RESUMEN
The effect of age on the plasticity of the putative peptide neurotransmitter substance P (SP) was examined in the rat superior cervical sympathetic ganglion. Explantation of ganglia from 6-month-old rats to serum-supplemented culture resulted in a tenfold increase in SP concentration, reproducing results previously obtained for ganglia from neonatal rats. Veratridine prevented the increase in SP concentration in adult ganglia, and tetrodotoxin blocked the veratridine effect, suggesting that membrane depolarization and sodium influx prevented the rise in the SP content of adult ganglia as well as of neonatal ganglia. However, the time courses of the increase in the amount of the peptide differed in neonatal and mature ganglia, suggesting that some aspects of regulation may differ in the two. The effects of aging on neural plasticity were further analyzed by explanting ganglia from 2-year-old rats. No significant increase in SP concentration was observed in these ganglia. Remarkable plasticity thus seems to persist in mature neurons but may be deficient in aged sympathetic neurons.
Asunto(s)
Ganglios Simpáticos/análisis , Plasticidad Neuronal , Neuronas/análisis , Sustancia P/análisis , Envejecimiento , Animales , Técnicas de Cultivo , Ganglios Simpáticos/citología , Ganglios Simpáticos/fisiología , Neuronas/fisiología , Ratas , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
Peptidergic-noradrenergic interactions were examined in explants of rat sympathetic superior cervical ganglia and in cultures of dissociated cells. The putative peptide transmitters substance P and somatostatin each increased the activity of the catecholamine-synthesizing enzyme tyrosine hydroxylase after 1 week of exposure in culture. Maximal increases occurred at 10(-7) molar for each peptide, and either increasing or decreasing the concentration reduced the effects. Similar increases in tyrosine hydroxylase were produced by a metabolically stable agonist of substance P, while a substance P antagonist prevented the effects of the agonist. The data suggest that the increased tyrosine hydroxylase activity was mediated by peptide interaction with specific substance P receptors and that peptides may modulate sympathetic catecholaminergic function.
Asunto(s)
Ganglios Simpáticos/enzimología , Somatostatina/farmacología , Sustancia P/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Bacitracina/farmacología , Captopril/farmacología , Células Cultivadas , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , RatasRESUMEN
Regulation of the putative peptide neurohumour [Leu]enkephalin and the catecholaminergic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyl-transferase was examined in the rat adrenal medulla in vivo and in vitro. Surgical denervation of the adrenal gland or pharmacologic blockade of synaptic transmission, treatments known to decrease catecholamine traits, increased [Leu]enkephalin content. Medullas explanted to culture exhibited a 50-fold rise in [Leu]enkephalin in 4 days, whereas tyrosine hydroxylase remained constant, and phenylethanolamine-N-methyltransferase decreased to a new baseline level. Veratridine-induced depolarization prevented the accumulation of [Leu]enkephalin, an effect that was blocked by tetrodotoxin, which antagonizes transmembrane Na+ influx. These studies suggest that enkephalinergic and catecholamine characters are differentially regulated by impulse activity and depolarization in the adrenal medulla.
Asunto(s)
Médula Suprarrenal/metabolismo , Catecolaminas/fisiología , Encefalina Leucina/fisiología , Médula Suprarrenal/efectos de los fármacos , Médula Suprarrenal/fisiología , Animales , Catecolaminas/metabolismo , Encefalina Leucina/metabolismo , Feniletanolamina N-Metiltransferasa/metabolismo , Ratas , Sodio/metabolismo , Transmisión Sináptica , Tetrodotoxina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Veratridina/farmacologíaRESUMEN
Regulation of the putative peptide neurotransmitter substance P was examined in the superior cervical sympathetic ganglion of the neonatal rat. Surgical decentralization (denervation) of the superior cervical ganglion increased ganglion substance P content. In cultured ganglia, the amount of substance P increased more than 50-fold after 48 hours, and this rise was dependent on protein and RNA synthesis. Veratridine prevented the increase in substance P in vitro, and tetrodotoxin blocked the veratridine effect; this suggests that sodium influx and membrane depolarization prevent substance P elevation. Immunohistochemical analysis of cultured ganglia indicated that substance P was present in the perikarya of principal sympathetic neurons and in ganglionic nerve processes. Transsynaptic impulses, through the mediation of postsynaptic sodium influx, may decrease substance P in sympathetic neurons.
