Expression of noradrenergic and cholinergic traits by sympathetic neurons cultured without serum

The ability to vary systematically the neuronal environment is one advantage afforded by the use of cell culture. Replacement of serum, a variable and undefined medium supplement, with known ingredients allows even greater control of culture conditions. We have studied biochemical and morphological properties related to neurotransmitter metabolism of rat sympathetic neurons cultured in a modified defined medium. Neuronal survival, ultrastructure, and expression of noradrenergic properties appear similar in serum-free and serum-supplemented cultures: small granular vesicles characteristic of norepinephrine storage were observed in both types of culture, and tyrosine hydroxylase activity, conversion of dopamine to norepinephrine, catecholamine production, and storage capacity are equivalent in serum-free and serum-containing cultures. Several of these properties were not exhibited at high levels in previous formulations of this defined medium. Acetylcholine production, however, was about 10-fold lower in serum-free compared to serum-supplemented cultures, consistent with the findings of lacovitti et al. (lacovitti, L., M. I. Johnson, T. H. Joh, and R. P. Bunge (1982) Neuroscience 7:2225-2239). Acetylcholine production can be induced under serum-free conditions by a previously characterized cholinergic inducing factor from heart cell conditioned medium. This responsiveness to serum-free heart cell conditioned medium indicates that serum-free cultures retain plasticity with respect to transmitter status, despite expression of noradrenergic characteristics, unlike cultured neurons of which the noradrenergic transmitter status is maintained by chronic depolarization. Thus, sympathetic neurons survive, express numerous differentiated properties, and display a novel transmitter status under serum-free conditions.     

BDNF and CNTF regulate cholinergic properties of sympathetic neurons through independent mechanisms

Cultured neonatal sympathetic neurons can synthesize and corelease norepinephrine (NE) and acetylcholine (ACh). Evoked release of NE has an excitatory effect on the beat rate of cocultured cardiac myocytes while ACh release results in myocyte inhibition. Here we show that the cholinergic properties of the neurons and the relative level of NE and ACh corelease are modulated by neurotrophic factors. Brain-derived neurotrophic factor (BDNF) rapidly promoted ACh release in the absence of cholinergic differentiation activity and even in neurons that were predominantly noradrenergic. This increase in the cholinergic component of sympathetic cotransmission was sufficient for myocytes to display an overall inhibitory response to neuronal stimulation. In contrast, short-term growth in ciliary neurotrophic factor (CNTF) resulted in the upregulation of cholinergic and downregulation of noradrenergic markers without an effect on normal excitatory neurotransmission. Only once the cells had acquired a cholinergic phenotype did CNTF acutely promote the evoked release of the cholinergic vesicle pool. The results of this study indicate that BDNF and CNTF, acting through independent pathways, modulate NE and ACh cotransmission to regulate the level of sympathetic excitation or inhibition of cardiac myocytes.     

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.     

Differential regulation of muscarinic and nicotinic cholinergic receptors and their mRNAs in cultured sympathetic neurons.

Mechanisms regulating expression of neuronal muscarinic and nicotinic receptors were examined in cultures of neonatal rat sympathetic neurons. Two factors known to stimulate cholinergic transmitter development in sympathetic neurons were examined for their effects on cholinergic receptor expression. A membrane associated factor (MANS46) and a diffusible factor produced by cultured rat fibroblasts (RFCM) each decreased muscarinic receptor number. By contrast, neither treatment altered levels of nicotinic receptors. Levels of muscarinic (m2) receptor mRNA were decreased by MANS but not by RFCM, indicating that effects of the two treatments were mediated by different mechanisms. Neither MANS nor RFCM altered levels of nicotinic alpha 3 or beta 2 mRNAs, consistent with the lack of change in numbers of nicotinic receptors. These observations indicate that receptor phenotype in developing neurons is subject to regulation by multiple epigenetic factors. Further, the same signals which regulate transmitter development may also regulate receptor expression in sympathetic neurons.     

