In human patients, loss-of-function mutations of the postsynaptic cell-adhesion molecule neuroligin-4 were repeatedly identified as monogenetic causes of autism. In mice, neuroligin-4 deletions caused autism-related behavioral impairments and subtle changes in synaptic transmission, and neuroligin-4 was found, at least in part, at glycinergic synapses. However, low expression levels precluded a comprehensive analysis of neuroligin-4 localization, and overexpression of neuroligin-4 puzzlingly impaired excitatory but not inhibitory synaptic function. As a result, the function of neuroligin-4 remains unclear, as does its relation to other neuroligins. To clarify these issues, we systematically examined the function of neuroligin-4, focusing on excitatory and inhibitory inputs to defined projection neurons of the mouse brainstem as central model synapses. We show that loss of neuroligin-4 causes a profound impairment of glycinergic but not glutamatergic synaptic transmission and a decrease in glycinergic synapse numbers. Thus, neuroligin-4 is essential for the organization and/or maintenance of glycinergic synapses.

We next asked whether other neuroligins contribute to glycinergic synaptic transmission in MNTB principal neurons. To address this question, we examined glycinergic synaptic responses after deletion of all neuroligins. Previous studies validated the efficiency of parvalbumin (PV)-driven Cre expression to delete floxed alleles in MNTB neurons at P12 ( Zhang et al., 2017 ). Therefore, we crossed previously generated PV-neuroligin-1/2/3 triple-KO mice ( Zhang and Südhof, 2016 ) with neuroligin-4 KO mice to delete all neuroligins in MNTB neurons (PV–neuroligin-123–neuroligin-4 KO, termed PV-Nlgn1234; Fig. 2 C ). We then studied the effects of the pan-neuroligin deletion in glycinergic synapses of MNTB principal neurons between P14 and P16 and compared these effects to those of the neuroligin-4 KO. As before, we recorded mIPSCs in the presence of 1 mM tetrodotoxin (TTX) at a high extracellular Ca 2+ concentration (10 mM). Strikingly, we found that the pan-neuroligin deletion reduced the mIPSC frequency (to ∼25% of controls) and additionally further decreased the mIPSC amplitude (to ∼40% of controls; Fig. 2, D and E ). Kinetic analyses again revealed no change in mIPSC rise times in PV-Nlgn1234 neurons but uncovered shorter mIPSC decay times ( Fig. 2, D and E ), indicating possible changes in subunit composition and/or mislocalization of GlyRs. This conclusion was strengthened by recordings of mIPSCs in MNTB neurons from pan-neuroligin quadruple-KO mice that were performed in the presence of 2 mM Ca 2+ , a more physiological concentration (Fig. S1). Specifically, in these mice, we detected the same mIPSC phenotype in 2 mM Ca 2+ as in 10 mM Ca 2+ , demonstrating that the high Ca 2+ concentration in itself did not elicit or distort the mIPSC phenotype. Thus, other neuroligins besides neuroligin-4 contribute to inhibitory transmission at glycinergic synapses in MNTB principal neurons. No change of mIPSC in PV-Nlgn123 (Fig. S1) suggested that the contributions of neuroligin-1 to -3 to glycinergic transmission become manifest only in the absence of neuroligin-4 KO.

To distinguish between these possibilities, we first analyzed the coefficient of variation of IPSC amplitudes, which depends largely but not exclusively on the presynaptic release probability. We uncovered a 100% increase in the coefficient of variation in neuroligin-4 KO MNTB principal neurons compared with control cells ( Fig. 1, D and E ). Although according to classical quantal analysis, this result indicates a decrease in presynaptic release probability ( Bekkers and Stevens, 1995 ), it could also potentially be because of an abnormal content or distribution of postsynaptic GlyRs, as suggested by studies in glutamatergic synapses ( Faber et al., 1992 ; Heine et al., 2008 ). To further explore the synaptic abnormalities in neuroligin-4 KO mice, we examined postsynaptic receptor content by measuring miniature IPSCs (mIPSCs). The mIPSC frequency broadly correlates with release probability (and other parameters), whereas the mIPSC amplitude is a measure of postsynaptic receptor content, providing an independent set of parameters to examine the cause of the massive decrease in glycinergic transmission induced by the neuroligin-4 KO. Because the mIPSC frequency in MNTB neurons was too low for accurate measurements in the presence of the standard 2 mM extracellular Ca 2+ and glycinergic synapses in the MNTB appear to exhibit little multivesicular release at higher Ca 2+ concentrations ( Lim et al., 2003 ), we increased the mIPSC frequency by elevating extracellular Ca 2+ from 2 mM to 10 mM. We found that under these conditions, the neuroligin-4 KO did not alter mIPSC frequency. Moreover, the neuroligin-4 KO caused a reduction in mIPSC amplitude (to ∼65% of control values) that was highly significant because of the reproducibility of mIPSC amplitude measurements but did not alter the rise and decay times of mIPSCs ( Fig. 2, A and B ). These data, viewed together, indicate that the loss of neuroligin-4 robustly decreases inhibitory glycinergic synaptic transmission in MNTB principal neurons primarily by reducing the postsynaptic receptor content.

