Sympathetic Ganglion Neurons Research Articles

Distribution pattern and chemical coding of neurons of the sympathetic chain ganglia supplying the descending colon in the pig

Sympathetic chain ganglia (SChG) neurons projecting to the descending colon of the pig were studied by means of retrograde tracing (Fast Blue, FB) and double-labelling immunofluorescence methods. FB was injected into the gut wall and after three weeks survival time the animals were transcardially perfused with paraformaldehyde and the bilateral sympathetic trunks were collected. The FB-positive neurons were localised only in the lumbar (L(1)-L(5)) ganglia of the sympathetic trunk and appeared either as small (30-50 microm in diameter) round-shaped perikarya forming clusters localised in caudal-ventral area or, rarely, as bigger (50-80 microm) and dispersed solitary irregular perikarya. Immunohistochemical staining revealed the catecholaminergic (tyrosine hydroxylase-/dopamine beta-hydroxylase-immunoreactive) character of the great majority of FB-positive neurons which preferentially co-expressed neuropeptide Y. In addition, none of the FB-positive perikarya was immunopositive to galanin, somatostatin, choline acetyltransferase, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, leu(5)-enkephalin, nitric oxide synthase, substance P and calcitonin-generelated peptide.

Vulnerability of peripheral catecholaminergic neurons to MPTP is not regulated by α-synuclein

Although generally considered a prototypical movement disorder, Parkinson's disease is commonly associated with a broad-spectrum of non-motor symptoms, including autonomic dysfunctions caused by significant alterations in catecholaminergic neurons of the peripheral sympathetic nervous system. Here we present evidence that α-synuclein is highly expressed by sympathetic ganglion neurons throughout embryonic and postnatal life and that it is found in tyrosine hydroxylase-positive sympathetic fibers innervating the heart of adult mice. However, mice deficient in α-synuclein do not exhibit any apparent alterations in sympathetic development. Sympathetic neurons isolated from mouse embryos and early postnatal mice are sensitive to the parkinsonian drug MPTP/MPP + and intoxication requires entry of the neurotoxin through the noradrenaline transporter. Furthermore, recovery of noradrenaline from cardiac sympathetic fibers is reduced in adult mice treated with MPTP systemically. However, MPP +-induced sympathetic neuron loss in vitro or MPTP-induced cardiac noradrenaline depletion in vivo is not modified in mice lacking α-synuclein. This is in clear contrast with the observation that dopaminergic neurons of the central nervous system are significantly less vulnerable to MPTP/MPP + in the absence of α-synuclein, suggesting different actions of this molecule in central and peripheral catecholaminergic neurons.

Phenotyping of Sympathetic Chain Ganglia (SChG) Neurons in Porcine Colitis

Colonic inflammation involves the changes in chemical coding of not only sensory but also enteric and sympathetic prevertebral neurons innervating inflamed target tissue. Since the sympathetic chain ganglia neurons (SChG) affect colonic function, the purpose of our study was to determine the precise location of the SChG neurons innervating the porcine descending colon, define their chemical coding and answer the question of whether paraformaldehyde-induced colitis in female pigs is able to change the chemical phenotype of the perikarya. Using the Fast Blue (FB) retrograde tracing, the greatest concentration of the (FB+) perikarya was revealed in L₃ ganglia. Applying double immunohistochemistry, we detected a strongly catecholaminergic character and in majority neuropeptide Y (NPY)-positive staining of the colon-projecting FB+ neurons both in the control and the inflamed animals. Furthermore, colonic inflammation caused the significant increase in density of galanin- (P<0.001), somatostatin- (P<0.005), leu5-enkephalin- (P<0.05), substance P- (P<0.005), and calcitonin gene-related peptide- (P<0.005) immunoreactive (-IR) nerve fibers surrounding the FB+ perikarya, while the density of nitric oxide synthase-IR and NPY-IR fibers remained unaffected compared to the control animals. The increase in visible immunofluorescent detection of neuropeptide-containing fibers could possibly be caused by enhanced demand and release of neuropeptides that modulate intestinal immune response and regulate the gastrointestinal activity through inflammatory mediators.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers

Superoxide anion (O 2 −•) production was previously reported to be increased in celiac ganglia (CG) during DOCA–salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons are not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22 phox and neuropeptide Y (NPY) were colocalized in all neurons. P22 phox was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22 phox and p47 phox were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22 phox and p47 phox was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O 2 −•-generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control.

