A Specific Neuroligin3-αNeurexin1 Code Regulates GABAergic Synaptic Function in Mouse Hippocampus

Synapse formation and regulation require interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here, we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn interactions generate distinct functional properties at synapses.


Introduction
In central synapses, cell adhesion molecules (CAMs) are major players in trans-synaptic interactions (de Wit & Ghosh, 2016) which serve a primary role in initiating synapse formation by directing contact between axonal and dendritic membranes. Emerging evidence suggests that transsynaptic interactions are also important for synapse identity, function, plasticity and maintenance (Biederer, Kaeser, & Blanpied, 2017;Campbell & Tyagarajan, 2019;. Numerous CAM variants exist due to large gene families and alternative splicing, generating a vast array of possible combinations of pre-and postsynaptic CAMs. Although some specific trans-synaptic interactions of CAMs have been reported to underlie distinct synaptic properties (Chih, Gollan, & Scheiffele, 2006;Fossati et al., 2019;Futai, Doty, Baek, Ryu, & Sheng, 2013), elucidating synaptic CAM complexes that dictate synapse identity and function remains a major challenge.
Nrxn-Nlgn interactions depend on Nrxn protein length (long form [α] vs short form [β]), splice insertions at AS4 of Nrxns and splice insertions of Nlgns. For example, Nlgn1 splice variants that have splice insertions at site B have higher binding affinities for βNrxn1-AS4 (βNrxn1 lacking alternative splice insertion at AS4) than for βNrxn1+AS4 (containing an alternative splice insertion at AS4) (Boucard, Chubykin, Comoletti, Taylor, & Sudhof, 2005;Koehnke et al., 2010;Reissner, Klose, Fairless, & Missler, 2008). However, it is largely unknown which Nrxn-Nlgn combination defines specific synapse functionality. We recently found that Nlgn3Δ, which lacks both of the A1 and A2 alternative splice insertions, is the major Nlgn3 splice isoform expressed in hippocampal CA1 pyramidal neurons and regulates both excitatory and inhibitory synaptic transmission (Uchigashima et al., 2020). However, to the best of our knowledge, the synapses at which Nlgn3Δ interacts with presynaptic Nrxn isoform(s) have not been identified.
In the present study, we show that Nlgn3 is selectively enriched at vesicular glutamate transporter 3-expressing (VGT3+) Cck+ inhibitory terminals in the hippocampal CA1 area. Gain-offunction and loss-of-function studies revealed that Nlgn3 regulates VGT3+ interneuron-mediated inhibitory synaptic transmission. Importantly, the effect of Nlgn3 on VGT3+ synapses was hampered by the deletion of all Nrxn genes in VGT3+ interneurons and rescued by the selective expression of αNrxn1+AS4 in VGT3+ interneurons. These results suggest that the trans-synaptic interaction between αNrxn1+AS4 and Nlgn3 underlies the input cell-dependent control of VGT3+ GABAergic synapses in the hippocampus.

Results
Nlgn3 is enriched at VGT3+ GABAergic synapses in the hippocampal CA1 region.
In a recent study, we demonstrated that Nlgn3 localizes at and regulates both inhibitory and excitatory synapses in the hippocampal CA1 area (Uchigashima et al., 2020). However, the distribution of Nlgn3 at different types of inhibitory synapses has not yet been addressed. Therefore, we first examined which GABAergic inhibitory synapses express Nlgn3 in the CA1 area by immunohistochemistry. Cck+, Pv+ and Sst+ interneurons are the primary inhibitory neurons in the hippocampus. Moreover, the cell bodies and dendritic shafts of CA1 pyramidal cells are targeted by Cck+ and Pv+, and Sst+ interneurons, respectively (Pelkey et al., 2017). Our Nlgn3 antibody with specific immunoreactivity was validated in Nlgn3 KO brain ( Figure 1A) and displayed a typical membrane protein distribution pattern in the hippocampus, as we recently reported ( Figure 1B, C) (Uchigashima et al., 2020).
Inhibitory synapses expressing Nlgn3 were identified by co-localization of Nlgn3 signals with vesicular inhibitory amino acid transporter (VIAAT) signals. Four different inhibitory axons/terminals were visualized by anti-VGT3 and -CB1 (markers for Cck+ interneurons) (Fruh et al., 2016), -Pv and -Sst antibodies. We found that punctate signals for Nlgn3 were frequently associated with GABAergic terminals co-labeled with VGT3 or CB1 ( Figure 1D-G and L) compared with Pv ( Figure 1H, I, and L) or Sst ( Figure 1J, K and L) in the CA1 pyramidal cell layer. These data strongly suggest that Nlgn3 is preferentially recruited to Cck+ GABAergic synapses, but not to Pv+ or Sst+ inhibitory synapses.