These data indicate that the MNTB is ideally suited to examine a potential role for neuroligin-4 in glycinergic versus glutamatergic synaptic transmission. Toward this end, we assessed synaptic responses elicited by stimulation of single axons synapsing onto MNTB principal neurons, using acute slices from P14 to P16 mice. We adjusted the stimulus intensity to obtain a >60% failure rate, a condition under which the probability of an IPSC in the MNTB neuron being evoked by multiple axon inputs is low ( Awatramani et al., 2004 , 2005 ). With this minimal stimulation protocol, we found that the neuroligin-4 KO reduced IPSC amplitudes to ∼35% of control amplitudes without affecting the rise and decay times of IPSCs ( Fig. 1, D and E ). Under these conditions, the neuroligin-4 KO did not significantly reduce strychnine-insensitive IPSC amplitudes (Fig. S1). These data indicate that neuroligin-4 is critical for glycinergic strychnine-sensitive, but not GABAergic, strychnine-insensitive inhibitory synaptic transmission in MNTB neurons. Here, neuroligin-4 could perform possible functions (1) in maintaining the presynaptic release probability, (2) in organizing the postsynaptic receptor content, and/or (3) in maintaining the number of synapses.

Using acute slices, we next performed patch-clamp recordings of inhibitory synaptic responses in principal neurons of the MNTB. Consistent with previous findings ( Awatramani et al., 2004 , 2005 ), we found that at P13–14, an age at which MNTB synapses are relatively mature, >80% of evoked inhibitory postsynaptic currents (IPSCs) recorded from MNTB principal neurons were blocked by the glycine receptor (GlyR) blocker strychnine and were thus glycinergic ( Fig. 1, B and C ).

In such measurements of presumptive synaptic puncta by immunocytochemistry, the puncta “size” is determined both by the amount of the targeted antigen in a synapse (e.g., in this case the GlyR content) and by the overall size of the synapse. Viewed together, the morphological and electrophysiological data demonstrate that neuroligin-4 is important for the control of glycinergic synapses and that other neuroligins further contribute to this control. The good correspondence between the reduction in evoked IPSC amplitude and glycinergic synapse numbers indicates that most of the neuroligin-4 KO phenotype is due to a loss of synapses.

We found that the neuroligin-4 KO caused a large reduction of the density of vGAT puncta (to ∼65% of controls) and that the PV-Nlgn1234 mice produced a further reduction in vGAT puncta density (to ∼50% of control values). Both the neuroligin-4 KO and PV-Nlgn1234 similarly significantly reduced vGAT puncta size (to ∼90% of controls; Figs. 3, A and B ; and S2). Because most GlyRs are tightly juxtaposed to vGAT-positive glycinergic nerve endings in MNTB principal neurons ( Trojanova et al., 2014 ), we measured both GlyR signals adjacent to vGAT puncta next to the principal neurons of the MNTB and GlyR signals throughout the MNTB sections. We found that the neuroligin-4 KO significantly reduced the density of GlyR puncta that were adjacent to vGAT puncta (to ∼50% of controls), a reduction that is slightly higher than the reduction in inhibitory synapse density observed by labeling for vGAT itself. The pan-neuroligin KO (PV-Nlgn1234) further reduced the GlyR puncta density to ∼20% of control values in MNTB neurons. In addition, we found that PV-Nlgn1234, but not neuroligin-4 KO, reduced GlyR puncta size to ∼65% of control values ( Fig. 3 C ). To test whether neuroligin-4 plays a general role in the synaptic maintenance of GlyRs, we measured total GlyR puncta density in the whole MNTB field and found it to be reduced to ∼65% of control values in neuroligin-4 KOs and to be further reduced to ∼50% of control values in PV-Nlgn1234 ( Fig. 3 D ). This indicates a general role of neuroligin-4 in regulating GlyR levels and/or glycinergic synapse density.

It is still possible, however, that neuroligin-4 redundantly contributes to excitatory synapses in MNTB principal neurons together with other neuroligins. We therefore tested the effect of the combined deletion of neuroligin-1/2/3/4 on excitatory synaptic transmission at the calyx of Held synapse. To be systematic, we measured AMPA receptor–mediated EPSCs and mEPSCs from PV-Nlgn1234, PV-Nlgn123, and respective littermate control mice. In both PV-Nlgn123 and PV-Nlgn1234 mice, EPSC amplitudes were reduced to ∼70% of control values and mEPSC amplitudes were decreased to ∼75% of control values (Figs. 5, A–F). This decrease was not associated with a change in synapse density or in the morphology of calyx terminals, as analyzed by immunostaining for synaptotagmin-2 as a specific presynaptic marker (Fig. 5, H and I; Sun et al., 2007). Viewed together, these results indicate that neuroligin-4 is dispensable for excitatory synaptic transmission at the calyx of Held synapse, because the observed impairments in EPSC and mEPSCs in PV-Nlgn1234 and PV-Nlgn123 are identical and likely originate from the deletion of neuroligin3 at the calyx of Held (Zhang et al., 2017).

