Coexpression of cholinergic and noradrenergic phenotypes in human and nonhuman autonomic nervous system

It has long been known that the sympathetic innervation of the sweat glands is cholinergic in most mammalian species and that, during development, rodent sympathetic cholinergic sweat gland innervation transiently expresses noradrenergic traits. We show here that some noradrenergic traits persist in cholinergic sympathetic innervation of the sweat glands in rodents but that lack of expression of the vesicular monoamine transporter renders these cells functionally nonnoradrenergic. Adult human sweat gland innervation, however, is not only cholinergic but coexpresses all of the proteins required for full noradrenergic function as well, including tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine beta-hydroxylase, and the vesicular monoamine transporter VMAT2. Thus, cholinergic/noradrenergic cotransmission is apparently a unique feature of the primate autonomic sympathetic nervous system. Furthermore, sympathetic neurons innervating specifically the cutaneous arteriovenous anastomoses (Hoyer-Grosser organs) in humans also possess a full cholinergic/noradrenergic cophenotype. Cholinergic/noradrenergic coexpression is absent from other portions of the human sympathetic nervous system but is extended in the parasympathetic nervous system to intrinsic neurons innervating the heart. These observations suggest a mode of autonomic regulation, based on corelease of norepinephrine and acetylcholine at parasympathocardiac, sudomotor, and selected vasomotor neuroeffector junctions, that is unique to the primate peripheral nervous system.     

Nerve growth factor expression in parasympathetic neurons: regulation by sympathetic innervation

Interactions between sympathetic and parasympathetic nerves are important in regulating visceral target function. Sympathetic nerves are closely apposed to, and form functional synapses with, parasympathetic axons in many effector organs. The molecular mechanisms responsible for these structural and functional interactions are unknown. We explored the possibility that Nerve Growth Factor (NGF) synthesis by parasympathetic neurons provides a mechanism by which sympathetic-parasympathetic interactions are established. Parasympathetic pterygopalatine ganglia NGF-gene expression was examined by in situ hybridization and protein content assessed by immunohistochemistry. Under control conditions, NGF mRNA was present in approximately 60% and NGF protein was in 40% of pterygopalatine parasympathetic neurons. Peripheral parasympathetic axons identified by vesicular acetylcholine transporter-immunoreactivity also displayed NGF immunoreactivity. To determine if sympathetic innervation regulates parasympathetic NGF expression, the ipsilateral superior cervical ganglion was excised. Thirty days postsympathectomy, the numbers of NGF mRNA-positive neurons were decreased to 38% and NGF immunoreactive neurons to 15%. This reduction was due to a loss of sympathetic nerve impulse activity, as similar reductions were achieved when superior cervical ganglia were deprived of preganglionic afferent input for 40 days. These findings provide evidence that normally NGF is synthesized by parasympathetic neurons and transported anterogradely to fibre terminals, where it may be available to sympathetic axons. Parasympathetic NGF expression, in turn, is augmented by impulse activity within (and presumably transmitter release from) sympathetic axons. It is suggested that parasympathetic NGF synthesis and its modulation by sympathetic innervation provides a molecular basis for establishment and maintenance of autonomic axo-axonal synaptic interactions.     

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.     

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.     

Chemical coding of the human gastrointestinal nervous system: cholinergic,VIPergic, and catecholaminergic phenotypes

The aim of this investigation was to identify the proportional neurochemical codes of enteric neurons and to determine the specific terminal fields of chemically defined nerve fibers in all parts of the human gastrointestinal (GI) tract. For this purpose, antibodies against the vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), serotonin (5-HT), vasoactive intestinal peptide (VIP), and protein gene product 9.5 (PGP 9.5) were used. For in situ hybridization (35)S-labeled VMAT1, VMAT2, and VAChT riboprobes were used. In all regions of the human GI tract, 50-70% of the neurons were cholinergic, as judged by staining for VAChT. The human gut unlike the rodent gut exhibits a cholinergic innervation, which is characterized by an extensive overlap with VIPergic innervation. Neurons containing VMAT2 constituted 14-20% of all intrinsic neurons in the upper GI tract, and there was an equal number of TH-positive neurons. In contrast, DBH was absent from intrinsic neurons. Cholinergic and monoaminergic phenotypes proved to be completely distinct phenotypes. In conclusion, the chemical coding of human enteric neurons reveals some similarities with that of other mammalian species, but also significant differences. VIP is a cholinergic cotransmitter in the intrinsic innervation of the human gut. The substantial overlap between VMAT2 and TH in enteric neurons indicates that the intrinsic catecholaminergic innervation is a stable component of the human GI tract throughout life. The absence of DBH from intrinsic catecholaminergic neurons indicates that these neurons have a dopaminergic phenotype.     

Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum.

During the development of sweat gland innervation, interactions with the target tissue induce a change from noradrenergic to cholinergic and peptidergic properties. To determine whether the change in neurotransmitter properties that occurs in the sweat gland innervation occurs more generally in sympathetic neurons, we identified a new target of cholinergic sympathetic neurons in rat, the periosteum, which is the connective tissue covering of bone, and characterized the development of periosteal innervation of the sternum. During development, sympathetic axons grow from thoracic sympathetic ganglia along rib periosteum to reach the sternum. All sympathetic axons displayed catecholaminergic properties when they reached the sternum, but these properties subsequently disappeared. Many axons lacked detectable immunoreactivities for vesicular acetylcholine transporter and vasoactive intestinal peptide when they reached the sternum and acquired them after arrival. To determine whether periosteum could direct changes in the neurotransmitter properties of sympathetic neurons that innervate it, we transplanted periosteum to the hairy skin, a noradrenergic sympathetic target. We found that the sympathetic innervation of the transplant underwent a noradrenergic to cholinergic and peptidergic change. These results suggest that periosteum, in addition to sweat glands, regulates the neurotransmitter properties of the sympathetic neurons that innervate it.     

Modulation of parasympathetic neuron phenotype and function by sympathetic innervation

Selective sympathetic nerve dysfunction occurs during aging and in certain disease states. Here, we review findings concerning the effects of chronic sympathetic denervation on parasympathetic innervation to orbital target tissues in the adult rat. Long-term sympathetic denervation was induced by excising the ipsilateral superior cervical ganglion for 5-6 weeks prior to analyses. Following sympathectomy, pterygopalatine ganglion parasympathetic neurons show reduced nitric oxide synthase protein in their somata and projections to vascular targets. Laser Doppler measurements of ocular blood flow indicate that sympathectomy is also accompanied by reduced nitrergic vasodilatation. In the superior tarsal muscle of the eyelid, parasympathetic varicosities, normally, are distant to smooth muscle cells but make axo-axonal contacts with sympathetic nerves, consistent with physiological evidence showing only prejunctional inhibitory effects on sympathetically mediated smooth muscle contraction. Following sympathectomy, parasympathetic varicosities proliferate and closely appose smooth muscle cells, and this is accompanied by establishment of parasympathetic-smooth muscle excitatory neurotransmission. Many pterygopalatine parasympathetic neurons normally contain nerve growth factor (NGF) protein and express NGF mRNA. However, following chronic sympathectomy or elimination of sympathetic impulse activity, NGF mRNA and protein are markedly reduced, indicating that sympathetic neurotransmission enhances NGF expression in parasympathetic neurons. Together, these findings portray a striking dependency of parasympathetic neurons on sympathetic nerves to maintain normal phenotype and function. Sympathetic influences on parasympathetic neurons may be mediated, in part, through axo-axonal synapses. NGF synthesis and release by parasympathetic neurons may represent a molecular basis underlying the formation of these synapses, and up-regulation of NGF synthesis by sympathetic nerve activity may act to reinforce these associations.     

