SUMMARY Brain function is shaped by postnatal experience and vulnerable to disruption of Methyl-CpG-binding protein, Mecp2, in multiple neurodevelopmental disorders. How Mecp2 contributes to the experience-dependent refinement of specific cortical circuits and their impairment remains unknown. We analyzed vision in gene-targeted mice and observed an initial normal development in the absence of Mecp2. Visual acuity then rapidly regressed after postnatal day P35–40 and cortical circuits largely fell silent by P55-60. Enhanced inhibitory gating and an excess of parvalbumin-positive, perisomatic input preceded the loss of vision. Both cortical function and inhibitory hyperconnectivity were strikingly rescued independent of Mecp2 by early sensory deprivation or genetic deletion of the excitatory NMDA receptor subunit, NR2A. Thus, vision is a sensitive biomarker of progressive cortical dysfunction and may guide novel, circuit-based therapies for Mecp2 deficiency.

Neuroligins (NL1-NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimuluscoding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.synaptogenesis | inhibitory transmission | visual processing I n rodents, postsynaptic adhesion proteins of the neuroligin family (NL1-NL4) are expressed throughout the central nervous system (CNS) (1-3) and essential for synapse organization and function (2-5). In vivo, each NL isoform localizes to specific synapse subpopulations, with NL1, NL2, and NL3 predominantly associating with glutamatergic, GABAergic, or both types of postsynapses, respectively (1, 6-9).Thus far, the distribution and function of the fourth NL isoform has remained unclear, despite the wide interest triggered by the causal link of specific loss-of-function mutations in NL4 to cases of autism, which led to the notion that aberrant synaptic transmission may cause autism spectrum disorders (ASDs) (10).We examined the distribution of NL4 in the mouse retina, a well-characterized region of the CNS with distinct, topographically organized glutamatergic, GABAergic, and glycinergic synapses, which has recently allowed us to characterize crucial aspects of NL2 distribution and function (8). Additionally, we assessed NL4 function by studying synaptic activity and visual processing in the NL4-deficient (NL4-KO; ref.3) mouse retina. Finally, we studied NL4 localization in the rest of the CNS and identified some of its key binding partners at the synapse. Results NL4 Is Localized to Glycinergic Postsynapses in the Retina.We characterized the distribution of NL4 by immunohistochemistry by using an isoform-specific antibody (3) (Fig. 1). A punctate labeling was detected in the inner plexiform layer (IPL) of wildtype (WT) but not NL4-KO retinae (Fig. 1A). NL4-positive puncta were abundant in the outer IPL but sparse in the rest of the IPL (Fig. 1A), which is reminiscent of glycine receptor (GlyR) distribution in the retina (11,12). Indeed, upon co...

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Gephyrin and collybistin are key components of GABA A receptor (GABA A R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA A R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA A R ␣2 and ␣3 subunit intracellular M3-M4 domain (but not ␣1, ␣4, ␣5, ␣6, ␤1-3, or ␥1-3) with gephyrin. Curiously, GABA A R ␣2, but not ␣3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA A R ␣2 also overlap at the start of the gephyrin E domain. This suggests that although GABA A R ␣3 interacts with gephyrin, GABA A R ␣2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA A R ␣2 and collybistin or GABA A R ␣2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA A R ␣2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA A R ␣2 is capable of "activating " collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA A R ␣2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA A R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with ␣2 subunit containing GABA A Rs, but not GlyRs or other GABA A R subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA A Rs containing the ␣2 subunit.The clustering proteins gephyrin (1) and collybistin (2, 3) are thought to represent key players in the synaptic clustering of both glycine receptors (GlyRs) 4 and GABA A Rs. Studies using gephyrin knock-out mice or mRNA knockdown (4 -7) have shown a loss of postsynaptic clustering of GlyRs and GABA A Rs containing ␣2 and ␥2 subunits. However, certain GABA A R subtypes still appear to cluster in neurons lacking gephyrin (6 -9). Hence, although the majority of GlyRs are likely to be clustered by gephyrin (10), exactly which GABA A R subtypes are subject to gephyrin-dependent clustering remains unclear. What is certain is that the subcellular localization of gephyrin is dependent on certain GABA A R subtypes. For example, targeted deletion of the GABA A R ␣1, ␣3, and ␥2 subunit genes results in a loss of synaptic gephyrin and GABA A R clusters (11-15), with cytoplasmic gephyrin aggregates indicating disrupted synaptic targeting. Although the interaction of the GlyR ␤ subunit with the gephyrin E domain has been well characterized (3,16,17), the same cannot be said for GABA A Rs. Different mechanisms have been proposed to explain the complex interactions of GABA A Rs with gephyrin, including alternative splicing (18), intermediate acces...