In the present study, we analyzed the function of neuroligin-4 in excitatory and inhibitory synapses by studying the effect of the neuroligin-4 KO on excitatory and inhibitory synaptic inputs onto the principal neurons of the MNTB, which is a central component of a well-defined auditory circuit in the brainstem. We found that the neuroligin-4 KO caused a large decrease in the amplitude of glycinergic IPSCs and a proportionally smaller decrease in the amplitude of mIPSCs (Figs. 1 and 2). These phenotypes were largely due to a loss of glycinergic synapses (Fig. 3) and specific for inhibitory glycinergic synapses, indicating that neuroligin-4 is essential for glycinergic synapses in mice. Although the neuroligin-4 KO phenotype of glycinergic synapse loss was the most severe single-neuroligin deletion phenotype observed in a direct comparison of mutant mice, it was nevertheless exacerbated by additional deletion of other neuroligins, indicating that, similar to other synapses, multiple neuroligins redundantly contribute to the organization of glycinergic synapses (Figs. 2 and 3). Based on the present data and earlier immunolocalization results (Hoon et al., 2011), we thus propose that the major function of neuroligin-4 in mice is to mediate the formation and/or maintenance of glycinergic synapses, with the decrease in mIPSC amplitude suggesting that this function includes organizing postsynaptic glycinergic receptors. An overall view emerges whereby neuroligin-1 functions primarily in excitatory synapses, neuroligin-2 in GABAergic and cholinergic synapses, neuroligin-3 in both excitatory and inhibitory synapses, and neuroligin-4 in glycinergic synapses and probably also in at least some types of GABAergic synapses. The reduced glycinergic synaptic transmission observed here may at least partially account for some of the behavioral abnormalities in neuroligin-4 KO mice, consistent with the proposal that a glycinergic impairment may be involved in the etiology of ASDs (Pilorge et al., 2016).

Our data raise three major overall issues that partly already emerged in earlier studies on neuroligins. First, the neuroligin-4 KO phenotype described here can be best accounted for by a loss of glycinergic synapses as evidenced by (1) the decrease in IPSC amplitude and the (2) the corresponding decrease in the density of synaptic puncta immunolabeled by antibodies GlyRs or by vGAT. However, a simple loss of glycinergic synapses as a cause of the neuroligin-4 KO phenotype does not account for the decrease in mIPSC amplitude and the modest decrease in GlyR-staining intensity per synaptic punctum, which indicate that, in addition to causing a loss of glycinergic synapses, the neuroligin-4 KO decreases the GlyR content of the remaining glycinergic synapses, most likely affecting GlyRα1 as the predominantly expressed GlyR subunit (Hruskova et al., 2012). This conclusion is consistent with the observation that GlyRα1 clusters are also selectively reduced (by ∼16%) in neuroligin-4 KO retina (Hoon et al., 2011). In addition, the neuroligin-4 KO greatly increased the coefficient of variation in evoked IPSCs. Although such an increase traditionally is interpreted as indicative of a change in release probability, such an increase could conceivably reflect a change in the postsynaptic organization of GlyRs, i.e., could be caused by a disorganization of the alignment of postsynaptic GlyR clusters with presynaptic release sites. Interestingly, the additional deletion of all neuroligins did not simply enhance the neuroligin-4 KO phenotype at glycinergic synapses, but it changed this phenotype. Deletion of all neuroligins not only further decreased the density of glycinergic synapses, resulting in a loss of >80% of synapses but also reduced the GlyR content of the remaining synapses by >30%. Viewed together, these findings raise the question whether the loss of synapses and disorganization of GlyRs induced by neuroligin deletions reflect two independent functions of neuroligins or whether they are the consequence of a single central function of neuroligins in organizing glycinergic synapse.

Second, it is becoming increasingly clear that the genetic loss- or gain-of-function phenotypes induced by neuroligin-1 or -3 mutations at excitatory synapses never appear to produce a decrease in synapse numbers, whereas the loss-of-function phenotypes induced by neuroligin-2 or -4 mutations at inhibitory synapses often cause a decrease in synapse density, as observed here for neuroligin-4 and glycinergic synapses, or in the prefrontal cortex for neuroligin-2 and GABAergic synapses (Liang et al., 2015). This general observation is mirrored by neurexin mutations whereby deletion of neurexins caused a loss of a defined subset of inhibitory synapses (Chen et al., 2017).