The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit functionhttps://doi.org/10.7554/eLife.22757.001
Specific synaptic connectivity and function are essential for the appropriate operation of neuronal circuits. A large degree of this structural and functional specificity is thought to be genetically encoded. For example, synaptic partners express matching pairs of adhesive factors or afferents are repelled from inappropriate targets through chemorepulsive signaling molecules (Sanes and Yamagata, 2009; Shen and Scheiffele, 2010). Gene families encoding large numbers of isoforms generated through multiple genes, alternative promoters and extensive alternative splicing, hold the potential to generate recognition tags for specific trans-synaptic interactions (Baudouin and Scheiffele, 2010; Reissner et al., 2013; Takahashi and Craig, 2013; Schreiner et al., 2014b; Li et al., 2015). However, given the difficulty of mapping endogenous splice isoform repertoires it is poorly understood how splice isoforms are differentially distributed across neuronal cell types.
Neurexins (Nrxn1,2,3) represent one gene family of highly diversified synaptic adhesion molecules. Through the use of alternative promoters (alpha and beta) and alternative splicing at up to six alternatively spliced segments (AS1-6) more than 1300 transcripts are generated that are expressed in the mature mouse nervous system (Schreiner et al., 2014a; Treutlein et al., 2014; Schreiner et al., 2015). Isoform diversity scales with the cellular complexity of brain regions and one purified cell population was shown to be strongly enriched for a subset of isoforms (Schreiner et al., 2014a). This indicated that at least some neurexin isoforms are enriched in a cell type-specific manner. Pairwise comparisons of relative transcript levels recovered from single cells suggested that individual cells within one cell type might exhibit more similar alternative exon usage than cells from divergent origins (Fuccillo et al., 2015). However, the actual alternative exon incorporation rates in interneuron and principal neuron populations have not been examined.
Importantly, individual splice insertions in the neurexin proteins control biochemical interactions with an array of synaptic ligands (Baudouin and Scheiffele, 2010; Reissner et al., 2013). Based on ectopic expression and knock-out experiments in mice it has been postulated that neurexin isoforms might contribute to an alternative splice code for selective synaptic interactions and differ in their tethering at neuronal synapses (Boucard et al., 2005; Chih et al., 2006; Graf et al., 2006; Fu and Huang, 2010; Futai et al., 2013; Aoto et al., 2015; Traunmüller et al., 2016). However, interpretation of such findings is complicated by the fact that the manipulations of isoforms were conducted in cells where endogenous isoform repertoires were unknown. In the human population mutations in Nrxn1,2, and 3 are associated with neurodevelopmental disorders such as autism and schizophrenia (Kim et al., 2008; Yan et al., 2008; Kirov et al., 2009; Rujescu et al., 2009; Gauthier et al., 2011; Vaags et al., 2012). Global deletion of the majority of Nrxn1 or Nrxn3 transcripts in mice or global perturbation of the Nrxn alternative splicing at AS4 disrupts function and plasticity of glutamatergic and GABAergic synapses (Missler et al., 2003; Etherton et al., 2009; Aoto et al., 2013; Traunmüller et al., 2016). However, the function of neurexin isoforms in interneurons has not been examined with targeted approaches.
In this study we uncover a major alternative splice isoform switch that distinguishes glutamatergic and GABAergic cell populations in the hippocampus. We demonstrate that Nrxn1,2,3α transcripts are commonly expressed in pyramidal cells and fast-spiking GABAergic interneurons expressing the calcium binding protein parvalbumin (PV+ cells). However, pyramidal and PV+ cells exhibit highly differential incorporation rates of alternative exons at AS4. This alternative splicing switch depends on the differential expression of RNA-binding proteins and coincides with the cell type specific expression of a neurexin splice isoform-specific ligand. Selective disruption of PV+ cell splice variants in mice results in functional and behavioral abnormalities. Thus, interneuron-specific alternative splicing of neurexins is important for normal circuit function.
To begin to assess the differential expression and functional relevance of neurexin isoforms in mouse neuron populations, we first examined the six primary Nrxn1,2,3α/β transcripts by in situ hybridization. Similar to what has been reported for rat hippocampus (Ullrich et al., 1995) we find significant expression of Nrxn1,2,3α transcripts in cornus ammonis (CA) pyramidal cells as well as presumptive interneurons (Figure 1—figure supplement 1A and B). To specifically interrogate Nrxn transcripts in genetically defined cell populations we tagged ribosomes in CA pyramidal cells and PV+ interneurons, a population of GABAergic, fast-spiking cells that encompasses chandelier and basket cells (Hu et al., 2014). We used a conditional HA-tagged Rpl22 allele (Sanz et al., 2009) crossed with CamK2cre (Tsien et al., 1996) and Pvalbcre drivers (Hippenmeyer et al., 2005), respectively (see Figure 1 and also Figure 1—figure supplement 2 for the selectivity of Rpl22-HA expression in the resulting CamK2Ribo and PVRibo mice). RiboTrap purifications (Heiman et al., 2014) of polysome-associated mRNAs from adolescent (P24-P28) CamK2Ribo or PVRibo mice yielded enrichment of mRNAs from the respective cell populations as confirmed by real-time quantitative PCR (qPCR). Thus, CamK2Ribo preparations showed enrichment of CamK2 mRNA and the CA1-specific marker Wsf1 (Wolfram syndrome 1) (Figure 1B) which is consistent with high cre-activity in the CA1 area (Figure 1A and Figure 1—figure supplement 2A) and a de-enrichment of interneuron and astrocyte markers. By contrast, PVRibo preparations were highly enriched in the common interneuron marker (Gad1) and in the mRNAs specifically expressed in fast-spiking basket cells (Pvalb, Erbb4) (Figure 1B and Figure 1—figure supplement 1B). Nrxn1,2,3 mRNAs were recovered in both CamK2Ribo and PVRibo cell-derived transcript preparations (note that Nrxn3β expression in mouse hippocampus is low and could not be reliably detected – see Figure 1—figure supplement 1A–C). Notably, amongst all neurexin transcripts Nrxn3α was most highly enriched in the PV-cell population (Figure 1C). PV-cell expression of Nrxn3α was further confirmed by dual labeling with in situ hybridization using Nrxn3α probes and immunostaining in mice where PV+ cells were genetically labelled with red fluorescent protein (Pvalbcre::Ai9Tom) (Figure 1D).
To quantitatively probe alternative exon incorporation rates in the Nrxn transcripts we used radioactive PCR amplification with primers flanking the alternatively spliced segments (AS2-AS6). Importantly, this method is not plagued by problems of differential PCR primer efficiencies that are encountered in isoform-specific real-time qPCR. We uncovered similar usage of alternative exons at AS3 across all preparations. Interestingly, Nrxn1 AS6 and Nrxn2 AS2 exhibited differential usage in PV− versus CamK2 cells. Moreover, for all three Nrxn transcripts (Nrxn1,2,3) the alternative exon incorporation rates at AS4 were remarkably divergent between the two cell populations (Figure 2B and C and Figure 2—figure supplement 1A). While in CamK2Ribo cells the cassette exon at AS4 was largely skipped there was a high level of alternative exon inclusion in PVRibo cells. Thus, highly selective, cell type-specific alternative exon incorporation rates of Nrxn mRNAs generate divergent Nrxn splice isoform repertoires in glutamatergic CA pyramidal cells and PV+ interneurons.
