Regulatory effects of nicotine on neurite outgrowth in rat superior cervical ganglia cells.

We have reported that nicotine has a neurotrophic action on peripheral adrenergic nerves in vivo, which is mediated by α7 nicotinic acetylcholine receptors (nAChRs). To clarify the possible mechanisms, the present study further investigated the effect of nicotine on neurite outgrowth in tyrosine hydroxylase (TH)-positive superior cervical ganglia (SCG) cells isolated from neonatal rats in vitro. Nicotine at low concentrations (0.01-0.3 mM) increased the number of neurite outgrowths in TH-immunopositive SCG cells, while high concentrations of nicotine (1-10 mM) gradually reduced it, and only 10 mM nicotine was markedly inhibited compared to the control. A 100 µM of nicotine-induced increase in neurite numbers depended on the exposure time and was inhibited by treatment with the nAChR antagonist hexamethonium (Hex) and α7 nAChR antagonist α-bungarotoxin (α-Bgtx). The nicotine (10 mM)-induced a significant decrease in neurite outgrowth in SCG, which was perfectly canceled by Hex to the control level but not by α-Bgtx. These results suggest that nicotine has a regulatory neurotrophic action mediated by both α7 nAChR and other subtypes in TH-positive SCG cells of rats.

Sympathetic nervous system contributes to orthodontic tooth movement by central neural regulation from hypothalamus.

Orthodontic tooth movement (OTM) is a specific treatment of malocclusion, whose regulation mechanism is still not clear. This study aimed to reveal the relationship between the sympathetic nervous system (SNS) and OTM through the construction of an OTM rat model through the utilization of orthodontic nickeltitanium coiled springs. The results indicated that the stimulation of SNS by dopamine significantly promote the OTM process represented by the much larger distance between the first and second molar compared with mere exertion of orthodontic force. Superior cervical ganglionectomy (SCGx) can alleviate this promotion effect, further proving the role of SNS in the process of OTM. Subsequently, the ability of orthodontic force to stimulate the center of the SNS was visualized by the tyrosin hydroxylase (TH) staining of neurons in ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC) of the hypothalamus, as well as the up-regulated expression of norepinephrine in local alveolar bone. Moreover, we also elucidated that the stimulation of SNS can promote osteoclast differentiation in periodontal ligament cells (PDLCs) and bone marrow-derived cells (BMCs) through regulation of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) system, thus promoting the OTM process. In conclusion, this study provided the first evidence for the involvement of the hypothalamus in the promotion effect of SNS on OTM. This work could provide a novel theoretical and experimental basis for further understanding of the molecular mechanism of OTM.

Sympathetic Effects of Internal Carotid Nerve Manipulation on Choroidal Vascularity and Related Measures.

Purpose: To investigate specific effects of denervation and stimulation of the internal carotid nerve (ICN) on the choroid and retina. Methods: Female Sprague Dawley rats underwent unilateral ICN transection (n = 20) or acute ICN electrical stimulation (n = 7). Rats in the denervation group were euthanized 6 weeks after nerve transection, and eyes were analyzed for changes in choroidal vascularity (via histomorphometry) or angiogenic growth factors and inflammatory markers (via ELISA). Rats in the stimulation group received acute ICN electrical stimulation with a bipolar cuff electrode over a range of stimulus amplitudes, frequencies, and pulse widths. Choroidal blood flow and pupil diameter were monitored before, during, and after stimulation. Results: Six weeks after unilateral ICN transection, sympathectomized choroids exhibited increased vascularity, defined as the percentage of choroidal surface area occupied by blood vessel lumina. Vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein levels in denervated choroids were 61% and 124% higher than in contralateral choroids, respectively. TNF-α levels in denervated retinas increased by 3.3-fold relative to levels in contralateral retinas. In animals undergoing acute ICN electrical stimulation, mydriasis and reduced choroidal blood flow were observed in the ipsilateral eye. The magnitude of the reduction in blood flow correlated positively with stimulus frequency. Conclusions: Modulation of ICN activity reveals a potential role of the ocular sympathetic system in regulating endpoints related to neovascular diseases of the eye.

