Leptin stimulates sympathetic axon outgrowth.

The neurohormone leptin regulates energy homeostasis. Circulating levels of leptin secreted by adipose tissue act on hypothalamic neurons in the brain leading to decreased appetite and increased energy expenditure. Although leptin signaling in the central nervous system (CNS) is fundamental to its ability to regulate the body's metabolic balance, leptin also has a variety of effects in many peripheral tissues including the heart, the liver, and the sympathetic nervous system. Leptin stimulation of the hypothalamus can stimulate glucose uptake via the sympathetic nervous system in heart, muscle, and brown adipose tissue. Leptin receptors (Ob-Rb) are also expressed by peripheral sympathetic neurons, but their functional role is not clear. In this study, we found that leptin stimulates axonal growth of both adult and neonatal sympathetic neurons in vitro. Leptin stimulates acute activation of the transcription factor STAT3 via phosphorylation of tyrosine 705. STAT3 phosphorylation is required for leptin-stimulated sympathetic axon outgrowth. Thus, circulating levels of leptin may enhance sympathetic nerve innervation of peripheral tissues.

Exocytosis and mobility of histamine vesicles exhibit no difference to that of histamine-negative vesicles in guinea pig superior cervical ganglion neurons.

Our previous studies have shown that histamine existed widely in the sympathetic nervous system and functioned differentially on the sympathetic nerve activation level. Therefore, in this study, we tried to find out whether it is the special exocytosis/recycling of histamine-containing vesicles that contribute to those differential histamine synaptic effects. By using N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide and histamine immunostaining methods, we confirmed that histamine was stored in small vesicles and found that the histamine-containing vesicles included the recycling pool and the reserve pool. However, we also found for the first time that the release and mobility kinetics of histamine-containing vesicles were identical to that of histamine-negative vesicles. In conclusion, these findings provide further characters of histamine as a sympathetic neurotransmitter.

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus.

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

Antibody-mediated impairment and homeostatic plasticity of autonomic ganglionic synaptic transmission.

Antibodies against ganglionic acetylcholine receptors (AChR) are implicated as the cause of autoimmune autonomic ganglionopathy (AAG). To characterize ganglionic neurotransmission in an animal model of AAG, evoked and spontaneous excitatory post-synaptic potentials (EPSP) were recorded from neurons in isolated mouse superior cervical ganglia (SCG). In vitro exposure of ganglia to IgG from AAG patients progressively inhibited synaptic transmission. After passive transfer of antibody to mice, evoked EPSP amplitude decreased, and some neurons showed no synaptic responses. EPSP amplitude recovered by day 7 despite persistence of ganglionic AChR antibody in the mouse serum. There was a more persistent (at least 14-day) reduction in miniature EPSP amplitude consistent with antibody-mediated reduction in post-synaptic AChR. Although the quantal size was reduced, a progressive increase in the frequency of spontaneous synaptic events occurred, suggesting a compensatory increase in presynaptic efficacy. The quantal size returned to baseline by 21 days while the frequency remained increased for at least four weeks. Ganglionic AChR antibodies cause an impairment of autonomic ganglionic synaptic transmission. Homeostatic plasticity in autonomic neurotransmission could help explain the spontaneous clinical recovery seen in some AAG patients and may also play an important role in regulating normal autonomic reflexes.

Subcellular localization of the antidepressant-sensitive norepinephrine transporter.

