Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered at the post-synaptic membrane by a strong interaction of the β subunit with the scaffold protein gephyrin. Even though GlyRβ mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas in the telencephalon, including hippocampus and striatum. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyRβ, we identified low-copy GlyR complexes at inhibitory synapses in different hippocampal regions. Dual-colour SMLM further revealed that the sparse GlyRs are integrated within the post-synaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results further highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 2

Inhibitory neurotransmission in the central nervous system (CNS) is largely mediated by glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) (Alvarez, 2017, Kasaragod & Schindelin, 2018). Both classes of receptor are widely expressed throughout the brain and the spinal cord, however, they have specific regional expression patterns. In the case of the GlyR, it is particularly abundant in spinal cord and brain stem where it plays an important role in the processing of sensory and motor information as well as the modulation of pain responses (Alvarez, 2017, Fenech et al., 2024). Although the expression of GlyRs is much lower in the brain (Maynard et al., 2021, Zeilhofer et al., 2005), glycinergic transmission is known to play a role in reward signalling and possibly pain associated responses (Adermark et al., 2011, Devoght et al., 2023, Fenech et al., 2024, Muñoz et al., 2018, San Martin et al., 2020). Both GABAARs and GlyRs are cys-loop pentameric chloride channels that are composed of different combinations of subunits. Each subunit contains a large N-terminal extracellular domain (ECD), four transmembrane domains (TM1-4), as well as a flexible intracellular domain (ICD) between TM3 and TM4 (Kasaragod & Schindelin, 2018). Pentameric GlyRs are assembled from five different subunits, α1-α4 and β. Homopentameric GlyRs composed only of α subunits are mostly found in the extra-synaptic plasma membrane. In contrast, heteropentameric receptors containing both α and β subunits accumulate at post-synaptic sites, due to a direct interaction between the ICD of the β subunits and gephyrin, the main scaffold protein at inhibitory synapses (Alvarez, 2017). In situ hybridisation studies have shown that the mRNA of the GlyRα1 and β subunits is highly expressed in spinal cord neurons (Ceder et al., 2024, Malosio et al., 1991). The GlyRβ transcript is also expressed in most brain regions, including olfactory bulb, cerebral cortex, hippocampus, and striatum (Ceder et al., 2024, Fujita et al., 1991, Malosio et al., 1991). Surprisingly, expression of GlyRβ protein appears to be exceedingly low in the telencephalon. For example, GlyR labelling was only detected at a few synapses in the hippocampus, mainly in the pyramidal layer (Danglot et al., 2004, Maynard et al., 2021, Weltzien et al., 2012). In line with this, electrophysiological measurements have failed to detect synaptic GlyR currents in the hippocampus (Chattipakorn & McMahon, 2002, Mori et .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 3 al., 2002, Song et al., 2006), with only a single study reporting evoked IPSCs in mouse hippocampal CA1pyramidal cells (Muller et al., 2013). The mRNA levels of the GlyR α subunits are generally low across the brain (Ceder et al., 2024, Malosio et al., 1991). Yet, surface expression of homopentameric GlyRs was demonstrated by electrophysiological recordings of glycine-induced currents in several regions including the hippocampus and dorsal striatum (Chattipakorn & McMahon, 2002, Comhair et al., 2018, Molchanova et al., 2017, Mori et al., 2002, Song et al., 2006). These extrasynaptic receptors are thought to play a role in the tonic inhibition of central neurons (Mori et al., 2002, Song et al., 2006). This situation appears to be different in the ventral striatum (nucleus accumbens), where glycinergic mIPSCs likely corresponding to heteropentameric GlyRs have been reported (Muñoz et al., 2018). To gain insight into the presence and distribution of GlyRs at synapses in the brain, in this study we made use of the high spatial resolution and extraordinary sensitivity of single molecule localisation microscopy (SMLM), which enabled us to detect individual GlyR complexes in different regions of the telencephalon, including the hippocampal formation and striatum. Using a knock-in (KI) mouse model that expresses endogenous mEos4b-tagged GlyRβ subunits (Maynard et al., 2021), we identify low copy numbers of GlyRs at inhibitory synapses. Dual-colour SMLM further demonstrated the integration of these GlyRs within the post-synaptic gephyrin scaffold, suggesting that they are important in assembly or maintenance of inhibitory synaptic structures in the brain.

Identification of low-copy synaptic GlyRs in mouse hippocampus The distribution of glycinergic synapses is mainly confined to the spinal cord and brain stem (Alvarez, 2017). While the presence of GlyRβ subunit mRNAs is well documented in different brain areas (Ceder et al., 2024, Fujita et al., 1991, Malosio et al., 1991); see also Supplementary Fig. S1), little is known about GlyRβ protein expression in the brain (Danglot et al., 2004, Maynard et al., 2021). Here, we exploited the high sensitivity and spatial resolution of SMLM to probe the expression of GlyRβ subunit at synapses in the brain, focusing on the hippocampus. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 4 Since no reliable antibodies against the β-subunit of the GlyR are available, we used a knock- in mouse model expressing endogenous mEos4b-tagged GlyRβ subunits (Maynard et al., 2021, Wiessler et al., 2024). Thin cryostat coronal sections (10 µm) were cut from homozygous Glrbeos/eos mouse brain and labelled with NeuN antibody and with Sylite, a small peptide probe against the inhibitory synaptic scaffold protein gephyrin (Khayenko et al., 2022). The analyses were conducted in the molecular layer of the dentate gyrus (DG), where the dendrites of granule cells receive synaptic inputs from the entorhinal cortex, and in the stratum radiatum of the CA3 and CA1 regions, where the apical dendrites of pyramidal cells make contact with Mossy fibers and Schaffer collaterals, respectively (Supplementary Fig. S1C). The different hippocampal subregions were identified in the green channel using the neuronal marker NeuN (not shown). Reference images of Sylite were taken in the far-red channel, followed by SMLM recordings of the green-to-red photoconvertible fluorescent protein mEos4b attached to the GlyRβ subunit (Fig. 1A). We observed very few single molecule detections during SMLM, indicating exceedingly low mEos4b-GlyRβ expression in the hippocampus. SMLM super-resolution images were reconstructed and analysed using Icy software to quantify the number of mEos4b detections per gephyrin cluster (Fig. 1B). We counted on average between 3 to 10 detections per synapse in all hippocampal sub-regions. This is about two orders of magnitude lower than in the spinal cord (p < 0.0001, nonparametric Kruskal–Wallis ANOVA with Dunn’s multiple comparison test), where we counted almost 1000 detections of mEos4b-GlyRβ per synapse (Fig. 1B, Supplementary Fig. S2). Along with the low detection numbers in the hippocampus, we did not see obvious differences between the sub-regions, with a significant difference only between the DG and CA1 regions (p = 0.002). To ascertain that what we saw in the hippocampus were indeed mEos4b detections and not imaging artefacts, we performed control experiments in hippocampal slices from wildtype mice not expressing mEos4b-tagged GlyRβ subunits. As expected, the mean number of detections at CA3 synapses was significantly lower in the negative control than in the Glrbeos/eos slices (Fig. 1B; p < 0.0001). Similarly, we recorded SMLM movies in Glrbeos/eos slices without 405 nm illumination. In the absence of UV, the mEos4b fluorescent protein is not converted into the red form, making this a stringent internal control. The number of detections per gephyrin cluster was again much lower than in the recordings with 405 nm .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 5 laser illumination (Fig. 1B; p < 0.0001). These data confirm that the non-specific background of detections, mainly outside of synapses, is sufficiently low to accurately identify and quantify endogenous mEos4b-GlyRβ subunits in the hippocampus of our knock-in mouse model. We also estimated the absolute number of GlyRs per synapse in the hippocampus. The number of mEos4b detections was converted into copy numbers by dividing the detections at synapses by the average number of detections of individual mEos4b-GlyRβ containing receptor complexes. This value was measured across CA3 slices, both at synapses as well as in the extrasynaptic region (see Methods). According to our quantification GlyRs are present at about a quarter of hippocampal synapses, with copy numbers most often in the single digits (Fig. 1C, Table 1). Only few synapses contain 10 or more GlyRs, and as many as 75% of hippocampal synapses do not contain any GlyRs at all. No obvious differences were noticed in the copy number of GlyRs between regions. Taken together, our findings demonstrate the existence of very few GlyR complexes at inhibitory synapses in the hippocampus. With an average of approximately one GlyR per synapse, this is about a hundredfold lower than in the spinal cord. Sub-synaptic distribution of GlyRs at hippocampal synapses At glycinergic synapses in the spinal cord the receptors are anchored in the post-synaptic membrane by gephyrin, a scaffold protein that binds with high affinity to the GlyRβ subunit, promoting the synaptic localisation of GlyRs (e.g. (Kasaragod & Schindelin, 2018, Kostrz et al., 2024, Maynard et al., 2021)). The organisation of inhibitory synapses in the hippocampus containing low-copy GlyRs has not yet been studied in detail (Danglot et al., 2004). To get information about the the nanoscale organisation of GlyRs within the synaptic structure, we carried out dual-colour SMLM of GlyRβ and gephyrin in brain slices of adult Glrbeos/eos mice using stochastic optical reconstruction microscopy (dSTORM) with organic fluorophores in a reducing buffer (Yang & Specht, 2020). Endogenous mEos4b-GlyRβ subunits were labelled with anti-mEos-AF647 nanobodies (NanoTag). To test the specificity of the nanobody, spinal cord slices were also labelled with gephyrin antibody (mAb7a, Synaptic Systems) and CF568-conjugated secondary antibody. Wide-field fluorescence images showed extensive co-localisation of all three channels .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 6 (mEos4b, anti-gephyrin, anti-mEos-AF647) in Glrbeos/eos slices; the non-specific background of the nanobody in wildtype animals was very low (Supplementary Fig. S3). For dual-colour SMLM, brain slices from Glrbeos/eos mice were labelled with anti-mEos- AF647 nanobody as well as with anti-gephyrin and CF680-conjugated anti-mouse secondary antibodies (Fig. 2A). The emitted light from the two far-red dyes was separated by spectral demixing, using a SAFe 360 nanoscope (Abbelight) equipped with a dichroic mirror at 700 nm and two sCMOS cameras for detection. In this imaging modality, the single molecule detections recorded simultaneously on the two cameras are attributed to one or the other far- red fluorophore based on their specific intensity ratio (see Methods; Supplementary Fig. S4). In line with our SMLM experiments with the photoconvertible fluorescent protein mEos4b in brain slices (Fig. 1), we observed sparse single molecule detections of anti-mEos-AF647 nanobody that generally co-localised with dense clusters of gephyrin detections, confirming the presence of mEos4b-GlyRβ at inhibitory synapses in the hippocampus (Fig. 2A). The two-colour SMLM pointillist images were analysed using the DBSCAN clustering tool in the NEO software to measure the distance between mEos4b-GlyRβ and the corresponding gephyrin clusters. According to our measurements, the Euclidean distance between the centre of mass (CM) of GlyRβ and gephyrin was 78 ± 61 nm (mean ± SD, n = 138 pairs of clusters), pointing to a close spatial relationship at inhibitory synapses (Fig. 2B). We also calculated the relative distance of the GlyRβ detections from the centre of mass of gephyrin, compared to the mean distance of the gephyrin detections from their own centre of mass as defined by the radius of gyration (RG) of the gephyrin clusters. Our findings showed that this ratio was below one for most clusters, indicating that the sparse GlyR complexes are well integrated within the postsynaptic gephyrin domains at hippocampal synapses (Fig. 2C). Differential expression of endogenous mEos4b-GlyRβ in dorsal and ventral striatum Several reports described the presence of glycinergic currents in the striatum (Molchanova et al., 2017, Muñoz et al., 2018). However, the localisation of GlyRs at synapses in this brain area remains controversial. Homopentameric GlyRα2 complexes are thought to mediate tonic inhibition in the dorsal striatum (Devoght et al., 2023, Molchanova et al., 2017), yet glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were detected in the nucleus accumbens, i.e. part of the ventral striatum (Muñoz et al., 2018). To resolve this issue, we .