Publisher Correction: Transcriptomic and neurochemical analysis of the stellate ganglia in mice highlights sex differences.

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Transcriptomic and neurochemical analysis of the stellate ganglia in mice highlights sex differences.

The stellate ganglia are the predominant source of sympathetic innervation to the heart. Remodeling of the nerves projecting to the heart has been observed in several cardiovascular diseases, however studies of adult stellate ganglia are limited. A profile of the baseline transcriptomic and neurochemical characteristics of the stellate ganglia in adult C57Bl6j mice, a common model for the study of cardiovascular diseases, may aid future investigations. We have generated a dataset of baseline measurements of mouse stellate ganglia using RNAseq, HPLC and mass spectrometry. Expression differences between male and female mice were identified. These differences included physiologically important genes for growth factors, receptors and ion channels. While the neurochemical profiles of male and female stellate ganglia were not different, minor differences in neurotransmitter content were identified in heart tissue.

Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac sympathetic innervation and prevents arrhythmias.

Millions of people suffer a myocardial infarction (MI) every year, and those who survive have increased risk of arrhythmias and sudden cardiac death. Recent clinical studies have identified sympathetic denervation as a predictor of increased arrhythmia susceptibility. Chondroitin sulfate proteoglycans present in the cardiac scar after MI prevent sympathetic reinnervation by binding the neuronal protein tyrosine phosphatase receptor σ (PTPσ). Here we show that the absence of PTPσ, or pharmacologic modulation of PTPσ by the novel intracellular sigma peptide (ISP) beginning 3 days after injury, restores sympathetic innervation to the scar and markedly reduces arrhythmia susceptibility. Using optical mapping we observe increased dispersion of action potential duration, supersensitivity to ß-adrenergic receptor stimulation and Ca(2+) mishandling following MI. Sympathetic reinnervation prevents these changes and renders hearts remarkably resistant to induced arrhythmias.

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.

Myocardial infarction stimulates galanin expression in cardiac sympathetic neurons.

Cardiac ischemia-reperfusion alters sympathetic neurotransmission in the heart, but little is known about its effect on neuropeptide expression in sympathetic neurons. Ischemia followed by reperfusion induces the production of inflammatory cytokines in the heart, including interleukin-6 and cardiotrophin-1. These cytokines and related molecules inhibit the expression of neuropeptide Y (NPY), and stimulate the expression of vasoactive intestinal peptide (VIP), substance P (SubP), and galanin (GAL) in cultured sympathetic neurons. Therefore, we quantified NPY, VIP, SubP, and GAL mRNA in neurons of the stellate ganglia 1 week after ischemia-reperfusion to determine if neuropeptide expression was altered in cardiac sympathetic neurons. NPY, VIP, and SubP mRNAs were unchanged compared to unoperated control animals, but GAL mRNA was increased significantly. The increased GAL mRNA was not accompanied by elevated GAL peptide content in the stellate ganglia. Galanin content was increased significantly in the heart, however, indicating that elevated GAL mRNA led to increased peptide production. GAL content was increased in the left ventricle below the coronary artery ligation, but was not increased significantly in the atria or the base of the heart above the ligation. The buildup of GAL specifically in the damaged left ventricle is consistent with previous reports that GAL is transported to regenerating nerve endings after axon damage.

Catecholamines are required for the acquisition of secretory responsiveness by sweat glands.