Asunto(s)
Ganglios Simpáticos/fisiología , Sustancia P/metabolismo , Potenciales de la Membrana , Neuronas/fisiología , Transmisión SinápticaRESUMEN
The use of molecular biological approaches has defined new mechanisms that store information in the mammalian nervous system. Environmental stimuli alter steady-state levels of messenger RNA species encoding neurotransmitters, thereby altering synaptic, neuronal, and network function over time. External or internal stimuli alter impulse activity, which alters membrane depolarization and selectively changes the expression of specific transmitter genes. These processes occur in diverse peripheral and central neurons, suggesting that information storage is widespread in the neuraxis. The temporal profile of any particular molecular mnemonic process is determined by specific kinetics of turnover and by the geometry of the neuron resulting in axonal transport of molecules to different synaptic arrays at different times. Generally, transmitters, the agents of millisecond-to-millisecond communication, are subject to relatively long-lasting changes in expression, ensuring that ongoing physiological function is translated into information storage.
Asunto(s)
Memoria/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Neurotransmisores/fisiología , Médula Suprarrenal/metabolismo , Animales , Encéfalo/fisiología , Sistema Nervioso/anatomía & histología , Sistema Nervioso/metabolismo , Neuronas/fisiología , Neurotransmisores/metabolismo , Sistema Nervioso Simpático/metabolismo , Sistema Nervioso Simpático/fisiología , Transcripción GenéticaRESUMEN
Contrary to long-held assumptions, recent work indicates that neurons may profoundly change transmitter status during development and maturity. For example, sympathetic neurons, classically regarded as exclusively noradrenergic or cholinergic, can also express putative peptide transmitters such as substance P. This neuronal plasticity is directly related to membrane depolarization and sodium ion influx. The same molecular mechanisms and plastic responses occur in mature as well as developing neurons. Further, contrary to traditional teaching, adult primary sensory neurons may express the catecholaminergic phenotype in vivo. Transmitter plasticity is not restricted to the peripheral nervous system: ongoing studies of the brain nucleus locus ceruleus in culture indicate that specific extracellular factors elicit marked transmitter changes. Consequently, neurotransmitter expression and metabolism are dynamic, changing processes, regulated by a variety of defined factors. Transmitter plasticity adds a newly recognized dimension of flexibility to nervous system function.
Asunto(s)
Sistema Nervioso/crecimiento & desarrollo , Plasticidad Neuronal , Neuronas/fisiología , Neurotransmisores/fisiología , Médula Suprarrenal/fisiología , Envejecimiento , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Catecolaminas/fisiología , Neuronas Aferentes/fisiologíaRESUMEN
Mechanisms regulating the content of the putative peptide transmitters, substance P and somatostatin, were examined in several neuronal populations in culture. Substance P levels increased more than 25-fold within 48 h in sympathetic neurons in the explanted rat superior cervical ganglion, and remained elevated for 4 weeks. Identity of the peptide was authenticated by combined high pressure liquid chromatography-radioimmunoassay. Veratridine prevented the increase of substance P in vitro, and tetrodotoxin blocked the veratridine effect, suggesting that sodium ion influx and membrane depolarization prevent peptide elevation. Veratridine (or potassium)-induced membrane depolarization released substance P into the culture medium through a calcium-dependent process. Consequently, at least some veratridine effects are attributable to release and subsequent depletion of ganglion peptide. However, the inhibitory effects of veratridine were far greater than could be accounted for by the quantity of peptide released, suggesting a separate influence on net synthesis (synthesis less catabolism) of substance P. Viewed in conjunction with previous in vivo studies, our observations suggest that trans-synaptic impulses, through the mediation of postsynaptic sodium flux, release substance P from sympathetic neurons and also regulate intracellular peptide metabolism. To determine whether the processes regulating substance P in sympathetic neurons reflect generalized mechanisms, a different peptide, somatostatin, was examined in sympathetic neurons; moreover, substance P was examined in a different neuronal population, special sensory neurons in the nodose ganglion. Substance P levels increased significantly in both sympathetic and sensory neurons after explantation, and somatostatin levels increased in sympathetic neurons. In each instance, the increase was dependent upon the presence of the calcium ions. Moreover, these increases were all prevented by veratridine, in a tetrodotoxin-sensitive manner. Our observations suggest that common regulatory mechanisms govern peptide transmitter metabolism in diverse neuronal populations.
Asunto(s)
Ganglios Simpáticos/metabolismo , Ganglio Nudoso/metabolismo , Somatostatina/metabolismo , Sustancia P/metabolismo , Transmisión Sináptica , Nervio Vago/metabolismo , Animales , Calcio/farmacología , Diferenciación Celular , Desnervación , Femenino , Ganglios Simpáticos/citología , Potenciales de la Membrana/efectos de los fármacos , Neuronas/metabolismo , Ganglio Nudoso/citología , Embarazo , Ratas , Ratas Endogámicas , Sinapsis/metabolismo , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
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.
