensii Karsch (BMK) scorpion venom in vivo and in vitro in order to understand the mechanism involved in the cardiovascular pressor effect of this venom. In conscious unrestrained rats, administration of 100 μg/kg i.v. BMK venom induced an increase in blood pressure, which was associated with a significant increase in plasma noradrenaline. In isolated atria, BMK also induced an increase in the stimulation-induced release of [3H]noradrenaline in a dose-dependent manner. The modulatory effect of agents acting at sympathetic prejunctional adrenoceptors on [3H]noradrenaline release was not altered by BMK venom administration. Finally, it was observed that 100 μg/mL BMK venom increased the intracellular calcium concentration in acutely dissociated sympathetic neurons from adult rat superior cervical ganglion. This action appeared to be mainly due to an influx of extracellular calcium. BMK venom induced a small rise in intracellular calcium in the absence of external calcium, indicating that it may also mobilize calcium from intracellular stores. The results observed in this study suggest that BMK venom may induce pressor responses by releasing noradrenaline from the sympathetic nerve terminals and that activation of neuronal calcium channels may be involved in that process.Key words: scorpion venom, noradrenaline release, presynaptic modulation, intracellular calcium.

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Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents

The Journal of Cell Biology ◽

10.1083/jcb.143.2.457 ◽

1998 ◽

Vol 143 (2) ◽

pp. 457-467 ◽

Cited By ~ 167

Author(s):

David S. Park ◽

Erick J. Morris ◽

Jaya Padmanabhan ◽

Michael L. Shelanski ◽

Herbert M. Geller ◽

...

Keyword(s):

Cortical Neurons ◽

Sindbis Virus ◽

Kinase Inhibitors ◽

Sympathetic Neurons ◽

Dominant Negative ◽

Cyclin Dependent Kinase ◽

Cyclin Dependent Kinases ◽

Dna Damaging Agents

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16ink4, p21waf/cip1, and p27kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

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Sympathetic neuroblasts undergo a developmental switch in trophic dependence

Development ◽

10.1242/dev.119.3.597 ◽

1993 ◽

Vol 119 (3) ◽

pp. 597-610 ◽

Cited By ~ 14

Author(s):

S.J. Birren ◽

L. Lo ◽

D.J. Anderson

Keyword(s):

Neurotrophic Factors ◽

Sympathetic Neurons ◽

Neurotrophin Receptor ◽

Neurotrophin 3 ◽

Bona Fide ◽

Neuronal Number ◽

High Doses ◽

Developmental Switch

Sympathetic neurons require NGF for survival, but it is not known when these cells first become dependent on neurotrophic factors. We have examined in vitro mitotically active sympathetic neuroblasts immuno-isolated from different embryonic stages, and have correlated this functional data with the expression of neurotrophin receptor mRNAs in vivo. Cells from E14.5 ganglia are supported by neurotrophin-3 (NT-3) in a serum-free medium, but not by NGF; NT-3 acts as a bona fide survival factor for these cells and not simply as a mitogen. By birth, sympathetic neurons are well-supported by NGF, whereas NT-3 supports survival only weakly and at very high doses. This change in neurotrophin-responsiveness is correlated with a reciprocal switch in the expression of trkC and trkA mRNAs by sympathetic neuroblasts in vivo. These data suggest that neurotrophic factors may control neuronal number at earlier stages of development than previously anticipated. They also suggest that the acquisition of NGF-dependence may occur, at least in part, through the loss of receptors for these interim survival factors.

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THE P53 FAMILY MEMBER, P63, REGULATES NEURAL PRECURSOR CELL SURVIVAL DURING CORTICAL DEVELOPMENT

Clinical & Investigative Medicine ◽

10.25011/cim.v31i4.4799 ◽

2008 ◽

Vol 31 (4) ◽

pp. 8

Author(s):

Sagar Dugani ◽

Annie Paquin ◽

Masashi Fujitani ◽

David R Kaplan ◽

Freda D Miller

Keyword(s):

Cell Death ◽

Sympathetic Neurons ◽

Cortical Development ◽

Cre Recombinase ◽

Cortical Plate ◽

P53 Family ◽

Knock Down ◽

Developing Neurons

Background: p63, a member of the p53 family of proteins, is involved in the regulation of naturally-occurring apoptosis in sympathetic neurons of the peripheral nervous system. Since data from our laboratory indicated that p63 is also expressed in stem cells and neurons within the developing brain, we hypothesized that p63 is involved in regulating the genesis and survival of developing neurons. Methods: As cortical neurogenesis is initiated at embryonic day 12, we knocked-down p63 levels in isolated murine cortical precursors byusing shRNA against p63 or by transfecting floxed-p63 precursors with Cre recombinase. We performed similar studies in vivo using in uteroelectroporation to express either p63 shRNA or Cre recombinase to acutely knockdown or genetically ablate p63. We then performed immunofluorescence for known markers of apoptosis, cell-division, and differentiation to assess the level of cell death, proliferation and neurogenesis. Results: Knock-down of p63 in vitro resulted in a 2-foldincrease in the death of precursors and neurons, associated with blunted neurogenesis but unaltered precursor proliferation. Coincident knock-down of p63 family members, p53, but not p73, rescued the elevated death suggesting that p63 and p53 antagonize each other to promote survival. Similar results were observed in vivo, where knockdown of p63 caused cell death and a decrease in the proportionof neurons in the cortical plate. Conclusions: These experiments indicate that p63 is required forthe survival of neural precursors and newly-born neurons, and for normal cortical development. Ongoing work will explore the environmental cues that regulate p63 during neurogenesis.

