Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms

Abstract

The kinetics of IPSCs influence many neuronal processes, such as the frequencies of oscillations and the duration of shunting inhibition. The subunit composition of recombinant GABA(A) receptors (GABA(A)Rs) strongly affects the deactivation kinetics of GABA-evoked currents. However, for GABAergic synapses, the relationship between subunit composition and IPSC decay is less clear. Here we addressed this by combining whole-cell recordings of miniature IPSCs (mIPSCs) and quantitative immunolocalization of synaptic GABA(A)R subunits. In cerebellar stellate, thalamic relay, and main olfactory bulb (MOB) deep short-axon cells of Wistar rats, the only synaptic α subunit was α1, and zolpidem-sensitive mIPSCs had weighted decay time constants (τ(w)) of 4-6 ms. Nucleus reticularis thalami neurons expressed only α3 as the synaptic α subunit and exhibited slow (τ(w) = 28 ms), zolpidem-insensitive mIPSCs. By contrast, MOB external tufted cells contained two α subunit types (α1 and α3) at their synapses. Quantitative analysis of multiple immunolabeled images revealed small within-cell, but large between-cell, variability in synaptic α1/α3 ratios. This corresponded to large cell-to-cell variability in the decay (τ(w) = 3-30 ms) and zolpidem sensitivity of mIPSCs. Currents evoked by rapid application of GABA to patches excised from HEK cells expressing different mixtures of α1 and α3 subunits displayed highly variable deactivation times that correlated with the α1/α3 cDNA ratio. Our results demonstrate that diversity in the decay of IPSCs can be generated by varying the expression of different GABA(A)R subunits that alone confer different decay kinetics, allowing the time course of inhibition to be tuned to individual cellular requirements.

Figure 1.

Immunofluorescent labeling for GABA_AR subunits in the main olfactory bulb. Low-magnification images of representative immunofluorescent reactions encompassing the various layers of the main olfactory bulb are shown. Bottom right, Triple labeling for the α1, β3, and γ2 subunits. Scale bar, 50 μm (applies to all panels). GL, Glomerular layer; MCL, mitral cell layer.

Figure 2.

Multiple immunofluorescent labeling for GABA_A receptor subunits in the inframitral layers of the main olfactory bulb. A, Immunoreactivity for the α1 subunit (green) delineates the somatodendritic membranes of a dSAC. α1-Immunopositive clusters (arrows) that also show labeling for the synaptic marker gephyrin (gray) are all immunonegative for the α3 subunit (red). α3-Immunopositive clusters (arrowheads) are negative for the α1 subunit. B, An α1-immunopositive dSAC is immunonegative for the α2 subunit (red). The α2 subunit-positive clusters originate from GCs. Immunolabeling for gephyrin (gray) indicates GABAergic synapses. α1 (arrows) and α2 (arrowheads) subunit-immunopositive puncta also contain gephyrin, demonstrating the synaptic enrichment of these GABA_AR subunits. C, D, α1-Immunopositive clusters (green) in a dSAC (arrows) contain strong immunolabeling for the β2 (C, red), but not for the β3 (D, red), subunit. Strong β3-immunopositive clusters belong to GCs. Scale bars: (in A, B) A–D, 5 μm. C and D are in the same magnification as A.

Figure 3.

mIPSC kinetics and pharmacology of main olfactory bulb dSACs. A, Continuous current recordings of mIPSCs from an EPL-dSAC (MDE471) before (black traces) and after (red traces) bath application of 100 nm zolpidem. B, Superimposed consecutive mIPSCs before (black, average in gray) and after (red, average in dark red) the application of 100 nm zolpidem. Right inset shows peak-scaled averaged mIPSCs, demonstrating that zolpidem slowed the decay of mIPSCs. Left inset illustrates a plot of mean current against current variance (peak-scaled nonstationary fluctuation analysis). Parabolic fit indicates a single-channel conductance (γ) of 31.5 pS and that 40 channels open at the peak of the averaged mIPSC (N_p). C, Peak-scaled averages of baseline recordings from 14 morphologically identified EPL-dSACs (gray) and the population mean trace (cyan). τ_w of the population mean and CV are indicated. D, Summary of the effects of ACSF (p = 0.37, paired t test, n = 4) or 100 nm zolpidem (p = 0.004, paired t test, n = 4) on the weighted decay time of mIPSCs.

Figure 4.