Role of pituitary adenylyl cyclase-activating polypeptide in intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Inhibition by ketamine and amphetamine analogs of the neurogenic nitrergic vasodilations in porcine basilar arteries.

The abuse of ketamine and amphetamine analogs is associated with incidence of hypertension and strokes involving activation of sympathetic activities. Large cerebral arteries at the base of the brain from several species receive dense sympathetic innervation which upon activation causes parasympathetic-nitrergic vasodilation with increased regional blood flow via axo-axonal interaction mechanism, serving as a protective mechanism to meet O2 demand in an acutely stressful situation. The present study was designed to examine effects of ketamine and amphetamine analogs on axo-axonal interaction-mediated neurogenic nitrergic vasodilation in porcine basilar arteries using techniques of blood-vessel myography, patch clamp and two-electrode voltage clamp, and calcium imaging. In U46619-contracted basilar arterial rings, nicotine (100µM) and electrical depolarization of nitrergic nerves by transmural nerve stimulation (TNS, 8Hz) elicited neurogenic nitrergic vasodilations. Ketamine and amphetamine analogs concentration-dependently inhibited nicotine-induced parasympathetic-nitrergic vasodilation without affecting that induced by TNS, nitroprusside or isoproterenol. Ketamine and amphetamine analogs also concentration-dependently blocked nicotine-induced inward currents in Xenopus oocytes expressing α3ß2-nicotinic acetylcholine receptors (nAChRs), and nicotine-induced inward currents as well as calcium influxes in rat superior cervical ganglion neurons. The potency in inhibiting both inward-currents and calcium influxes is ketamine>methamphetamine>hydroxyamphetamine. These results indicate that ketamine and amphetamine analogs, by blocking nAChRs located on cerebral perivascular sympathetic nerves, reduce nicotine-induced, axo-axonal interaction mechanism-mediated neurogenic dilation of the basilar arteries. Chronic abuse of these drugs, therefore, may interfere with normal sympathetic-parasympathetic interaction mechanism resulting in diminished neurogenic vasodilation and, possibly, normal blood flow in the brainstem.

Muscarinic inhibition of nicotinic transmission in rat sympathetic neurons and adrenal chromaffin cells.

Little is known about the interactions between nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine-induced nAChR currents in sympathetic superior cervical ganglion neurons and adrenal chromaffin cells. The muscarine-induced inhibition (MiI) substantially reduced ACh-induced membrane currents through nAChRs and quantal neurotransmitter release. The MiI was time- and temperature-dependent. The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2), suggested, instead of a direct open-channel blockade, an intracellular metabotropic process. The effects of GTP-γ-S, GDP-ß-S and pertussis toxin suggested that MiI was mediated by G-protein signalling. Inhibitors of protein kinase C (bisindolymaleimide-Bis), protein kinase A (H89) and PIP2 depletion attenuated the MiI, indicating that a second messenger pathway is involved in this process. Taken together, these data suggest that mAChRs negatively modulated nAChRs via a G-protein-mediated second messenger pathway. The time dependence suggests that MiI may provide a novel mechanism for post-synaptic adaptation in all cells/neurons and synapses expressing both types of AChRs.

Virtual leak channels modulate firing dynamics and synaptic integration in rat sympathetic neurons: implications for ganglionic transmission in vivo.