Nlgn3 regulates inhibitory synaptic transmission at VGT3+ GABAergic synapses.
To determine whether Nlgn3 has specific roles at VGT3+ inhibitory synapses, we assessed the effect of overexpressing Nlgn3Δ, the major Nlgn3 splice isoform expressed in CA1 pyramidal neurons (Uchigashima et al., 2020), on input-specific inhibitory transmission. To distinguish a subset of GABAergic synapses and evoke cell-specific synaptic transmission, we generated three cell typespecific fluorescent lines by crossing VGT3 cre , Sst cre or Pv cre with a TdTomato (RFP) reporter line, producing respectively, VGT3/RFP, Sst/RFP and Pv/RFP mouse lines. TdTomato-expressing cells in each of the three fluorescent mouse lines were distributed in the CA1 in a layer-dependent manner ( Figure S1). We evaluated the effect of Nlgn3Δ overexpression (OE) on unitary inhibitory postsynaptic currents (uIPSCs) by triple whole-cell recordings using organotypic slice cultures from each mouse line. Two to three days after transfection of Nlgn3Δ or EGFP control by biolistic gene gun, currentand voltage-clamp recordings were conducted from a presynaptic RFP+ interneuron and postsynaptic EGFP or EGFP/Nlgn3Δ-positive and -negative postsynaptic pyramidal neurons, respectively ( Figure   2A). RFP interneurons expressing VGT3 in the pyramidal cell layer and stratum (st.) radiatum, Pv in the st. pyramidale and Sst in the st. oriens, were chosen ( Figure S1). Unitary inhibitory postsynaptic currents (uIPSCs) were evoked by inducing action potentials in RFP+ neurons. The amplitude and paired-pulse ratio (PPR), monitoring release probability, of uIPSCs and synaptic connectivity were compared between Nlgn3Δ-transfected and -untransfected neurons (Figure 2B-E). Importantly, VGT3+ inhibitory interneurons displayed clear potentiation of uIPSCs onto CA1 pyramidal neurons overexpressing Nlgn3Δ. Paired action potentials (APs) of VGT3+ neurons with short intervals (50 ms) induced paired-pulse depression (PPD) of uIPSCs. Nlgn3Δ displayed reduced PPD compared with untransfected neurons, consistent with previous work (Futai et al., 2007;Shipman et al., 2011;Uchigashima et al., 2020) (Figure 2D). As PPR inversely correlates with presynaptic release probability, these results suggest that Nlgn3Δ OE can facilitate presynaptic GABA release. In contrast, Nlgn3Δ OE reduced uIPSCs in Pv+ inhibitory synaptic transmission, but had no effect on PPR as reported previously (Figure 3A-D) (Horn & Nicoll, 2018). Lastly, Nlgn3Δ OE did not alter uIPSCs or PPR mediated by Sst+ interneurons (Figure 3E-H). No effect of biolistic transfection with EGFP alone was found on uIPSC amplitude, PPR or connection probability at Pv+ and Sst+ inhibitory synapses.
The above results strongly suggest that Nlgn3 modifies inhibitory synaptic function depending on the type of presynaptic interneuron with which it interacts. Interestingly, Nlgn3Δ OE did not increase synaptic connectivity (Figure 2E), suggesting that i) Nlgn3 regulates pre-existing inhibitory inputs on postsynaptic neurons and/or ii) postsynaptic Nlgn3Δ OE is not sufficient to induce new synapse formation.
We next tested the impact of acute Nlgn3 knockdown (KD) on inhibitory synaptic transmission.
Organotypic slice cultures prepared from C57BL/6J mice were biolistically transfected with shRNA against Nlgn3 (shNlgn3#1), which exhibits over 90% knockdown efficiency specific to Nlgn3 isoforms ( Figure S2A), or control shRNA (shCntl). Transfection was performed at days in vitro (DIV) one and recordings were performed 7-9 days later to measure inhibitory synaptic transmission mediated by three different synaptic inputs (Figure 4). Compared with untransfected neurons, shNlgn3#1-  Figure 4D, H, L) between transfected and untransfected neurons. Taken together, these data suggest that Nlgn3 is required for synaptic transmission specifically at VGT3+ GABAergic synapses in an input cell-dependent manner.