BACKGROUND: Reuptake of synaptic norepinephrine (NE) via the antidepressant-sensitive NE transporter (NET) supports efficient noradrenergic signaling and presynaptic NE homeostasis. Limited, and somewhat contradictory, information currently describes the axonal transport and localization of NET in neurons. RESULTS: We elucidate NET localization in brain and superior cervical ganglion (SCG) neurons, aided by a new NET monoclonal antibody, subcellular immunoisolation techniques and quantitative immunofluorescence approaches. We present evidence that axonal NET extensively colocalizes with syntaxin 1A, and to a limited degree with SCAMP2 and synaptophysin. Intracellular NET in SCG axons and boutons also quantitatively segregates from the vesicular monoamine transporter 2 (VMAT2), findings corroborated by organelle isolation studies. At the surface of SCG boutons, NET resides in both lipid raft and non-lipid raft subdomains and colocalizes with syntaxin 1A. CONCLUSION: Our findings support the hypothesis that SCG NET is segregated prior to transport from the cell body from proteins comprising large dense core vesicles. Once localized to presynaptic boutons, NET does not recycle via VMAT2-positive, small dense core vesicles. Finally, once NET reaches presynaptic plasma membranes, the transporter localizes to syntaxin 1A-rich plasma membrane domains, with a portion found in cholera toxin-demarcated lipid rafts. Our findings indicate that activity-dependent insertion of NET into the SCG plasma membrane derives from vesicles distinct from those that deliver NE. Moreover, NET is localized in presynaptic membranes in a manner that can take advantage of regulatory processes targeting lipid raft subdomains.

Silent synapses sit and wait for a better day.

Synaptic activity is thought to be critical for synaptic stabilization. In this issue of Neuron, Krishnaswamy and Cooper show that nicotinic synapses on autonomic neurons remain intact without synaptic activity. Postsynaptic responses are required, however, for presynaptic terminals to acquire the high-affinity choline transporter necessary for high-frequency transmission.

Hybrid synapse formation between spinal motoneurons and superior cervical ganglion neurons in vitro: a study of the functional reconstruction of visceral organs.

Spinal motoneurons (SMNs) were co-cultured with labeled superior cervical ganglion neurons (SCGNs) in complete medium on coverslips prepared by the microisland technique. "Hybrid" synapses formation between SMNs and SCGNs was examined by double immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), and electrophysiological assays. The number of synaptic puncta was significantly higher in co-cultured cells compared to controls (P<0.01). Spontaneous synaptic currents (SSCs) were also significantly increased in SCGNs within the co-cultured population of cells (P<0.01). These SSCs could be blocked by a specific nicotinic receptor blocker, but not by a glutamate receptor antagonist. These observations clearly demonstrate the functional synapses formation increased in our co-culture system, suggesting that the "hybrid" synapse formation occurred between somatic and automatic neurons. Our data also indicate that acetylcholine and nicotinic receptors may be involved in mediating these processes.

High-frequency stimuli preferentially release large dense-core vesicles located in the proximity of nonspecialized zones of the presynaptic membrane in sympathetic ganglia.

We characterized the effect of a brief high-frequency stimulus on the number, distribution, and optical density of large dense-core vesicles (LDCVs) in the nerve terminals of the rat superior cervical ganglia. From 4.21+/-0.37 LDCVs/bouton detected in control nerve terminals, a stimulus of 40 Hz for 1 min released 41% of LDCVs, decreasing their number to 2.48+/-0.14 LDCVs/bouton (p=0.0009). In control ganglia, most dense vesicles were located close to the plasma membrane (at

Calpain-mediated cleavage of collapsin response mediator protein(CRMP)-2 during neurite degeneration in mice.