KEY POINTS: The synaptic organization of paravertebral sympathetic ganglia enables them to relay activity from the spinal cord to the periphery and thereby control autonomic functions, including blood pressure and body temperature. The present experiments were done to reconcile conflicting observations in tissue culture, intact isolated ganglia and living animals. By recording intracellularly from dissociated neurons and intact ganglia, we found that when electrode damage makes cells leaky it could profoundly distort cellular excitability and the integration of synaptic potentials. The experiments relied on the dynamic clamp method, which allows the creation of virtual ion channels by injecting current into a cell based upon a mathematical model and using rapid feedback between the model and cell. The results support the hypothesis that sympathetic ganglia can produce a 2.4-fold amplification of presynaptic activity. This could aid understanding of the neural hyperactivity that is believed to drive high blood pressure in some patients. ABSTRACT: The excitability of rat sympathetic neurons and integration of nicotinic EPSPs were compared in primary cell culture and in the acutely isolated intact superior cervical ganglion using whole cell patch electrode recordings. When repetitive firing was classified by Hodgkin's criteria in cultured cells, 18% displayed tonic class 1 excitability, 36% displayed adapting class 2 excitability and 46% displayed phasic class 3 excitability. In the intact ganglion, 71% of cells were class 1 and 29% were class 2. This diverges from microelectrode reports that nearly 100% of superior cervical ganglion neurons show phasic class 3 firing. The hypothesis that the disparity between patch and microelectrode data arises from a shunt conductance was tested using the dynamic clamp in cell culture. Non-depolarizing shunts of 3-10 nS converted cells from classes 1 and 2 to class 3 dynamics with current-voltage relations that replicated microelectrode data. Primary and secondary EPSPs recorded from the intact superior cervical ganglion were modelled as virtual synapses in cell culture using the dynamic clamp. Stimulating sympathetic neurons with virtual synaptic activity, designed to replicate in vivo recordings of EPSPs in muscle vasoconstrictor neurons, produced a 2.4-fold amplification of presynaptic activity. This gain in postsynaptic output did not differ between neurons displaying the three classes of excitability. Mimicry of microelectrode damage by virtual leak channels reduced and eventually obliterated synaptic gain by inhibiting summation of subthreshold EPSPs. These results provide a framework for interpreting sympathetic activity recorded from intact animals and support the hypothesis that paravertebral ganglia function as activity-dependent amplifiers of spinal output from preganglionic circuitry.

Nicotine facilitates reinnervation of phenol-injured perivascular adrenergic nerves in the rat mesenteric resistance artery.

Nicotine has been shown to have neuroprotective and neurotrophic actions in the central nervous system. To elucidate the peripheral neurotrophic effects of nicotine, we determined whether nicotine affected the reinnervation of mesenteric perivascular nerves following a topical phenol treatment. A topical phenol treatment was applied to the superior mesenteric artery proximal to the abdominal aorta in Wistar rats. We examined the immunohistochemistry of the distal small arteries 7 days after the treatment. The topical phenol treatment markedly reduced the density of tyrosine hydroxylase (TH)-LI and calcitonin gene-related peptide (CGRP)-LI fibers in these arteries. The administration of nicotine at a dose of 3 mg/kg/day (1.5 mg/kg/injection, twice a day), but not once a day or its continuous infusion using a mini-pump significantly increased the density of TH-LI nerves without affecting CGRP-LI nerves. A pretreatment with nicotinic acetylcholine receptor antagonists hexamethonium, mecamylamine, and methyllycaconitine, but not dextrometorphan, canceled the TH-LI nerve reinnervation induced by nicotine. Nicotine significantly increased NGF levels in the superior cervical ganglia (SCG) and mesenteric arteries, but not in the dorsal root ganglia, and also up-regulated the expression of NGF receptors (TrkA) in the SCG, which were canceled by hexamethonium. These results suggested that nicotine exhibited neurotrophic effects that facilitated the reinnervation of adrenergic TH-LI nerves by activating α7 nicotinic acetylcholine receptor and NGF in the SCG.

Force-induced Adrb2 in periodontal ligament cells promotes tooth movement.