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 7 quantified the presence of GlyRβ-containing synaptic receptors in Glrbeos/eos animals using SMLM. We determined the number of detections of mEos4b-GlyRβ per gephyrin cluster, as well as the copy number of GlyRs in the dorsal and in the ventral striatum in Glrbeos/eos mice at postnatal day 40. Coronal cryo-sections of 10 µm thickness were immunolabeled for NeuN to distinguish the two subregions of the striatum. Gephyrin was labelled with the Sylite probe in order to identify inhibitory synapses (Fig. 3A). The average number of mEos4b-GlyRβ detections per gephyrin cluster was significantly higher in the ventral striatum compared to the dorsal striatum (136 ± 5 versus 25 ± 1 detections per synapse, mean ± SEM, n = 3309 and 2352, respectively; p < 0.0001, nonparametric Mann-Whitney test; Fig. 3B). Conversion of the detection numbers into receptor copy numbers revealed that about 40% of synapses in the ventral striatum contain at least 10 heteropentameric GlyR complexes, many more than in the dorsal striatum (p < 0.0001, Kruskal-Wallis and Dunn’s multiple comparison test; Fig. 3C, Table 1). The same quantification was done in the spinal cord, where we found significantly higher copy numbers of receptor complexes containing mEos4b-GlyRβ (p < 0.0001 against both striatal regions). With an average of 120 ± 5 receptor complexes per synaptic cluster (mean ± SEM, n = 4006 synapses, Table 1), these values are close to previous estimates (Maynard et al., 2021). Our findings show that the numbers of mEos4b-GlyRβ subunits are much higher at synapses in the striatum compared to synapses in the hippocampal formation, and that their distribution in the striatum is area-specific. This observation is supported by electrophysiological recordings of glycinergic synaptic currents in the nucleus accumbens (Muñoz et al., 2018) as opposed to mainly extrasynaptic tonic currents in the dorsal striatum (Molchanova et al., 2017). Miniature synaptic currents in ventral striatum recorded with whole cell patch-clamp Since the published recordings of glycinergic currents in the striatum were performed under different conditions, i.e. different striatal sub-regions, mouse strains and ages (Molchanova et al., 2017, Muñoz et al., 2018), we wanted to verify the area-specific functional differences in a single experimental setting. We pharmacologically isolated glycinergic mIPSCs and determined their frequency and amplitudes in brain slices of both ventral and dorsal striatum .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 8 of C57BL/6J mice at postnatal day 35 to 41 using whole cell patch clamp (Fig. 4). mIPSCs are considered a measure of functional synapses because they are generated by the spontaneous release of synaptic vesicles from presynaptic terminals. Our data confirm the presence of glycinergic mIPSCs in medium spiny neurons (MSNs) in the ventral striatum and the near complete absence of glycinergic mIPSCs in dorsal striatal MSNs (p < 0.05; Fig. 4D). With an amplitude of 17.2 ± 2.9 pA (mean ± SEM, n= 5; Fig. 4C) and a frequency of 0.12 ± 0.03 Hz (Fig. 4D), the mIPSCs recordings in the ventral striatum are similar to the results of an earlier study of the nucleus accumbens (Muñoz et al., 2018). Our electrophysiological data also match the differences in synaptic GlyR copy numbers obtained by quantitative SMLM (Fig. 3C). Expression of recombinant GlyR subunits in cultured hippocampal neurons Synaptic GlyRs are heteropentamers composed of α (α1–α4) and β subunits, and their assembly and synaptic targeting requires the presence of both types of subunit (Alvarez, 2017). Since the β transcript appears to be expressed at high levels in most neurons including in the hippocampus (Supplementary Fig. S1), we hypothesised that the expression of α subunits may be limiting factor controlling the number of synaptic GlyRs. We therefore expressed different recombinant GlyR subunits in cultured hippocampal neurons and analysed their accumulation at inhibitory synapses. Cells were infected with lentivirus expressing mEos4b-tagged GlyR subunits α1, α2 or β at day in vitro 3 (DIV3), fixed at DIV16, and stained with Sylite to identify synaptic gephyrin clusters. We found co-localisation of mEos4b and Sylite puncta in neurons infected with either of the virus constructs, indicating that some cultured hippocampal neurons express both α and β endogenous receptor subunits (Fig. 5A). However, synaptic GlyR puncta were found in relatively few neurons despite using high virus titers. This means that in most cultured hippocampal neurons low mRNA levels of α and β transcripts likely limit the expression of GlyRs. Interestingly, the mEos4b-GlyRα1 construct was expressed at higher levels than the other subunits and generally had a punctate appearance in most neurons (Fig. 5A). Since the majority of these puncta did not co-localise with gephyrin, they likely represent extrasynaptic clusters of homopentameric receptors composed of GlyRα1 subunits, similar to the clustering .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 9 of GlyRα3L observed in HEK cells (Notelaers et al., 2012). In contrast, expression of GlyRα2 produced a diffuse labelling with only weak synaptic puncta. The distribution of the GlyR complexes at synapses and in the extrasynaptic membrane was confirmed by super- resolution imaging of the mEos4b-tagged receptor subunits (Fig. 5B). In line with the above obervations, we found a mainly clustered distribution of GlyRα1, both at synapses as well as in the extrasynaptic membrane. The GlyRα2 subunit had a large extrasynaptic receptor population, likely composed of diffusely distributed GlyRα2 homopentamers, and only few synaptic heteropentamers. Finally, the mEos4b-GlyRβ subunit was expressed only in a few cells, where it had a predominantly synaptic localisation, in line with its direct interaction with gephyrin.

