Neuroligins Mediate Excitatory and Inhibitory Synapse Formation

Abstract

The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1␤. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses.Synapse formation is a tightly regulated process that involves the recruitment of specific cell adhesion molecules and scaffolding proteins to newly formed contacts between an axon and a dendrite (1-3). In the brain, excitatory and inhibitory synaptic transmission is mainly mediated by two neurotransmitters: glutamate, which is released at excitatory glutamatergic synaptic contacts, and GABA, 1 which is released at inhibitory GABAergic synapses. Initial transformation of a contact to either an excitatory or inhibitory synapse is thought to be controlled by spatial and temporal changes in protein content. This process is critical because an appropriate balance between excitatory and inhibitory synapses is required for proper neuronal excitability and function (2, 4 -6). However, molecular events that control differentiation of a contact into either an excitatory or inhibitory synapse remain unknown.The postsynaptic density protein, PSD-95, is a molecule that is exclusively localized to glutamatergic synapses and regulates clustering of AMPA receptors through association with stargazin (7, 8). Through its third PDZ (PSD-95/Dlg/ZO-1) homology domain, PSD-95 also recruits neuroligin-1 (NLG1), a cell adhesion molecule involved in synapse formation (9 -11). These findings indicate that association of PSD-95 with NLG1 is involved in excitatory synapse development. Recent work by Prange et al. (12) showed that NLG1 can drive both excitatory and inhibitory presynaptic contact formation. These results suggested that NLGs are involved in inhibitory synapse formation. Our work also showed that the effects of NLG1 on postsynaptic differentiation were less dramatic. Overexpression of NLG1 modestly increased the total number of excitatory posts...

N-cadherin and Neuroligins Cooperate to Regulate Synapse Formation in Hippocampal Cultures

“…Whereas NL2 is enriched at GABAergic synapses, NL1, NL3, and NL4 have previously been shown to localize mainly at glutamatergic synapses (20 -23). In stark contrast to what has been shown for cadherins, overexpression of NLs in cultured hippocampal neurons dramatically enhances the formation of glutamatergic and GABAergic synapses and also increases spine number (20,24,25). NLs are not only sufficient to induce synapse formation but are also required for the development of synapses.…”

“…Although NL2 is preferentially localized to inhibitory synapses, NL2 expression has also been shown to induce the formation of glutamatergic synapses (24,27,28). In addition, NL2 distribution shifts from inhibitory to excitatory contacts upon altered expression of postsynaptic scaffolding proteins (20,32,33). Overexpression of N-cadherin in wild-type cells was insufficient to enhance NL2/VGlut-1 colocalization, nor did overexpression of N-cadherin alone enhance synapse density.…”

“…Because NL2 has also been implicated in the formation of glutamatergic synapses (32), we next determined whether N-cadherin overexpression can rescue the NL1 knockdown phenotype by recruiting NL2, which in turn induces synapse formation. Previous work has shown that NL2 can shift from GABAergic to glutamatergic synapses following knockdown of the postsynaptic inhibitory scaffold protein gephyrin (32) or overexpression of the excitatory postsynaptic scaffold molecule PSD-95 (20). Cells were transfected with control siRNA, NL1 siRNA, or N-cadherin-CFP alone or cotransfected with NL1 siRNA and N-cadherin-CFP and then immunolabeled for NL2 and VGlut-1 (Fig.…”

Gephyrin Regulates GABAergic and Glutamatergic Synaptic Transmission in Hippocampal Cell Cultures

“…Interestingly, increasing the expression level of PSD-95, the scaffold molecule that directly binds NLG1, caused an enhancement of the glutamatergic innervation at the expense of the GABAergic one. This effect was accompanied by the recruitment of NLG2 to glutamatergic synapses (11)(12)(13). Moreover, the recent demonstration of a direct interaction between NLG2 and gephyrin (14) suggests a role for this protein in regulating transynaptic signaling at inhibitory connections.…”

“…NLG1 is enriched at glutamatergic synapses (8,9), whereas NLG2 is preferentially associated with GABAergic connections (10). Overexpression of NLGs has been shown to increase the number of GABAergic and glutamatergic synaptic contacts (11). Interestingly, increasing the expression level of PSD-95, the scaffold molecule that directly binds NLG1, caused an enhancement of the glutamatergic innervation at the expense of the GABAergic one.…”