Article Figures & Data
Figures
- Figure 4.
2meSATP did not increase IPSC frequency in CA3 pyramidal neurons from the P2Y1-deficient mice. A, Membrane current traces recorded with CsCl-based internal solution in the presence of kynureic acid (1 mm) from a CA3 pyramidal neuron from a rat (top traces), a wild-type mouse (WT; middle traces), and a P2Y1-deficient mouse (P2Y1-/-; bottom traces). Traces obtained before (left) and after (right) 2meSATP (100 μm) application. The holding potential was -70 mV. B, The time course of the changes in IPSC frequency with 2meSATP application. Mean and SE of nine neurons from eight slices from four wild-type mice (open circles) and 14 neurons from eight slices from four P2Y1-deficient mice (filled circles). IPSC frequency became significantly different (p < 0.05; ANOVA) between in the wild-type and in the P2Y1-deficient mice during the period indicated by the horizontal bar below the plots. C, Left, Summary of the effects of 2meSATP on IPSC frequency from wild-type mice in the absence and presence of MRS2179 (30 μm). *p < 0.05; **p < 0.01; NS, not significantly different (ANOVA); n = 8 from four wild-type mice. Right, Summary of the effects of 2meSATP on IPSC frequency recorded in P2Y1-deficient mice. NS, Not significantly different (ANOVA); n = 14 from four P2Y1-deficient mice.
- Figure 1.
ATP increased IPSC frequency and decreased EPSC frequency. A, Simultaneous recording of EPSCs and IPSCs from a CA3 pyramidal neuron. ATP (1 mm) was applied at the horizontal bar. a-c are the time-extended traces taken at the points a-c in the trace in A. IPSCs and EPSCs (outward and inward events, respectively) are marked with open circles above the trace and filled circles below the trace, respectively. B, The time course of the changes in frequencies of IPSC and EPSC (top and bottom, respectively) with ATP application. Each point and vertical bar represents mean and SE of 22 neurons, onto which ATP (1 mm) was applied. The frequency became significantly different (p < 0.05; paired t test) from the preapplication value at the times marked with * at the bottom of plots. C, Summary of the effects of ATP on the frequencies of IPSC (left) and EPSC (right). **p < 0.01 (ANOVA); n = 22.
- Figure 2.
Effects of purinoceptor agonists on IPSC frequency. A, The membrane current traces recorded before (left) and near the peak of the effect after agonist application (right). ATP (1 mm), 2meSATP (100 μm), and 2meSADP (100 μm), but not adenosine (1 mm), increased the IPSC frequency (recorded as outward events). B, Summary of the effects of the purinoceptor agonists ATP (100 μm, n = 12; 300 μm, n = 7; 1 mm, n = 22), 2meSATP (100 μm, n = 12; 300 μm, n = 5), 2meSADP (100 μm, n = 4), ATPγS (100 μm, n = 5; 300 μm, n = 5), ADP (100 μm, n = 4; 300 μm, n = 4; 1 mm, n = 5), α, β-meATP (100 μm, n = 6; 300 μm, n = 5; 1 mm, n = 8), and adenosine (100 μm, n = 10; 300 μm, n = 5; 1 mm, n = 6) on IPSC frequency. Ordinate, Changes in the IPSC frequency expressed as the percentage of the preapplication value; they are from the same set of neurons shown in supplemental Figure 2 A (available at www.jneurosci.org as supplemental material). *p < 0.05; **p < 0.01 (Mann-Whitney U test).
- Figure 3.
Effects of purinoceptor antagonists on the increase in IPSC frequency. A1, The time course of the changes in PSC frequency with 2meSATP (100 μm) in the absence of MRS2179 (open circles), in the presence of MRS 2179 (30 μm; filled triangles), and after 30 min of washout (open squares). Each point and vertical bar represent mean and SE of four neurons. A2, Summary of the effect of MRS2179 on the increase in PSC frequency with 2meSATP. *p < 0.05; **p < 0.01; NS, not significantly different (ANOVA); n = 4. B, The summary of the effect of PPADS (40 μm) on the increase in IPSC frequency with 2meSATP. *p < 0.05; NS, not significantly different (ANOVA); n = 5. C, Summary of the effect of TNP-ATP (10 μm) on the effect of 2meSATP on the IPSC frequency. *p < 0.05; NS, not significantly different (ANOVA); n = 5. D, The summary of the effect of DPCPX on the effects of ATP (1 mm) on IPSC frequency; they are from the same set of neurons shown in supplemental Figure 2B (available at www.jneurosci.org as supplemental material). *p < 0.05; **p < 0.01; NS, not significantly different (ANOVA); n = 6.
- Figure 5.
2meSATP excited interneurons in the stratum oriens in the CA3. A, An example of a pyramidal (PYR) neuron. Left trace, The membrane potential response to a 20 pA depolarizing current injection. Middle, An IR-DIC image of the CA3 region, from which the pyramidal neuron (PYR) showing the responses in A and C was recorded (the cell with the patch pipette tip). Scale bar, 50 μm. Right, A confocal image of the pyramidal neuron and the tip of a patch pipette filled with AlexaFluor 568. Scale bar, 50 μm. B, Left trace, Membrane potential response to a 40 pA depolarizing current injection. Middle, An IR-DIC image showing the interneuron (IN) recorded in the stratum oriens (the cell with the patch pipette tip). Scale bar, 50 μm. Right, A confocal image of the interneuron and the tip of a patch pipette filled with AlexaFluor 568. Scale bar, 50 μm. C, PYR, Membrane currents of the CA3 pyramidal neuron recorded with low-Cl internal solution at a holding potential of 0 mV. 2meSATP (100 μm) was applied at the horizontal bar. Consecutive time-extended versions of the membrane current are shown as insets above. IN1, The membrane potential of the interneuron shown in B. IN2, The membrane current of the interneuron in the stratum oriens. The holding potential was -70 mV. Note that the increase in IPSC frequency (PYR), the action potential generation (IN1), and the inward current (IN2) in response to 2meSATP were of highly similar time course.
- Figure 6.
2meSATP elicited MRS2179-sensitive and voltage-dependent inward currents in the interneurons. A1, Effect of MRS2179 (30 μm) on the inward current generated by 2meSATP (100 μm; horizontal bar). A whole-cell perforated-patch recorded from an interneuron in the absence (control) and presence (+MRS2179) of MRS2179 and after 30 min of washout. The holding potential was -70 mV. A2, Summary of the effect of MRS2179 (30 μm). **p < 0.01 (ANOVA); n = 4. B, Current-voltage relationship of the current generated in response to 2meSATP application (I2meSATP) in the presence of TTX (1 μm). B1, Top, Original traces showing the membrane current in response to a ramp command (shown as inset) before (1) and during (2) 2meSATP application. The cells were first kept at +30 mV and hyperpolarized to -120 mV at a velocity of 150 mV/sec. Bottom, Membrane current during the ramp command before (solid curve, 1) and during (broken curve, 2) 2meSATP. The two curves crossed each other at the potential indicated with an arrow. B2, Summary of the current-voltage relationship of I2meSATP as measured by subtracting the ramp-command responses. Mean and SE of three interneurons.
- Figure 7.
Increases in IPSC frequency by 2meSATP did not involve ionotropic and metabotropic glutamate receptors. A, Summary of the effect of ionotropic glutamate receptor antagonist kynureic acid (1 mm) on the increase in IPSC frequency with 2meSATP (100 μm). **p < 0.01; NS, not significantly different (ANOVA); n = 5. B, Summary of the effect of metabotropic glutamate receptor antagonist MCPG (500 μm) on the effects of 2meSATP on IPSC frequency. **p < 0.01; NS, not significantly different (ANOVA); n = 5.
- Figure 8.
Imaging of [Ca2+]i in the CA3 slice with simultaneous recording of the postsynaptic current of a pyramidal cell. A1, Top trace, PSC frequency of a CA3 pyramidal neuron recorded with CsCl-based internal solution at a holding potential of -70 mV; bottom traces, relative changes in Fluo-4 fluorescence of 10 representing cells shown at the bottom of A2. For the classification of types 1-3, see Results. 2meSATP (100 μm) and 20 mm K+ solution were applied at the respective horizontal bars. The ACSF was replaced by the Ca2+-free ACSF during the period indicated with the horizontal bar denoted 0 mm Ca2+. A2, Top, An IR-DIC image showing the location of the CA3 peri-pyramidal layer in which [Ca2+]i imaging was made. Note a patch pipette on a pyramidal neuron in the center. Bottom, A merged image of two images: one showing cells responding to 2meSATP in red and another showing cells responding to 20 mm K+ in green. This projected confocal image indicates the same region as in the IR-DIC image of the top panel. Images of the cells responding to each solution were obtained by subtracting the fluorescence intensity of each pixel acquired before the application from that at the peak of the PSC effect (pairs of arrows in red and green at the bottom of A1). Note that two cells in the merged image in A2 are in yellow (white arrowheads), indicating that they responded to both 2meSATP and 20 mm K+ (type 1 cells in A1). Scale bar, 50 μm. B1, Relationship between changes in the fluorescence intensity (ΔF/F0) in response to 20 mm K+ (abscissa) and 2meSATP (ordinate). Three ovals in different colors indicate groups of neurons classified according to the responses to two solutions (see Results). B2, The relative location of cells analyzed. Abscissa indicates the distance of the cells from the borders of the pyramidal layers. The vertical dashed line in the left and right indicate the border between the pyramidal layer and the S.R. and S.O., respectively. C, The summary of changes in Fluo-4 fluorescence (ΔF/F0) of types 1-3 cells in response to 20 mm K+ and 2meSATP in the absence of extracellular Ca2+, in the presence of MRS2179 (30 μm), and its washout. Numbers in parentheses indicate the number of cells from six CA3 slices. Bars and vertical lines indicate mean and SE, respectively.