Detection of heteropentameric GlyR complexes at hippocampal synapses using SMLM Making use of the ultra-sensitivity of single molecule localisation microscopy (SMLM) this study describes the presence of very low copy numbers of GlyRs at synapses throughout the hippocampus. Key to this was the use of highly specific labelling of synaptic receptors using a knock-in model expressing endogenous mEos4b-GlyRβ that was used either directly for SMLM through the photoconversion of the mEos4b fluorescent protein or as an antigen that was recognised by a specific anti-mEos nanobody. To positively identify the GlyRβ signals, it was necessary to reduce as much as possible the non-specific background of detections that invariably occurs in SMLM recordings. In the case of mEos4b imaging, this was done by acquiring a negative control movie without photoconversion of the fluorophore. In other words, the photo-physical properties of the fluorophore can be used to identify them with some certainty (Wulffele et al., 2022). Since we focussed on GlyRs at inhibitory synapses, the co-localisation of the detections with the synaptic gephyrin scaffold provided additional assurance that single mEos4b fluorophores could be identified in 10 µm thick slices of brain tissue. These data illustrate the power of SMLM to detect rare or sparsely distributed target molecules in complex samples, in addition to the high spatial resolution afforded by super- resolution imaging. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 10 Another strength of SMLM is the possibility to gain access to absolute molecule numbers. Since a fluorophore can be detected several times during the recording, the number of detections has to be translated into the number of emitting fluorophores (Patrizio & Specht, 2016). This is generally more straightforward with fluorescent proteins such as mEos4b that are irreversibly bleached after a number of detections (Wulffele et al., 2022), instead of organic fluorophores (e.g. AF647) that can blink over extended periods. In our case, the use of a knock-in mouse (Glrbeos/eos) expressing recombinant mEos4b-GlyRβ made it possible to estimate the real copy number of endogenous GlyRs at synapses (Maynard et al., 2021). To ensure accurate readings, SMLM movies were recorded until all fluorophores were exhausted (typically 5000 frames in hippocampus and as many as 15000 in the spinal cord). Our quantification in spinal cord indicated that there are on average 120 GlyRs per synapse (Table 1). This result is similar to earlier measurements, in which we calculated median values of 114 and 238 GlyRs at synapses in the dorsal and the ventral horn of the spinal cord, respectively (Maynard et al., 2021). The slightly lower values compared to the earlier study could be related to the lower age of the animals (40 days instead of 2 or 10 months), or to the greater thickness of the sample (10 µm versus 2 µm slices) that affects the signal to noise ratio of the detections. Furthermore, a simpler approach was used in the current study to translate detections into copy numbers. We divided the number of detections at synapses by the number of clusters of detections in sparsely labelled regions in the CA3 that contain mostly individual and spatially separated GlyRs (including extrasynaptic receptors). The advantage of this estimation is that it is independent of the stoichiometry of heteropentameric GlyRs that remains controversial (e.g. (Durisic et al., 2012, Durisic et al., 2014, Maynard et al., 2021, Patrizio et al., 2017, Yu et al., 2021, Zhu & Gouaux, 2021)). However, our quantification could underestimate the GlyR copy numbers, because a certain fraction of fluorescent proteins like mEos4b is often not functional or not detected under the given imaging conditions, partly due to their complex photophysical properties (e.g. (Durisic et al., 2014, Patrizio et al., 2017, Wulffele et al., 2022). As opposed to spinal cord synapses, the numbers of GlyRs at hippocampal synapses are very low. In most cases, only a single cluster of detections (i.e. a single heteropentameric GlyR complex) was present at these synapses (Table 1). Yet, the existing GlyRs appear to be well .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 11 integrated into the gephyrin domain as judged by dual-colour super-resolution microscopy, pointing to a possible role at synapses (see below). Discrepancies between transcription and protein expression of GlyRs in the brain Our study addresses a long-standing debate on the presence of GlyR expression in the brain. According to many reports including recent transcriptomic analyses, GlyRβ mRNA is highly expressed in neurons throughout the telencephalon including the hippocampus (Ceder et al., 2024, Fujita et al., 1991, Malosio et al., 1991) (see also Fig. S1). This is also true for the human brain, where high GlyRβ mRNA levels are detected in all sub-regions of the hippocampus (https://www.proteinatlas.org/ENSG00000109738-GLRB/brain; (Karlsson et al., 2021)). In spite of this, the protein expression of GlyRβ receptor subunits in the hippocampus is very low, as judged by our analysis of synaptic (heteropentameric) GlyR complexes in mEos4b-GlyRβ knock-in animals. What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits. The presence of transcripts of the GlyR subunits α1, α2 and α3 in the brain has been demonstrated (Ceder et al., 2024, Malosio et al., 1991), in line with the existence of extrasynaptic (homopentameric) GlyR complexes (Chattipakorn & McMahon, 2002, Molchanova et al., 2017, Mori et al., 2002, Song et al., 2006). Our re-analysis of transcriptomic data confirms that the GlyRα1 mRNA levels are low in the telencephalon, and increase towards dorsal regions of the brain (Fig. S1A), in parallel to the expression of GlyRβ protein at inhibitory synapses (Maynard et al., 2021). This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both, α1 and β transcripts. To test this hypothesis, we expressed recombinant GlyR α and β subunits in cultured hippocampal neurons. The expression of mEos4b-GlyRβ resulted in synaptic receptors only in very few neurons, suggesting that in the majority of cells the β transcript it is not the limiting element for efficient cell-surface expression of heteropentameric GlyR complexes. In contrast, lentiviral infection with an mEos4b-GlyRα1 construct resulted in efficient surface expression of GlyRα1 clusters in a large proportion of cultured neurons, in some of which it co-localised with the synaptic gephyrin scaffold. mEos4b-GlyRα2 expression was detected only in few .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 12 neurons and was mostly extrasynaptic. These observations indicate that GlyRα1 (and possibly GlyRα3) could have a particular role in the assembly and forward trafficking of heteropentameric GlyRs towards the plasma membrane. Possible roles of low-copy GlyRs at brain synapses The low number of GlyRs at hippocampal synapses begs the question what their role may be. Experiments in GlyRα2 knock-out animals have shown that GlyRs help maintain the excitatory / inhibitory balance in the dorsal striatum (Devoght et al., 2023). However, these receptors are likely to be extrasynaptic GlyRα2 homopentamers. On the other hand, the contribution of a single synaptic GlyR to the chloride influx during inhibitory neurotransmission is probably not significant. In line with this interpretation, glycinergic miniature IPSCs in hippocampal slices are not generally detected (Chattipakorn & McMahon, 2002, Mori et al., 2002, Song et al., 2006), but see (Muller et al., 2013)). The same is true for the putamen (dorsal striatum), where whole-cell currents were recorded in response to glycine application (Molchanova et al., 2017). Again, our quantification indicates that GlyR copy numbers in this area are low (Table 1). Synaptic GlyRs are more numerous at synapses in the nucleus accumbens (ventral striatum), and indeed, our electrophysiological data support this finding in agreement with earlier studies (Muñoz et al., 2018). Another possible role of low-copy GlyRs at synapses in the hippocampus, dorsal striatum and maybe other regions of the telencephalon could be a structural one. This concept is based on the high affinity of the GlyRβ-gephyrin interaction (Kasaragod & Schindelin, 2018) that stabilises the receptor as well as the gephyrin scaffold at inhibitory synapses (Chapdelaine et al., 2021). GABAAR subunits have a much lower affinity for gephyrin and cannot provide the same level of stability (e.g. (Kostrz et al., 2024, Maric et al., 2014, Maric et al., 2011)). However, GABAARs could probably be recruited efficiently to an existing gephyrin scaffold. Our two-colour SMLM data of endogenous mEos4b-GlyRβ and gephyrin at hippocampal synapses confirms that despite their low number the GlyRs are integral components of the postsynaptic gephyrin domain in support of a structural role. In conclusion, our data positively identify the presence of individual GlyR complexes at inhibitory synapses in the brain. This is drastically different to the situation in the spinal cord and to a lesser extent in the nucleus accumbens, where GlyRs are abundant and densely .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 13 clustered at most inhibitory synapses. While it is therefore reasonable to classify different inhibitory synapses as mainly glycinergic or GABAergic, it should be noted that this is a simplification that does not account for the full diversity of inhibitory synapses that may assemble in a continuum of mixed compositions across the entire dynamic range.

