Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival, growth, and differentiation of catecholamine production

To study the effect of nerve growth factor (NGF) on neuronal survival, growth, and differentiation, cultures of dissociated neonatal rat sympathetic neurons virtually free of other cell types were maintained for 3-4 wk. In the absence of NGF, the neurons did not survive for more than a day. Increased levels of NGF increased neuronal survival and growth (total protein and total lipid phosphate); saturation occurred at 0.5 microgram/ml 7S NGF. Neuronal differentiation examined by measuring catecholamine (CA) production from tyrosine also depended on the level of NGF in the culture medium. As the NGF concentration was raised, CA production per neuron, per nanogram protein, or per picomole lipid phosphate increased until saturation was achieved between 1 and 5 microgram/ml 7S NGF. Thus, NGF induces neuronal survival, growth, and differentiation of CA production in a dose-dependent fashion. Neuronal growth and differentiation were quantitatively compared in the presence of the high and low molecular weight forms of NGF; no significant functional differences were found.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Developmental studies

Adrenergic sympathetic neurons were grown for 4 wk in submaximal and saturating concentrations of nerve growth factor (NGF) in the virtual absence of non-neuronal cells. In 0.2 or 5 microgram/ml 7S NGF, the neurons gradually decreased in number during the first week, although fewer neurons died at the higher level. No significant change in cell number was observed thereafter. Total neuronal protein, a measure of cell growth, increased linearly with age in both concentrations of NGF. At each age, neurons in high NGF exhibited greater growth per cell than those in low NGF. The ability of neurons to produce catecholamine (CA) increased dramatically during the second and third weeks in both concentrations of NGF, and along a similar time-course, although neurons in submaximal NGF developed a lesser capacity for CA production. As neurons developed in culture, they became less dependent on NGF for survival and CA production, but even in older cultures, approximately 50% of the neurons died when NGF was withdrawn.     

Conditions increasing the adrenergic properties of dissociated chick superior cervical ganglion neurons grown in long-term culture

Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.     

Intracellular Storage and Evoked Release of Acetylcholine from Postnatal Rat Basal Forebrain Cholinergic Neurons in Culture with Nerve Growth Factor

Cholinergic neurons from the septum area, the vertical limb of the diagonal band of Broca, and the nucleus basalis of Meynert of postnatal 13-day-old rats were cultured with or without nerve growth factor (NGF) conditions. Total choline acetyltransferase (ChAT) activities, acetylcholine (ACh) contents, and survival numbers of cholinergic neurons in culture from each of three distinct regions were increased by NGF treatment, but little difference was found in cellular ChAT activities and ACh contents obtained in cultures with or without NGF. The result shows that NGF promotes the survival of cholinergic neurons from 13-day-old rats. Furthermore, the release of ACh from cultured neurons was investigated. The cells cultured with NGF showed a larger increase of the high K+-evoked ACh release than those cultured without NGF. However, NGF had no effect on spontaneous release. This suggests that NGF could regenerate and sustain the stimulation-evoked release mechanisms of ACh in cultured cholinergic neurons from postnatal rats.     

神经生长因子分化后的PC12 细胞对乙酰胆碱的敏感性及其特性

目的和方法：采用全细胞膜片钳技术观察神经生长因子（NGF）分化后的PC12细胞对乙酰胆碱（ACh)的敏感性,并对ACh诱发电流（IACh)的特性进行分析。结果：NGF处理后的PC12乐仅形态上向交感神经元分化,而且具有电学兴奋性,它对ACh敏感性比未分化前显著提高。药理学鉴定表明PC12上的IACh是由烟碱受体（nAChR)引起的,具有明显的失敏特性。宏观IACh呈内向整流和浓度依赖性。结论：PC12细胞培养方便,同源性好,加入NGF后向交感神经元分化,且其具有神经元烟碱受体,可以作为交感神经元烟碱受体研究的很好的模型系统。     

Regulation of cholinergic gene expression by nerve growth factor depends on the phosphatidylinositol-3'-kinase pathway

