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.     

Single-channel and whole-cell currents evoked by acetylcholine in dissociated sympathetic neurons of the rat

Single acetylcholine-activated channels have been recorded from neurons dissociated from the sympathetic chain of 17-21 day old rats. The mean single channel conductance is 35 pS in normal medium containing 1 mM calcium, and 51 pS in the absence of calcium. The measured current amplitudes are about five times more variable than at the frog endplate, at least in part because the current, while the channel is open, is much noisier than when it is shut. Single activations of the receptor by acetylcholine (ACh) produce a burst of openings; the distribution of the burst length has two components, the longer of which is of primary importance in synaptic transmission. Whole-cell currents, in response to ACh (up to 30 microM), show strong inward rectification with no outward current being detectable. This phenomenon is similar whether the intracellular ion is sodium or cesium, whether or not divalent cations are present, and whether or not atropine is present. Nevertheless, outward single-channel currents (of normal conductance) are detectable in isolated outside-out patches.     

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of muscle-conditioned medium

The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.     

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons

Brain Cell Biology - Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both...     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture. I. Cholinergic transmission

Sympathetic neurons, dissociated from superior cervical ganglia of newborn rats, and skeletal muscle cells were grown together in mass cultures containing many neurons (ca. 1000–3000) and myotubes, and in microcultures containing only one to three neurons and one or a few myotubes. When these neurons grow under the influence of certain nonneuronal cells many of them acquire cholinergic functions; in the absence of this influence they remain adrenergic. In the present study, the influence of the skeletal muscle cells was so effective that under certain conditions more than 75% of the neurons expressed cholinergic function as judged by their ability to form excitatory cholinergic synapses with myotubes (from rat and chick) and with each other. Stimulation of single neurons often gave rise in the myotubes to simple (direct) postsynaptic potentials (ejp's) and/or complex responses comprising a burst of ejp's that evoked one or more spikes; it appeared that these complex responses involved the activation of interneuronal pathways. In microcultures, a single neuron often made cholinergic synapses with itself (“autapse”) and/or with another neuron as well as with one or more myotubes. The nicotinic blocking agents, tubocurare (dTC), α-bungarotoxin (α-BuTx), and hexamethonium (C₆), attenuated or abolished the ejp's at moderate concentrations; the muscarinic blocker, atropine, was effective only at high concentrations. At several neuron-myotube junctions, the acetylcholine (ACh) receptors had dTC sensitivity similar to adult extrajunctional receptors; however, when different junctions were pooled the average dTC sensitivity was intermediate between that of adult end plate and extrajunctional receptors. The junctional C₆ sensitivity was much higher than expected from the action of the drug at the adult mammalian end plate. As in other studies, chemical transmission from neuron to neuron was also nicotinic cholinergic, but the nicotinic receptors on the myotubes were pharmacologically distinct from those on the neurons.     

Effects of estrone on quisqualate-induced toxicity in primary cultures of rat cortical neurons.

Estrogens exert protective effects against neurotoxic changes induced by over-activation of ionotrophic glutamate receptors, whereas little is known about their interaction with changes mediated by metabotropic glutamate receptors. We evaluated effects of estrone on quisqualate (QA)-induced toxicity in neuronal cell cultures on 7 and 12 day in vitro (DIV). Twenty four hour exposure to QA (150 microM and 300 microM) significantly decreased cell survival in 7 day old cultures, but the 12 day old cultures were more resistant to its toxicity. DNQX (10 microM), an AMPA/kainate receptor antagonist, partly attenuated the toxic effects of QA, whereas LY 367 385 (100 microM), a selective mGluR1a antagonist, completely reversed the above effect. QA did not activate, but suppressed spontaneous caspase-3-like activity. Estrone (100 nM and 500 nM) attenuated QA-mediated neurotoxic effects independently of estrogen receptors, as indicated with ICI 182, 780 and without affecting the caspase-3-like activity. At early stage of development in vitro (7 DIV) toxic effects of QA were more profound and mediated mainly by metabotropic glutamate receptors of group I, whereas later (12 DIV) they were mediated mostly by ionotropic AMPA/kainate receptors. The toxic effects of QA were partly accompanied by anti-apoptotic action against spontaneous caspase-3-like activity, possibly due to modulation of neuronal plasticity.     

