Development and regulation of substance P in sensory neurons in vitro

Adler, Joshua E.; Kessler, John A.; Black, Ira B.

doi:10.1016/0012-1606(84)90206-9

Cited by 44 publications

(33 citation statements)

References 30 publications

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“…In DRG neurons cultured before the establishment of functional connections with target tissues, the addition of muscle, skin, brain, or spinal cord reduced the number of substance P-IR neurons (Barakat-Walter et al, 1991). NGF given to newborn rats stimulates a dose-dependent increase in substance P content in DRG in vivo (Otten et al, 1980) and in adult DRG cultures , but NGF does not seem to alter significantly the number of sensory neurons that express the peptide (Adler et al, 1984;. In combination, these observations suggest that target-derived signals can influence some sensory phenotypic properties.…”

Section: Discussionmentioning

confidence: 94%

“…Thus, although functional subtypes in mature animals show significant neurotrophin specificity, the embryonic neurons used in the present experiments do not display those subtypes. NGF is thought to regulate the level of neuropeptides already expressed by the neuron rather than to induce de novo expression of those neuropeptides (Kessler and Black, 1980;Otten et al, 1980;Kessler and Black, 1981;Adler et al, 1984;Lindsay and Harmar, 1989;Inaishi et al, 1992;Davies, 1994;Snider, 1994;Bothwell, 1995). Furthermore, although the NGF receptor trkA is downregulated in many small DRG neurons postnatally, the CGRP-IR neuronal phenotype is specified before the downregulation occurs (D. Molliver and W. Snider, personal communication).…”

Section: Discussionmentioning

confidence: 99%

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The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

Hall

Hickman

et al. 1997

J. Neurosci.

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Adult sensory neurons differ chemically, morphologically, and functionally, but the factors that generate their diversity remain unclear. For example, neuropeptides are generally found in small neurons, whereas abundant neurofilament is common in large neurons. Neurons containing the neuropeptides calcitonin gene-related peptide (CGRP) or substance P were quantified using immunohistochemistry in rat lumbar dorsal root ganglion (DRG) at times before and after sensory neurons contact central and peripheral targets in vivo. No neurons in the newly formed DRG expressed neuropeptide or neuropeptide mRNA, but neuropeptides were detectable about the time that axons connect with peripheral targets. To determine the requirement for target in neuropeptide regulation, embryonic DRG neurons were isolated at times before central and peripheral connections had formed, placed in culture, and immunocytochemically assayed for CGRP and substance P. Cultured neurons expressed neuropeptides with a time course and in proportions similar to those in vivo. Thus, some neurons in the embryonic DRG seem to be intrinsically specified to later express CGRP and substance P. The percentage of CGRP-immunoreactive neurons was not changed by cell density, non-neuronal cells, neurotrophins in addition to nerve growth factor (NGF), or antibody inactivation of neurotrophin-3 in the presence of NGF. To test the role of extrinsic cues on CGRP expression, DRG neurons were co-cultured with potential target tissues. Coculture with a rat epidermal or smooth muscle cell line increased the proportion of CGRP-containing neurons, whereas primary skeletal muscle and 3T3 cells had no effects. Thus, multiple appropriate sensory neuron phenotypes arise in a regulated fashion in cultured neurons isolated before target connections have formed, and some candidate target tissues can modulate that intrinsic expression pattern. Key words: neuropeptides; substance P; calcitonin generelated peptide; neurofilament; nociceptive; sensory ganglion; target tissue; skin; neurotrophinA striking feature of the adult vertebrate sensory nervous system is the diversity of neuronal cell types that perform together to maintain homeostasis and convey information about the environment. During development, specific neurons must be generated in appropriate locations and connect discretely to both peripheral targets and the CNS for functional integrity. Thus, it is important to understand not only how the phenotype is generated but also how neurons match targets. This study examines the developmental regulation of sensory neurons containing calcitonin generelated peptide (CGRP) that predominantly contact visceral and cutaneous peripheral target end organs in vivo.Adult rat sensory neurons can express neuromodulatory neuropeptides, but the factors that regulate their initial expression remain unknown. Cutaneous afferents appear in rat proximal hindlimb on embryonic days 14 -15 (E14 -E15) and in skin of distal toes at E16 -E17 (Reynolds et al

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

Hall

Hickman

et al. 1997

J. Neurosci.

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“…Because neonatal DRG neurons undergo apoptosis in the absence of NGF, neurite growth experiments were conducted in the presence of the neurotrophin (31). To assess the signaling effects of ATP␥S independent of NGF, we used DRG neurons derived from 4-week old mice, which do not require NGF for survival (32).…”

Section: Genetic or Sirna Depletion Of P2y2 Receptors Eliminates Atp␥mentioning

confidence: 99%

P2Y ₂ receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

Arthur

Akassoglou²,

Insel³

2005

Proc. Natl. Acad. Sci. U.S.A.

