# The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin

Sven Wagner, Choongku Lee, Lucia Rojas, Christian G Specht, Jeongseop

Rhee, Nils Brose, Theofilos Papadopoulos

► To cite this version:

Sven Wagner, Choongku Lee, Lucia Rojas, Christian G Specht, Jeongseop Rhee, et al.. The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin. Journal of Biological Chemistry, 2021, 296, pp.100709. 10.1016/j.jbc.2021.100709. hal-04252772

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# The α3 subunit of GABA~A~ receptors promotes formation of inhibitory synapses in the absence of collybistin

Received for publication, October 20, 2020, and in revised form, April 14, 2021
https://doi.org/10.1016/j.jbc.2021.100709

Sven Wagner¹, ChoongKu Lee¹, Lucia Rojas¹, Christian G. Specht², JeongSeop Rhee¹, Nils Brose¹, and Theofilos Papadopoulos³,*

From the ¹Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany; ²Diseases and Hormones of the Nervous System (DHNS), Inserm U1195, Université Paris-Saclay, Le Kremlin-Bicêtre, France; and

³Department of Molecular Biology, Universitätsmedizin Göttingen, Göttingen, Germany

Edited by Phyllis Hanson

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. Collybistin (CB), a brain-specific guanine nucleotide exchange factor, is essential for the formation of γ-aminobutyric acidergic (GABAergic) postsynapses in defined regions of the mammalian forebrain, including the hippocampus and basolateral amygdala. This process depends on a direct interaction of CB with the scaffolding protein gephyrin, which leads to the redistribution of gephyrin into submembranous clusters at nascent inhibitory synapses. Strikingly, synaptic clustering of gephyrin and GABA~A~ type A receptors (GABA~ARs) in several brain regions, including the cerebral cortex and certain thalamic areas, is unperturbed in CB-deficient mice, indicating that the formation of a substantial subset of inhibitory postsynapses must be controlled by gephyrin-interacting proteins other than CB. Previous studies indicated that the α3 subunit of GABA~ARs~ (GABA~AR-α3~) binds directly and with high affinity to gephyrin. Here, we provide evidence (i) that a homooligomeric GABA~AR-α3~A343W mutant induces the formation of submembranous gephyrin clusters independently of CB in COS-7 cells, (ii) that gephyrin clustering is unaltered in the neuronal subpopulations endogenously expressing the GABA~AR-α3~ in CB-deficient brains, and (iii) that exogenous expression of GABA~AR-α3~ partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons. Our results identify an important role of GABA~AR-α3~ in promoting gephyrin-mediated and CB-independent formation of inhibitory postsynapses.

Fast chemical synaptic transmission between neurons requires the orchestrated clustering of ionotropic neurotransmitter receptors in the postsynaptic plasma membrane. At γ-aminobutyric acidergic (GABAergic) postsynapses, inhibitory neurotransmission is mediated by γ-aminobutyric acid (GABA) acting through GABA type A receptors (GABA~ARs; for review, see (1)). These receptors are

heteropentameric GABA-gated chloride channels that belong to the Cys-loop ligand-gated ion channel superfamily (2). They are encoded by 19 different genes, which are grouped into eight subclasses based on their sequence homologies (α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3). Different subunit combinations of two α subunits, two β subunits, and a single γ subunit or δ subunit exist in distinct GABA~AR~ subtypes (3). The subsets of GABA~ARs~ at synapses are typically composed of two α1 subunits, α2 subunits, or α3 subunits together with two β2 subunits or β3 subunits and a single γ2 subunit (4, 5). The γ2 is essential for the postsynaptic clustering of GABA~ARs~ (6).

Apart from the cognate GABA~ARs~, core components of many GABAergic postsynapses are cell adhesion proteins, such as the neuroligins (NLs), particularly NL-2 and NL-4 (7, 8), the scaffolding protein gephyrin (9), and the guanine nucleotide exchange factor collybistin (CB, also known as ARHGEF9) (10), which together control GABA~AR~ recruitment to synapses. The synaptic localization of gephyrin depends on the presence of certain GABA~AR~ subtypes. For example, targeted deletion of the GABA~AR-α1~, GABA~AR-α2~, GABA~AR-α3~, or GABA~AR-γ2~ subunit results in a loss of synaptic gephyrin clusters (11–17). Vice versa, ablation of gephyrin expression by either antisense depletion in cultured neurons or by gene KO in mice prevents the clustering of glycine receptors (GlyRs) (9, 18) and α2- or γ2-containing GABA~ARs~ at developing post-synaptic sites (6, 19, 20). However, certain GABA~AR~ subunits were previously found at synapses despite the absence of gephyrin. In the spinal cord, synaptic GABA~AR-α1~ localization is unaltered upon gephyrin KO, whereas the levels of α2, α3, β2/3, and γ2 at synapses are significantly reduced in gephyrin KOs (21). Specific effects of gephyrin depletion on receptor subtypes containing the α2 subunits and γ2 subunits have also been described in cultured hippocampal neurons, whereas synaptic clustering of GABA~AR-α1~ is unaffected by gephyrin KO (22).

An essential component of many, but not all, GABAergic synapses is the gephyrin-interacting protein CB (10, 23), which is a brain-specific guanine nucleotide exchange factor for the small GTPases Cdc42 and TC10 (24, 25). Genetic deletion of CB in mice leads to a massive reduction of synaptic gephyrin and γ2 subunit-containing GABA~ARs~ in certain regions of the

<footnote>* For correspondence: Theofilos Papadopoulos, theofilos.papadopoulos@med.uni-goettingen.de.</footnote>
CB-independent synapse formation

forebrain, including the hippocampus and basolateral amygdala (26, 27). This loss of inhibitory postsynaptic proteins is accompanied by a substantial decrease in hippocampal GABAergic neurotransmission and significant changes in hippocampal synaptic plasticity, leading to impaired spatial learning and increased anxiety levels (26). In contrast, postsynaptic clustering of gephyrin and GlyRs in the brainstem and spinal cord and glycinec neurotransmission in these regions are not altered by CB KO (26). In line with the functional defects observed in CB KO mice, mutations in the human CB homolog, hPEM-2, cause intellectual disability, epilepsy, and autism spectrum disorder (28-40). Strikingly though, GABAergic synapses in the cerebral cortex, certain thalamic areas, and other brain regions are unaffected by CB KO (26). This indicates that a substantial subset of GABAergic postsynapses is formed independently of CB and that the formation of those postsynapses must be controlled by gephyrin-interacting proteins other than CB. The identification of corresponding CB-independent mechanisms involved in the formation of inhibitory synapses is essential for a comprehensive understanding of inhibitory synapses and circuits in the brain, the identification of pathological mechanisms that cause epileptiform activity, intellectual disability, or autism spectrum disorder in GABAergic synaptopathies, and for the development of appropriate therapeutic strategies.

In addition to CB, gephyrin binds directly and with high affinity to the β subunit of GlyRs (41–45). Previous studies showed that gephyrin and GlyRs colocalize not only at the postsynaptic plasma membrane but also at the intracellular transport vesicles (46), and that active microtubule-dependent motor protein complexes interact and comigrate with GlyR-fusion and gephyrin-fusion proteins through neurite processes (47–49). Gephyrin also binds directly to certain α subunits of GABAARs, albeit with lower affinities than to the GlyR-β subunit (50–55). More recently, the α2 subunit of GABAARs was shown to bind directly to both, gephyrin and CB (53–55). However, these studies were based on *in vitro* analyses using recombinantly expressed and purified intracellular loops or small peptides of receptor-binding domains that cannot fully account for the complexity of interactions between multiple components *in cellula*. One of the main obstacles in analyzing the interactions of individual GABAAR-α subunits with gephyrin in heterologous cells is that all of them accumulate in intracellular compartments upon their over-expression and do not travel to the cell surface alone (56, 57). Their cell surface expression requires their coassembly with β subunits and γ subunits in the endoplasmic reticulum (56, 57). Gephyrin has been previously shown to bind not only to α but also to β subunits of GABAARs (58). Thus, it is difficult to study and characterize the significance of individual GABAAR subunits for the submembranous clustering of gephyrin in cells separately, since coexpression of α subunits, β subunits, and γ subunits and their cell-surface localization as heteropentamers is required. Interestingly, a previous study found that the substitution of a single amino acid residue in the transmembrane domain (TMD) of the GABAAR-α1 subunit is sufficient to bypass the stringent assembly rules, permitting

access of homomeric GABAAR-α1 complexes to the cell surface without the need for subunit coassembly (59).

Based on these findings (59), we mutated the corresponding TMD residues to enable cell surface assembly of homomeric α1 subunits, α2 subunits, or α3 subunits of GABAARs and analyzed the interaction of individual α subunits with gephyrin *in cellula*. We made three major observations: (i) the α3 subunit of GABAARs induces clustering of gephyrin at the plasma membrane independently of CB or additional accessory proteins. (ii) In contrast to α2 subunit containing GABAARs, the subcellular colocalization and synaptic clustering of GABAAR-α3 and gephyrin is unchanged in CB KO mice, pointing to an important role of the α3 subunit in priming gephyrin-mediated and CB-independent formation of GABAergic synapses. (iii) Recombinant expression of the α3 subunit partially rescues the impaired clustering of gephyrin in cultured CB KO hippocampal neurons, indicating that enhancing GABAAR-α3 function might be a potential tool toward improving therapeutic strategies that target GABAergic synapse dysfunction caused by loss of or aberrant CB function.