Heterogeneous expression of SNAP-25 and synaptic vesicle proteins by central and peripheral inputs to sympathetic neurons

Neurons in prevertebral sympathetic ganglia receive convergent synaptic inputs from peripheral enteric neurons in addition to inputs from spinal preganglionic neurons. Although all inputs are functionally cholinergic, inputs from these two sources have distinctive neurochemical and functional profiles. We used multiple-labeling immunofluorescence, quantitative confocal microscopy, ultrastructural immunocytochemistry, and intracellular electrophysiologic recordings to examine whether populations of inputs to the guinea pig coeliac ganglion express different levels of synaptic proteins that could influence synaptic strength. Boutons of enteric intestinofugal inputs, identified by immunoreactivity to vasoactive intestinal peptide, showed considerable heterogeneity in their immunoreactivity to synaptosome-associated protein of 25 kDa (SNAP-25), synapsin, synaptophysin, choline acetyltransferase, and vesicular acetylcholine transporter. Mean levels of immunoreactivity to these proteins were significantly lower in terminals of intestinofugal inputs compared with terminals of spinal preganglionic inputs. Nevertheless, many boutons with undetectable levels of SNAP-25 immunoreactivity formed morphologically normal synapses with target neurons. Treatment with botulinum neurotoxin type A (20-50 nM for 2 hours in vitro) generated significant cleavage of SNAP-25 and produced similar dose- and time-dependent inhibitions of synaptic transmission from all classes of inputs, regardless of their mean level of SNAP-25 expression. The simplest interpretation of these results is that only synaptic boutons with detectable levels of SNAP-25 immunoreactivity contribute significantly to fast cholinergic transmission. Consequently, the low synaptic strength of intestinofugal inputs to final motor neurons in sympathetic pathways may be due in part to the low proportion of their boutons that express SNAP-25 and other synaptic proteins.     

Neuropeptides in the sympathetic system: Presence,plasticity, modulation,and implications

Neuropeptides are ubiquitous in the sympathetic system and modulate transmission at the levels of the intermediolateral cell column, sympathetic ganglia, and neuroeffector junctions. Several neuropeptide-containing pathways from the hypothalamus and medulla modulate excitability of preganglionic neurons. Neuropeptides coexist with norepinephrine or acetylcholine in subpopulations of chemically coded, target-specific sympathetic ganglion neurons. Neuropeptide Y is colocalized in adrenergic vasconstrictor neurons, whereas vasoactive intestinal polypeptide is colocalized in cholinergic sudomotor neurons. Neuropeptide expression is plastic; during development, neurons that switch from a noradrenergic to a cholinergic phenotype increase expression of vasoactive intestinal polypeptide, somatostatin, and substance P. Preganglionic inputs increase neuropeptide Y and inhibit substance P expression. Sympathetic denervation produces sprouting of sensory fibers containing substance P and calcitonin gene–related peptide in target tissues. Neuropeptides from preganglionic fibers (e.g., enkephalin) and primary afferents (e.g., substance P, vasoactive intestinal polypeptide) modulate transmission in sympathetic ganglia. Neuropeptide Y produces vasoconstrction, prejunctional inhibition of norepinephrine release, and postjunctional potentiation of norepinephrine effects. Plasma neuropeptide Y increase during intense sympathoexcitation, hypertension, and pheochromocytoma. Dystrophic neurites containing neuropeptide Y occur in human sympathetic ganglia during aging, diabetes, and dysautonomia. Sympathetic neuropeptides may thus have important clinical implications.     

Sympathetic cholinergic target innervation requires GDNF family receptor GFR alpha 2

Many cholinergic parasympathetic and enteric neurons require neurturin signaling through GDNF family receptor GFRalpha2 for target innervation. Since a distinct minority of sympathetic neurons are cholinergic, we examined whether GFRalpha2 is important for their development. We detected GFRalpha2 in neonatal sympathetic cholinergic neurons and neurturin mRNA in their target tissues, sweat glands in footpads, and periosteum. Lack of GFRalpha2 in mice did not affect the number of sympathetic cholinergic neurons, but their soma size was decreased in comparison to wild types. In adult and in 3-week-old GFRalpha2 knockout mice, the density of sympathetic cholinergic innervation was reduced by 50-70% in the sweat glands, and was completely absent in the periosteum. Sympathetic noradrenergic innervation of blood vessels in the footpads was unchanged. The density of sympathetic axons in sweat glands was unaffected at postnatal day P4 reflecting successful growth into the target area. Our results indicate that the cholinergic subpopulation of sympathetic neurons requires GFRalpha2 signaling for soma size and for growth or maintenance of target innervation. Thus, neurturin may be a general target-derived innervation factor for postganglionic cholinergic neurons in all parts of the autonomic nervous system.     

Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion

Parasympathetic neurons of the ciliary ganglion are innervated by preganglionic cholinergic neurons whose cell bodies lie in the brain stem; the ganglion cells in turn provide cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympathetic neurons of the superior cervical ganglion. Using immunocytochemical and histochemical techniques, we have examined the ciliary ganglion of adult rats for the expression of cholinergic and noradrenergic properties. As expected, the postganglionic ciliary neurons possessed detectable levels of choline acetyltransferase immunoreactivity (ChAT-IR). Unexpectedly, many ciliary neurons also exhibited immunoreactivity for tyrosine hydroxylase (TH-IR). Some had dopamine beta-hydroxylase-like (DBH-IR) immunoreactivity, but none contained detectable catecholamines, even after treatment with nialamide and L-DOPA. A sparse plexus of fibers exhibiting faint TH-IR was present in the irises of acutely sympathectomized rats. The terminals of preganglionic axons in the ciliary ganglion exhibited not only immunoreactivity for ChAT, but also for TH and contained stores of endogenous catecholamine. Neither ciliary neurons nor their preganglionic innervation accumulated detectable stores of exogenous catecholamines. Rats sympathectomized as neonates by treatment with 6-hydroxydopamine subsequently had a greater proportion of neurons possessing detectable TH-IR in the ciliary ganglion; both the TH-IR perikarya and their axons in the iris were more intensely immunofluorescent. TH-IR was present in the ciliary neuron cell bodies of mouse, guinea pig, and ferret. These species, however, lacked detectable TH-IR or catecholamine stores in preganglionic terminals. These observations indicate that mature, functionally cholinergic neurons from 2 different embryonic origins, postganglionic ciliary neurons derived from the neural crest and preganglionic neurons derived from the neural tube, display several catecholaminergic properties.     

Cholinergic vesicular transporters in progressive supranuclear palsy

OBJECTIVE: To determine the status of cholinergic and monoaminergic vesicular transporter binding sites in progressive supranuclear palsy (PSP). METHODS: The authors determined autoradiographically the regional expression of acetylcholine vesicular transporter (VAChT) and monoamine vesicular transporter type 2 (VMAT2) binding sites in postmortem basal ganglia samples from subjects with PSP. Comparison neurochemical measures included choline acetyltransferase (ChAT) enzyme activity and benzodiazepine (BZ) binding sites. RESULTS: VAChT expressions and ChAT activities in caudate nucleus and putamen were markedly decreased in PSP, whereas BZ binding was unaffected, consistent with selective losses of striatal cholinergic interneurons. VMAT2 density was reduced significantly in the caudate nucleus, putamen, and substantia nigra pars compacta, consistent with degeneration of dopaminergic nigrostriatal projection neurons in PSP. In the globus pallidus, BZ receptor binding sites were reduced, whereas VMAT2 and VAChT binding sites were unchanged, indicating losses of intrinsic pallidal neurons and synapses. CONCLUSIONS: These results confirm selective and marked degenerations of basal ganglia cholinergic and dopaminergic terminals in PSP. Striatal VAChT reduction may provide a unique neurochemical imaging marker for distinction of PSP from other types of basal ganglia neurodegeneration.     

Origin of neuronal nitric oxide synthase (NOS)-immunoreactive fibers in guinea pig parasympathetic cardiac ganglia

