Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line

Abstract

Development of neuroprotective strategies for peripheral neuropathies requires high-throughput drug screening assays with appropriate cell types. Currently, immortalized dorsal root ganglion (DRG) sensory neuronal cell lines that maintain nociceptive sensory neuronal properties are not available. We generated immortalized DRG neuronal lines from embryonic day 14.5 rats. Here, we show that one of the immortalized DRG neuronal lines, 50B11, has the properties of a nociceptive neuron. When differentiated in the presence of forskolin, these cells extend long neurites, express neuronal markers, and generate action potentials. They express receptors and markers of small-diameter sensory neurons and upregulate appropriate receptor populations when grown in the presence of glial cell line-derived neurotrophic factor or nerve growth factor. Furthermore, they express capsaicin receptor transient receptor potential vanilloid family-1 (TRPV-1) and respond to capsaicin with increases in intracellular calcium. In a 96-well plate format, these neurons show a decline in ATP levels when exposed to dideoxycytosine (ddC) in a proper time- and dose-dependent manner. This ddC-induced reduction in ATP levels correlates with axonal degeneration. The immortalized DRG neuronal cell line 50B11 can be used for high-throughput drug screening for neuroprotective agents for axonal degeneration and antinociceptive drugs that block TRPV-1.

Figure 1

Immortalized DRG neuronal cells extend neurites, express neuronal markers, and generate action potentials after differentiation. Phase contrast images of (A) undifferentiated and (B) differentiated 50B11 cells show extension of axons 4 h after differentiation with forskolin. Twenty-four hours after differentiation, 50B11 cells stain with (C) antineurofilament and (D) anti-βIII-tubulin antibodies. 4′,6-diamidino-2-phenylindole counterstain shows nuclei. Scale bar = 20 μm. (E) A representative action potential is seen in a differentiated 50B11 cell after application of a depolarizing current.

Figure 2

The 50B11 neuronal line expresses markers of small-diameter sensory neurons. Immunofluorescence images of 50B11 cells stained with (A) isolectin B4 and (B) anticalcitonin gene–related protein. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole. Scale bar = 20 μm. (C) Changes in expression of mRNA for p75, Trk-A, c-ret, and GDNF family receptor alpha-1 in the presence of forskolin (FSK), nerve growth factor, and glial cell line–derived neurotrophic factor (GDNF) compared with baseline levels of undifferentiated 50B11 cells. n = 6–8/group, error bars denote SEM, *p < 0.05 compared with baseline, **p < 0.05 FSK + GDNF vs. FSK + nerve growth factor.

Figure 3

The 50B11 neuronal line expresses nociceptive markers and respond to capsaicin. (A) Immunofluorescence image of 50B11 cells stained with anti-transient receptor potential vanilloid family-1 (TRPV-1) antibody. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole. Scale bar = 25 μm. (B) Fold change in TRPV-1 mRNA in response to differentiation and neurotrophic factor treatment is seen. n = 6–8/group, error bars denote SEM, *p < 0.05 compared with baseline, **p < 0.05 forskolin (FSK) + glial cell line–derived neurotrophic factor vs. FSK + nerve growth factor. (C) Measurements of intracellular calcium levels of undifferentiated 50B11 with vehicle control treatment (red line), after differentiation with FSK with (green), and without (blue) capsazepine pretreatment. Each line represents the average of three to five fields of cells with multiple cells in each view. Single arrow indicate the time at which capsazepine was added and double arrows indicate the time at which capsaicin was added.

Figure 4

Capsaicin-induced neurotoxicity. The 50B11 cells were grown in 96-well plates and treated with varying doses of (A) capsaicin and (B) capsaicin + capsazepine for 24 h; cellular ATP levels were measured and expressed as a percentage of control cultures. n = 8/condition, error bars denote SEM, *p < 0.05 compared with controls.

Figure 5

Neurotoxicity assay suitable for high-throughput screening. The 50B11 cells were grown in 96-well plates and treated with varying doses of (A) dideoxycytosine (ddC) and ddC + GPI-1046 for 24 h; cellular ATP levels were measured and expressed as a percentage of control cultures. n = 8/ condition, error bars denote SEM, *p < 0.05 compared with controls. In validation experiments, 50B11 cells were differentiated and allowed to extend their neurites 24 h. (B) Then, they were treated with ddC or ddC + GPI-1046 for another 24 h, and total neurite lengths were measured. n = 6/condition, error bars denote SEM, *p < 0.05 compared with controls.