The Journal of Neuroscience, December 1, 2004, ():
Direct Excitation of Inhibitory Interneurons by Extracellular ATP Mediated by P2Y1 Receptors in the Hippocampal Slice
J. Neurosci. Kawamura et al.
24: 10835
Supplemental data
Decrease in EPSC frequency by ATP
In contrast to the increased IPSC frequency, the decrease in EPSC frequency by ATP is likely to be mediated by adenosine A1 receptors activated by adenosine produced through extracellular breakdown of applied ATP, as described in numerous other studies in acute slices (Dunwiddie et al., 1997; Cunha et al., 1998; Masino et al., 2002).
This conclusion is based on the following four types of results. First, not only ATP but also adenosine, ATP?S and ADP significantly decreased the EPSC frequency (86.6 ± 4.9% inhibition by 100 µM adenosine; n=10; P<0.01; 80.1 ± 8.6% inhibition by 100 µM ATP?S; n=5; P<0.01; 78.2 ± 15.5% inhibition by 100 µM ADP; n=4; P<0.05; Mann-Whitney’s U test; supplemental figure 2A). In contrast, 2meSATP, 2meSADP and ??meATP were not effective (6.9 ± 16.0% inhibition by 100 µM 2meSATP; n=12; not significant; 4.7 ± 16.8% inhibition by 100 µM 2meSADP; n=4; not significant; 7.9 ± 15.5% increase by 100 µM ??meATP; n=6; not significant; Mann-Whitney’s U-test; supplemental figure 2A). These agonist profiles imply that P2 receptors are not involved in the increase in IPSC frequency, rather, they suggest an involvement of P1 receptors. (Ralevic and Burnstock, 1998). Second, a selective antagonist of A1 receptors, DPCPX (1 µM), significantly reduced the effect of ATP on EPSC frequency (in the absence of DPCPX, 0.42 ± 0.05 Hz to 0.08 ± 0.02 Hz; in the presence of DPCPX, 0.44 ± 0.11 Hz to 0.32 ± 0.08 Hz; n=6; P<0.05; supplemental figure 2B). In addition, PPADS, a P2 receptor selective antagonist, did not significantly modify the effect of ATP on EPSC frequency (in the absence of PPDAS, 0.96 ± 0.23 Hz to 0.22 ± 0.05 Hz; in the presence of PPADS, 0.97 ± 0.26 Hz to 0.41 ± 0.18 Hz; P=0.33). Third, ??meADP (100 µM), an inhibitor of the 5'-ecto-nucleotidase, which is primarily responsible for the ATP-to-adenosine conversion in the hippocampus (Cunha et al., 1998; Braun et al., 1998) significantly reduced the effect of ATP on EPSC frequency, but not that on IPSC frequency. In the absence of ?,?meADP, ATP and adenosine significantly reduced the EPSC frequency to 49.8 ± 9.5% (n=4) and 30.8 ± 4.4% (n=3), respectively. In the presence of ?,?meADP (100 µM), the ATP-induced reduction in EPSC frequency was no longer significant (reduced only to 89.9 ± 30.9%), whereas that induced by adenosine was still significant (reduced to 37.3 ± 3.2%). Fourth, ATP required a longer time to attain the maximal effect than adenosine (the largest decrease in EPSC frequency with adenosine appeared at 34.8 ± 9.7 s, range = 11 s - 92 s, after application, whereas that with ATP was at 61.6 ± 7.5 s, range, 28 - 90 s; n=8; P<0.05; a comparison made in neurons onto which both ATP and adenosine were applied), a result consistent with an interpretation that the extracellular conversion of ATP requires additional time (Kato and Shigetomi, 2001).
Files in this Data Supplement:
- supplemental material
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Simultaneous recording of EPSCs and IPSCs. A1, whole-cell current of a CA3 pyramidal neuron. Outward (marked with open circles) and inward (filled circles) postsynaptic events were simultaneously recorded. A2, superimposed traces of the inward (filled circle; n=3) and outward (open circle; n=6) events sampled from the traces in A1. B, effect of CNQX (10 ?M) and bicuculline (10 ?M) on IPSC and EPSC. 1, original traces; 2, summary of the effect of CNQX and bicuculline on IPSC and EPSC frequency. *, P<0.05; **, P<0.01; NS, not significantly different (ANOVA); n=3. C, left, traces recorded at distinct holding potentials. Right, the I-V relationship of IPSCs (open circles and solid line) and EPSCs (filled circles and dashed line). Each point and vertical bar indicates mean and SE of the event amplitude. D, amplitude distribution histogram of IPSCs and EPSCs simultaneously recorded in a neuron. Constructed using 100 IPSCs and 100 EPSCs that were randomly sampled.
- supplemental material
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Effects of purinoceptor agonists and antagonist on EPSC frequency.
A1, the membrane currents before (traces in left) and after (traces in right) application of agonists. adenosine (1 mM) and ATP (1 mM), but not 2meSATP (100 ?M) and ?,?-meATP (1 mM), decreased EPSC (marked with filled circles) frequency. A2, summary of the effects of the purinoceptor agonists, ATP, 2meSATP, 2meSADP, ATP?S, ADP, ?,?-meATP and adenosine on EPSC frequency; they are from the same set of neurons in Fig. 2A2. Ordinate, decrease in EPSC frequency by each agonist expressed as the percentage of the pre-application value. *, P<0.05; **, P<0.01 (Mann-Whitney’s U test). B1, membrane currents recorded from a CA3 pyramidal neuron with low-Cl internal solution at a holding potential of -60 mV before (left) and after (right) ATP (1 mM) application in the absence (above) and presence (bottom) of DPCPX (1 ?M). B2, summary of the effect of DPCPX of effects of ATP on EPSC frequency. *, P<0.05; **, P<0.01; NS, not significantly different (ANOVA); n=6.
