Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.     

Structural specificity of peptides influencing neuronal survival during development

Vasoactive intestinal peptide (VIP) has been shown to increase the survival of developing neurons grown in dissociated spinal cord cultures. This result was evident when synaptic activity was blocked with tetrodotoxin (TTX) during a critical period of development (days 7-21 after plating). Other neuropeptides, with a close sequence homology to VIP, have now been tested for their effects on neuronal survival in culture. Within the critical period, the survival of spinal cord neurons was significantly decreased (30-35%) after incubation with 1 nM peptide histidyl-isoleucine amide (PHI-27) or 0.1 nM growth hormone releasing factor (GRF). Neuronal cell death produced by these peptides did not exceed that observed from tetrodotoxin treatment alone. Secretin had no detectable effect on neuronal survival at any of the concentrations tested. In tetrodotoxin-treated cultures, PHI-27 and GRF prevented the neuronal cell death produced by TTX, but only at concentrations greater than 0.1 microM. In contrast, VIP significantly increased neuronal survival at concentrations less than 0.01 nM. The presence of 0.1 nM PHI-27 significantly decreased the effectiveness of VIP in preventing TTX-mediated neuronal cell death. Addition of PHI-27 or VIP, with or without TTX, to one month-old cultures produced no significant change in the number of neurons compared to control cultures. These studies indicate that the survival-promoting effect of VIP is highly structure-dependent and that this action appears to be confined to a critical period of development.     

VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro

Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VIP) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VIP. Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIP10-28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina.     

Head and trunk neural crest in vitro: Autonomic neuron differentiation

Isolated cultures of premigratory neural crest cells were used to study the initial stages of autonomic neuron development. Autonomic neurons are phenotypically characterized on the basis of their neurotransmitter synthetic enzymes, dopamine β-hydroxylase (DBH) and choline acetyltransferase (CAT). DBH converts dopamine to norepinephrine in noradrenergic neurons while CAT synthesizes acetylcholine from choline in cholinergic neurons. Activities of both enzymes were detected in isolated cultures of trunk neural crest and head neural crest. DBH was detected at all culture ages examined (from 1 to 20 days) whereas CAT activity was first detected only after 5 days in vitro. While specific enzyme activity of DBH peaks on Day 6 and specific enzyme activity of CAT peaks on Day 10, absolute activity for both enzymes increases throughout the 20-day culture period. DBH and CAT develop in vitro without any spinal presynaptic input, without typical target tissue interactions (such as blood vascular elements or heart tissue), and without addition of conditioned medium factors.     

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: The role of cholinergic differentiation factor/leukemia inhibitory factor

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum Against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy. 1994 John Wiley & Sons, Inc.     

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine Regulation of Nerve Growth Factor-Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts

The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum-free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), and transforming growth factor-beta 1. In contrast, ACM induced choline acetyltransferase expression in septal cholinergic neurons via nerve growth factor (NGF)-dependent and -independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF-mediated cholinergic neurotrophic activity in ACM. A combination of IL-1 beta and TNF alpha significantly enhanced choline acetyltransferase activity in septal neurons co-cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara-C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL-1 beta and TNF alpha had no effect on glutamate decarboxylase activity in septal neurons co-cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin-derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum-free defined medium with Ara-C. The cytokines also enhanced NGF-mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF-type and non-NGF-type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF-type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF-sensitive neurons in the central and peripheral nervous systems.     

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Cholinergic fiber growth in co-cultures of CNS tissue.

In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of fetal mouse spinal cord-dorsal root ganglion explants to study the factors underlying selective connections in vitro

Dorsal root ganglion (DRG) afferents selectively project to dorsal spinal cord (SC) explants when grown in a medium containing serum or a serum-free medium (CDM) containing galactose compounds. SC-DRG explants grown in CDM retain their gross morphological characteristics over several months in vitro, greatly facilitating the mapping of the sensory afferents within given regions of the cord explant. Explants grown in CDM without the addition of galactose show no such selective preference for dorsal cord, and terminate equally throughout the cord. This lack of selectivity also occurs in galactose-grown SC-DRG provided tetrodotoxin (TTX) is added to the medium sufficient to block all recordable spontaneous bioelectric activity (SBA), viz., action potential discharges and slow waves. Gangliosides are galactose-containing compounds found in neural membranes which may be involved in cell-cell recognition/adhesion. The addition of a mixture of gangliosides to the CDM results in the preferential restriction of the sensory afferents within the dorsal cord regions even in the presence of TTX. It is concluded that gangliosides may be able to compensate for the absence of SBA during periods of development associated with the formation of selective interneuronal connections.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.     

Production of GABA by cultured hippocampal glial cells

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.     