Neurexin alternative splicing at AS4 is regulated by the STAR-family of RNA-binding proteins, in particular the protein Slm2 which regulates skipping of the alternative exon (Iijima et al., 2011; Ehrmann et al., 2013; Iijima et al., 2014; Traunmüller et al., 2014, 2016). Thus, we tested whether cell type-specific alternative splicing at AS4 may be a consequence of differential expression of Slm2 in pyramidal versus PV+ cells that are targeted in the CamK2cre and Pvalbcre transgenic lines, respectively. As reported previously (Stoss et al., 2004; Iijima et al., 2014), we observed high expression of Slm2 protein in pyramidal cells, and more than 90% of CamK2Ribo-positive cells were Slm2-positive (Figure 2E). By contrast, 75% of cells marked in Pvalbcre::Ai9Tom mice in hippocampus area CA1 do not express Slm2 (Figure 2E). In particular, cells within or close to the pyramidal cell layer were Slm2-negative (Figure 2D and E - note that the percentage of Slm2-expressing ‘PV+ cells’ depends on the anatomical position within the hippocampus and the use of genetic versus antibody-labeling to define ‘PV+ cells’, Figure 2—figure supplement 1D and E). We further explored differential expression of STAR-family RNA binding proteins by real-time qPCR on polysome-associated transcripts from CamK2Ribo and PVRibo mice (Figure 2F). Consistent with the immunohistochemistry results, we observed an enrichment of Slm2 transcripts in the CamK2Ribo population and a de-enrichment in the PVRibo preparations (similarly the Slm2 paralogues Slm1 and Sam68 were de-enriched). Given that global ablation of Slm2 results in a significant loss of the skipped (AS4-) neurexin isoforms (Ehrmann et al., 2013; Traunmüller et al., 2014), this strongly suggests that differential expression of Slm2 in pyramidal versus PV+ cells drives the cell type-specific skipping of alternative exons at AS4 in pyramidal cells of the hippocampus. Finally, we observed that ectopic expression of Slm2 in cells that normally do not express high levels of STAR proteins (cerebellar granule cells in culture) was sufficient to shift alternative splicing in favor of Nrxn AS4- isoforms (Figure 2—figure supplement 2).
Neurexin AS4+ and AS4- isoforms differ in biochemical interactions with several synaptic ligands. In particular, AS4+ isoforms bind to a class of extracellular ligands called cerebellins (Cbln1-4) (Uemura et al., 2010; Ito-Ishida et al., 2012). We hypothesized that neurexin alternative splicing and Cbln expression might coincide. We detected an enrichment of Cbln4-encoding transcripts in the PV+ cell population (Figure 3A). By contrast, the CamK2Ribo population was de-enriched for both, Cbln2 and Cbln4 transcripts (note that Cbln1 and Cbln3 show very low expression in the mouse hippocampus as compared to the cerebellum, Figure 3—figure supplement 1A). Thus, pyramidal neurons in the hippocampus express high levels of Slm2 which drives production of AS4- splice isoforms, and there are low levels of Cbln2 and 4. Conversely, the PV+ cell population largely lacks STAR-family RNA-binding proteins (Sam68, Slm1, Slm2), exhibit high levels of alternative exon inclusion at AS4, and the cells co-express Cbln4. Despite the similarities between Cbln1,2, and 4 it has remained unclear to what extent Cbln4 interacts with Nrx AS4+ proteins. In fact, binding to Nrx3 AS4+ isoforms has been reported to be low or not detectable (Joo et al., 2011; Matsuda and Yuzaki, 2011; Wei et al., 2012). Using in vitro binding assays with Cbln4 from conditioned media we confirmed binding to cells expressing Nrx1α AS4+ but significantly lower binding to Nrx3 AS4+ isoforms (Figure 3B–D). We then tested another assay configuration where Cbln proteins and Nrx isoforms are co-expressed in the same cell (Figure 3E). In this assay, Cbln1 or 4 were strongly retained at the cell surface of cells co-expressing Cbln1 or Cbln4 together with Nrx3 AS4+ isoforms but not Nrx3 AS4- (Figure 3F and G). Thus, Cbln4 indeed associates not only with Nrx1α but also Nrx3α proteins in a AS4 splice-isoform-specific manner (further studies will be required to confirm whether these interactions are direct or whether they may involve additional linker proteins).
The observation that AS4+ isoforms are highly expressed in PV+ cells is notable since AS4+ variants of beta-neurexin exhibit a synaptogenic activity preferentially for GABAergic over glutamatergic postsynaptic structures in cell culture assays (Chih et al., 2006; Graf et al., 2006). However, the majority of neurexin proteins in vivo are alpha isoforms (Schreiner et al., 2015) and the impact of AS4+ insertions on the synaptogenic activity of alpha neurexins has not been examined. Thus, we tested whether the presence or absence of AS4 insertions might modify the synaptogenic activity of Nrxα proteins. Inclusion of the 30 amino acid AS4+ insertion elevated the action of Nrx3α proteins towards GABAergic postsynaptic sites (Figure 3H and I and Figure 3—figure supplement 1C. Note that for Nrx1α we did not observe the same impact of the AS4+ insertions, Figure 3—figure supplement 1C–E). Co-expression of Cbln4 with the Nrx isoforms neither enhanced nor inhibited the synaptogenic activity in this assay (Figure 3H and I). Thus, this function either does not directly involve Cbln4 or there is sufficient endogenous Cbln4 expressed in the neuronal cultured to transduce a neurexin signal towards the interaction partners at GABAergic postsynaptic sites.
To assess whether the molecular program identified here is functionally relevant we generated Nrxn3 AS4 splice isoform-specific conditional knock-out mice in which exon 21 (which encodes the alternative insertion at AS4) is flanked by loxP sites (Figure 4A). Upon germline ablation we observed a complete loss of exon 21-containing Nrxn3 transcripts (Figure 4B). Loss of Nrx3 AS4+ protein isoforms was confirmed by serial reaction monitoring mass-spectrometry assays (Schreiner et al., 2015) designed to specifically quantify expression of AS4+ isoforms (Figure 4C). Importantly, alternative splicing in Nrxn1 and 2 as well as total Nrx1,2,3 protein levels were unchanged (Figure 4B and D). Thus, the Nrxn3 exon 21 mutation specifically alters the splice isoform identity but not total protein or transcript neurexin levels in the hippocampus.
We then conditionally ablated exon 21 selectively in PV+ cells using Pvalbcre mice (Nrxn3ex21flox::Pvalbcre, in the following referred to as Nrxn3ex21ΔPV). Considering that neurexin proteins derived from the Nrxn1,2,3 genes may exhibit overlapping and/or redundant functions, we combined Nrxn3ex21ΔPV mutants with conditional Nrxn1ex21flox knock-out mice (Traunmüller et al., 2016). The conditional Nrxn3ex21ΔPV single mutant mice and Nrxn1/3ex21ΔPV double mutant mice were born at Mendelian ratios and were viable and fertile (Figure 4—figure supplement 1). Overall, hippocampal anatomy and density of PV-immunoreactive cells was not detectably changed, indicating that the mutations did not result in severe developmental defects (Figure 4E). In the conditional single and double knock-out mice there was no change in the density of perisomatic synapses detected by immunostaining for the postsynaptic GABAergic marker Neuroligin2 (NL2) and synaptotagmin2 (Syt2) (which selectively marks PV+ cell terminals [Sommeijer and Levelt, 2012]) (Figure 5A and B and Figure 5—figure supplement 1A and B). Moreover, we did not detect any change in inhibitory synapses formed between PV+ interneurons as identified as Syt2-positive structures formed onto PV+ somata (Figure 5—figure supplement 2).
The ultrastructure of perisomatic PV+ interneurons output synapses formed onto pyramidal cells (identified based on location and ultrastructural characteristics [Takács et al., 2015]) was unaltered in the conditional single and double knock-out mice (Figure 5C–F and Figure 5—figure supplement 1C–F). Moreover, frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) recorded from CA1 neurons in conditional single and double knock-out mice were unchanged (Figure 5G and H and Figure 5—figure supplement 1G and H). These findings indicate that basal inhibitory synaptic transmission in the CA1 pyramidal cells and synaptic structure are not severely affected by the ablation of the PV-cell specific Nrxn1 and Nrxn3 AS4+ isoforms.
As a more general read-out for the function of inhibitory networks in the hippocampus, we scored the density of c-fos immunoreactive cells in hippocampal sub-regions DG, CA3, CA1 (Figure 6A). These experiments revealed a significant elevation in the density of c-fos+ cells in CA3 regions of the Nrxn1/3ex21ΔPV mice (Figure 6B), indicating that network activity is elevated upon ablation of the PV-cell-specific Nrxn splice insertions. Given these alterations in hippocampal network activity, we tested whether Nrxn1/3ex21ΔPV mice display behavioral deficits using a novel object recognition task which – at least in part – relies on hippocampal function (Cohen and Stackman, 2015). In this task, control mice exhibit a significant preference for exploring novel over familiar objects. By contrast, we observed that Nrxn1/3ex21ΔPV mutant mice spent equal time exploring familiar and novel objects, indicating a defect in short-term memory (Figure 6C and D). These defects were not a consequence of general behavioral disruptions as the mice showed similar total object exploration times, normal locomotion, and no signs of anxiety-related behaviors (Figure 6E–K). Thus, PV-cell-specific loss of Nrxn1 and 3 AS4+ splice variants results in impairment in neuronal circuit function.
