Fast P2X receptor-mediated excitatory postsynaptic current (EPSC) was identified in the pyramidal neurones of layers II/III and V of somatosensory cortex in slices obtained from the brain of 17- to 22-day-old rats. Animals were killed according to UK legislation. The EPSCs were elicited by electrical stimulation of vertical axons originating from layer IVÐVI neurones at 0.1 Hz in the presence of 20 mM bicuculline. When the glutamatergic EPSC was blocked by the saturating concentrations of glutamate receptor inhibitors NBQX and D-AP5, a small EPSC component was recorded from 90 % of neurones tested. The amplitude of residual EPSC averaged 9.7 ± 7.1 % (n = 44, all data are presented as means ± S.D.) of total EPSC amplitude measured at a holding potential of -80 mV. Doubling the concentration of glutamatergic antagonists did not affect the amplitude of residual EPSC (rEPSC), indicating that it was not due to incomplete inhibition of glutamate receptors, thus representing an activation of non-glutamatergic ionotropic receptors. To verify the absence of chloride conductance contribution to rEPSC we replaced caesium chloride in the intrapipette solution with caesium gluconate. Neither amplitude nor currentÐvoltage relationship of rEPSC were changed after substitution of intracellular Cl– ions for gluconate. The reversal potential of rEPSC recorded using the caesium gluconate-based pipette solution was 15.5 ± 4 mV (n = 7), the corresponding values for caesium chloride-based pipette solution was 15 ± 5 mV (n = 7). This result demonstrates negligible contribution of chloride conductance to rEPSC.
The rEPSC was not affected by selective blockers of nicotinic (hexamethonium) and serotonin (Y25130) receptors, although it was reversibly inhibited by antagonists of P2X receptors (NF023, NF279 and PPADS). The specific P2X receptor blocker NF023 (10 mM) reduced the amplitude of rEPSC by 73 ± 22 % (n = 7). The inhibitory effects of NF279 (2 mM) and PPADS (30 mM) were 61 ± 18 % (n = 10) and 51 ± 9 % (n = 8), respectively.
An application of ATP (10 mM) or α,β-methylene ATP (10 mM) to pyramidal neurones, acutely isolated from cortical slices, evoked inward currents (30Ð200 pA) in 65 % of cells tested. ATP-mediated currents were reversibly blocked by P2X antagonists. This inhibition was significant but not complete: PPADS (30 mM), and suramin (50 mM) reduced the amplitude of ATP-induced currents by 48 ± 8 % (n = 6) and 53 ± 14 % (n = 5), respectively. The voltage dependence of ATP-induced currents was almost similar to the purinergic rEPSC. The values of reversal potential measured at extracellular calcium concentrations of 2 and 5 mM were 12.5 ± 0.7 and 17.1 ± 1.1 mV (n = 5). The relative calcium permeability (PCa/PCs) of P2X receptors was 12.3 as estimated from reversal potential of ATP-induced current measured at different extracellular calcium concentrations.
We concluded that P2X purinoreceptors participate in synaptic transmission in neocortex. In cortical pyramidal neurones P2X receptors may provide a significant Ca2+ entry at resting membrane potentials, and hence an ATP-activated component of synaptic transmission may play an important role in cortical pyramidal neurone function.
This research was supported by The Wellcome Trust.
All procedures accord with current UK legislation.