The sympathetic innervation of sweat glands undergoes a developmental change in transmitter phenotype from catecholaminergic to cholinergic. Acetylcholine elicits sweating and is necessary for development and maintenance of secretory responsiveness, the ability of glands to produce sweat after nerve stimulation or agonist administration. To determine whether catecholamines play a role in the development or function of this system, we examined the onset of secretory responsiveness in two transgenic mouse lines, one albino and the other pigmented, that lack tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Although both lines lack TH, their catecholamine levels differ because tyrosinase in pigmented mice serves as an alternative source for catecholamine synthesis (Rios et al., 1999). At postnatal day 21 (P21), 28 glands on average are active in interdigital hind footpads of albino TH wild-type mice. In contrast, fewer than one gland is active in albino TH null mice, which lack catecholamines in gland innervation. Treatment of albino TH null mice with DOPA, a catecholamine precursor, from P11 to P21 increases the number of active glands to 14. Pigmented TH null mice, which have faint catecholamine fluorescence in the developing gland innervation, possess 12 active glands at P21, indicating that catecholamines made via tyrosinase, albeit reduced from wild-type levels, support development of responsiveness. Gland formation and the appearance of cholinergic markers occur normally in albino TH null mice, suggesting that catecholamines act directly on gland cells to trigger their final differentiation and to induce responsiveness. Thus, catecholamines, like acetylcholine, are essential for the development of secretory responsiveness.

Norepinephrine transporter expression in cholinergic sympathetic neurons: differential regulation of membrane and vesicular transporters.

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development.

Catecholamine synthesis is mediated by tyrosinase in the absence of tyrosine hydroxylase.

Catecholamine neurotransmitters are synthesized by hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-Dopa) by tyrosine hydroxylase (TH). The elimination of TH in both pigmented and albino mice described here, like pigmented TH-null mice reported previously (Kobayashi et al., 1995; Zhou et al., 1995), demonstrates the unequivocal requirement for catecholamines during embryonic development. Although the lack of TH is fatal, TH-null embryos can be rescued by administration of catecholamine precursors to pregnant dams. Once born, TH-null pups can survive without further treatment until weaning. Given the relatively rapid half-life of catecholamines, we expected to find none in postnatal TH-null pups. Despite the fact that the TH-null pups lack TH and have not been supplemented with catecholamine precursers, catecholamines are readily detected in our pigmented line of TH-null mice by glyoxylic acid-induced histofluorescence at postnatal day 7 (P7) and P15 and quantitatively at P15 in sympathetically innervated peripheral organs, in sympathetic ganglia, in adrenal glands, and in brains. Between 2 and 22% of wild-type catecholamine concentrations are found in these tissues in mutant pigmented mice. To ascertain the source of the catecholamine, we examined postnatal TH-null albino mice that lack tyrosinase, another enzyme that converts tyrosine to L-Dopa but does so during melanin synthesis. In contrast to the pigmented TH-null mice, catecholamine histofluorescence is undetectable in postnatal albino mutants, and the catecholamine content of TH-null pups lacking tyrosinase is 18% or less than that of TH-null mice with tyrosinase. Thus, these extraordinary circumstances reveal that tyrosinase serves as an alternative pathway to supply catecholamines.

A sweat gland-derived differentiation activity acts through known cytokine signaling pathways.

The sympathetic innervation of sweat glands undergoes a target-induced noradrenergic to cholinergic/peptidergic switch during development. Similar changes are induced in cultured sympathetic neurons by sweat gland cells or by one of the following cytokines: leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these is the sweat gland-derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIF receptor beta (LIFRbeta) and gp130 and well defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity was a member of the LIF/CNTF cytokine family, we tested whether it acted via these same receptors and signal cascades. Blockade of LIFRbeta inhibited the sweat gland differentiation activity in neuron/gland co-cultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFRbeta and gp130. An inhibitor (CGX) of molecules that bind the CNTFRalpha, which is required for CNTF signaling, did not affect the gland-derived differentiation activity. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased vasoactive intestinal peptide mRNA, STAT3 dimerization, and DNA binding, and stimulation of transcription from the vasoactive intestinal peptide cytokine-responsive element. Thus, the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a family member.

Differential regulation of adrenergic receptor development by sympathetic innervation.