Comparison of the effects of elevated K+ ions and muscle-conditioned medium on the neurotransmitter phenotype of cultured sympathetic neurons

Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons. We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures. A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold. In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development. The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules. Conversely, K+ ions (30-40 mM) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold. Cultures grown with CM in high K+ medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K+ medium suggesting that CM and K+ ions had antagonistic effects on the expression of these enzymes. However, K+ ions did not affect the development of AADC in these cultures. CM suppressed in a reversible manner the development of the 16 S form of AcChE. In the presence of 40 mM K+, the rate of development of AcChE was reduced. In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed. The effect of elevated K+ ions on the percentage of 16 S AcChE was rapidly reversible. It is concluded that CM and elevated K+ ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.     

Interferon induces astrocyte maturation causing an increase in cholinergic properties of cultured human spinal cord cells

It has been well established that interferon-gamma (IFN-gamma) can modify the immune status of cells in the central nervous system (CNS) by inducing major histocompatibility antigens. Furthermore, it has been shown that endogenous IFN can be produced in the brain following viral infection and a form of IFN-alpha/beta can be produced by astrocytes in culture. Here we show that IFN can induce astrocyte maturation and alter neurotransmitter properties in cultured CNS neurons at a given developmental stage. IFN causes a dose-dependent increase in choline acetyltransferase activity and glial fibrillary acidic protein (GFAP) immunoreactivity in cultures of human embryonic spinal cord neurons. The GABAergic activity and the Thy1 immunoreactivity remain unchanged. IFN-gamma does not act directly on the neurons but via the nonneuronal cells, probably the astrocytes, which in turn stimulate the cholinergic traits. These studies could be important for demonstrating an action of the immune system on glial cell maturation and on the neurotransmitter phenotype expression in CNS neurons.     

Differential regulation of cholinergic and peptidergic development in the rat striatum in culture

The development of substance P, somatostatin, and choline acetyltransferase activity was examined in embryonic rat striatum in vivo and in culture. The study was undertaken to help define mechanisms by which diverse neurotransmitter phenotypes may be regulated within the same structure in the brain. Choline acetyltransferase (CAT) was present in striatum before gestational Day 13.5 (E13.5), and enzyme levels increased continually between E13.5 and birth. By contrast, substance P (SP) and somatostatin (SS) did not develop in vivo until E15, and peptide levels fluctuated between E15 and birth, indicating that striatal peptidergic and cholinergic development were regulated differently. To define mechanisms mediating the differential regulation of striatal peptidergic and cholinergic neurons, neurotransmitter development was examined in embryonic striatum in vitro. Cultured striatal neurons from E13.5 embryos expressed substance P and somatostatin de novo after several days in culture, and peptide levels and CAT activity increased significantly in vitro. Each transmitter phenotype was regulated in vitro by a different constellation of environmental factors, and many factors differentially influenced SP, SS, and CAT development. For example, coculture of striatum with a target tissue, the ventral mesencephalon (substantia nigra), increased CAT activity and SP levels but had no significant effect on levels of SS. Moreover, there were widely differing effects on CAT, SP, and SS development of medium conditioned by exposure to a variety of cell types, indicating that the three transmitter systems were regulated by different soluble factors. Potassium-induced membrane depolarization also exerted different effects on the different transmitter traits, elevating CAT activity but decreasing SP and SS. Finally, insulin was required for the survival of SP-containing neurons, but not for the survival of SS- or CAT-containing neurons, indicating that the survival of different populations of striatal neurons was dependent upon different factors. Our observations suggest that different populations of neurons in the striatum are regulated by different mechanisms, so that alterations in the environment may produce strikingly diverse responses in the development of different phenotypic traits within the same structure.     

The role of extracellular signals in the differentiation of cholinergic neurons from the CNS and PNS in culture

Rat skeletal muscle cells release in culture a macromolecule which stimulates by 25-100 fold the development of choline acetyltransferase (CAT) in cultures of new-born rat sympathetic neurons. This "cholinergic factor" impaired the development of three norepinephrine synthesizing enzymes and of acetylcholinesterase (AChE) in these cultures. The 16S form of AChE failed to develop in cultures grown with the factor, but amounted to 30-40% in 3-week old cultures grown in its absence. Using the development of CAT activity in sympathetic neuron cultures as an assay, the cholinergic factor has been partially purified in 6 steps, and its hydrodynamic parameters determined. The effects of this factor on sympathetic neurotransmitter choice were qualitatively reproduced by 1-10 mM Na butyrate. The cholinergic factor increased CAT activity and decreased AChE in neuron cultures from new-born rat nodose ganglia. The factor also stimulated CAT activity in rat embryo (E14) spinal cord cultures, but stimulated the development of AChE in these cultures.