Brigitte Raynaud

The use of a tyrosine-hydroxylase cDNA probe to study the neurotransmitter plasticity of rat sympathetic neurons in culture

We have compared quantitatively the effects of muscle-conditioned medium (CM) and elevated K+ concentration (40 mM) on the enzymatic activity of tyrosine hydroxylase (TH) and on TH-mRNA levels in primary cultures of rat sympathetic neurons. Northern blot analysis of RNA from cultured neurons with a 32P-labeled rat TH-cDNA probe was performed. The probe hybridized strongly with a single RNA species of 1.9 kb, similar in size to the TH-mRNA from PC12 pheochromocytoma cells. In agreement with earlier data both CM and a partially purified factor from CM increased choline acetyltransferase activity up to 200-fold and depressed TH activity by 2- to 7-fold in cultured sympathetic neurons. These effects were accompanied by a decrease in TH-mRNA level, which correlated with the decrease in TH activity. On the other hand, a culture medium supplemented with 40 mM KCl caused a 1.5- to 5-fold increase in TH activity, which was accompanied by an increase in TH-mRNA level of the same order of magnitude. As a working hypothesis, we suggest that CM and neuronal depolarization control the transcription of the TH gene in an antagonistic manner.

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.

Aromatic L-amino acid decarboxylase-immunoreactive structures in human midbrain, pons, and medulla.

The objective of the present study was to determine with precision the localization of neurons and fibers immunoreactive (ir) for aromatic L-amino acid decarboxylase (AADC), the second-step enzyme responsible for conversion of L-dihydroxyphenylalanine (L-DOPA) to dopamine (DA) and 5-hydroxytryptophan (5-HTP) to serotonin (5-hydroxytryptamine: 5-HT) in the midbrain, pons, and medulla oblongata of the adult human brain. Intense AADC immunoreactivity was observed in a large number of presumptive 5-HT neuronal cell bodies distributed in all of the raphe nuclei, as well as in regions outside the raphe nuclei such as the ventral portions of the pons and medulla. Moderate to strong immunoreaction was observable in presumptive DA cells in the mesencephalic reticular formation, substantia nigra, and ventral tegmental area of Tsai, as well as in presumptive noradrenergic (NA) cells, which were aggregated in the locus coeruleus and dispersed in the subcoeruleus nuclei. In the medulla oblongata, immunoreaction of moderate intensity was distributed in the mid and ventrolateral portions of the intermediate reticular nucleus, which constitutes the oblique plate of A1/C1 presumptive adrenergic and/or NA neurons. The dorsal vagal AADC-ir neurons were fewer in number and stained more weakly than cells immunoreactive for tyrosine hydroxylase (TH). AADC immunoreactivity was not identified in an aggregate of TH-ir neurons lying in the gelatinous subnucleus of the solitary nucleus, a restricted region just rostroventral to the area postrema. Nonaminergic AADC-positive neurons (D neurons), which are abundant in the rat and cat midbrain, pons, and medulla, were hardly detectable in homologous regions in the human brain, although they were clearly distinguishable in the forebrain.

Induction of the vesicular monoamine transporter by elevated potassium concentration in cultures of rat sympathetic neurons

The expression of the vesicular monoamine transporter was studied in newborn rat sympathetic neurons and compared to that of the catecholamine biosynthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase. The vesicular monoamine transporter was assayed using the specific ligand [3H]dihydrotetrabenazine. In cultures grown for 10 days in the presence of 35 mM K+, tyrosine hydroxylase activity and the density of [3H]dihydrotetrabenazine binding sites were increased by a similar 2-3-fold factor, while dopamine-beta-hydroxylase activity and protein level were unchanged. Under these conditions, choline acetyltransferase activity was depressed by 90%. The induction of the vesicular monoamine transporter by high K+ was dependent upon Ca2+ entry through slow calcium channels since it was inhibited by the diphenylbutylpiperidine antagonist fluspirilene and by 20 mM Mg2+, and was enhanced by the dihydropyridine agonist, Bay K8644. The induction of the vesicular monoamine transporter by neuronal depolarization indicates the existence of a Ca2(+)-dependent mechanism of coregulation for this intrinsic component of monoaminergic synaptic vesicles and tyrosine hydroxylase. On the other hand, the apparent absence of dopamine-beta-hydroxylase induction is probably due to the continuous secretion of this intravesicular enzyme by the depolarized sympathetic neurons, an effect already observed in trans-synaptically stimulated adult sympathetic ganglion and adrenal medulla.

