Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin

Abstract

Background: Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated.

Results: Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X(3) and TRPV1 receptors after 1-4 days in culture (together with their more frequent co-localization), while P2X(2) ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca(2+) imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist alpha, beta-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X(3) receptors (selectively antagonized by A-317491) and heteromeric P2X(2/3) receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X(3) receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X(3) receptors.

Conclusion: Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.

Figure 1

Survival of neurons in TG ganglia or in culture. Survival of neurons (calculated as neuron density expressed as number of β-tubulinIII positive cells per unit area) at 1–4 days in culture. Data are normalized with respect to those at 1 day (n = 250). *: P < 0.05.

Figure 2

Real time RT-PCR and western immunoblot of ganglia and cultures. A, real time RT-PCR of ganglia and cultures from rat and mouse. Ordinate: relative increment with respect to the ganglion products. For each receptor, amplification values were normalized with β-tubulinIII mRNA levels and compared with the ganglion mRNA levels. GAPDH amplification control was the same in all reactions. B, western immunoblots of equal amounts of neuronal protein lysates (β-tubulinIII) derived from ganglia or culture. Immunodetection of P2X₃, P2X₂ and TRPV1 mature proteins revealed proper migration (a) in accordance with Vulchanova et al.[53] for the predominant P2X₃ form (57 kDa), with Newbolt et al. [54] for the P2X₂ mature receptor (65 kDa) and with Kedei et al. [55] for the TRPV1 mature receptor (120 kDa). Panel B b shows P2X₃ native (45 kDa) and intermediate polypeptides (up to 50 kDa) detected one d in culture (1) but not in the ganglion (T) as reported by Nicke et al. [56] C, relative optical density values of mature receptors at 1–4 days in culture (normalized with respect to β-tubulinIII) and compared to ganglion values. For all experiments shown in A-C n = 3 animals for ganglion or day in culture datapoint (each point is mean ± SEM). *: P < 0.05; **: P < 0.01.

Figure 3

P2X and TRPV1 immunoreactivity in ganglia and cultures. A, example of P2X₃ receptor expression by β-tubulinIII labeled neurons in a fixed ganglion (left; calibration bar = 50 μm). After one day in culture, medium-size neurons that are also labeled by β-tubulinIII are immunoreactive for P2X₃ (middle) or TRPV1 (right; calibration bar = 50 μm) receptors. B, percentage of neurons (β-tubulinIII positive) immunoreactive for P2X₃, P2X₂ and TRPV1 in the ganglion and at different d in culture, for rat (left) and mouse (right; n = 3 animals in all cases). The number of P2X₃ and TRPV1 immunoreactive neurons increase significantly after dissection and remain constant in culture, while P2X₂ receptor labeled neurons remain unchanged. *: P < 0.05 for indicated pair of histograms. Values at later d in culture are also significantly different from ganglia. C, comparison of TRPV1 receptor immunoreactivity of rat (left) or mouse (right) ganglia. Note larger number of immunoreactive neurons in the rat tissue. Calibration bars = 50 μm.

Figure 4

Receptor co-localization investigated with double immunofluorescence. Percent of neurons showing double immunoreactivity for pairs of P2X₃, P2X₂ and TRPV1 as indicated (rat data are on the left while mouse ones are on the right). The "reference" receptor type is taken as 100%. Data from ganglia (about 1,000 neurons) or at different d in culture (about 500 cells per culture) are shown with differently shaded histograms. n = 3 animals for each bar. *: P < 0.05. Values at later d in culture are also significantly different from ganglia. Further details are in the legend to Fig. 3.

Figure 5

Calcium imaging indicates functional expression of P2X and TRPV1 receptors. A, examples of Ca²⁺ transients in mouse TG neurons activated by 2 s application of 10 μM α, β-meATP or 1 μM capsaicin. a, example of cell responding to α, β-meATP only ('purinergic phenotype'); b, responding to capsaicin only ('vanilloid phenotype'); c, responding to both agonists ('mixed phenotype'). All cells respond to pulse application of KCl. B, number of neurons sensitive to α, β-meATP (regardless of their response to capsaicin), to capsaicin (regardless of their response to α, β-meATP) or to both agonists, for mouse (n = 346; left) and rat (n = 41; right) TG neurons. Neurons (kept in culture for 24 h) were identified by their responsiveness to 15 mM KCl (1 s), while satellite cells and fibroblasts did not respond to this agent. Note that the percent of cells responding to capsaicin was higher in the rat (55%) than in the mouse (33%).

Figure 6

Characteristics of pain receptors of cultured TG neurons. A, examples of currents recorded from rat and mouse TG neurons in culture (24 h) during application of α, β-meATP (10 μM, 2 s; upper row) and capsaicin (10 μM, 2 s for rat; 1 μM, 2 s for mouse; bottom row). B, log dose-response curves for rat (filled symbols; n = 9) and mouse (grey symbols; n = 12) neurons cultured for 24 h. Both potency and efficacy of α, β-meATP were similar for rat and mouse. C, distribution of values of the residual current (I_residual) present at the end of an application of α, β-meATP (10 μM, 2 s) in rat and mouse neurons cultured for 24 h, expressed as a fraction of the peak current (I_peak; n = 24 and 58 for rat and mouse, respectively). Inset shows an example of the mixed type of current recorded from a subset of rat TG neurons. Dashed line indicates the 5% arbitrary threshold above which currents were considered to be mixed.

Figure 7

Functional characterization of rat and mouse TG neurons. A, fraction of cells responding to α, β-meATP (independently from their response to capsaicin), to capsaicin (independently from their response to α, β-meATP) or to both agonists for rat (left panel; n = 33) and mouse (right panel; n = 50). B, persistence of the responsiveness of rat TG neurons in culture to α, β-meATP and capsaicin. a, Proportion of cells responding to α, β-meATP (black bars; n = 7, 7, 10 cell cultures for the 1^st, 2^nd and 3^rd day, respectively) and capsaicin (grey bars; n = 7, 8 and 8 cell cultures for the 1^st, 2^nd and 3^rd day). b, peak amplitude of currents elicited by α, β-meATP (black bars; n = 22, 11 and 31 cells for the 1^st, 2^nd and 3^rd day, respectively) and capsaicin (grey bars; n = 16, 13 and 10 cells for the 1^st, 2^nd and 3^rd day). Only cells responding to the agonist were included in the current analysis.

Figure 8

Modulation of receptor function by chronically applied NGF or 5-HT. A, Ca²⁺ imaging of single neurons shows percent increase in mouse TG cells responsive to α, β-meATP (a) in control or after application of 50 ng/ml NGF (24 h; n = 6 culture dishes). In 6 sister cultures there was no significant change as far as responses to capsaicin (b) were concerned. *: P < 0.05. B, a, patch clamp current records show increased amplitude of mouse responses to α, β-meATP after 24 h NGF treatment, while responses to capsaicin remained equiamplitude. B, b, histograms summarizing the significant (*: P < 0.05) rise in α, β-meATP evoked current amplitude (n = 26) without significant change in capsaicin responses (n = 14). Data are expressed as % of control amplitude in sister cultures. C, 5-HT (10 μM; 24 h) upregulates the amplitude of rat capsaicin current without affecting responses to α, β-meATP (a). C,b shows significant rise in the peak current induced by capsaicin (n = 31) with no change in the α, β-meATP-evoked current (n = 31). Data are expressed as % of control amplitude in sister cultures. *: P < 0.05.