Multiple immunofluorescent labeling for GABA_A receptor subunits in the VPL and the nRT. A, Neither α1 (green) nor α2 (red) subunit immunoreactivity is detected in the nRT in strong gephyrin immunoreactive clusters (double arrowheads). B, Strong, punctate α3 subunit immunoreactivity (red) is observed in the nRT, showing intense colocalization with gephyrin (arrowheads). C, Immunoreactivity for the β1 subunit (green) is not detected in the nRT (identified by the α3 immunoreactivity, red) or in the adjacent VPL, despite strong labeling in the dentate gyrus in the same section (inset). D, GABA_AR α1 (red) and β2 (green) subunits colocalize in the VPL, but are both absent from the nRT. E, GABA_AR β3 subunit (green) immunoreactivity in the nRT is strong and punctate, but it is much weaker in the adjacent α1 subunit-positive (red) VPL. F, G, High-magnification images indicate the lack of β1 immunoreactivity in nRT neurons covered by strong α3-immunoreactive puncta (F, arrowheads), whereas both α3 and β1 subunit-immunoreactive puncta are observed on somatic profiles in hippocampal interneurons (G). Scale bars: A, B, F, G, 5 μm; C, D, 50 μm; E, 20 μm. Scale bar in A applies to B. ml, Molecular layer of the dentate gyrus; VPL, ventral posterolateral thalamic nucleus.

Figure 5.

mIPSC kinetics and pharmacology in identified nRT cells. A, Continuous current recordings of mIPSCs from an nRT cell (MDE816) before (black traces) and after (red traces) the bath application of 100 nm zolpidem. B, Superimposed, consecutive mIPSC traces before (black, average in gray) and after (red, average in dark red) 100 nm zolpidem application. Peak-scaled averages (right-hand inset) show the lack of effect of zolpidem. C, Peak-scaled averages of baseline recordings from 22 morphologically identified nRT cells (gray) and the population mean average trace (cyan). τ_w of the population mean and CV are indicated. D, Summary data showing the effects of ACSF (p = 0.33, paired t test; n = 3) and 100 nm zolpidem (p = 0.60, paired t test; n = 5) on mIPSC mean τ_w. E, A biocytin-filled nRT cell (red) is located within the parvalbumin-immunoreactive nRT (green). F, Higher-magnification view of the boxed region in E. Scale bars: E, 500 μm; F, 100 μm. fi, Fimbria; CPu, caudate–putamen; LGP, lateral globus pallidus; ic, internal capsule; VA, ventral anterior thalamic nucleus.

Figure 6.

Multiple immunofluorescent labeling for GABA_AR subunits in the supramitral layers of the main olfactory bulb. A, GABA_AR α1 subunit immunoreactivity (green) in the glomerular layer delineates the somatic membrane of many juxtaglomerular cells but does not colocalize with the α2 subunit (red). Punctate gephyrin labeling (gray) is associated with both the α1 (arrows) and α2 (arrowheads) subunits. B, GABA_AR α1 (green) and α3 (red) subunit immunoreactivity in the glomerular layer show highly variable colocalization, such that puncta on individual somatic and dendritic membranes in the overlay appear as various shades of green (cells 3 and 4), orange (cells 2 and 5), yellow (cell 1), and red (cell 6). Scale bars: A, 5 μm; B, 10 μm.

Figure 7.

Quantitative analysis of immunofluorescent labeling of synaptic GABA_ARs in ETCs. A, Different ETCs show a range of α3/α1 subunit ratios visualized by the different color hues between green (cells 6, 7, and 8), yellow (cells 4 and 5), orange (cells 2 and 3), and red (cell 1). B, Plot of average α3 subunit against average α1 subunit immunofluorescence intensity of all puncta for each of 29 ETCs. Note the large heterogeneity in the intensities of both subunits, and note that cells occupy the entire parameter space; no clear clusters are evident. C, Distribution of the α3/α1 intensity ratio for 29 ETCs (black bars). Distributions of individual puncta from two cells are shown in the yellow and green histograms. As an example, the small variance (CV = 0.15) of the yellow distribution indicates that the synapse-to-synapse variability in the α3/α1 subunit ratio within a single cell is low. The large variance (CV = 1.35) of the total distribution shown in black demonstrates the large cell-to-cell variability in the α3/α1 ratio. Scale bar, 5 μm.

Figure 8.

mIPSC kinetics and pharmacology in ETCs. A, C, Continuous current recordings of mIPSCs from two ETCs (left, MDE599; right, MDE542) before (black traces) and after (red traces) the bath application of 100 nm zolpidem. B, D, Superimposed consecutive mIPSC traces before (black, average in gray) and after (red, average in dark red) 100 nm zolpidem application. Insets show peak-scaled averaged mIPSCs of baseline (gray) and zolpidem (dark red) of each cell for comparison. Note that the decay of the fast mIPSCs in B is prolonged by zolpidem, whereas that of the slow mIPSC in D is affected to a much smaller extent. E, Superimposed peak-scaled averages of baseline recordings from 54 ETCs (gray) and the population mean average trace (cyan). τ_w of the population mean and the CV are indicated. F, Summary data showing the effects of ACSF (p = 0.16, paired t test, n = 12) and 100 nm zolpidem (p < 0.001, paired t test, n = 13) on mIPSC τ_w.

Figure 9.