Primary culture of hippocampal neurons Hippocampal neurons were prepared from wild-type mice with Swiss background, at embryonic day E17.5. Experiments were performed according to the European directive on the protection of animals used for scientific purposes (2010/63/EU) and the regulations of the local veterinary authority (Inserm UMS44-Bicêtre, license G94043013). The mice came from excess production for another project, meaning that no animals were generated for the current project. Pregnant mice were put to death by cervical dislocation and the embryos collected by caesarean section and decapitated. Hippocampi were rapidly dissected in cold Hank’s Balanced Salt Solution (HBSS, Gibco, #14180-046) containing 20 mM HEPES (Gibco, #15630-056) and incubated at 37°C for 15 min in dissection medium containing 0.25% trypsin (Gibco #15090-046). After trypsinisation, hippocampi were washed twice in plating medium composed of Minimal Essential Medium (MEM) containing Earle’s Balanced Salts (EBSS) (Cytiva, #SH30244.01), 2 mM GlutaMAX (Gibco, #35050-038), 1 mM sodium pyruvate (Thermo Fisher Scientific, #11360-039), and 10% heat inactivated horse serum (Gibco, #26050-088). The hippocampal tissue was then triturated in plating medium containing 0.3 mg/ml DNase I (Merck, #11284932001). Neurons were seeded at a concentration of 3.4 x 105/cm2 in 12-well plates (Thermo Fisher, #150628) on round glass coverslips (type 1.5, 18 mm diameter; Marienfeld, #0112580) that were pre-coated with poly- D,L-ornithine (Merck, #P8638). The medium was replaced 4 hours after plating with maintenance medium: Neurobasal medium (Gibco, #21103-049) containing B-27 Supplement (Gibco, #17504-044) and 2 mM GlutaMAX. Once a week, 300 µL of fresh maintenance medium was added. Hippocampal neurons were typically infected with 20 µl of lentivirus stocks at DIV3 and fixed for immunocytochemistry at day in vitro DIV16. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 14 Lentivirus expression constructs The following lentivirus constructs were used for the expression of mEos4b-tagged GlyR subunits: FU-mEos4b-GlyRα1, expressing the coding sequence (cds) of rat Glra1 isoform a (UniProt P07727-1) (Patrizio et al., 2017); FU-mEos4b-GlyRα3L containing the long isoform of human GLRA3 (UniProt O75311-1) (Patrizio et al., 2017); and FU-mEos4b- GlyRβ-bis the cds of human GLRB (UniProt P48167-1) (Grunewald et al., 2018). The receptor sequence in construct FU-mEos4b-GlyRβ-bis (excluding the signal peptide and mEos4b sequence) was replaced with the cds of human GLRA2 (UniProt P23416) to generate the expression construct FU-mEos4b-GlyRα2. For virus production, HEK-293 tsA201 cells were co-transfected with equal amounts (5 µg each) of the replicon plasmid and the three helper plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G (Addgene #12251, #12253, #12259) using lipofectamine 2000 (Invitrogen, #11668- 019). Cells were cultured in maintenance medium (see above) supplemented with 5 U/ml penicillin and 5 µg/ml streptomycin at 37°C / 5% CO2 for 24 h, at which point the medium was exchanged. The culture medium containing lentivirus was collected at 48-55 hours, filtered with a pore size of 0.45 µm, and frozed as aliquots at -70°C. Immunocytochemistry in cultured neurons Hippocampal neurons were fixed at DIV16 with 4% w/v PFA and 1% w/v sucrose in 0.1 M phosphate buffer, pH 7.4 for 10 min. After three washes in PBS, the cells were permeabilized in PBS, pH 7.4 containing 0.25% Triton X-100 and 4% w/v bovine serum albumin (BSA) (Sigma, #A7030) for 10 min and then blocked in PBS containing 4% w/v BSA for 1 h. Neurons were incubated for 1h with the 200 nM gephyrin marker Sylite (Khayenko et al., 2022) in PBS containing 1% w/v BSA. The cells were rinsed twice in PBS and kept in PBS overnight at 4°C until imaging. Sample preparation of spinal cord and brain slices for SMLM of endogenous mEos4b- GlyRβ Brain and spinal cord tissue from homozygous mEos4b-GlyRβ knock-in (KI) mice (Glrbeos/eos, mouse strain C57BL/6N-Glrbtm1Ics, backcrossed into C57BL/6J, accession number MGI:6331106 (Maynard et al., 2021)) was recovered from an earlier project (Wiessler et al., 2024). In this mouse strain the β-subunit of the GlyR is tagged at its N- .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 15 terminus with the photoconvertible mEos4b fluorescent protein. The chosen animals did not carry any genomic modification other than Glrbeos/eos. For control experiments brain tissue from a wild-type Swiss mouse was used. Freshly frozen spinal cords and brains from male and female mice at postnatal day 40 were embedded in OCT (Pink Neg-50, Thermo Fisher Scientific, #6502P) and coronal slices of a nominal thickness of 10 µm were cut on a cryostat (Leica, Wetzlar, Germany, #CM3050S) with a chamber temperature of -23°C. The slices were collected on SuperFrost Plus glass slides (Epredia, #J7800AMNZ), fixed with 2% w/v PFA and 0.5% w/v sucrose in PBS pH 7.4 for 10 min and rinsed three times for 5 min in PBS at room temperature (RT). Afterwards, the slices were permeabilized and blocked in PBS containing 4% w/v BSA (A7030, Sigma) and 0.25% v/v Triton X100 for 15 min. Optionally, the slices were labelled overnight with a polyclonal antibody against NeuN (raised in chicken, Synaptic Systems, #266006, 1:2000) at 4°C, followed by 4 h of AF488-conjugated donkey anti chicken secondary antibody (Jackson ImmunoResearch, #703-545-155, 1:2000) at RT. The gephyrin-specific peptide probe Sylite (Khayenko et al., 2022) was applied at a final concentration of 50 nM in PBS containing 1% w/v BSA for 1 h at RT. After two washes of 5 min in PBS, slices were mounted in PBS, covered with a glass coverslip (type 1.5), sealed with PicoDent Twinsil Speed (#1300-1002) and kept overnight at 4°C or directly used for SMLM imaging of the mEos4b photoconvertible protein. Single molecule localisation microscopy (SMLM) of mEos4b fluorescent protein SMLM imaging of mEos4b-tagged GlyRs was performed using an ELYRA PS.1 microscope setup (Zeiss, Jena, Germany). Samples were placed on the motorized stage of an Axio Observer.Z1 SR inverted microscope and imaged with a Plan-Apochromat 63x / NA 1.4 oil immersion objective, with an additional 1.6x lens in the emission path. Images were captured with an Andor iXon 897 back-thinned EMCCD camera (16 bit, 512 x 512 pixels, 16 µm pixel size, QE 90%, set at -60°C working temperature), resulting in an image pixel size of 160 nm.