This study was conducted to determine the origin(s) of neuronal nitric oxide synthase-immunoreactive (NOS-IR) fibers within guinea pig atrial whole-mount preparations containing the cardiac ganglia. Intrinsic NOS-IR cardiac neurons exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they were cholinergic as well as nitrergic. Comparison of control versus 72-hour explant culture preparations indicated that most of the nitrergic fibers within cardiac ganglia were extrinsic. The extrinsic NOS-IR fibers were not IR for ChAT (marker of preganglionic parasympathetic neurons), tyrosine hydroxylase (marker of catecholaminergic sympathetic postganglionic axons), or calcitonin gene-related peptide (CGRP) (marker of afferent fibers). Separate NOS-IR and ChAT-IR neurons were present within medullary regions containing the cardiovascular regulatory nuclei (nucleus ambiguus and dorsal motor nucleus of the vagus), but no cells were found that exhibited both NOS immunoreactivity and ChAT immunoreactivity. The small size and location of the medullary NOS-IR neurons suggested they were probably interneurons. Only an occasional sympathetic postganglionic cell in the stellate ganglion complex exhibited NOS immunoreactivity. NOS-IR cells were present in dorsal root ganglia (thoracic 1-5), but these typically also exhibited CGRP immunoreactivity. NOS-IR cells were also present in the nodose ganglia, but only some exhibited CGRP immunoreactivity. We concluded that virtually all the extrinsic NOS-IR nerve fibers represented an afferent fiber input that was separate from the substance P (SP)/CGRP-containing population of sensory fibers. Furthermore, much of this NOS innervation is probably derived from the nodose ganglia.     

Neurochemical characterization of extrinsic innervation of the guinea pig rectum

The presence of markers for parasympathetic, sympathetic, and glutamatergic or peptidergic sensory innervation was investigated by using in vitro tracing with biotinamide, combined with immunohistochemistry, to characterise quantitatively extrinsic axons to myenteric ganglia of the guinea pig rectum. Of biotinamide-filled varicose axons, 3.6 +/- 1.3% were immunoreactive for tyrosine hydroxylase (TH) and 16.0 +/- 4.8% for vesicular acetylcholine transporter (VAChT). TH and vesicular monoamine transporter (VMAT1) showed high coexistence (83-100%), indicating that varicosities lacking TH immunoreactivity also lacked VMAT1. VAChT was detectable in 77% of choline acetyltransferase (ChAT)-immunoreactive varicosities. Calcitonin gene-related peptide (CGRP) was detected in 5.3 +/- 1.6% of biotinamide-labeled varicosities, the vesicular glutamate transporter (VGluT) 1 in 2.8 +/- 0.8%, and VGluT2 in 11.3 +/- 4.2% of varicosities of extrinsic origin. Varicosities from the same axon showed consistent immunoreactivity. A novel type of nerve ending was identified, with branching, flattened lamellar endings, similar to the intraganglionic laminar endings (IGLEs) of the proximal gut. Rectal IGLEs were frequently immunoreactive for VGluT1 and VGluT2. Thus most varicose axons of extrinsic origin, which innervate rectal myenteric ganglia, lack detectable levels of immunoreactivity for TH, VMAT1, VAChT, ChAT, VGluT1/2, or CGRP, under conditions in which these markers are readily detectable in other axons. Although some unlabeled varicosities may belong to afferent axons that lack detectable CGRP or VGluT1/2 in the periphery, this suggests that a large proportion of axons do not release any of the major autonomic or sensory transmitters. We speculate that this may vary under particular circumstances, for example, inflammation or obstruction of the gut.     

Neurotrophic factors acutely alter the sorting of the vesicular acetyl choline transporter and the vesicular monoamine transporter 2 in bimodal sympathetic neurons

Individual sympathetic neurons in co-cultures with cardiac myocytes store acetylcholine and noradrenaline in two different populations of synaptic vesicles and release both neurotransmitters from single presynaptic terminals. Neurotrophic factors selectively and acutely stimulate differential release of the two types of neurotransmitters from these bimodal neurons. Here we investigated the acute effects of neurotrophic factors on two pivotal marker proteins for catecholaminergic and cholinergic synaptic vesicle populations: the vesicular monoamine transporter 2 and the vesicular acetylcholine transporter. We observed that separation of the two fluorescence labeled transporters is not restricted to the varicosities, but can also be observed in the neurites as well as in the cell soma. Application of nerve growth factor, brain-derived neurotrophic factor and ciliary neuronotrophic factor caused acute alterations in transporter segregation. These results point to a novel function of neurotrophic factors during the short-term regulation of synaptic protein sorting in neurons.     