Results

Cell surface expression of the GABAAR-α3 subunit induces CB-independent formation of GFP-gephyrin submembranous microclusters

Gephyrin was previously shown to interact with different affinities with the intracellular loops of GABA<sub>A</sub> α1 subunit, α2 subunit, and α3 subunit (50–55). Moreover, GABA<sub>A</sub> α3 subunit displays the highest *in vitro* gephyrin affinity of all α subunits, with an estimated K<sub>D</sub> of 5.3 µM for the full-length intracellular loop (53, 55). A previous study indicated that just a single substitution of a TMD alanine (A) residue with tryptophan (W) in the GABAAR-α1 subunit (Fig. 1A; substitution of the boxed A with W) permits access of homomeric α1 subunit GABAARs to the cell surface (59). Since this A residue is located within a highly conserved sequence in the M3 of the α1 subunit, α2 subunit, and α3 subunit (Fig. 1A), we performed the A/W substitution for all three subunits. This allowed us to study interactions of individual α subunits with gephyrin *in cellula*.

First, we expressed the individual WT α subunits and their corresponding A/W mutants in COS-7 cells in the absence of GFP-gephyrin (Fig. 1, B–G) and analyzed their subcellular distribution after fixation and cell permeabilization. Total (intracellular + surface) expressions were comparable between individual mutants and their corresponding WT proteins. Next, we coexpressed the individual α subunits or their corresponding A/W mutants together with GFP-gephyrin and analyzed their subcellular distribution and colocalization upon fixation and cell permeabilization (Fig. 1, H–M). GFP-gephyrin expressed alone (not shown) or together with WT α1 subunit, α2 subunit, or α3 subunit (Fig. 1, H–J) formed intracellular aggregates, as described previously (10, 41). In cells coexpressing GABAAR-α1<sup>WT</sup>, we consistently found no colocalization with intracellular GFP-gephyrin aggregates
CB-independent synapse formation

(Fig. 1H). Furthermore, analyses of fluorescence intensity plots (obtained by measuring the intensity profile along a straight line connecting GFP-gephyrin puncta; Fig. 1H; right panel) and Mander's coefficients for the whole cells (M1 and M2; Fig. 1N) indicated no correlation between the GFP-gephyrin aggregates and the α¹WT signals. In contrast, intracellular GFP-gephyrin aggregates were found to be colocalized with both, GABA<sub>A</sub>R-α2WT and GABA<sub>A</sub>R-α3WT (Fig. 1, I and J). Moreover, fluorescence intensity plots and Mander's coefficients indicated good correlation between GFP-gephyrin and α2WT or α3WT signals, respectively (Fig. 1, I and J; right panels; Fig. 1N). In cells expressing the α¹A<sup>317W</sup> mutant (Fig. 1K), no colocalization with GFP-gephyrin aggregates was observed, which is in line with the results obtained with α¹WT (Fig. 1N). Similar to α2WT and α3WT, the α2A<sup>318W</sup> (Fig. 1L) and α3A<sup>343W</sup> (Fig. 1M) mutants colocalized with intracellular GFP-gephyrin aggregates, and corresponding fluorescence intensity plots indicated a clear overlap between the highest intensity peaks in both channels (Fig. 1, L and M; right panels). In addition, Mander's coefficients for the whole cells indicated good correlations between GFP-gephyrin and α2A<sup>318W</sup> or α3A<sup>343W</sup> signals, respectively (Fig. 1N).

More importantly, expression of α3A343W, and to a much lesser extent of the α2A318W mutant, led to the formation of numerous smaller GFP-gephyrin clusters (so-called microclusters; indicated by arrows in Fig. 1, L and M). Accordingly, the mean total number of GFP-gephyrin microclusters (0.05–0.3 µm²) was significantly increased in COS-7 cells coexpressing GABAAR-α3A343W (20.6 ± 11.9; Fig. 1O), as compared with cells expressing GABAAR-α3WT (2.3 ± 2.6). In contrast, changes in the numbers of GFP-gephyrin microclusters in cells coexpressing GABAAR-α1A317W (0.9 ± 1.6) or GABAAR-α2A318W (4.4 ± 4.4) did not reach statistical significance, as compared with cells coexpressing GABAAR-α1WT (0.9 ± 1.28) or GABAAR-α2WT (1.3 ± 1.56), respectively (Fig. 1O).

Previous studies showed that GFP-gephyrin intracellular
aggregates can be disassembled, so that gephyrin is recruited
to membrane-associated microclusters, if a constitutively
active splice variant of CB lacking the N-terminal SH3 domain,
or if intrinsically inactive CB variants containing the N-ter-
minal SH3 domain along with CB activators, such as NL-2, the
α2 subunit of GABAARs, or the small Rho-like GTPase TC10,
are coexpressed (7, 10, 25, 60, 61). However, in all previous
studies, the presence of CB for redistributing gephyrin into
microclusters in heterologous cells was obligatory. In contrast,
our findings here indicate that mutations enabling cell surface
expression of the GABAAR-α3 or GABAAR-α2 subunit induce
gephyrin microcluster formation in the absence of CB.

Hannan and Smart (59) showed that the A/W mutation
enables homomeric GABA<sub>A</sub>R-α1 subunits to traffic to the cell
surface. To unequivocally demonstrate that the corresponding
A/W mutations in the α2 and α3 subunits display a similar cell
surface expression as α1, we again coexpressed the A/W
mutants of the α1, α2, or α3 subunits together with GFP-
gephyrin and analyzed their immunoreactivities upon fixa-
tion in nonpermeabilized cells using subunit-specific anti-
bodies against extracellular epitopes. This revealed that the

A/W mutations enabled all three α subunits to reach the cell surface (Fig. 2, A–C). As expected for nonpermeabilized cells, no colocalization between intracellular GFP-gephyrin aggregates and the immunoreactivities of GABA<sub>A</sub>R-α subunits were observed (see overlays in Fig. 2, A–C). In addition, fluorescence intensity profiles along a straight line through the GFP-gephyrin puncta indicated no correlation between the intracellular GFP-gephyrin aggregates (marked with A) and surface-labeled α subunits (Fig. 2, A–C; right panel). In contrast, GABA<sub>A</sub>R-α3A343W immunoreactivities, but not those of GABA<sub>A</sub>R-α1A317W or GABA<sub>A</sub>R-α2A318W, were partially colocalized with submembranous GFP-gephyrin microclusters (marked with M in the fluorescence intensity scans of Fig. 2, A–C). In addition, analyses of Mander’s coefficients for the whole cells (M1 and M2; Fig. 2D) indicated no correlation between the GFP-gephyrin fluorescence and the α1A317W or the α2A318W surface signal but partial and significantly increased colocalization of the GFP-gephyrin signal with surface-localized mScarlet-α3A343W (Fig. 2D).

Together, the results shown in Figures 1 and 2 demonstrate that surface expression of homomeric GABA<sub>A</sub>R-α3, the α subunit with the highest binding affinity for gephyrin as compared with α1 or α2 (55), induces the formation of sub-membranous GFP-gephyrin microclusters. Thus, α3 is the first GABA<sub>A</sub>R subunit shown to be capable of inducing sub-membranous gephyrin clustering in the absence of CB.

CB-dependent clustering of gephyrin in neurons endogenously
expressing the GABA<sub>A</sub>R-α2 subunit

Previous studies indicated that the α2 subunit of GABAARs
binds with high affinity to CB and with low affinity to gephyrin
(53–55). In contrast, the α3 subunit of GABAARs binds to
gephyrin with the highest affinity estimated for GABAAR
subunits but does not interact with CB (53, 55). Whereas the
immunoreactivity of the GABAAR-α1 subunit is the most
abundant and ubiquitous across the brain, the immunoreac-
tivities of GABAAR-α2 and GABAAR-α3 subunits have more
restricted, although distinct, distributions, being expressed at
high levels only in specific brain areas (62). For example,
GABAAR-α2 is expressed throughout the hippocampus, but
not in the reticular thalamic nuclei (nRT), whereas GABAAR-
α3 is abundantly expressed in nRT, an area rich in GABAergic
interneurons (63) but less so or not at all in the different
hippocampal areas (62). The respective distributions of the α2
and α3 subunits also indicate that they occur mostly in distinct
neuronal populations (62).

Previous studies indicated that CB deficiency leads to the
loss of gephyrin clusters at a specific set of GABAARs and in
distinct neuronal subpopulations of the mammalian forebrain
(26, 27). However, in these studies, gephyrin and GABAARs
were measured separately, disregarding the colocalization be-
tween gephyrin and specific GABAAR subunits in CB-deficient
mice. As CB, in addition to its interaction with gephyrin (10),
binds with high affinity to the GABAAR-α2 subunit, but not to
the GABAAR-α3 subunit (55), we analyzed the densities and
colocalization of gephyrin with α2 or α3 in different neuronal
subpopulations with abundant to moderate expression of the
CB-independent synapse formation