Rat sweat glands provide an interesting model system for a developmental study of adrenergic receptor expression because their sympathetic innervation undergoes a switch from a nonadrenergic to cholinergic and peptidergic phenotype. alpha 1B, alpha 2B, and beta 2 receptors are expressed in rat footpads; alpha 1 and beta 2 receptors are localized specifically to sweat glands, and alpha 2 receptors also are expressed in other tissues. alpha 1 and, to a lesser extent, beta 2 receptors decrease during development, whereas alpha 2 levels remain relatively constant. Decreased receptor expression is accompanied by the loss of alpha 1-stimulated inositol phosphate accumulation, but no change in beta-stimulated cAMP production. The number of alpha 1 and beta 2 receptors decreases after P21, when the sympathetic innervation no longer produces catecholamines. Neonatal sympathectomy causes a partial failure of alpha 1 downregulation, but has no effect on beta 2 or alpha 2 receptor levels. Therefore, at least two distinct mechanisms regulate development of adrenergic receptors in sweat glands. Innervation-independent processes control developmental expression of alpha 1, beta 2, and alpha 2 receptors, and an additional, innervation-dependent mechanism influences expression of alpha 1 receptors. Denervation at postnatal day 20, when the sympathetic innervation is cholinergic and peptidergic, results in retention of alpha 1 receptors, but cholinergic blockade begun at P20 does not. These results indicate that regulation of receptor expression in sweat glands is complex, and suggest that the innervation-dependent factors that decrease alpha 1 levels during development act through a nonadrenergic, noncholinergic mechanism.

Cardiotrophin-1 is not the sweat gland-derived differentiation factor.

Sympathetic neurons innervating sweat glands undergo a target-directed switch in neurotransmitter properties. Although the factor responsible for inducing this switch has not been identified, it appears to be a member of the neuropoietic cytokine family. Cardiotrophin-1 (CT-1), a new family member, was analyzed to determine whether it was a relevant factor. CT-1 induced choline acetyl-transferase and vasoactive intestinal peptide in cultured sympathetic neurons, and RT/PCR amplified CT-1 mRNA from footpad total RNA. The differentiation activity of CT-1 was blocked by CT-1 antiserum. The activity in sweat gland extracts and cultures was not, however, suggesting that CT-1 is not the sweat gland-derived factor.

Production of sweat gland cholinergic differentiation factor depends on innervation.

Sympathetic neurons innervating sweat glands undergo a target-directed developmental switch in neurotransmitter properties. Using cultured sympathetic neurons as a bioassay for cholinergic differentiation factors, we and others found that extracts containing soluble proteins from developing and adult footpads caused the same changes in transmitter properties in sympathetic neurons in vitro that the target does in vivo. In the present studies, using footpads from Tabby mutant mice that lack sweat glands, we found that the presence of sweat glands is correlated with the presence of cholinergic differentiation activity in footpad extracts. We examined the conditions necessary for secretion of differentiation activity from primary cultures of sweat gland cells. Surprisingly, sweat gland cells cultured alone do not produce or secrete cholinergic differentiation activity. When grown in the presence of sympathetic neurons, however, gland cells induce cholinergic function, increase vasoactive intestinal peptide content, and reduce catecholamine production in the neurons. Medium conditioned by sweat gland/neuron cocultures has a similar effect on the transmitter properties of cultured sympathetic neurons, indicating that the target influence on phenotype is mediated by a secreted factor(s). The innervation-dependence of cholinergic differentiation factor production provides evidence that reciprocal interactions between neurons and sweat glands are necessary for acquisition of the mature transmitter phenotype.

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Noradrenergic regulation of cholinergic differentiation.

When the sympathetic nerves that innervate rat sweat glands reach their targets, they are induced to switch from using norepinephrine as their neurotransmitter to acetylcholine. Catecholamines (such as norepinephrine) released by nerves growing to the sweat gland induce this phenotypic conversion by stimulating production of a cholinergic differentiation factor [sweat gland factor (SGF)] by gland cells. Here, culture of gland cells with sympathetic, but not sensory, neurons induced SGF production. Blockage of alpha 1- or beta-adrenergic receptors prevented acquisition of the cholinergic phenotype in sympathetic neurons co-cultured with sweat glands, and sweat glands from sympathectomized animals lacked SGF. Thus, reciprocal instructive interactions, mediated in part by small molecule neurotransmitters, direct the development of this synapse.

