Mouse Superior Cervical Ganglion Research Articles

Sympathetic neurons secrete retrogradely transported TrkA on extracellular vesicles

Proper wiring of the peripheral nervous system relies on neurotrophic signaling via nerve growth factor (NGF). NGF secreted by target organs (i.e. eye) binds to the TrkA receptor expressed on the distal axons of postganglionic neurons. Upon binding, TrkA is internalized into a signaling endosome and retrogradely trafficked back to the soma and into the dendrites to promote cell survival and postsynaptic maturation, respectively. Much progress has been made in recent years to define the fate of the retrogradely trafficked TrkA signaling endosome, yet it has not been fully characterized. Here we investigate extracellular vesicles (EVs) as a novel route of neurotrophic signaling. Using the mouse superior cervical ganglion (SCG) as a model, we isolate EVs derived from sympathetic cultures and characterize them using immunoblot assays, nanoparticle tracking analysis, and cryo-electron microscopy. Furthermore, using a compartmentalized culture system, we find that TrkA derived from endosomes originating in the distal axon can be detected on EVs secreted from the somatodendritic domain. In addition, inhibition of classic TrkA downstream pathways, specifically in somatodendritic compartments, greatly decreases TrkA packaging into EVs. Our results suggest a novel trafficking route for TrkA: it can travel long distances to the cell body, be packaged into EVs, and be secreted. Secretion of TrkA via EVs appears to be regulated by its own downstream effector cascades, raising intriguing future questions about novel functionalities associated with TrkA+ EVs.

Antiarrhythmic calcium channel blocker verapamil inhibits trek currents in sympathetic neurons.

Background and Purpose: Verapamil, a drug widely used in certain cardiac pathologies, exert its therapeutic effect mainly through the blockade of cardiac L-type calcium channels. However, we also know that both voltage-dependent and certain potassium channels are blocked by verapamil. Because sympathetic neurons of the superior cervical ganglion (SCG) are known to express a good variety of potassium currents, and to finely tune cardiac activity, we speculated that the effect of verapamil on these SCG potassium channels could explain part of the therapeutic action of this drug. To address this question, we decided to study, the effects of verapamil on three different potassium currents observed in SCG neurons: delayed rectifier, A-type and TREK (a subfamily of K2P channels) currents. We also investigated the effect of verapamil on the electrical behavior of sympathetic SCG neurons. Experimental Approach: We employed the Patch-Clamp technique to mouse SCG neurons in culture. Key Results: We found that verapamil depolarizes of the resting membrane potential of SCG neurons. Moreover, we demonstrated that this drug also inhibits A-type potassium currents. Finally, and most importantly, we revealed that the current driven through TREK channels is also inhibited in the presence of verapamil. Conclusion and Implications: We have shown that verapamil causes a clear alteration of excitability in sympathetic nerve cells. This fact undoubtedly leads to an alteration of the sympathetic-parasympathetic balance which may affect cardiac function. Therefore, we propose that these possible peripheral alterations in the autonomic system should be taken into consideration in the prescription of this drug.

Cannabidiol activates neuronal Kv7 channels.

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

Contribution of KCNQ and TREK Channels to the Resting Membrane Potential in Sympathetic Neurons at Physiological Temperature.

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

Characterization of a cell bridge variant connecting the nodose and superior cervical ganglia in the mouse: Prevalence, anatomical features, and practical implications.