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Surface expression of the GABA<sub>A</sub>R-α3 subunit induces formation of GFP-gephyrin microclusters in COS-7 cells. A, alignment of a highly conserved primary sequence within the M3 domains of mouse α1, mouse α2, and rat α3 subunits of GABA<sub>A</sub>Rs used in this study. The boxed alanine (A) residue replaced by tryptophan (W) in α1 has been previously shown to induce surface expression of homomeric GABA<sub>A</sub>-α1 receptors (59). In the current study, the corresponding A residues of α1, α2, and α3 subunits were replaced by W, respectively. B–G, images of COS-7 cells expressing the WT α1, α2, and mScarlet-α3 subunits (B–D) or their corresponding A/W mutants (E–G). Total expression (intracellular + surface) of the different receptor subunits was visualized by cell permeabilization and immunostaining with subunit-specific antibodies (α1 and α2) or by the fused mScarlet fluorescent protein (α3). The scale bars represent 10 µm. H–M, images of COS-7 cells coexpressing GFP-gephyrin together with the WT α1, α2, or mScarlet-α3 (H–J) or their corresponding A/W mutants (K–M). Right panels indicate fluorescence intensity scans over the yellow lines in H–J and K–M, respectively (GFP-gephyrin, green; GABA<sub>A</sub>R subunits, red). Note that the highest peaks of α2 and mScarlet-α3 intensities, but not those of α1, colocalize with GFP-gephyrin. In addition, note that the mScarlet-α3 A343W mutant, and to a much lesser extent the α2 A318W mutant, induces the formation of GFP-gephyrin microclusters (indicated by arrows in L and M). The scale bars represent 10 µm. N, the degree of colocalization between the red (α subunit signal in permeabilized cells) and green (GFP-gephyrin) signals was statistically analyzed for the whole cells and expressed with the Mander's colocalization coefficients M1 and M2. M1 represents the fraction of the particular receptor subunit or its corresponding mutant, overlapping with GFP-gephyrin. M2 represents the fraction of GFP-gephyrin overlapping with the α subunits, as indicated. All calculations for the Mander's coefficients were performed by the ImageJ software, as described in *Experimental procedures* section. Data represent means ± SD of N = 10 cells per condition. ***p < 0.001; unpaired two-tailed Student's t test. O, total</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2. The GABA<sub>A</sub>R-α3<sup>A343W</sup> mutant partially redistributes GFP-gephyrin into submembranous microclusters in COS-7 cells.** A–C, images of COS-7 cells coexpressing GFP-gephyrin together with α1 A317W, α2 A318W, or the mScarlet-α3 A343W mutant, as indicated. Surface expression of the different receptor subunits in unpermeabilized cells was visualized by subunit-specific antibodies (α1, α2, or α3) binding to extracellular epitopes. Right panels indicate fluorescence intensity scans over the yellow lines in A–C, respectively (GFP-gephyrin, green; GABA<sub>A</sub>R subunits, red). Note that the highest peaks of mScarlet-α3 intensities, but not that of α1 or α2, colocalize with GFP-gephyrin submembranous microclusters (peaks marked with M). In contrast, intracellular GFP-gephyrin aggregates (peaks marked with A) do not colocalize with the immunoreactivities of the α subunits in unpermeabilized cells. The scale bar represents 10 µm. D, the degree of colocalization between the red (α1<sup>A317W</sup>, α3<sup>A318W</sup>, or α3<sup>A343W</sup> immunoreactivities in nonpermeabilized cells) and green (GFP-gephyrin) signals was statistically analyzed for the whole cells and expressed with the Mander's colocalization coefficients M1 and M2; M1 represents the fraction of the α subunit mutants overlapping with GFP-gephyrin. M2 represents the fraction of GFP-gephyrin overlapping with α1<sup>A317W</sup>, α3<sup>A318W</sup>, or α3<sup>A343W</sup>, as indicated. Data represent means ± SD of N = 10 cells per condition. **p* < 0.01; unpaired two-tailed Student's *t* test. GABA<sub>A</sub>R-α3, α3 subunit of GABA type A receptor.</figcaption></figure>

individual subunits. For GABA<sub>A</sub>R-α2 subunit, we focused our
analysis on two hippocampal regions with abundant expres-
sion of this subunit, the hilus of the dentate gyrus (DG) and
stratum radiatum (SR) of the cornu ammonis 1 (CA1) hip-
 pocampal area (Fig. 3). In line with previous studies (26, 27,
64), the densities of gephyrin-immunoreactive puncta were
significantly reduced in the brains of CB KO mice, as
compared with their WT littermates (Fig. 3C; DG hilus: CB
KO, 0.16 ± 0.02 puncta/100 µm³ versus WT, 1.60 ± 0.09
puncta/100 µm³; Fig. 3I; CA1 SR: CB KO, 0.06 ± 0.04 puncta/
100 µm³ versus WT, 2.36 ± 0.73 puncta/100 µm³). The
apparent sizes of the remaining gephyrin puncta in the DG
hilus of CB KO animals were comparable to WT values
(Fig. 3D; CB KO, 2.36 ± 0.68 µm² versus WT, 2.72 ± 0.13 µm²).
However, in the CA1 SR, the mean size of gephyrin puncta was
significantly increased, as compared with controls (Fig. 3J; CB
KO, 2.57 ± 0.16 µm² versus WT, 2.11 ± 0.16 µm²). As previ-
ously shown (26), this is probably because of the accumulation
of gephyrin in intracellular aggregates within the somata of
isolated CA1 neurons. GABA<sub>A</sub>Rs containing the α2 subunit

were previously shown to depend on both gephyrin and CB for
postsynaptic localization (6, 19, 55). In line with this, the mean
density of GABA<sub>A</sub>R-α2 immunoreactive puncta was signifi-
cantly reduced in the hilus of CB KO mice as compared with
controls (Fig. 3E; CB KO, 0.12 ± 0.07 puncta/100 µm³ versus
WT, 2.92 ± 0.53 puncta/100 µm³). In contrast, in the CA1 SR,
the GABA<sub>A</sub>R-α2 densities were similar between groups
(Fig. 3K; CB KO, 2.05 ± 1.69 puncta/100 µm³ versus WT,
1.84 ± 1.24 puncta/100 µm³), even though the apparent sizes
of the α2 subunit puncta were significantly reduced in the CA1
SR, as compared with WT (Fig. 3L; CB KO, 1.83 ± 0.08 µm²
versus WT, 2.34 ± 0.09 µm²). Moreover, the analysis of
gephyrin-immunoreactive puncta colocalized with GABA<sub>A</sub>R-
α2 puncta indicated a strong reduction in the fraction of
colocalized puncta in the brains of CB KO animals, as
compared with those of WT littermates (Fig. 3M).

The aforementioned data indicate that CB is indispensable
for the clustering and/or stabilization of gephyrin and α2
subunit containing GABA<sub>A</sub>Rs, as well as for the colocalization
of the two proteins at synapses.

numbers of GFP-gephyrin microclusters and aggregates from images of COS-7 cells coexpressing GFP-gephyrin together with α1 (WT or the A317W mutant), α2 (WT or the A318W mutant), or mScarlet-α3 (WT or the A343W mutant), as indicated. Counted puncta in N = 10 cells per condition were binned according to their size. *Insets*, relative fractions of microclusters (0.05–0.3 µm²; *left*) and aggregates (>1 µm²; *right*). Data in the insets represent means ± SD of N = 10 cells per condition. ***p* < 0.001; unpaired two-tailed Student's *t* test. GABA<sub>A</sub>R-α3, α3 subunit of GABA type A receptor.
<figure><img src="image_5.png" /><figcaption>CB-independent synapse formation</figcaption></figure>

**Figure 3.** The clustering of GABA~A~R-α2 in the hilus of the DG and CA1 SR of the hippocampus depends on both CB and gephyrin. A and B, representative snapshots of confocal 3D images of gephyrin/GABA~A~R-α2 colabeling in the hilus of the DG in slices derived from 8-week-old CB WT mice (A) or their CB KO littermates (B). Slices were processed for confocal microscopy, and images were generated using the Imaris software, as described in *Experimental procedures* section. In agreement with previous studies (26, 64), a strong reduction of both, gephyrin and α2 punctate immunoreactivities was observed in CB KO slices, as compared with controls. The scale bars represent 10 µm. Scale frames within the 3D images are composed of 5 × 5 µm squares. C–F, quantifications of gephyrin (C and D) and GABA~A~R-α2 (E and F) immunoreactive densities (C and E) and apparent sizes of puncta (D and F) in the hilus of 8-week-old CB WT mice (black) or their CB KO littermates (red). Each bar corresponds to mean values (±SD) obtained with sections from three individual brains per genotype. ***p < 0.001; unpaired two-tailed Student's t test. G and H, representative 3D images of gephyrin/GABA~A~R-α2 costainings in the hippocampal CA1 SR in slices derived from 8-week-old CB WT mice (G) or their CB KO littermates (H). The scale bar represents 10 µm. I and L, quantifications of gephyrin (I and J) and GABA~A~R-α2 (K and L) immunoreactive puncta densities (I and K) and sizes (J and K) in the CA1 SR of 8-week-old CB WT mice (black) or their CB KO littermates (red). Note the strong reduction of gephyrin punctate immunoreactivity (I) and the reduction in the size of GABA~A~R-α2 puncta (L) in CB KO slices, as compared with controls. Each bar corresponds to mean values (±SD) obtained with sections from N = 3 individual brains per genotype. *p < 0.05, **p < 0.01; unpaired two-tailed Student's t test. M, quantifications of the percentages of gephyrin-immunoreactive puncta colocalized with GABA~A~R-α2 in the hilus of DG and CA1 SR of 8-week-old CB WT mice (black) or their CB KO littermates (red). Each bar corresponds to mean values (±SD) obtained with sections from N = 3 individual brains per genotype. *p < 0.05, **p < 0.01; unpaired two-tailed Student's t test. CA1, cornu ammonis 1;CB, collybistin; DG, dentate gyrus; GABA~A~R-α2, α2 subunit of GABA type A receptor; SR, stratum radiatum.
### CB-independent clustering of gephyrin in neurons endogenously expressing the GABA~A~R-α3 subunit

To analyze the clustering of GABA~A~R-α3 in the absence of CB, we focused on two regions, the hilus of the DG, in which a small population of neurons were found to express high levels of α3, and the nRT, the region with the highest expression of GABA~A~R-α3 in the mouse brain (15, 16, 62). In contrast to GABA~A~R-α2, which is abundantly expressed in the hilus, the α3 immunoreactivity in this area is only sparse and restricted to few hilar neurons. Hilar areas encompassing neurons expressing the α3 subunit were selected in slices derived from CB KO and WT animals, and the densities of both gephyrin and α3 puncta were quantified. Again, we consistently found a strong reduction in the density of gephyrin puncta in the hilus of CB KO mice, as compared with WT controls (Fig. 4, A–C; CB KO, 0.92 ± 0.11 puncta/100 µm³ versus WT, 5.09 ± 1.96 puncta/100 µm³). In contrast, GABA~A~R-α3 puncta densities (Fig. 4E; CB KO, 1.19 ± 0.54 puncta/100 µm³ versus WT, 1.49 ± 0.45 puncta/100 µm³) and apparent sizes (Fig. 4F; CB KO, 3.67 ± 0.54 µm² versus WT, 3.37 ± 0.33 µm²) in the hilus were comparable between groups. Furthermore, the percentages of gephyrin puncta colocalized with α3 puncta were not significantly different between groups (Fig. 4M, left). Interestingly, the percentage of gephyrin colocalized with α3 in the hilus of CB KO mice was markedly increased, as compared with WT, even though this effect did not reach significance (Fig. 4M, left; CB KO, 48.08 ± 7.56% versus WT, 28.83 ± 10.29%, *p* = 0.059). This result indicates that approximately half of the remaining gephyrin puncta in the hilus of CB KO animals are located at postsynapses of neurons expressing the GABA~A~R-α3 subunit (also compare *overlays* in Fig. 4, A and B).