Nerve growth factor (NGF) exerts anti-apoptotic, trophic and differentiating actions on sympathetic neurons and cholinergic cells of the basal forebrain and activates the expression of genes regulating the synthesis and storage of the neurotransmitter acetylcholine (ACh). We have been studying the intracellular signaling pathways involved in this process. Although, in the rat pheochromocytoma cell line PC12, NGF strongly activates the mitogen-activated protein kinase (MAPK) pathway, prolonged inhibition of MAPK kinase (MEK) activity by PD98059 or U0126 did not affect the ability of NGF to up-regulate choline acetyltransferase (ChAT) or to increase intracellular ACh levels. In contrast, the treatment with the phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002, but not with its inactive analogue LY303511, completely abolished the NGF-induced production of ACh. Inhibition of PI3K also eliminated the NGF effect on the intracellular ACh level in primary cultures of septal neurons from E18 mouse embryos. Blocking the PI3K pathway prevented the activation of cholinergic gene expression, as demonstrated in RT/PCR assays and in transient transfections of PC12 cells with cholinergic locus promoter-luciferase reporter constructs. These results indicate that the PI3K pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation.     

Sympathetic neurons synthesize and secrete pro‐nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF‐immunoreactive proteins synthesized by cultured NGF‐dependent and ‐independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro‐NGF protein. These findings suggest that a potential NGF‐sympathetic neuron autocrine loop may exist in this prototypic target‐dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival. © 2003 Wiley Periodicals, Inc. J Neurobiol 38–53, 2003     

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.     

Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium-Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC₅₀ of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.     

Nerve Growth Factor and glucocorticoids regulate phenotypic expression in cultured chromaffin cells from adult rhesus monkeys

Adrenal chromaffin cells and sympathetic neurons are both derivatives of the neural crest. Despite their morphological and functional differences, chromaffin cells retain some developmental plasticity and if treated with Nerve Growth Factor (NGF), can express certain characteristics of sympathetic neurons. However, there is some age and species variability in the response of chromaffin cells to NGF: in general chromaffin cells from adult animals are not considered to be dependent on NGF for survival, and chromaffin cells from adults of several species fail to respond to NGF in vitro by growing neurites. This is in contrast to the dramatic effects of NGF on chromaffin cells from perinatal rats. We have examined the requirements of chromaffin cells from adult rhesus monkeys to survive, to proliferate, and to express a neuronal morphology in vitro. NGF greatly enhances the proportion of rhesus chromaffin cells that form neurites and the length of the neurites that are formed, but the conversion to a neuronal phenotype is more limited than in chromaffin cells cultured from young rats. NGF also enhances rhesus chromaffin cell survival, but fails to stimulate their proliferation, in contrast to its effect on perinatal rat cells [18]. Glucocorticoid hormones (GCs) specifically antagonize the effects of NGF on neuritic outgrowth while promoting chromaffin cell survival. Thus adrenal chromaffin cells from rhesus monkeys retain a degree of developmental plasticity even in the adult animal.     

Sympathetic neurons synthesize and secrete pro-nerve growth factor protein

Postmitotic sympathetic neuronal survival is dependent upon nerve growth factor (NGF) provided by peripheral targets, and this dependency serves as a central tenet of the neurotrophic hypothesis. In some other systems, NGF has been shown to play an autocrine role, although the pervasiveness and significance of this phenomenon within the nervous system remain unclear. We show here that rat sympathetic neurons synthesize and secrete NGF. NGF mRNA is expressed in nearly half of superior cervical ganglion sympathetic neurons at embryonic day 17, rising to over 90% in the early postnatal period, and declining in the adult. Neuronal immunoreactivity is reduced when retrograde transport is interrupted by axotomy, but persists in a subpopulation of neurons despite diminished mRNA expression, suggesting that intrinsic protein synthesis occurs. Cultured neonatal neurons express NGF mRNA, which is maintained even when they are undergoing apoptosis. To determine which NGF isoforms are secreted, we performed metabolic labeling and immunoprecipitation of NGF-immunoreactive proteins synthesized by cultured NGF-dependent and -independent neurons. Conditioned medium contained high molecular weight NGF precursor proteins, which varied depending upon the state of NGF dependence. Mature NGF was undetectable by these methods. High molecular weight NGF isoforms were also detected in ganglion homogenates, and persisted at diminished levels following axotomy. We conclude that sympathetic neurons express NGF mRNA, and synthesize and secrete pro-NGF protein. These findings suggest that a potential NGF-sympathetic neuron autocrine loop may exist in this prototypic target-dependent system, but that the secreted forms of this neurotrophin apparently do not support neuronal survival.     

