Article Figures & Data
Figures
- Figure 1.
Activation of high-affinity binding KAR subunit KA2 by low nanomolar concentrations of KA modulates IsAHP in CA3 pyramidal neurons. A, IsAHP amplitude (mean ± SEM) plotted against time (n = 4), showing a reduction evoked by bath perfusion of 5 and 50 nm KA (open squares and filled circles, respectively). A gentle rundown was observed when no KA was applied (open circles). Insets show sample traces of IsAHP before and after KA application (average of 5 consecutive traces). B, Dose–response curve for KA-induced inhibition of IsAHP and relative changes in somatic holding current obtained in wild-type mice (a given KA concentration was tested in at least 3 cells; n = 21). Data from KA-induced inhibition of IsAHP were fitted with the least-square method, which gave an EC50 of 6 nm. Inhibition of IsAHP occurred at low doses of KA (5–50 nm) without a significant shift in somatic holding current. C, Average of five consecutive traces taken before and after application of 50 nm KA in GluR6–/– and KA2–/– mice. D, Dose–response curves for KA-induced inhibition of IsAHP obtained in GluR6–/– and KA2–/– knock-out mice (n = 4 cells for each concentration, from 5 GluR6–/– and 5 KA2–/– mice). The dose–response relationship obtained with low concentrations of KA in wild-type was replotted for comparison. CA3 pyramidal cells were held in voltage clamp at –60 mV with a K-methylsulfate-based solution, in the presence of TTX (1μm), TEA (5 mm), PTX (100μm), CGP 55845 (5 μm), APV (50 μm), LY 341495 (100 μm), and GYKI 53655 (50 μm), to block voltage-dependent Na2+ and K+ channels, GABAA and GABAB receptors, NMDA receptors, metabotropic glutamate receptors, and AMPA receptors. The bar charts show mean ± SEM values. *p < 0.05. Ctrl, Control.
- Figure 2.
The KA2 subunit is responsible for the modulation of IsAHP in CA3 pyramidal cells. A, Immunostaining of hippocampal sections from wild-type, GluR6–/–, and KA2–/– mice with anti-GluR6/7 and anti-KA2 antibodies. Both antibodies labeled stratum lucidum but not stratum radiatum and show a loss of KA2 immunoreactivity in the CA3 region in GluR6–/– mice. B, Western blot analysis of KA2 proteins in hippocampal membranes prepared from wild-type, GluR6–/–, and KA2 transgenic mice. To quantify the relative amount of proteins in each lane, Western blots were probed with a tubulin antibody. KA2 protein was not detected in KA2–/– mice and greatly reduced in GluR6–/– mice (87% of the wild-type amount). C, Using an anti-GluR6 antibody, a G-protein labeled by the anti-Gαq/11 antibody was coimmunoprecipitated with the KA2 and GluR6 subunit in wild-type and GluR5–/– mice and not in KA2–/– mice.
- Figure 3.
Endogenous glutamate inhibits IsAHP via KARs. A, Left, Sample recordings showing the prominent facilitation of EPSCs evoked by trains of stimuli (20 Hz) applied to the dentate gyrus (d.g.) in the absence (Ctrl) and presence of GYKI 53655. Blocking AMPA/KA receptors with GYKI 53655 (50 μm) revealed the characteristic slow KAR-EPSCs, with frequency-dependent facilitation of postsynaptic currents in CA3 pyramidal neurons (average of 5 traces). Right, Superimposed traces showing consecutive sample recordings of KAR-EPSCs in the presence of GYKI 53655 (50 μm), induced by trains of stimuli (20 Hz) at 1 s interval, in the absence (Ctrl), presence (LCCG-1), and after the washout of LCCG-1 (10 μm). These characteristics point to KAR-EPSCs with a physiological and pharmacological profile typical of mossy fiber synapses. B, Time course of IsAHP amplitude (n = 14) in whole-cell mode showing a reversible decrease after 20 Hz tetanic stimulation in the dentate gyrus as indicated by the arrows. After obtaining a stable IsAHP, three trains of five stimuli (20 Hz) were delivered at 1 s interval, and IsAHP was resumed 350 ms later. Insets show (top) sample recordings of IsAHP before (average of 3 traces), 350 ms after the