Modulation of eIPSCs by Ca2+ elevation in astrocytes
Hippocampal slices were bulk loaded with calcium-fluorescence-indicator fluo-4 AM and caged Ca2+
, NP-EGTA AM. Uncaging NP-EGTA was used to control [Ca2+
in astrocytes. NP-EGTA releases Ca2+
upon uncaging by lowering its affinity with Ca2+
(Ellis-Davies and Kaplan, 1994
). We have showed previously that Ca2+
uncaging in astrocytes induces a decrease in the frequency of mIPSCs in neighboring interneurons and that blocking AMPA/kainate and NMDA glutamate receptors pharmacologically has no effect on this presynaptic inhibition. Here, we test whether eIPSCs are also affected by Ca2+
elevation in astrocytes. Whole-cell recordings were made from interneurons in stratum radiatum in CA1 region. The interneurons were voltage-clamped at −60 mV. AMPA/kainate and NMDA receptor antagonists CNQX (50 μM) and CPP (5 μM) were included to block evoked excitatory postsynaptic currents (EPSCs). Monosynaptic eIPSCs were elicited at 0.2 Hz by focal electrical stimulation. The fine-tipped stimulating electrode was placed within 100 μm of one of the processes of the recorded interneurons. The cell bodies of the astrocytes chosen for uncaging NP-EGTA were within 30 μm of proximal dendrites, which were within 100 μm of the soma of the recorded interneurons (Liu et al., 2004
After a stable, baseline, whole-cell recording was achieved, an astrocyte near the interneuron process (<30 μm) was targeted and stimulated with a train of 12 UV pulses (0.1 Hz, 2 minutes), the resultant fluorescence change was monitored and eIPSCs were recorded simultaneously. Uncaging NP-EGTA produced a stepwise increase in [Ca2+]i in the astrocyte (). The astrocyte Ca2+ elevation (mean peak ΔF/F0 = 204 ± 18%, n = 9) was accompanied by a significant reduction in the amplitude of the eIPSCs in 53% (9 of 17) of interneurons (responder, ). The mean amplitude of eIPSCs during Ca2+ uncaging was 73 ± 5% of baseline control (n = 9, P < 0.001, paired t-test), and recovered after the uncaging to 97 ± 3% of baseline control (P > 0.3). During the depression the coefficient of variation (CV; SD/mean) of the eIPSCs increased from 0.32 ± 0.03 to 0.38 ± 0.03 (n = 9, P < 0.01), which is consistent with the depression of eIPSCs being mediated by a decrease in the probability of GABA release. The time-course of the decrease in the amplitude of eIPSCs in interneurons followed that of Ca2+ elevation in astrocytes (). Ca2+ uncaging in astrocytes (peak ΔF/F0 = 194 ± 23%, n = 8) had no significant effect on the amplitude of eIPSCs in 47% (8 of 17) of interneurons (non-responder). The average amplitude of eIPSCs was 96 ± 3% of baseline level (n = 8, P > 0.3). There was no significant changes in CV (control: 0.30 ± 0.03; uncaging: 0.31 ± 0.03; n = 8). The responder and non-responder were classified by testing whether the amplitude of eIPSCs was significantly different during Ca2+ uncaging from the pre-uncaging level (paired t-test). When experiments from both groups were averaged, the depression of eIPSCs during Ca2+ uncaging was still significantly different from the pre-uncaging level (83 ± 4% of control, n = 17, P < 0.01). The peak ΔF/F0 levels between the two groups are not significantly different (P > 0.5). Thus, the difference between responder and non-responder can not be explained by different Ca2+ levels in astrocytes produced by Ca2+ uncaging.
Fig. 1 Depression of eIPSCs in interneurons by Ca2+ uncaging in astrocytes. (A) Images from an astrocyte before, during and after stimulation with a train of twelve UV laser pulses (0.1 Hz) to uncage NP-EGTA. Scale bar, 10 μm. (B) Upper: Ca2+ uncaging (more ...)
The anatomical proximity of the stimulated astrocytes with the dendrites of the recorded interneurons is required for astrocyte-mediated depression of sIPSCs. In astrocytes that are 60–100 μm from the dendrites of recorded interneurons, Ca2+ uncaging elevated [Ca2+]i in astrocytes (peak ΔF/F0 = 199 ± 24%, n = 7) but did not significantly alter the amplitude of eIPSCs (96 ± 5% of baseline, n = 7, P > 0.4, paired t-test). Therefore, only astrocytes within 30 μm of the proximal dendrites of recorded interneurons were chosen for the following experiments.