Asunto(s)
Encéfalo/fisiología , Comunicación Celular , Regulación de la Expresión Génica , Hipocampo/fisiología , Factores de Crecimiento Nervioso/genética , Neuroglía/fisiología , Médula Espinal/fisiología , Animales , Animales Recién Nacidos , División Celular , Membrana Celular/fisiología , Células Cultivadas , Neuroglía/citología , ARN Mensajero/genética , RatasRESUMEN
High cell density, with attendant aggregation, selectively increases expression of substance P (SP) and choline acetyltransferase (ChAT) in virtually pure neonatal sympathetic neuronal cultures. To investigate the specific role of cell contact in selective transmitter expression, SP content and ChAT activity were examined in such cultures under various conditions. At high neuronal density SP content, detectable 6 h after plating, doubled during the first two culture days and subsequently increased more than 10-fold. Similarly, ChAT activity appeared de novo after two days and rose rapidly thereafter. The increases closely paralleled perikaryal aggregation, suggesting that cell contact might be the critical factor. Moreover, interference with aggregation physically, using methylcellulose, or chemically, using tunicamycin, inhibited the increases in SP content and ChAT activity without affecting neuronal survival. Thus, cell contact appears to mediate the expression of ChAT and the rise of SP in high-density neuronal cultures. To determine whether interaction of membrane component(s) elicited the rises in ChAT activity and SP content, membranes extracted from the neonatal superior cervical ganglion (SCG) were added to cultures of varying densities. After 3 days in high-density cultures, membranes doubled the increases in ChAT and SP. Moreover, even in lower-density cultures, membranes elicited the appearance of ChAT activity. Specificity was defined by examining membranes extracted from a variety of neonatal rat tissues. Dorsal root ganglia membranes were most effective in stimulating ChAT, followed by membranes from the SCG, kidney and brain. Membranes derived from the adrenal gland, liver and spinal cord had no effect. Our findings suggest that interaction of cell membrane components regulates phenotypic expression in aggregating neurons.
Asunto(s)
Colina O-Acetiltransferasa/metabolismo , Ganglios Simpáticos/fisiología , Sustancia P/metabolismo , Animales , Animales Recién Nacidos , Agregación Celular , Comunicación Celular , Células Cultivadas , Fenotipo , Ratas , Ratas Endogámicas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Transsynaptic activity differentially regulates biosynthesis of sympathoadrenal catecholamines and co-localized opiate peptides in the rat. We determined whether similar mechanisms were operative during development. Adrenal Leu-enkephalin (LEU), was first detected at E16.5, then increased 5-fold during maturation from birth to adulthood while adrenal weight increased 10-fold. Since medullary cells do not divide after the first postnatal week, this represents a specific maturational increase in LEU content per chromaffin cell. In adult medullae, decreasing transsynaptic activity through adrenal denervation or explantation results in a 30-50-fold increase in LEU. In contrast, LEU levels in denervated or explanted medullae from neonatal rats (less than or equal to 10 days) do not. Prolonged denervation (day 5-21) prevented even the normal maturational increase in LEU. However, depolarizing medullae with KCl lowered LEU levels at all ages tested with an increased magnitude of effect after 10 days postnatal age. Specific deficits in signal-transduction mechanisms or immaturity of opiate biosynthetic pathways may account for these observations. Thus, during development, adrenal opiate peptides are not under transsynaptic control yet require presynaptic terminals to mature normally. Therefore, like catecholamines, co-localized adrenal opiate peptides require presynaptic regulatory signals to achieve normal development and function.
