Cultures Of Sympathetic Neurons Research Articles

Innervated adrenomedullary microphysiological system to model nicotine and opioid exposure.

Transition to extrauterine life results in a surge of catecholamines necessary for increased cardiovascular, respiratory, and metabolic activity. Mechanisms mediating adrenomedullary catecholamine release are poorly understood. Important mechanistic insight is provided by newborns delivered by cesarean section or subjected to prenatal nicotine or opioid exposure, demonstrating impaired release of adrenomedullary catecholamines. To investigate mechanisms regulating adrenomedullary innervation, we developed compartmentalized 3D microphysiological systems (MPS) by exploiting GelPins, capillary pressure barriers between cell-laden hydrogels. The MPS comprises discrete cultures of adrenal chromaffin cells and preganglionic sympathetic neurons within a contiguous bioengineered microtissue. Using this model, we demonstrate that adrenal chromaffin innervation plays a critical role in hypoxia-mediated catecholamine release. Opioids and nicotine were shown to affect adrenal chromaffin cell response to a reduced oxygen environment, but neurogenic control mechanisms remained intact. GelPin containing MPS represent an inexpensive and highly adaptable approach to study innervated organ systems and improve drug screening platforms.

Membrane Depolarization Inhibits BIMEL Upregulation but Prevents Neuronal Apoptosis Primarily by Increasing Cellular GSH Levels.

Sympathetic neurons deprived of nerve growth factor (NGF) die by apoptosis. Chronic depolarization with elevated concentrations of extracellular potassium ([K+]E) supports long-term survival of these and other types of neurons in culture. While depolarization has long been used to support neuronal cultures, little is known about the mechanism. We explored how chronic depolarization of NGF-deprived mouse sympathetic neurons in culture blocks apoptotic death. First, we determined the effects of elevated [K+]E on proapoptotic BH3-only proteins reported to be upregulated in sympathetic neurons after NGF withdrawal. Upregulation of BIMEL was blocked by depolarization while upregulation of PUMA was not. BMF levels did not increase after NGF withdrawal, and elevated [K+]E had no effect on its expression. dp5/HRK was not detectable. A large increase in production of mitochondria-derived reactive species (RS), including reactive oxygen species (ROS), occurs in NGF-deprived sympathetic neurons. Suppressing these RS prevents cytochrome c release from mitochondria and apoptosis. The addition of high [K+]E to cultures rapidly blocked increased RS and cytochrome c release. Elevated [K+]E caused an increase of the cellular antioxidant glutathione (GSH). Preventing this increase prevented the elevated [K+]E from blocking cytochrome c release and death. While suppression of BIMEL upregulation by elevated [K+]E may contribute to high [K+]E pro-survival effects, we conclude that elevated [K+]E prevents apoptotic death of NGF-deprived sympathetic neurons primarily via an antioxidant mechanism.

Axon Degeneration Assays in Superior Cervical Ganglion Explant Cultures.

The ability of peripheral nervous system neurons to extend long, axon-like neurites in vitro makes them ideally suited for studies on mechanisms of axon survival and degeneration. In this chapter, we describe how to prepare explant cultures of sympathetic neurons of the superior cervical ganglion (SCG). We also describe how to induce and assess axon degeneration with an injury or a chemical insult.

Microinjection of Superior Cervical Ganglion Neurons for Studying Axon Degeneration.

Primary cultures of neurons of the peripheral nervous system have been successfully used for studying many aspects of neuronal development and survival, including investigations into the mechanisms of axon degeneration. In this chapter, we describe how to prepare and microinject dissociated cultures of sympathetic neurons of the superior cervical ganglion (SCG) specifically for use in highly controlled and targeted assays of axon survival and degeneration.

C-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

Millisecond Ca2+ dynamics activate multiple protein cascades for synaptic vesicle control.

