Cyclic AMP regulates substance P expression in developing and mature spinal sensory neurons.

The tachykinin, substance P, has long been associated with transmission of noxious stimuli. However, relatively little is known about signal transduction pathways subserving peptidergic regulation in sensory neurons. To investigate whether cyclic AMP (cAMP) could be a potential second messenger subserving substance P expression, dorsal root ganglion neurons were grown in culture in the presence of agents that increase content of cAMP. In developing neurons, forskolin increased substance P content and survival almost threefold. Anti-nerve growth factor (NGF) blocked the effect of NGF but not forskolin, suggesting that increased cAMP acts directly and not via increased secretion of NGF from Schwann cells and fibroblasts. In adult neurons, which do not require supplemental trophic factors for survival, NGF and forskolin had similar effects on substance P levels. Neither agent had any effect on somatostatin content of either developing or mature sensory neurons. 8-bromo cAMP and isobutyl methylxanthine duplicated the action of forskolin. Further, all three agents increased expression of preprotachykinin mRNA. Forskolin appeared to increase both total and neuron-specific expression of message as well as the number of neurons expressing mRNA. Our results suggest that cAMP directly regulates substance P content in sensory neurons from adult and neonatal rats.

Age-dependent differential regulation of sensory neuropeptides by glial cell line-derived neurotrophic factor.

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.

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.

Regulation of neurotransmitter expression by a membrane-derived factor.

Cell-cell contact appears to play a critical role in the expression of transmitter traits in developing neurons. We have previously shown that cell membrane contact induces the de novo appearance of choline acetyltransferase (CAT) in virtually pure cultures of dissociated sympathetic neurons. A membrane-associated CAT-inducing factor has been extracted and purified 5000-fold. This factor exerts differential effects on transmitter traits in cultured sympathetic neurons. After 3 days in vitro, neurons exposed to the factor contained 40-fold higher levels of the neuropeptide substance P than controls. Somatostatin exhibited a similar dramatic elevation. In contrast, the factor had no effect on leucine-enkephalin. Further, the specific activity of tyrosine hydroxylase was reduced to 5% of control activity in treated cultures. These effects occurred in the absence of any increases in cell number. Thus, it appears that cell contact via membrane-associated factors may exert differential effects on phenotypic expression.

Partial purification and characterization of a membrane-derived factor regulating neurotransmitter phenotypic expression.

Cell membrane contact induces the de novo expression of choline O-acetyltransferase (CAT; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) activity in cultures of virtually pure neonatal rat dissociated sympathetic neurons. To identify molecular mechanisms underlying membrane-associated CAT induction, the responsible membrane component was characterized and partially purified. Substantial CAT-inducing activity was found in membranes from adult rat spinal cord and sensory and sympathetic ganglia. Whole brain membranes demonstrated significantly less activity. CAT induction in sympathetic neurons in response to spinal cord membranes was linear with respect to time, after an initial 6-hr lag. It was also linear with respect to concentrations of spinal cord protein from 2 to 100 micrograms per ml. CAT-inducing activity was extracted from spinal cord membranes by incubation with 100 mM NaCl and was purified approximately 5000-fold by DEAE ion-exchange and gel filtration chromatography. The active factor appears to be an extrinsic protein with an apparent molecular mass of 27 kDa. It is inactivated by trypsin and chymotrypsin but is moderately thermostable, retaining activity at 60 degrees C but not at 90 degrees C.

Neuronal aggregation and neurotransmitter regulation: partial purification and characterization of a membrane-derived factor.

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.

Development of transsynaptic regulation of adrenal enkephalin.

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.

Glucocorticoids regulate adrenal opiate peptides.

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.

Depolarizing influences regulate preprotachykinin mRNA in sympathetic neurons.