In the nRT, the expression of GABA~A~Rs containing the α2 subunit was previously estimated to be very low to absent, whereas GABA~A~R-α3 is abundantly expressed (62). In this region, CB deficiency had no effect on the densities (Fig. 4, I and K; gephyrin CB KO, 2.69 ± 1.71 puncta/100 µm³ versus gephyrin WT, 3.64 ± 1.70 puncta/100 µm³; α3 CB KO, 2.1 ± 0.39 puncta/100 µm³ versus α3 WT, 2.94 ± 0.82 puncta/100 µm³) or on their apparent sizes (Fig. 4, J and L; gephyrin CB KO, 2.45 ± 0.30 µm² versus gephyrin WT, 2.73 ± 0.73 µm²; α3 CB KO, 4.69 ± 0.97 µm² versus α3 WT, 3.84 ± 0.15 µm²) of gephyrin and GABA~A~R-α3 immunoreactive puncta. In addition, colocalization analysis of gephyrin and α3 puncta revealed no significant differences between groups in the nRT (Fig. 4M, right).

These findings reveal no differences between CB KO brains and controls regarding the clustering of gephyrin and GABA~A~R-α3 in certain brain regions or neuronal subpopulations.

Furthermore, to particularly test whether CB deficiency affects the clustering of GABA~A~R-α3 at synaptic sites, we performed costainings for α3 and the vesicular inhibitory amino acid transporter (VIAAT), a well-characterized marker for inhibitory presynapses (65). Again, our quantifications in the hilus of the DG and nRT revealed no differences in the

densities and sizes of both GABA~A~R-α3 and VIAAT-immunoreactive puncta between groups (Fig. 5, A–L). Furthermore, the percentages of postsynaptic GABA~A~R-α3 puncta in the nRT apposed to VIAAT were similar between groups (Fig. 5M, right). In contrast, the percentage of postsynaptic α3 apposed to VIAAT was significantly increased in the hilus of CB KO animals, as compared with their WT littermates (Fig. 5M, left; CB KO, 77.11 ± 6.33% versus WT, 54.26 ± 7.09%).

This result indicates that in the absence of GABA~A~Rs containing the α2 subunit in the hilus of CB KO mice, the synaptic clustering of the α3 subunit is increased.

### Partial rescue of gephyrin clustering in cultured CB KO hippocampal neurons exogenously expressing the GABA~A~R-α3 subunit

In the different areas of the rodent hippocampus, the expression of endogenous GABA~A~R-α3 is very low to absent (62), and previous studies on CB KO animals revealed the most severe defects in gephyrin and GABA~A~R clustering in these areas (26, 27). This indicates a possible compensation of CB loss by GABA~A~R-α3, in accord with our finding of CB-independent clustering of gephyrin in neuronal subpopulations that endogenously express the α3 subunit. We therefore studied the effects of recombinantly expressing GABA~A~R-α3 in cultured hippocampal CB KO neurons, which normally express very low levels of or no GABA~A~R-α3. We transfected cultured hippocampal neurons of CB KO embryos or their WT littermates on *day in vitro* 4 (DIV 4) with either mScarlet alone or with mScarlet-GABA~A~R-α3. At DIV 14, the neurons were fixed, permeabilized, and labeled with antibodies against GABA~A~R-α3 and endogenous gephyrin (Fig. 6, A–D). As previously described (62), the endogenous immunoreactivity of GABA~A~R-α3 in WT and CB KO neurons expressing mScarlet alone (high-intensity magnification panels of Fig. 6, A and B) was very low to absent. In agreement with a previous study (26), the densities of gephyrin puncta were significantly reduced in the dendrites of cultured hippocampal CB KO neurons expressing mScarlet alone, as compared with CB WT controls (Fig. 6, A, B, and E; CB KO/mScarlet, 2.15 ± 0.87 puncta/40 µm versus WT/mScarlet, 12.9 ± 2.97 puncta/40 µm). Similarly, the apparent sizes of gephyrin-immunoreactive puncta in CB KO neurons expressing mScarlet alone were reduced, as compared with controls (Fig. 6, A, B, and F; CB KO/mScarlet, 0.067 ± 0.016 µm² versus WT/mScarlet, 0.098 ± 0.013 µm²). Overexpression of mScarlet-GABA~A~R-α3WT in both CB KO neurons and WT controls led to the colocalization of the dendritic mScarlet-α3WT signal with gephyrin and increased the level of GABA~A~R-α3 immunoreactivity (indicated by *arrowheads* in the zoomed panels of Fig. 6, C and D). Analysis of the Mander's coefficients M1 and M2 for selected dendritic segments showed good correlation of the mScarlet-α3WT signal and the endogenous gephyrin signal in the dendrites of CB WT neurons (Fig. 6G; left). In CB KO neurons, we observed clear differences between M1 and M2 (Fig. 6G; right). The total mScarlet-α3WT signal overlapping
<figure><img src="image_6.png" /><figcaption>**Figure 4.** CB-independent clustering of gephyrin and GABA<sub>A</sub>R-α3 in certain neuronal subpopulations with high endogenous GABA<sub>A</sub>R-α3 expression. A and B, representative confocal 3D images of gephyrin/GABA<sub>A</sub>R-α3 colabeling in the hilus of the DG in slices derived from 8-week-old CB WT mice (A) or their CB KO littermates (B). Note the strong reduction of gephyrin puncta in CB KO animals, as described previously (26). In contrast, the punctate immunoreactivity of GABA<sub>A</sub>R-α3 was similar between groups. In addition, note that a significant amount of the remaining gephyrin puncta in the hilus of the CB KO brains colocalize with GABA<sub>A</sub>R-α3 puncta. The scale bar represents 10 µm. Scale frames within the 3D images are composed of 5 × 5 µm squares. C–F, quantifications of gephyrin (C and D) and GABA<sub>A</sub>R-α3 (E and F) immunoreactive puncta densities (C and E) and sizes (D and F) in the hilus of 8-week-old CB WT mice (black) and their CB KO littermates (red). Each bar corresponds to mean values (±SD) obtained with sections from three individual brains per genotype. \*p < 0.05; unpaired two-tailed Student's t test. G and H, representative confocal 3D images of gephyrin/GABA<sub>A</sub>R-α3 dual labeling in the nRT in slices derived from 8-week-old CB WT mice (G) or their CB KO littermates (H). The scale bar represents 10 µm. I–L, quantifications of gephyrin (I and J) and GABA<sub>A</sub>R-α3 (K and L) immunoreactive puncta densities (I and K) and sizes (J and K) in the nRT of 8-week-old CB WT mice (black) or their CB KO littermates (red). Note that both gephyrin (I and J) and GABA<sub>A</sub>R-α3 (K and L) values are similar between groups. Each bar corresponds to mean values (±SD) obtained with sections from N = 3 individual brains per genotype. M, quantifications of the percentages of gephyrin-immunoreactive puncta colocalized with GABA<sub>A</sub>R-α3 in the hilus of DG and nRT of 8-week-old CB WT mice (black) or their CB KO littermates (red). Note that colocalization percentages are not significantly different between CB KO animals (red) and their WT littermates (black). Each bar corresponds to mean values (±SD) obtained with sections from N = 3 individual brains per genotype. CB, collybistin; DG, dentate gyrus; GABA<sub>A</sub>R-α3, α3 subunit of GABA type A receptor; nRT, reticular thalamic nuclei.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 5.** CB-independent postsynaptic clustering of GABA~A~R-α3. A and B, representative confocal 3D images of GABA~A~R-α3/VIAAT costainings through the hilus of the DG in slices derived from 8-week-old CB WT mice (A) or their CB KO littermates (B). The scale bar represents 10 µm. C–F, quantification of the densities (C and E) and the apparent sizes (D and F) of GABA~A~R-α3 (C and D) and VIAAT (E and F) puncta in the hilus of 8-week-old WT (black) and CB KO mice (red). Note that both GABA~A~R-α3 (C and D) and VIAAT (E and F) values are similar between groups. G and H, GABA~A~R-α3/VIAAT labeling in the nRT in slices from 8-week-old CB WT mice (G) and their CB KO littermates (H). The scale bar represents 10 µm. Scale frames within the 3D images are composed of 5 × 5 µm squares. I–L, quantification of GABA~A~R-α3 (I and J) and VIAAT (K and L) immunoreactive puncta densities (I and K) and sizes (J and K) in the nRT of CB WT mice (black) or their CB KO littermates (red). Note that both GABA~A~R-α3 (I and J) and VIAAT (K and L) values are similar between groups (mean ± SD, slices from N = 3 brains per genotype). M, quantification of the fraction of GABA~A~R-α3 immunoreactive puncta apposed to VIAAT in the hilus of DG and nRT of 8-week-old mice. Note that the percentage of postsynaptic GABA~A~R-α3 puncta is significantly higher in the hilus of CB KO animals (red), as compared with the WT control group (black). Means ± SD from N = 3 brains per genotype. CB, collybistin; DG, dentate gyrus; GABA~A~R-α3, α3 subunit of GABA type A receptor; nRT, reticular thalamic nuclei; VIAAT, vesicular inhibitory amino acid transporter.</figcaption></figure>

with the gephyrin signal (M1) was significantly reduced in CB KO neurons, as compared with the corresponding M1 values of CB WT cells (Fig. 6G; M1 of CB KO: 0.27 ± 0.1 versus M1 of CB WT: 0.65 ± 0.21). In contrast, the total dendritic gephyrin signal overlapping with the mScarlet-α3<sup>WT</sup> signal (M2) was significantly increased in CB KO neurons, as compared with