In this study we identify an alternative splicing switch that distinguishes principal and PV+ interneurons in the hippocampus. First, we demonstrate that alpha neurexin transcripts are commonly expressed in pyramidal cells as well as PV+ interneurons. We then show that alternative splicing at the alternatively spliced segment four (AS4) is a major driver for the generation of divergent neurexin isoforms in PV+ cells and pyramidal cells. Differential alternative splicing emerges from differential expression of the RNA-binding protein Slm2 that drives selective production of AS4- splice isoforms in pyramidal cells. By contrast, Slm2 is absent from the majority of PV+ cells which produce AS4+ isoforms and co-express the splice isoform-specific ligand Cbln4. Finally, we provide evidence that selective disruption of the PV+ cell-associated AS4+ isoforms results in increased hippocampal network activity and impairs short-term memory, demonstrating that the cell type-specific alternative splice variants are indeed relevant for circuit function.
Previous work provided evidence for cell type-specific repertoires of full-length neurexin transcripts (Schreiner et al., 2014a). Pairwise comparison of alternative exon amplification indicated similar exon amplification from single cells within one class of cells (Fuccillo et al., 2015). However, these approaches did not provide actual exon incorporation rates across cell types which are a prerequisite for understanding the logic of cell type-specific function of endogenous isoforms.
Our observation that AS4 insertion-containing Nrxn mRNAs are dominant in PV+ cells and abundant in the entire hippocampus was surprising as it had been previously suggested that these Nrxn3 AS4+ isoforms make up less than 10% of Nrxn3 in the mouse hippocampus (Aoto et al., 2013). We note that the estimates of exon incorporation rates based on extrapolation from several independent qPCR assays can make it difficult to reliably assess isoform contents due to differential primer efficiencies. The high abundance of AS4+ isoforms in the hippocampus reported in the present study is supported by radioactive and semi-quantitative PCR assays, as well as previous mass-spectrometric assays that probe Nrx3 AS4+ variants on the protein level (Figure 2 and [Ehrmann et al., 2013; Traunmüller et al., 2014; Schreiner et al., 2015]). Thus, we conclude that these variants are indeed significantly expressed. More importantly, we demonstrate that they are specifically enriched in interneurons but largely absent from pyramidal cells in the hippocampus.
The highly selective alternative splicing choices in the neurexin pre-mRNAs suggest that they result from neuronal cell-type specific expression of alternative splicing factors. The high expression of Nrxn1,2,3 AS4- variants in pyramidal cells discovered here correlates with the high expression of Slm2 protein in pyramidal cells and the near complete loss of these variants in Slm2 knock-out mice (Ehrmann et al., 2013; Traunmüller et al., 2014). Moreover, we demonstrate that Slm2 is absent from the majority of hippocampal CA1 interneurons marked in the Pvalbcre::Ai9Tom mouse line. Correspondingly, these cells generate high levels of AS4+ neurexin isoforms. It remains to be tested whether the absence of STAR-family RNA binding proteins is sufficient to direct alternative splicing to the inclusion of AS4+ or whether other, yet unidentified alternative splicing factors contribute to this alternative splicing choice. Finally, we demonstrate that this selective expression of AS4+ variants coincides with PV+ cell enrichment of Cbln4, an AS4+ specific neurexin ligand. Interestingly, the differential expression of AS4 isoforms in PV versus principal cells is also in part observed for cortical cell populations (Figure 2—figure supplement 1E). In additional RiboTrap purifications from somatostatin-positive interneurons in the hippocampus we also observed an enrichment of Nrxn AS4+ isoforms as compared to principal cells (Figure 2—figure supplement 1F) along with Slm2-negative somatostatin-expressing cells in the hippocampus (in particular in the hilus, Figure 2—figure supplement 1D). Thus, our study uncovers a hippocampal interneuron-specific gene expression program that consists of neurexin isoforms and ligands in the hippocampus.
The difficulty of assigning cell type-specific Nrxn transcripts and splice isoform expression has been a major impediment for the interpretation of functional studies. Global knock-out studies demonstrated that neurexin alpha triple knock-out mice show a 40% reduction in the density of presumptive inhibitory synapses and defects in synaptic transmission in the brain stem of perinatal mice (Missler et al., 2003). Moreover, simultaneous global ablation of all Nrxn3 transcripts results in brain area-specifc alterations in synaptic transmission (Aoto et al., 2015). In these studies it was unknown which endogenous primary transcripts and which splice isoforms are expressed and the knock-out manipulations were not cell type-specific. The data presented here demonstrate that pyramidal and PV+ cells share primary Nrxn1,2,3 transcripts but that the endogenous neurexin isoforms differ dramatically in their alternative splicing regulation at AS4.
In cellular assays, we demonstrate that the AS4+ splice insertion in Nrx3α selectively increases Nrx3 activity towards assembly of GABAergic postsynaptic structures and that AS4+ variants bind to the extracellular ligand Cbln4 that is co-expressed in PV+ cells. In the mouse cerebellum, Neurexin-Cbln1 interactions have been demonstrated to be essential for the formation and stability of granule cell-Purkinje cell synapses (Uemura et al., 2010; Ito-Ishida et al., 2012). Similar to granule cells, PV+ cells in the hippocampus express a Cbln family protein (Cbln4) and neurexin AS4+ isoforms which interact with Cblns. However, genetic disruption of Nrxn1 and 3 AS4 insertions did not result in morphological alterations of PV+ cell output synapses detectable at the level of light or electron microscopy. Structural functions of a Nrx1/3-Cbln4 pair, analogous to the cerebellar system, may be compensated by the remaining Nrx2 AS4+ isoforms or conditional ablation of the AS4 insertions with the Pvalbcre line may not be complete when PV+ cell synapses form during postnatal development. Alternatively, the essential roles for Nrx1/3-Cbln4 complexes in PV+ cells may differ from what is observed for Nrx-Cbln1 complexes in the cerebellum. Regardless, the increased c-fos+ expression in the hippocampus of Nrxn1/3ex21ΔPV mice indicates that the AS4+ isoforms have important functions and presumably reflects an impairment in PV+ cell mediated inhibition in hippocampal circuits. Moreover, the impairment of short-term memory further supports the notion that neurexin AS4+ isoforms have important functions in the cells targeted by the Pvalbcre line in the hippocampus. Dysfunction of PV+ interneuron networks has been postulated to be a major contributor to the pathophysiology of schizophrenia and autism (Del Pino et al., 2013; Gogolla et al., 2014). Thus, we speculate that part of the neuronal circuit deficits resulting from Nrxn1 and 3 mutations in disease states may directly impair output synapses of the PV-interneuron network.
All procedures involving animals were approved by and performed in accordance with the guidelines of the Kantonales Veterinäramt Basel-Stadt (Licences 2272, 2293, 2599). Nrxn3ex21Δ conditional knock-out mice were generated in collaboration with Dr. Siu-Pok Yeh at the University of Connecticut Gene Targeting Facility. The mice carry loxP sites on either side of exon 21 of the Nrxn3 gene. A targeting vector was designed with a loxP site placed 718 bp upstream of exon 21 of mouse Nrxn3 and a loxP-Frt-PGKneo-Frt cassette inserted 840 bp downstream of the same exon. The 5’ and 3’ homology arms were approximately 5 kb and 3 kb in length, respectively, and the targeting construct contained herpes simplex virus thymidine kinase as negative selectable marker in the 3' arm of the targeting vector. The construct was electroporated into ES cells derived from F1(129Sv/B6) embryos. Correctly targeted ES cells were then used to generate chimeric mice. Male chimera were mated with ROSA26-Flpe females (Jax stock no: 003946), which had been backcrossed over five generations with C57Bl6J, to remove the PGKneo cassette. The resulting heterozygotes were intercrossed to produce homozygotes. Mice that are homozygous for this allele are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Germline ablation was created by crossing with CMV-cre mice (Jax stock no: 006054)(Schwenk et al., 1995). Nrxn1ex21Δ conditional knock-out mice were previously described (Traunmüller et al., 2016). Rpl22-HA (RiboTag) mice (Sanz et al., 2009), Pvalbcre mice (Hippenmeyer et al., 2005), SSTcre mice (Taniguchi et al., 2011), CamK2cre mice (Tsien et al., 1996) and Ai9Tom (Madisen et al., 2010) mice were obtained from Jackson Laboratories (Jax stock no: 011029, 017320, 005359, 007909 respectively).
