Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses
- Neuro-Bicêtre, Inserm U1195, Université Paris-Saclay, France
- UHasselt, Neurophysiology Laboratory, BIOMED Research Institute, Belgium
Figures
Figure 1
Glycine receptor (GlyR) gene expression in mouse brain.
(A) mRNA expression of GlyR subunits α1, α2, α3, and β in different brain regions of 2-month-old C57BL/6J mice (sorted from frontal regions on the left to dorsal on the right). RNA-seq data were retrieved from Human Protein Atlas (HPA, https://www.proteinatlas.org/) and are expressed as normalised transcripts per million (nTPM). The hippocampus includes the subregions of the cornu ammonis (CA) and the dentate gyrus (DG). (B) UMAP projection (using RPCA reduction) of single-cell transcriptomic data derived from four mouse brain regions: hippocampus (HIP-CA), dorsal striatum (STRd), ventral striatum (STRv), and medulla (see Methods for details on data acquisition and processing). Each panel shows the expression of a different GlyR subunit (Glra1, Glra2, Glra3, or Glrb), visualised with a colour scale from low (yellow) to high (purple). Clusters corresponding to major cell types (neurons, oligodendrocytes, oligodendrocyte precursor cells [OPCs], astrocytes, and microglia) are labelled in the first panel. (C) Schematic diagram of hippocampus. The red squares represent the hippocampal sub-regions in which SMLM recordings were taken; the molecular layer of the DG and the stratum radiatum of CA3 and CA1.
Figure 2 with 1 supplement
Single molecule localisation microscopy (SMLM) of mEos4b-GlyRβ subunits in the hippocampus.
(A) Single molecule detections of mEos4b-GlyRβ (red SMLM pointillist image) in the dentate gyrus (DG), CA1 and CA3 regions of the hippocampus in 10 µm cryostat sections of the knock-in mouse line Glrbeos/eos at postnatal day 40 (red pointillist images). Inhibitory synapses were identified in epifluorescence images using the gephyrin marker Sylite (cyan). Scale bar: 5 µm. (B) Mean number of mEos4b-GlyRβ detections per synaptic gephyrin cluster in spinal cord (n = 1265 synaptic clusters from 8 fields of view) and brain slices (n > 5000 clusters from 9 fields of view for each region) from N = 3 independent experiments corresponding to 3 Glrbeos/eos mice (N = 2 for spinal cord). Recordings were also made in the CA3 of wildtype mice not expressing endogenous mEos4b-GlyRβ (negative control; n = 1424 clusters, 3 fields of view, 2 GlrbWT/WT mice) and in Glrbeos/eos hippocampal slices without photoconversion of mEos4b (no UV, n = 1214 clusters, 9 fields of view, 3 animals). A pixel shift control with flipped images was done with the same dataset (Glrbeos/eos, CA3; n = 1978 clusters, 9 fields of view, 3 animals). Data are shown as mean ± SD. Levels of significance were determined using a Kruskal-Wallis test with Dunn’s multiple comparison test: ***p < 0.0001. (C) Cumulative distribution representing the estimated copy number of mEos4b-GlyRβ complexes per synapse in DG (grey line), CA1 (black line), and CA3 (red line) regions. Copy numbers were background-corrected by subtracting the value obtained for the negative control in wildtype slices.
Figure 2—figure supplement 1
Single molecule localisation microscopy (SMLM) of endogenous mEos4b-GlyRβ in spinal cord.
Single molecule detections of mEos4b-GlyRβ (in red) were recorded in spinal cord slices of Glrbeos/eos mice at postnatal day 40 (n = 1265 clusters, 8 fields of view, 2 mice) and labelled with Sylite gephyrin marker (cyan). Scale bar: 5 µm.
Figure 3 with 2 supplements
Dual-colour single molecule localisation microscopy (SMLM) of glycine receptor (GlyR) and gephyrin at inhibitory hippocampal synapses.