Δ9-tetrahydrocannabinol increases activity of tyrosine hydroxylase in cultured fetal mesencephalic neurons

The exposure of pregnant rats to Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive constituent ofCannabis sativa, during gestation and lactation, affects the gene expression and the activity of tyrosine hydroxylase (TH) in the brain of their offspring, measured at fetal and early postnatal ages, when the expression of this enzyme plays an important role in neural development. In the present article, we have examined whether Δ⁹-THC is able to affect TH activity in cultured mesencephalic neurons obtained from fetuses at gestational d 14. Thus, TH activity increased approximately twofold in cells obtained from naive fetuses when exposed for 24 h to medium containing Δ⁹-THC. In addition, TH activity was also approx twofold higher in cells obtained from fetuses exposed daily to Δ⁹-THC from d 5 of gestation than in cells obtained from control fetuses, when both were exposed to basal media. This effect of Δ⁹-THC on TH activity seems to be produced via the activation of cannabinoid receptors, in particular the CB₁ subtype, which would presumably be located in these cells. This is because the exposure to medium containing both Δ⁹-THC and SR141716A, a specific antagonist for CB₁ receptors, abolished the effect observed with Δ⁹-THC alone. SR141716A alone was without effect on TH activity. Collectively, our results support the notion that Δ⁹-THC increased TH activity in cultured mesencephalic neurons, as previously observed in vivo, and that this effect was produced by activation of CB₁ receptors, which seem to be operative at these early ages. All this points to a role for the endogenous cannabimimetic system in brain development. Part of this work has been previously presented in abstract form at the 24th Meeting of the Federation of European Biochemical Societies, Barcelona, Spain, July, 1996.     

Tyrosine hydroxylase and norepinephrine transporter in sympathetic ganglia of female rats vary with reproductive state

In females, sympathetic activity varies with changes in reproductive status, but whether expression of proteins critical to the function of sympathetic neurons is also altered is unknown. Therefore, the present study tested the hypothesis that, in rat adrenal gland and superior cervical ganglia, the expression of tyrosine hydroxylase (TH) and the norepinephrine transporter (NET), measured using Western analysis, are changed during pregnancy and the estrous cycle. Compared to diestrus, pregnancy increased TH levels in both superior cervical ganglia and adrenal gland. Pregnancy was also associated with decreased NET levels in the superior cervical ganglia, but increased levels in the adrenal gland. Relative to diestrus, the pattern of changes of TH and the NET in rats during proestrus was generally similar to changes observed during pregnancy. To assess whether gonadal hormones were involved, ovariectomized rats were also studied and changes in serum estrogen and progesterone were assayed in a subset of animals in all groups. Variations in TH and the NET among all groups did not correlate with changes in either estrogen or progesterone, suggesting that the steroids were not exclusively responsible. In conclusion, reproductive status alters the expression of TH and the NET in adrenal gland and superior cervical ganglia of female rats, which could significantly influence the function of the sympathetic nervous system. However, the mechanism for these changes does not depend solely on changes in estrogen or progesterone.     

Harmonal regulation of adult sympathetic neurons: the effects of castration on tyrosine hydroxylase activity

The effects of the hormone testosterone on neurotransmitter synthesis in peripheral sympathetic ganglia were examined in adult male Sprague-Dawley rats. Tyrosine hydroxylase (T-OH), the rate limiting enzyme in catecholamine biosynthesis was examined in the hypogastric (HG), coeliac (CG), and superior cervical ganglion (SCG) subsequent to castration. Initial studies indicated that 2 weeks after surgery, HG T-OH activity fell to approximately 30% of control. In order to more clearly define the pattern of testosterone effects, HG was examined 1, 2 and 4 weeks after surgery. T-OH activity was 67%, 50% and 11% of control at these 3 respective time points, and the observed alteration in T-OH activity appeared to parallel changes in the size of pelvic target organs. Similar hormonal effects did not occur in other peripheral sympathetic ganglia; T-OH activity was unchanged in SCG and CG when examined 1 month after castration. Enzyme activity was restored following replacement therapy with testosterone, whereas the neural metabolite 17-beta estradiol was without effect. The recovery in T-OH activity was associated with partial recovery of target organ size. These studies suggest that hormonal factors regulate neurotransmitter synthesizing enzymes in adult sympathetic neurons and may do so via consequences of alterations in target organs. These observations parallel similar events in the developing nervous system.     

Astrocytes induce dendritic development in cultured sympathetic neurons

Sympathetic neurons in culture require the influence of Schwann cells in order to develop dendrites comparable to those seen in vivo. This study demonstrates that astrocytes induce dendritic development in greater than 90% of sympathetic neurons after two weeks of co-culture. We conclude that dendrite inducing factors are distributed on both central and peripheral glia.     