In situ hybridizations were performed as previously described (Schaeren-Wiemers and Gerfin-Moser, 1993). DNA constructs encoding probes for primary neurexin transcripts contained SP6 and T7 promoters at 5’- or 3’-end, respectively:
Templates for in vitro transcription using SP6-polymerase (sense probe) or T7-polymerase (anti-sense probe) were amplified by PCR using ISP-SP6-5' (5’-CTATCGATTTAGGTGACACTATAGAAG-3’) and ISP-T7-3' (5’-GAATTGTAATACGACTCACTATAGGGA-3’) primers.
For the dual labeling of Nrxn3α in PV+ cells in CA1 of Pvalbcre::Ai9Tom mice, the in situ hybridization with Nrxn3α probes was performed first. To identify the td Tomato PV+ cells, the brain sections were incubated with anti-RFP. The imaging was done using a Nikon Eclipse E800 (Plan Apo 40X/0.95)
Custom antibodies to Slm2 were previously described (Iijima et al., 2011). The following commercially available antibodies were used: anti-c-Fos (K-25, Santa Cruz), anti-Gapdh (D16H11, Cell Signaling), anti-Gephyrin (mAb7a, Synaptic Systems), anti-GFP (affinity purified, homemade), anti-HA (11867431001, Roche), anti-His6 (A190-114A, Bethyl), anti-Myc (9E10, Invitrogen), anti-NeuN (MAB377, Chemicon), anti-Neuroligin2 (sc-1487, Santa Cruz), anti-pan Nrx (affinity purified, homemade), anti-Parvalbumin (PVG214, Swant), anti-PSD95 (51–6900, Invitrogen Novus), anti-RFP (600-401-379, Rockland) anti- Synaptotagmin2 (Znp1, Zebrafish International Resource), anti-Tubulin (E7, Developmental Studies Hybridoma Bank),anti-V5 (C-9, Santa Cruz).
Selected reaction monitoring (SRM) for quantification of neurexin protein levels was performed as previously described (Schreiner et al., 2015).
For Ribotag purifications the procedure of Heiman and colleagues for affinity-purification of polysomes (Heiman et al., 2014) was modified as follows:
Brains of mice between (postnatal day 24 to 28) were dissected in ice-cold PBS and four hippocampi (two animals per condition) were lysed in 500 µL of homogenization buffer containing 100 mM KCl, 50 mM Tris-HCl pH 7.4, 12 mM MgCl2, 100 ug/mL cycloheximide (Sigma), 1 mg/mL heparin (Sigma),1x complete mini, EDTA-free protease inhibitor cocktail (Roche), 200 units/mL RNasin© plus inhibitor (Promega) and 1 mM DTT (Sigma). The lysate was centrifuged at 2’000xg for 10 min. Igepal-CA380 was then added to the supernatant to a final concentration of 1%. After 5 min incubation on ice, the lysate was centrifuged at 12’000xg for 10 min. Anti-HA coupled magnetic beads (Pierce) were added to the supernatant in the following concentrations: 25 µL/mL for CamK2Ribo and 15 µL/mL of beads for PVRibo samples, respectively. Incubation was performed at 4°C for 4 hr. The beads were washed four times in washing buffer containing 300 mM KCl, 1% Igepal-CA380, 50 mM Tris-HCl, pH7,4, 12 mM MgCl2, 100 µg/mL Cycloheximide (Sigma) and 1 mM DTT(Sigma). The beads were eluted in 350 µL of RLT plus buffer (Qiagen). The RNA purification was performed using RNeasy mini plus kit following the manufacturers’ instructions. 30–50 ng of total RNA was reverse transcribed using random hexamers and ImProm II Reverse Transcriptase (Promega).
For the misexpression of Slm2, granules cells were infected with either AAV encoding for Slm2-2A-Venus YFP or GFP containing the human synapsin promoter at day in vitro three and were harvested at day in vitro 14.
Quantitative PCR was performed on a StepOnePlus qPCR system (Applied Biosystems). To assess Nrxn expression level gene expression assays (see Table 1, Table 2) were used with TaqMan Fast Universal Master Mix (Applied Biosystems) and comparative CT method. The mRNA levels were normalized to β-actin mRNA or to pan-Nrxn mRNA. To determine the enrichment fold of mRNA purification, DNA oligonucleotides were used with FastStart Universal SYBR Green Master (Rox) (Roche) and comparative CT method (see Table 3). For each assay, two technical replicates were performed and the mean was calculated.
Standard PCR reactions were performed using 5X Firepol Master mix (Solis BioDyne). Radioactive semi-quantitative PCR was performed with γ-32P 5’ end-labelled primers (Hartmann) using 5X Firepol mastermix (Solis Biodyne). For the quantification of the radioactive semi-quantitative PCR (see Table 4), the gel image was acquired with Typhon FLA 700 (GE Healthcare). The PCR band intensity was analyzed using Fiji software. The background intensity was subtracted from the mean intensity of each PCR band and then divided by the number of GTP and CTP in PCR product (MeanNorm). To calculate the % of inclusion the following formula was used: MeanNormAS+/[(Mean NormAS4+)+(Mean NormAS4−)].
Animals (male and female) from postnatal day 24 to 26 were transcardially perfused with fixative (4% paraformaldehyde/15% picric acid in 100 mM phosphate buffer, pH 7.4). The brains were post-fixed overnight in same fixative at 4°C and incubated in 30% sucrose in PBS for two nights at 4°C and frozen at −80°C. Tissue was sectioned at 30 µm on a cryostat for synapses quantification and 35 µm for HA. Floating sections were immunostained following standard procedures for HA immunostaining. For synapse quantitative analysis, sections were treated with pepsin before staining, as described (Panzanelli et al., 2011). Briefly, sections were incubated in prewarmed 0.1M phosphate buffer at 37°C for 10 min and then in 0.15 mg/mL pepsin (Dako) dissolved in 0.2M HCl for 10 min. Images for synapse quantification were acquired at room temperature on an upright LSM700 confocal microscope (Zeiss) using 63x Apochromat objectives and controlled by Zen 2010 software.
For quantification of synaptic markers, three to four brain sections were used per genotype. Additionally, four to six confocal planes within the stratum pyramidale of CA1 were acquired per section. Using Metamorph software (Molecular Devices) area of somata were manually drawn to measure the area. Synaptotagmin2 and perisomatic Neuroligin2 punctae were counted manually in separated channels and normalized to 100 µm2. For each genotype the average synapses punctae for each animal was calculated. Control littermates mice carried the floxed alleles but were Cre negative (n = 6 mice for control and five mice for Nrxn3ex21ΔPV and four mice each for control and Nrxn1/3ex21ΔPV).
The quantification of the number of PV+ cells was also done using an upright LSM700 confocal microscope (Zeiss) using 10X objectives. Stacks of 22 µm with (2.2 µm step size) were acquired.
Images for assessing the Rpl22-HA expression in the CamK2Ribo and PVRibo were acquired at room temperature on an inverted LSM500 confocal microscope (Zeiss) using 10x and 20 x objectives.
To quantify the number of PV inputs on PV+ interneurons, brain sections from mice aged between 5 to 8 weeks were stained for Synaptotagmin2 and Parvalbumin (n = 4 mice for each genotype). The images of dorso-medial hippocampi were acquired on a LIS-spinning disk confocal system (40X/1.3 NA objective, 0.1 µm step size). Four separate fields of the hippocampal regions of interest were acquired in two separate brain sections per animal. The density of Synaptotagmin2 on PV+ interneurons was manually counted using Fiji software. The average synapses punctae for each animal was calculated.
For Slm2 immunolabelling, animals (male and female postnatal day 25–30) were transcardially perfused with fixative (4% PFA in 100 mM Phosphate Buffer, pH = 7.2). Tissue was sectioned at 50 µm in PBS on a vibratome. Images of dorso-medial hippocampi were acquired at room temperature on a confocal microscope using 40X Apochromat objectives, which were controlled by Zen 2010 software (1 µm step sizes). For assessment of Slm2 expression in Pvalbcre::Ai9Tom (n = 5 mice) and SSTcre::Ai9tom (n = 4 mice) positive interneurons, Imaris was used. Briefly, Slm2 intensity levels were characterized in tdTomato-positive cells and clustered into Slm2 negative, high and intermediate levels. Assessment of Slm2 expression in pyramidal cells was done in the dorso-medial hippocampi of CamK2Ribo animals (n = 4 mice). Hippocampal slices were stained for HA (for Rpl22-HA expression) and Slm2. Overlap between Rpl22-HA and Slm2 was determined by analysis of single planes in Fijii (Image J). Slm2 background intensity levels were determined by Slm2 immunostaining in the dentate gyrus and Slm2KO mice.