CB WT controls (Fig. 6G; M2 of CB KO: 0.65 ± 0.14 versus M2 of CB WT: 0.49 ± 0.2). In CB KO neurons expressing mScarlet-GABA~A~R-α3<sup>WT</sup>, densities and sizes of dendritic gephyrin puncta remained significantly reduced, as compared with WT/mScarlet-α3<sup>WT</sup> controls (Fig. 6, C–F). Nonetheless, expression of mScarlet-GABA~A~R-α3<sup>WT</sup> partially rescued the
CB-independent synapse formation

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Overexpression of mScarlet-GABA~A~R-α3 partially rescues the impaired gephyrin clustering in dissociated hippocampal neurons derived from CB KO mice. A–C, cultured hippocampal neurons from E18 CB KO mouse embryos (B and D) were transfected at DIV 4 with either mScarlet alone (B) or mScarlet-GABA~A~R-α3~WT~(D). At DIV 14, the neurons were fixed and immunostained for gephyrin and GABA~A~R-α3. Cultured hippocampal neurons from E18 CB WT littermates, transfected with either mScarlet alone (A) or mScarlet-GABA~A~R-α3~WT~(C), served as controls. Right panels show (from top to bottom) high-intensity magnifications of gephyrin immunoreactivities, mScarlet fluorescent signals, GABA~A~R-α3 immunoreactivities, and overlays of the boxed areas of A–D, respectively. Arrowheads indicate selected colocalized puncta in the three different channels. The scale bars represent 10 µm. *E* and *F*, quantifications of dendritic gephyrin-immunoreactive puncta densities (*E*) and sizes (*F*) in DIV 14 neurons from CB WT and CB KO animals, treated as indicated. In agreement with a previous study (26), gephyrin punctate staining was significantly reduced in the dendrites of CB KO neurons, as compared with controls. Note that overexpression of mScarlet-GABA~A~R-α3 partially rescues the impaired gephyrin clustering, as compared with CB KO neurons overexpressing mScarlet alone. Bars correspond to mean values (±SD) obtained from N = 10 individual neurons and n = 20 dendritic segments per condition and genotype. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001; unpaired two-tailed Student's t test. *G*, the degree of colocalization between the red (mScarlet-α3~WT~) and green (endogenous gephyrin) signals was statistically analyzed in the dendrites and expressed with the Mander's colocalization coefficients M1 and M2. M1 represents the fraction of mScarlet-α3~WT~ (red) overlapping with gephyrin (green). M2 represents the fraction of gephyrin overlapping with mScarlet-α3~WT~. All calculations for the Mander's coefficients were performed by the ImageJ software, as described in *Experimental procedures* section. Data represent means ± SD of 12 dendritic segments per condition. \* *p* < 0.05, \*\* *p* < 0.001; unpaired two-tailed Student's t test. *CB*, collybistin; DIV 4, *day in vitro* 4; E18, embryonic day 18; GABA~A~R-α3, α3 subunit of GABA type A receptor.</figcaption></figure>

synaptic gephyrin immunoreactivity, as compared with CB KO neurons expressing mScarlet alone (Fig. 6, B, D, and E; mean density: 6.1 ± 1.62 puncta/40 µm; Fig. 6, B, D, and F; mean size: 0.08 ± 0.014 µm²).

These data indicate that the expression of exogenous GABA~A~R-α3 in cultured hippocampal CB KO neurons partially rescues the impaired clustering of gephyrin observed in CB KO cultures and significantly increases the
colocalization of nascent gephyrin-immunoreactive puncta with mScarlet-α3<sup>WT</sup> containing GABA<sub>A</sub>Rs, as compared with controls.

To determine the physiological consequences of the mScarlet-α3WT overexpression in CB KO neurons, we recorded GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in dissociated DIV 14-16 hippocampal neurons derived from CB KO embryos or their WT littermates, which were transfected at DIV 4 with either mScarlet alone or with mScarlet-α3WT. Interestingly, mean mIPSC amplitudes, frequencies, and rise times were not significantly different between CB WT and CB KO control neurons expressing mScarlet alone (Fig. 7, A–D; amplitudes: CB WT, 38.7 ± 14.04 pA versus CB KO, 32.08 ± 14.01 pA; frequencies: CB WT, 1.67 ± 1.26 Hz versus CB KO, 2.15 ± 1.43 Hz; rise times: CB WT, 0.67 ± 0.12 ms versus CB KO, 0.73 ± 0.13 ms). As

previous recordings of GABAergic mIPSCs from CA1 pyramidal neurons in slices derived from CB KO mice and their WT littermates showed significantly reduced mean mIPSC amplitudes, frequencies, and rise times in CB KOs compared with controls (26), these results reveal qualitative differences in the composition of synaptic GABA<sub>A</sub>Rs in the dendrites of cultured hippocampal neurons, as compared with the *in vivo* situation. Accordingly, it was shown previously that the postnatal expression of GABA<sub>A</sub>R subunit mRNAs in the mammalian brain, including the hippocampus, exhibits a unique temporal and regional developmental profile *in vivo*, which may be altered in hippocampal cultures *in vitro* (66). In line with this, a previous study showed that in cultures of hippocampal gephyrin KO neurons, GABAergic transmission and expression of the gephyrin-independent α1 containing GABA<sub>A</sub>Rs were unchanged, as compared with WT controls

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Functional analysis of mScarlet-GABA<sub>A</sub>R-α3 expressing cultured hippocampal neurons. A, representative mIPSC traces recorded at a holding potential of -70 mV in the presence of 300 nM TTX from CB WT or CB KO cultured hippocampal neurons expressing either mScarlet alone or mScarlet-α3WT, as indicated. B and C, mean mIPSC amplitudes (B) and frequencies (C) in CB WT or CB KO neurons expressing mScarlet alone or mScarlet-α3WT, as indicated. Note that overexpression of mScarlet-α3WT led to reduced mIPSC amplitudes and frequencies in both, WT and KO cells, compared with cells expressing mScarlet alone. Furthermore, note increased ratios of change in both mean amplitudes and frequencies of CB KO neurons, as compared with WT cells. D, representative mIPSC events (top), and mean values of times to peak (20–80% rise time) of mIPSC events, as indicated. E, representative events (top) and mean currents induced by exogenous application of 100 µM glutamate in cultured hippocampal neurons, as indicated. F, representative events (top) and mean currents induced by exogenous application of 10 µM GABA in cultured hippocampal neurons, as indicated. G, mean ratios of steady state/peak currents from GABA-induced responses recorded in CB WT or CB KO cultured hippocampal neurons expressing either mScarlet alone or mScarlet-α3WT, as indicated. H, representative events (top) and mean values of time to peak (20–80% rise time) of GABA-induced responses in cultured hippocampal neurons, as indicated. Data represent means ± SD of 14 to 27 cells per condition. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001; unpaired two-tailed Student's t test. Error estimates for ratios given in (B) and (C) were obtained by bootstrap analysis using 10,000 bootstrap resamples. CB, collybistin; GABA, γ-aminobutyric acid; GABA<sub>A</sub>R-α3, α3 subunit of GABA type A receptor; mIPSC, miniature inhibitory postsynaptic current; TTX, tetrodotoxin.</figcaption></figure>
CB-independent synapse formation

(22). Thus, the high expression of α1-containing receptors in cultured neurons may mask the differences in dendritic inhibition between CB KOs and WT controls in our experiments.

Overexpression of mScarlet-α3WT led to a reduction in the size of mIPSC events and slowed down their kinetics (see exemplary traces in Fig. 7A). In WT hippocampal neurons, mean mIPSC amplitudes (24.12 ± 12.19 pA) were significantly reduced compared with WT cells expressing mScarlet alone (Fig. 7B). Furthermore, mScarlet-α3WT-expressing neurons showed delayed events. In order to ensure that only real mIPSCs were recorded, events with a time-to-peak duration of more than 3 ms were excluded from our analysis. This type of analysis reported a significant reduction in the mean mIPSC frequencies of CB WT cells expressing mScarlet-α3WT (0.59 ± 0.67 Hz) as compared with CB WT cells expressing mScarlet alone (Fig. 7, A–C).

Similarly, CB KO neurons expressing mScarlet-α3WT showed significantly reduced mean mIPSC frequencies (1.33 ± 0.85 Hz). mIPSC amplitudes were also reduced (25.13 ± 13.84 pA), but this change did not reach statistical significance, as compared with CB KO cells expressing mScarlet alone (Fig. 7, A–C). In addition, mScarlet-α3WT-expressing neurons showed significantly increased mean rise times (CB WT, 0.82 ± 0.23 ms; CB KO, 0.83 ± 0.14 ms), as compared with neurons expressing mScarlet alone (Fig. 7D). Interestingly, a comparison of the ratios of change (mScarlet-α3WT/mScarlet) for both mean mIPSC amplitudes and mIPSC frequencies between CB WT and KO neurons yielded values closer to 1 in CB KO neurons, as compared with WT controls (Fig. 7B; ratios of change in mean amplitudes: CB WT, 0.62 ± 0.08 versus CB KO, 0.78 ± 0.13; Fig. 7C; ratios of change in mean frequencies: CB WT, 0.35 ± 0.1 versus CB KO, 0.62 ± 0.13). However, the indicated tendencies of higher ratios of change in CB KO cells for both, mIPSC amplitudes and mIPSC frequencies, were not statistically significant, compared with CB WT cells ($p > 0.05$ for both sets of data). Nevertheless, as synaptic localization of α3 subunit-containing GABAARs depends on the presence of a gephyrin scaffold at inhibitory postsynaptic sites (21), these tendencies of higher ratios of change in CB KO cells (Fig. 7, B and C) indicate that the relative amount of clustered gephyrin, capable of binding mScarlet-α3WT at synapses, was partially increased in CB KO neurons expressing mScarlet-α3WT, as compared with CB KO neurons expressing mScarlet alone.