The sympathetic nervous system (SNS) regulates bone resorption through ß-2 adrenergic receptor (Adrb2). In orthodontic tooth movement (OTM), mechanical force induces and regulates alveolar bone remodeling. Compressive force-associated osteoclast differentiation and alveolar bone resorption are the rate-limiting steps of tooth movement. However, whether mechanical force can activate Adrb2 and thus contribute to OTM remains unknown. In this study, orthodontic nickel-titanium springs were applied to the upper first molars of rats and Adrb1/2(-/-) mice to confirm the role of SNS and Adrb2 in OTM. The results showed that blockage of SNS activity in the jawbones of rats by means of superior cervical ganglion ectomy reduced OTM distance from 860 to 540 µm after 14 d of force application. In addition, the injection of nonselective Adrb2 agonist isoproterenol activated the downstream signaling of SNS to accelerate OTM from 300 to 540 µm after 7 d of force application. Adrb1/2(-/-) mice showed significantly reduced OTM distance (19.5 µm) compared with the wild-type mice (107.6 µm) after 7 d of force application. Histopathologic analysis showed that the number of Adrb2-positive cells increased in the compressive region of periodontal ligament after orthodontic force was applied on rats. Mechanistically, mechanical compressive force upregulated Adrb2 expression in primary-cultured human periodontal ligament cells (PDLCs) through the elevation of intracellular Ca(2+) concentration. Activation of Adrb2 in PDLCs increased the RANKL/OPG ratio and promoted the peripheral blood mononuclear cell differentiation to osteoclasts in the cocultured system. Upregulation of Adrb2 in PDLCs promoted osteoclastogenesis, which accelerated OTM through Adrb2-enhanced bone resorption. In summary, this study suggests that mechanical force-induced Adrb2 activation in PDLCs contributes to SNS-regulated OTM.

Nerve growth factor sensitizes adult sympathetic neurons to the proinflammatory peptide bradykinin.

Levels of nerve growth factor (NGF) are elevated in inflamed tissues. In sensory neurons, increases in NGF augment neuronal sensitivity (sensitization) to noxious stimuli. Here, we hypothesized that NGF also sensitizes sympathetic neurons to proinflammatory stimuli. We cultured superior cervical ganglion (SCG) neurons from adult male Sprague Dawley rats with or without added NGF and compared their responsiveness to bradykinin, a proinflammatory peptide. The NGF-cultured neurons exhibited significant depolarization, bursts of action potentials, and Ca(2+) elevations after bradykinin application, whereas neurons cultured without NGF showed only slight changes in membrane potential and cytoplasmic Ca(2+) levels. The NGF effect, which requires trkA receptors, takes hours to develop and days to reverse. We addressed the ionic mechanisms underlying this sensitization. NGF did not alter bradykinin-induced M-current inhibition or phosphatidylinositol 4,5-bisphosphate hydrolysis. Maxi-K channel-mediated current evoked by depolarizations was reduced by 50% by culturing neurons in NGF. Application of iberiotoxin or paxilline, blockers of Maxi-K channels, mimicked NGF treatment and sensitized neurons to bradykinin application. A calcium channel blocker also mimicked NGF treatment. We found that NGF reduces Maxi-K channel opening by decreasing the activity of nifedipine-sensitive calcium channels. In conclusion, culture in NGF reduces the activity of L-type calcium channels, and secondarily, the calcium-sensitive activity of Maxi-K channels, rendering sympathetic neurons electrically hyper-responsive to bradykinin.

Substance P plays an important role in cell adhesion molecule 1-mediated nerve-pancreatic islet α cell interaction.

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca(2+) concentration ([Ca(2+)]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6(siRNA-CADM1) cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6(siRNA-CADM1) cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6(siRNA-CADM1) cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve-α cell interaction. Pretreatment with CP99,994 (0.1µg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.

Parasympathetic influences on emmetropization in chicks: evidence for different mechanisms in form deprivation vs negative lens-induced myopia.

