A mosaic of functional kainate receptors in hippocampal interneurons

Abstract

Although some physiological functions of kainate receptors (KARs) still remain unclear, recent advances have highlighted a role in synaptic physiology. In hippocampal slices, kainate depresses GABA-mediated synaptic inhibition and increases the firing rate of interneurons. However, the sensitivity to agonists of these responses differs, suggesting that the presynaptic and somatic KARs have a distinct molecular composition. Hippocampal interneurons express several distinct KAR subunits that can assemble into heteromeric receptors with a variety of pharmacological properties and that, in principle, could fulfill different roles. To address which receptor types mediate each of the effects of kainate in interneurons, we used new compounds and mice deficient for specific KAR subunits. In a recombinant assay, 5-carboxyl-2,4-di-benzamido-benzoic acid (NS3763) acted exclusively on homomeric glutamate receptor subunit 5 (GluR5), whereas 3S,4aR,6S,8aR-6-((4-carboxyphenyl)methyl) 1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY382884) antagonized homomeric GluR5 and any heteromeric combination containing GluR5 subunits. In hippocampal slices, LY382884, but not NS3763, was able to prevent kainate-induced depression of evoked IPSC. In contrast, neither prevented the concomitant increase in spontaneous IPSC frequency. The selectivity of these compounds was seen additionally in knock-out mice, such that they were inactive in GluR5-/- mice but completely effective in GluR6-/- mice. Our data indicate that in wild-type mice, CA1 interneurons express heteromeric GluR6 -KA2 receptors in their somatic compartments and GluR5-GluR6 or GluR5-KA2 at presynaptic terminals. However, functional compensation appears to take place in the null mutants, a new pharmacological profile emerging more compatible with the activity of homomeric receptors in both compartments: GluR5 in GluR6-/- mice and GluR6 in GluR5-/- mice.

Figure 1.

Sensitivity of homomeric and heteromeric kainate receptors to the NS3763 and LY382884 antagonists. A, Examples of current responses in HEK293 cells transiently transfected with GluR5 and either KA2 or GluR6 and in cells stably transfected with GluR5 alone. Inset, Response induced by ATPA (100 μm), revealing the presence of heteromeric GluR5-GluR6 receptors in the membrane. The rapid application of 1 mm glutamate in the presence (gray trace) and absence (black trace) of the antagonist is denoted by horizontal bars. B, Summary of the effect of 10 μm NS3763 or LY383884 on kainate receptors of different subunit composition. The amplitudes of the responses in the presence of the antagonist are expressed as a percentage of the responses obtained by the application of 1 mm glutamate in its absence. The values are the mean ± SEM of 4-12 separate experiments. C, Heteromeric GluR6 -KA2 but not homomeric GluR6 receptors are sensitive to micromolar concentrations of the agonist ATPA.

Figure 2.

Effect of kainate receptor activation on spontaneous and evoked IPSCs recorded from CA1 pyramidal cells. A, IPSCs were evoked by paired pulses applied to the stratum oriens (arrows; 40 msec interval), in the presence of antagonists of NMDA (APV; 50 μm) and AMPA (LY303070; 25 μm) receptors. Black and gray traces correspond to readings before and during kainate (3 μm) perfusion, respectively. B, The evoked IPSCs are superimposed after subtracting the holding current. C, Time course of evoked IPSC amplitudes (filled circles) and frequency (unfilled circles), before, during, and after kainate application. Points are the mean ± SEM of 14 experiments. D, Mean basal frequency of spontaneous IPSCs in wild-type and GluR5 or GluR6 KO mice. The spontaneous IPSC frequency was computed for a 3 min segment in each cell from 6-14 separate experiments. The columns represent the mean ± SEM.

Figure 3.

Effect of antagonists on the increase in the kainate-induced frequency of spontaneous IPSCs in wild-type and KO mice. A, Representative recordings before (black traces) and during (gray traces) kainate (3 μm) application in the presence and absence of NS3763 (3 μm). Traces correspond to different neurons because kainate was applied just once per slice. B, Increase in spontaneous IPSC frequency induced by kainate and ATPA in wild-type and kainate receptor KO mice. Bars are the averages of data obtained from 5-14 experiments. C, Effect of NS3763 and LY382448 on the kainate-induced increase in spontaneous IPSC frequency. The dotted line represents the effect of kainate (3 μm) in the absence of antagonist. ^*p < 0.05; ^**p < 0.005, two-tailed Student's unpaired t test.

Figure 4.

Effect of LY38288 on ATPA-induced currents in hippocampal interneurons. A, Inward currents induced by bath-applied ATPA (10 μm) in hippocampal interneurons before (black trace), during (dark gray trace), and after (light gray trace) application of LY382884 (10 μm). LY382884 was introduced in the chamber at least 4 min before the agonist. Rec, Recovery. B, Summary of the ATPA-induced responses in the presence of LY382884. The values are the mean ± SEM expressed as a percentage of control responses (4 slices).

Figure 5.

Effect of kainate receptor antagonists on the kainate-induced reduction of evoked IPSC amplitude in wild type and KO mice. A, Representative evoked IPSCs before (black records) and during (gray traces) application of kainate (3 μm) in the presence and absence of NS3763 (3 μm). Recordings are from different neurons because kainate was applied just once per slice. B, Inhibitory effects of kainate and S-ATPA on the amplitude of evoked IPSCs in wild-type and KO animals. C, Effect of kainate receptor antagonists on the kainate-induced inhibition of evoked IPSCs. The dotted line represents the inhibitory effect induced by kainate in the absence of antagonists. Bars correspond to the mean ± SEM of 3-14 experiments. ^*p < 0.05, two-tailed Student's unpaired t test.

Figure 6.

The KA2 subunit is downregulated in GluR5- and GluR6-deficient mice. Western blot analysis of hippocampal homogenates showed that the protein levels of the KA2 subunit were much lower in both GluR5- and GluR6-deficient mice compared with WT mice. The signals obtained by antibodies directed toward N-terminal (α-KA2_Nterm) (A) or C-terminal (α-KA2_Cterm) (B) epitopes of KA2 were assessed by densitometric analysis. Densitometry profiles of immunoblots are shown for each case. β-Actin was used as a loading control in each case. Calibration: KA2 blots, 0.02 arbitrary units; β-actin blots, 0.2 arbitrary units.

Figure 7.

Diagram summarizing the deducted subunit composition of somatic and presynaptic kainate receptors in CA1 interneurons from wild-type and KO mice. The table at the bottom summarizes the main activity of agonists and antagonists on the two parameters studied. ↑ indicates that the agonist stimulates the receptor, where as ↓ indicates that the antagonist significantly reduced receptor activation; - indicates that the compound was inactive in antagonizing or stimulating receptors. Each color represents a particular pharmacological profile. Note that although inhibition of evoked IPSCs (i.e., presynaptic activity) presents a profile compatible with both heteromeric GluR5-GluR6 (green) and GluR5-KA2 (cream) receptors, the behavior of the IPSC frequency (i.e., somatodendritic receptor activity) only fits with the heteromeric GluR6 -KA2 profile (light blue). The situation is drastically altered in KO mice, in which the corresponding pharmacological profiles exclusively match profiles of homomeric receptors (yellow or blue).