For reliable transmission at chemical synapses, neurotransmitters must be released dynamically in response to neuronal activity in the form of action potentials. Stable synaptic transmission is dependent on the efficacy of transmitter release and the rate of resupplying synaptic vesicles to their release sites. Accurate regulation is conferred by proteins sensing Ca2+ entering through voltage-gated Ca2+ channels opened by an action potential. Presynaptic Ca2+ concentration changes are dynamic functions in space and time, with wide fluctuations associated with different rates of neuronal activity. Thus, regulation of transmitter release includes reactions involving multiple Ca2+-dependent proteins, each operating over a specific time window. Classically, studies of presynaptic proteins function favored large invertebrate presynaptic terminals. I have established a useful mammalian synapse model based on sympathetic neurons in culture. This review summarizes the use of this model synapse to study the roles of presynaptic proteins in neuronal activity for the control of transmitter release efficacy and synaptic vesicle recycling.

Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons

During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as 'competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death.

Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons.

During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as 'competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death.

Neurotrophin-dependent plasticity of neurotransmitter segregation in the rat superior cervical ganglion in vivo.

Neurons are able to segregate transmitters to different axon endings. Segregation is a plastic neuronal feature; it can be modulated by synaptic environment. We have demonstrated that neurotrophin and other cellular factors regulate segregation in sympathetic neurons in culture. Herein we tested the hypothesis that sympathetic neurons in vivo are also capable to exhibit neurotrophin-dependent plasticity of segregation. To explore the effect of neurotrophin on segregation, we reduced ganglionic NGF content by the transection of postganglionic nerves (axotomy) of the superior cervical ganglia. By immunohistochemistry, Western blot, and PCR analyses, we explored the effect of axotomy on the NGF and BDNF content of ganglionic neurons, and on the segregation extent of vesicular acetylcholine transporter (VAChT) and methionine enkephalin (mENK) in pre-ganglionic varicosities. We analyzed NGF-dependence of the changes found by applying exogenous NGF. Axotomy reduced ganglionic NGF and BDNF content, increased NGF transcripts, and increased VAChT-mENK segregation. Axotomy also increased the number of VAChT immunopositive varicosities, and caused the appearance of a population of VAChT-, mENK- or SV2-containing varicosities lacking Synaptophysin (Syn). Administration of NGF prevented changes in NGF content, kept NGF transcripts increased, and counteracted changes in segregation and in the number of cholinergic varicosities. The exogenous NGF did not preclude change in BDNF content or in the occurrence of the VAChT- or mENK-containing varicosities lacking Syn. Data demonstrate that segregation of transmitters in vivo is plastic and it is modulated by environmental signals like NGF. We propose a possible functional correlate of segregation plasticity in the sympathetic ganglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 832-846, 2016.

Dendrite Complexity of Sympathetic Neurons Is Controlled during Postnatal Development by BMP Signaling

Dendrite development is controlled by the interplay of intrinsic and extrinsic signals affecting initiation, growth, and maintenance of complex dendrites. Bone morphogenetic proteins (BMPs) stimulate dendrite growth in cultures of sympathetic, cortical, and hippocampal neurons but it was unclear whether BMPs control dendrite morphology in vivo. Using a conditional knock-out strategy to eliminate Bmpr1a and Smad4 in immature noradrenergic sympathetic neurons we now show that dendrite length, complexity, and neuron cell body size are reduced in adult mice deficient of Bmpr1a. The combined deletion of Bmpr1a and Bmpr1b causes no further decrease in dendritic features. Sympathetic neurons devoid of Bmpr1a/1b display normal Smad1/5/8 phosphorylation, which suggests that Smad-independent signaling paths are involved in dendritic growth control downstream of BMPR1A/B. Indeed, in the Smad4 conditional knock-out dendrite and cell body size are not affected and dendrite complexity and number are increased. Together, these results demonstrate an in vivo function for BMPs in the generation of mature sympathetic neuron dendrites. BMPR1 signaling controls dendrite complexity postnatally during the major dendritic growth period of sympathetic neurons.