In order to ensure that the CB WT and KO neurons expressing mScarlet-α3WT were healthy, as compared with neurons expressing mScarlet alone, we exogenously applied glutamic acid (100 µM) or GABA (10 µM) to activate all surface (synaptic + extrasynaptic) glutamate receptors and GABAARs (Fig. 7, E and F). No significant changes in the mean amplitudes of glutamate-induced (Fig. 7E) and GABA-induced (Fig. 7F) currents were apparent between the two genotypes and different groups (Fig. 7, E and F). Furthermore, the kinetics of glutamate-induced currents was similar between the two genotypes and experimental conditions (see representative traces in Fig. 7E). In contrast, the desensitization of GABA-induced currents was faster in cells of both genotypes

expressing mScarlet, as compared with the cells expressing mScarlet-α3WT (see representative traces in Fig. 7F). This indicates that the incorporation of mScarlet-α3WT into functional pentameric GABAARs at synapses changed the kinetics of spontaneous desensitization of open channels. These results are in line with previous studies showing that the substitution of α3 for α1 in heteropentameric GABAARs reduces the apparent activating-site affinity and slows activation, desensitization, and deactivation of macroscopic currents (56, 67). Together, the unaltered kinetics of glutamate-induced currents and the mScarlet-α3WT-dependent changes in the kinetics of GABA-induced currents indicate that the reduced mean mIPSC amplitudes and frequencies, as well as the increased rise times observed in CB WT and CB KO neurons expressing mScarlet-α3WT (Fig. 7, A–D), are due to the replacement of gephyrin-dependent α3 subunit for gephyrin-independent α1-containing GABAARs at inhibitory postsynaptic sites. In line with this, the ratios of desensitization (steady state/peak; Fig. 7G) and the rise times (Fig. 7H) of GABA-induced currents were significantly increased in CB WT and CB KO neurons expressing mScarlet-α3WT, as compared with neurons expressing mScarlet alone (Fig. 7, G and H). Together, the electrophysiological analyses of mIPSCs and GABA-induced currents indicated a significant incorporation of mScarlet-α3WT-containing GABAARs at synapses of both CB KO and CB WT neurons. Furthermore, the data shown in Figures 6 and 7 indicate that an increase in gephyrin clustering at synapses does not unconditionally correlate with an increase in GABAAR kinetics that are strongly dependent on their subunit composition. As α3 subunit-containing receptors depend on gephyrin for their postsynaptic clustering (21), however, our results indirectly indicate that overexpression of mScarlet-α3WT is associated with an increase of gephyrin scaffolds at postsynaptic sites in the dendrites of CB KO neurons. Furthermore, the tendencies of higher ratios of change (mScarlet-α3WT/mScarlet) of the mean mIPSC amplitudes and frequencies in CB KO, compared with WT neurons, confirm our immunocytochemical data (Fig. 6) that the expression of α3 subunits in cultured hippocampal neurons leads to a partial rescue of the gephyrin clustering.

Discussion

In the present study, we identified an important role of the α3 subunit of GABAARs in priming CB-independent and gephyrin-mediated formation of GABAergic synapses in specific regions of the mammalian forebrain. Evidence for a GABAAR-α3-dependent formation of certain inhibitory postsynapses in the absence of CB is provided by the following findings: (i) a homooligomeric GABAAR-α3A343W mutant, which is trafficked to the cell surface, induces submembranous gephyrin clusters independently of CB in COS-7 cells. (ii) Gephyrin clustering is unaltered in those neuronal subpopulations of CB-deficient brains that endogenously express the GABAAR-α3. (iii) Exogenous expression of GABAAR-α3 partially rescues impaired gephyrin clustering in CB-deficient hippocampal neurons.
## Gephyrin clustering by GABA~A~R-α3

Homooligomeric mutant GABA~A~R-α3~A~343W induces sub-membranous gephyrin clusters independently of CB in COS-7 cells. In all previous studies (7, 10, 25, 60, 61), the presence of CB was obligatory for the redistribution of gephyrin into such microclusters. In contrast, our findings clearly show that a mutation enabling cell surface expression of the GABA~A~R-α3 (59) induces formation of gephyrin microclusters in the absence of CB, and that these microclusters partially colocalized with GABA~A~R-α3 at the plasma membrane. Gephyrin binds directly to several GABA~A~R-α subunits, albeit with lower affinities than to the GlyR-β subunit (50–55). More recently, the α2 subunit of GABA~ARs~ was shown to directly bind to both, gephyrin (with low affinity) and CB (with high affinity) (55), indicating that the α2 subunit is an essential determinant of CB activation and membrane anchoring at certain post-synapses (55, 60). In contrast, the GABA~A~R-α3 does not bind to CB but displays the highest *in vitro* gephyrin affinity of all α subunits with an estimated K~D~ of 5.3 µM for the full-length intracellular loop (53, 55).

In agreement with these studies, we consistently found that the α2 and α3 subunits, but not the α1 subunit, of GABA~ARs~ colocalized with intracellular GFP-gephyrin aggregates (>1 µm²) in COS-7 cells. Interestingly, both the β subunit of GlyRs and the α3 subunit of GABA~ARs~ strongly interact with gephyrin by occupying overlapping sites in the gephyrin E domain (54). Furthermore, a previous study indicated that glycinergic neurotransmission and clustering of GlyRs is unaffected in the spinal cord and brainstem of CB KO mice (26). Accordingly, the strong interaction of gephyrin with GlyRs and α3 subunit-containing GABA~ARs~ indicates that equivalent CB-independent mechanisms may modulate gephyrin-mediated clustering of GlyRs and α3-containing GABA~ARs~ at inhibitory synapses. In line with this, a recent study showed that gephyrin with its dimerizing E domain binds to the cytoplasmic loops of the GlyR-β subunit or the GABA~A~R-α3 subunit, and that the multivalent nature of GABA~ARs~ or GlyRs can polymerize gephyrin into a sheet-like assembly *via* phase separation (68). More importantly, this phase separation (and likely clustering at inhibitory synapses) of the gephyrin complexes with GlyRs or α3-containing GABA~ARs~ is regulated *via* phosphorylation of the gephyrin C domain (linker domain) or *via* binding of proteins, such as the dynein light chain, to the C domain of gephyrin (68).

## CB-independent gephyrin clustering in GABA~A~R-α3 expressing neurons

In agreement with previous data showing a high-affinity interaction between GABA~A~R-α2 and CB (55), the present study indicates that CB ablation causes significant defects in the clustering and colocalization of gephyrin and α2 subunit-containing GABA~A~R isoforms. However, areas or neuronal subpopulations with prominent expression of GABA~A~R-α3s, for example, the nRT, do not exhibit altered clustering and colocalization of gephyrin and GABA~A~R-α3. Given that GABA~A~R-α2 binds strongly to CB, but only with low affinity to

gephyrin (55), a type of coincidence detection at GABAergic postsynapses as that previously proposed (69), where coinciding signals are amplified by a cooperative action of two different ligands at two different binding sites, might be significantly impaired in GABA~A~R-α2 expressing neurons of CB KO mice. In contrast, GABA~A~R-α3 binds strongly to gephyrin but not to CB (55). Thus, in the absence of CB, the interaction of GABA~A~R-α3 with additional, currently unknown, regulatory proteins in certain subpopulations of GABAergic postsynapses appears to be sufficient to retain postsynaptic clustering of gephyrin.

Different GABA~A~R subtypes display differential regional distributions (62). The most prevalent subunit combination is the triplet α1/β2,3/γ2, detected in numerous cell types throughout the brain, whereas other triplets such as the α2/β2,3/γ2 and α3/β2,3/γ2 were identified only in discrete cell populations (62). In the hilus of the DG, the α2 subunit of GABA~ARs~ dominates at inhibitory postsynaptic sites. Here, CB deficiency leads to a strong reduction of postsynaptic gephyrin and α2 cluster densities and sizes in these areas. The majority of the remaining gephyrin clusters in the hilus of CB KO animals colocalize with GABA~ARs~ in neurons expressing the α3 subunit. The sparse distribution of these GABA~A~R-α3 expressing neurons, with their dendritic trees restricted next to the border of the granule cell layer, indicates that they are highly differentiated groups of interneurons (70, 71). However, additional work will be required to specify the morphological and electrophysiological identity of the hilar interneurons that are insensitive to CB deficiency with regard to gephyrin and GABA~A~R-α3 postsynaptic clustering.