UV flashings to astrocytes did not produce appreciable change in Ca2+ fluorescence when hippocampal slices from the same rat brains are loaded with fluo-4 alone (ΔF/F0 = 3 ± 2%; n = 9). This indicates that the astrocyte [Ca2+]i elevation during the uncaging is not due to either photo-damage or unspecific effects produced by the UV laser. Under this condition, UV flashing to astrocytes had no significant effect on the amplitude of eIPSCs recorded from interneurons in these slices (98 ± 3% of control, n = 9, P > 0.3, ). Pre-treatment of the slices with BAPTA-AM (10 μM for 20–30 minutes) abolished the uncaging-induced Ca2+ increase in astrocytes (peak ΔF/F0 = 9±3%, n = 8) and the associated depression of eIPSCs in interneurons (96 ± 4% of preuncaging level, n = 8, P > 0.5). These results indicate that the depression of eIPSCs in interneurons is likely to be caused by Ca2+ elevation in astrocytes.
In agreement with previous studies (Porter and McCarthy, 1996
), we found that high frequency stimulation (50–100 Hz, 1 second) caused robust increases in [Ca2+
in 3–20 astrocytes, but either single or paired-pulse electrical stimulation at low frequency (0.1 Hz) used in this study did not (data not shown). This observation indicates that [Ca2+
in astrocytes is not affected by the electrical stimulation used to elicit eIPSCs.
The fluo-4 fluorescence was present only in astrocytes, not in neurons, from slices prepared from 11–15-day-old rats. We have showed previously that astrocytes are also loaded preferentially with NP-EGTA in NP-EGTA AM bulk-loaded slices, the neuronal loading of NP-EGTA is insignificant (Liu et al., 2004
). First, we made perforated whole-cell recording from pyramidal neurons in the coloaded slices. Pyramidal neurons in CA1 region were voltage-clamped at −60 mV. UV flashing to pyramidal neurons failed to induce Ca2+
currents in NP-EGTA AM bulk-loaded slices, but uncaging NP-EGTA loaded into pyramidal neurons through conventional whole-cell recording induced such currents from pyramidal neurons in the same slices (120 ± 19 pA, n
= 7, ). Second, when fura-2 AM rather than fluo-4 AM was used for coloading with NP-EGTA AM in some slices, UV uncaging at astrocytes caused Ca2+
elevation, but uncaging at either pyramidal neurons or interneurons did not. Unlike fluo-4, fura-2 could still be loaded into some pyramidal neurons and interneurons in the hippocampal slices through bulk loading. These two lines of evidence indicate that NP-EGTA is loaded selectively into astrocytes but not neurons (Liu et al., 2004
). The UV-laser beam was focused through a 40× water-immersion object onto astrocytes with a diameter of ~15 μm. The preferential loading of NP-EGTA into astrocytes and targeted UV stimulation ensure that only astrocytes, not neurons, are stimulated during uncaging NP-EGTA.
Fig. 2 Neuronal loading of NP-EGTA in NP-EGTA-AM-loaded slices is insignificant. (A1) A pyramidal neuron was loaded with potassium salts of NP-EGTA (2 mM, pre-loaded with 2 mM Ca2+) and fluo-4 (0.1 mM) through conventional whole-cell recording. Uncaging NP-EGTA (more ...)
Pharmacological activation of group II/III mGluRs on presynaptic terminals of interneurons
Next we investigated the mechanism that underlie the depression of eIPSCs in interneurons produced by elevation of [Ca2+
in astrocytes. Ca2+
-dependent release of glutamate is reported to account for several neuronal responses (Araque et al., 1998b
; Araque et al., 1998a
; Bezzi et al., 1998
; Kang et al., 1998
; Pasti et al., 2001
; Fiacco and McCarthy, 2004
; Liu et al., 2004
). Group II/III mGluRs are expressed on presynaptic terminals and that activation of the receptors reduces transmitter release from many excitatory and inhibitory synapses (Conn and Pin, 1997
; Scanziani et al., 1998
; Semyanov and Kullmann, 2000
). We asked whether the observed depression of eIPSCs is due to activation of presynaptic mGluRs induced by the Ca2+
-dependent release of glutamate from astrocytes.
We first established whether presynaptic group II/III mGluRs are present on presynaptic terminals of the inter-neurons using a conventional pharmacological approach. Paired-pulse stimulation with an inter-pulse interval of 50 ms was used to test whether there is an effect at presynaptic or postsynaptic sites. Bath application of 0.5 μM DCG IV, a selective group II mGluR agonist (Hayashi et al., 1993
), decreased the mean amplitude of the first eIPSCs to 43 ± 4% of that of baseline (n
= 8, P
< 0.001, paired t
-test). The paired-pulse ratio (PPR), calculated as the ratio of the amplitude of the second eIPSCs to that of the first eIPSCs, increased significantly (P
< 0.05). Co-application of 300 μM CPPG, the most potent group II/III mGluR antagonist described (Toms et al., 1996
), with the agonist, reversed the agonist-induced inhibition (86 ± 3% of control). CPPG also reversed the DCG IV-induced change in PPR ().
Fig. 3 Selective group II/III mGluR agonists depress eIPSCs in interneurons. (A) The group II mGluR agonist DCG IV (0.5 μM) decreases the amplitude of eIPSCs (left), accompanied by an increase in the paired-pulse ratio (PPR; right, n=8). Both effects (more ...)
Similarly, application of a selective agonist of the group III mGluRs, L-AP4 (50 μM) (Scanziani et al., 1998
), reduced the mean amplitude of the first eIPSCs to 44 ± 7% of that of baseline (n
= 7, P
< 0.001, paired t
-test). PPR was increased significantly by L-AP4
< 0.05). Co-application of 300 μM CPPG and L-AP4
reversed the agonist-induced inhibition (98 ± 5% of control). CPPG also reversed the L-AP4
-induced change in PPR (). Application of CPPG alone did not significantly change the mean amplitude of baseline eIPSCs (103 ± 3% of control, n
= 7), which indicates that group II/III mGluRs are not activated tonically under resting conditions (). Therefore, CPPG was chosen for the following experiment to test whether astrocyte-mediated depression of eIPSCs and mIPSCs is caused by activation of presynaptic mGluRs.
Activation of group II/III mGluRs mediates astrocyte-induced depression of eIPSCs
If astrocytes release glutamate, activate presynaptic group II/III mGluRs and depress GABA release, then CPPG, a potent blocker of group II/III mGluRs, should block the astrocyte-mediated depression of eIPSCs and mIPSCs. We, therefore, examined whether Ca2+ uncaging in astrocytes depresses eIPSCs and mIPSCs in the presence of CPPG. Using the protocol described previously, we uncaged NP-EGTA in astrocytes and recorded eIPSCs in interneurons at a holding potential of −60 mV. As shown in , uncaging NP-EGTA in the presence of CPPG (300 μM) had no significant effect on the amplitude of eIPSCs in any of the eight interneurons tested (101 ± 3% of pre-uncaging level, P > 0.5, paired t-test). CPPG had no significant effect on the uncaging-induced Ca2+ elevation in astrocytes; the peak ΔF/F0 during uncaging (215 ± 22%, n = 8) in the presence of CPPG was not significantly different from that in the absence of CPPG (P > 0.5).
Fig. 4 The group II/III mGluR antagonist CPPG blocks the depression of eIPSCs induced by uncaging NP-EGTA in astrocytes. (A) In the presence of AMPA/kainate and NMDA receptor antagonists (50 μM CNQX and 5 μM CPP) and Group II/III mGluR antagonist (more ...)