OE.
Our results suggest that Nlgn3 specifically translocates to VGT3+ inhibitory terminals and regulates inhibitory synaptic transmission. Postsynaptic Nlgns couple with presynaptic Nrxns to form trans-synaptic protein complexes that regulate synapse formation and function . Does inputspecific Nlgn3Δ ( Figure 2C) require presynaptic Nrxn proteins? To address this question, we generated VGT3 neuron-specific triple Nrxn knockout with TdTomato reporter gene mouse line (Nrxn1/2/3 f/f /VGT3 cre /TdTomato: NrxnTKO/VGT3/RFP). This mouse line is fertile with KO of Nrxn1, 2 and 3 specifically in TdTomato-positive VGT3+ neurons ( Figure 5A). We transfected Nlgn3Δ in NrxnTKO/VGT3/RFP slice cultures and performed triple whole-cell recordings as described above using VGT3/RFP slice cultures (Figure 2B-E). VGT3+ neurons lacking Nrxns induced synaptic release regardless of Nlgn3Δ gene transfection, indicating that presynaptic Nrxns in VGT3+ interneurons are not essential for synaptogenesis. Importantly, we observed no enhancement of uIPSC amplitude in Nlgn3-overexpressed neurons compared with untransfected pyramidal neurons ( Figure 5B-E). These results strongly suggest that presynaptic Nrxn proteins are necessary for regulating inhibitory synaptic transmission through postsynaptic Nlgn3. αNrxn1 and βNrxn3 mRNAs are highly expressed in VGT3+ interneurons.
Our results above clearly suggest that presynaptic Nrxn proteins are important for the function of postsynaptic Nlgn3Δ. We therefore hypothesized that Nrxn isoforms highly expressed in VGT3+ interneurons functionally couple with postsynaptic Nlgn3Δ. To address this hypothesis, we examined the mRNA expression patterns of α and β isoforms of Nrxn1-3 in hippocampal CA1 interneurons by fluorescent in situ hybridization (FISH). The specificities of cRNA probes for 6 Nrxn isoforms were validated recently (Uchigashima, Cheung, Suh, Watanabe, & Futai, 2019). mRNAs encoding all Nrxn isoforms, except βNrxn1, were detected not only in the st. pyramidale but also in scattered cells across all layers (Figure S3A-F). Importantly, αNrxn1 and βNrxn3 mRNAs appeared to be enriched in scattered cells within the st. radiatum or pyramidale (arrows in Figure 6A-C and G-I), where VGT3+ interneurons are dominantly distributed compared with Pv+ and Sst+ interneurons (Pelkey et al., 2017). Double FISH signals were twice as strong for αNrxn1 and βNrxn3 mRNAs in VGT3+ ( Figure   6A, G, J) interneurons than in Pv+ ( Figure 6B, H, J) and Sst+ ( Figure 6C, I and J) interneurons. In contrast, there were no differences in the signal intensities for the remaining Nrxn isoforms between VGT3+ and other interneurons ( Figure 6D-F and J, S3). These findings suggest that αNrxn1 and βNrxn3 mRNAs are highly expressed in VGT3+ interneurons compared with Pv+ or Sst+ interneurons.
Our rescue approach suggests that αNrxn+AS4 regulates inhibitory synaptic transmission with Nlgn3Δ. The gene structures of α and βNrxns are similar, therefore, different αNrxn+AS4 isoform(s) may be able to functionally substitute αNrxn1+AS4. Our FISH results indicate that VGT3+, Nlgn3Δ had any effect on inhibitory synaptic transmission. These results strongly suggest that αNrxn1+AS4, but not αNrxn3+AS4, has a unique code in the extracellular domain important for synaptic function with Nlgn3Δ.