Additional Files
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 �M adenosine; n=10; P <_0.01 _80.1="_80.1" _="_" _8.6="_8.6" inhibition="inhibition" by="by" _100="_100" m="m" atps="atps" n="8;" p0.01="p0.01" _78.2="_78.2" _15.5="_15.5" adp="adp" p0.05="p0.05" mann-whitneys="mann-whitneys" u="u" test="test" supplemental="supplemental" figure="figure" _2a.="_2a." in="in" contrast="contrast" _2mesatp="_2mesatp" _2mesadp="_2mesadp" and="and" meatp="meatp" were="were" not="not" effective="effective" _6.9="_6.9" _16.0="_16.0" significant="significant" _4.7="_4.7" _16.8="_16.8" _7.9="_7.9" increase="increase" u-test="u-test" these="these" agonist="agonist" profiles="profiles" imply="imply" that="that" p2="p2" receptors="receptors" are="are" involved="involved" the="the" ipsc="ipsc" frequency="frequency" rather="rather" they="they" suggest="suggest" an="an" involvement="involvement" of="of" p1="p1" receptors.="receptors." ralevic="ralevic" burnstock="burnstock" _1998.="_1998." second="second" a="a" selective="selective" antagonist="antagonist" a1="a1" dpcpx="dpcpx" _1="_1" significantly="significantly" reduced="reduced" effect="effect" atp="atp" on="on" epsc="epsc" absence="absence" _0.42="_0.42" _0.05="_0.05" hz="hz" to="to" _0.08="_0.08" _0.02="_0.02" presence="presence" _0.44="_0.44" _0.11="_0.11" _0.32="_0.32" _2b.="_2b." addition="addition" ppads="ppads" receptor="receptor" did="did" modify="modify" ppdas="ppdas" _0.96="_0.96" _0.23="_0.23" _0.22="_0.22" _0.97="_0.97" _0.26="_0.26" _0.41="_0.41" _0.18="_0.18" p="0.33)." third="third" meadp="meadp" inhibitor="inhibitor" _5-ecto-nucleotidase="_5-ecto-nucleotidase" which="which" is="is" primarily="primarily" responsible="responsible" for="for" atp-to-adenosine="atp-to-adenosine" conversion="conversion" hippocampus="hippocampus" cunha="cunha" et="et" al.="al." _1998="_1998" braun="braun" but="but" frequency.="frequency." adenosine="adenosine" _49.8="_49.8" _9.5="_9.5" _30.8="_30.8" _4.4="_4.4" respectively.="respectively." atp-induced="atp-induced" reduction="reduction" was="was" no="no" longer="longer" only="only" _89.9="_89.9" _30.9="_30.9" whereas="whereas" induced="induced" still="still" _37.3="_37.3" _3.2.="_3.2." fourth="fourth" required="required" time="time" attain="attain" maximal="maximal" than="than" largest="largest" decrease="decrease" with="with" appeared="appeared" at="at" _34.8="_34.8" _9.7="_9.7" s="s" range="range" _-="_-" _92="_92" after="after" application="application" _61.6="_61.6" _7.5="_7.5" _28="_28" _90="_90" comparison="comparison" made="made" neurons="neurons" onto="onto" both="both" applied="applied" result="result" consistent="consistent" interpretation="interpretation" extracellular="extracellular" requires="requires" additional="additional" kato="kato" shigetomi="shigetomi" _2001.="_2001." _--="_--" end="end" desc="desc" dc1="dc1">
Files in this Data Supplement:
- supplemental material - 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 _="_" p0.01="p0.01" ns="ns" not="not" significantly="significantly" different="different" anova="anova" n="3." c="c" left="left" traces="traces" recorded="recorded" at="at" distinct="distinct" holding="holding" potentials.="potentials." right="right" the="the" i-v="i-v" relationship="relationship" of="of" ipscs="ipscs" open="open" circles="circles" and="and" solid="solid" line="line" epscs="epscs" filled="filled" dashed="dashed" line.="line." each="each" point="point" vertical="vertical" bar="bar" indicates="indicates" mean="mean" se="se" event="event" amplitude.="amplitude." d="d" amplitude="amplitude" distribution="distribution" histogram="histogram" simultaneously="simultaneously" in="in" a="a" neuron.="neuron." constructed="constructed" using="using" _100="_100" that="that" were="were" randomly="randomly" sampled.--="sampled.--" end="end" desc="desc" supp.1.gif="supp.1.gif" _--="_--">
- supplemental material - 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 _="_" p0.01="p0.01" mann-whitneys="mann-whitneys" u="u" test.="test." b1="b1" membrane="membrane" currents="currents" recorded="recorded" from="from" a="a" ca3="ca3" pyramidal="pyramidal" neuron="neuron" with="with" low-cl="low-cl" internal="internal" solution="solution" at="at" holding="holding" potential="potential" of="of" _-60="_-60" mv="mv" before="before" left="left" and="and" after="after" right="right" atp="atp" _1="_1" mm="mm" application="application" in="in" the="the" absence="absence" above="above" presence="presence" bottom="bottom" dpcpx="dpcpx" m.="m." b2="b2" summary="summary" effect="effect" effects="effects" on="on" epsc="epsc" frequency.="frequency." p0.05="p0.05" ns="ns" not="not" significantly="significantly" different="different" anova="anova" n="6." _--="_--" end="end" desc="desc" supp.2.gif="supp.2.gif">