Potassium-stimulated purine release by cultured sympathetic neurons

Environmental factors can influence cultured sympathetic neurons to acquire several different neurotransmitter phenotypes. Cholinergic and noradrenergic transmitter status can be influenced by heart cell conditioned medium, chronic depolarization (Patterson, P. H. (1978) Annu. Rev. Neurosci. 1:1-17), and rat serum (Wolinsky, E. J., and P. H. Patterson, (1985) J. Neurosci. 5:1509-1512); formation of electrical synapses can be induced by insulin (Wolinsky, E. J., H. Patterson, and A. L. Willard (1985) J. Neurosci., 5:1675-1679). Purine release has also been proposed as a possible transmission mode for sympathetic neurons (Potter, D. D., E. J. Furshpan, and S. C. Landis (1983) Fred. Proc. 42:1626-1632), and as such, it is another candidate for environmental modulation. In this report, we assess the ability of sympathetic neuron cultures grown with and without serum to release metabolically labeled tritriated purine compounds in response to depolarization. Exposure to 54 mM potassium stimulated release of adenosine, inosine, and hypoxanthine from both serum-supplemented and defined-medium cultures. However, depolarization-stimulated release of adenine nucleotides was observed only from serum-supplemented cultures and not from serum-free cultures. The release of adenine nucleotides from serum-containing cultures is affected by divalent cations in the manner expected for a neurosecretory process. The failure of serum-free cultures to release detectable adenine nucleotides raises the possibility that they do not share with, or that they differ from, serum-supplemented cultures in the purinergic aspect of the multiple transmission modes available to sympathetic neurons, and that this difference may be due to effects of the culture medium.     

Lithium decreases Cl--ATPase activity and increases intracellular Cl- concentration in cultured rat hippocampal neurons

Under the conditions of stimulated phosphatidylinositol turnover (0. 1 mM carbachol plus 20 mM KCl), LiCl (0.1-10 mM) reduced the activity of Cl--ATPase in cultured rat hippocampal neurons without affecting Na+/K+- or anion-insensitive Mg2+-ATPase. This inhibition of Cl--ATPase was attenuated by the addition of 0.5 mM inositol to culture media. The intracellular Cl- concentrations of the LiCl-treated neurons increased in an inositol-sensitive manner.     

Tyrosine hydroxylase activity in discrete brain regions of depression model rats

Tyrosine hydroxylase (TH) activity was measured in discrete brain regions of rats during short-term forced running stress (FRS). TH activity was also determined in a depression-like state and in a recovered state after a long-term FRS. Under the short-term FRS, the TH activity showed a significant increase in the locus ceruleus, certain limbic regions and tuberoinfundibular system. In the depression-like state, however, there was a significant decrease in the locus ceruleus and certain limbic regions, but a significant increase was seen in the median eminence. The TH activity in recovered rats showed no difference from the level in the controls. These findings demonstrate an adaptive increase in the TH activity in relation to stress, and may also indicate a failure of adaptation in the depression-like state.     

Tyrosine hydroxylase containing neurons lacking aromatic amino acid decarboxylase in the hamster brain

We have recently described populations of tyrosine hydroxylase-immunoreactive neurons in the hamster brain in regions not known to contain catecholamine cell bodies. In the present study, the nature of the tyrosine hydroxylase immunoreactivity in the hamster brain was determined. In addition, these tyrosine hydroxylase-immunoreactive cell groups were examined for their ability to express aromatic amino acid decarboxylase. Immunohistochemistry with two different antibodies to tyrosine hydroxylase identified immunoreactive cell bodies in regions known to contain catecholamine neurons, including the substantia nigra and locus ceruleus. In addition, tyrosine hydroxylase-immunoreactive neurons were observed in other regions, including the basal forebrain, inferior colliculus, lateral parabrachial nucleus, and dorsal motor nucleus of the vagus. Western blotting indicated that hamster brain contained only one immunoreactive molecule, very similar in size to rat tyrosine hydroxylase. Thus it is likely that the immunohistochemical studies stained authentic hamster tyrosine hydroxylase. Indeed, in situ hybridization studies using a synthetic oligonucleotide probe against tyrosine hydroxylase mRNA resulted in specific and heavy labelling of these novel tyrosine hydroxylase-immunoreactive neurons. When adjacent sections were stained with antibodies to aromatic amino acid decarboxylase, known catecholamine cell groups were stained. However, the novel tyrosine hydroxylase cell groups did not display any aromatic amino acid decarboxylase immunoreactivity. These results suggest that neurons are present in the hamster brain that are able to hydroxylate tyrosine to L-DOPA, but that lack the ability to decarboxylate aromatic amino acids to produce dopamine or other catecholamines.     

