Proper circuit function in the mammalian brain relies upon cell-cell communication through two main types of chemical synapses: glutamatergic (excitatory) and GABAergic (inhibitory). In the cortex, activity of excitatory pyramidal neurons is tightly controlled by inhibitory interneurons, the dysfunction of which has been linked to disorders such as autism and epilepsy (Ferguson and Gao, 2018; Takano and Sawai, 2019). Therefore, it is critical to understand the mechanisms by which GABAergic interneurons form proper synaptic connections with both glutamatergic pyramidal neurons and GABAergic interneurons.

Synapse formation is a complex process occurring in several steps. First, initial contact between a pre- and postsynaptic neuron occurs, followed by adhesion and stabilization of this point of contact. Subsequently, synaptic machinery accumulates in both the presynaptic active zone and postsynaptic specialization, specifically: neurotransmitter release machinery, scaffolding proteins, neurotransmitter receptors, and other required signaling molecules (reviewed in Batool et al., 2019; McAllister, 2007; Scheiffele, 2003). Numerous families of secreted or membrane attached ligand/receptor pairs and cell adhesion molecules (CAMs), expressed in neurons and in glia are implicated in synapse development and function (Sudhof, 2018). While it is well-established that molecules such as Ephs/ephrins, neuroligins/neurexins, Nrg1/ErbB4, FGF/FGFR, CAMs, and Semaphorins/Plexins regulate synapse formation, it is an outstanding challenge to determine precisely which steps of synapse formation these molecules control, as well as whether they exert their functions from the presynaptic or postsynaptic neuron or both (Craig and Kang, 2007; Dabrowski et al., 2015; Hruska and Dalva, 2012; Koropouli and Kolodkin, 2014; Luo et al., 2021; Sudhof, 2018; Terauchi et al., 2010).

Importantly, it has also been a challenge to define mechanisms that differ between glutamatergic and GABAergic synapse formation, in part because few synaptogenic molecules have been identified that selectively promote formation of GABAergic, but not glutamatergic, synapses. One exception to this is that Sema4D signaling through its receptor Plexin-B1 regulates GABAergic, but not glutamatergic, synapse formation. We previously demonstrated that application of the extracellular domain of Sema4D either in vitro or in vivo caused the rapid formation (2 h) of functional, GABAergic synapses (Adel et al., 2023; Kuzirian et al., 2013). Using a live imaging approach we showed that Sema4D application affects the behavior of GFP-labeled gephyrin which is a postsynaptic scaffolding protein at GABAergic synapses. Taken together, our data favor a model whereby Sema4D/Plexin-B1 signaling acts at the recruitment step in synapse formation to localize synaptic components to the nascent synapse.

In addition, recent advances from our group demonstrated the therapeutic potential of selectively targeting GABAergic synapse formation and function in the case of neurological disorders involving hyperexcitability, underscoring the importance of delineating these processes (Acker et al., 2018; Adel et al., 2023). For example, we previously found that application of the Sema4D extracellular domain to neuronal cultures increased GABAergic synapse density within 30 min, increased inhibitory tone within 2 h of application, and increased resistance to seizure in various rodent models of epilepsy (Adel et al., 2023; Kuzirian et al., 2013). The selective, rapid, and functional effects of Sema4D motivated our further investigation into the mechanisms of synapse development mediated by Semaphorin/Plexin signaling.

Semaphorins, a 20-member family of secreted and membrane-attached ligands in mammals, contain a conserved extracellular Sema domain through which they bind to the extracellular Sema domain of Plexin receptors (reviewed in Alto and Terman, 2017; Junqueira Alves et al., 2019; Koropouli and Kolodkin, 2014; Tamagnone and Comoglio, 2000; Yazdani and Terman, 2006). Plexins are a family of 9 transmembrane receptors with a wide range of functions including regulation of cell migration and proliferation (reviewed in Tamagnone et al., 1999; Toledano and Neufeld, 2023). Class 4 Semaphorin dimers bind to Plexin-B receptors to regulate numerous functions in the developing nervous system, such as neural tube closure, cerebellar cell migration and differentiation, and actin cytoskeleton remodeling (Deng et al., 2007; Friedel et al., 2007; Oinuma et al., 2010; Oinuma et al., 2003; Worzfeld et al., 2014).

In the past decade, we established novel roles for Plexin-B receptors in synapse formation. The Plexin-B1 receptor promotes GABAergic synapse formation upon activation by the Sema4D-ECD (Fig. 1A, B) while having no effect on glutamatergic synapse formation (Kuzirian et al., 2013; McDermott et al., 2018). We also observed that Plexin-B1 was required in both the presynaptic interneuron and postsynaptic pyramidal cell for Sema4D-dependent GABAergic synapse formation (Kuzirian et al., 2013; McDermott et al., 2018). However, while addition of exogenous Sema4D ligand promotes GABAergic synapse formation via Plexin-B1, we failed to observe a decrease in GABAergic synapse density upon Plexin-B1 knockout in the absence of exogenous ligand (Fig. 1C) (also see Fig. 4 in (Kuzirian et al., 2013). Based on this observation we hypothesized that additional Plexin-B receptors may also play a role in GABAergic synapse formation.

In support of this hypothesis, in situ hybridization studies revealed that Plexin-B1 and Plexin-B2 are expressed in both presynaptic inhibitory interneurons and in postsynaptic excitatory neurons in the hippocampus (McDermott et al., 2018). In addition, using RNAi-mediated gene knockdown in cultured hippocampal neurons, we showed that Plexin-B2 is required in the postsynaptic, pyramidal neuron for proper GABAergic synapse formation (Fig. 1D) while having no effect on glutamatergic synapse formation (McDermott et al., 2018). Further, addition of soluble, Sema4D-ECD to hippocampal cultures in which Plexin-B2 expression was knocked down in the postsynaptic neuron resulted in increased GABAergic synapse density relative to Plexin-B2 knockdown neurons treated with control protein (Fig. 1E), presumably due to signaling through the Plexin-B1 receptor. This result suggests that Plexin-B1 and Plexin-B2-dependent signaling can play different roles in GABAergic synapse formation.

Here, we sought to reveal overlapping and distinct functions of Plexin-B1 and Plexin-B2 receptors in GABAergic synapse formation and uncover their underlying molecular mechanisms. We also sought to determine if Plexin-B2 is required in presynaptic interneurons for proper GABAergic synapse formation. First, we interrogate the role of Plexin-B2 expressed in parvalbumin-positive (PV) cells in the hippocampus in GABAergic synapse formation. We next ask if Plexin-B1 and Plexin-B2 function redundantly in GABAergic synapse formation. Finally, we perform a structure-function analysis to identify roles for distinct Plexin-B1 and Plexin-B2 molecular domains in driving formation of GABAergic synapses. Our data reveal unique roles for different Plexin-B receptor domains in GABAergic synapse formation and provide new insights into the precise signaling configurations by which Plexin-B receptors mediate this process.