Developmental changes in P2X purinoceptors on glycinergic presynaptic nerve terminals projecting to rat substantia gelatinosa neurones

Abstract

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Figure 1. Glycinergic mIPSCs

A, typical spontaneous miniature postsynaptic currents (mIPSCs) observed before, during and after the application of 10⁻⁶m strychnine in the presence of 3 × 10⁻⁷m TTX, 3 × 10⁻⁶m CNQX, 10⁻⁵m AP5 and 3 × 10⁻⁶m bicuculline. Insets indicate the current recordings with an expanded time scale. Ba, typical traces recorded from a neurone at various holding potentials. Bb, the I–V relationship in which each point is the mean of 4 neurones.

Figure 2. ATP- and αβ-me-ATP-elicited multiple responses

A, the incidence of four kinds of ATP-elicited responses including only presynaptic action (a), presynaptic and postsynaptic actions (b), only postsynaptic action (c) and no action (d) in P28–30 neurones. B, the incidence of four kinds of αβ-me-ATP-elicited responses including only presynaptic action (a), presynaptic and postsynaptic actions (b), only postsynaptic action (c) and no action (d). Note that αβ-me-ATP-elicited postsynaptic currents were more quickly desensitized than ATP-elicited postsynaptic currents.

Figure 3. Developmental changes of agonist-elicited presynaptic responses

A, typical traces of mIPSCs induced by application of 10⁻⁴m ATP, 2MeS-ATP and αβ-me-ATP. All recordings were obtained from a single neurone at different developmental stages. B, each column is the mean of 5–12 neurones and normalized to the respective control. *P < 0.05.

Figure 4. Time-dependent recovery of ATP- and αβ-me-ATP-elicited presynaptic responses

Aa, typical traces of mIPSCs induced by repeated applications of 10⁻⁴m ATP at different time intervals. Ab, summary of time-dependent recovery of ATP- and αβ-me-ATP-elicited presynaptic responses. Note that ATP and αβ-me-ATP actions on mIPSC frequency were fully recovered after an interval of 20 min. B, the reproducibility of ATP- or αβ-me-ATP-elicited presynaptic responses. Ba, successive applications of 10⁻⁴mαβ-me-ATP at 19 min intervals increased mIPSC frequency in a reproducible manner. The number of events every 10 s is plotted. Bb, each column is the mean of 4 neurones and normalized to the respective control.

Figure 5. Concentration-response relationships for ATP and its agonists

Concentration-response relationships for ATP, 2MeS-ATP and αβ-me-ATP in P10–12, P16–18 and P28–30 neurones, respectively. All points (n = 5–12 neurones) are normalized to the respective control. The continuous lines represent the fit of the Hill function (see Methods).

Figure 6. Effects of Cd²⁺ and Ca²⁺-free external solution on ATP- and αβ-me-ATP-elicited glycinergic mIPSC frequency facilitation

Aa, typical traces of mIPSCs observed from a P28–30 neurone after the application of 10⁻⁴m ATP or αβ-me-ATP in the presence of 10⁻⁴m Cd²⁺. Ab, each column is the mean of 5 neurones. All columns are normalized to the control. *P < 0.05, **P < 0.01. B and C, typical traces of mIPSCs observed from a P28–30 neurone in the Ca²⁺-free external solution after the application of 10⁻⁴m ATP (Ba) and αβ-me-ATP (Ca). Pooled data for ATP (Bb, n = 5) and αβ-me-ATP (Cb, n = 4) are presented. Each column is normalized to the control. *P < 0.05, **P < 0.01.

Figure 7. Effect of thapsigargin on ATP- and αβ-me-ATP-elicited glycinergic mIPSC frequency facilitation

A and B, typical traces of mIPSCs recorded during the application of 10⁻⁴m ATP (A) and αβ-me-ATP (B) in the absence (a) and presence (b) of 10⁻⁶m thapsigargin. C, each column is the mean of 5 neurones. All columns are normalized to the control. *P < 0.05.

Figure 8. Effects of P2X receptor antagonists on ATP- and αβ-me-ATP-elicited postsynaptic currents

A, typical traces of 10⁻⁴m ATP-elicited postsynaptic currents in the absence of any antagonists (a), and in the presence of 10⁻⁷m TNP-ATP (b) and both 10⁻⁷m TNP-ATP and 10⁻⁵m PPADS (c). In d, ATP-elicited postsynaptic current was clearly recovered after washing out antagonists. B, typical traces of 10⁻⁴mαβ-me-ATP-elicited postsynaptic currents in the absence of any antagonists (a), and in the presence of 10⁻⁷m TNP-ATP (b). In c, αβ-me-ATP-elicited postsynaptic current was clearly recovered after washing out TNP-ATP.

Figure 9. Effects of P2X receptor antagonists on ATP- and αβ-me-ATP-elicited presynaptic responses

A, typical traces of mIPSCs observed after the application of 10⁻⁴m ATP in the presence of either 10⁻⁵m PPADS (a) or 10⁻⁷m TNP-ATP (b). Ac, each column is the mean of 5 neurones. All columns are normalized to their respective control. Note that ATP-induced facilitation of mIPSCs was completely blocked by PPADS, but partially blocked by TNP-ATP. *P < 0.05. B, typical traces of mIPSCs observed after the application of 10⁻⁴mαβ-me-ATP in the presence of PPADS (a) or TNP-ATP (b). Bc, each column is the mean of 4 neurones and normalized to its respective control. Note that αβ-me-ATP-induced facilitation of mIPSCs was completely blocked by either TNP-ATP or PPADS. *P < 0.05.

Figure 10. Effect of αβ-me-ATP on glycinergic eIPSCs

All recordings were performed in the slice preparation using the whole cell patch clamp technique. The incubation medium contained 10⁻⁵m CNQX, 10⁻⁵m AP5 and 10⁻⁵m bicuculline, but not TTX. A, typical recordings of eIPSCs obtained from P10–12 neurones (left) and P28–30 neurones (right). B, each column is the mean of 4 (P10–12) and 6 (P28–30) neurones and was normalized to its respective control. *P < 0.05. Ca, typical traces of mIPSCs observed before and during the application of 10⁻⁵mαβ-me-ATP. Cb, cumulative probability plots for inter-event interval (left) and amplitude (right) of spontaneous IPSCs obtained from the same neurone. P values indicate the results of K-S test for frequency and amplitude (908 events for control and 1518 events for αβ-me-ATP).