Insulin promotes electrical coupling between cultured sympathetic neurons

Placing neurons in tissue culture is one way to study how environmental factors affect their differentiation. Replacement of serum-supplementation of the culture medium with defined ingredients extends the experimenter's control of the culture environment; however it also introduces additional potential influences. In this report, we confirm the observation of Higgins and Burton (Higgins, D., and H. Burton (1982) Neuroscience 7:2241-2253) of increased frequency of electrical coupling in serum-free compared to serum-supplemented cultures of rat sympathetic neurons. In addition, experiments were performed to determine whether this effect results from the removal of serum or from the addition of the defined medium components to the culture environment. The results of testing individual ingredients of the defined medium recipe adapted for use on sympathetic neurons (Bottenstein, J.E., and G. H. Sato (1979) Proc. Natl. Acad. Sci. U. S. A. 76:514-517) show that insulin is capable of inducing electrical coupling in serum-free cultures. Thus, the formation of electrical synapses by sympathetic neurons can be hormonally regulated.     

Fos activation in cultured tyrosine hydroxylase and oxytocin immunoreactive neurons

Fos and other immediate early gene products are used as markers for neuronal activity. We identified Fos immunocytochemically after KCl-induced depolarization of cultured hypothalamic neurons. Five-day cultures were treated for 1 h with 50 mM KCl or media and fixed at 0, 0.5, 1, 2, and 4 h post-treatment. Sequential immunocytochemistry was performed to identify Fos immunoreactivity in tyrosine hydroxylase (TH)-immunoreactive(-ir) or oxytocin (OT)-ir neurons. Activated neurons [brown cells (TH-ir or OT-ir) with purple nuclei (Fos-ir)] were counted microscopically. KCl treatment resulted in an increased percentage of Fos-ir TH-ir and OT-ir neurons over control levels over the time course examined. Fos-ir peaked in TH-ir neurons at 0.5 to 1 h posttreatment, with levels returning to baseline at 4 h. Fos-ir in OT-ir neurons peaked at 2 h, and remained elevated at 4 h, showing prolonged activation. These results demonstrate that KCl-induced depolarization of cultured hypothalamic neurons increases Fos with a different time course in TH-ir vs. OT-ir neurons.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Tyrosine hydroxylase activity: regional and subcellular distribution in the brain

The ratio of soluble to membrane-bound tyrosine hydroxylase activity was determined in the cell bodies and nerve endings of the nigro-striatal pathway (substantia nigra and caudate-putamen, respectively) and of the dorsal NE pathway (locus coeruleus and hippocampus-cortex, respectively) as well as in the hypothalamus which contains both CA cell bodies and nerve endings. Regional dissection was accomplished by a new procedure which allows for the rapid and consistent dissection of discrete brain areas.The cell body and nerve ending regions obtained by this procedure were found to be distinguishable with respect to subcellular distribution of tyrosine hydroxylase activity independent of the absolute amount of tyrosine hydroxylase. Locus coeruleus and substantia nigra showed a significantly greater proportion of soluble to membrane-bound tyrosine hydroxylase activity than did the caudate-putamen and hippocampus-cortex, where nerve endings predominate. The ratio for the hypothalamus is consistent with the presence of both CA cell bodies and nerve endings in this region. The necessity to distinguish between relatively discrete cell body and nerve ending regions as well as between NE and DA pathways is discussed as a prerequisite for the adequate characterization of the effects of experimental manipulations.     

Ultrastructural cytochemistry of monoamines in cultured human fetal sympathetic neurons

The presence and storage of adrenergic neurotransmitter (monoamines) in cultured human fetal sympathetic neurons was investigated by chromate-dichromate cytochemistry, formaldehyde-induced fluorescence and potassium permanganate fixation. Monoamines were specifically identified in the neurons by the presence of an electron dense precipitate following cytochemical treatment. Reaction product was found in cell somas and processes in all chromate—dichromate treated cultures. The size range and morphology of the precipitate indicated a vesicular storage site within large dense core vesicles. Neurons fluoresced after treatment with formaldehyde vapors, further confirming the presence of monoamines. When potassium permanganate was employed as the fixative, occasional positive dense core vesicles were found but their frequency was greatly reduced from that seen in the chromate-dichromate treated cultures. These findings show that cultured human fetal sympathetic neurons retain an adrenergic phenotype during long-term serum-free culture. In addition, the storage site for the adrenergic neurotransmitter in the developing neuron is within large dense core vesicles. The lack of dense core vesicles in potassium permanganate fixed material is believed to be due to the depletion of monoamines during fixation.