Potentiation of the glutamatergic synaptic input to rat locus coeruleus neurons by P2X7 receptors

Abstract

Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg(2+)-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons.

Keywords: Adenosine 5′-triphosphate; Locus coeruleus; Miniature excitatory postsynaptic currents; P2X7 receptors; Presynaptic modulation; Spontaneous excitatory postsynaptic currents.

Fig. 1

Effects of AP5 and CNQX on the amplitude and frequency of sEPSCs in rat LC neurons. The holding potential in this and all following experiments was −80 mV. A Representative recording of sEPSCs and their abolition by a combination of the NMDA- and non-NMDA receptor antagonists AP5 (50 μM) and CNQX (10 μM), respectively. B Percentage change by these antagonists of the normalised sEPSC amplitude and frequency, expressed as mean ± S.E.M. values (n = 4). *P < 0.05; statistically significant differences from the control data, 8–10 and 13–15 min after drug application

Fig. 2

Facilitation by 2-MeSATP of the sEPSC frequency, but not amplitude in rat LC neurons. Aa Representative recordings of sEPSCs before (Ab, left panel) and 8–10 min after (Bb, right panel) starting superfusion with 2-MeSATP (300 μM). Note the different time-scales of recording in Aa and Ab as well as the transient inward current induced by 2-MeSATP in Aa. Change of the absolute amplitude (Ba) and frequency (Bb) of the sEPSCs before, during and after the application of 2-MeSATP (mean ± S.E.M. of seven experiments). The time-period elapsing from the beginning of superfusion with 2-MeSATP is indicated. ^#P < 0.05; statistically significant difference from the control value

Fig. 3

Antagonism by PPADS of the 2-MeSATP-induced change in frequency of sEPSCs in rat LC neurons. Aa Representative recording of sEPSCs and blockade of the 2-MeSATP (300 μM)-induced frequency potentiation by PPADS (30 μM). Ab Representative recording of sEPSCs and no change of the 2-MeSATP (300 μM)-induced frequency potentiation by reactive blue 2 (100 μM). Percentage 2-MeSATP-induced changes of the amplitude (Ba) and frequency (Bb) of sEPSCs in the presence of PPADS, reactive blue 2 (RB2) and suramin, or their absence, 8–10 min after agonist application. Mean ± S.E.M. of four to seven experiments. *P < 0.05; statistically significant differences from zero. ^#P < 0.05; statistically significant differences from the effect of 2-MeSATP alone

Fig. 4

Facilitation by ATP and no effect by α,β-meATP and UTP of the sEPSC frequency in rat LC neurons. Representative recordings of the increase of the sEPSC frequency by ATP (300 μM; Aa), but not α,β-meATP (100 μM; Ab) and UTP (100 μM; Ac). Percentage agonist-induced changes of the amplitude (Ba) and frequency (Bb) of sEPSCs, 8-10 min after agonist application. Mean ± S.E.M. of three to five experiments. *P < 0.05; statistically significant differences from zero. ^#P < 0.05; statistically significant differences from the effect of ATP alone

Fig. 5

Effects of BzATP on the amplitude, frequency and time-course of sEPSCs in rat LC neurons. A Representative recording of sEPSCs before, during and after a 10-min superfusion with BzATP (300 μM). Note the stability of the holding current in spite of the presence of BzATP. B Representative recordings of two sEPSCs of similar amplitude under control conditions (left panel) and in the presence of BzATP (right panel). The decay phases of the amplitudes were fitted according to a monoexponential function to calculate the offset time constants (τ_off). There was no change of the offset time-constant 8–10 min after the application of BzATP, when compared with the control values (for the mean onset and offset time constants see the “Results” section). Mean ± S.E.M. of seven experiments. C Plot of the number of events against the amplitude (Ca) and inter-event interval (Cb) in the cell shown in A and B. Statistically significant shift by BzATP of the control plot of cumulative probability against the inter-event interval (Cb, inset). Change of the absolute amplitude (Da) and frequency (Db) of the sEPSCs before, 8–10 min after the application of BzATP, and a further 8–10 min of washout. Mean ± S.E.M. of seven experiments. ^#P < 0.05; statistically significant difference from the control value

Fig. 6

Blockade by P2 receptor antagonists, selective astrocytic poisons, and external Mg²⁺ of the BzATP-induced change in frequency of sEPSCs in rat LC neurons. Representative recordings of sEPSCs and antagonism by BBG (1 μM; Aa) and fluorocitric acid (100 μM; Ab) of the effect of BzATP (300 μM). Percentage BzATP-induced changes of the amplitude (Ba) and frequency (Bb) of sEPSCs in the presence of PPADS (30 μM) or Brilliant Blue G (BBG; 1 μM), or their absence, 8–10 min after starting agonist application. Some brain slices were incubated for 2 h in a fluorocitric acid (FCA; 100 μM)- or aminoadipic acid (AAA; 100 μM)-containing medium, before starting superfusion in the recording chamber; control measurements of the BzATP effect were made on brain slices incubated for a comparable time in drug-free ACSF. The BzATP-induced change in amplitude and frequency in a normal Mg²⁺-containing superfusion medium is shown as the last in each series of columns. Mean ± S.E.M. of five to six experiments. *P < 0.05; statistically significant differences from zero. ^#P < 0.05; statistically significant differences from the effect of BzATP alone

Fig. 7

Effects of BzATP on the amplitude and frequency of mEPSCs in rat LC neurons. mEPSCs were measured in the continuous presence of tetrodotoxin (0.5 μM). Aa Representative recordings of sEPSCs before (Ab, left panel) and 8–10 min after (Ab, right panel) starting superfusion with BzATP (300 μM). Note the different time-scales of recording in Aa and Ab as well as the stability of the holding current in spite of the application of BzATP in Aa. B Plot of the number of events against the amplitude (Ba) and inter-event intervals (Bb) in the cell shown in Aa and Ab. Statistically significant shift by BzATP of the control plot of cumulative probability against the inter-event interval (Bb, inset). Change of the absolute amplitude (Ca) and frequency (Cb) of the mEPSCs before, 8–10 min after starting the application of BzATP, and a further 8–10-min of washout. Mean ± S.E.M. of five experiments. The time-period elapsing from the beginning of superfusion with BzATP was as in Aa and Ab. ^#P < 0.05; statistically significant difference from the control value

Fig. 8

Effects of capsaicin on the amplitude and frequency of sEPSCs in rat LC neurons; interaction with capsazepine and PPADS. Aa Representative recordings of sEPSCs before, and 8–10 min after starting superfusion with capsaicin (10 μM), as well as after a further 8–10 min of washout. Ab Representative recordings of sEPSCs before, and 8–10 min after starting superfusion with capsaicin (10 μM), as well as after a further 8–10 min of washout, in the continuous presence of capsazepine (30 μM). Change of the absolute amplitude (Ba) and frequency (Bb) of the sEPSCs before, during and after the application of capsaicin, in the absence or presence of capsazepine or PPADS (30 μM). Mean ± S.E.M. of six to eight experiments. ^#P < 0.05; statistically significant difference from the control value