Discussion
Synaptic protein-protein interactions are critical for the development, maturation and survival of neurons. However, it is technically challenging to physiologically characterize trans-synaptic CAM protein interactions in two different neurons due to the difficulty in identifying synaptically-connected neuronal pairs in the brain. Co-culture approaches consisting of non-neuronal cells transfected with different Nrxn splice isoforms and dissociated neurons expressing endogenous Nlgns (or expressing Nrxn-binding proteins in non-neuronal cells and observing their interactions with endogenous Nrxns) have begun to elucidate the roles of trans-synaptic Nrxn/Nlgn isoforms on the clustering of pre-/postsynaptic molecules (Chih et al., 2006;Kang, Zhang, Dobie, Wu, & Craig, 2008;Ko et al., 2009;Nam & Chen, 2005;Scheiffele, Fan, Choih, Fetter, & Serafini, 2000). However, this approach is limited to primary cultures and cannot address whether these trans-synaptic interactions are sufficient to induce functional synapse diversification. To fully understand the physiological roles of trans-synaptic molecules, one must be able to manipulate the expression of these molecules in pre-and postsynaptic neurons simultaneously followed by determination of the functional consequences of such manipulation. Using our newly developed gene electroporation method that enables us to transfect genes in minor cell types such as specific inhibitory interneurons (Keener et al., 2020), we demonstrated for the first time that αNrxn1+AS4 and Nlgn3Δ, which are endogenously expressed in VGT3+ inhibitory and CA1 pyramidal neurons ( Figure 7D) (Futai et al., 2007;Shipman et al., 2011;Uchigashima et al., 2020), respectively, form a specific code that dictates inhibitory synaptic transmission.
It has been reported that Nlgn proteins regulate inhibitory synaptic transmission in an input cell-specific manner. For example, a Nlgn2 KO mouse line displays deficits in fast-spiking but not Sst+ interneuron-mediated inhibitory synaptic transmission in the somatosensory cortex (Gibson, Huber, & Sudhof, 2009). Furthermore, Nlgn3 KO mice and the Nlgn3 R451C knock-in mutant line, which mimics a human autism mutation, showed Pv+ or Cck+ input-specific abnormal inhibitory synaptic transmission in the hippocampal CA1 region (Foldy et al., 2013). Therefore, the function of postsynaptic Nlgns are determined by the type of presynaptic inputs it receives, supporting the intriguing hypothesis that specific Nrxn-Nlgn binding regulates synaptic function. Our gene expression and functional assays highlight that αNrxn1 is abundantly expressed in VGT3+ interneurons compared with other interneurons ( Figure 6J) and αNrxn1+AS4 is the dominant αNrxn1 splice isoform expressed in VGT3+ interneurons ( Figure 7C) which regulates inhibitory synaptic transmission with Nlgn3Δ ( Figure 8C). On the other hand, our FISH experiments, which do not distinguish the AS4 insertion, detected αNrxn1 at low levels in Pv+ and Sst+ interneurons ( Figure 6J).
The expression of αNrxn1+AS4 was also previously confirmed in Pv+ interneurons (Fuccillo et al., 2015). Considering the lower expression of Nlgn3 at Pv+ inhibitory synapses (Figure 1L), other postsynaptic mechanism(s) might exist to regulate inhibitory synaptic transmission with αNrxn1+AS4 at Pv+ inhibitory synapses.
However, our results clearly showed that αNrxn1+AS4, but not the other Nrxn variants we analyzed, contributes to Nlgn3Δ-mediated function at VGT3+ synapses. Similarly, αNrxn1-AS4 is known to specifically interact with Nlgn2 and enhances inhibitory synaptic transmission (Futai et al., 2013). This gap between biochemical binding affinities and physiological synaptic functions is likely due to interactions of Nrxn and Nlgn with other synaptic proteins. In this regard, our study dissects physiological Nrxn-Nlgn interactions at synapses compared with biochemical analyses of purified Nrxn and Nlgn proteins.
The AS4 is the most important splice insertion site in Nrxns to change the affinity to postsynaptic binding partners, including Nlgn . A splice insertion at AS4 of βNrxn1 can weaken the interaction with Nlgns (Koehnke et al., 2010). Indeed, we have reported that βNrxn1-AS4 but not βNrxn1+AS4 increases synaptic transmission via interaction with Nlgn1 (Futai et al., 2013). In contrast, we found that an insertion in AS4 of αNrxn1 can increase inhibitory synaptic transmission at VGT3+ synapses via trans-synaptic interaction with Nlgn3Δ. It is particularly interesting that only αNrxn1+AS4, but not αNrxn3+AS4, encoded uIPSC enhancement with postsynaptic Nlgn3Δ ( Figure   8C and 9F). These results suggest that structural differences beyond the AS4 site exist between these two αNrxns. Since the structures of the major Nrxn domains, such as LNS and EGFA-EGFC, are similar between αNrxns, one possible interpretation is that differential alternative splicing events occurring at other AS sites may regulate binding with Nlgn3Δ. Further structural and functional analyses targeting these AS events could reveal a novel AS structure that dictates Nrxn-Nlgn codes on synaptic function. Moreover, it will be important to address whether αNrxn1+AS1, αNrxn3+AS4 and βNrxn3-AS4, which are highly expressed in VGT3+ neurons (Figure 7), can encode specific synaptic functions when interacting with other postsynaptic Nrxn-binding partners such as Nlgn2, CST-3 and IgSF21 (Pettem et al., 2013;Tanabe et al., 2017;Um et al., 2014).
