Specific Neuroligin3–αNeurexin1 signaling regulates GABAergic synaptic function in mouse hippocampus

  1. Motokazu Uchigashima
  2. Kohtarou Konno
  3. Emily Demchak
  4. Amy Cheung
  5. Takuya Watanabe
  6. David G Keener
  7. Manabu Abe
  8. Timmy Le
  9. Kenji Sakimura
  10. Toshikuni Sasaoka
  11. Takeshi Uemura
  12. Yuka Imamura Kawasawa
  13. Masahiko Watanabe
  14. Kensuke Futai

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Abstract

Synapse formation and regulation require signaling 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 signaling generates distinct functional properties at synapses.

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  1. Version published to 10.7554/elife.59545 on eLife
  2. eLife

    ###Reviewer #3:

    General assessment:

    Futai and colleagues present an extremely elegant study in hippocampal CA1 slice cultures which combines cell type-specific expression of inhibitory synapse markers and conditional deletion of neurexins (Nrxn), knockdown of neuroligin3 (Nlgn3) and rescue experiments with defined splice variants of Nrxn by biolistic transfection with paired patch-clamp recordings. They find that synaptic transmission between inhibitory cholecystokinin(&VGT3)-positive interneurons (VGT3+) and CA1 pyramidal neurons depends specifically on the combination of presynaptic Nrxn1α with insert in splice site #4 and postsynaptic Nlgn3 without A1&A2 splice inserts.

    Major concerns:

    1.) The role of neurexins for transmission at the VGT3+ interneuron-to-CA1-pyramidal cell synapse remains unclear: The authors claim that Nrxn are important for the transmission at the VGT3+ synapse. However, I do not see the necessary experiment to substantiate such a general claim, for example, by comparing VGT3+synapses of control/undeleted to deleted NrxnTKO slices. Figure 5 rather shows that Nrxn is required to mediate the effect of overexpression of transfected Nlgn3Δ in CA1 neurons but this might be due to the overexpression itself. Thus, this effect would be more convincing if lack of Nrxn at VGT3+ synapses caused the opposite result on uIPSCs.

    2.) The idea of "A Specific Neuroligin3-αNeurexin1 Code ..." implies to most readers that a direct interaction between these two molecules is involved because numerous biochemical papers in the field have used the term splice code to refer to a hierarchy of binding affinities between Nrxn and Nlgn variants. However, such preferential binding of αNrxn1+AS4 to Nlgn3Δ is neither shown in the manuscript, nor is it likely: The authors report that „αNrxn1+AS4 and βNrxn3-AS4 are the unique Nrxn genes expressed in VGT3+ neurons compared with PV+ and Sst+ neurons" (Figure 6 & 7) but demonstrate that of these two isoforms, only αNrxn1+AS4 transfected into VGT3+ interneurons mediated the effect of Nlgn3Δ overexpressed in pyramidal neurons (Figure 8). If binding or direct physical interaction was involved in the mechanism, βNrxn3-AS4 should have performed better than αNrxn1+AS4 because both the LNS5-EFG-LNS6 cassette and the insert in AS4 reduced the affinity. The surprise is shared by the authors themselves in the last paragraph of the Results section (p.12). At least to me, it appears that additional pre- or postsynaptic molecules, or a different mechanism altogether, are involved in mediating the effect of αNrxn1+AS4&Nlgn3Δ on VGT3+ synapses.

    3.) To actually prove the specificity of the impact of Nlgn3Δ splice variant on inhibitory transmission from VGT3+ interneurons (Figure 2), an important control is missing: Another Nlgn3 variant, in which the A inserts are present, should be tested in the overexpression experiment. I do acknowledge that the authors compared different Nlgn3 variants in a recent paper (Uchigashima et al., 2020, JBC) in a related setting but no data exist for the proposed specificity of the Nlgn3Δ splice variant at VGT3+ synapses as far as I can see.

  3. eLife

    ###Reviewer #2:

    Motokazu et. al., identified a specific Nlgn-Nrxn pair that regulates GABAergic synapse function in a subset of interneurons. This is a really interesting study, in which they use complicated techniques to dissect NLGN3 and NRXN function. The authors performed elaborate experiments from a single cell level to a tissue level that support their conclusions. Overall the data appear of high quality and reliable, but the study would benefit from some clarification of text and figures.

    1. They are doing overexpression experiments on a WT background, so it's impossible to know if this effect is from homodimers of NLGN3 or heterodimers of NLGN3 with either NLGN1 or NLGN2. Perhaps the authors could discuss this caveat in the manuscript.