Non-neuronal cell conditioned medium stimulates peptidergic expression in sympathetic and sensory neurons in vitro

Regulation of peptide neurotransmitter metabolism was examined in dissociated cell cultures of neonatal rat sympathetic and sensory ganglia. Previous studies have shown that pineal gland conditioned medium (PCM) influences substance P (SP) and somatostatin (SS) metabolism in sympathetic neurons in vitro. The present study examines mechanisms mediating these effects, and compares the actions of PCM on sympathetic and sensory neurons. PCM treatment increased SP levels in a dose-dependent manner without altering SS content of sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Conversely, treatment of pure sympathetic neuron cultures resulted in a dose-dependent increase in SS, while SP was virtually undetectable at all doses. By contrast, dorsal root ganglion, trigeminal ganglion, and nondose ganglion sensory neurons contained SP both in the presence and absence of ganglion non-neuronal cells. Moreover, in each of these neuronal populations treatment with PCM increased SP levels both in the presence and in the absence of ganglion non-neuronal cells. These observations suggest that ganglion non-neuronal cells are necessary for sympathetic but not sensory neuron expression of SP. Moreover, PCM apparently stimulates SP in neurons which already contain the peptide, but the factor cannot foster de novo expression of the phenotype. PCM also influenced other transmitter traits in sympathetic neurons, suggesting linkage between mechanisms regulating peptides and other transmitters. In cultures containing both sympathetic neurons and non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) activity as well as SP, and decreased tyrosine hydroxylase (TOH) activity. By contrast, PCM treatment of pure sympathetic neuron cultures led to parallel increases in SS and TOH activity with negligible levels of SP and CHAC. These observations suggest that in sympathetic neurons, SS may be linked with noradrenergic expression, while SP is associated with cholinergic development, although more data are required to confirm this relationship. Moreover, there may be a reciprocal relationship between SP and SS expression by sympathetic neurons analogous to previous observations regarding cholinergic-noradrenergic expression (P. H. Patterson and L. L. Y. Chun, Proc. Natl. Acad. Sci. USA 71, 3607-3610, 1974; Dev. Biol. 56, 263-280, 1977). Consequently, neurotransmitter phenotypic expression is a complex process in which the environment regulates a balance among multiple transmitters.     

Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem

Serotonin neurons in 14-d embryonic rat brain stem were identified by peroxidase-antiperoxidase immunocytochemistry with an affinity-purified antiserotonin antibody. Brain-stem tissue was dissected from 14- or 15- d embryonic rats, dissociated and grown in cell culture for up to 5 wk, and serotonin neurons were identified by immunocytochemistry. Within 24 h of plating, serotonin immunoreactivity was present in 3.3% of neurons. Immunoreactivity in neuronal cell bodies decreased with time, whereas staining of processes increased. The number of serotonin- immunoreactive neurons remained constant at 3-5% over the first 14 d in culture. From 14 to 28 d, the total number of neurons decreased with little change in the number of serotonin neurons, such that, by day 28 in culture, up to 36% of surviving neurons exhibited serotonin immunoreactivity. Similar percentages of cultured brain stem neurons accumulating 3H-serotonin were identified by autoradiography. Uptake was abolished by the serotonin-uptake inhibitor, clomipramine, but was unaffected by excess norepinephrine, or by the norepinephrine-uptake inhibitor, maprotiline. Synthesis of 3H-serotonin was detected after incubation of cultures with 3H-tryptophan, and newly synthesized serotonin was released by potassium depolarization in a calcium- dependent manner. More than 95% of serotonin neurons were destroyed after incubation of cultures with 5,6-dihydroxytryptamine. Brain-stem cultures contained virtually no neurons with the ability to accumulate 3H-norepinephrine or 3H-dopamine. Approximately 40% of brain-stem neurons were labeled with gamma-aminobutyric acid (3H-GABA). However, there was almost no overlap in the surface area of neurons accumulating 3H-serotonin or 3H-GABA.     

Effects of antibiotics on protein synthesis and degradation in primary cultures of rat hepatocytes

Summary In primary hepatocyte cultures, maintained in a protein-free medium, streptomycin, penicillin, and Garamycin (gentamicin) all inhibited protein synthesis at concentrations above 0.1 mM. Some inhibition was also observed with the fungicide Mycostatin at 100 U/ml. Hepatocytic protein degradation was markedly inhibited by penicillin at concentrations above 0.1 mM, whereas streptomycin and Garamycin only showed slight inhibition at concentrations in excess of 1 mM. None of the antibiotics had any detectable effect on the structural integrity (viability) of the cells. The work was supported by grants from The Norwegian Cancer Society and The Norwegian Council for Science and the Humanities     

Developmental changes in the neurotransmitter properties of dissociated sympathetic neurons: a cytochemical study of the effects of medium

Sympathetic principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in several culture conditions shown previously to affect the transmitter status of the neurons. In three of these conditions the neurons are known to develop adrenergic functions over a 3- to 4-week period; in a fourth condition, they develop predominantly cholinergic functions. In this ultrastructural study, the transmitter status of the neurons during development in the several different media was examined after permanganate fixation which causes a granular precipitate in synaptic vesicles containing norepinephrine (small granular vesicles or SGV). It was found that as early as 4 days after plating, synapses and varicosities were present. In all four conditions, all of the terminals contained numerous SGV, indicating that the neurons both synthesize and store norepinephrine. Under “adrenergic” growth conditions, the terminals remained adrenergic in appearance during further development. Under “cholinergic” conditions, terminals of cholinergic appearance were present as early as 7 days and their incidence increased with time. Although the cholinergic terminals contained little or no endogenous norepinephrine, many were initially able to take up and store exogenous catecholamine. These results indicate that the dissociated sympathetic neurons of newborn rats which survive in culture acquired adrenergic transmitter functions early. Under “cholinergic” culture conditions, the neurons lose the ability to synthesize detectable quantities of norepinephrine; the ability to take up and store detectable quantities of exogenous catecholamines disappears more slowly.     