images in the green channel were taken with a 488 nm excitation laser (nominal output 300 mW) and a band pass (BP) 495-575 nm (+ LP 750) emission filter. For the far-red channel we used a 642 nm excitation laser (nominal output 150 mW) and a long pass (LP) 655 nm emission filter. SMLM recordings were performed exploiting the properties of the fluorescent protein mEos4 that is photoconverted from green to red state upon UV illumination (405 nm laser, nominal power 50 mW) and image acquisition in the red channel (561 nm laser, nominal power 200 mW, emission filter BP 570-650 + LP 750). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 16 Regarding the experiments in hippocampal cultures (data in Fig. 5), infected neurons expressing mEos4b-tagged GlyR subunits were identified in the green channel, and single

images of 100 ms exposure were taken with the 488 nm excitation laser set at 2% and the 405 nm laser at 1% of the maximal power, and a camera gain of 100. For Sylite we used the far-red channel, taking one image of 100 ms with the 642 nm laser at 10% output and a camera gain of 10. As SMLM movie of 10000 frames was then recorded at 20 Hz streamed aquisition (50 ms frames) with constant 561 nm laser illumination at 100% output and a gain 300. The photoconversion of mEos4b-tagged GlyR subunits (from green to red) was done by continuous 405 nm laser illumination that was gradually increased from 0.01 to 4% intensity. For the detection of endogenous mEos4b-GlyRβ in brain slices (data in Fig. 1 and 3), we used the following acquisition parameters: Hippocampal and striatal regions were identified using NeuN immunolabelling in the green channel (AF488). A single image of 50 ms was taken with 488 nm illumation, 0.2% laser intensity, and a camera gain of 200 to record the NeuN immunofluorescence. Sylite was detected in the far-red channel, taking one image of 100 ms at 642 nm (5% output, camera gain 100). A first SMLM recording of 2000 frames at 20 Hz was done in the red channel with 561 nm laser illumination at 80%, a camera gain of 300, and without 405 nm laser illumination. Under these conditions, mEos4b is not converted into the active (red) form. Afterwards, a second SMLM movie of 5000 frames was taken with the same settings, but with the addition of continuous 405 nm laser illumination that was gradually increased from 0.01 to 3% intensity. During these recordings, the total population of mEos4b molecules is converted (and bleached), since only very sparse detections are seen at the end of the movie. Glycinergic synapses in spinal cord slices (Supplementary Fig. S2) were identified as bright puncta of (unconverted) mEos4b-GlyRβ subunits in the green channel, and reference images were taken (green, mEos4b: 100 ms acquistion with the 488 laser at 0.2% power, camera gain 200; far-red, Sylite: 100 ms, 642 laser at 3%, gain 200 ms). SMLM movies of 15000 frames were recorded with the same settings as before with a continuous 405 nm laser illumination, gradually increasing from 0.01 to 5% intensity to ensure the complete conversion of mEos4b by the end of the experiment. SMLM image analysis of mEos4b fluorescent protein SMLM movies were processed with Zen software (Zeiss, Zen 2012 SP5 FP3 black, 64 bit) using a peak mask size of 7 pixels, a peak intensity of 6 and excluding the overlapping .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 17 molecules. The single molecule localisations were corrected for x/y-drift with model-based algorithm (without fiducial markers). A rendered super-resolution image was reconstructed from the pointillist image, in which each detection is represented with a two-dimensional Gaussian distribution with a width corresponding to its point spread function (PSF) and a 10 nm pixel size and saved in tiff format. Inhibitory synapses in spinal cord and brain slices were identified using the gephyrin-specific peptide marker Sylite (Khayenko et al., 2022). First, the Sylite reference images were adjusted to a 10 nm pixel size by multiplying the pixel number by a factor of 16. We then detected Sylite puncta using the spot detector plugin in Icy (de Chaumont et al., 2012) with the following parameters: bright spots over dark background were detected at scale 5 set to a sensitivity of 100, and the size filtering between 400 and 10000 pixels. The obtained regions of interest (ROI) of Sylite were then employed as mask to measure the intensity of the mEos4b-GlyRβ signals in the rendered SMLM image. Where necessary, the alignment of the

image and the rendered SMLM image were manually adjusted in ImageJ. The output data consist in a excel table containing the total intensity of Sylite and mEos4b-GlyRβ in each ROI. To convert the mEos4b-GlyRβ signals into detection numbers, the obtained total intensity values per ROI were divided by the total intensity of single mEos4b detections in areas of the image with sparse labelling. A similar approach was used to estimate the copy numbers of mEos4b-GlyRβ subunits per gephyrin cluster in the CA3 region of the hippocampus. Rendered SMLM images were segmented in Icy with the parameters: detection of bright spots over dark background, scale 3 and 4 at a sensitivity of 100, and filtering between a size of 400–10000 pixels. Due to the low mEos4b-GlyRβ density in CA3, the obtained ROIs were considered to be clusters arising from single mEos4b fluorophores. The average number of detections per cluster was calculated by dividing the total intensity of the cluster by the total intensity of a single detection (see above). This value was used as conversion factor to translate detection numbers into molecule numbers and was applied to all SMLM images from spinal cord, hippocampus and striatum. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 18 Immunostaining of brain tissue slices used dual-colour SMLM with organic fluorophores Frozen brain tissue from Glrbeos/eos mice (Maynard et al., 2021, Wiessler et al., 2024) was cut into 10 µm thick coronal cryostat sections and processed as described above. For dual-colour SMLM with organic fluorophores, the slices were labelled with primary antibodies against gephyrin (mAb7a mouse monoclonal, Synaptic Systems, #147011, 1:1000 dilution) and mEos protein (AF647-conjugated FluoTag-X2, NanoTag Biotechnologies; #N3102-AF647- L, 1:500) overnight at 4°C, followed by secondary goat anti-mouse antibody coupled with a single CF680 dye (Biotium, #20817, 1:1000) for 4 h at RT. After washing in PBS, the slices were kept overnight at 4°C and imaged the next day. Dual-colour SMLM acquisition and image processing The cover glasses were placed in a closed Ludin chamber (Life Imaging Services) and, through the perfusion holes, was added a homemade dSTORM buffer (Yang & Specht, 2020) that was prepared as follows: A suspension containing 200 µg catalase (from bovine liver, Merck, # C30 1003493507) was washed three times with 1 ml of cold PBS and collected by centrifugation at 12000 g at 4°C for 1 min. After removing the supernatant, the catalase crystals were resuspended in 1 ml of PBS and incubated at 37°C with agitation for 30 min. The final dSTORM buffer was composed of PBS pH7.4, containing 50 mM cysteamine hydrochloride (= β-mercaptoethylamine, MEA, Merck, #M6500), 250 mM glucose (Merck, #G7021), 0.5 mg/ml glucose oxidase (from Aspergillus niger, Merck, #G2133-10KU) and 40 µg/ml dissolved catalase. Prior to use, the dSTORM buffer was degassed with N2, transferred into a syringe and kept on ice. SMLM experiments were carried with an Abbelight nanoscope (SAFe 360 Nexus SD) installed on the Zeiss Elyra PS1 setup described above, using two sCMOS cameras (Hamamatsu Orca-Fusion BT) for simultaneous dual-colour imaging. Before the acquisitions, the two cameras were aligned using 0.1 µm TetraSpeck beads (Invitrogen, #T-7279) deposited on a glass coverslip. The Ludin chamber with the tissue slices in dSTORM buffer was then placed on the stage of the inverted microscope and imaged with a Plan-Apochromat 100x / NA 1.46 oil-immersion objective without additional magnification. The far red AF647 and CF680 dyes were excited with a 640 nm laser of 500 mW nominal power (Oxxius LPX- 640-500) using adaptable scanning of the excitation region (ASTER). SMLM images were .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 19 acquired using NEOimaging software v.2.17.1 with the following parameters: 10000 images (image size 256 x 256 pixels and 97 nm pixel size) of 50 ms exposure were taken in the far- red channel with 80% laser power and a field of excitation set at 10%. Fluorophore blinking was gradually adjusted with a 405 nm laser (LBX-405-100, nominal output 100 mW), increasing the power from zero to 2% intensity along the recording. The emitted wavelengths were separated into two light paths with a 700 nm dichroic mirror for simultaneous dual- colour imaging and filtered with a band pass from 669 nm to 741 nm. STORM images were processed with NEO analysis software (Abbelight v.39). The raw tiff movies from the transmitted camera (> 700 nm) and the reflected camera (< 700 nm) were loaded and processes sequentially. Single fluorophore signals were detected using temporal mean subtraction with a sliding window of 50 frames, and fit with a Gaussian distribution. The detection coordinates in both channels were corrected for x/y drift and saved as coordinate tables. For spectral demixing and image reconstruction (Supplementary Fig. S4), the detections from the two cameras were imported in the NEO 3D viewer, superimposed and automatically re-aligned. The intensity ratios for each detection were calculated according to the formula Ireflected / (Ireflected + Itransmitted), and we selected for each dye (AF647 and CF680) the inferior and superior cut-offs (0.25-0.32 for AF647); 0.42-0.70 for CF680). After removing the detections with a localisation precision above 25 nm the two spectrally demixed data were then saved as coordinate tables as well as in the form of rendered super-resolution images with a normalized Gaussian representation of each detection and a pixel size of 10 nm. The sub-synaptic distribution of GlyRs (AF647-labelled mEos4b-GlyRβ) and gephyrin (mAb7a-CF680) in hippocampal synapses was investigated by DBSCAN cluster analysis implemented in NEO 3D viewer, using the following parameters: a radius of ε = 200 nm and n ≥ 5 neighbours for the AF647 detections (GlyRβ); and ε = 80 nm and n ≥ 50 neighbours for CF680 (gephyrin). We exported the data table containing the filtered clusters and their coordinates, and calculated the Euclidian distance between the centre of mass (CM) of the corresponding GlyR and gephyrin clusters, as well as the ratio of the GlyRβ-gephyrin distances divided by the radius of gyration (RG) of the gephyrin cluster. Values <1 indicate that the GlyRβ detections are closer to the CM of gephyrin than the dispersion (RG) of the .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 20 gephyrin detections themselves, suggesting that the GlyRs are integrated within the postsynaptic gephyrin cluster. Confocal microscopy For the control experiments shown in Fig. S3, spinal cord slices from Glrbeos/eos and wildtype animals (GlrbWT/WT) were labelled with AF647-conjugated nanobody (1:1000, NanoTag) and with an antibody against gephyrin (mouse anti-gephyrin mAb7a, Synaptic Systems, 1:1000), followed by a donkey anti-mouse secondary antibody coupled with CF568 (Sigma #SAB4600075, 1:1000). Confocal images were taken on an SP8 microscope (Leica) using a 63x oil immersion objective and a Hybrid detector (HyD3). Acquisition of images (512 x 512 px, 16 bit) with a pixel size of 120 nm in the x/y plane and 1.27 µm in z (pinhole 1) was done in the three channel, green (mEos4b), red (CF568) and far-red (AF647). Statistical analyses Statistical analyses and graphing were performed using GraphPad Prism v.9. Data are represented as mean ± SD (standard deviation) or mean ± SEM (standard error of the mean) as indicated. Statistical significance was calculated using a nonparametric two-tailed Mann- Whitney test, or a Kruskal-Wallis test (one-way ANOVA) with a post-hoc Dunn’s multiple comparison test. Preparation of acute brain slices Adult C57BL/6J mice (postnatal day 35-41) were anesthetized with isoflurane and decapitated. The brains were quickly removed on ice and glued to the prechilled stage of a vibratome (Leica VT 1200S). Coronal brain sections containing both dorsal and ventral striatum (150 µm) were sliced in ice-cold cutting solution (140 mM choline chloride, 26 mM NaHCO3, 10 mM glucose, 7 mM MgCl2, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, saturated with 95% O2 and 5% CO2). Brain slices were placed in recovery solution (120 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose, 26 mM NaHCO3, 1.2 mM NaH2PO4, saturated with 95% O2 and 5% CO2) for 45 minutes at 32°C. Slices were kept at 32°C and used 1-4 h after slicing. Electrophysiology Acute brain slices were placed in the recording chamber and perfused with oxygenated aCSF (124 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 10 mM glucose, 1 mM MgCl2, 26 mM NaHCO3, .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 21 1.2 mM NaH2PO4, saturated with 95% O2 and 5% CO2, pH 7.4, at room temperature). MSNs in the dorsal or ventral striatum were visualized using 4x air and 40x water immersion