Effects of nerve growth factor on splenic norepinephrine and pineal N-acetyl-transferase in neonate rats exposed to alcohol in utero: Neuroimmune correlates

Prenatal alcohol exposure (FAE) has been associated with multiple anomalies, including a selective developmental delay of sympathetic innervation in lymphoid organs. Sympathetic neurons require nerve growth factor (NGF) for their development and maintenance, and recent evidence has suggested that alcohol impairs the synthesis and/or biological activity of NGF in selected central and peripheral neurons. Thus, the present study examined the hypothesis that NGF administration to FAE rats during early postnatal development would reverse some of the peripheral sympathetic deficits. Neonate rats, FAE and the corresponding control cohorts, received daily treatments of NGF or cytochrome C (0.3 mg/kg; s.c.) for various time intervals, and were killed 24hr or 10 days after the last treatment. The measured parameters included norepinephrine (NE) concentrations in the spleen and heart, which receive noradrenergic innervation from the coeliac ganglion and the superior cervical ganglion (SCG), respectively. In addition, we measured the activity of pineal N-acetyltransferase (NAT), the rate-limiting enzyme of melatonin biosynthesis, which depends on sympathetic innervation from the SCG. The data show that chronic, but not acute, NGF treatments reversed the FAE-related deficits in splenic NE concentrations as well as in pineal NAT activity in a time- and age-dependent manner. Sympathetic neurons play an important role in immune modulation. Thus, the altered splenic NE levels and pineal NAT activity may play a role in immune deficits associated with exposure to alcohol in utero.     

Functional implications of the noradrenergic-cholinergic switch induced by retinoic acid in NB69 neuroblastoma cells

Some neuroblastoma cell lines change their neurotransmitter phenotype from noradrenergic to cholinergic under retinoic acid treatment. Such "neurotransmitter switch" seems to be a consequence of changes in the expression and activity of the biosynthetic machinery for both neurotransmitters. In this study, we have characterized this "neurotransmitter switch" induced by retinoic acid in a human neuroblastoma cell line (NB69) showing catecholaminergic characteristics. Retinoic acid treatment reduced tyrosine hydroxylase activity and noradrenaline levels in NB69 cells but did not modify the expression of this enzyme. Moreover, the calcium-dependent release of [(3)H]noradrenaline in control cells was highly reduced by retinoic acid treatment. On the other hand, NB69 cells treated with retinoic acid enhanced the expression of choline acetyltransferase and acquired the capability to release [(3)H]acetylcholine in a calcium-dependent way. In addition, we found that the expression of the vesicular monoamine transporter 2 (VMAT2) and the vesicular acetylcholine transporter (VAChT) was increased in those cells treated with retinoic acid. Immunostaining revealed that retinoic acid treatment changed the cellular distribution of both vesicular monoamine transporter 2 and vesicular acetylcholine transporter. In conclusion, retinoic acid induces a noradrenergic to cholinergic switch in NB69 cells by acting at several levels of the neurotransmitter phenotypic expression.     

Cardiovascular dysautonomia in Parkinson disease: from pathophysiology to pathogenesis