Precocious induction of arginase in primary cultures of fetal rat hepatocytes

Summary Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis. This work was supported by the Institut National Scientifique et de la Recherche Médicale through Grant 85.80.117.     

Interactions between dissociated rat sympathetic neurons and skeletal muscle cells developing in cell culture: II. Synaptic mechanisms

Dissociated rat sympathetic neurons and skeletal myotubes were grown in mass cultures and microcultures as described in the accompanying paper (C. A. Nurse, 1981, Develop. Biol.88, 55–70). Excitatory synaptic interactions developed between neuron and neuron and between neuron and myotube. Electrical coupling occurred rarely. More often, the interaction was chemical and as shown in the accompanying paper, cholinergic. At the chemical neuronmyotube junctions, spontaneous miniature potentials (mejp's), sensitive to d-tubocurarine, occurred infrequently (1–20/min) and their discharge appeared random; their amplitude distribution was skew at all ages (up to ca. 4 weeks) even when the myotube was innervated by a single neuron in microculture. The evoked postsynaptic potentials (ejp's) in the myotubes were sensitive in conventional ways to the extracellular calcium (Ca_o) and magnesium (Mg_o) concentrations, and several tests suggested that transmission was quantal. In a few cases where a single neuron innervated a myotube in microculture, the estimated mean quantal unit size (assuming “Poisson” release) was similar to the mode of the spontaneous mejp amplitude histogram, suggesting that many of the spontaneous units were similar to the evoked units. At several junctions the quantal content m_o, estimated by the “failure” method, varied nonlinearly with Ca_o over the range 0.2–1.2 mM; data could be fitted by a power relation where the power ranged from 2.6 to 5.2.     

Effects of triiodothyronine (T3) on differentiation of rat cortical neurons in primary cultures

Some of the events which characterize neuronal terminal differentiation have been studied in rat cortical neurons cultured in a selective synthetic medium for a period which corresponds to terminal brain maturation in vivo. In particular, we have studied the effect of T3 on the synthesis of nuclear proteins and the expression of the mRNAs which encode different variants of T3 nuclear receptors (c erb A proteins). We have shown that: a) T3 stimulates the turnover of nuclear proteins, with a more evident effect on the non-histone component; b) for the whole lifespan of cultures the predominant form of c erb A mRNA is the 2 variant (which encodes a protein unable to bind T3); whatever the function of 2 protein this finding suggests that its predominance on 1 is settled very early during mammalian brain maturation.     

Induced neurotransmitter release from primary cultures of rat brain neurons

Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4–5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated ³H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release ³H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca²⁺ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10–11 days $\text{in vitro}$. The fully expressed release capacity (20–30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.     

Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium

Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm-associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage.     

Cell-to-cell contacts in primary cultures of dissociated chicken embryonic brain

Summary In the elasmobranch fish, Scyllium stellare, a complex group of cells protrudes into the cavity of the mesencephalic ventricle of the optic tectum. It consists of six to seven large spherical perikarya which resemble neurons of the mesencephalic nucleus of the Vth cranial nerve. The bundled processes of these cells form a stalk connecting the protrusion with the brain tissue. The protrusion is located in the region where the mesencephalic ventricle joins the cerebral aqueduct. This complex was not found in all specimens examined in the present study. The functional role of this peculiar group of cells, which contain dense core granules and are bathed in the cerebrospinal fluid, is open to discussion.     

Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons

Although the cellular and molecular mechanisms underlying the delayed-type hypersensitivity (DTH) reaction have been investigated, the functions of infiltrating leukocytes and skin resident cells in the elicitation phase of the DTH reaction are not completely understood. To gain more insight into the role of these cells in the DTH reaction, we identified about 250 cDNA fragments showing elevated expression during the DNCB-induced guinea pig skin DTH reaction by differential display analysis. Characterization of 50 of them led to the identification of 28 genes whose expression was elevated in the DNCB-induced DTH reactive tissue. Sequencing of the 28 cDNA fragments and homology search analysis demonstrated that 10 of them represented known genes, some of which, in particular elafin (an elastase inhibitor) and ferritin, are considered to play roles in the DTH reaction. The other 18 fragments are probably derived from unknown genes. Cloning of the cDNAs of one of these genes indicated that it is that for guinea pig tryptophanyl-tRNA synthetase (WRS), a protein found to be induced by interferon-gamma and upregulated during the late stages of mononuclear phagocyte maturation in vitro. Strong induction of the WRS gene during the DTH reaction suggests its involvement in the in vivo immune response.