For analysis of overlap between Pvalbcre::Ai9Tom positive interneurons and antibody labelled PV neurons, dorso-medial hippocampal slices were stained for Parvalbumin (n = 5 mice, postnatal day 25–30). Co-labelling of genetic td Tomato and the PV antibody was assessed in stacks of ~30 µm (1 µm step sizes) with Fijii (Image J).
Images were assembled using Adobe Photoshop and Illustrator Software.
Statistical analyses were done with Prism software (Graphpad software). The image acquisition and analysis were done blinded with respect to the genotype of the animals.
Animals (postnatal day 24–25, Nrxn3ex21ΔPV, Nrxn1/3ex21ΔPV and respective control littermates which carried the floxed alleles but were Cre negative) were transcardially perfused with fixative (2% paraformaldehyde, 2% glutaraldehyde in 100 mM phosphate buffer [pH 7.4] and brains were postfixed for 1 hr. Tissues were sectioned coronally at 60 µm thickness in PBS on a vibratome. Sections from the dorso-medial hippocampi were analyzed for each genotype. Sections were washed in 0.1 M cacodylate buffer [pH 7.4], postfixed in 0.1 M reduced osmium (1.5% K4Fe(CN)6, 1% OsO4 in water) and embedded in Epon resin. Images were acquired on a Transmission Electron Microscope (Fei Morgagni, 268D). Quantification of the number and distribution of vesicles was performed using Reconstruct software (http://synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm). All image acquisition and analysis was done blinded with respect to the genotype of the animals. Independent data sets were collected from four mice for each genotype. PV-terminals were identified by their localization on the neuron plasma membrane and the presence of large mitochondria. The vesicles and the active zones were manually drawn. The shortest distance from the vesicle membrane to the active zone membrane was then calculated and all vesicles at distances of less than 200 nm were taken into account.
Co-culture assays were performed essentially as previously described (Scheiffele et al., 2000). Hippocampal neurons isolated from E16 mouse embryos were seeded at a density of 40.000 cells / well (24 well plates) on cover slips coated with poly-D-lysine. At DIV19 HEK293 (ATCC, Cell line was used within 2 years of being purchased from ATCC. No Mycoplasma contamination was detected in bi-annual tests of the cultures) cells transfected with neurexin-alpha constructs (with or without insertion at AS4) were added at a density of 6.000 cells / well. Three days after co-culture mixed cultures were fixed with 4% PFA/4% Sucrose in PBS for 10 min at RT, washed 2x with PBS, and blocked and permeabilized in blocking solution (10% normal donkey serum/2% BSA/0.1% Triton-X100 in PBS) for 1 hr at RT. Blocking solution was removed and cover slips were incubated with primary antibodies anti-Gephyrin (mAb7a, Synaptic Systems) diluted 1:2000 in blocking solution overnight. Subsequently cover slips were washed twice with PBS and incubated with secondary antibodies (anti-mouse Alexa-564/1:750 in blocking solution) for 2 hr at room temperature, washed 4x with PBS and mounted on glass slides for microscopy.
Pictures (40x per experiment and condition) were acquired with a Nikon Eclipse E800 with an 60x objective (Plan Apo 60XA/1.40, oil) from fields containing GFP positive cells (transfected HEK-cells) and analyzed with ImageJ software as followed: (i) gephyrin positive areas co-localized with GFP-positive area were extracted using Colocalization Highlighter plugin, (ii) extracted gephyrin positive area as well as GFP positive area were calculated using particle analysis plugin, (iii) percentage of gephyrin positive area to total GFP area were calculated.
For Cbln binding assay COS7 (ATCC, Cell line was used within 2 years of being purchased from ATCC. No Mycoplasma contamination was detected in bi-annual tests of the cultures) cells grown in six well plates in DMEM medium supplemented with 10% FBS were transfected with full-length HA-Nrx1α, HA-Nrx3α (with or without insertion at the AS4), HA-Nrx or mock transfected. 24 hr post-transfection cells were trypsinized and distributed on 48-well plate (six well each construct / plate). On the next day cells were washed with warm DMEM. Conditioned medium containing Cbln1, 2 and 4 was collected from HEK293 cells and were added to Nrx-expressing cells. Plates were incubated for 3 hr at 37°C, washed 3x with cold DMEM and fixed with 4%PFA for 10 min. Fixed cells were washed 1x with PBS and blocked with 5% milk in PBS for 1 hr. After blocking cells were washed with PBS and incubated with primary antibody (mouse anti-V5-probe, or rat anti-HA, 1:2000, Roche) diluted in 1% dry milk in PBS at 4°C overnight. On the next day cells were washed 3x with PBS and HRP-conjugated antibody (1:2000, anti-Mouse-HRP or 1:4000, anti-Rat-HRP for detection of V5 or HA-antibodies, respectively) was added for 2 hr at RT. Subsequently, cells were washed 4x with PBS, and 100 µl/well Ultra-TMB substrate (Pierce) was added to the well. The reaction was stopped by addition of 1N NaCl, and the absorption was measured at 450 nm in an iMark microplate reader (Biorad).
For Cbln surface retention assays COS7 cells were co-transfected with HA-tagged Nrx1/3-full –length and Cbln-Myc constructs. 24 hr post-transfection cells were trypsinized and distributed on 96 well plates (six well each construct / plate). On the next day cells were fixed and cell surface bound Cbln and surface expressed Nrx were probed as described.
For acute slice recording, postnatal day 23–30 mice were anesthetized with isoflurane and rapidly decapitated. Three hundred micrometer thick sagittal sections were cut in sucrose substituted artificial cerebrospinal fluid (ACSF) that consisted of 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose, 75 mM sucrose, 0.5 mM CaCl2, 7 mM MgCl2. Slices were allowed to recover at 34°C for 1 hr and then maintained at room temperature in the same sucrose ACSF. For whole-cell recordings, slices were perfused with 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, 25 mM glucose, 4 uM AP5, 2 uM GYKI. For all experiments, whole-cell recordings were digitized at 10 kHz and filtered at 2 kHz. Whole-cell patch-clamp recordings of CA1 pyramidal cells were done using 2.7–3.5 U pipettes and filled with an internal solution that contained 170 mM CsCl, 10 mM Hepes, 0.5 mM QX314, 2 mM Mg-ATP, 0.5 mM NaGTP, 2 mM EGTA, 318 mOsm, pH 7.27. The cells were held at a holding potential of –70 mV. For mini recordings, slices were also perfused with 500 nM TTX. The mIPSCs were detected using Igor and the mIPSCs were detected using macros written by Dr. Taschenberger and modified by Dr. Kochubey.
Behavioral testing was done with littermate control (carrying floxed alleles but were Cre negative) and Nrxn1/3 ex21ΔPV males which were aged between 6 and 15 weeks. Behavioral tests were done with two mouse cohorts (five mice per genotype in each cohort). Before each behavioral test, mice were allowed to acclimate in the behavioral room for at least 30 min. In each tasks, arena and objects were cleaned with 70% ethanol between trials.
One day before the novel object recognition test, mice were placed in an open field arena, where they were allowed to explore freely 7 min the arena. Explorative behaviors were recorded by a Noldus Camera. Using the video tracking software Ethovision 10, the distance traveled, velocity and time spent in the center of arena were determined. This experiment was used as a habituation for the novel object recognition test.
For the novel object recognition test, mice were placed in a squared arena (50x50×25 cm). The recordings were acquired with a Canon camera. Animals were allowed to explore two identical small Falcon tissue culture flasks filled with sand for 5 min. After a 1 hr inter-trial interval, one flask was replaced with a tower of Lego bricks and duration of interaction was assessed in a 5 min trial. Mouse behaviors were acquired with a Canon camera and exploration time for each object was measured manually. Preferences for the novel object was expressed as a Discrimination Index (DI): (time novel object− time familiar object)/(time novel object+time familiar object). If a mouse exhibited less than 2 s in the total time exploring the objects (time novel object+time familiar object), it was excluded from the analysis. Object exploration was defined as the orientation of the mouse snout toward the object, sniffing or touching with the snout within 2 cm proximity. Leaning, climbing, looking over or biting the objects were not considered as exploration time. The position of the objects in the test was counterbalanced between the animals in a group.
