Synapse formation and clustering of neuroligin-2 in the absence of GABA _A receptors

Abstract

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABAA␣1 knockout (KO) mice express transiently the ␣3 subunit, leading to the assembly of functional GABAA receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the ␣3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABAA receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABAA receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.gephyrin ͉ Purkinje cell ͉ synaptogenesis T he identification of the factors that are critical for the assembly, maturation, and stabilization of synaptic connections remains a central puzzle in developmental neuroscience. Studies on cultured neurons have been invaluable for uncovering the molecular mechanisms of synaptogenesis (1, 2). Unfortunately, such studies can be influenced by varying experimental conditions, and their results often have not been replicated by in vivo analyses of gene knockout (KO) mice (3). As an alternative approach, gene deletion techniques allow investigating the in vivo function of genes that are believed to be critical for synapse formation (4-8). However, the premature death of newborn mutant mice has often prevented long-term analyses of synapse maturation and function. In the present study, we took advantage of a KO model that survives up to adulthood without a major phenotype, despite extensive loss of GABA-mediated function in specific populations of neurons.Specifically, we investigated the in vivo effects of a selective silencing of GABAergic transmission on the formation and longterm stability of GABAergic synapses, which provide the major inhibitory control over neuronal activity in the brain (9). By analogy to glutamatergic synapses, assembly of GABAergic synapses is believed to involve selective trans-synaptic interactions between adhesion molecules and cytosolic interactions with scaffolding proteins (2). Neuroligin-2 (NL2) is a postsynaptic adhesion molecule that localizes at GABAergic synapses and triggers synapse formation by interacting with presynaptic neurexins (10, 11). NL2 belongs to a family of related proteins also comprising NL1, NL3, and NL4 (12). Importantly, NL1 is targeted to glutamatergic synapses (13,14), suggesting that different NLs may play an important role in specifying distinct types of synapse and in determining a balance between n...

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression

“…The specificity of MGL antibodies has been verified by blank labeling in global MGL-KO brains in our previous studies (22,35). By double immunofluorescence for MGL and carbonic anhydrase 8 (Car8), a marker of PCs (38), punctate MGL labeling was prominent in the neuropil of the molecular layer (ML), but was absent in Car8-labeled PC dendrites and somata (Fig. 1A).…”

Molecular and functional heterogeneity of GABAergic synapses

“…5) In contrast, the DGC is not altered in neurons lacking functional GABAergic transmission or gephyrin clusters (Fig. 4D, G) [105,74], indicating that its formation and postsynaptic localization is not dependent on GABA A R or gephyrin. 6) Preservation of NL2 clustering in the absence of GABA A R and/or gephyrin suggests a mandatory association between NL2 and S-SCAM/β-dystroglycan complex (Fig.…”

“…4) Gephyrin clustering, but not NL2 clustering, requires the presence of postsynaptic GABA A R (Fig. 4C-D) [105], in particular those containing the α2 subunit (Fig. 4G) [74].…”

Neuroligin 2 Controls the Maturation of GABAergic Synapses and Information Processing in the Retina

“…In the brainstem, where GABA and glycine are often coreleased, lack of NL2 leads to deficits in both GABAergic and glycinergic neurotransmission (G. Aramuni and W. Zhang, personal communication). Furthermore, NL2 clusters are detected at very early stages of inhibitory postsynapse differentiation (Varoqueaux et al, 2004;Patrizi et al, 2008), and in vitro studies have suggested that NL2 may contribute to the recruitment of GABA A receptors (Dong et al, 2007). Thus, there is emerging evidence for a central role of NL2 in organizing inhibitory synapse assembly and function.…”