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Elucidating the role of P63 during development of the mammalian nervous systems

Clinical & Investigative Medicine ◽

10.25011/cim.v30i4.2848 ◽

2007 ◽

Vol 30 (4) ◽

pp. 79

Author(s):

Sagar Dugani ◽

Annie Paquin ◽

David R. Kaplan ◽

Freda D. Miller

Keyword(s):

Stem Cells ◽

Cell Death ◽

Cortical Neurons ◽

Sympathetic Neurons ◽

Fold Increase ◽

P53 Family ◽

Ongoing Work ◽

Cortical Precursors

Background: The protein p63, a recently discovered member of the p53 family of proteins, is implicated in the maintenance and differentiation of stem cells in the epidermis and is involved in the regulation of naturally-occurring cell death in sympathetic neurons of the peripheral nervous system. Since initial data from our laboratory indicated that p63 is also widely expressed in stem cells and neurons within the developing brain, we assessed its involvement in regulating the genesis and survival of developing cortical neurons. As neurogenesis is initiated at embryonic day 12 (E12), we isolated cortical precursors from p63-/- embryos at E14 and cultured them for 2 days in vitro (DIV). Methods: Based on immunocytochemistry to known markers of apoptosis and neurons, we assessed the level of cell death and neurogenesis. Results: Compared to p63+/+ cortical precursors, p63-/- precursors from littermates showed a 50 % reduction in neuronal death, as assessed by the apoptosis marker, cleaved caspase 3. Interestingly, the proportion of neurons and astrocyte precursors, the latter identified by S100b was also reduced in p63-/- embryos, as compared to p63+/+ littermates. Conclusions: These results suggest that p63 may be involved in the regulation of cell survival and in the differentiation of precursors into neurons and astrocytes. To assess the former, we overexpressed TAp63a, a full-length isoform of p63, in E12/13 cortical precursors and assessed the level of cell death after 2 DIV. Compared to control cells, cells transfected with TAp63a demonstrated a 2-fold increase in cell death. Ongoing work will characterize p63 involvement in differentiation of precursor cells into neurons and astrocytes. To assess if these findings are relevant in vivo, we will use p63flox,flox X Nextin-Cre mice, which have p63 specifically ablated in neural precursors. We will analyze the survival, proliferation, and fate of these p63-/- cells. Together, these studies will help to determine a role for p63 in neural proliferation and apoptosis, processes central to development and response to injury.

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Multiple mechanisms regulate sympathetic neuronal phenotype

Development ◽

10.1242/dev.121.8.2361 ◽

1995 ◽

Vol 121 (8) ◽

pp. 2361-2371

Author(s):

A.K. Hall ◽

S.E. MacPhedran

Keyword(s):

Sympathetic Neurons ◽

Intrinsic Property ◽

Sympathetic Ganglia ◽

Environmental Cues ◽

Peripheral Target ◽

Neuronal Phenotype ◽

Adult Rat ◽

Cervical Ganglion

Adult rat sympathetic neurons can possess specific neuropeptides utilized as cotransmitters along with norepinephrine, but the factors that regulate their expression remain unknown. 60% of adult rat superior cervical ganglion (SCG) neurons express neuropeptide Y (NPY) in vivo. To determine whether the restricted expression was an intrinsic property of sympathetic ganglia, we examined if embryonic sympathetic precursors gave rise to NPY immunoreactive (-IR) neurons in vitro. After one week in culture, 60% of neurons derived from the E14.5 rat SCG were NPY-IR. Thus, ganglia isolated before peripheral target contact or preganglionic innervation were capable of regulating NPY expression both in the number of neurons with NPY and in the developmental timing of NPY expression. To determine if the restricted expression of NPY was a reflection of neuroblasts committed to an NPY fate, SCG precursors were labeled with a replication incompetent retrovirus carrying lacZ, and NPY expression in lacZ-labeled clones examined after one week. Two thirds of neuronal clones obtained were uniformly NPY-IR; that is, all neurons in a clone either possessed or lacked NPY. One-third of the neuronal clones were mixed and contained both neurons with and without NPY. We provide a novel demonstration that both lineage and environmental cues contribute to neuropeptide phenotype.

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