of an upright Zeiss microscope (Axioskop 2FS Plus). A P-1000 micropipette puller (Sutter Instruments) was used to prepare filament-containing borosilicate glass patch pipettes (Hilgenberg GmbH) with a resistance of 4-5 MΩ. Patch pipettes were filled with internal solution (120 mM KCl, 4 mM MgCl2, 10 mM HEPES, 10 mM BAPTA, 5 mM Lidocaine N- ethyl bromide, 0.5 mM Na2GTP, 2 mM Na2ATP, adjusted to 280 mOsm, pH 7.4). Cells were recorded using voltage-clamp at a holding potential of -60 mV in whole cell configuration with a sampling rate of 10 kHz. During recordings, aCSF bath perfusion was applied for 1 minute. To pharmacologically isolate glycinergic mIPSCs, blockers of AMPA receptors (DNQX, 10 µM), nicotinic receptors (DHBE, 0.1 µM), NMDA receptors (L-689560, 5 µM), action potential firing (tetradotoxin, 0.5 µM) and GABAARs (bicuculline, 10 µM) were added to aCSF and recorded for 5 minutes. To confirm that these mIPSCs were indeed glycinergic currents, strychnine was added at a concentration of 1 µM to the cocktail of blockers and recorded for an additional 1 minute. All recordings were acquired using a Multiclamp 700B amplifier (Axon Instruments), stored using 1440A Digidata (Axon Instruments), and analysed using Clampfit 10.7.0.3 (Axon Instruments) and NeuroExpress 24.c.16 (Szücs, 2022). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 22

We thank Hans Maric (Virchow Center, University of Würzburg) for the Sylite probe, Carmen Villmann and Natascha Schäfer (Universitätsklinikum Würzburg) for the collection of brain and spinal cord tissue, Nicolas Froger (MAPREG, France) for the supply of mouse embryos, Elisabeth Piccart and Petra Bex (UHasselt, Belgium) for the brain slice patch clamp training, and Michael Schumacher (NeuroBicêtre, Inserm, UPSaclay) for his generous help with the setting up of the super-resolution imaging platform. We kindly acknowledge the support from Zeiss and Abbelight. Author contributions Experimentation: SC, YV Data analysis: SC, YV, SYA Experimental tools and techniques: CM, BB Project design: SC, BB, CSP Writing of the manuscript: SC, CSP Editing of the manuscript: YV, SYA, BB Funding acquisition: BB, CSP Funding This research was funded by the Agence Nationale de la Recherche (ANR-20-CE11-0002, InVivoNanoSpin, to CGS) and Fonds voor Wetenschappelijk Onderzoek (11N1422N, V419721N, to BB). SC was supported by a postdoctoral contract through the ANR projects InVivoNanoSpin and NeurATRIS (ANR-11-INBS-0011). The SMLM setup was acquired with the support of the Fédération pour la Recherche sur le Cerveau (FRC/Neurodon, opération Rotary – Espoir en Tête, to CGS), and an ERM equipment grant from UPSaclay (to CGS). Conflict of interest statement The authors declare no conflict of interest. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 23

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Nature 599: 513-517 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 25 Figures and tables Figure 1. SMLM of mEos4b-GlyRβ subunits in the hippocampus. (A) Single molecule detections of mEos4b-GlyRβ (red 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. Inhibitory synapses were identified using the gephyrin marker Sylite (cyan). Scale bar 5 µm. (B) 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 Glrbeos/eos mice (N = 3). 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). Data are shown as mean ± SD. Levels of significance were determined using nonparametric Kruskal–Wallis ANOVA 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. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 26 Figure 2. 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 = 3 independent experiments using 3 Glrbeos/eos animals. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 27 Figure 3. 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 (N = 3). 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 for ventral and dorsal striatum, respectively, from 9 fields of view per sub-region, N = 3 animals). Data are shown as mean ± SEM. Levels of significance determined using a nonparametric unpaired 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). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 28 Figure 4. 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 mIPSCs in ventral and dorsal MSNs (mean ± SEM; n=5 cells from 3 animals in ventral striatum and 5 cells from 4 animals in dorsal striatum). Levels of significance determined using a nonparametric unpaired one-tailed Mann-Whitney test (** p < 0.05). .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint 29 Figure 5. Expression of recombinant GlyR subunits in cultured hippocampal neurons. (A) Cultured mouse embryonic hippocampal neurons (E17.5) were infected with lentivirus expressing mEos4b-tagged GlyR subunits α1, α2 or β (red), fixed at DIV17 and stained for gephyrin (Sylite marker, cyan). Scale: 10 µm. (B) SMLM pointillist images showing the photoconverted mEos4b detections. Dense clusters of synaptic (red arrowheads) and extrasynaptic receptors (red cross) are indicated. Diffusely distributed extrasynaptic GlyR complexes (red circle) are seen as small clusters of detections resulting from the repetitive detection of single mEos4b fluorophores. Scale: 2 µm. N = 3 independent experiments. Table 1. Estimated copy number of mEos4b-GlyRβ containing receptor complexes at inhibitory synapses in different regions of the CNS. 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 0.91 CA1 0.73 ± 0.05 0 – 4 0.23 CA3 1.53 ± 0.30 0 – 6 0.24 DG 0.67 ± 0.18 0 – 2 0.19 Dorsal striatum 3.33 ± 0.19 0 – 15 0.48 Ventral striatum 26.46 ± 1.48 0 – 113 0.76 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted January 7, 2025. ; https://doi.org/10.1101/2025.01.07.631736doi: bioRxiv preprint