Caspase‐generated neoepitopes as markers of axonal degeneration in neural development and injury

Neuronal death, axonal degeneration, and synapse loss are hallmarks of central nervous system development and injury. Commonly employed markers of apoptosis are TUNEL and immunohistochemistry for activated caspases, but these often fail to report axonal degeneration associated with somatic apoptosis. Axonal degeneration is classically detected by silver degeneration stains, but these have limitations (non‐selectivity, minimal reproducibility and incompatibility with co‐labeling). We demonstrate that antibodies to caspase‐cleaved protein substrates beta‐actin (cleaved by caspase 3 at D244) and alpha‐tubulin (cleaved by caspase 6 at D438) enable superior visualization of neurite degeneration. These antibodies label apoptotic cell bodies and neurites during embryonic and postnatal developmental pruning. In addition, they reveal injured neurons and neurites after induction of apoptosis in a mouse model of fetal alcohol syndrome and in sympathetic neuron cultures induced to undergo apoptosis after nerve growth factor withdrawal. Finally, the markers may have clinical utility, as they highlight degenerating neurites in neonatal hypoxic/ischemic injury and in adult patients with multiple sclerosis. Ongoing work aims to identify novel compartment‐specific neoepitopes that will allow monitoring of somatodendritic, axonal, and synaptic remodeling and degeneration. Supported by NS075869.

A HaloTag® method for assessing the retrograde axonal transport of the p75 neurotrophin receptor and other proteins in compartmented cultures of rat sympathetic neurons

We have adapted HaloTag® (HT) technology for use in compartmented cultures of rat sympathetic neurons in order to provide a technique that can be broadly applied to studies of the retrograde transport of molecules that play roles in neurotrophin signaling. Transfected neurons expressing HT protein alone, HT protein fused to the p75 neurotrophin receptor (p75NTR) or HT protein fused to tubulin α-1B were maintained in compartmented cultures in which cell bodies and proximal axons of rat sympathetic neurons reside in proximal compartments and their distal axons extend into distal compartments. HT ligand containing a fluorescent tetramethylrhodamine (TMR) label was applied either in the distal compartments or the proximal compartments, and the transport of labeled proteins was assayed by gel fluorescence imaging and TMR immunoblot. HT protein expressed alone displayed little or no retrograde transport. HT protein fused to either the intracellular C-terminus or the extracellular N-terminus of p75NTR was retrogradely transported. The retrograde transport of p75NTR was augmented when the distal axons were provided with nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or antibodies to BDNF. The anterograde transport of HT protein fused to the N-terminus of tubulin α-1B was also demonstrated. We conclude that retrograde transport of HT fusion proteins provides a powerful and novel approach in studies of axonal transport.

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive (1-3), and influences the types and distribution of these inputs (4-6). Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models (7), and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders (8-10) and neurodegenerative diseases (11-13). Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases. Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture (14,15). However, the addition of either bone morphogenetic protein-7 (BMP-7) (16,17) or Matrigel (18) to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons (19) that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies (17,18,20,21). Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth (17,18). Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

Axonal Neuropathy-associated TRPV4 Regulates Neurotrophic Factor-derived Axonal Growth

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission

Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin typeA) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin typeE). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.

Electrophysiological Properties of Cultured Sympathetic Neurons of the Rat Superior Cervical Ganglion

We obtained a viable culture of sympathetic neurons of the superior cervical ganglion of neonatal rats. Electrophysiological experiments (patch-clamp in the whole-cell configuration) showed that the cultured neurons generated action potentials after intercellular stimulation by long-lasting pulses of depolarizing current. In some neurons, background impulse electrical activity and evoked postsynaptic currents suppressed by applications of benzohexonium were observed.

Vascular endothelial growth factor protects post-ganglionic sympathetic neurones from the detrimental effects of hydrogen peroxide by increasing catalase

Vascular production of hydrogen peroxide (H(2)O(2)) is implicated in the development and progression of vascular disease. Hydrogen peroxide also promotes neuronal degeneration, which suggests that vascular H(2)O(2) would promote degeneration of perivascular sympathetic nerves. Vascular cells also produce vascular endothelial growth factor (VEGF), which could protect perivascular nerves from the detrimental effects of H(2)O(2) . The aim of this study was to test these hypotheses. The effects of H(2)O(2) and VEGF on neuronal survival and noradrenaline uptake were studied in cultures of rat post-ganglionic sympathetic neurones. Western analyses of catalase and growth associated protein 43 were performed and reactive oxygen species (ROS) were measured using the fluorescent indicator 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate, acetyl ester. Hydrogen peroxide (30 μm) decreased the survival of post-ganglionic sympathetic neurones (57.8 ± 4.8% of control) and decreased noradrenaline uptake into the neurones (14 ± 6% of control). Hyperglycaemia, which is known to increase H(2)O(2), also decreased survival (31.4 ± 12% of control) and noradrenaline uptake (42 ± 18.4% of control). VEGF reduced the effects of H(2)O(2) (94.3 ± 12% of control) and hyperglycaemia (83.5 ± 23.6% of control) on survival. VEGF increased catalase, a primary determinant of intracellular concentrations of H(2)O(2) , and decreased H(2)O(2) -induced increases in ROS. These results indicate that VEGF protects post-ganglionic sympathetic neurones from the detrimental effects of H(2)O(2). Our data suggest that an increase in catalase is the mechanisms underlying this neuroprotection.

