P2X7 receptors in satellite glial cells mediate high functional expression of P2X3 receptors in immature dorsal root ganglion neurons

Abstract

Background: The purinergic P2X3 receptor (P2X3R) expressed in the dorsal root ganglion (DRG) sensory neuron and the P2X7 receptor (P2X7R) expressed in the surrounding satellite glial cell (SGC) are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats.

Findings: We immunostained DRGs of immature rats and found that P2X3Rs were expressed only in neurons and P2X7Rs were expressed only in SGCs. Western blot analyses indicated that P2X3R expression decreased while P2X7R expression increased with the age of rats. Electrophysiological studies showed that the number of DRG neurons responding to the stimulation of the P2XR agonist, α,β-meATP, was higher and the amplitudes of α,β-meATP-induced depolarizations were larger in immature DRG neurons. As a result, P2X3R-mediated flinching responses were much more pronounced in immature rats than those found in adult rats. When we reduced P2X7R expression with P2X7R-siRNA in postnatal and adult rats, P2X3R-mediated flinch responses were greatly enhanced in both rat populations.

Conclusions: These results show that the P2X7R expression increases as rats age. In addition, P2X7Rs in SGCs exert inhibitory control on the P2X3R expression and function in sensory neurons of immature rats, just as observed in adult rats. Regulation of P2X7R expression is likely an effective way to control P2X3R activity and manage pain relief in infants.

Figure 1

P2X3Rs are expressed in a higher percentage of DRG neurons in immature rats. (A) Immunocytochemical staining of P2X3Rs and P2X7Rs in DRGs of P6 and adult rats. P2X3R labels (red) were found only in neurons; P2X7R labels (green) were found only in SGCs. (Scale bars: P2X3R, P6 = 40 μm, Adult = 50 μm; P2X7R, P6 = 25 μm, Adult = 30 μm) (B) Percentage of DRG neurons expressing P2X3Rs decreases significantly during postnatal development (P6 rats: 74.83 ± 2.03%, n = 3; adult rats: 43.45 ± 5.03%, n = 3. Student t-test, *P < 0.05)

Figure 2

High P2X3R-mediated activity in DRG neurons of immature rats. (A) α,β-meATP induced significant depolarizations in DRG neurons of immature rats. In this example, α,β-meATP evoked 8.6 mV depolarization in a P4 DRG neuron. The depolarization was reversibly blocked by the P2X antagonist, TNP-ATP (1 μM). (B) α,β-meATP induced a much reduced, i.e., 2 mV, depolarization in an adult DRG neuron. (C) The percentage of DRG neurons responded to α,β-meATP was high in immature rats and greatly reduced in adult rats (P3-P14 rats: 55.0%, adult rats: 15.4%. Fisher exact test, *P < 0.05). The amplitude of depolarizations induced by α,β-meATP was reduced as rats matured (P3-P14 rats: 7.0 ± 0.8 mV, n = 11; adult rats: 2.0 ± 0 mV, n = 2. Mann-Whitney test, *P < 0.05)

Figure 3

P2X3R expression decreases and P2X7R expression increases with rat age. Western blot analyses of P2X3Rs and P2X7Rs were conducted on the DRG protein samples isolated from P6, P15 and adult rats. P2X3R and P2X7R expressions were expressed in relative intensity with respect to those of adult rats, which was set at 1.0. During postnatal development, the P2X7R expression increased (P6/Adult: 0.42 ± 0.05; P15/adult 0.64 ± 0.06; n = 3-5), while the P2X3R expression decreased (P6/adult: 1.71 ± 0.28; P15/adult: 1.42 ± 0.11; n = 5) (One-way ANOVA, *P < 0.05)

Figure 4

P2X7R-P2X3R inhibitory control exists in immature rats. (A) P2X7R-siRNA treatment down-regulated P2X7R expression while increased P2X3R expression in P15-16 rats. P2X3R and P2X7R expressions in P2X7R-siRNA rats were expressed in relative intensity with respect to those in Con-siRNA rats, which was set at 1.0. (B) The α,β-meATP induced much more pronounced P2X3R-mediated flinching responses in con-siRNA immature rats than in adult rats. In both immature and adult rats, α,β-meATP evoked significantly larger flinching responses in P2X7R-siRNA rats than in con-siRNA rats.