    2. The authors see an increase in IPSCs when o/e NLGN3 in pyramidal neurons when Sst+ neurons were stimulated using optogenetics, whereas Horn and Nicoll did not see any changes. Horn and Nicoll used NLGN3A2 (including A2 insert) and in this study the construct doesn't have A1 or A2 insert. Perhaps they can discuss if the A2 insert can potentially be the culprit for the discrepancy if this is a potential confounding factor.

    Additional comments:

    1. In Fig. 1, please indicate Fig. 1C in the legends and make a box for the enlarged region in the lower magnificent image. It would be better to take out the busy alphabetic labels (E1, E2, E3, etc.).

    2. Please increase text font size for the sample traces in all figures.

    3. Authors showed quantitative graphs showing connectivity but definition of the connectivity is not well explained. More detailed explanation for how they quantified the connectivity should be addressed in methods.

    4. In Fig. 5A, it would be more accurate to normalize KO Nrxns to WT Nrxns (set WT Nrnx as 100 %). The current Fig. 5A graph looks like Nrxn3 is not dominant in WT mice (~ 0.1 %) but the Fig. 7B graph shows Nrxn3 is dominant. Is there a discrepancy or perhaps add an explanation?

    5. In Fig. 6J, the graphs for βNrxn1, αNrxn2, and βNrxn2 can go together with the image data in Figure S3.

    6. Authors should explain and provide more information about the parameters of the Fig. 7A plot in the main text and legends. Correct the missed indication of Fig. 2B in the results text.

    7. What if presynaptic αNrxn1+AS4 couples with Nlgn3 KD or NlgnTKO condition? What would be the expectation?

  4. eLife

    ###Reviewer #1:

    Gaining insights into synapse-type specific regulatory mechanisms is of significant general interest. Yet, substantial concerns need to be addressed to improve this study.

    Major points:

    1. The authors state that Nlgn3 is particularly enriched at VGT3+ synapses. This is based on the colocalization of immunolabeling for Nlgn3 and each of the markers for three interneurons types as well as with the general inhibitory synapse marker VIAAT in Figure 1. If the intensity of marker labeling is not similar across interneuron types and if imaged fields are not comparable, the use of Nlgn3 co-labeling to assess a preferential localization is compromised. This concern is apparent e.g. in the example image for SST+ synapses with its weak labeling (Fig 1J) and needs to be addressed.

    2. The data in Figure 5 support that the lack of presynaptic Nrxns 1/2/3 abolishes the potentiation effect of VGT3+ inhibitory synaptic transmission by Nlgn3Δ. To interpret these data, the authors also need to show whether the Nrxn triple KO in VGT3+ cells affects the amplitude of uIPSCs in postsynaptic control neurons expressing Nlgns at endogenous level.

    3. The physiological findings are based on paired recordings where genetically labeled VGT3+ interneurons are stimulated. These cells are sparse and heterogeneously distributed in CA1 (Pelkey et al., Physiological Reviews 2017). Given the issues with Cre driver lines, a more thorough analysis is needed to establish that bona fide VGT3+ interneurons contribute to the reported findings. The scattered distribution of the individual RFP+ cells in single-cell RNAseq data (Fig 7a) adds to this concern about cell identity. The only relevant evidence presented is the IHC analysis in Fig S1A-C but it does not include probes for other interneuron types in the CA1 that shows the specificity of the VGT3+ label.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary

    This study by Uchigashima et al. investigates to what extent Neurexin-Neuroligin interactions define synapse functions in an inhibitory microcircuit in the hippocampal CA1. The authors propose that Neuroligin-3 and Neurexin-1α regulate inhibitory synaptic transmission at synapses formed by vGluT3-positive (VGT3+) interneurons on CA1 hippocampal pyramidal neurons. This is based on (1) immunohistochemical data that localize Nlgn3 to synapses of VGT3+ interneurons, (2) the regulation of unitary inhibitory postsynaptic currents by Nlgn3 overexpression or knockdown in postsynaptic pyramidal neurons in organotypic slices from VGT3+ reporter mice, and (3) the finding that Nrxn deletion in VGT3+ interneurons prevents the effect of Nlgn3 overexpression in postsynaptic neurons. Single-cell RNA sequencing and in situ hybridization are presented to show that Nrxn1α and Nrxn3beta mRNAs are prominent Nrxn isoforms in VGT3+ interneurons, and Nrxn1α SS4 rescued Nrxn deletion effects. With some additional critical controls and a more careful interpretation of the presumed mechanism, the manuscript would make a highly interesting contribution to the field of synapse specification by synaptic cell adhesion molecules.

  6. Version published to 10.1101/2020.05.27.119024 on bioRxiv