107

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Neurotrophins are essential for neuronal differentiation, but the onset and the intensity of neurotrophin signaling within the neuronal microenvironment are poorly understood. We tested the hypothesis that extracellular nucleotides and their cognate receptors regulate neurotrophin-mediated differentiation. We found that 5 -O-(3-thio)triphosphate (ATP␥S) activation of the G protein-coupled receptor P2Y2 in the presence of nerve growth factor leads to the colocalization and association of tyrosine receptor kinase A and P2Y2 receptors and is required for enhanced neuronal differentiation. Consistent with these effects, ATP␥S promotes phosphorylation of tyrosine receptor kinase A, early response kinase 1͞2, and p38, thereby enhancing sensitivity to nerve growth factor and accelerating neurite formation in both PC12 cells and dorsal root ganglion neurons. Genetic or small interfering RNA depletion of P2Y2 receptors abolished the ATP␥S-mediated increase in neuronal differentiation. Moreover, in vivo injection of ATP␥S into the sciatic nerve increased growth-associated protein-43 (GAP-43), a marker for axonal growth, in wild-type but not P2Y2 ؊/؊ mice. The interactions of tyrosine kinase-and P2Y 2-signaling pathways provide a paradigm for the regulation of neuronal differentiation and suggest a role for P2Y 2 as a morphogen receptor that potentiates neurotrophin signaling in neuronal development and regeneration.neurotrophin signaling ͉ P2Y2 ͉ tyrosine receptor kinase A N eurite formation, a process extending from the cell soma and led by a growth cone, is a primary morphological event in neuronal differentiation, ultimately facilitating synaptic connections by neurons (1). Neuronal regeneration depends on the formation of neurites to repair injured or lost connections. These neuronal growth events require appropriate spatial and temporal expression and action of both initiation signals and promoter molecules (2). Neurotrophins, such as nerve growth factor (NGF), are key regulators of neuronal differentiation. NGF is released by target tissues, initiates neurite generation, maintains neuronal survival, prevents apoptosis, and promotes synapse formation (3-5). NGF signaling via the tyrosine receptor kinase A (TrkA) leads to stimulation of early response kinase 1͞2 (ERK1͞2) via Ras͞Raf pathways, which is required for differentiation, as well as the activation of the survival kinase, Akt (6). The regulation of NGF͞ TrkA signaling is determined by availability of NGF, as well as TrkA activation, characterized by receptor autophosphorylation, internalization, and retrograde transport from axons to cell bodies (7). Recent evidence has shown that receptor crosstalk is an important mechanism for regulation of neurotrophin signaling (6). The NGF͞ TrkA-signaling pathway converges with pain-related ion channels to regulate NGF-mediated heat sensitivity of sensory neurons (5) and with adenosine A 2A receptors to regulate neuronal survival (8). The contributions of other signal transduction pathways integrated with NGF͞TrkA signali...

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“…All experimental and control cultures were grown for 1 week. Substance P is expressed as pg per dish (mean ± SEM); choline acetyltransferase activity is expressed as nmol of product per dish per hr (mean SEM); 0 time, 6 hr after seeding. characters are, in fact, present in the same sympathetic neurons under a variety of conditions (11,15).…”

Section: Discussionmentioning

confidence: 99%

“…Methods of dissociation and culture have been described (4 Extraction and Assay Procedures for Substance P, Tyrosine Hydroxylase, and Choline Acetyltransferase. Substance P was extracted from cultures and quantified by radioimmunoassay as described (6). For tyrosine hydroxylase extraction, cultures were rinsed with ice-cold Puck's saline G, following which 200 ,ul of 20 mM potassium phosphate buffer, pH 7.6/0.2% Triton X-100 was added.…”

Section: Methodsmentioning

confidence: 99%

Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.

Adler¹,

Black²

1985

Proc. Natl. Acad. Sci. U.S.A.

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The effects of cell density and aggregation on expression of transmitter traits were examined in dissociated, pure sympathetic neuron cultures, grown in fully defmed, serum-free medium. After 1 week at a density of 7-8 x 103 neurons per 35-mm dish, moderate levels of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) activity and substance P were detected. When neuron density was increased 4-fold, a 4-fold increase in tyrosine hydroxylase activity was observed; i.e., there was no change in tyrosine hydroxylase activity per neuron. In contrast, substance P increased 30-fold, corresponding to a 7-fold increase in substance P per neuron.Choline O-acetyltransferase (EC 2.3.1.6) activity, not detected at low cell densities, was first detectable at a concentration of 15,000 neurons per dish and increased 6-fold when this cell concentration was doubled. Medium conditioned by highdensity cultures failed to reproduce these effects on low-density cultures, suggesting that diffusible factors are not involved in thedensity-dependentdifferential regulation. Time-lapse phasecontrast microscopy of high-density cultures showed neuronal migration and progressive aggregation, which did not occur in low-density cultures. Our observations suggest that cell contact may mediate differential expression of transmitter traits.Development of the nervous system consists of a set of seemingly discrete, reproducible processes including cellular migration, aggregation, transmitter phenotypic expression, and synaptogenesis. Although a number of these processes have been defined in some detail, potential mechanistic relationships among the processes are unclear. For example, it is well recognized that aggregation generates the stable formation of nuclei in the brain and ganglia in the periphery. What, however, is the relationship of aggregation to transmitter expression? One hint may derive from observations in the embryonic rat in vivo: initial expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) and dopamine-f3-hydroxylase (dopamine ,-monooxygenase, EC 1.14.17.1) and of catecholamines coincides with cellular aggregation to form the primitive sympathetic ganglia (1). The temporal association of cellular aggregation and transmitter expression raised the possibility that these processes are causally related. This possibility has gained indirect support from the observations that growth of bovine adrenal chromaffin cells (2) or PC12 rat pheochromocytoma cells (3) in high-density cell culture selectively increased tyrosine hydroxylase specific activity.To examine the relationship of cell aggregation and transmitter expression, we are growing virtually pure, neonatal rat sympathetic neurons in fully defined, serum-free medium at various cell densities. We now report that increasing cell density (with attendant neuronal aggregation) differentially affects levels of tyrosine hydroxylase; choline O-acetyltransferase (EC 2.3.1.6), a cholinergic enzyme; and substance P, a ...

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Development and regulation of substance P in sensory neurons in vitro

Cited by 44 publications

References 30 publications

The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

P2Y ₂ receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.

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Development and regulation of substance P in sensory neurons in vitro

Cited by 44 publications

References 30 publications

The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

The Generation of Neuronal Heterogeneity in a Rat Sensory Ganglion

P2Y 2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation

Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.

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P2Y ₂ receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation