Astrocyte-mediated activation of neuronal kainate receptors

Abstract

Exogenous kainate receptor agonists have been shown to modulate inhibitory synaptic transmission in the hippocampus, but the pathways involved in physiological activation of the receptors remain largely unknown. Accumulating evidence indicates that astrocytes can release glutamate in a Ca(2+)-dependent manner and signal to neighboring neurons. We tested the hypothesis that astrocyte-derived glutamate activates kainate receptors on hippocampal interneurons. We report here that elevation of intracellular Ca(2+) in astrocytes, induced by uncaging Ca(2+), o-nitrophenyl-EGTA, increased action potential-driven spontaneous inhibitory postsynaptic currents in nearby interneurons in rat hippocampal slices. This effect was blocked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptor antagonists, but not by selective AMPA receptor or N-methyl-d-aspartate receptor antagonists. This pharmacological profile indicates that kainate receptors were activated during Ca(2+) elevation in astrocytes. Kainate receptors containing the GluR5 subunit seemed to mediate the observed effect because a selective GluR5-containing kainate receptor antagonist blocked the changes in sIPSCs induced by Ca(2+) uncaging, and bath application of a selective GluR5-containing receptor agonist robustly potentiated sIPSCs. When tetrodotoxin was included to block action potentials, Ca(2+) uncaging induced a small decrease in the frequency of miniature inhibitory postsynaptic currents, which was not affected by AMPA/kainate receptor antagonists. Our data suggest that an astrocyte-derived, nonsynaptic source of glutamate represents a signaling pathway that can activate neuronal kainate receptors. By modulating the activity of interneurons, astrocytes may play a critical role in circuit function of hippocampus.

Fig. 1.

Ca²⁺ uncaging in astrocytes. (A₁) An astrocyte was stimulated with a train of 12 UV laser pulses (0.1 Hz) to uncage NP-EGTA. Images were taken before, during, and after the UV pulses, as indicated. Scale bar in recovery, 10 μm. (A₂) Time course of changes in calcium fluorescence, ΔF/F₀, of the experiment shown in A₁. (B) The Ca²⁺ uncaging-induced change in ΔF/F₀ was significantly attenuated by CPA (10 μM), which depletes internal Ca²⁺ stores. (C) Preloading with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate AM (BAPTA-AM; 10 μM), a calcium chelator, prevented the calcium change induced by Ca²⁺ uncaging. (D) UV pulses had no effect on ΔF/F₀ when slices were loaded with fluo-4 alone (No NP-EGTA). (E) Averaged peak values of ΔF/F₀ under the various conditions; n = 8-11 for each group.

Fig. 2.

Effects of Ca²⁺ uncaging on sIPSCs in interneurons. (A) Ca²⁺ uncaging produced a change of ΔF/F₀ in an astrocyte (Upper) and a reversible increase in the frequency of sIPSCs in nearby interneuron (Lower). (B) Traces are sIPSCs taken from A but shown in an expanded time scale. (C and D) Cumulative frequency plots show that Ca²⁺ uncaging has no significant effect on the amplitude distribution (P > 0.1, Kolmogorov-Smirnov test, C) but shifts the interevent interval toward the left (P < 0.001, Kolmogorov-Smirnov test, D). (E) Normalized frequency of sIPSCs under different conditions. No NP-EGTA indicates that slices were loaded with fluo-4 alone. ^*, P < 0.05, uncaging versus other groups by ANOVA with Dunnett's test; n = 8-11 for each group.

Fig. 3.

Effects of ionotropic glutamate receptor antagonists on the uncaging-induced increase in the frequency of sIPSCs. Pooled data of normalized frequency of sIPSCs during Ca²⁺ uncaging. The data for uncaging was taken from that shown in Fig. 2E for comparison. NBQX and CNQX (50 μM) each prevented the increased frequency of sIPSCs in response to Ca²⁺ uncaging, whereas the selective AMPA antagonist SYM 2206 and the selective NMDA receptor antagonist CPP did not. ^*, P < 0.05 compared with control, NBQX, or CNQX groups by ANOVA with Dunnett's test, n = 8-9 for each group.

Fig. 4.

Effects of selective GluR5-containing kainate receptor agonist ATPA and antagonist LY293558 (LY) on sIPSCs. ATPA (1 μM) increased the frequency of sIPSCs. LY (30 μM) had no effect on the frequency of sIPSCs but prevented ATPA and Ca²⁺ uncaging-induced increases. ^*, P < 0.001, ATPA versus other groups by ANOVA with Dunnett's test, n = 8 or 9 for each group.

Fig. 5.

Modulation of sIPSCs by uncaging NP-EGTA loaded through whole-cell recording. (A Upper) A representative trace showing the time course of Ca²⁺ transients (ΔF/F₀) induced by Ca²⁺ uncaging. Ca²⁺/NP-EGTA (1 mM/2 mM) was loaded into the astrocyte for 3 min before uncaging. (A Lower) Ca²⁺ uncaging produced a reversible increase in the frequency of sIPSCs in interneurons. (B) Pooled data show the effect of NP-EGTA loading, uncaging, and glutamate infusion on the frequency of sIPSCs in interneurons. Whole-cell loading of glutamate and/or NP-EGTA had no effect on sIPSCs. After loading 1 mM Ca²⁺/2 mM NP-EGTA for 3 min in astrocytes, Ca²⁺ uncaging produced a significant increase in the frequency of sIPSCs, which was blocked by the GluR5-containing kainate receptor antagonist LY293558 (LY, 30 μM). After loading Ca²⁺/NP-EGTA for 10 min, Ca²⁺ uncaging had no significant effect on sIPSCs (Left). The time-dependent rundown of the responses was mainly due to the washout of glutamate in whole-cell recording, because including 1 mM glutamate in the recording solution significantly retarded the rundown (Center). Uncaging NP-EGTA alone (in the absence of added Ca²⁺) had no effect on sIPSCs (Right). The number of cells tested in each group is shown in parentheses. ^*, P < 0.05; ^**, P < 0.01 compared with control or loading groups by ANOVA with Dunnett's test.