Deletion of Neuroligins from Astrocytes Does Not Detectably Alter Synapse Numbers or Astrocyte Cytoarchitecture by Maturity
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eLife Assessment
This fundamental study examines whether synaptic cell adhesion molecules neuroligin 1-3 resident on astrocytes, rather than neurons, exert effect on synaptic structure and function. With compelling evidence, the authors report that deletion of neuroligins 1-3 specifically in astrocytes does not alter synapse formation or astrocyte morphology in the hippocampus or visual cortex. This study highlights the specific role of neuronal neuroligins rather than their astrocytic counterparts in synaptogenesis.
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Abstract
Astrocytes perform multifarious roles in the formation, regulation, and function of synapses in the brain, but the mechanisms involved are incompletely understood. Interestingly, astrocytes abundantly express neuroligins, postsynaptic adhesion molecules that function as synaptic organizers by binding to presynaptic neurexins. Here we examined the function of neuroligins in astrocytes with a rigorous genetic approach that uses the conditional deletion of all major neuroligins ( Nlgn1-3 ) in astrocytes in vivo and complemented this approach by a genetic deletion of neuroligins in glia cells that are co-cultured with human neurons. Our results show that early postnatal deletion of neuroligins from astrocytes in vivo has no detectable effect on cortical or hippocampal synapses and does not alter the cytoarchitecture of astrocytes when evaluated in young adult mice. Moreover, deletion of astrocytic neuroligins in co-cultures of human neurons produced no detectable consequences for the formation and function of synapses. Thus, astrocytic neuroligins are unlikely to fundamentally shape synapse formation or astrocyte morphogenesis but likely perform other important roles that remain to be discovered.
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eLife Assessment
This fundamental study examines whether synaptic cell adhesion molecules neuroligin 1-3 resident on astrocytes, rather than neurons, exert effect on synaptic structure and function. With compelling evidence, the authors report that deletion of neuroligins 1-3 specifically in astrocytes does not alter synapse formation or astrocyte morphology in the hippocampus or visual cortex. This study highlights the specific role of neuronal neuroligins rather than their astrocytic counterparts in synaptogenesis.
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Reviewer #1 (Public review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative …
Reviewer #1 (Public review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative western blot analyses, imaging and electrophysiology. They also characterize the specificity of the cre line they used. Overall, this is a very clear and strong paper that is supported by rigorous experiments. The discussion considers the findings carefully in relation to past work. This paper is of high importance, because it now raises the fundamental question of exactly what neuroligins 1-3 are actually doing in astrocytes. In addition, it enriches our understanding of the mechanisms by which astrocytes participate in synapse formation and function. The paper is very clear, well written and well illustrated with raw and average data.
Comments on revisions:
My previous comments have been addressed. I have no additional points to make and congratulate the authors.
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Reviewer #2 (Public review):
In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin (Nlgn) family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate …
Reviewer #2 (Public review):
In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin (Nlgn) family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Nlgn1-3 specifically from astrocytes in mice, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. To further extend this finding, the authors additionally analyzed human neurons co-cultured with mouse glia lacking expression of Nlgn1-4. No difference in excitatory synaptic transmission was observed between neurons cultured in the present of wildtype vs. Nlgn1-4 conditional knockout glia. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.
Overall, this is a strong study that addresses a fundamental and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, Nlgn1-3 are efficiently deleted from astrocytes in vivo, and that this deletion does not lead to major alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes. While the co-culture experiments are somewhat more difficult to interpret due to lack of a control for the effect of wildtype mouse astrocytes on human neurons, they are also consistent with the notion that deletion of Nlgn1-4 from astrocytes has no consequences for the function of excitatory synapses. Together, the data from this study provide compelling and important evidence that, whatever the role of astrocytic Neuroligins may be, they do not contribute substantially to synapse formation or function under the conditions investigated.
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Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for …
Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative western blot analyses, imaging and electrophysiology. They also characterize the specificity of the cre line they used. Overall, this is a very clear and strong paper that is supported by rigorous experiments. The discussion considers the findings carefully in relation to past work. This paper is of high importance, because it now raises the fundamental question of exactly what neuroligins 1-3 are actually doing in astrocytes. In addition, it enriches our understanding of the mechanisms by which astrocytes participate in synapse formation and function. The paper is very clear, well written and well illustrated with raw and average data.
We thank the reviewer for the balanced and informative summary.
Reviewer #2 (Public Review):
In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Neuroligins 1, 2 and 3 specifically from astrocytes, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.