Augmentation of Cav1 channel current and action potential duration after uptake of S100A1 in sympathetic ganglion neurons

S100A1, a 21-kDa dimeric Ca2+-binding protein of the EF-hand type, is expressed in cardiomyocytes and is an important regulator of heart function. During ischemia, cardiomyocytes secrete S100A1 to the extracellular space. Although the effects of extracellular S100A1 have been documented in cardiomyocytes, it is unclear whether S100A1 exerts modulatory effects on other tissues in proximity with cardiac cells. Therefore, we sought to investigate the effects of exogenous S100A1 on Ca2+ signals and electrical properties of superior cervical ganglion (SCG) neurons. Immunostaining and Western blot assays indicated no endogenous S100A1 in SCG neurons. Cultured SCG neurons took up S100A1 when it was present in the extracellular medium. Inside the cell exogenous S100A1 localized in a punctate pattern throughout the cytoplasm but was excluded from the nuclei. S100A1 partially colocalized with markers for both receptor- and non-receptor-mediated endocytosis, indicating that in SCG neurons multiple endocytotic pathways are involved in S100A1 internalization. In compartmentalized SCG cultures, axonal projections were capable of uptake and transport of S100A1 toward the neuronal somas. Exogenous S100A1 applied either extra- or intracellularly enhanced Cav1 channel currents in a PKA-dependent manner, prolonged action potentials, and amplified action potential-induced Ca2+ transients. NMR chemical shift perturbation of Ca2+-S100A1 in the presence of a peptide from the regulatory subunit of PKA verifies that S100A1 directly interacts with PKA, and that this interaction likely occurs in the hydrophobic binding pocket of Ca2+-S100A1. Our results suggest the hypothesis that in sympathetic neurons exogenous S100A1 may lead to an increase of sympathetic output.

S1.5 Sympathetic and parasympathetic adult rat pelvic ganglion neurons utilize distinct cyclic nucleotide signaling pathways to mediate Semaphorin 3A-induced growth cone collapse

After nerve injury, appropriate target reconnection is required for successful functional regeneration, but this can be limited by aberrant reinnervation of inappropriate targets. These processes may recapitulate developmental events during which both attractive and repulsive guidance factors determine axonal targeting. Semaphorins constitute a major family of guidance molecules that typically signal to repel axons from inappropriate targets. Semaphorin 3A (Sema3A) has been shown to guide many sensory projections, and has been linked to motor and autonomic nerve pathfinding in development.

A case of primary erythermalgia with encephalopathy

Sirs, Primary erythermalgia is an autosomal dominant disorder characterized by burning pain in the limbs [10]. Encephalopathic symptoms associated with primary erythermalgia have previously been reported but are not well understood [9]. A 19-year-old woman has been followed for primary erythermalgia from childhood. Her sister had a similar phenotype and died as a result of uncontrolled sepsis. Her father was also affected, but less severely. A mutation in SCN9A has been reported in this family [3] (family 4, exon 5, c.647T [ C, p.F216S). Her symptoms, which have become more intense over time, could not be relieved by sympathectomy, lidocaine injection, or morphine, but they improved moderately when treated with oral mexiletine combined with ice applied to her legs. At the end of 2007, the mexiletine was replaced by a low dose of clonazepam and paracetamol because of continuing discomfort. Two months later, she became drowsy and mentally confused, presenting with auditory and visual hallucinations, deep psychomotor slowdown, and cerebellar ataxia without focal deficit or pyramidal syndrome. Hypothermia (31 C), hypoglycemia (3.65 mmol l), and hypercalcemia (2.66 mmol l) were observed. Despite our advice, she continued to use ice to reduce the pain. A cerebrospinal fluid analysis and the cerebral magnetic resonance imaging (MRI) examination did not reveal any abnormalities. However, the electroencephalography (EEG) showed aspecific encephalopathic features. Following admission to hospital, the patient was treated with a combination of risperidone, clonazepam, oral steroid, and mexiletine, which improved the erythermalgia, encephalopathic symptoms, and laboratory anomalies. The hypothermia disappeared. The combined drug treatment was stopped within a few days, but mexiletine therapy was continued. A clue to the pathogenesis of primary erythermalgia was first reported by Yang et al. [11] and then confirmed by Drenth et al. [3], both of whom identified mutations in SCN9A, which encodes the Nav1.7 sodium channel. These mutations, such as p.F216S [1], cause a hyperpolarizing shift in the voltage dependence of channel activation, which allows the channel to be activated by smaller than normal depolarizations. In humans, Nav1.7 channels are selectively expressed in the peripheral nervous system within the dorsal root ganglion and sympathetic ganglion neurons [4]. The hyperexcitability produced by these SCN9A mutations appears to account, at least in part, for the pain experienced by patients [4]. However, our patient also presented symptoms of encephalopathy, hypothermia, and hypoglycemia. To date, the pathomechanism of this erythermalgiaassociated encephalopathy remains obscure. Two cases of primary erythermalgia associated with encephalopathy have been reported previously [6, 9]. One of these [9] was very similar to ours: a 15-year-old boy who developed hypothermia, somnolence, and ataxic gait. However, in this case, the brain MRI revealed increased signal intensity in the splenium of the corpus callosum, which is in contrast to our normal MRI findings, although the EEG was very J. Seneschal A. Taieb Department of Dermatology and Pediatric Dermatology, Saint Andre and Pellegrin Hospitals, Bordeaux University Hospitals, 1 Rue Jean Burguet, 33000 Bordeaux, France e-mail: julien.seneschal@free.fr

Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels "tunes" heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the generation and survival of various DRG neuron classes, in particular proprioceptors. Its importance for peripheral projections and central connectivity of proprioceptors demonstrates the significance of NT signalling for integrating responsive neurons in neural networks. The molecular targets of NT3 signalling in proprioceptor differentiation remain to be characterized. In sympathetic ganglia, NGF signalling regulates dendritic development and axonal projections. Its role in the specification of other neuronal properties is less well analysed. In vitro analysis suggests the involvement of NT signalling in the choice between the noradrenergic and cholinergic transmitter phenotype, in the expression of various classes of ion channels and for target connectivity. In vivo analysis is required to show the degree to which NT signalling regulates these sympathetic neuron properties in developing embryos and postnatally.

Early- and late-onset inherited erythromelalgia: genotype–phenotype correlation

Inherited erythromelalgia (IEM), an autosomal dominant disorder characterized by severe burning pain in response to mild warmth, has been shown to be caused by gain-of-function mutations of sodium channel Na(v)1.7 which is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Almost all physiologically characterized cases of IEM have been associated with onset in early childhood. Here, we report the voltage-clamp and current-clamp analysis of a new Na(v)1.7 mutation, Q10R, in a patient with clinical onset of erythromelalgia in the second decade. We show that the mutation in this patient hyperpolarizes activation by only -5.3 mV, a smaller shift than seen with early-onset erythromelalgia mutations, but similar to that of I136V, another mutation that is linked to delayed-onset IEM. Using current-clamp, we show that the expression of Q10R induces hyperexcitability in DRG neurons, but produces an increase in excitability that is smaller than the change produced by I848T, an early-onset erythromelalgia mutation. Our analysis suggests a genotype-phenotype relationship at three levels (clinical, cellular and molecular/ion channel), with mutations that produce smaller effects on sodium channel activation being associated with a smaller degree of DRG neuron excitability and later onset of clinical signs.

Uptake Of S100A1 And Augmentation Of Cav1 Channel Current And Action Potential Duration In Sympathetic Ganglion Neurons

S100A1, a ∼20 kD dimeric Ca2+-binding protein of the EF-hand type, is highly expressed in cardiomyocytes and is considered an important regulator of heart function. During ischemia, cardiomyocytes release S100A1 to the extracellular space. Although the effects of extracellular S100A1 have been documented in cardiomyocytes, it is unclear whether S100A1 exerts modulatory effects on other tissues in close proximity with cardiac cells. Neurons from the cervical ganglion chain extend postgaglionic axons that innervate the heart. Therefore, we sought to investigate the effects of exogenous S100A1 on Ca2+ signals and electrical properties of principal sympathetic ganglion neurons (SGNs) from the superior cervical ganglion. Immunostaining and western blot analysis did not detected any endogenous S100A1 in SGNs from normal mice. Cultured adult SGNs took up fluorescent S100A1 when it was present in the extracellular media. Inside the cell exogenous fluorescent S100A1 localized primarily in a punctuate pattern throughout the cytoplasm and axoplasm, but was excluded from the nuclei. Time lapse imaging and FRAP experiments reveal axonal transport of S100A1 puncta, presumably endosomes. In compartmentalized (Campenot) SGN cultures, axonal projections were capable of uptake and transport of S100A1 towards the neuronal somas. Exogenous S100A1 enhanced Cav1 channel currents, increased the amplitude of action potential-evoked Ca2+ transients and prolonged the action potentials. Our results, showing enhanced somatic Ca2+ entry, larger cytosolic Ca2+ transients, and prolongation of action potential duration, suggest the hypothesis that S100A1 released from heart muscle cells may be taken up by sympathetic neurons, leading to an increase of sympathetic output to the heart. Supported by NIH, Grants R01-N5042839, R01-AR055099.