(A) Dual-colour SMLM using spectral demixing of endogenous mEos4b-GlyRβ labelled with anti-mEos-AF647 nanobody (red, NanoTag), and mouse anti-gephyrin (mAb7a, Synaptic Systems) and CF680-conjugated secondary anti-mouse antibodies (cyan) in hippocampal slices of the Glrbeos/eos knock-in mouse line at postnatal day 40. Scale bar: 500 nm. (B) Euclidean distance between the centre of mass (CM) of the anti-mEos-AF647 nanobody (GlyRβ) and gephyrin (mAb7a-CF680) detections of corresponding clusters. (C) Distance of the CM of the anti-mEos-AF647 detections from the CM of gephyrin relative to the radius of gyration (RG) of the gephyrin cluster along the x and y axes (△x/RGx, △y/RGy). N = 2 independent experiments corresponding to 2 Glrbeos/eos animals.
Figure 3—figure supplement 1
Specificity of the anti-mEos-AF647 nanobody.
Spinal cord slices from Glrbeos/eos knock-in and wildtype mice (GlrbWT/WT) were labelled with anti-mEos-AF647 nanobody (AF647-conjugated FluoTag-X2, NanoTag Biotechnologies; #N3102-AF647-L) and with antibodies against gephyrin (mouse anti-gephyrin mAb7a, Synaptic Systems, secondary anti-mouse IgG coupled with CF568). Both the mEos4b-GlyRβ (green) and the anti-mEos-AF647 signals (cyan) co-localised closely with the synaptic gephyrin clusters in Glrbeos/eos slices. mEos immunoreactivity was absent in GlrbWT/WT, showing only minimal non-specific background in conventional fluorescence microscope images. Scale bar: 5 µm.
Figure 3—figure supplement 2
Dual-colour single molecule localisation microscopy (SMLM) using spectral demixing.
(A) Overview of the transmitted camera (CAM T) of endogenous mEos4b-GlyRβ (labelled with anti-mEos-AF647 nanobody) and gephyrin (labelled with mAb7a and CF680-conjugated anti-mouse antibodies) in hippocampal slices of the Glrbeos/eos knock-in mouse line at postnatal day 40. Scale bar: 5 µm. (B) Single detections from the reflected (CAM R) and transmitted camera (CAM T) in frame (fr) 4693. Scale bar: 2 µm. (C) AF647 (red) and CF680 (cyan) far-red dyes were separated by spectral demixing, selecting for each fluorophore the inferior and superior cut-offs (0.25–0.32 for AF647; 0.42–0.70 for CF680) of the intensity ratios [Ireflected/ (Ireflected + Itransmitted)]. (D) Dual-colour rendered SMLM images of glycine receptors (GlyRs) labelled with anti-mEos-AF647 nanobodies (red) and gephyrin (cyan, mAb7a-CF680) at hippocampal synapses after spectral demixing. Scale bar: 2 µm. (E) Overlay of the demixed images shown in D. Scale bar: 2 µm. (F) Spectral demixing histogram from a control experiment in which only gephyrin (CF680) was labelled, producing a single peak of intensity ratios. When the same cut-offs as in C were applied, the number of detections in the AF647 channel was significantly reduced (n = 7 recordings, Mann-Whitney [MW] test, p < 0.01), despite a small number of detections in which the colour was incorrectly attributed. (G) Dual-colour rendered SMLM image of gephyrin labelled with mAb7a-CF680 (cyan) in the absence of the AF647-conjugated nanobody. Scale bar: 2 µm.
Figure 4
Quantitative single molecule localisation microscopy (SMLM) of endogenous glycine receptors (GlyRs) at synapses in the striatum.
(A) Super-resolution imaging of mEos4b-GlyRβ subunits (in red) in the dorsal and ventral striatum of Glrbeos/eos knock-in mice. Cryostat slices were labelled with the gephyrin marker Sylite (cyan). Scale bar: 5 μm. (B) Single molecule detection numbers of mEos4b-GlyRβ per gephyrin cluster (n=2985 and 2719 clusters for ventral and dorsal striatum, respectively, from 6 fields of view per sub-region and N = 3 independent experiments from three animals; mean ± SEM; two-tailed Mann-Whitney test: ***p < 0.0001). (C) Cumulative distribution of the estimated number of mEos4b-GlyRβ containing receptor complexes per synapse in the dorsal striatum (grey line), ventral striatum (black line), and spinal cord (red line). Copy numbers are background-corrected (see Table 1).
Figure 5
Glycinergic miniature inhibitory postsynaptic currents (mIPSCs) of medium spiny neurons (MSNs) in ventral but not in dorsal striatum.
