Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

Abstract

Background: Nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception.

Results: Compared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K+-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the Emax without altering the EC50 for either compound.

Conclusions: Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system.

Figure 1

Experimental design for neuronal counting. 12 slides were utilized in these experiments, with 4 slides for each growth factor. The antibodies used on each slide are shown to the right of the slide along with the wavelength for the corresponding secondary antibody. For the 2 matching wells for each slide (growth factor concentration), the neuron numbers were averaged to give one observation (since the wells were not derived from independent cultures). Each slide contained a no growth factor control (blue), hence n = 9 for this condition. All other growth factors at 1 (i.e. NGF, red), 10 (i.e. GDNF, green) or 100 ng/ml (i.e. BDNF, yellow) are n = 3. The same slides were then utilized to ascertain the proportions of neurons expressing CGRP- or TRPV1-immunoreactivity or IB₄-binding, as described in Methods. This figure refers to Figures 2 and 3 and Table 1.

Figure 2

Representative 20X photomicrographs of growth factor-treated TG neurons. TG cultures were fixed and labeled for NF-H-immunoreactivity (green), and the 20X photomicrographs depicted here are representative of the 20 images taken per slide for each condition for neuron counting. The upper frame shows the no growth factor treated control with corresponding growth factor treatments shown for BDNF, GDNF and NGF at 1, 10 and 100 ng/ml concentrations.

Figure 3

NGF, GDNF and BDNF and TG neuronal surival. All NF-H-immunoreactive neurons were counted for each growth factor-treated TG culture (n = 9 no growth factor; n = 3 all other conditions) and compared with the control (no growth factor-treated) TG cultures to assess neuronal survival at day 5 post-plating (* p < 0.05, *** p < 0.001).

Figure 4

Assessment of TRPV1 mRNA levels by Realtime PCR. Levels of TRPV1 mRNA are depicted as fold change compared with no growth factor-treated cultures for each growth factor condition normalized to its corresponding GAPDH mRNA levels (n = 3).

Figure 5

Assessment of TRPV1 protein by Western Blot. Panel A, 20 μg total protein was electrophoretically seperated per lane and transferred to membranes that were subsequently probed with an anti-TRPV1 antibody and then reprobed for standardization with β-actin (X = no growth factor; N 100 = NGF 100 ng/ml; G 100 = GDNF 100 ng/ml). Image is of a representative Western blot. Immunoreactivity to a protein corresponding to the size of the glycosyated form of TRPV1 was detected at ~130 kD. Panel B depicts alterations in TRPV1 protein levels standardized to β-actin (* p < 0.05, n = 3)

Figure 6

50 mM K+ -evoked CGRP release and total CGRP content in TG cultures. Panel A illustrates the effect of growth factor treatment on 50 mM K+ -evoked CGRP release, while panel B shows the data standardized to the number of CGRP-positive neurons per condition as a proportion of the no growth factor-treated cultures. Panel C shows total CGRP content by growth factor treatment, and again, panel D shows this data standardized to CGRP-positive neurons, as stated above (*** p < 0.001, n = 6).

Figure 7

Representative 20X photomicropgraphs of colocalization of sensory neurons markers with NF-H in growth factor-treated TG neurons. Immunoreactivity for CGRP and TRPV1 and staining for IB₄-binding sites (red) was assessed following immunocytochemistry for NF-H (green) to assess the proportion of neurons expressing these population markers. Representative 20X photomicrographs of each growth factor at 100 ng/ml for CGRP (left), TRPV1 (middle) and IB₄ (right) are shown as well as control (no growth factor-treated) TG cultures (top panels). All cell bodies containing both NF-H-immunoreactivity and CGRP- or TRPV1-immunoreactivity or IB₄-binding appear yellow from the overlay of the red with green. In no cases were neurons observed that contained CGRP- or TRPV1-immunoreactivity or IB₄-binding without the co-presence of NF-H-immunoreactivity.

Figure 8

Growth factor treatment increases CAP (100 nM)-evoked CGRP release in TG cultures. Panel A illustrates the total amount of CGRP released by 10 min treatment with capsaicin for each growth factor. Panel B illustrates the same data standardized to the number of TRPV1-positive neurons under each growth factor condition (** p < 0.01, *** p < 0.01 for comparisons to no growth factor treated controls; ## p < 0.01, ### p < 0.001 for comparisons to NGF at the equivalent growth factor concentration by two-way ANOVA with Bonferroni post-test; n = 6).

Figure 9

NGF and GDNF alter capsaicin-evoked CGRP release. Concentration-response functions (molar, log units) are shown for capsaicin-evoked CGRP release in TG cultures maintained in 100 ng/ml NGF, 100 ng/ml GNDF or no growth factor conditions (n = 6). Panel A illustrates the maximum relative effect of capsaicin under each condition matched to the maximum evoked-CGRP release for that given condition. Panel B depicts the CAP-evoked CGRP release data in terms of CGRP released in fmoles in the presence or absence of NGF or GDNF to illustrate the magnitude of increases in E_max and the biphasic nature of the capsaicin-evoked CGRP concentration-response function.

Figure 10

The effect of growth factor treatment on AEA- and ACEA-evoked CGRP release. TG neurons were grown in the presence of the indicated concentrations of NGF (panel A), GDNF (panel B) or BDNF (panel C) for five days and then exposed to 100 nM CAP, 30 μM AEA or 30 μM ACEA for 10 min, and the released CGRP was quantified (n = 12). Panel D, TG neurons were grown in the absence of growth factors or in the presence of 100 ng/ml NGF or GDNF for 5 days and exposed to the indicated concentrations of AEA (molar, log units) to assess CGRP release (### p < 0.001; GDNF and NGF vs. no growth factor; two-way ANOVA, Bonferroni post-test, n = 6).

Figure 11

Equation used to normalize CGRP release. "No GF" refers to no growth factor controls and "GF condition" refers to any given growth factor treated condition