Overall, this is a strong study that addresses an important and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, no alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes, are observed.
We are also grateful for this reviewer’s constructive comments.
One caveat to this study is that the authors do not directly provide evidence that their Tamoxifen-inducible conditional deletion paradigm does indeed result in efficient deletion of all three Neuroligins from astrocytes. Using a Cre-dependent tdTomato reporter line, they show that tdTomato expression is efficiently induced by the current paradigm, and they refer to a prior study showing efficient deletion of Neuroligins from neurons using the same conditional Nlgn1-3 mouse lines but a different Cre driver strategy. However, neither of these approaches directly provide evidence that all three Neuroligins are indeed deleted from astrocytes in the current study. In contrast, Stogsdill et al. employed FACS and qPCR to directly quantify the loss of Nlgn2 mRNA from astrocytes. This leaves the current Golf et al. study somewhat vulnerable to the criticism, however unlikely, that their lack of synaptic effects may be a consequence of incomplete Neuroligin deletion, rather than a true lack of effect of astrocytic Neuroligins.
The concern is valid. In the original submission of this paper, we did not establish that the Cre recombinase we used actually deleted neuroligins in astrocytes. We have now addressed this issue in the revised paper with new experiments as described below.
However, the reviewer’s impression that the Stogsdill et al. paper confirmed full deletion of Nlgn2 is a misunderstanding of the data in that paper. The reviewer is correct that Stogsdill et al. performed FACS to test the efficacy of the GLAST-Cre mediated deletion of Nlgn2-flox mice, followed by qRT-PCR comparing heterozygous with homozygous mutant mice. With their approach, no wild-type control could be used, as these would lack reporter expression. However, this experiment does NOT allow conclusions about the degree of recombination, both overall recombination (i.e. recombination in all astrocytes regardless of TdT+) and recombination in TdT+ astrocytes because it doesn’t quantify recombination. To quantify the degree of recombination, the paper would have had to perform genomic PCR measurements.
The problem with the data on the degree of recombination in the Stogsdill et al. (2017) paper, as we understand them, is two-fold.
First, the GLAST-Cre line only targets ~40-70% of astrocytes, at least as evidenced by highly sensitive Cre-reporter mice in a variety of studies using this Cre line. The 40-70% variation is likely due to differences in the reporter mice and the tamoxifen injection schedule used. In comparison, we are targeting most astrocytes using the Aldh1l1-CreERT2 mice. Moreover, GLAST-Cre mice exhibit neuronal off-targeting, consistent with at least some of the remaining Nlgn2 qRT-PCR signal in the FACS-sorted cells. As we describe next, this signal also likely comes from astrocytes where recombination was incomplete This is the reason why we, like everyone else, are now using the Aldh1l1-Cre line that has been shown to be more efficient both in terms of the overall targeting of astrocytes (i.e. nearly complete) and the level of recombination observed in reporter(+) astrocytes.
Second, Stogsdill et al. detected a significant decrease in the Nlgn2 qRT-PCR signal in the FACS-sorted homozygous Nlgn2 KO cells compared to the heterozygous Nlgn2 KO cells but the Nlgn2 qRT-PCR signal was still quite large. The data is presented as normalized to the HET condition. As a result, we don’t know the true level of gene deletion (i.e. compared to TdT- astrocytes). For example, based on the Stogsdill et al. data the HET manipulation could have induced only a 20% reduction in Nlgn2 mRNA levels in TdT(+) astrocytes, in which case the KO would have produced a 40% reduction in Nlgn2 mRNA in TdT(+) astrocytes. Moreover, it is possible based on our own experience with the GLAST-Cre line, that the reporter may also not turn on in some astrocytes where other alleles have been independently recombined – just as some astrocytes that are Td(+) would still be wild-type or heterozygous for Nlgn2. Thus, it is impossible to calculate the actual percentage of recombination from these data, even in TdT(+) cells, absent of PCR of genomic DNA from isolated cells. Alternatively, comparison of mRNA levels using primers sensitive to floxed sequences in wild-type controls versus cKO mice would have also yielded a much better idea of the recombination efficiency.
In summary, it is unclear whether the Nlgn2 deletion in the Stogsdill et al. paper was substantial or marginal – it is simply impossible to tell.