Muscarinic Stimulation Promotes Cultured Purkinje Cell Survival: A Role for Acetylcholine in Cerebellar Development?

Abstract: The survival and development of cerebellar neurons are under the control of interacting epigenetic signals. In the present study, we have examined interactive effects of nerve growth factor (NGF) and acetylcholine on in vitro cerebellar Purkinje cell survival. In initial experiments, dissociated rat cerebellar cultures were grown for 6–7 days in the presence of NGF and the stable cholinergic agonist carbachol. Simultaneous exposure to carbachol and NGF selectively increased Purkinje cell number, whereas neither agent was effective when tested alone. The increase in survival was blocked by the muscarinic antagonists atropine (0.1 µ M ) and pirenzepine (10 n M ), but not by methoctramine (25 n M ). Nicotine had no effect on survival when tested alone or in combination with NGF. The cerebellar cultures exhibited cholinergic neuronal traits: high-affinity choline uptake, and choline acetyltransferase and acetylcholinesterase activities. To determine whether transmitter produced in vitro triggers Purkinje responsiveness to NGF, cells were exposed to physostigmine, an acetylcholinesterase inhibitor. Physostigmine alone induced an atropine-sensitive increase in cell survival that was enhanced in the presence of NGF. These data suggest that the early expression of cholinergic traits plays a role in Purkinje development. Activation of muscarinic receptors triggers enhanced Purkinje survival in the presence of NGF.     

Concentration-dependent regulation of neuronal gene expression by nerve growth factor

NGF is a neurotrophic protein that promotes the survival, growth, and differentiation of developing sympathetic neurons. To directly determine the effects of different concentrations of NGF on neuronal gene expression, we examined mRNAs encoding the p75 low-affinity NGF (LNGF) receptor, T alpha 1 alpha-tubulin (T alpha 1), and tyrosine hydroxylase (TH) in pure cultures of rat sympathetic neurons from postnatal day 1 superior cervical ganglia. Studies of the timecourse of gene expression during 2 wk in culture indicated that a 5-d incubation period would be optimal for the concentration-effect studies. Analysis of RNA isolated from neurons cultured in 2-200 ng/ml 2.5S NGF for 5 d revealed that, as the NGF concentration increased, neurons expressed correspondingly increased levels of all three mRNAs. Both LNGF receptor and TH mRNAs increased seven-fold, and T alpha 1 mRNA increased four-fold in neurons cultured in 200 versus 10 ng/ml NGF. In contrast, T26 alpha-tubulin mRNA, which is constitutively expressed, did not alter as a function of NGF concentration. When neurons were initially cultured in 10 ng/ml NGF for 5 d, and then 200 ng/ml NGF was added, LNGF receptor, T alpha 1, and TH mRNAs all increased within 48 h. The timecourse of induction differed: T alpha 1 mRNA was maximal by 5 h, whereas LNGF receptor and TH mRNAs first began to increase at 12 h after the NGF increase. These experiments show that NGF regulates expression of a subset of mRNAs important to neuronal growth and differentiation over a broad concentration range, suggesting that the effects of NGF may be mediated by more than just a single receptor operating at one fixed affinity. These results also suggest a mechanism for coupling neuronal synthesis of axonal proteins to increases in size of the innervated target territory during growth of the organism.     

Both nerve growth factor and high K concentrations support the survival of chick embryo sympathetic neurons : Evidence for a common mechanism of action

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K⁺ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K⁺, and survival was not increased when both NGF and K⁺ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K⁺ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K⁺ and NGF for the survival of cultured embryonic neurons.     