The kinetics of mIPSCs do not correlate with the dendritic morphology of ETCs. A, B, Neurolucida reconstructions of representative ETCs with fast mIPSC kinetics that lack (B, MDE758, orange cell) or that have (A, MDE798, green cell) basal dendrites in the EPL. C, D, Examples of ETCs with slow mIPSC kinetics are shown without (C, MDE750, cyan cell) and with (D, MDE795, blue cell) basal dendrites. Central inset shows peak-scaled peak-aligned averaged mIPSCs of the color-coded cells with τ_w indicated. Scale bars: 25 μm. GL, Glomerular layer.

Figure 10.

Immunofluorescent localization of the α3 subunit in the MOB. A, Low-magnification view of the distribution of the α3 subunit in the MOB. Note the large difference in intensity between ETCs and GCs. Within the GCL, the signal strength increases from superficial to deep zones. B, C, High-magnification views of double-immunofluorescent labeling for the α3 and β3 subunits in superficial (B) and deep (C) GCL. Note that there are many more α3 subunit-immunopositive clusters in the deep GCL than in the superficial part and that all these α3 subunit-positive clusters colocalize with the β3 subunit. Scale bars: A, 50 μm; B, C, 5 μm. Scale bar in B applies to C.

Figure 11.

Properties of mIPSCs in MOB GCs. A, Continuous current recordings from a GC (MDE525) before (black traces) and after (red traces) bath application of 100 nm zolpidem. B, Superimposed consecutive mIPSCs before (black, average in gray) and after (red, average in dark red) 100 nm zolpidem application. Inset shows peak-scaled averages, demonstrating the lack of zolpidem effect on mIPSC kinetics. C, The population mean average mIPSC trace (cyan) is superimposed on the peak-scaled averaged mIPSCs from 48 GCs (gray). τ_w of the population mean and the CV are indicated. D, τ_w of the mIPSCs shows a positive correlation with the distance of the GC somata from the MCL; the deeper the somatic location, the slower the mIPSC τ_w. The correlation coefficient of a linear fit (black line) to the data is indicated. MCL, Mitral cell layer.

Figure 12.

Deactivation kinetics of recombinant GABA_A receptors expressed in HEK cells following transfection of α1, α3, or both α1 and α3 cDNA. A, Representative current evoked by 1 ms application of 1 mm GABA to a patch from an HEK cell expressing α1β3γ2L receptors. The black line is a superimposed triple exponential fit; the gray trace above is the residual. The inset shows the initial part of the record. B, The global average of normalized α1 currents (n = 6); the gray fill denotes the SEM. Inset shows the same trace on an expanded amplitude scale (record truncated) and slower time base. C, Same as A but for a current evoked in a patch from an HEK cell expressing α3β3γ2L receptors (black line is fit with four exponential components). D, Global average from nine α3β3γ2L patches (inset, same as in B). E, Three representative currents evoked in patches from HEK cells transfected with α1 and α3 cDNAs at a ratio of 1:1.5. The inset shows the average currents on an expanded amplitude scale (records truncated) and slower time base. F, Global averages of normalized currents for α1 and α3 cotransfected at ratios of 1:1, 1:1.5, and 1:2 (n = 7, 20, and 12, respectively). Inset shows the average currents on a different scale (same as in B, D), together with the α1 (blue) and α3 (red) averages for comparison. G, Box plots showing τ_w,deact for different α subunits. Boxes indicate 25–75th percentiles, and whiskers indicate the 10–90th percentiles; horizontal red bars denote median, and crosses denote mean values. Green circles indicate individual values. Asterisks indicate significant difference from α3β3γ2L receptors (**p < 0.01, Tukey's HSD test). H, Plot showing τ_w,deact values for all patches. For those cells transfected with both α1 and α3 cDNAs (open symbols), k-means clustering of pooled data identified two clusters (open blue and open red symbols) with means of 52 and 139 ms. These clusters overlapped with the α1-only (filled blue symbols) and α3-only groups (filled red symbols), respectively.

Figure 13.

Summary of the kinetics and zolpidem sensitivity of mIPSCs recorded from a variety of nerve cells under identical conditions. A, Peak-scaled, peak-aligned population average traces demonstrating the different kinetics underlying different GABA_AR α subunit expression in different cell types. Note that the ETC average (bright green) falls between the other population averages (i.e., red-orange and blue-black). Two individual ETCs with fast (MDE578, light green) and slow (MDE795, dark green) decay kinetics are also shown. Similarly, fast (MDE950, light blue) and slow (MDE890, dark blue) individual GCs are shown. The two individual cells (GC and ETC) represent the ∼15th and ∼85th percentiles of the population. B, Plot of the effect of 100 nm zolpidem as a function of the initial τ_w of mIPSCs. Zolpidem strongly affects the decay of mIPSCs in cells with fast kinetics [cerebellar interneurons (INs), MOB EPL-dSACs] but has no effect on cells with slow kinetics (nRT cells). The effect of zolpidem on the decay of MOB ETC mIPSCs depends on the initial decay time constant of the mIPSCs.