While autonomic ganglia have been extensively studied in rats instead of mice, there is renewed interest in the anatomy of the mouse autonomic nervous system. This study examined the prevalence and anatomical features of a cell bridge linking two autonomic ganglia of the neck, namely, the nodose ganglion (NG) and the superior cervical ganglion (SCG) in a cohort of C57BL/6J mice. We identified a cell bridge between the NG and the cranial pole of the SCG. This cell bridge was tubular shaped with an average length and width of 700 and 240 μm, respectively. The cell bridge was frequently unilateral and significantly more prevalent in the ganglionic masses from males (38%) than females (21%). On each of its extremities, it contained a mixed of vagal afferents and postganglionic sympathetic neurons. The two populations of neurons abruptly replaced each other in the middle of the cell bridge. We examined the mRNA expression for selected autonomic markers in samples of the NG with or without cell bridge. Our results indicated that the cell bridge was enriched in both markers of postganglionic sympathetic and vagal afferents neurons. Lastly, using FluoroGold microinjection into the NG, we found that the existence of a cell bridge may occasionally lead to the inadvertent contamination of the SCG. In summary, this study describes the anatomy of a cell bridge variant consisting of the fusion of the mouse NG and SCG. The practical implications of our observations are discussed with respect to studies of the mouse vagal afferents, an area of research of increasing popularity.

PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons.

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

The effects of sympathetic nerve damage on satellite glial cells in the mouse superior cervical ganglion.

Neurons in sensory, sympathetic, and parasympathetic ganglia are surrounded by satellite glial cell (SGCs). There is little information on the effects of nerve damage on SGCs in autonomic ganglia. We studied the consequences of damage to sympathetic nerve terminals by 6-hydroxydopamine (6-OHDA) on SGCs in the mouse superior cervical ganglia (Sup-CG). Immunostaining revealed that at 1-30 d post-6-OHDA injection, SGCs in Sup-CG were activated, as assayed by upregulation of glial fibrillary acidic protein. Intracellular labeling showed that dye coupling between SGCs around different neurons increased 4-6-fold 1-14 d after 6-OHDA injection. Behavioral testing 1-7 d post-6-OHDA showed that withdrawal threshold to tactile stimulation of the hind paws was reduced by 65-85%, consistent with hypersensitivity. A single intraperitoneal injection of the gap junction blocker carbenoxolone restored normal tactile thresholds in 6-OHDA-treated mice, suggesting a contribution of SGC gap junctions to pain. Using calcium imaging we found that after 6-OHDA treatment responses of SGCs to ATP were increased by about 30% compared with controls, but responses to ACh were reduced by 48%. The same experiments for SGCs in trigeminal ganglia from 6-OHDA injected mice showed no difference from controls, confirming that 6-OHDA acted selectively on sympathetic nerves. However, systemic inflammation induced by lipopolysaccharide did not affect SGCs of Sup-CG, but did influence SGCs in trigeminal ganglia in the same manner as 6-OHDA did on SGCs in Sup-CG. We conclude that even though SGCs in sympathetic and sensory ganglia are morphologically similar, they are quite different functionally, particularly after damage.

Localization of CGRP and VEGF mRNAs in the mouse superior cervical ganglion during pre- and postnatal development.

The neuropeptide calcitonin gene-related peptide (CGRP) mediates inflammation and head pain by influencing the functional vascular blood supply. CGRP is a well-characterized mediator of receptor-regulated neurotransmitter release. However, knowledge regarding the role of CGRP during the development of the superior cervical ganglion (SCG) is limited. In the present study, we observed the localization of CGRP and vascular endothelial growth factor (VEGFA) mRNAs during prenatal development at embryonic day 14.5 (E14.5), E17.5 and postnatal day 1 (P1) using in situ hybridization. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels were detected at E17.5. In contrast, the antisense probe for VEGF-A was detected by in situ hybridization in gradually increasing intensity from E14.5 to P1. The differences in the expression of these two markers revealed specific characteristics related to CGRP concentration and release compared to those of VEGF-A during development. The correlation between CGRP and VEGF-A may influence functional stress and the vascular blood supply during prenatal and postnatal development.

Development of mouse superior cervical ganglion during prenatal and postnatal stages.