In nRT neurons, which selectively express the α3 subunit, none of the other five α subunits were detected in brain slices derived from α3-KO mice (15). Moreover, clustering of gephyrin was disrupted in these cells (15), providing additional evidence that in the neuronal subpopulation of the nRT, the GABA~A~R-α3, but not CB, constitutes one of the major determinants for gephyrin postsynaptic clustering. In the CA1 pyramidal neurons, which express both α1 and α2, but not the α3 subunit of GABA~ARs~, α2 KO mice show significantly reduced gephyrin postsynaptic clustering but preserved peri-somatic inhibition, which was proposed to be mediated by gephyrin-independent α1-containing GABA~ARs~ (17, 21, 72). In the current study, our immunolabeling analyses on slices derived from CB KO animals revealed qualitative differences in the clustering of α2-containing GABA~ARs~ in the CA1 SR that were not detected in previous studies (26), mainly because of the lack of high-resolution microscopy techniques and processing software. Whereas gephyrin immunoreactivity was strikingly reduced in the CA1 SR, our statistical analyses of the GABA~A~R-α2 immunoreactive puncta in 3D confocal images processed with the Imaris software (Bitplane) indicated significant differences in the apparent cluster sizes, but not in the densities of the α2 subunit in the CA1 SR of CB KO mice, as compared with controls. In contrast, in DG hilar neurons of CB KO mice, the strong reduction in gephyrin clustering was accompanied by an equivalent reduction in the clustering of GABA~A~R-α2. Together, these findings indicate that in
## CB-independent synapse formation

particular hippocampal areas, such as the CA1 SR, the densities of GABA~A~R-α2 remain unaltered in the absence of both postsynaptic gephyrin and CB. The reason for the unchanged densities of α2 in CA1 pyramidal cells of CB KO mice is currently unknown but may reflect the activation of compensatory mechanisms and the involvement of scaffolding proteins other than gephyrin, such as dystrophin and its associated glycoprotein complex (17, 73, 74), in the clustering and stabilization of GABA~ARs at certain GABAergic postsynapses.

### GABA~A~R-α3 compensates the effects of CB loss on gephyrin clustering

In cultured hippocampal neurons prepared from embryonic day 18 (E18) mouse embryos, pyramidal neurons account for the vast majority of the total neuronal population, because in late-stage embryos, the generation of pyramidal cells is essentially complete, but the generation of dentate granule cells has not yet begun (75). Our study demonstrates CB-independent clustering of gephyrin in neuronal subpopulations that endogenously express GABA~A~R-α3. In contrast, in regions such as the CA1 SR, where α1 and α2, but not α3, are expressed, gephyrin-immunoreactive puncta are strikingly reduced in CB KO animals. This prompted us to study the effects of recombinantly expressing the GABA~A~R-α3 subunit in cultured pyramidal cells derived from the brains of CB KO embryos, as compared with WT controls.

Our functional analyses of control CB WT and CB KO neurons expressing mScarlet alone indicated no significant differences in mean mIPSC amplitudes, frequencies, and rise times between genotypes. This result is in line with a previous study showing that in cultured hippocampal neurons from gephyrin KO mice, GABAergic neurotransmission is unaffected, probably because of the high expression of GABA~ARs containing the gephyrin-independent α1 subunit (22). Overexpression of mScarlet-GABA~A~R-α3~WT in both CB WT and CB KO neurons led to a significant reduction in the size of the mIPSC events and slowed down their kinetics. This is probably because of the replacement of α1/β2,3/γ2 for mScarlet-α3/β2,3/γ2 receptors at inhibitory postsynaptic sites, revealing a dependence of the GABA~A~R gating kinetics on the α subunit isoform, as previously shown (67). As α3-containing GABA~ARs depend on gephyrin for their synaptic localization (21), the mScarlet-α3-induced changes in the GABA~A~R kinetics of CB KO neurons predicted the presence of a gephyrin scaffold at inhibitory postsynaptic sites.

Indeed, overexpression of mScarlet-GABA~A~R-α3 in CB KO neurons partially rescued the defects in dendritic gephyrin clustering. Both densities and sizes of gephyrin clusters were significantly increased in the dendrites of CB KO neurons expressing mScarlet-α3~WT, compared with CB KO neurons expressing mScarlet alone. Moreover, the Mander's coefficients indicated that the increased fraction of gephyrin-immunoreactive puncta in the dendrites of CB KO neurons overlapped with the mScarlet-α3~WT signal. As mentioned previously, this result is in line with a recent study showing

that the binding of GABA~A~R-α3 to the E domain of gephyrin induces clustering of gephyrin-GABA~A~R complexes at inhibitory synapses (68). However, densities and sizes of gephyrin clusters in CB KO cells expressing mScarlet-α3~WT remained significantly reduced, compared with CB WT neurons expressing mScarlet-α3~WT, meaning that the rescue of gephyrin clustering in CB KO pyramidal neurons was partial. Accordingly, the ratios of change (mScarlet-α3~WT/mScarlet) of both mean mIPSC amplitudes and mIPSC frequencies were less pronounced in CB KO neurons than in WT controls, suggesting that the fraction of clustered gephyrin was in fact partially increased upon overexpression of mScarlet-α3~WT.

In view of the previously postulated function of the α subunits of GABA~ARs as prime mediators of specific targeting (76), different GABA~AR-α subunits may bind directly or indirectly to different regulatory proteins. In the absence of CB, the interaction of GABA~A~R-α3 with gephyrin in hippocampal pyramidal neurons may require the involvement of other, currently unknown, regulatory proteins. The predominant expression of the α3 subunit of GABA~ARs in GABAergic interneurons, as opposed to glutamatergic neurons (62, 77), indicates a more important role of α3-containing GABA~ARs in localizing gephyrin at postsynaptic sites of interneuronal synapses. Thus, in glutamatergic pyramidal cells, the molecular machinery required for GABA~A~R-α3-mediated stabilization of gephyrin scaffolds at postsynaptic sites may be incomplete. Nevertheless, the partial rescue of the gephyrin clustering observed in cultured CB KO pyramidal neurons upon GABA~A~R-α3 overexpression indicates that manipulating expression of the α3 subunit in neuronal circuits affected in the absence of CB may be a potential target for developing novel therapeutic tools.

Loss of CB in mice results in increased levels of anxiety and impaired spatial learning (26). Accordingly, several mutations of the CB gene (ARHGEF9; Online Mendelian Inheritance in Man number: 300429) in patients have been implicated in epilepsy, X-linked intellectual disability, aggressive behavior, anxiety and, in one case, with hyperexplexia (29–31, 34, 35, 42, 78). Intriguingly, the anxiolytic activity of diazepam (79) is mediated primarily by GABA~ARs α2/β/γ2 (80) and under conditions of high receptor occupancy, also by GABA~ARs containing the α3 subunit (81, 82). One promising strategy in mouse models that has recently gained substantial attention is the use of viral vectors, particularly adeno-associated viral vectors, to introduce therapeutic tools in a circuit-specific manner (83). Thus, in cases in which directly targeting the mutated CB gene is unsuccessful, adeno-associated viral-mediated GABA~A~R-α3 expression in specific circuits affecting anxiety levels may be a promising new therapeutic strategy.

## Experimental procedures

### Animals

The generation and characterization of CB KO mice has been previously described (26). All mice used in the current study were housed in the Max Planck Institute of Experimental Medicine Animal Care Facility in Göttingen, and all
CB-independent synapse formation

procedures were approved by the Institutional Animal Care
and Use Committee.

cDNA constructs

The GFP-gephyrin plasmid has been described previously (84). The pcDNA3.1-gabra1 and pcDNA3.1-gabra2 constructs were kindly provided by Mr Yoshimitsu Sasa, Director of the Wako Administrative Division of the RIKEN Institute. The mScarlet_C1 vector and a FUGW-mScarlet-Gabra3, in which the GABAAR-α3 subunit was tagged with mScarlet after the signal peptide was generated by C. G. S. This construct contained an undesired point mutation (I342M). Upon cloning of the mScarlet-Gabra3 complementary DNA (cDNA) into the XbaI/EcoRI sites of the pKH3-vector (Addgene), correction of the undesired point mutation (M342 → I) in the pKH3-mScarlet-Gabra3 plasmid, as well as the introduction of the A317W, A318W, or A343W point mutations in the pcDNA3.1-gabra1, pcDNA3.1-gabra2, or the pKH3-mScarlet-Gabra3 plasmid, respectively, were performed by site-directed mutagenesis using the QuikChange protocol (Stratagene). Sequencing of the full open reading frames was performed in each case to validate constructs.

Antibodies

The following primary antibodies were used for immuno-
histochemistry and immunocytochemistry: monoclonal mouse
antigephyrin (mAb7a; Connex; 1:2000); polyclonal rabbit anti-
GABAAR-α1 (AB5592; Chemicon; 1:1000), polyclonal rabbit
anti-GABAAR-α2 (224103; Synaptic Systems; 1:1000), poly-
clonal rabbit anti-GABAAR-α3 (224303; Synaptic Systems;
1:1000), and polyclonal guinea pig anti-VIAAT (131004; Syn-
aptic Systems; 1:1000). The following secondary antibodies
were used for immunohistochemistry and immunocytochem-
istry: Alexa Fluor 488, 555, or 633, goat antimouse, goat anti-
rabbit or goat antiguinea pig (Invitrogen; 1:2000). For nuclear
stainings of brain slices, 4',6-diamidino-2-phenylindole dihy-
drochloride (D1306; ThermoFisher Scientific) was used.