β-Adrenoceptor Blockers Increase Cardiac Sympathetic Innervation by Inhibiting Autoreceptor Suppression of Axon Growth

β-Adrenoceptor antagonists are used widely to reduce cardiovascular sympathetic tone, but withdrawal is accompanied by sympathetic hyperactivity. Receptor supersensitivity accounts for some but not all aspects of this withdrawal syndrome. Therefore, we investigated effects of β-blockers on sympathetic innervation. Rats received infusions of adrenergic receptor blockers or saline for 1 week. The nonselective β-blocker propranolol and the β(1)-antagonist metoprolol both increased myocardial sympathetic axon density. At 2 d after propranolol discontinuation, β-receptor sensitivity and responsiveness to isoproterenol were similar to controls. However, tyramine-induced mobilization of norepinephrine stores produced elevated ventricular contractility consistent with enhanced sympathetic neuroeffector properties. In addition, rats undergoing discontinuation showed exaggerated increases in mean arterial pressure in response to air puff or noise startle. In sympathetic neuronal cell cultures, both propranolol and metoprolol increased axon outgrowth but the β(2)-blocker ICI 118551 did not. Norepinephrine synthesis suppression by α-methyl-p-tyrosine also increased sprouting and concurrent dobutamine administration reduced it, confirming that locally synthesized norepinephrine inhibits outgrowth via β(1)-adrenoceptors. Immunohistochemistry revealed β(1)-adrenoceptor protein on sympathetic axon terminations. In rats with coronary artery ligation, propranolol reversed heart failure-induced ventricular myocardial sympathetic axon depletion, but did not affect infarct-associated sympathetic hyperinnervation. We conclude that sympathetic neurons possess β(1)-autoreceptors that negatively regulate axon outgrowth. Chronic β-adrenoceptor blockade disrupts this feedback system, leading to ventricular sympathetic axon proliferation and increased neuroeffector gain, which are likely to contribute to β-blocker withdrawal syndrome.

An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons.

mRNA localization is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. To identify mRNAs localized in subcellular compartments of developing neurons, we took an original approach that combines compartmentalized cultures of rat sympathetic neurons and sequential analysis of gene expression (SAGE). Unexpectedly, the most abundant transcript in axons was mRNA for myo-inositol monophosphatase-1 (Impa1), a key enzyme that regulates the inositol cycle and the main target of lithium in neurons. A novel localization element within the 3' untranslated region of Impa1 mRNA specifically targeted Impa1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to nerve growth factor (NGF). Selective silencing of IMPA1 synthesis in axons decreased nuclear CREB activation and induced axonal degeneration. These results provide insights into mRNA transport in axons and reveal a new NGF-responsive localization element that directs the targeting and local translation of an axonal transcript.

Production of compartmented cultures of rat sympathetic neurons

The compartmented culture, in which primary neurons plated in a proximal compartment send their axons under silicone grease barriers and into left and right distal compartments, has enhanced the experimental capabilities of neuronal cultures. Treatments can be applied separately to cell bodies/proximal axons or distal axons, and cell bodies/proximal axons and distal axons can be separately harvested and analyzed. Distal axons can be axotomized, and the neurons can be studied while their axons regenerate. Construction of the culture dishes requires 3 h for 48 cultures, and preparing the neurons also requires 3 h. Compartmented cultures provide enough cellular material for biochemical analyses such as immunoblotting. The uses of compartmented cultures have included studies of neurotrophic factor retrograde signaling, axonal transport, and axonal protein and lipid biosynthesis. Here we focus on sympathetic neurons cultured from neonatal rats and provide protocols for the production and some of the uses of compartmented cultures.