We have been studying mechanisms regulating neurotransmitter plasticity in sympathetic neurons. Neurons of the rat superior cervical ganglion (SCG) synthesize multiple putative transmitters, including the peptide substance P (SP). We have now examined steady-state levels of the mRNA encoding preprotachykinin (PPT), the SP precursor. A cloned cDNA probe was used to examine regulation mRNA levels in culture and in vivo. In RNA gel blot experiments, a single band (1.1 kilobases long) was observed in all cases in which an RNA was detected. A low level of PPT mRNA was detected by RNase protection assay in uncultured ganglia, suggesting that the low levels of SP previously observed in the normal ganglion in vivo are synthesized locally. When ganglia were maintained in culture, with consequent denervation, the steady-state level of PPT mRNA increased by 25-fold over the first 24 hr, and the high level was maintained for at least 7 days. RNase protection experiments indicated that the major message in the SCG is the beta-PPT mRNA, encoding both SP and neurokinin A peptide regions. Accumulation of the PPT mRNA in cultured ganglia was sharply inhibited by the depolarizing agent veratridine, and this effect was blocked by tetrodotoxin. Therefore, one form of neuronal plasticity, change in neurotransmitter metabolism, is regulated at least in part by altering steady-state levels of specific mRNA. More generally, extracellular signals may contribute to neuronal plasticity through changes in gene expression.

Biochemistry of information storage in the nervous system.

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.

Catecholaminergic primary sensory neurons: autonomic targets and mechanisms of transmitter regulation.

Contrary to traditional teaching, mammalian primary sensory neurons may express catecholaminergic (CA) neurotransmitter characteristics in vivo. Sensory neurons in the nodose, petrosal, and dorsal root ganglia of rats express tyrosine hydroxylase, the rate-limiting enzyme in CA biosynthesis, and formaldehyde-induced CA fluorescence, in addition to other CA traits. These findings suggest that catecholamines may function as sensory as well as autonomic motor (e.g., sympathetic) neurotransmitters. Most CA cells in the petrosal ganglion project peripherally to the carotid body, which indicates a striking correlation between CA expression in sensory neurons and the pattern of sensory innervation. Inasmuch as petrosal ganglion afferents make synaptic contact with chemoreceptive glomus cells in the carotid body, it is likely that CA sensory neurons in the ganglion transmit chemoreceptor information to the brain stem. Comparison with sympathetic neurons indicates that some mechanisms of CA regulation, such as altered activity of tyrosine hydroxylase in response to depolarizing stimuli, are shared among sensory and traditional CA populations. Other mechanisms, including trophic regulation, appear to be distinct. Therefore, despite expression of common phenotypic traits, CA expression in diverse populations of peripheral neurons is not necessarily associated with a common repertoire of regulatory mechanisms.

Membrane contact regulates transmitter phenotypic expression.

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.

Expression and regulation of tyrosine hydroxylase in adult sensory neurons in culture: effects of elevated potassium and nerve growth factor.

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)

Depolarization regulates adrenal preproenkephalin mRNA.

The regulation of neuropeptide gene expression has been investigated by using rat adrenal medullae grown in explant culture. After 3 days in culture the (now denervated) explants exhibited a 10-fold increase in leucine-enkephalin (leu-EK) content. Inhibition of protein synthesis with cycloheximide completely blocked the rise, whereas inhibition of RNA synthesis with actinomycin D or alpha-amanitin inhibited the increase by 50%. Inhibition of DNA synthesis with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside) had no discernible effect. To determine whether the rise in leu-EK was associated with an increase in specific mRNA coding for the opiate peptide precursor, blot hybridization analysis was performed. A single species of preproenkephalin mRNA was detected after various culture periods. The amount of mRNA increased 34-fold after 2 days in culture and 74-fold after 4 days. Consequently, the rise in mRNA levels preceded the increase in the amount of leu-EK. Depolarization of the adrenal medullae with either elevated potassium or veratridine, which prevents leu-EK accumulation, inhibited the increase in the amount of preproenkephalin mRNA. Moreover, the effect of veratridine was blocked by tetrodotoxin, suggesting that transmembrane sodium ion influx affects the increase in the amount of message. Our studies suggest that elevation of leu-EK in explanted (denervated) medullae is associated with increased amounts of mRNA coding for the peptide precursor and that these processes can be regulated by depolarization.

Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.

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.

Neurotransmitter plasticity at the molecular level.

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.

Plasticity of substance P in mature and aged sympathetic neurons in culture.

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.

Impulse activity differentially regulates [Leu]enkephalin and catecholamine characters in the adrenal medulla.

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.