NADPH-diaphorase-positive neurons in sympathetic ganglia during postnatal ontogenesis

The locations and morphometric characteristics of NADPH-diaphorase (NADPH-d)-positive neurons were identified in the cranial cervical (CCG), stellate (SG), and celiac (CG) ganglia in neonatal rats, mice, and cats and animals aged 10, 20, 30, 60, and 180 days. No NADPH-d-positive neurons were found in rats and mice in any of the age groups studied. In kittens, the majority of NADPH-d-positive neurons were located in the SG, with fewer in the CCG and only occasional neurons in the CG, regardless of age. The proportion of NADPH-d-positive neurons in the SG increased during the first 20 days of life and decreased after 30 days, to the end of the second month of life. The proportion of NADPH-d-reactive neurons in the CCG and CG did not change during ontogenesis. The mean sizes of NADPH-d-positive neurons in different ganglia in animals of the same age were not significantly different. These data lead to the conclusion that the development of NADPH-d-positive neurons with age occurs heterochronously and is complete by the end of the second month of life.

Neuritic dystrophy and neuronopathy in Akita ( Ins2Akita) diabetic mouse sympathetic ganglia

Diabetic autonomic neuropathy is a debilitating, poorly studied complication of diabetes. Our previous studies of non-obese diabetic (NOD) and related mouse models identified rapidly developing, dramatic pathology in prevertebral sympathetic ganglia; however, once diabetic, the mice did not survive for extended periods needed to examine the ability of therapeutic agents to correct established neuropathy. In the current manuscript we show that the Akita ( Ins2 Akita ) mouse is a robust model of diabetic sympathetic autonomic neuropathy with unambiguous, spontaneous, rapidly-developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. Akita mice diabetic for 2, 4 or 8 months of diabetes progressively developed markedly swollen axons and dendrites (“neuritic dystrophy”) in the prevertebral superior mesenteric (SMG) and celiac ganglia (CG). Comparable changes failed to develop in the superior cervical ganglia (SCG) of the Akita mouse or in any ganglia of non-diabetic mice. Morphometric studies demonstrate an overall increase in presynaptic axon terminal cross sectional area, including those without any ultrastructural features of dystrophy. Neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum. These changes result in the loss of a third of neurons in the CG over the course of 8 months of diabetes. The extended survival of diabetic mice and robust pathologic findings provide a clinically relevant paradigm that will facilitate the analysis of novel therapeutic agents on the reversal of autonomic neuropathy.

Asymmetric post-natal development of superior cervical ganglion of paca (Agouti paca)

Functional asymmetry has been reported in sympathetic ganglia. Although there are few studies reporting on body side-related morphoquantitative changes in sympathetic ganglion neurons, none of them have used design-based stereological methods to address this issue during post-natal development. We therefore aimed at detecting possible asymmetry-related effects on the quantitative structure of the superior cervical ganglion (SCG) from pacas during ageing, using very precise design-based stereological methods. Forty (twenty left and twenty right) SCG from twenty male pacas were studied at four different ages, i.e. newborn, young, adult and aged animals. By using design-based stereological methods the total volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated, using the vertical nucleator. The main findings of this study were: (1) the right SCG from aged pacas has more mononucleate and binucleate neurons than the left SCG in all other combinations of body side and animal age, showing the effect of the interaction between asymmetry (right side) and animal age, and (2) right SCG neurons (mono and binucleate) are bigger than the left SCG neurons (mono and binucleate), irrespective of the animal age. This shows, therefore, the exclusive effect of asymmetry (right side). At the time of writing there is still no conclusive explanation for some SCG quantitative changes exclusively assigned to asymmetry (right side) and those assigned to the interaction between asymmetry (right side) and senescence in pacas. We therefore suggest that forthcoming studies should focus on the functional consequences of SCG structural asymmetry during post-natal development. Another interesting investigation would be to examine the interaction between ganglia and their innervation targets using anterograde and retrograde neurotracers. Would differences in the size of target organs explain ganglia structural asymmetry?