The neuropeptide calcitonin gene‐related peptide (CGRP) is a well‐characterized mediator of receptor‐regulated neurotransmitter release. However, little is known between CGRP during the development of the superior cervical ganglion (SCG). In the present study, we observed the localization of the CGRP mRNA using in situ hybridization during development from the embryonic stage to after birth day 14.5 (E14.5), E17.5 and postnatal day 1 (P1). The different ratio was found in SCG ganglion cells and shrunken cells. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels of positive CGRP ganglion cells were detected at E17.5. The specific characters of CGRP concentration and release were occurred during development. The CGRP may influence the functional stress during prenatal and postnatal development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cholinergic responses of satellite glial cells in the superior cervical ganglia

Satellite glial cells (SGCs) surround the neurons in sympathetic ganglia and are believed to make important contributions to the function of the ganglia under normal and pathological conditions. It has been proposed that SGCs communicate chemically with the neurons, but little is known about their pharmacological properties and there is no information on whether they respond to acetylcholine (ACh), which is the major neurotransmitter in these ganglia. We used calcium imaging to examine responses of SGCs in the mouse superior cervical ganglion to ACh. The SGCs responded to ACh (0.01–2 mM) with an elevation of intracellular Ca2+, which appeared to be due to direct action on these cells, as the response persisted in the presence of the nerve blocker tetrodotoxin (1 μM). The response was largely inhibited by atropine, indicating an action on muscarinic ACh receptors. In contrast to this, sensory ganglia (nodose and trigeminal) were not sensitive to ACh. Incubation of the ganglia in ACh (0.5 or 1 mM) increased the expression of glial fibrillay acidic protein, which is a marker for glial activation. Such incubation also increased the electrical coupling of SGCs, which is known to occur in sensory ganglia following injury. We conclude that SGCs in the superior cervical ganglia display muscarinic ACh receptors, which enable them to communicate chemically with the sympathetic neurons.

Acute and chronic effects of clozapine on cholinergic transmission in cultured mouse superior cervical ganglion neurons

Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia, possibly through modulation of the cholinergic system. However, little is known about specific underlying mechanisms. We investigated the acute and chronic effects of clozapine on cholinergic synaptic transmission in cultured superior cervical ganglion (SCG) neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected and were reversibly inhibited by the nicotinic receptor antagonist d-tubocurarine, confirming that the synaptic responses were primarily mediated by nicotinic receptors. Bath application of clozapine at therapeutic concentrations rapidly and reversely inhibited both the amplitude and frequency of sEPSCs in a concentration-dependent manner, without changing either rise or decay time, suggesting that clozapine effects have both presynaptic and postsynaptic origins. The acute effects of clozapine on sEPSCs were recapitulated by chronic treatment of SCG cultures with similar concentrations of clozapine, as clozapine treatment for 4 d reduced the frequency and amplitude of sEPSCs without affecting their kinetics. Cell survival analysis indicated that SCG neuron cell counts after chronic clozapine treatment were comparable to the control group. These results demonstrate that therapeutic concentrations of clozapine suppress nicotinic synaptic transmission in SCG cholinergic synapses, a simple in vitro preparation of cholinergic transmission.

How Microfluidics Can Help to Understand and Promote Nerve Healing after Injury: A Neurobiological Microfluidic Device with Electrophysiological Functionality