For the elevated plus maze, mice were placed at the junction of four arms (35 cm x 6 cm, 74 cm above the ground) of the maze and the behavior was recorded by a camera (Canon) for 5 min. Entries and duration in each arm were measured manually.
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Sacha B NelsonReviewing Editor; Brandeis University, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]
Thank you for submitting your work entitled "An alternative splicing switch shapes neurexin repertoires in principal neurons versus interneurons of the hippocampus" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom, Sacha B Nelson (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Joris De Wit (Reviewer #2).
Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that we will not be able to publish your manuscript in eLife in its current form.
The editorial policy at eLife requires that manuscripts requiring additional experimental work that would take more than approximately two months be rejected. There is also no separate "soft reject" or "reject-resubmit" category recognized. In this case, however, all three reviewers recognized the merits of the study and support publication, but also felt clarification, and importantly, additional experiments were likely needed to support the claims made in the paper. Assuming the results of these experiments are not already in hand, they would likely take several months to complete.
The Reviewing Editor has identified the following key points for revision with input from the other reviewers and Senior Editor:
1) All three reviewers felt that the absence of a synaptic deficit significantly reduced the impact of the paper and made it difficult to relate the behavioral phenotype to molecular differences between hippocampal cell types identified. Further attempts to address this are warranted. The problem is that there is no evidence tying the behavioral phenotype to the cells on which the rest of the paper focuses. The tie could be made by seeking a synaptic phenotype, but if the authors have some other way of making the link, that would be fine too.
2) Two of the reviewers felt that the relationship between cerebellin expression and neurexin function was not pursued adequately. Some additional experiments were proposed but it is also possible that this concern could be addressed with results on hand and textual clarification or by removing this section.
3) An additional concern of one reviewer was the lack of direct evidence linking Slm2 levels and AS4 splicing in these cells. While it would improve the manuscript to address this with additional manipulations this is not required for acceptance.
Since the discovery that neurexins undergo extensive alternative splicing, scientists have speculated that cell type specific splicing is important for cell type specific patterns of connectivity. Several studies have provided evidence that multiple splice variants are expressed and some studies have argued that this expression is cell type specific, but this is the first study I am aware of to directly test the consequence of a cell type specific knockout of a specific isoform.
The authors first show that Pvalb interneurons selectively express the AS4+ isoform and that this is likely due to the fact that they lack expression of the Slm2 RNA binding protein. They also show that heterologous expression of this splice variant in culture enhances formation of inhibitory synapses. They then use a conditional allele to selectively delete this isoform in Pvalb+ cells in mice and show a behavioral deficit in a working memory task.
The results presented are convincing and are important because they directly test key aspects of the neurexin splice code hypothesis. Unfortunately, the paper is missing a phenotype at the synaptic level. Somatic inhibitory synapses onto pyramidal neurons appear normal and mIPSCs are not affected (nor are numbers of Pvalb neurons). But the findings – that the potentially relevant ligand (Cbln4) is expressed in the same Pvalb cells as the NrxAs4+ transcripts, and that these Nrx transcripts enhance inhibitory synapses – argue that the relevant synaptic deficit to look for are inhibitory synapses between Pvalb cells. At least in the neocortex, these are the dominant source of inhibition of these interneurons and disruption of this synapse might destabilize the persistent activity hypothesized to underlie working memory. Therefore, it is disappointing that the authors did not look for anatomical and physiological evidence of reduced inhibition between Pvalb interneurons. If the authors did look for this and did not find it, this negative result is an important one that should be included. (As an aside, SST neurons also provide some inhibitory input to Pvalb neurons in some forebrain structures).
I think the paper is probably important enough to publish even without this synaptic punchline, but it would have greatly increased impact with this additional data if this is feasible.
Nguyen et al. investigate neurexin (Nrxn) isoform diversity in specific neuronal cell types and uncover a major, cell type-specific difference in Nrxn alternative splice site 4 (AS4) usage. They find that parvalbumin (PV)-positive interneurons exhibit a high level of AS4 inclusion (AS4+), whereas pyramidal cells are preferentially AS4-. The AS4 inclusion in α Nrxns increases their ability to induce inhibitory postsynaptic specializations in cell culture. In addition, expression of AS4+ Nrxns in PVs coincides with the secreted Nrxn ligand Cbln4, which specifically binds to AS4+ Nrxns. Thus, pyramidal and PV neurons have differential molecular repertoires of Nrxn isoforms and Nrxn ligands. The authors then test the effects of deleting Nrxn1 and Nrxn3 AS4 in PV neurons and find no changes in PV synapse structure or function, but do find changes in short-term memory.
Overall this is a well-performed study that will be of broad interest to the field. Deciphering how cell type-specific repertoires of molecularly diverse cell surface proteins are generated and contribute to the formation of precisely wired neural circuits is an important and timely topic. I have only a few comments/suggestions that need to be addressed before publication.
1) The authors show a correlation between low PV neuron expression of the STAR-family RNA-binding protein Slm2, which regulates AS4 skipping, and high levels of AS4+ Nrxn isoforms in these cells. The manuscript would be strengthened by putting the causal relation between absence of Slm2 and cell type-specific exon skipping to the test. One experiment to test this would be to use AAV to misexpress Slm2 specifically in PV cells in a Cre-dependent manner and analyze whether this causes a shift towards more AS4-Nrxn isoforms. Another experiment would be to analyze Nrxn isoform repertoires in pyramidal cells of CamK2 Ribotrap mice crossed with Slm2 KO mice.
2) The interaction of Cbln4 with Nrxn AS4+ shown in Figure 3, and the functional relevance thereof, should be further clarified. In Figure 3D, Cbln4 shows little binding to Nrxn3α AS4+ and Cbln1 shows some binding to Nrxn3α AS4+, whereas in 3F Cbln4 shows binding to Nrxn3α AS4+ to a similar degree as Cbln1. How can the apparent difference in Cbln binding to Nrxn between Figure 3D and F (COS cell binding vs surface retention) be explained, as both experiments measure Cbln at the surface? Does binding of Cbln4 to Nrxn AS4+ affect the induction of postsynaptic inhibitory specializations by AS4+ α Nrxn? In Figure 3C and D, are Cbln1, which binds most strongly to Nrxna AS4+, and Cbln3 also expressed in PV neurons? Finally, is expression of Cbln4 in PV neurons perhaps dependent on an Nrxn AS4+ repertoire?
3) The differential expression of Nrxn splice variants in principal vs. PV interneurons is well characterized, and the correlation of the AS4+ variant in PV cells with low Slm2 expression and high Clbn4 expression is striking. However, there is a large gap between these findings and the finding that short-term memory is impaired in the mice with PV-specific deletion of Nrxn1/3 AS4, which limits the impact of the manuscript. The link between PV neuron-specific Nrxn splicing, absence of obvious synaptic abnormalities in PV synaptic boutons and the observed behavioral impairments is not clear and raises questions. Are PV neurons known to play a role in the behavioral task assayed? Does this task also rely on PV micro-circuits outside the hippocampus and do PV neurons in those circuits also display enriched expression of AS4+? In other words, is the preferential use of AS4+ in PV cells a global phenomenon or might it be brain region-specific? A further exploration of synaptic defects in PV neurons might help to bridge the current gap between PV-specific Nrxn isoform repertoires and behavioral defects. In addition to analyzing spontaneous synaptic transmission, the authors could analyze evoked inhibitory responses and look at plasticity of PV-pyramidal neuron synapses. This would strengthen the impact of their findings. I understand the current focus is on PV synaptic terminals, but the authors could also explore whether excitatory inputs onto PV neurons might be affected in Nrxn1/3ex21Pv mice.
In a series of papers, Scheiffele and colleagues have analyzed alternative splicing in neurons, with particular emphasis on the neurexin and neuroligin adhesion molecules and the STAR family of splicing factors (Chih, 2006; Ijima 2011, 2014; Schreiner, 014a, 2015; Traunmuller, 2014, 2016). Here, Nguyen et al. continue this analysis, focusing on differences in α-neurexin splicing between inhibitory (PV+) and pyramidal (CamK2+) neurons in hippocampus. Using state-of-the-art methods, including RiboTrap purification of RNA from genetically identified cell types and quantitative analysis of exon usage, they show high inclusion rate of AS4 in neurexins in inhibitory relative to pyramidal neurons. Their previous work had shown that the STAR family member Slm2 leads to AS4 exclusion, and sure enough, PV+ cells have lower levels of Slm2 than pyramids. They then go on to assess the consequences of AS4 inclusion/exclusion in two ways. First they confirm and extend previous work showing that AS4 affects the relative activity of neurexins on inhibitory and excitatory synaptogenesis, as well as its binding to cerebellins. Second, they show that selective deletion of the AS4 exon from neurexins 1 and 3 in PV-expressing cells degrades performance of mice in an object recognition task, which is generally interpreted as indicating impairment in short-term memory.