Loss of AKAP150 perturbs distinct neuronal processes in mice

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate context-dependent neuronal signaling events in vivo.

Nuclear Factor κB Signaling Either Stimulates or Inhibits Neurite Growth Depending on the Phosphorylation Status of p65/RelA

Nuclear factor kappaB (NF-kappaB) signaling is known to promote neurite growth from developing sensory neurons and to enhance the size and complexity of pyramidal neuron dendritic arbors in the developing cerebral cortex. In marked contrast, here we show that NF-kappaB signaling can also exert a potent inhibitory influence on neurite growth in certain neurons, and can either promote or inhibit neurite growth in the same neurons depending on the mechanism of NF-kappaB activation. In neonatal superior cervical ganglion sympathetic neurons, enhancing NF-kappaB transcriptional activity by overexpressing either the p65 NF-kappaB subunit or the IkappaB kinase-beta (IKKbeta) subunit of the IkappaB kinase complex, or by tumor necrosis factor alpha (TNFalpha) treatment, strongly inhibits neurite growth. Paradoxically in neonatal nodose ganglion sensory neurons, enhancing NF-kappaB transcriptional activity by p65/p50 overexpression increases neurite growth, whereas enhancing NF-kappaB transcriptional activity by IKKbeta overexpression inhibits neurite growth. In addition to activating NF-kappaB, IKKbeta overexpression leads to phosphorylation of p65 on serine 536. Blockade of serine 536 phosphorylation by a S536A-p65 mutant protein prevents the growth-inhibitory effects of IKKbeta overexpression in both sensory and sympathetic neurons and the growth-inhibitory effects of TNFalpha on sympathetic neurons. Furthermore, expression of a p65 S536D phosphomimetic mutant inhibits neurite growth from sensory neurons. These results demonstrate that NF-kappaB can either stimulate or inhibit neurite growth in developing neurons depending on the phosphorylation status of p65.

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.

Development of the NADPH-diaphorase-positive neurons in the sympathetic ganglia

Changes in the distribution of NADPH-diaphorase (NADPH-d) were studied in neurons of the superior cervical ganglion (SCG), stellate ganglion (SG) and celiac ganglia (CG) in newborn, 10-, 20-day-old, 1-month-old, 2-month-old and 6-month-old rats, mice and kittens. NADPH-d-positive neurons were revealed in all sympathetic ganglia in kittens but not in rodents from birth onwards. In kittens, the largest population of NADPH-d-positive cells was found in the SG, the smallest in the SCG (<1%) and we observed only a few cells in the CG. The proportion of NADPH-d-positive cells in the SG increased from 3.1 +/- 0.15% in newborn kittens to 9.3 +/- 0.63% in 20-day-old animals and decreased further from 8.1 +/- 0.75% in 30-day-old kittens to 3.4 +/- 0.54% in 2-month-old animals. The content of NADPH-d-positive cells in the CG and SCG did not change during development. There were no differences in cross-sectional area between neurons located in different ganglia of animals from the same age group under study. We conclude that the development of NADPH-d-positive neurons in different sympathetic ganglia has its own time dynamics and is completed by the end of the second month of life.

Loss of the Prader‐Willi syndrome protein necdin causes defective migration, axonal outgrowth, and survival of embryonic sympathetic neurons

Prader-Willi syndrome is a neurodevelopmental disorder marked by abnormalities in feeding, drinking, thermoregulation, intestinal motility, and reproduction, suggesting disruption of the autonomic nervous system. Necdin, one of several proteins genetically inactivated in individuals with Prader-Willi syndrome, is important for the differentiation of central and sensory neurons. We now show that formation, migration, and survival of sympathetic superior cervical ganglion neurons are impaired in Ndn-null embryos. We observed reduced innervation of superior cervical ganglion target organs, including the submandibular gland, parotid gland, and nasal mucosa. While the formation of other sympathetic chain ganglia is unaffected, axonal extension is impaired throughout the sympathetic nervous system. These results demonstrate a novel role for necdin in cellular migration, in addition to its roles in survival and axon outgrowth. Furthermore, reduced sympathetic function provides a plausible explanation for deficiencies of salivary gland function in individuals with congenital necdin deficiency consequent to Prader-Willi syndrome.

Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive Gα Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.