Ciliary ganglionectomy inhibits the development of myopia in chicks (Schmid et al., 1999), but has no effect on the compensatory responses to spectacle lenses (Schmid and Wildsoet, 1996). This study was done to assess the potential influence of the other parasympathetic input to the choroid, the pterygopalatine ganglia, on the choroidal and axial responses to retinal defocus, and to form deprivation. 4-5 week-old chicks had one of the following surgeries to one eye: (1) Section (X) of the autonomic part of cranial N VII (input to the pterygopalatine ganglia) (PPGX, n = 16), (2) PPGX plus ciliary ganglionectomy (PPG/CGX, n = 23) or (3) PPGX plus superior cervical ganglionectomy (PPG/SCGX, n = 10). Experimental eyes were fitted with positive or negative lenses, or diffusers, several days after surgery. In one group of PPG/CGX, eyes did not wear any devices (n = 8). Intact (no surgery) controls were done for all visual manipulations (lenses or diffusers). Sham surgeries were done for the PPG/CGX condition (n = 4). Ocular dimensions were measured using A-scan ultrasonography prior to the surgery, 5 days later when visual devices were placed on the eyes, at the end of lens- or diffuser-wear, and in the case of diffusers, 4 days after diffuser removal to look at "recovery". Refractive errors were measured using a Hartinger's refractometer. IOP was measured in 7 PPG/CGX birds 7d after surgery. PPGX/CGX resulted in choroidal thickening (125 µm) and a decrease in IOP over one week post-surgery. It also prevented the development of myopia in response to form deprivation (X vs intact: 0.2 D vs -4.1 D; p < 0.005), by preventing the increase in axial elongation (250 µm vs 670 µm/5d; p < 0.005). In fact, growth rate slowed below normal (X vs fellow eyes: 250 µm vs 489 µm/5d; p = 0.002). By contrast, there were no effects of this lesion on the development of myopia in response to negative lenses (X vs intact: -5.4 D vs -5.3 D). All three lesions inhibited the compensatory choroidal thickening in response to myopic defocus (ANOVA, p = 0.0008), but had no effect on the thinning response to hyperopic defocus. These results argue for different underlying mechanisms for the growth responses to form deprivation vs negative lens wear. They also imply that choroidal thickening and thinning are not opposing elements of a single mechanism.

Effect of chronic cervical ganglionectomy on the spontaneous variability of internal carotid blood flow in the conscious rat.

The role of sympathetic innervation in the control of spontaneous fluctuations of cerebral blood flow is still poorly understood. In conscious, unrestrained rats, blood flow velocity (pulsed Doppler) was measured in both internal carotid arteries 1 week after either excision of the right superior cervical ganglion (n = 8) or sham surgery (n = 6). Using Fourier-based techniques, spectral power of each carotid blood flow (CBF) was computed over the whole recording period (246 min), which was segmented into nine consecutive 27.3 min periods. Variability of CBF (spectral power) was ∼40% higher (P < 0.02) on the denervated than on the intact side at frequencies <1 Hz. Coherence between left and right CBFs was similar in the two groups of rats, except in the 0.01-0.1 Hz frequency range where it was lower (P < 0.05) in rats with unilateral sympathectomy (0.54 ± 0.03) than in intact rats (0.74 ± 0.06). In this frequency range, mathematically removing the influence of arterial pressure had little effect on coherence between CBFs in both groups of rats, so that coherence remained significantly lower in rats with unilateral sympathectomy (0.52 ± 0.03) than in intact rats (0.70 ± 0.06). This study indicates that sympathetic innervation has an overall buffering influence on CBF variability. This modulatory role is especially important in a frequency range corresponding to slow fluctuations of CBF (lasting from 10 to 100 s), which are essentially unrelated to fluctuations of arterial pressure.

Sympathetic activation increases basilar arterial blood flow in normotensive but not hypertensive rats.

The close apposition between sympathetic and parasympathetic nerve terminals in the adventitia of cerebral arteries provides morphological evidence that sympathetic nerve activation causes parasympathetic nitrergic vasodilation via a sympathetic-parasympathetic interaction mechanism. The decreased parasympathetic nerve terminals in basilar arteries (BA) of spontaneously hypertensive rat (SHR) and renovascular hypertensive rats (RHR) compared with Wistar-Kyoto rats (WKY), therefore, would diminish this axo-axonal interaction-mediated neurogenic vasodilation in hypertension. Increased basilar arterial blood flow (BABF) via axo-axonal interaction during sympathetic activation was, therefore, examined in anesthetized rats by laser-Doppler flowmetry. Electrical stimulation (ES) of sympathetic nerves originating in superior cervical ganglion (SCG) and topical nicotine (10-30 µM) onto BA of WKY significantly increased BABF. Both increases were inhibited by tetrodotoxin, 7-nitroindazole (neuronal nitric oxide synthase inhibitor), and ICI-118,551 (ß(2)-adrenoceptor antagonist), but not by atenolol (ß(1)-adrenoceptor antagonist). Topical norepinephrine onto BA also increased BABF, which was abolished by atenolol combined with 7-nitroindazole or ICI-118,551. Similar results were found in prehypertensive SHR. However, in adult SHR and RHR, ES of sympathetic nerves or topical nicotine caused minimum or no increase of BABF. It is concluded that excitation of sympathetic nerves to BA in WKY causes parasympathetic nitrergic vasodilation with increased BABF. This finding indicates an endowed functional neurogenic mechanism for increasing the BABF or brain stem blood flow in coping with increased local sympathetic activities in acutely stressful situations such as the "fight-or-flight response." This increased blood flow in defensive mechanism diminishes in genetic and nongenetic hypertensive rats due most likely to decreased parasympathetic nitrergic nerve terminals.