Development of trophic interactions in the vertebrate peripheral nervous system

During embryogenesis, the neurons of vertebrate sympathetic and sensory ganglia become dependent on neurotrophic factors, derived from their targets, for survival and maintenance of differentiated functions. Many of these interactions are mediated by the neurotrophins NGF, BDNF, and NT3 and the receptor tyrosine kinases encoded by genes of thetrk family. Both sympathetic and sensory neurons undergo developmental changes in their responsiveness to NGF, the first neurotrophin to be identified and characterized. Subpopulations of sensory neurons do not require NGF for survival, but respond instead to BDNF or NT3 with enhanced survival. In addition to their classic effects on neuron survival, neurotrophins influence the differentiation and proliferation of neural crest-derived neuronal precursors. In both sympathetic and sensory systems, production of neurotrophins by target cells and expression of neurotrophin receptors by neurons are correlated temporally and spatially with innervation patterns. In vitro, embryonic sympathetic neurons require exposure to environmental cues, such as basic FGF and retinoic acid to acquire neurotrophin-responsiveness; in contrast, embryonic sensory neurons acquire neurotrophin-responsiveness on schedule in the absence of these molecules.     

Effects of neurotransmitters and peptides on phospholipid hydrolysis in sympathetic and sensory neurons

The effects of neurotransmitters and peptides on phosphoinositide hydrolysis were studied by measuring [3H]inositol monophosphate ([3H]IP) and protein kinase C (PKC) activity in the sympathetic and sensory neuronal cultures of the chick embryo. [3H]IP was increased in sympathetic neurons by acetylcholine (ACh), muscarine, serotonin (5-HT), and vasoactive intestinal polypeptide. ACh, muscarine, 5-HT, and bradykinin increased [3H]IP in sensory neuronal cultures. Dopamine, norepinephrine, histamine, and nerve growth factor did not stimulate [3H]IP formation in both cultures. ACh and phorbol 12,13-dibutyrate (PDB) increased the PKC activity by two- to sevenfold in the particulate fraction of both cultures. In sympathetic neurons, PKC activity was increased in the particulate fraction; activity in the cytosolic fraction was not affected. There was a 50% decline in the protein kinase C activity of the cytosolic fraction after PDB and ACh treatment of sensory cultures. The decline in PKC activity in the cytosolic fraction was attributed to the presence of nonneuronal cells in sensory cultures. To confirm this, the enzyme activity was determined in tissues that contain a heterogeneous population of cells. PDB activated PKC in the adrenal medulla and the brain of the rat. In both tissues there was a 65% decline in the PKC activity of the cytosolic fraction and about a 75% increase in the particulate fraction. We conclude that the mechanism of activation of protein kinase C in pure cultures of sympathetic neurons is different than in tissues containing a mixed population of neurons and nonneuronal cells.     

Ionic behaviors and neuronal survival in developing ganglia. III. Studies with embryonic chick sympathetic neurons

We have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na⁺,K⁺-pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na⁺ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13–E18), NGF becomes less and less required for that control as the neurons gain a self-sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.     

Human Skeletal Muscle Cells Synthesise a Neuronotrophic Factor Reactive with Spinal Neurons

Retrograde trophic influences originating in the skeletal musculature have been postulated to be involved in regulating survival and differentiation of embryonic motor neurons and reactive terminal sprouting of mature motor fibres. We have previously described the use of a quantitative immunoassay for neurofilament protein to bioassay in vitro the cell-type-specific neuronotrophic activity of nerve growth factor (NGF) on sensory ganglion neurons. In the present study, the effect of media conditioned by adult human muscle cells (MCM) on the in vitro development of chicken spinal neurons has been studied using a similar approach. Significant increases in neurofilament protein levels in 7-day chicken embryonic spinal cord cultures were found with doses of MCM protein as low as 0.4 microgram/ml, with a dose-response relationship yielding maximal and half-maximal effects at 4 and 1 microgram/ml, respectively. Maximal increases in neurofilament protein levels were associated with an approximate two-fold increase in neuronal cell survival. MCM also induced increases in choline acetyltransferase activity in chick spinal cord cultures. In both the absence and presence of NGF, MCM did not increase neurofilament protein expression in primary cultures of sensory neurons.     

Studies on rat sympathetic neurons developing in cell culture. III. Cholinergic transmission.

Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.'s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.'s in size and time course; the ACh potentials and the e.p.s.p.'s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.'s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.