Asunto(s)
Médula Suprarrenal/crecimiento & desarrollo , Envejecimiento/fisiología , Encefalina Leucina/biosíntesis , Sinapsis/fisiología , Médula Suprarrenal/inervación , Médula Suprarrenal/metabolismo , Animales , Animales Recién Nacidos , Desnervación , Ratas , Ratas EndogámicasRESUMEN
Although glucocorticoids and impulse activity are well-recognized mediators of adrenal catecholamine biosynthesis, the effects of these signals on the colocalized opiate peptide system is only presently emerging. Since it is generally agreed that impulse activity regulates adrenal opiate peptides, in the present report we sought to determine whether adrenal opiates are also subject to hormonal control. Pharmacological destruction of the adrenal cortex resulted in a decrease in baseline Leu-enkephalin levels in vivo. This suggested a tonic regulatory effect of adrenal cortical steroids on enkephalin pathways. To further examine the role of glucocorticoid hormones in regulating enkephalin biosynthesis in a more dynamic system, medullae were grown as explants where peptide levels typically rise 30- to 50-fold above baseline. Explanted medullae required medium supplemented with dexamethasone or corticosterone to achieve maximal levels of Leu-enkephalin in a dose-dependent fashion. The effects of glucocorticoid treatment were blocked by specific glucocorticoid receptor antagonists or by inhibition of receptor translocation to the nucleus. Since enkephalin levels rose in cultured medullae (even in the absence of added glucocorticoids), glucocorticoid-independent regulatory mechanisms may also play a role in this model. Based on this and previous results, it appears that adrenal opiate peptides, like catecholamines, are subject to dual hormonal and transsynaptic regulatory influences. The interaction of these two regulatory mechanisms may serve an adaptive role in modulating complex biochemical and behavioral responses with exquisite precision.
Asunto(s)
Médula Suprarrenal/metabolismo , Encefalina Leucina/metabolismo , Glucocorticoides/fisiología , Médula Suprarrenal/efectos de los fármacos , Animales , Células Cultivadas , Corticosterona/farmacología , Corticosterona/fisiología , Cortodoxona/farmacología , Dexametasona/análogos & derivados , Dexametasona/farmacología , Dexametasona/fisiología , Glucocorticoides/antagonistas & inhibidores , Glucocorticoides/farmacología , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacología , Técnicas In Vitro , Masculino , Ratas , Ratas Endogámicas , Receptores de Glucocorticoides/efectos de los fármacosRESUMEN
To determine whether similar molecular mechanisms regulate the same proteins in diverse neuronal populations, the present study compared regulation of tyrosine hydroxylase (TOH) in placodal sensory and neural crest-derived sympathetic neurons in tissue culture. Long-term explant cultures of adult nodose and petrosal sensory ganglia (NPG) contained abundant TOH-immunoreactive neurons and exhibited TOH catalytic activity, as in vivo. After an initial decline during the first week of culture, enzyme activity was maintained at a stable plateau of 60% of zero time values for at least 3 weeks. However, exposure of 2-week-old cultures to depolarizing concentrations of potassium (K+; 40 mM) increased TOH activity approximately two-fold; total protein was unchanged, suggesting that the rise was due to increased TOH specific activity. Therefore, membrane depolarization in vitro appears to regulate this specific catecholaminergic (CA) trait in sensory, as in sympathetic CA cells. In sympathetic neurons, NGF regulates TOH activity throughout life. In marked contrast, TOH activity in adult NPG cultures was unchanged in the presence of 0, 10 or 100 units NGF/ml or in the presence of high concentrations of antiserum against the beta-subunit of NGF. Adult sympathetic neurons, however, grown under identical conditions, exhibited a 5- to 10-fold rise in TOH activity in the presence of NGF. Thus, unlike sympathetics, CA metabolism in adult NPG neurons is not regulated by NGF in vitro; NGF is therefore unlikely to mediate target effects on CA metabolism in placodal sensory neurons in vivo. Our findings indicate that certain mechanisms of CA regulation are shared by placodal sensory and neural crest-derived sympathetic neurons, whereas others are not.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Catecolaminas/metabolismo , Nervios Craneales/metabolismo , Factores de Crecimiento Nervioso/fisiología , Neuronas Aferentes/metabolismo , Potasio/fisiología , Tirosina 3-Monooxigenasa/metabolismo , Animales , Femenino , Nervio Glosofaríngeo/metabolismo , Histocitoquímica , Inmunoquímica , Técnicas In Vitro , Potenciales de la Membrana , Ganglio Nudoso/metabolismo , Ratas , Ratas EndogámicasRESUMEN
We previously observed that substance P (SP) levels in the rat superior cervical ganglion (SCG) rise sharply when the ganglion is maintained in vitro. To define the cellular localization of SP, sections of cultured SCG were stained for the possible dual presence of SP and tyrosine hydroxylase (TOH). Immunoreactivity to both SP and TOH was present in the majority of principal neurons. This observation suggests that principal sympathetic neurons can express simultaneously both the noradrenergic and SP-peptidergic phenotypes.