Most previous studies on patterns and roles of alternative splicing have been performed at the level of brain regions, whereas direct evidence of cell type-specific isoform expression and function has been limited. In this regard, the work of Nguyen et al. is an important contribution to our understanding of molecular mechanisms that distinguish cell types and their functions. There are, however, some gaps in the analysis that deserve attention.
1) The authors emphasize splicing and bioactivities of Nrxn3 in much of the text, then perform the genetic analysis only on Nrxn1/3 double mutants. This leads to the obvious question of why Nrxn3 single mutants weren't analyzed, or at least compared to the doubles. In fact, I think this seeming inconsistency is more a matter of style than substance. If the authors gave similar weight to the two genes in the splicing analysis, everything would be more logical. Some additional data on Nrxn1 will be needed in the binding and bioactivity sections, unless it can be cited from previous papers.
2) It would be helpful to include data on pyramidal cells in Figure 2E, for comparison with the data on interneurons.
3) The data on cerebellins is somewhat peripheral to the main point, but since the authors have chosen to include it, they need to expand it a bit. Are cerebellins believed to act in the same cells as neurexins or to be transsynaptic ligands? What does the "association" shown in Figure 3E and 3F mean? The authors seem to suggest it is not direct binding. A minor related point is that the reader may be led to believe that selective cerebellin expression is going to figure in the phenotype, whereas in fact it is just an interesting observation. This should be clarified to avoid confusion and disappointment.
4) Perhaps the most important problem is with the behavioral analysis in vivo. The authors are making the case that inclusion of Nrxn1/3 exon 21 in hippocampal interneurons is essential for optimal hippocampal operation. Yet their cre line excises these exons from large numbers of interneurons throughout the brain and spinal cord, as well as large populations of sensory neurons and even some non-neuronal cells. Moreover, they fail to identify any physiological or morphological defects in the interneuronal synapses that could be responsible for driving the behavioral change. Thus, I'm not sure it is justifiable to relate the behavioral results in Figure 5 to the neurons analyzed in Figures 1-4.https://doi.org/10.7554/eLife.22757.020
[Editors’ note: the author responses to the first round of peer review follow.]
[…] The results presented are convincing and are important because they directly test key aspects of the neurexin splice code hypothesis. Unfortunately, the paper is missing a phenotype at the synaptic level. Somatic inhibitory synapses onto pyramidal neurons appear normal and mIPSCs are not affected (nor are numbers of Pvalb neurons). But the findings – that the potentially relevant ligand (Cbln4) is expressed in the same Pvalb cells as the NrxAs4+ transcripts, and that these Nrx transcripts enhance inhibitory synapses – argue that the relevant synaptic deficit to look for are inhibitory synapses between Pvalb cells. At least in the neocortex, these are the dominant source of inhibition of these interneurons and disruption of this synapse might destabilize the persistent activity hypothesized to underlie working memory. Therefore, it is disappointing that the authors did not look for anatomical and physiological evidence of reduced inhibition between Pvalb interneurons. If the authors did look for this and did not find it, this negative result is an important one that should be included. (As an aside, SST neurons also provide some inhibitory input to Pvalb neurons in some forebrain structures).
We thank the reviewer for this suggestion. We have now quantified the density of Synaptotagmin-2 positive (PV-neuron) terminals formed onto parvalbumin-positive interneurons in control and Nrxn1/3ex21 conditional knock-out mice. We did not find any significant alteration, indicating that there is not a major change in these synapses. This new data is now included in Figure 5—figure supplement 2.
[…] 1) The authors show a correlation between low PV neuron expression of the STAR-family RNA-binding protein Slm2, which regulates AS4 skipping, and high levels of AS4+ Nrxn isoforms in these cells. The manuscript would be strengthened by putting the causal relation between absence of Slm2 and cell type-specific exon skipping to the test. One experiment to test this would be to use AAV to misexpress Slm2 specifically in PV cells in a Cre-dependent manner and analyze whether this causes a shift towards more AS4- Nrxn isoforms.
We have now performed a similar experiment to test whether Slm2 is sufficient to drive the expression of Nrxn AS4- isoforms when ectopically expressed. We drove expression of Slm2 in cerebellar granule cells which are Slm2-negative and primarily express Nrxn AS4+ variants. Ectopic expression of Slm2 was sufficient to shift splicing towards expression of AS4- variants. This data is now included in Figure 2—figure supplement 2.
Another experiment would be to analyze Nrxn isoform repertoires in pyramidal cells of CamK2 Ribotrap mice crossed with Slm2 KO mice.
This is a good suggestion. Unfortunately, we currently did not have this genotype combined in our mouse colony. Given that this experiment requires combination of four alleles (CaMK2cre, Rpl22-HA, and two Slm2 KO alleles) we were not able to perform this experiment in the allotted timeframe.
2) The interaction of Cbln4 with Nrxn AS4+ shown in Figure 3, and the functional relevance thereof, should be further clarified. In Figure 3D, Cbln4 shows little binding to Nrxn3α AS4+ and Cbln1 shows some binding to Nrxn3α AS4+, whereas in 3F Cbln4 shows binding to Nrxn3α AS4+ to a similar degree as Cbln1. How can the apparent difference in Cbln binding to Nrxn between Figure 3D and 3F (COS cell binding vs surface retention) be explained, as both experiments measure Cbln at the surface?
We apologize for not having explained the difference between these two experiments appropriately in the original manuscript. Figure 3B-D showed experiments were Neurexin was expressed in cells and Cbln proteins expressed in a separate batch of cells and secreted into the medium were added to the Neurexin-expressing cells to assess surface binding. In a second set of assays (Figure 3E-G) we co-expressed Cbln proteins and neurexins in the same cells (as in parvalbumin-positive interneurons both proteins are co-expressed). In this second assay configuration Cbln4 shows significantly higher interaction with Nrx3αAS4+ isoforms. This may resolve some of the conflicting data that was found in the previous literature. To clarify the two assay configurations used in our study we now included cartoons for illustration (Figure 3B and 3E).
Does binding of Cbln4 to Nrxn AS4+ affect the induction of postsynaptic inhibitory specializations by AS4+ α Nrxn? In Figure 3C and D, are Cbln1, which binds most strongly to Nrxna AS4+, and Cbln3 also expressed in PV neurons? Finally, is expression of Cbln4 in PV neurons perhaps dependent on an Nrxn AS4+ repertoire?
We have tested directly whether co-expression of Cbln4 together with NrxnAS4+ modifies the induction of postsynaptic inhibitory specializations. We do not find a significant difference in this assay. We note that presence of endogenous Cbln4 in the neuronal culture may conceal a function for Cbln4. Loss of function studies for Cbln4 will be required to resolve this. The new results are now shown in Figure 3E and Figure 3—figure supplement 1D and 1E.
Regarding the expression of Cbln1 and 3 in PV+ cells we note that both transcripts show only very low expression in hippocampus when probed with qPCR assays. The same assays show robust detection in the cerebellum (new data in Figure 3—figure supplement 1A). Thus, we interpret our observation that Cbln1 and 3 transcripts are poorly or not at all detected in hippocampal PV+ cells as an indication that they are not significantly expressed and are unlikely to play a major role in these cells. This conclusion is consistent with previous in situ hybridization data in the literature (Miura et al., European Journal of Neuroscience, 2006).
3) The differential expression of Nrxn splice variants in principal vs. PV interneurons is well characterized, and the correlation of the AS4+ variant in PV cells with low Slm2 expression and high Clbn4 expression is striking. However, there is a large gap between these findings and the finding that short-term memory is impaired in the mice with PV-specific deletion of Nrxn1/3 AS4, which limits the impact of the manuscript. The link between PV neuron-specific Nrxn splicing, absence of obvious synaptic abnormalities in PV synaptic boutons and the observed behavioral impairments is not clear and raises questions. Are PV neurons known to play a role in the behavioral task assayed? Does this task also rely on PV micro-circuits outside the hippocampus?