first series of trains (single trace), and after recovery; (bottom) average of five KAR-EPSCs evoked by the trains of stimuli. C, Effect of perforant-pathway (p.path) activation on IsAHP. The same protocol as that applied for dentate stimulation was used to activate perforant-path synapses. AMPA receptors were not blocked, and IsAHP was resumed 350 ms after the last train. The amplitude of IsAHP plotted against time illustrates a nonsignificant decrease after trains of stimuli were delivered at the perforant path as indicated by the arrows. Sample traces (top) show IsAHP before (average of 3 traces) and 350 ms after the last train of stimuli (single trace); average of five EPSC traces (bottom) evoked by the trains of stimuli. D, Summary chart showing that increasing the interval between trains of stimuli and the test pulse for IsAHP induction from 350 ms to 5 s resulted in IsAHP inhibition (0.5 s delay, 24 ± 4%, n = 5; 2 s delay, 25 ± 4%, n = 4; 5 s delay, 23 ± 5%, n = 3), which showed no significant difference from that obtained with a delay of 350 ms (p > 0.1; n = 6). E, Increased stimulus frequency yields greater inhibition ofIsAHP. The bar chart shows IsAHP inhibition in each condition (average of 3–4 cells per condition). The antagonist mixture contained TEA (5 mm), PTX (100μm), APV (50μm), CGP 55845 (5 μm), LY 341495 (100 μm), and GYKI 53655 (50 μm), except in C, in which GYKI 53655 was omitted. *p < 0.05; **p < 0.01.
- Figure 4.
Pharmacological features of IsAHP modulation by synaptically released glutamate from mossy fibers. A, Plot of IsAHP amplitude showing that bath application of NBQX (25 μm) blocked poststimulus depression of IsAHP (whole-cell recordings; n = 7). Top traces show sample recordings of IsAHP in the presence of NBQX, before and after trains of stimuli were delivered to the dentate granule cell layer (d.g.). KAR-EPSCs were concomitantly blocked by NBQX as illustrated in middle inset. B, Plot of IsAHP amplitude showing that preincubation of slices with calphostin C (Calph C; 1μm; n = 5) reduced poststimulus modulation of IsAHP without affecting KAR-EPSCs. Insets, Top, Sample recordings of IsAHP before and after stimulus trains in the presence of calphostin C; bottom, KAR-EPSCs in the presence of calphostin C. C, D, Plot of IsAHP amplitude showing that preincubation of slices with U73122(10μm; n = 5) or U73433(10μm; n = 5) reduced and unaffected poststimulus modulation of IsAHP, respectively, without affecting KAR-EPSCs. Insets, Top, Sample recordings of IsAHP before and after stimulus trains in the presence of U73122 and U73433, respectively; bottom, KAR-EPSCs in the presence of U73122 and U73433, respectively. E, Effect of GDP-β-s (0.5 mm; filled circles) and GTP-γ-s (0.1 mm; open circles) on IsAHP amplitude during trains of stimuli applied to the dentategyrus.F, Bar chart summarizing the magnitude of stimulus-induced IsAHP inhibition obtained with perforated-patch and whole-cell recordings, as well as the effect of different pharmacological treatments on IsAHP modulation. **p < 0.01.
- Figure 5.
Regulation of spike frequency and excitability by synaptic KARs in CA3 pyramidal cells. A, Sample traces evoked by a 1 s depolarizing pulse taken from wild-type and KA2–/– mice, before (left) and after (right) trains of stimuli applied in the dentate gyrus (d.g.) (3 trains of 5 stimuli; 20 Hz). Middle traces illustrate KAR-EPSPs evoked by a single train of stimuli. An increase in the number of spikes after trains of stimuli was observed in wild-type mice only. Membrane potential was –60 mV. B, Bar chart shows the average number of spikes (mean ± SEM) before and after trains of stimuli, indicating a significant increase in excitability in wild-type (n = 4) but not KA2–/– (n = 4 cells, from 3 mice) mice. The increase in spike number observed in wild-type mice returned to baseline value 30 s after stimulation (recovery). **p < 0.01.
- Figure 6.