The role of Ca2+ channels of the L-type in neurotransmitter plasticity of cultured sympathetic neurons

We have studied the effects of Ca2+ antagonists and agonists on the development of choline acetyltransferase (ChAT), tyrosine hydroxylase (TOH) and acetylcholinesterase (AChE) in cultures of rat sympathetic neurons maintained for 6-9 days in low K+ (5 mM) or high K+ (35 mM) medium. Previous experiments have shown that high K+ medium increases TOH activity and TOH-mRNA level up to 3.5-fold and depresses the development of AChE, in particular of its asymmetric A12 form. Moreover, high K+ medium inhibits ChAT induction by 90% in muscle-conditioned medium (Raynaud et al., Dev. Biol., 119 (1987) 305-312; 121 (1987) 548-558). None of the Ca2+ antagonists tested affected the development of ChAT, TOH or AChE in low K+ medium. In high K+ medium, nitrendipine (3 microM) or fluspirilene (1 microM) fully restored ChAT induction by conditioned medium to the level observed in low K+ medium. Other drugs (1 microM) gave partial reversion: flunarizine greater than (+)-PN 200-110 greater than (-)-D-888 greater than cinnarizine = lidoflazine. On the other hand, ChAT induction was not restored by a calmodulin inhibitor, calmidazolium (1 microM). Fluspirilene, PN 200-110, and nitrendipine also totally abolished TOH induction by high K+ medium; fluspirilene (1 microM) suppressed the inhibitory effect of high K+ medium on AChE development and restored the development of A12 AChE. Conditioned medium also depresses AChE and blocks the development of A12 AChE (Swerts et al., Dev. Biol., 103 (1984) 230-234), but these effects were insensitive to fluspirilene. The Ca2+ agonist Bay K 8644 (1 microM) potentiated the effects of elevated K+ on both ChAT and TOH. The data suggest that the effects of long-term depolarization on ChAT, TOH and AChE are mediated by Ca2+ entry specifically through voltage-sensitive channels of the L-type. Our results on cultured sympathetic neurons raise the possibility that Ca2+ antagonists, which are widely used clinically, may affect the expression of neurotransmitter phenotypic traits in vivo and interfere with trans-synaptic induction of enzymes.

Chapter 1 Choline acetyltransferase: a molecular genetic approach

Publisher Summary This chapter discusses the recent findings in the study of choline acetyltransferase (ChAT), following cloning of mammalian ChAT and discusses potential directions of research. ChAT constitutes the only specific marker available to date for cholinergic neurons. The paucity of the enzyme, whose purification from rat brain required about a 10 6 -fold enrichment, has limited its biochemical and structural analysis. The choice of the starting material to isolate a particular cDNA is crucial when dealing with proteins, such as ChAT, which are expressed at a very low abundance. The ability to differentially express various forms of the enzyme that exhibit distinct specific activities represents an attractive means of regulating neurotransmitter availability at particular synapses. Such a possible diversity could also be developmentally regulated or be tissue-specific. The porcine and rat ChAT complementary DNA (cDNA) hybridize with human ChAT mRNA, which will facilitate the isolation of the human gene. These probes will be more appropriate for analyzing the regulation of the expression of the ChAT gene under various physiological conditions and in pathological situations such as Alzheimers disease and amyotrophic lateral sclerosis, for which there is no animal model.

Dopaminergic neurons in the cat dorsal motor nucleus of the vagus, demonstrated by dopamine, AADC and TH immunohistochemistry

In the rostral part of the dorsal motor nucleus of the vagus of the cat, neurons do not contain histochemically detectable catecholamines, even though many perikarya contain both intense aromatic L-amino acid decarboxylase (AADC) immunoreactivity and strong monoamine oxidase enzymatic activity. Similarly located perikarya have distinct immunoreactivities to tyrosine hydroxylase (TH) and dopamine after treatment with colchicine. Since inhibition of monoamine oxidase fails to reveal dopamine in these cells, its absence in non-colchicine-treated animals cannot be due to rapid deamination. It appears that dopamine is synthesized by TH and AADC in dorsal motor vagal cells and is then rapidly transported from the perikarya.

Studies of the Neurotransmitter Plasticity of Cultured Rat Sympathetic Neurons at the Molecular Level

Cultures of sympathetic neurons from new-born rats constitute an attractive model to study the triggering and modulation of the expression of neurotransmitter phenotypic traits during neuronal differentiation. These neurons can express a variety of neurotransmitters and neuropeptides, and experiments performed in vivo or in cultures have led to insights on the molecular mechanisms of this phenotypic plasticity. In particular, several extracellular cues have been identified, which modify the expression of cholinergic and catecholaminergic characters in these cultured neurons (for a review, see Patterson, 1978): conditioned medium (CM) by certain non-neuronal cells induces the biosynthesis of acetylcholine in such cultures, and depress that of catecholamines (Patterson and Chun, 1977). On the other hand, neuronal depolarization fosters the development of noradrenergic characteristics and depresses acetylcholine (ACh) biosynthesis (Walicke et al., 1977; Walicke and Patterson, 1981). In addition, neuronal depolarization inhibits the development of substance P in rat sympathetic neurons, both in vivo and in culture (Kessler et al., 1981; Adler and Black, 1984).