The sympathetic neurotransmitter switch depends on the nuclear matrix protein Satb2.

Sympathetic neurons can switch their neurotransmitter phenotype from noradrenergic to cholinergic on exposure to neuropoietic cytokines in vitro and in vivo. Here, we provide evidence that this transspecification is regulated by the chromatin architecture protein Satb2. Treatment with the neuropoietic cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor rapidly and strongly increases Satb2 transcript and protein levels in cultures of rat superior cervical ganglia neurons. Knockdown of endogenous Satb2 by short interfering RNA prevents the upregulation of choline acetyltransferase (Chat) and vesicular acetylcholine transporter (Vacht) by CNTF as well as the loss of norepinephrine transporter (Net). Conversely, overexpression of Satb2 in the noradrenergic sympathetic phenotype results in a marked increase of Chat and Vacht expression and reduced Net mRNA levels in the absence of neuropoietic cytokines. Chromatin immunoprecipitation analysis in primary sympathetic neurons reveals that Satb2 binds to matrix attachment regions (MARs) within the Chat locus. In vivo, in the rat stellate ganglion, Satb2 is expressed exclusively in sudomotor cholinergic neurons innervating the sweat glands and only after establishment of contact between neurons and target. These findings demonstrate a function of the MAR-binding protein Satb2 in growth factor-dependent neurotransmitter plasticity in postmitotic neurons.

Venus fly trap domain of mGluR1 functions as a dominant negative against group I mGluR signaling.

Metabotropic glutamate receptors (mGluRs) form covalently linked homodimers and contain large, N-terminal extracellular ligand binding, "venus fly trap" (VFT) domains. These domains, when expressed separately, are secreted as disulfide linked dimers and can dimerize with full-length receptors. mGluR splice variants have been described that contain only this domain, but the consequences of their interaction on receptor signaling have not been explored. Here it is shown that an mGluR1 mutant containing only the VFT is retained on the cell surface when a full-length receptor is co-expressed. Further, when expressed in rat superior cervical ganglion (SCG) neurons and modulation of native calcium currents is used as an assay for receptor activity, the VFT acts as a dominant negative with respect to mGluR1 signaling. Although full-length mGluR1 and mGluR5 are not known to heterodimerize, the mGluR5 VFT partially occludes mGluR1 signaling and the mGluR1 VFT potently occludes mGluR5 signaling in SCG neurons. In addition, an mGluR1 point mutant, mGluR1 C140G, which cannot covalently dimerize, functions like the wild-type receptor when expressed alone. The C140G mutant is inhibited by the mGluR1 VFT construct but does not retain the mGluR1 VFT on the cell surface, suggesting that the loss of C140 renders the interaction reversible. Finally, a peptide designed to disrupt mGluR1 dimerization reduced signaling through the C140G mutant receptor, but only when applied intracellularly for several hours, indicating that loss of signaling requires disruption of dimerization prior to plasma membrane insertion.

The differential axonal degradation of Ret accounts for cell-type-specific function of glial cell line-derived neurotrophic factor as a retrograde survival factor.