Absence of synaptic modulation of IsAHP in GluR6–/– and KA2–/– mice. A, B, Sample traces showing the average of five consecutive EPSCs taken from GluR6–/– and KA2–/– mice, before (Ctrl) and after addition of GYKI 53655 (50μm). Trains of EPSCs displayed facilitation and disappeared during application of GYKI 53655 in GluR6–/– mice, confirming that they were mediated by AMPA receptors. In slices from KA2–/– mice, trains of stimuli elicited KAR-EPSCs with marked summation typical of mossy fiber responses. C, D, Plots of IsAHP amplitude showing the lack of synaptic modulation of IsAHP in GluR6–/– (n = 5) and KA2–/– (n = 6) mice. Insets show IsAHP before (averages of 3 traces) and after (single trace) the first series of trains. E, The decrease in IsAHP amplitude after trains was 7 ± 2% (n = 5 cells from 3 mice; p < 0.05 compared with wild-type mice) and 5 ± 3% (n = 6 cells from 3 mice; p < 0.01), in GluR6–/– and KA2–/–, respectively, compared with that obtained in wild-type mice. *p < 0.05; **p < 0.01. Ctrl, Control; d.g., dentate gyrus.
- Figure 7.
Pharmacological manipulation of ionotropic and metabotropic functions of synaptic KARs. A, Occlusion of the metabotropic action of KARs by low concentration of KA (5 nm). Top, Sample traces (average of 5 traces) showing mossy fiber KAR-EPSCs without (Ctrl) and in the presence of KA. KA (5 nm) moderately reduced KAR-EPSC amplitude. Bottom, Plot showing little effect of mossy fiber synaptic modulation of IsAHP in the presence of KA (5 nm)(n = 3). B, Inhibition of KAR-EPSCsbyKyn(1mm) and lack of effecton IsAHP modulation.Top, Sample traces (average of 5 traces) show KAR-EPSCs in a CA3 pyramidal cell without (Ctrl) and with kynurenic acid (1 mm). KAR-EPSCs were primarily inhibited by kynurenic acid (n = 4). Bottom, Plot of IsAHP amplitude in the presence of kynurenic acid (1 mm), before (Ctrl) and after stimulus trains (Kyn, recovery) showing a reversible modulation. Insets in A and B show sample recordings of IsAHP before (average of 3 traces) and after (single trace) stimulus trains. C, Bar charts summarizing the effect of KA (5 nm) and kynurenic acid (1 mm) on the magnitude of mossy fiber synaptic IsAHP inhibition and KAR-EPSCs. *p < 0.05; **p < 0.01. d.g., Dentate gyrus.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental Figure 1: Modulation of IsAHP by KAR activation and the involvement of a metabotropic pathway. (A) Input resistance (mean � s.e.m.) plotted against time (n = 4), showing no change evoked by bath perfusion of 50 nM KA. (B) Sample traces show the average of 5 consecutive IsAHP recordings in neurons prior and after application of 50 nM KA in the presence of NBQX (50 �M). Summary bar chart showing that prior application of 20 �M CNQX (n = 3) or 50 �M NBQX (n = 5) blocked the effect of 50 nM KA on IsAHP. (C) KA-induced inhibition of IsAHP requires PTX-sensitive G-protein coupling and PKC activation. Bar chart illustrating the attenuated KA-induced inhibition of IsAHP in slices treated with calphostin C (1 �M, 2 � 6 hours; inhibition of 26 � 7 %, n = 5) or perfused with NEM (50 �M, 20 minutes; inhibition of 28 � 12 %, n = 9). Sample traces show the average of 5 consecutive traces prior and after application of 50 nM KA in neurons recorded from calphostin C or NEM-treated slices. (D) Bar charts of IsAHP characteristics obtained from wild-type, GluR6-/- (n = 9) and KA2-/- (n = 10) mice showing no significant differences in peak amplitude (pA), decay time constant (s) and charge (nC). Recordings were obtained with TTX (1 �M), TEA (5 mM), PTX (100 �M), APV (50 �M), CGP 55845 (5 �M), LY 341495 (100 �M), and GYKI 53655 (50 �M). The bar charts show mean � s.e.m values. * p < 0.05.