Isolation and characterization of a novel cDNA which identifies both neural-specific and ubiquitously expressed GS alpha mRNAs.

Heterotrimeric G proteins consisting of alpha, beta, and gamma subunits couple sensory, hormone, and neurotransmitter receptors to intracellular and transmembrane effectors. Several splicing variants of the GS (the G protein that stimulates adenylyl cyclase) alpha subunit (GS alpha) have been described. Some of these couple receptors to stimulation of adenylyl cyclase and Ca2+ channels, whereas others encode truncated proteins whose functions are not currently defined. We describe a 1321N1 human astrocytoma cDNA clone for a novel GS alpha isoform isolated from astrocytoma cells (G(astro)) that is identical to GS alpha-1 with the exception of a novel 5' sequence extending into the previously described exon 1 of GS alpha, a single base change, and an alternative polyadenylation site. Analysis by northern blotting and reverse transcription/PCR confirms the presence of an mRNA corresponding to this cDNA in astrocytoma cells. Additional northern analysis indicates that G(astro) recognizes two novel GS alpha mRNAs in the rat: a 2.0-kb mRNA expressed only in neural and neuroendocrine tissues and a 1.8-kb mRNA that is ubiquitously expressed. Functional analysis of G(astro) is complicated by the apparent insertion of alphoid satellite DNA into the transcription unit. The resulting cDNA encodes a truncated protein that may be translated from the methionine in exon 2 as previously described.

Multiple second-messenger pathways mediate agonist regulation of muscarinic receptor mRNA expression.

Muscarinic acetylcholine receptors in the embryonic chicken heart undergo agonist-induced internalization followed by decreases in both receptor number and mRNA expression. Muscarinic agonists cause both inhibition of adenylyl cyclase and activation of phospholipase C in chick heart cells. Treatment of cells with islet activating protein, which blocks coupling of muscarinic receptors to adenylyl cyclase but not phospholipase C, blocks muscarinic receptor-mediated regulation of receptor mRNA levels. Incubation of cells with the partial agonist pilocarpine, which causes inhibition of adenylyl cyclase but not stimulation of phospholipase C, induces less down-regulation of receptor mRNA levels than agonist which regulate both second-messenger systems. Thus, both second-messenger pathways are required for maximal regulation of muscarinic receptor mRNA levels in response to receptor activation. We also demonstrate that the regulation of receptor mRNA by agonist plays an important role in modulating the rate of recovery of muscarinic acetylcholine receptor number following agonist-induced down-regulation.

Regulation of expression and function of muscarinic receptors.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Regulation of muscarinic acetylcholine receptor mRNA expression by activation of homologous and heterologous receptors.

Muscarinic acetylcholine receptors (mAChR) in the embryonic chicken heart undergo agonist-induced internalization and a subsequent decrease in receptor number (downregulation). Cloning studies have identified two subtypes of mAChR expressed in the embryonic chicken heart, the cm2 and cm4 receptors. We report here that persistent activation of the mAChR in cultured chicken heart cells with the cholinergic agonist carbachol causes significant decreases in the levels of both cm2 and cm4 mRNA, as measured by solution hybridization analyses. The half-lives of the cm2 and cm4 mRNAs are not altered by agonist treatment, indicating that agonist most likely regulates mRNA levels by regulating the rate of gene transcription. Activation of mAChR in chicken heart causes both inhibition of adenylate cyclase activity and stimulation of phospholipase C activity. To test whether changes in the levels of intracellular second messengers were involved in the changes in mAChR mRNAs observed following agonist exposure, we determined the effects of incubation with agonists for the A1 adenosine receptors (which inhibit adenylate cyclase in chicken heart) and angiotensin II receptors (which stimulate phospholipase C) on mAChR receptor number and mRNA levels. Activation of these pathways together through heterologous receptors resulted in decreased mAChR number and mRNA levels, although these changes were not as large as those seen with direct activation of the mAChR. These results suggest that regulation of adenylate cyclase and phospholipase C activities may be involved in the regulation of mAChR gene expression.