Axon or dendrite degeneration involves activation of the ubiquitin-proteasome system, failure to maintain neuritic ATP levels, microtubule fragmentation and a mitochondrial permeability transition that occur independently of the somal death programs. To gain further insight into the neurite degeneration mechanims we have compared two-dimensional gel electrophoresis patterns of neurite proteins from suprior cervical ganglia during degeneration caused by nerve growth factor (NGF) deprivation. We show here that collapsin response mediator protein (CRMP)-2 and CMRP-4 protein patterns were altered during beading formation, an early hallmark of neurite degeneration, prior to neurite fragmentation, the final stage of degeneration. Western blotting using a monoclonal antibody against CRMP-2 shows that the native form (64 kDa) was cleaved to generate a truncated form (58 kDa). No cleavage of CRMP-2 or -4 occurred in NGF-deprived neurites from Wld(s) (Wallerian degeneration slow) mutant mice in which neurite degeneration is markedly delayed. Using different protease inhibitors, purified calpain 1 protein and calpain 1-specific siRNA, we have demonstrated that CRMP-2 is a substrate for calpain 1. Indeed, caplain activity was activated at an early phase of neuronal degeneration in cerebellar granule neurons, and down-regulation of caplain 1 expression suppressed CRMP-2 cleavage. Furthermore, this cleavage occurred after vinblastine treatment or in vitro Wallerian degeneration, suggesting that it represents a common step in the process of dying neurites. CRMP-2 and -4 play a pivotal role in axonal growth and transport, and the C-terminus region of CRMP-2 is essential for its binding to kinesin-1. Hence, this cleavage will render them dysfunctional and subject to autophagic processing associated with beading formation, as evidenced by the finding that the truncated form was localized in the beadings.

Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis.

West Nile virus (WNV) has emerged as a significant cause of epidemic viral encephalitis and flaccid limb paralysis, yet the mechanism by which it enters the CNS remains uncertain. We used compartmentalized neuron cultures to demonstrate that WNV spreads in both retrograde and anterograde directions via axonal transport. Transneuronal spread of WNV required axonal release of viral particles and was blocked by addition of a therapeutic neutralizing antibody. To test the physiologic significance of axonal transport in vivo, we directly inoculated the sciatic nerve of hamsters with WNV. Intrasciatic infection resulted in paralysis of the hind limb ipsilateral but not contralateral to the injection site. Limb paralysis was blocked either by surgical transection of the sciatic nerve or treatment with the therapeutic neutralizing antibody. Collectively, these studies establish that WNV undergoes bidirectional spread in neurons and that axonal transport promotes viral entry into the CNS and acute limb paralysis. Moreover, antibody therapeutics directly inhibit transneuronal spread of WNV infection and prevent the development of paralysis in vivo.

Wide distribution and subcellular localization of histamine in sympathetic nervous systems of different species.

Previous studies have demonstrated that histamine (HA) acts as a neurotransmitter in the cardiac sympathetic nervous system of the guinea pig. The aim of the current study was to examine whether HA widely exists in the sympathetic nervous systems of other species and the subcellular localization of HA in sympathetic terminals. An immunofluorescence histochemical multiple-staining technique and anterograde tracing method were employed to visualize the colocalization of HA and norepinephrine (NE) in sympathetic ganglion and nerve fibers in different species. Pre-embedding immunoelectron microscopy was used to observe the subcellular distribution of HA in sympathetic nerve terminals. Under the confocal microscope, coexistence of NE and HA was displayed in the superior cervical ganglion and celiac ganglion neurons of the mouse and dog as well as in the vas deferens, mesenteric artery axon, and varicosities of the mouse and guinea pig. Furthermore, colocalization of NE and HA in cardiac sympathetic axons and varicosities was labeled by biotinylated dextranamine injected into the superior cervical ganglion of the guinea pig. By electron microscopy, HA-like high-density immunoreactive products were seen in the small vesicles of the guinea pig vas deferens. These results provide direct cellular and subcellular morphological evidence for the colocalization of HA and NE in sympathetic ganglion and nerve fibers, and support that HA is classified as a neurotransmitter in sympathetic neurons.

Scanning electron microscopic observations on the third ventricular floor of the rat following cervical sympathectomy.

Various investigators have shown that unilateral ganglionectomy or transection of the internal and external carotid nerves leads to a regenerative response in the ipsilateral superior cervical ganglion and to uninjured mature sympathetic neurons sprouting into bilaterally innervated shared target organs. In this study changes in the supraependymal neuronal network following unilateral and bilateral cervical sympathectomy on the infundibular floor of the third ventricle were studied by scanning electron microscopy in comparison with normal and sham-operated control animals. After unilateral cervical sympathectomy there was a great increase in the number of varicose nerve fibres on the infundibular floor as compared to the normal and sham-operated control animals. Not only was there an increase in the number of nerve fibres, but also their varicosities were substantially larger than those normally present on the ependymal surface. This study indicates the possible sympathetic projections from the superior cervical ganglia to the ependymal surface of the third cerebral ventricle.

