SMLM of mEos4b-GlyRβ subunits in the hippocampus.

(A) Single molecule detections of mEos4b-GlyRβ (red SMLM pointillist image) in the 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 = 9733 synaptic clusters from 10 fields of view) and brain slices (n > 5000 clusters from 9 fields of view for each region) from N = 3 independent experiments corresponding to three Glrbeos/eos mice. Recordings were also made in the CA3 of wildtype mice not expressing endogenous mEos4b-GlyRβ (negative control; n = 3637 clusters, 3 fields of view, 2 GlrbWT/WT mice) and in Glrbeos/eos hippocampal slices without photoconversion of mEos4b (no UV, n = 3301 clusters, 9 fields of view, 3 animals). A pixel shift control with flipped images was done with the same dataset (Glrbeos/eos, CA3; n = 3301 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.

Estimated copy numbers of mEos4b-GlyRβ containing heteropentameric GlyR complexes at inhibitory synapses in different regions of the CNS of Glrbeos/eos KI mice (background-corrected, see Methods).

Dual-colour SMLM of 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: 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-AF647detections from the CM of gephyrin relative to the radius of gyration (RG) of the gephyrin cluster along the x and y axes (Δx/RGx, Δy/RGy). N = 2 independent experiments corresponding with 2 Glrbeos/eos animals.

Quantitative SMLM of endogenous 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: 5 µm. (B) Single molecule detection numbers of mEos4b-GlyRβ per gephyrin cluster (n = 3309 and 2352 clusters for ventral and dorsal striatum, respectively, from 9 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).

Glycinergic mIPSCs of MSNs in ventral but not in dorsal striatum.

(A-B) Representative ventral and dorsal current traces recorded using whole cell patch clamp in medium spiny neurons (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).

Expression of recombinant 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 DIV17 and stained for gephyrin (Sylite marker, cyan). Bottom images: uninfected control neurons. Scale: 10 μm. (B) 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: 2μm. (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 = 4 independent experiments (i.e. cultures). The mean is indicated as 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; 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.