We present a microfluidic device that has been designed to study the neuroregeneration of nerve tissue after injury. Reconnection between neurons of the peripheral nervous system after injury or dissection is essential for regaining the full motoric and sensory capabilities. The neuroregeneration of damaged axons and the functional reconnection to a target cell is a complex processes, where (1) chemical guidance and (2) topographic guidance of the neurite growth cone play a crucial role. To identify new chemical guidance substances and to understand how geometry guides neurite growth, we have developed a microfluidic in-vitro system for studying neuronal cell cultures. The microchannels act as artificial guidance conduits for the growth of nerve processes (dendrites, axons), also named neurites in the following text. The microstructured system consists of a microelectrode array with sub-10 µm microelectrodes for recording neuronal signals plus a microfluidic attachement. This microfluidic attachment has 2 macrochannels with a width above 100 µm that can accommodate entire cells. Both macrochannels are connected by up to 60 microchannels which are 100 to 500 µm long but only 10 µm wide and 2 µm high, so that cell somata cannot enter the microchannels while cell processes (axons, dendrites) are small enough to enter the channel. A neuronal culture of mouse superior cervical ganglion (SCG) is seeded in the source macrochannel, while the target macrochannel stays cell-free and is filled cell medium with neurotrophic factors added. These soluble neurotrophic factors act as chemical guidance cues for the neurites and have been under debate as enhancers of axon regeneration. In our microfluidic system the neurites can reach the target macrochannel by growing through the small microchannels that are too small for entire cell somata. This topographic guidance provided by the microchannels mimics’ topographic guidance of the endoneurial channel formed by Schwann cells for a reconnecting neuron. We will present the design considerations as well as the actual fabrication of this microfluidic system on the microelectrode system. Simulations on the diffusion of chemical entities through the microchannels will be presented. Finally, images of the neurites grown through the microchannels will be shown and prove the research concept. Neurotrophic factors are also under debate for accelerating the functional recovery, i.e. the recovery of the neuron to propagate action potentials along the neurite. With our microfluidic setup the microelectrodes of the microelectrode array have been aligned such that every microchannel contains at least one (up to four) microelectrodes. By implementing microelectrodes inside of the microfluidic channels also the electrophysiological activity of the reconnecting neurites can be monitored. This electrical activity of neurons is a measure for the health status of a neuron. With the microelectrodes providing electrochemical sensor functionality to the microfluidic channels, this important question could be answered. We will present the effect, that various gradients of neurotrophic factors have on the growth and electrical activity of growing neurons. Finally, a recommendation will be given on the optimum concentration for the addition of nerve growth factor in order to promote a faster growth of electrophysiologicallly active neurites. The potential to use this microfluidic platform for identifying new nerve growth promotors and for quantifying the effects will be discussed. Concepts for advancing this microfluidic system further as cell culture reactors as well as for other interdisciplinary areas including neurobiological and medical applications will be presented. Figure 1

Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation.

BackgroundNerve growth factor (NGF) is the prototypical target-derived neurotrophic factor required for sympathetic neuron survival and for the growth and ramification of sympathetic axons within most but not all sympathetic targets. This implies the operation of additional target-derived factors for regulating terminal sympathetic axon growth and branching.ResultsHere report that growth differentiation factor 5 (GDF5), a widely expressed member of the transforming growth factor beta (TGFβ) superfamily required for limb development, promoted axon growth from mouse superior cervical ganglion (SCG) neurons independently of NGF and enhanced axon growth in combination with NGF. GDF5 had no effect on neuronal survival and influenced axon growth during a narrow window of postnatal development when sympathetic axons are ramifying extensively in their targets in vivo. SCG neurons expressed all receptors capable of participating in GDF5 signaling at this stage of development. Using compartment cultures, we demonstrated that GDF5 exerted its growth promoting effect by acting directly on axons and by initiating retrograde canonical Smad signalling to the nucleus. GDF5 is synthesized in sympathetic targets, and examination of several anatomically circumscribed tissues in Gdf5 null mice revealed regional deficits in sympathetic innervation. There was a marked, highly significant reduction in the sympathetic innervation density of the iris, a smaller though significant reduction in the trachea, but no reduction in the submandibular salivary gland. There was no reduction in the number of neurons in the SCG.ConclusionsThese findings show that GDF5 is a novel target-derived factor that promotes sympathetic axon growth and branching and makes a distinctive regional contribution to the establishment of sympathetic innervation, but unlike NGF, plays no role in regulating sympathetic neuron survival.

Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

Regulation of SPRY3 by X chromosome and PAR2-linked promoters in an autism susceptibility region.