To separate eIPSCs from evoked EPSCs, AMPA/kainate and NMDA receptor antagonists CNQX (50 μM) and CPP (5 μM) were included in ACSF in the above recordings. These antagonists should block the activation of all ionotropic glutamate receptors including kainate receptors. Activation of either AMPA/NMDA receptors or kainate receptors by glutamate released from astrocytes modulates synaptic transmission (Araque et al., 1998a
; Kang et al., 1998
; Liu et al., 2004
). We tested the effects of Ca2+
uncaging in astrocytes on eIPSCs in the absence of CNQX and CPP in the ACSF. To separate eIPSCs from evoked EPSCs, we clamped interneurons at the reversal potential of EPSCs, 0 mV. The intracellular solution contained 10 mM CsCl and 130 mM CsCH3
to increase the driving force for eIPSCs. Evoked IPSCs appeared outward under this condition. Uncaging NP-EGTA in astrocytes significantly reduced the amplitude of eIPSCs in five out of nine interneurons tested (76 ± 6% of control, n
= 5, P
< 0.01, paired t
-test, ). The mean peak ΔF/F0
during the uncaging in astrocytes was 201 ± 25% (n
= 5). The depression of eIPSCs was accompanied by an increase in CV from 0.28 ± 0.02 to 0.35 ± 0.03 (P
< 0.01, paired t
-test), which is consistent with a presynaptic mechanism (Bekkers and Stevens, 1990
; Mitchell and Silver, 2000b
). In the remaining four interneurons, Ca2+
uncaging in astrocytes (peak ΔF/F0
= 197 ± 27%, n
= 4) had no significant effect on eIPSCs (99 ± 3%, P
> 0.5, paired t
-test) and did not affect the CV of eIPSCs (baseline 0.30 ± 0.04; uncaging 0.29 ± 0.03). Averaging experiments from the responsive and non-responsive interneurons, the depression of eIPSCs during Ca2+
uncaging is still significantly different from the pre-uncaging level (86 ± 4% of control, n
= 9, P
< 0.05, ). The depression of eIPSCs in the absence of CNQX and CPP is not significantly different from that obtained in the presence of CNQX and CPP (P
> 0.2, ANOVA). In the absence of CNQX and CPP, the depression of eIPSCs and increases in the frequency of spontaneous IPSCs occur in the same interneurons (data not shown). The increase in the frequency of sIPSCs is caused by the activation of kainate receptors, as described in our previous study (Liu et al., 2004
In separate experiments, CPPG (300 μM) was included in ACSF to block group II/III mGluR receptors. Uncaging NP-EGTA in astrocytes caused Ca2+ elevation (ΔF/F0 = 192 ± 20%; n = 9), but had no significant effect on eIPSCs in any of the nine interneurons tested () The mean amplitude of eIPSCs was 97 ± 3% of that of pre-uncaging level (P > 0.3, ). Thus, exclusion of CNQX and CPP from the ACSF did not unmask any significant modulation of eIPSCs caused by astrocyte activation of AMPA/kainate and NMDA receptors. The depression of eIPSCs in interneurons by astrocytes is likely to be caused by Ca2+-dependent release of glutamate and the subsequent activation of presynaptic group II/III mGluRs, which is blocked by CPPG.
Activation of mGluRs also mediates the presynaptic modulation of mIPSCs
We showed previously that Ca2+
uncaging in astrocytes causes a small, significant decrease in the frequency of mIPSCs in interneurons (Liu et al., 2004
). The amplitude distribution of mIPSCs is not affected by the uncaging, which indicates that the depression of mIPSCs has a presynaptic origin. We demonstrated further that pharmacological blocking of AMPA/kainate and NMDA receptors by CNQX and CPP had no effect on the uncaging-induced depression of mIPSCs (Liu et al., 2004
). We suspect that depression of mIPSCs shares a similar mechanism with depression of eIPSCs. We, therefore, tested whether CPPG blocks the effect of Ca2+
uncaging on mIPSCs.
We recorded mIPSCs from interneurons in the presence of TTX (0.5 μM) to block action potentials. CNQX (50 μM) and CPP (5 μM) were also included into ACSF to block miniature EPSCs. As reported in our previous study (Liu et al., 2004
uncaging in astrocytes (peak ΔF/F0
= 197 ± 18%; n
= 7) produces a significant depression of mIPSCs in neighboring interneurons. The frequency of mIPSCs during the uncaging was 78 ± 3% of baseline frequency (n
= 7; P
< 0.05, paired t
-test; ). The amplitude distribution of mIPSCs remained unchanged (), which indicates that a presynaptic mechanism is involved.
Fig. 5 Activation of presynaptic group II/III mGluRs mediates the Ca2+ uncaging-induced decrease in the frequency of mIPSCs. (A) Ca2+ uncaging in an astrocyte decreases the frequency of mIPSCs in a neighboring interneuron. (B) In the presence of CPPG (300 μM), (more ...)
We tested the effect of CPPG (300 μM) on the uncaging-induced mIPSCs. Bath application of CPPG had no significant effect on baseline frequency of mIPSCs (103 ± 4%, n = 8, P > 0.5; paired-t test, ), but it prevented the uncaging-induced depression of mIPSCs (). The mean frequency of mIPSCs during uncaging was 98 ± 6% of that of the pre-uncaging level (n = 8; P > 0.5; paired t-test, ). The mean peak δDF/F0 during uncaging was 189 ± 15%, which is not significantly different from that in the absence of CPPG (P > 0.1, ANOVA).