Macro- and microstructure of the superior cervical ganglion in dogs, cats and horses during maturation.

The superior cervical ganglion (SCG) provides sympathetic input to the head and neck, its relation with mandible, submandibular glands, eyes (second and third order control) and pineal gland being demonstrated in laboratory animals. In addition, the SCG's role in some neuropathies can be clearly seen in Horner's syndrome. In spite of several studies published involving rats and mice, there is little morphological descriptive and comparative data of SCG from large mammals. Thus, we investigated the SCG's macro- and microstructural organization in medium (dogs and cats) and large animals (horses) during a very specific period of the post-natal development, namely maturation (from young to adults). The SCG of dogs, cats and horses were spindle shaped and located deeply into the bifurcation of the common carotid artery, close to the distal vagus ganglion and more related to the internal carotid artery in dogs and horses, and to the occipital artery in cats. As to macromorphometrical data, that is ganglion length, there was a 23.6% increase from young to adult dogs, a 1.8% increase from young to adult cats and finally a 34% increase from young to adult horses. Histologically, the SCG's microstructure was quite similar between young and adult animals and among the 3 species. The SCG was divided into distinct compartments (ganglion units) by capsular septa of connective tissue. Inside each ganglion unit the most prominent cellular elements were ganglion neurons, glial cells and small intensely fluorescent cells, comprising the ganglion's morphological triad. Given this morphological arrangement, that is a summation of all ganglion units, SCG from dogs, cats and horses are better characterized as a ganglion complex rather than following the classical ganglion concept. During maturation (from young to adults) there was a 32.7% increase in the SCG's connective capsule in dogs, a 25.8% increase in cats and a 33.2% increase in horses. There was an age-related increase in the neuronal profile size in the SCG from young to adult animals, that is a 1.6-fold, 1.9-fold and 1.6-fold increase in dogs, cats and horses, respectively. On the other hand, there was an age-related decrease in the nuclear profile size of SCG neurons from young to adult animals (0.9-fold, 0.7-fold and 0.8-fold in dogs, cats and horses, respectively). Ganglion connective capsule is composed of 2 or 3 layers of collagen fibres in juxtaposition and, as observed in light microscopy and independently of the animal's age, ganglion neurons were organised in ganglionic units containing the same morphological triad seen in light microscopy.

Size and number of binucleate and mononucleate superior cervical ganglion neurons in young capybaras.

The total number of neurons in the superior cervical ganglion (SCG) of adult capybaras is known from a previous study, where a marked occurrence of binucleate neurons (13%) was also noted. Here, distribution, number and fate of binucleate neurons were examined in younger, developing capybaras, aged 3 months. The mean neuronal cross-sectional area was 575.2 microm2 for mononucleate neurons and 806.8 microm2 in binucleate neurons. Frequency of binucleate neurons was about 36%. The mean ganglion volume was about 190 mm3 in young capybaras and the mean neuronal density was about 9,517 neurons/mm3. The total number of neurons per ganglion was about 1.81 mill. Neuronal cell bodies constituted 22.5% of the ganglion volume and the average neuronal volume was 23,600 microm3. By comparing the present data with those previously published the conclusion is drawn that the maturation period was characterized by the following points: a 26% remarkable decrease in neuronal density which was significant (P < 0.05) and a significant 16% (P < 0.05) decrease in the total number of SCG neurons accompanied by a 23% decrease in the total number of SCG binucleate neurons.

The spermatogenic Ig superfamily/synaptic cell adhesion molecule mast-cell adhesion molecule promotes interaction with nerves.

Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. The molecules that sustain this association have not been identified. Because synaptic cell adhesion molecule (SynCAM), alternatively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be a candidate. To examine this possibility, mast cells with or without SgIGSF/SynCAM were cocultured with superior cervical ganglion neurons that express SgIGSF/SynCAM, and the number of mast cells attached to neurites was counted. The attachment of mast cells with SgIGSF/SynCAM, i.e., bone marrow-derived mast cells (BMMC) from wild-type mice, was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/SynCAM, i.e., BMMC from microphthalmia transcription factor-deficient mice and BMMC-derived cell line IC-2 cells, were defective in attachment to neurite, and transfection with SgIGSF/SynCAM normalized this. When the nerves were specifically activated by scorpion venom, one-quarter of the attached IC-2 cells mobilized Ca(2+) after a few dozen seconds, and ectopic SgIGSF/SynCAM doubled this proportion. At points of contact between neurites and wild-type BMMC, SgIGSF/SynCAM was locally concentrated in both neurites and BMMC. SgIGSF/SynCAM on mast cells appeared to predominantly mediate attachment and promote communication with nerves.

Sympathetic innervation of the tongue in rats.

We employed a glyoxylic catecholaminergic histofluorescence method to study the sympathetic innervation present in the rat tongue. One percent neutral red was used as a counterstain. Many noradrenergic fibers were demonstrated around the blood vessels, muscles, glands and submucosa of the tongue, but not in the epithelium or papilla. In a group of rats following neurectomy, the superior cervical ganglia (SCG) were removed unilaterally or bilaterally. Changes in sympathetic innervation of the tongue were examined 14 days after SCG ganglionectomy. In those animals after unilateral SCG ganglionectomy, we found no noradrenergic histofluorescence in the ipsilateral anterior 2/3 of the tongue, although some scant fluorescence was found in the ipsilateral posterior 1/3 of the tongue. However, no noradrenergic histofluorescence could be observed in animal's bilateral SCG ganglionectomies. Our results indicate that sympathetic innervation of the tongue in rats originates in the SCG, with some cross-innervation of the sympathetic fibers occurring in the posterior 1/3 of the tongue.

Nicotinic acetylcholine receptor subtypes in the rat sympathetic ganglion: pharmacological characterization, subcellular distribution and effect of pre- and postganglionic nerve crush.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in autonomic ganglia, which innervate and control the activity of most visceral organs. By combining ultrastructural, immunocytochemical, and pharmacological analyses, we characterized the nAChR subtypes in the rat superior cervical ganglion (SCG) and the effect of pre- and postganglionic nerve crush on their number in the ganglion and their distribution at the intraganglionic synapses. Binding with radioactive nicotinic ligands, immunoprecipitation, and immunolocalization experiments revealed the presence of different nAChR subtypes: those containing the alpha3 subunit associated with beta4 and/or beta2 subunits that bind 3H-Epibatidine with high affinity, and those containing the alpha7 subunit that bind 125I-alphaBungarotoxin. After postganglionic nerve crush, the number of nicotinic receptors and immunopositive intraganglionic synapses for each nAChR subunit strongly decreased. Both the number of nAChRs and immunoreactivity recovered 26 days after injury, when regenerating postganglionic fibers had reinnervated the peripheral target organs, as shown by the restoration of tyrosine hydroxylase immunoreactivity in the iris. This observation and the lack of any effect of preganglionic nerve crush on the number of nicotinic receptors suggest that the peripheral targets affect the organization of intraganglionic synapses in adult SCG.

Expression of basic fibroblast growth factor isoforms in postmitotic sympathetic neurons: synthesis, intracellular localization and involvement in karyokinesis.