Deletion of Nrxns or Nlgns has been reported to have little effect on synapse formation (Chanda, Hale, Zhang, Wernig, & Sudhof, 2017;Chen, Jiang, Zhang, Gokce, & Sudhof, 2017;Varoqueaux et al., 2006). Our manipulation of the expression levels of either presynaptic Nrxns or postsynaptic Nlgn3 consistently did not affect synaptic connectivity, a measurement of the number of active synapses (Figure 2, 3 and 6). However, Nlgn3Δ formed new synapses only when αNrxn1+AS4 was simultaneously expressed in VGT3+ interneurons ( Figure 8E). This suggests that trans-synaptic interactions of Nrxns and Nlgns may control not only synapse function but also synapse number, while the lack of Nrxns or Nlgns on either side of the synapse can be compensated by other CAM interactions. In this context, it is important to address whether the subcellular localization of Nrxn and Nlgn proteins is regulated by synaptic activity, such as homeostatic synaptic plasticity (Mao et al., 2018).
Note that our results, demonstrating the role of postsynaptic Nlgn3Δ in CA1 pyramidal neurons on inhibitory synaptic transmission, have some inconsistencies with a previously published study, which found that Nlgn3 differentially regulates Pv+ and Sst+ interneuron inhibitory synapses (Horn & Nicoll, 2018). Horn and Nicoll reported that OE of human Nlgn3A2 reduced Pv+ and increased Sst+ inhibitory synaptic transmission. The former finding is consistent with our results in Figure 3B, displaying that Nlgn3Δ OE reduces Pv+ uIPSC amplitude. This may suggest that Pv+ neurons have a unique trans-synaptic regulatory mechanism compared with other interneurons and that Nlgn3Δ OE may disrupt endogenous GABA A R complexes at Pv+ inhibitory synapses. In contrast, Nlgn3Δ OE did not increase Sst+ uIPSCs (Figure 3F), as observed in Horn & Nicoll, 2018. This difference might be due to variations in experimental approaches including the Nlgn3 clone used (human Nlgn3A2 versus mouse Nlgn3Δ slice isoform) and the duration of transgene or shRNA expression in hippocampal CA1 pyramidal neurons. Additionally, Horn and Nicoll crossed Pv cre and Sst cre , the same cre lines we tested, with Ai32 mice, a cre-dependent channelrhodopsin line (Madisen et al., 2012), to evoke Pv+ or Sst+ neuron-mediated synaptic transmission, respectively (Horn & Nicoll, 2018). However, our current and previous studies for mouse line validation indicate that while PV cre and Sst cre lines exhibit highly specific cre expression in these cell types, these lines have leaky cre expression in other cell type(s) (Figure S1, yellow arrow heads) (Mao et al., 2015). Therefore, lightevoked activation of non-specific cell types may contribute to the inconsistent results in synaptic transmission.
Electrophysiology: Whole-cell voltage-and current-clamp recordings were performed on postsynaptic and presynaptic neurons, respectively. Nlgn3Δ construct or shRNAs were transfected at DIV6-9 or DIV2 and subjected to recordings at two to three or five to twelve days after transfection, respectively. The extracellular solution for recording consisted of (in mM): 119 NaCl, 2.     ). The number of tested slice cultures is the same as that of cell pairs. n.s., not significant.
Mann-Whitney U test.  and 21/10). The number of tested slice cultures is the same as that of cell pairs. n.s., not significant.
Untrans Trans A N l g n 3 Δ N l g n 3 N l g n 3 N l g n 1 N l g n 2 A 1 A 2 A 2 N l g n 3 Δ N l g n 3 N l g n 3 N l g n 1 N l g n 2 A 1 A 2 A 2 ** ** *** ** **** *** B Figure Figure S4