Asunto(s)
Ganglios Simpáticos/enzimología , Regulación de la Expresión Génica , Norepinefrina/metabolismo , Fenotipo , Sustancia P/metabolismo , Animales , Técnicas de Cultivo , Técnica del Anticuerpo Fluorescente , Fibras Nerviosas/enzimología , Neuronas/enzimología , Ratas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Cell membrane contact induces marked differential changes in neurotransmitter expression. In cultures of virtually pure dissociated sympathetic neurons, when such contact is provided by either high cell densities or addition of membranes derived from specific tissues, there is a marked increase in cell-specific content of substance P and de novo induction of choline acetyltransferase. To identify molecular mechanisms underlying regulation of transmitter expression by neuronal aggregation and membrane contact, we have begun to isolate and characterize a membrane-associated factor responsible for stimulation of choline acetyltransferase activity. The factor was found in substantial quantities in membranes from adult rat spinal cord as well as from sympathetic and sensory ganglia. Ionic mechanisms were employed to extract transmitter-inducing activity from spinal cord membranes in soluble form. The solubilized factor was then partially purified by ion exchange and gel filtration chromatography. It appears to be an extrinsic (non-integral) protein with an apparent molecular weight of 27. It is inactivated by trypsin and chymotrypsin, but is only moderately sensitive to heat inactivation, retaining activity at 60 degrees C but not at 90 degrees C. Neuronal perikaryal contact via aggregation represents a critical mechanism by which neurons themselves may influence phenotypic expression. Membrane localization of the factor provides a means by which cell contact may regulate transmitter expression.
Asunto(s)
Agregación Celular , Colina O-Acetiltransferasa/metabolismo , Ganglios Simpáticos/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Factores de Crecimiento Nervioso/aislamiento & purificación , Médula Espinal/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ganglios Simpáticos/citología , Ganglios Simpáticos/efectos de los fármacos , Proteínas de la Membrana/farmacología , Proteínas de la Membrana/fisiología , Peso Molecular , Factores de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/fisiología , Ratas , Ratas EndogámicasRESUMEN
To test further the hypothesis that cycloheximide (CXM)-induced amnesia is due in part to its effects on the central adrenergic system, a comparison was made in mice of the effects of the antibiotic and of 6-hydroxydopamine (6-OHDA) on memory of a one-trial passive avoidance task. Both drugs produced anmesia 24 hr after training but unlike CXM, 6-OHDA had no effect on memory 20 min after training.
Asunto(s)
Reacción de Prevención/efectos de los fármacos , Cicloheximida/farmacología , Hidroxidopaminas/farmacología , Memoria/efectos de los fármacos , Animales , Química Encefálica/efectos de los fármacos , Depresión Química , Dopamina/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Norepinefrina/análisisAsunto(s)
Comunicación Celular , Diferenciación Celular , Membrana Celular/fisiología , Ganglios Simpáticos/citología , Regulación de la Expresión Génica , Fenotipo , Animales , Agregación Celular , Recuento de Células , Colina O-Acetiltransferasa/metabolismo , Técnicas de Cultivo , Neuronas/citología , Ratas , Ratas Endogámicas , Sustancia P/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Glial cell line-derived neurotrophic factor markedly enhances survival of neonatal dorsal root sensory neurons in vitro, an effect seen even in the presence of anti-nerve growth factor. Furthermore, it increases levels of substance P, inducing more than a sixfold rise that is maximal at 10 ng/ml. At the same dose, it potentiates the action of nerve growth factor on substance P but not on survival. Neither factor increases somatostatin content in neonatal neurons. Although its effect on substance P diminishes with age, glial cell line-derived neurotrophic factor dramatically increases somatostatin levels in neurons from adult rats. Glial cell line-derived neurotrophic factor is therefore the second trophic factor found to promote survival and regulate substance P in neonatal sensory neurons. More significant is that it is the first and sole neurotrophic factor reported to regulate somatostatin in sensory neurons at any age, with its effect restricted to the adult. These results suggest mechanisms for differential regulation of somatostatin versus substance P in nociceptive pathways.
Asunto(s)
Proteínas del Tejido Nervioso/farmacología , Neuronas Aferentes/metabolismo , Fármacos Neuroprotectores/farmacología , Somatostatina/metabolismo , Sustancia P/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factor Neurotrófico Ciliar , Relación Dosis-Respuesta a Droga , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
The effects of cell density and aggregation on expression of transmitter traits were examined in dissociated, pure sympathetic neuron cultures, grown in fully defined, serum-free medium. After 1 week at a density of 7-8 X 10(3) 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 high-density cultures failed to reproduce these effects on low-density cultures, suggesting that diffusible factors are not involved in the density-dependent differential regulation. Time-lapse phase-contrast 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.