Sprouty proteins are regulators of cell growth and branching morphogenesis. Unlike mouse Spry3, which is X-linked, human SPRY3 maps to the pseudoautosomal region 2; however, the human Y-linked allele is not expressed due to epigenetic silencing by an unknown mechanism. SPRY3 maps adjacent to X-linked Trimethyllysine hydroxylase epsilon (TMLHE), recently identified as an autism susceptibility gene. We report that Spry3 is highly expressed in central and peripheral nervous system ganglion cells in mouse and human, including cerebellar Purkinje cells and retinal ganglion cells. Transient over-expression or knockdown of Spry3 in cultured mouse superior cervical ganglion cells inhibits and promotes, respectively, neurite growth and branching. A 0.7 kb gene fragment spanning the human SPRY3 transcriptional start site recapitulates the endogenous Spry3-expression pattern in LacZ reporter mice. In the human and mouse the SPRY3 promoter contains an AG-rich repeat and we found co-expression, and promoter binding and/or regulation of SPRY3 expression by transcription factors MAZ, EGR1, ZNF263 and PAX6. We identified eight alleles of the human SPRY3 promoter repeat in Caucasians, and similar allele frequencies in autism families. We characterized multiple SPRY3 transcripts originating at two CpG islands in the X-linked F8A3-TMLHE region, suggesting X chromosome regulation of SPRY3. These findings provide an explanation for differential regulation of X and Y-linked SPRY3 alleles. In addition, the presence of a SPRY3 transcript exon in a previously described X chromosome deletion associated with autism, and the cerebellar interlobular variation in Spry3 expression coincident with the reported pattern of Purkinje cell loss in autism, suggest SPRY3 as a candidate susceptibility locus for autism.

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons.

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M-currents and voltage-dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M-current inhibition. Here, we report the presence of a riluzole-activated current (IRIL ) that flows through the TREK-2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine-M (Oxo-M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single-channel TREK-2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2-aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET-18-OCH3. Finally, the phosphatidylinositol-3-kinase/phosphatidylinositol-4-kinase inhibitor wortmannin strongly attenuated the IRIL , whereas blocking phosphatidylinositol 4,5-bisphosphate (PIP2 ) depletion consistently prevented IRIL inhibition by Oxo-M. These results demonstrate that TREK-2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC-dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M-current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.

Neuron Culture from Mouse Superior Cervical Ganglion.

The rodent superior cervical ganglion (SCG) is a useful and readily accessible source of neurons for studying the mechanisms of sympathetic nervous system (SNS) development and growth in vitro. The sympathetic nervous system (SNS) of early postnatal animals undergoes a great deal of remodeling and development; thus, neurons taken from mice at this age are primed to re-grow and establish synaptic connections after in situ removal. The stereotypic location and size of the SCG make it ideal for rapid isolation and dissociation. The protocol described here details the requirements for the dissection, culture and differentiation of SCG neurons. The protocol is suitable for culturing neurons from late embryonic gestation to approximately postnatal day 3. The culture technique discussed below utilizes glass coverslips for the microscopic examination of fixed cells.

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 Ca2+ concentration ([Ca2+]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6siRNA-CADM1 cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6siRNA-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 αTC6siRNA-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.

Reversing Roles for Innervation

Tumor necrosis factor (TNF) receptor in target tissues may guide the innervation of TNF-positive sympathetic axons in postnatal development.

ProNGF promotes neurite growth from a subset of NGF-dependent neurons by a p75NTR-dependent mechanism

The somatosensory and sympathetic innervation of the vertebrate head is derived principally from the neurons of trigeminal and superior cervical ganglia (SCG), respectively. During development, the survival of both populations of neurons and the terminal growth and branching of their axons in the tissues they innervate is regulated by the supply of nerve growth factor (NGF) produced by these tissues. NGF is derived by proteolytic cleavage of a large precursor protein, proNGF, which is recognised to possess distinctive biological functions. Here, we show that proNGF promotes profuse neurite growth and branching from cultured postnatal mouse SCG neurons. In marked contrast, proNGF does not promote the growth of trigeminal neurites. Studies using compartment cultures demonstrated that proNGF acts locally on SCG neurites to promote growth. The neurite growth-promoting effect of proNGF is not observed in SCG neurons cultured from p75NTR-deficient mice, and proNGF does not phosphorylate the NGF receptor tyrosine kinase TrkA. These findings suggest that proNGF selectively promotes the growth of neurites from a subset of NGF-responsive neurons by a p75NTR-dependent mechanism during postnatal development when the axons of these neurons are ramifying within their targets in vivo.