Low and high molecular weight isoforms of the mitogen and multifunctional cytokine basic fibroblast growth factor (FGF-2) are up-regulated in neurons and glial cells in response to peripheral nerve lesion. While synthesis, regulation and functions of FGF-2 in non-neuronal cells are well established, the significance of neuronal FGF-2 remains to be investigated in the peripheral nervous system. Therefore, the expression, intracellular localization and possible effects of FGF-2 isoforms were analyzed in primary sympathetic neurons derived from the rat superior cervical ganglion. FGF-2 is detected in the nucleus and in perinuclear Golgi fields of early postnatal neurons which also express mRNA and protein for the FGF receptor type 1. Biolistic transfection of plasmids encoding FGF-2 isoforms fused to fluorescent proteins demonstrates nuclear targeting of 18 kDa FGF-2 and 23 kDa FGF-2 with prominent accumulation in the nucleolus of neurons. Neither overexpression nor treatment with FGF-2 isoforms promotes survival of sympathetic neurons deprived of nerve growth factor; however, neuronal transfection of the high molecular weight FGF-2 isoform in dissociated and slice cultures results in a bi- or multinuclear phenotype. The present study provides evidence for neuronal synthesis and targeting of FGF-2 to the nucleus and Golgi apparatus supporting a dual role of FGF-2 in the nucleus and secretory pathway of sympathetic neurons.

Block of slow axonal transport and axonal growth by brefeldin A in compartmented cultures of rat sympathetic neurons.

Disruption of the Golgi by brefeldin A (BFA) has been reported to block fast axonal transport and axonal growth. We used compartmented cultures of rat sympathetic neurons to investigate its effects on slow axonal transport. BFA (1 micro g/ml) applied to cell bodies/proximal axons for 6-20 h disrupted the Golgi, reversibly blocked axonal growth, and reversibly blocked anterograde transport of all proteins, including tubulin. The retrograde transport of nerve growth factor (NGF) was also blocked. The phosphorylation of Erk1 and Erk2 in response to NGF was unaffected after 6 h of treatment with BFA, suggesting that the block of axonal transport was specific and direct. Consistent with its principal site of action at the Golgi, no effects were observed when BFA was applied only to the distal axons. Block of fast anterograde and retrograde axonal transport is consistent with the role of the Golgi in supplying transport vesicles. Block of slow axonal transport was surprising, and further results indicated that transport of tubulin en route along the axon was arrested by application of BFA to the cell bodies, suggesting that a continuous supply of anterograde transport vesicles from the Golgi is required to maintain slow axonal transport of cytoskeletal proteins.

Co-localization of histamine and dopamine-beta-hydroxylase in sympathetic ganglion and release of histamine from cardiac sympathetic terminals of guinea-pig.

1. The aim of this study was to investigate the co-localization of histamine and dopamine-beta-hydroxylase in the superior cervical ganglion of guinea-pig and release of histamine from cardiac sympathetic terminals in guinea-pig isolated atrium. 2. Histidine decarboxylase (a histamine-synthesizing enzyme) mRNA signals were detected in the neurones of superior cervical ganglion of guinea-pig by in situ hybridization. The results of double-labelled immunofluorescence further confirmed the co-localization of histamine and dopamine-beta-hydroxylase in the large principle neurons and small intensely fluorescent cells in the superior cervical ganglion. The immunoreactivities of both histamine and dopamine-beta-hydroxylase were significantly attenuated after 6-hydroxydopamine-induced lesion of sympathetic nerves. 3. The refractory electrical field stimulation caused the release of histamine from cardiac sympathetic terminals of guinea-pig isolated atria (112.14 +/- 40.34 ng x ml(-1)), which was significantly attenuated to 35 +/- 15.57 ng x ml(-1) by reserpine pretreatment. Following administering compound 48/80, a mast cell degranulator, electrical field stimulation induced a dramatic increase of endogenous histamine release from isolated atria (303.57 +/-72.93 ng x ml(-1)). When compound 48/80 was added to the reserpine-treated atria, the release of histamine induced by field stimulation was decreased to 207.14 +/- 76.39 ng x ml(-1). 4 These results provide novel evidence that histamine co-exists with noradrenaline in sympathetic nerves and might act as a neurotransmitter to modulate sympathetic neurotransmission.