Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus

Abstract

In cerebellar granule cells, delta subunit-containing GABA(A) receptors are found exclusively at extrasynaptic sites, but their subcellular distribution in other brain areas is poorly understood. We examined the anatomical localization and physiological activation of these receptors in adult mouse dentate gyrus granule cells. Immunocytochemistry revealed a high density of delta subunits in the molecular layer and a much lower density in the cell body layer. At the ultrastructural level, immunogold-labeled delta subunits were found at the edge of symmetric synapses on granule cell dendrites. Functional correlates of this perisynaptic localization were obtained by comparing inhibitory responses in delta subunit-deficient (delta-/-) and wild-type (wt) mice. In whole-cell recordings at 22 degrees C, the weighted decay time constants (tau(w)) of spontaneous IPSCs (sIPSCs) were significantly longer in wt mice but were similar at 34 degrees C, reflecting the role of temperature-dependent GABA uptake in shaping sIPSC decay. IPSCs evoked by minimal stimulation (eIPSCs) near the somata had similar tau(w) in delta-/- and wt mice, but eIPSCs elicited from dendritic sites decayed significantly more slowly in wt mice, consistent with a higher density of delta subunit-containing receptors in the molecular layer. The tau(w) of dendritic eIPSCs of wt mice were shortened by ZnCl2 (10 microm), reflecting the high Zn2+ sensitivity of delta subunit-containing GABA(A) receptors, and were prolonged by the GAT-1 GABA transporter inhibitor NO711 (10 microm). Our results demonstrate a perisynaptic localization of delta subunit-containing GABA(A) receptors and indicate that these receptors can be activated by GABA overspill in the molecular layer.

Figure 1.

Distribution of δ and α2 subunits at the light microscopic level. A, Moderate diffuse immunolabeling of the δ subunit is found in the molecular layer (M) of the dentate gyrus, and only light labeling is evident in the granule cell layer (G). Light diffuse labeling is present in the dendritic layers of CA1, but very little labeling is detected in CA3 and the hilus (H). Moderate to lightly labeled interneurons are present throughout the hippocampus. B, C, Immunoreactivity for both δ (B) and α2 (C) subunits is relatively strong in the molecular layer (M) of the dentate gyrus. In the granule cell layer (G), labeling of the δ subunits is low around the cell bodies of the granule cells (B). This contrasts with much stronger labeling of the α2 subunit in the granule cell layer, where the labeling outlines the contours of many granule cell somata (C). In B, several putative interneurons in the granule cell and molecular layers are moderately labeled for the δ subunit. Scale bars: A, 300 μm; B, C, 25 μm.

Figure 2.

Immunogold labeling of the δ and α2 subunits of the GABA_A receptor. Gold particles that indicate the location of the δ subunit (A-D, arrows) were present on or near the plasma membrane of dendrites (D) that were in contact with axon terminals (T). Gold particles were localized primarily at perisynaptic sites, just outside or at the outer edge of symmetric synapses (A, D, solid arrowheads). In contrast, immunogold labeling for the α2 subunit was observed directly at symmetric synapses (E, F, solid arrowheads). No labeling for either the δ or α2 subunit was observed at asymmetric synapses on spines (A, F, open arrowheads). Scale bars, 0.2 μm.

Figure 3.

sIPSC conductance and voltage dependence of τ_w in wt andδ-/- granule cells. A, Each trace is the average of >100 sIPSCs recorded at five different holding potentials of -70, -40, -20, +20, and +40 mV, respectively. B, Left panel, The sIPSC conductance was calculated from linear fits to the peak amplitude versus holding potential plots. The average peak amplitudes were obtained by the method of highest amplitude count matching (for details, see Results) to avoid the bias introduced by losing events to the noise when the holding potential was closer to E_Cl. There was no significant difference between the slope conductances of sIPSCs recorded in the two genotypes (0.90 ± 0.04 pS in wt vs 0.78 ± 0.05 pS in δ-/-). Right panel, The voltage dependence of τ_w was also comparable between wt and δ-/- granule cells. The number of cells is n = 10 for wt and n = 9 for the δ-/-.

Figure 4.

Temperature dependence of sIPSC recorded in wt and δ-/- granule cells. A, Normalized and superimposed sIPSC averages from >100 events in wt (gray trace) and in δ-/- (black trace) mice at 22° and 34°C, respectively. B, The weighted decay time constants are slower in wt (n = 10) than in δ-/- (n = 9) mice at 22°C (^*p < 0.01; t test), whereas they are similar at 34°C (wt, n = 5; δ-/-, n = 6). C, sIPSC amplitudes are also temperature dependent but not significantly different between wt (n = 10 at 22°C; n = 5 at 34°C) and δ-/- (n = 9 at 22°C; n = 6 at 34°C) mice.

Figure 5.

The effect of Zn²⁺ on sIPSC kinetics in wt and δ-/-. A, At 34°C, ZnCl₂ (10 μm) had no significant effect on the decay time constants of sIPSCs recorded in either wt (n = 5) or δ-/- (n = 6) granule cells. B, At 22°C, perfusion of 10 μm ZnCl₂ reduced the weighted sIPSC decay time constant in wt (n = 7; ^*p < 0.01; paired two-tailed t test) to a value similar to that found in δ-/- mice at the same temperature (Fig. 4B). In contrast, 10 μm ZnCl₂ does not significantly affect the decay time constants of sIPSCs in δ-/- neurons (n = 5; p > 0.05; paired two-tailed t test). Raw traces are averages of 60-120 sIPSCs.

Figure 6.

The effect of Zn²⁺ on eIPSCs elicited from somatic and dendritic stimulation sites in wt and δ-/- granule cells. A, Stimulating electrodes positioned near the soma (Soma) and in the molecular layer (Dendrite) were used to elicit eIPSCs by minimal stimulation. Traces are averages of >30 events from wt and δ-/- slices. The rightmost panels show normalized events at expanded time scales to illustrate the effect of 10 μm ZnCl₂ (Zn²⁺) on eIPSCs elicited from both sites in wt and on dendritically evoked events in wt and δ-/- granule cells. B, Summary data for five experiments each. Weighted decay time constants of somatic eIPSCs are comparable between wt and δ-/- granule cells (p > 0.05; two-tailed t test), and Zn²⁺ does not affect the decays of somatic eIPSCs in wt neurons (p > 0.05; paired two-tailed t test). In contrast, dendritically evoked eIPSCs in wt granule cells decay significantly faster in the presence of ZnCl₂ (10 μm) than under control conditions (^*p < 0.01; paired two-tailed t test). The dendritically evoked eIPSCs of δ-/- mice decay significantly faster than those recorded in wt granule cells (Table 2) and are unaffected by Zn²⁺ (p > 0.05; paired two-tailed t test).

Figure 7.

Effect of the GAT-1 GABA transporter inhibitor NO711 (10 μm) on somatically and dendritically elicited eIPSCs in wt and δ-/- granule cells. A, Averaged somatic (Soma) eIPSCs in wt and dendritic (Dendrite) eIPSCs in δ-/- (>30 each) recorded at 34°C are not affected by NO711. Only the dendritic eIPSCs of the wt are prolonged by the uptake blocker. B, Summary of weighted decay time constants of eIPSCs in four experiments each. Perfusion of 10 μm NO711 had no effect on the decays of somatic eIPSCs of wt or on the decays of dendritic eIPSCs in δ-/- granule cells (p > 0.05; paired two-tailed t test). In contrast, NO711 further prolonged (191% of pre-drug control) the already long, weighted decay time constants of dendritic eIPSCs of wt granule cells (^*p < 0.01; paired two-tailed t test).