[53] Long-term culture of dissociated sympathetic neurons

doi:10.1016/S0076-6879(79)58174-9

Methods in Enzymology

Volume 58, 1979, Pages 574-584

https://doi.org/10.1016/S0076-6879(79)58174-9 Get rights and content

Publisher Summary

This chapter discusses long-term primary culture of dissociated neurons, a technique that proved to be a valuable tool in studies of neuronal development and function. Such cultures offer the potential for systematic manipulation of the fluid medium surrounding the cells. These neuron-alone cultures are particularly well suited to biochemical analysis as the properties of any function under study are directly attributable to the neurons being cultured, without complicating contamination from other cell types. In dissociated cell culture under appropriate conditions, sympathetic neurons exhibit a normal developmental differentiation and maturation into adrenergic neurons. Medium conditioned by incubation on cultures of appropriate non-neuronal cells, when added to neuron-alone cultures produces an increase in neuronal cholinergic characteristics with a concomitant decrease in adrenergic characteristics. The extracellular environment is critically important in determining the differentiated fate of sympathetically derived neurons.

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PC12 cells that stably overexpress ERK5 shRNA or flag-ankrd1 were cultured in the presence of G418. Sympathetic neurons were dissociated from the superior cervical ganglia (SCG) of newborn rat pups as described [21,22]. Both sexes of rat pups were used without regard to sex.
Extracellular signal-regulated kinases (ERKs) play important roles in proliferation, differentiation and gene expression. In our previous study, we demonstrated that both ERK5 and ERK1/2 were responsible for neurite outgrowth and tyrosine hydroxylase (TH) expression in rat pheochromocytoma cells (PC12) (J Biol Chem 284, 23,564–23,573, 2009). However, the functional differences between ERK5 and ERK1/2 signaling in neural differentiation remain unclear. In the present study, we show that ERK5, but not ERK1/2 regulates TH levels in rat sympathetic neurons. Furthermore, microarray analysis performed in PC12 cells using ERK5 and ERK1/2-specific inhibitors, identified ankyrin repeat domain 1 (ankrd1) as an ERK5-dependent and ERK1/2-independent gene. Here, we report a novel role of the ERK5/ankrd1 signaling in regulating TH levels and catecholamine biosynthesis. Ankrd1 mRNA was induced by nerve growth factor in time- and concentration-dependent manners. TH levels were reduced by ankrd1 knockdown with no changes in the mRNA levels, suggesting that ankrd1 was involved in stabilization of TH protein. Interestingly, ubiquitination of TH was enhanced and catecholamine biosynthesis was reduced by ankrd1 knockdown. Finally, we examined the relationship of ERK5 to TH levels in human adrenal pheochromocytomas. Whereas TH levels were correlated with ERK5 levels in normal adrenal medullas, ERK5 was down-regulated and TH was up-regulated in pheochromocytomas, indicating that TH levels are regulated by alternative mechanisms in tumors. Taken together, ERK5 signaling is required for catecholamine biosynthesis during neural differentiation, in part to induce ankrd1, and to maintain appropriate TH levels. This pathway is disrupted in pathological conditions.
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We focus here on our own current methods. Similar or alternative methods for culturing these neuronal cell types, which include detailed descriptions of the tissue dissection and culturing procedures, can be found elsewhere (Goslin, Hannelore, & Banker, 1998; Hawrot & Patterson, 1979; He & Baas, 2003; Higgins, Lein, Osterhout, & Johnson, 1991; Johnson & Argiro, 1983; Johnson, Iacovitti, Higgins, Bunge, & Burton, 1981; Kaech & Banker, 2006; Kleitman, Wood, & Bunge, 1998; Mahanthappa & Patterson, 1998). To establish and maintain primary neuronal cultures, we use fine dissecting instruments, a binocular dissecting microscope capable of 8–25x magnification, a horizontal laminar flow hood, and a tissue culture incubator with temperature and CO2 control as well as active or passive humidification.
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Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at Ca_V1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca²⁺/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is γCaMKII, its phosphorylation at Thr287 by βCaMKII protects the Ca²⁺/CaM signal, and CaN triggers its nuclear translocation. Both βCaMKII and CaN act in close proximity to Ca_V1 channels, supporting their dominance, whereas γCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca²⁺/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of Ca_V1, γCaMKII, βCaMKII, and CaN in multiple neuropsychiatric disorders.