The object recognition task is known to rely on the hippocampus but also the entorhinal cortex (Cohen & Stackman, Behav. Brain Res., 2015). Donato et al. (Nature, 2013) demonstrated for area CA3 of the hippocampus that PV+ neurons activation decreases whereas PV-neuron inhibition increases performance of mice in the novel object recognition task.
And do PV neurons in those circuits also display enriched expression of AS4+? In other words, is the preferential use of AS4+ in PV cells a global phenomenon or might it be brain region-specific?
To further explore splicing regulation at AS4 we performed RiboTrap purifications from neocortical neurons. In our splicing analysis we find that – similar to the hippocampus – CamK2-positive neurons preferentially express AS4- whereas parvalbumin-positive interneurons preferentially express AS4+ (particularly for Nrxn3). Thus, the preferential exon use reported for the hippocampus is shared in neocortical cells. This new data is included in Figure 2—figure supplement 1E.
A further exploration of synaptic defects in PV neurons might help to bridge the current gap between PV-specific Nrxn isoform repertoires and behavioral defects. In addition to analyzing spontaneous synaptic transmission, the authors could analyze evoked inhibitory responses and look at plasticity of PV-pyramidal neuron synapses. This would strengthen the impact of their findings. I understand the current focus is on PV synaptic terminals, but the authors could also explore whether excitatory inputs onto PV neurons might be affected in Nrxn1/3ex21Pv mice.
We appreciate that identifying a synaptic alteration that might be responsible or at least correlate with the behavioral phenotype would greatly strengthen this work. We have initiated some paired recordings in the knock-out mice, however, these experiments are very time-consuming and have not yet yielded novel insights.
To explore further whether the behavioral alterations coincide with alterations in neuronal network activity in the hippocampus, we compared c-fos-immunoreactivity in control and Nrxn1/3ex21 conditional knock-out mice. We observed a significant increase in the density of c-fos+ cells in area CA3 of the mutant mice as well as a trend towards increased density of c-fos+ cells in the dentate gyrus and area CA1. This indicates that parvalbumin-neuron–specific ablation of the AS4+ insertions results in an overall increase in network activity in the hippocampus. These new results are now presented in Figure 6A and 6B.
[…] 1) The authors emphasize splicing and bioactivities of Nrxn3 in much of the text, then perform the genetic analysis only on Nrxn1/3 double mutants. This leads to the obvious question of why Nrxn3 single mutants weren't analyzed, or at least compared to the doubles. In fact, I think this seeming inconsistency is more a matter of style than substance. If the authors gave similar weight to the two genes in the splicing analysis, everything would be more logical.
The reason that we did emphasize Nrxn3 is that Nrxn3 transcripts were most strongly enriched in the parvalbumin-RiboTrap preparations. However, clearly Nrxn1 and 2 are also expressed in these cell preparations. We now included data on the Nrxn3 single mutants, including weight and Mendelian frequency (Figure 4—figure supplement 1), anatomical analysis (Figure 5—figure supplement 1A-F) and data from mIPSC recordings (Figure 5—figure supplement 1G, H). We do not detect any significant changes in these single mutants.
Some additional data on Nrxn1 will be needed in the binding and bioactivity sections, unless it can be cited from previous papers.
The manuscript did contain data on the Cbln4-Nrx1 interactions. We apologize if this was not sufficiently visible in the previous submission. This previous data is presented in Figure 3H and 3I. In addition, we now included data on induction of gephyrin-positive structures by Nrx1α isoforms in presence and absence of Cbln4 (Figure 3—figure supplement 3D and 3E). For both, Nrx1α and Nrx3α we do not observe a significant alteration in the synaptogenic activity with co-expression of Cbln4.
2) It would be helpful to include data on pyramidal cells in Figure 2E, for comparison with the data on interneurons.
We have now quantified the percentage of cells marked in the CamK2Ribo mice that express Slm2. We find that Slm2 is expressed in >90% of CamK2Ribo-positive cells (Figure 2E).
3) The data on cerebellins is somewhat peripheral to the main point, but since the authors have chosen to include it, they need to expand it a bit. Are cerebellins believed to act in the same cells as neurexins or to be transsynaptic ligands?
Our expression data demonstrates that Cbln4 is significantly enriched in PV+cells as compared to Camk2-positive cells. This means, Nrxn AS4+ isoforms and Cbln4 are co-expressed and indicates that they are transported to the PV-cell surface and then might engage postsynaptic ligands. This model is analogous to the action of Nrxn-Cbln1 complexes in the mouse cerebellum.
What does the "association" shown in Figure 3E and 3F mean? The authors seem to suggest it is not direct binding. A minor related point is that the reader may be led to believe that selective cerebellin expression is going to figure in the phenotype, whereas in fact it is just an interesting observation. This should be clarified to avoid confusion and disappointment.
The two assays presented in Figure 3 are cell binding assays. While the interactions that we see are consistent with direct Nrxn-Cbln interactions we cannot completely exclude that additional co-factors contribute to the interaction. We have now clarified this in the text (subsection “AS4 + splice insertions selectively enhance the function of neurexins towards GABAergic postsynaptic components”, first paragraph). We have also clarified that it remains to be shown whether Cbln4 mediates any of the functions of Nrxn AS4+ isoforms in PV+ cells.
4) Perhaps the most important problem is with the behavioral analysis in vivo. The authors are making the case that inclusion of Nrxn1/3 exon 21 in hippocampal interneurons is essential for optimal hippocampal operation. Yet their cre line excises these exons from large numbers of interneurons throughout the brain and spinal cord, as well as large populations of sensory neurons and even some non-neuronal cells. Moreover, they fail to identify any physiological or morphological defects in the interneuronal synapses that could be responsible for driving the behavioral change. Thus, I'm not sure it is justifiable to relate the behavioral results in Figure 5 to the neurons analyzed in Figures 1-4.
We agree with the reviewer that it is unclear whether the conditional ablation of Nrxn1/3 AS4 insertions in PVcre-positive cells modifies the behavior due to an alteration in hippocampal interneurons or other cells that are targeted by the PVcre line. The object recognition test relies in part on normal hippocampal function and performance of mice in this test has been linked to the activity of PV+-cells in the hippocampus (Donato et al., Nature, 2013). To assess whether hippocampal network activity might be altered in Nrxn1/3 conditional knock-out mice we measured the density of c-fos+ cells in the hippocampus. We find that the density of c-fos+ cells is elevated in the mutant mice as compared to littermate controls (Figure 6A and 6B). Thus, PV-cell-specific deletion of exon21 in Nrxn1 and 3 indeed results in a modified hippocampal network. Whether this modification is causal for the altered behavior of the mice remains to be explored. We have now extended the Discussion regarding this limitation of our study.https://doi.org/10.7554/eLife.22757.021
- Peter Scheiffele
- Peter Scheiffele
- Peter Scheiffele
- Peter Scheiffele
- Lisa Traunmüller
- Dietmar Schreiner
- Thi-Minh Nguyen
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank members of the Scheiffele Lab for support and constructive discussions. We are grateful to Siu-Pok Yee and the University of Connecticut Gene Targeting Facility for help with the generation of conditional knock-out mice. We thank Andrea Gomez for initial electrophysiology recordings, Elisabetta Furlanis for sharing RNA preparations and Oriane Mauger for data analysis advices. We also thank Markus Rüegg for kindly sharing CamK2cre animals and Ursula Sauder for excellent assistance with the ultrastructural analysis. TMN was supported by a fellowship from the Werner-Siemens Foundation/International PhD program Fellowship for Excellence. DS was financially supported by a grant from the Forschungsfond of the University of Basel and a FP7 Marie-Curie Mobility Fellowship from the FP7 of the European Union. LT was supported by the Boehringer Ingelheim Fonds. This work was supported by funds to PS from the Swiss National Science Foundation, the National Competence Centre for Research NCCR-SYNAPSY, the European Research Council (ERC-Advanced grant SPLICECODE), EU-AIMS which receives support from the Innovative Medicines Initiative Joint Undertaking of the EU FP7, and the Kanton Basel-Stadt.
Animal experimentation: All animal procedures were reviewed and approved by the Kantonales Veterinäramt Basel-Stadt (Licences 2272, 2293, 2599). The Procedures were performed in strict accordance to the guidelines and every effort was mode to minimize suffering of the animals and to minimize animal numbers (either by replacement or optimization of procedures).
- Sacha B Nelson, Brandeis University, United States
© 2016, Nguyen et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.