Transfections and immunocytochemistry of COS-7 cells

COS-7 cells (CRL-1651) were purchased from American
Type Culture Collection (LGC Standards GmbH). The cells
were tested independently and certified to be free of myco-
plasma. Transfections and immunocytochemistry were per-
formed as described previously (36, 61). The transfection
parameters were optimized in order to achieve low and com-
parable expression levels of all recombinant proteins analyzed.
COS-7 cells were plated in 24-well plates on 12-mm coverslips,
and 200 ng of each cDNA were used per well. The empty pcDNA
3.1 vector was used to equalize the total amount of DNA per
transfection to 400 ng. Cells were transfected with Lipofect-
amine 2000 (Invitrogen) following the manufacturer's protocol,
fixed 16 h after transfection, and stained, as described previously
(25). Images were collected either with an Axio-Imager Z1
equipped with a Zeiss apochromat 63× objective and an Apo-
tome module (Zeiss) or an inverse Leica DMIRE2 microscope
equipped with a 63× oil-immersion objective and connected to a

Leica TCS SP2 AOBS confocal laser scanning setup (Leica
Microsystems). ImageJ (https://imagej.nih.gov/ij/) was used to
analyze immunolabeling from images processed with stan-
dardized intensity thresholding. Briefly, the images were opened
with ImageJ and, upon splitting the channels, the same intensity
thresholds on the different GFP-gephyrin and GABAAR
channels were constantly applied. Subsequently, the images
were converted to a mask, single cells were selected using the
"freehand selection" tool, and particle numbers and sizes be-
tween 0.05 and 20 µm² were automatically analyzed by using the
"Analyze Particles" plugin of the ImageJ software. Colocaliza-
tion analysis was performed using the "Coloc2" plugin
embedded in the ImageJ software. Analysis was performed on
preselected whole cells. Each image was split into its respective
red, green, and blue channels, and thresholding and mask con-
version were performed as described previously. Mander's co-
efficients, M1 (representing the fraction of red signal
[GABAARs] overlapping with green signal [GFP-gephyrin]) and
M2 (representing the fraction of green signal overlapping with
red signal) were calculated to determine the degree of overlap
between the corresponding channels. Fluorescence intensity
scans were obtained by drawing a straight line across GFP-
gephyrin aggregates and microclusters of a cell in the green
channel and measuring the intensity profile along the selected
line in the green and red channel (GABAARs) using the "Plot
Profile" plugin embedded in ImageJ.

Transfections and immunocytochemistry of cultured
hippocampal neurons

Cultures of hippocampal neurons were prepared from E18
CB KO mice and their WT littermates, as previously described
(85). Neurons were transfected at DIV 4 using the CalPhos
mammalian transfection kit (Clontech). Immunocytochem-
istry was performed as previously described (36, 61, 86).
Multichannel image stacks of fluorescently stained neurons
were acquired on an inverse Leica DMIRE2 microscope con-
nected to a Leica TCS SP2 AOBS confocal laser scanning setup
(Leica Microsystems) with constant settings, with a 63× oil-
immersion objective and a zoom factor of 2. The image
stacks were processed identically using the ImageJ software
package (https://imagej.nih.gov/ij/). Briefly, "lei" files of the
stacks were imported as Bio-Formats. Stacks of the different
neurons were opened individually, autoscaled, and viewed with
"Hyperstack." The maximal intensity single channels of each
image were obtained by using the "Z-Project" plugin
embedded in the ImageJ software. Upon standardized
thresholding and mask conversion, 40 µm dendritic segments
were selected, and particles ≥0.05 µm² as well as particle sizes
were automatically analyzed by using the "Analyze Particles"
plugin of the ImageJ software. Mander's coefficients, M1
(representing the fraction of red signal [mScarlet-α3WT]
overlapping with green signal [endogenous gephyrin]) and M2
(representing the fraction of green signal overlapping with red
signal), were calculated to determine the degree of overlap
between the corresponding regions of detected signals at
default settings. Region(s) of interest were defined around
dendritic segments in the red channels (mScarlet-α3WT).
# CB-independent synapse formation

## Electrophysiology on cultured hippocampal neurons

Cultures of hippocampal neurons were prepared from E18 CB KO mice and their WT littermates, as previously described (85). Neurons were transfected at DIV 4 using the CalPhos mammalian transfection kit (Clontech) and grown for 14 to 16 DIV before electrophysiological analyses. Neurons were whole cell voltage clamped at -70 mV using a Multiclamp 700 B (Axon Instruments, Molecular Devices)/EPSC10 (HEKA electronics) amplifiers under the control of the pClamp software (Axon Instruments, Molecular Devices)/Patchmaster2 (HEKA electronics). Pipette resistances ranged between 3 and 4 MΩ, and only neurons with series resistances ≤12 MΩ were used for the analysis. Recordings of mIPSC were performed in the presence of 300 nM tetrodotoxin (Tocris Bioscience) and 10 µM 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (Tocris Bioscience) at holding potential -70 mV (87).

Cultured neurons were constantly supplied with fresh extracellular bathing solution (140 mM NaCl, 4 mM CaCl₂, 4 mM KCl, 10 mM Hepes, 24 mM MgCl₂, 10 mM glucose, adjusted to pH 7.3, and ~310 msOsmol/l) via a perfusion system. Neurotransmitter agonists (100 µM glutamate or 10 µM GABA) were applied using a fast flow application system. The intracellular solution contained 136 mM KCl, 17.8 mM Hepes, 15 mM phosphocreatine, 1 mM EGTA, 0.6 mM MgCl₂, 0.3 mM Na-GTP, 4 mM Mg-ATP, and 5 U/ml creatine phosphokinase, adjusted to pH 7.4 (~320 mOsmol/l). All experiments were carried out at room temperature.

All electrophysiology data were analyzed using Axograph (version 1.5.4) software (Axograph Scientific).

## Immunohistochemistry

Immunolabeling of brain slices of CB KO mice and WT littermates was performed as previously described (64). Eight-week-old mice were deeply anesthetized and decapitated. The brains were immediately removed, cut sagitally in two equal halves, and frozen on dry ice. Sagittal cryostat sections (14 µm) were fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 10 min at 4 °C, washed twice for 2 min in PBS and once with sodium citrate buffer (10 mM sodium citrate, 0.05% [v/v] Tween-20, pH 8.0). Subsequently, the sections were immersed in a preheated staining dish containing sodium citrate buffer and incubated for 30 min at 95 °C. After allowing the slides to cool down at room temperature for 20 min, sections were rinsed twice for 2 min in PBS, permeabilized with 0.3% (w/v) Triton X-100, 4% (v/v) goat serum in PBS, blocked for 3 h with 10% (v/v) goat serum in PBS, and incubated overnight at 4 °C with primary antibodies, and for 1 h with secondary antibodies at appropriate dilutions in PBS/10% (v/v) goat serum. Finally, the sections were incubated for 5 min in 2 nM 4',6-diamidino-2-phenylindole in PBS, washed four times for 2 min in PBS, and mounted with Aqua Poly/Mount (Polysciences). Multi-channel image stacks of fluorescently stained brain slices were acquired on an inverse Leica DMIRE2 microscope connected to a Leica TCS SP2 AOBS confocal laser scanning setup (Leica Microsystems) with constant settings, with a 63× oil-

immersion objective and a zoom factor of 4. The image stacks were analyzed with the Imaris 9.6 software package. The fluorescently tagged protein puncta were modeled in 3D with ellipsoidal objects (x and y = 350 nm; z = 500 nm), whose creation parameters were kept constant during analysis of WT and KO slices. Briefly, parameters such as initial seed-point dimensions (x and y = 100 nm), quality (270 arbitrary units), and region threshold (20 arbitrary units) were kept constant. Quality of the spots (signal) describes the intensity of the channel Gaussian filtered by three-fourth of spot radius. Statistical parameters of the resulting spots (puncta) were extracted, including total spot number, diameter (minor axis length), spot volume, and fluorescence intensity. The spot density (spots per 100 µm³) was calculated on the basis of the total number of spots and the total volume of the image. Spots originating from different fluorescent stains were analyzed by the colocalized spots feature of Imaris (distance threshold = 1). The colocalizing spots were normalized to the total spot number to derive the percent of colocalizing spots.

## Statistics

Experimental data were evaluated by investigators blind to experimental conditions. Statistical significance was tested by using an unpaired two-tailed Student's t test. The required sample sizes (n) were estimated based on previous experiences with similar experiments (26, 36, 64). Values are represented as means ± SD. Asterisks indicate significant differences (*p* < 0.05; **p** < 0.01; and ***p** < 0.001); ns indicates no significant difference. The statistics were evaluated using the GraphPad Prism software (GraphPad Software). Error estimates for ratios given in Figure 7, B and C were obtained by bootstrap analysis using 10,000 bootstrap resamples. Statistical significance of the shift in ratios observed between CB WT and CB KO cultures was assessed by permutation tests using 10,000 random permutations.

## Data availability

All data are contained within the article.

**Acknowledgments**—We thank Mr Yoshimitsu Sasa, Director of the Wako Administrative Division of the RIKEN Institute, for providing plasmids. We thank Dr Miso Mitkovski (Light Microscopy Facility, MPI of Experimental Medicine, Göttingen) for support and advice regarding the Imaris software. We thank Dr Christoph van Riesen (Universitätsmedizin Göttingen) and Dr Holger Taschenberger (MPI of Experimental Medicine, Göttingen) for assistance on statistics.

**Author contributions**—S. W. and T. P. performed the experiments with the exception of the electrophysiological recordings, which were designed and performed by C. K. L., L. R., and J. S. R. Correspondingly, S. W., T. P., C. K. L., L. R., and J. S. R. analyzed data. C. G. S. provided constructs and critically reviewed the article. T. P. wrote the article. N. B. and J. S. R. critically reviewed the article. All authors critically reviewed the results and approved the final version of the article.
**Funding and additional information**—This work was supported by the Deutsche Forschungsgemeinschaft (grant PA 2087/1-4 to T. P.).

**Conflict of interest**—The authors declare that they have no conflicts of interest with the contents of this article.

**Abbreviations**—The abbreviations used are: CA1, cornu ammonis 1; CB, collybistin; cDNA, complementary DNA; DG, dentate gyrus; DIV 4, day in vitro 4; E18, embryonic day 18; GABA, γ-aminobutyric acid; GABAergic, γ-aminobutyric acidergic; GABAAR, GABA type A receptor; GABAAR-α3, α3 subunit of GABAAR; GlyR, glycine receptor; mIPSC, miniature inhibitory postsynaptic current; NL, neuroligin; nRT, reticular thalamic nuclei; SR, stratum radiatum; TMD, transmembrane domain; VIAAT, vesicular inhibitory amino acid transporter.

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