Hierarchically structured nerve guidance channels based on poly-3-hydroxybutyrate enhance oriented axonal outgrowth
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To inhibit glial cell proliferation 0.24 μg ml−1 1-β-d-arabinofuranosylcytosine (Sigma) was added to the medium 24 h after seeding. SCG were cultured in Neurobasal A Medium (Life Technologies) supplemented with 1% N2-supplement (Gibco), Pen/Strep (200 U ml−1 and 200 mg ml−1), 0.7% B27 (Life Technologies), l-glutamine (2 mM), 10% FCS (Sigma) and NGF (100 ng ml−1, BD Bioscience) as previously described [47]. In order to allow Schwann cell growth and development, 1-β-d-arabinofuranosylcytosine was not added to SCG medium.
Traumatic peripheral nerve lesions can cause local anesthesia, paralysis and loss of autonomic control. Reconstruction using engineered nerve guidance conduits (NGCs) is rarely successful due to the sub-optimal characteristics of the conduits. To address the demands of clinical practice, we developed a hierarchically structured NGC from slowly resorbing poly(3-hydroxybutyric acid) (P3HB). The NGC consists of a permeable single-lumen tube and melt-spun fibrillar lumen fillers. Permeable tubes were constructed from P3HB/poly(ɛ-caprolactone) (PCL) blends or poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) (P(3HB-co-4HB)). Polyvinylpyrrolidone was used as a porogen in solvent-free thermoplastic processing, followed by selective polymer leaching. All tested material compositions showed hydrolytic degradation after 16 weeks in phosphate buffered saline, whereas P3HB/PCL tubes maintained mechanical strength compared to (P(3HB-co-4HB)). The porous scaffolds allowed diffusion of large molecules (∼70 kDa). In vitro studies demonstrated that mouse fibroblasts survived and proliferated inside closed porous tubes. An in vitro model of axonal regeneration using dorsal root ganglia and sympathetic cervical ganglia demonstrated that the NGCs successfully supported neuron survival and neurite outgrowth. The introduction of fibrillar lumen fillers promoted oriented neurite growth and coating with extracellular matrix proteins further increased ganglia attachment and cell migration. In this study we show that P3HB-based NGCs scaffolds have potential in long gap peripheral nerve repair strategies.
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Activity-dependent gene expression triggered by Ca²⁺ entry into neurons is critical for learning and memory, but whether specific sources of Ca²⁺ act distinctly or merely supply Ca²⁺ to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca_V1 and Ca_V2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca_V1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca²⁺ to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca_V2 channels must elevate [Ca²⁺]_i microns away and promote CaMKII aggregation at Ca_V1 channels. Consequently, Ca_V2 channels are ∼10-fold less effective in signaling to the nucleus than are Ca_V1 channels for the same bulk [Ca²⁺]_i increase. Furthermore, Ca_V2-mediated Ca²⁺ rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca²⁺, further attenuating Ca_V2-mediated gene expression.
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In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTβ2. In mice, CTβ2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTβ2 plays an important role in the nervous system. We, therefore, investigated the importance of CTβ2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTβ2, but not CTα, mRNA and protein. The brains of mice lacking CTβ2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTβ2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTβ2 deficiency in distal axons reduced the incorporation of [³H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTβ2 is the major CT isoform involved in PC synthesis in axons. Axons of CTβ2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTβ2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.

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[53] Long-term culture of dissociated sympathetic neurons

Publisher Summary

Dev. Biol.

Brain Res.

Exp. Cell Res.

Dev. Biol.

Brain Res.

Brain Res.

Brain Res.

Annu. Rev. Neurosci.

J. Neurocytol.

Lab. Invest.

J. Natl. Cancer Inst.