Signs or symptoms of impaired autonomic regulation of circulation often attend Parkinson disease (PD). This review covers biomarkers and mechanisms of autonomic cardiovascular abnormalities in PD and related alpha-synucleinopathies. The clearest clinical laboratory correlate of dysautonomia in PD is loss of myocardial noradrenergic innervation, detected by cardiac sympathetic neuroimaging. About 30-40% of PD patients have orthostatic hypotension (OH), defined as a persistent, consistent fall in systolic blood pressure of at least 20 mmHg or diastolic blood pressure of at least 10 mmHg within 3 min of change in position from supine to standing. Neuroimaging evidence of cardiac sympathetic denervation is universal in PD with OH (PD+OH). In PD without OH about half the patients have diffuse left ventricular myocardial sympathetic denervation, a substantial minority have partial denervation confined to the inferolateral or apical walls, and a small number have normal innervation. Among patients with partial denervation the neuronal loss invariably progresses over time, and in those with normal innervation at least some loss eventually becomes evident. Thus, cardiac sympathetic denervation in PD occurs independently of the movement disorder. PD+OH also entails extra-cardiac noradrenergic denervation, but this is not as severe as in pure autonomic failure. PD+OH patients have failure of both the parasympathetic and sympathetic components of the arterial baroreflex. OH in PD therefore seems to reflect a "triple whammy" of cardiac and extra-cardiac noradrenergic denervation and baroreflex failure. In contrast, most patients with multiple system atrophy, which can resemble PD+OH clinically, do not have evidence for cardiac or extra-cardiac noradrenergic denervation. Catecholamines in the neuronal cytoplasm are potentially toxic, via spontaneous and enzyme-catalyzed oxidation. Normally cytoplasmic catecholamines are efficiently taken up into vesicles via the vesicular monoamine transporter. The recent finding of decreased vesicular uptake in Lewy body diseases therefore suggests a pathogenetic mechanism for loss of catecholaminergic neurons in the periphery and brain. Parkinson disease (PD) is one of the most common chronic neurodegenerative diseases of the elderly, and it is likely that as populations age PD will become even more prevalent and more of a public health burden. Severe depletion of dopaminergic neurons of the nigrostriatal system characterizes and likely produces the movement disorder (rest tremor, slowness of movement, rigid muscle tone, and postural instability) in PD. Over the past two decades, compelling evidence has accrued that PD also involves loss of noradrenergic neurons in the heart. This finding supports the view that loss of catecholaminergic neurons, both in the nigrostriatal system and the heart, is fundamental in PD. By the time PD manifests clinically, most of the nigrostriatal dopaminergic neurons are already lost. Identifying laboratory measures-biomarkers-of the disease process is therefore crucial for advances in treatment and prevention. Deposition of the protein, alpha-synuclein, in the form of Lewy bodies in catecholaminergic neurons is a pathologic hallmark of PD. Alpha-synucleinopathy in autonomic neurons may occur early in the pathogenetic process. The timing of cardiac noradrenergic denervation in PD is therefore a key issue. This review updates the field of autonomic cardiovascular abnormalities in PD and related disorders, with emphasis on relationships among striatal dopamine depletion, sympathetic noradrenergic denervation, and alpha-synucleinopathy.     

Cardiac ischemia-reperfusion regulates sympathetic neuropeptide expression through gp130-dependent and independent mechanisms

Cardiac function is regulated by a balance of sympathetic and parasympathetic transmission. Neuropeptide Y (NPY) and galanin (GAL) released from cardiac sympathetic neurons inhibits parasympathetic transmission in the heart. Sympathetic peptides may contribute to autonomic imbalance, which is characterized by increased sympathetic and decreased parasympathetic transmission and contributes to life threatening cardiovascular pathologies. Several gp130 cytokines are increased in the heart after myocardial infarction (MI), and these cytokines stimulate neuropeptide expression in sympathetic neurons. We used mice whose sympathetic neurons lack the gp130 receptor (gp130^DBH-Cre/lox mice) to ask if cytokine activation of gp130 regulated neuropeptide expression in cardiac sympathetic nerves after MI. Myocardial infarction decreased NPY mRNA through a gp130 independent mechanism and increased VIP and PACAP mRNA via gp130, while GAL mRNA was unchanged. Immunohistochemistry revealed a gp130-dependent increase in PACAP38 in cells of the stellate ganglion after MI, and PACAP was detected in pre-ganglionic fibers of all genotypes and surgical groups. VIP was identified in a few sympathetic nerve fibers in all genotypes and surgical groups. GAL and PACAP38 were not detected in sham hearts, but peptide immunoreactivity was high in the infarct three days after MI. Surprisingly, peptides were abundant in cells that co-labeled with macrophage markers F4/80 and MAC2, but were not detected in sympathetic axons. PACAP protects cardiac myocytes from apoptosis, and GAL stimulates axon regeneration in addition to inhibiting parasympathetic transmission. Thus, these peptides may play an important role in cardiac and neuronal remodeling after ischemia-reperfusion.