(A–B) Representative ventral and dorsal current traces recorded using whole-cell patch clamp in MSNs in the ventral (A) and dorsal striatum (B). The top traces show spontaneous postsynaptic current (sPSCs) recorded during aCSF application to confirm whole-cell recording. The middle traces show the pharmacologically isolated glycinergic mIPSCs during the application of aCSF containing blockers (10 µM DNQX, 0.1 µM DHBE, 5 µM L-689560, 0.5 µM tetradotoxin, and 10 µM bicuculline) present in the ventral striatum and absent in dorsal striatum. Blocking the mIPSCs by 1 µM strychnine confirms their glycinergic identity. (C) Quantification of the amplitude and (D) frequency of glycinergic mIPSCs in ventral and dorsal MSNs (mean ± SEM; n = 5 cells from 3 animals in ventral striatum and n = 5 cells from 4 animals in dorsal striatum). Levels of significance determined using a one-tailed Mann-Whitney test (**p < 0.05).
Figure 6
Expression of recombinant glycine receptor (GlyR) subunits in cultured hippocampal neurons.
(A) Cultured mouse embryonic hippocampal neurons (E17.5) were transduced with lentivirus expressing mEos4b-tagged GlyR subunits α1, α2, or β (red), fixed at day in vitro 17 (DIV17) and stained for gephyrin (Sylite marker, cyan). Bottom images: uninfected control neurons. Scale bar: 10 μm. (B) Single molecule localisation microscopy (SMLM) pointillist images showing the photoconverted mEos4b detections. Dense clusters of synaptic (red arrowheads) and extrasynaptic receptors (red crosses) are indicated. Diffusely distributed extrasynaptic GlyR complexes (red circle) are seen as small clusters of detections resulting from the repetitive detection of a single mEos4b fluorophore. Scale bar: 2 μm. (C) Quantification of the percentage of infected neurons displaying mEos4b-positive GlyR clusters that co-localise with synaptic gephyrin clusters. Each data point represents one coverslip of cultured neurons (n=7 coverslips per condition, corresponding to 105 cells for GlyRα1, 114 cells for GlyRα2, and 109 cells for GlyRβ, from N = 3 independent experiments, i.e. cultures). The mean is indicated as a horizontal line. (D) Quantification of the total fluorescence intensity of mEos4b-tagged GlyR subunits at Sylite puncta in infected neurons and uninfected controls. The integrated mEos4b fluorescence (left graph) and integrated Sylite fluorescence (right) was measured for every Sylite-positive punctum and the median calculated per cell (n = 21 cells for GlyRα1; 9 for GlyRα2; 33 for GlyRβ, and 18 control cells, from N = 3 experiments; mean ± SD; Kruskal-Wallis [KW] test. *p < 0.05; **p < 0.01; ***p < 0.0001; n.s., not significant). The camera offset was corrected using the minimum pixel intensity in each channel. The signal in the mEos4b channel in the control cultures represents the fluorescence background.
Author response image 1
Left: Lentivirus expression of mEos4b-GlyRa1 in fixed and non-permeabilised hippocampal neurons (mEos4b signal).
Right: Surface labelling of the recombinant subunit with anti-Eos nanoboby (AF647).
Tables
Table 1
Estimated copy numbers of mEos4b-GlyRβ containing heteropentameric glycine receptors (GlyR) complexes at inhibitory synapses in different regions of the central nervous system (CNS) of Glrbeos/eos knock-in (KI) mice (background-corrected, see Methods).
| CNS region | GlyR copy number(mean ± SEM) | Range of GlyR copies(5–95 percentile) | Fraction of GlyR-positive synapses (≥0.5 copies) |
|---|---|---|---|
| Spinal cord | 120 ± 5 | 0 – 470 | 0.89 |
| CA1 | 0.34 ± 0.05 | 0 – 3 | 0.18 |
| CA3 | 1.11 ± 0.30 | 0 – 5 | 0.18 |
| DG | 0.35 ± 0.18 | 0 – 2 | 0.13 |
| Dorsal striatum | 3.00 ± 0.18 | 0 – 15 | 0.43 |
| Ventral striatum | 26.10 ± 1.48 | 0 – 112 | 0.73 |
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Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses
eLife 14:RP109447.
https://doi.org/10.7554/eLife.109447.2