Glial cell line-derived neurotrophic factor (GDNF) is a neuronal growth factor critical for the development and maintenance of central and peripheral neurons. GDNF is expressed in targets of innervation and provides support to several populations of large, projection neurons. To determine whether GDNF promotes retrograde survival over long axonal distances to cell bodies, we used a compartmentalized culture system. GDNF supported only modest and transient survival of postnatal sympathetic neurons when applied to their distal axons, in contrast to dorsal root ganglion (DRG) sensory neurons in which GDNF promoted survival equally well from either distal axons or cell bodies. Ret, the receptor tyrosine kinase for GDNF, underwent rapid proteasomal degradation in the axons of sympathetic neurons. Interestingly, the level of activated Ret in DRG neurons was sustained in the axons and also appeared in the cell bodies, suggesting that Ret was not degraded in sensory axons and was retrogradely transported. Pharmacologic inhibition of proteasomes only in the distal axons of sympathetic neurons caused an accumulation of activated Ret in both the axons and cell bodies during GDNF stimulation. Furthermore, exposure of the distal axons of sympathetic neurons to both GDNF and proteasome inhibitors, but neither one alone, promoted robust survival, identical to GDNF applied directly to the cell bodies. This differential responsiveness of sympathetic and sensory neurons to target-derived GDNF was attributable to the differential expression and degradation of the Ret9 and Ret51 isoforms. Therefore, the local degradation of Ret in axons dictates whether GDNF family ligands act as retrograde survival factors.

Increased sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling via P2X2/3 receptor in rat superior cervical ganglia.

The restructuring of cardiac innervation after myocardial ischemic injury, which is often associated with an increased sympathoexcitatory reflex characterized by an increase in blood pressure and sympathetic nerve activity. This study observed whether P2X(2/3) receptor in rat superior cervical ganglion (SCG) played a role in the increased sympathoexcitatory reflex induced by myocardial ischemic nociceptive signaling. The findings showed that the systolic blood pressure, heart rate and respiration in the myocardial ischemic rats were higher than those in control rats. The content of adenosine 5'-triphosphate (ATP) from myocardial ischemic group was higher than that from control group. After myocardial ischemic rats were treated with selective P2X(2/3) receptor antagonist A-317491, the content of ATP in SCG was reduced. Coexpression value of P2X(2), P2X(3) and tyrosine hydroxylase (TH) in the SCG and myocardial tissues of myocardial ischemic group was increased significantly, compared with that in the SCG and myocardial tissues of control group. The expression value of P2X(2), P2X(3) and TH in the SCG and myocardial tissues of myocardial ischemic rats treated with A-317491 was decreased. The amplitude of the currents was much larger in myocardial ischemic group than that obtained in control group after administration of ATP with the same concentration. After the myocardial ischemic rats were treated with A-317491, ATP-activated currents were reduced. The myocardial ischemic injury induced an increase in the expression of P2X(2/3) receptor colocalization with TH and a hypersensitivity state of SCG neurons to ATP, which led to the increased sympathoexcitatory reflex.

The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.

The conditioning lesion effect on sympathetic neurite outgrowth is dependent on gp130 cytokines.

Sympathetic neurons, like sensory neurons, increase neurite outgrowth after a conditioning lesion. Studies in leukemia inhibitory factor (LIF) knockout animals showed that the conditioning lesion effect in sensory neurons is dependent in part on this cytokine; however, similar studies on sympathetic neurons revealed no such effect. Comparable studies with sensory neurons taken from mice lacking the related cytokine interleukin-6 (IL-6) have yielded conflicting results. LIF and IL-6 belong to a family of cytokines known as the gp130 family because they act on receptors containing the subunit gp130. In sympathetic ganglia, axotomy leads to increases in mRNA for four of these cytokines (LIF, IL-6, IL-11, and oncostatin M). To test the role of this family of cytokines as a whole in the conditioning lesion response in sympathetic neurons, mice in which gp130 was selectively eliminated in noradrenergic neurons were studied. The postganglionic axons of the SCG were transected, and 7days later the ganglia were removed and neurite outgrowth was measured in explant and dissociated cell cultures. In both systems, neurons from wild type animals showed enhanced growth after a conditioning lesion. In contrast, no enhancement occurred in neurons from mutant animals. This lack of stimulation of outgrowth occurred despite an increase in expression of activating transcription factor 3 (ATF3) in the mutant mice. These studies demonstrate that stimulation of enhanced growth of sympathetic neurons after a conditioning lesion is dependent on gp130 cytokine signaling and is blocked in the absence of signaling by these cytokines in spite of an increase in ATF3.