Reviewer #3 (Public Review):
This study investigates the roles of astrocytes in the regulation of synapse development and astrocyte morphology using conditional KO mice carrying mutations of three neuroligins1-3 in astrocytes with the deletion starting at two different time points (P1 and P10/11). The authors use morphological, electrophysiological, and cell-biological approaches and find that there are no differences in synapse formation and astrocyte cytoarchitecture in the mutant hippocampus and visual cortex. These results differ from the previous results (Stogsdill et al., 2017), although the authors make several discussion points on how the differences could have been induced. This study provides important information on how astrocytes and neurons interact with each other to coordinate neural development and function. The experiments were well-designed, and the data are of high quality.
We also thank this reviewer for helpful comments!
Recommendations for the authors:
This project was meant to rigorously test the intriguing overall question whether neuroligins, which are abundantly expressed in astrocytes, regulate synapse formation as astrocytic synapse organizers. The goal of the paper was NOT to confirm or dispute the conclusion by Stogsdill et al. (Nature 2017) that Nlgn2 expressed in astrocytes is essential for excitatory synapse formation and that astrocytic Nlgn1-3 are required for proper astrocyte morphogenesis. Instead, the project was meant to address the much broader question whether the abundant expression of any neuroligin, not just Nlgn2, in astrocytes is essential for neuronal excitatory or inhibitory synapse formation and/or for the astrocyte cytoarchitecture. We felt that this was an important question independent of the Stogsdill et al. paper. We analyzed in our experiments young adult mice, a timepoint that was chosen deliberately to avoid the possibility of observing a possible developmental delay rather than a fundamental function that extends beyond development.
We do recognize that the conclusion by Stogsdill et al. (2017) that Nlgn2 expression in astrocytes is essential for excitatory synapse formation was very exciting to the field but contradicted a large literature demonstrating that Nlgn2 protein is exclusively localized to inhibitory synapses and absent from excitatory synapses (to name just a few papers, see Graf et al., Cell 2004; Varoqueaux et al., Eur. J. Cell Biol. 2004; Patrizi et al., PNAS 2008; Hoon et al., J. Neurosci. 2009). In addition, the conclusion of Stogsdill et al. that astrocytic Nlgn2 specifically drove excitatory synapse formation was at odds with previous findings documenting that the constitutive deletion of Nlgn2 in all cells, including astrocytes, has no effect on excitatory synapse numbers (again, to name a few papers, see Varoqueaux et al., Neuron 2006; Blundell et al., Genes Brain Behav. 2008; Poulopoulos et al., Neuron 2009; Gibson et al., J. Neurosci. 2009). These contradictions conferred further urgency to our project, but please note that this project was primarily driven by our curiosity about the function of astrocytic neuroligins, not by a fruitless desire to test the validity of one particular Nature paper.
The general goal of our paper notwithstanding, few papers from our lab have received as much attention and as many negative comments on social media as this paper when it was published as a preprint. Because we take these criticisms seriously, we have over the last year performed extensive additional experiments to ensure that our findings are well founded. We feel that, on balance, our data are incompatible with the notion that astrocytic neuroligins play a fundamental role in excitatory synapse formation but are consistent with other prior findings obtained with neuroligin KO mice. In the new data we added to the paper, we not only characterized the Cre-mediated deletion of neuroligins in depth, but also employed an independent second system -human neurons cultured on mouse glia- to further validate our conclusions as described below. Although we believe that our results are incompatible with the notion that astrocytic neuroligins fundamentally regulate excitatory or inhibitory synapse formation, we also conclude with regret that we still don’t know what astrocytic neuroligins actually do. Thus, the function of astrocytic neuroligins, as there surely must be one, remains a mystery.
Finally, there are many possible explanations for the discrepancies between our conclusions and those of Stogsdill et al. as described in our paper. Most of these explanations are technical and may explain why not only our, but also the results of many other previous studies from multiple labs, are inconsistent with the conclusions by Stogsdill et al. (2017), as discussed in detail in the revised paper.
Reviewer #1 (Recommendations For The Authors):
The paper is very clear and well written. I have only one comment and that is to increase the sizes of Figs 2, 4 and 6 so that the imaging panels can be seen more clearly. Also, although I know the n numbers are provided in the figure legends, the authors may help the reader by providing them in the results when key data and findings are reported.
We agree and have followed the reviewer’s suggestions as best as we could.
Reviewer #2 (Recommendations For The Authors):
(1) Given the strength and importance of the claims that the authors make, I would highly recommend adding some quantitative evidence regarding the efficacy of deletion in astrocytes, e.g. using the same strategy as in Stogsdill et al. As unlikely as it may be that Neuroligin deletion is in fact incomplete, this possibility cannot be excluded unless directly measured. To avoid future discussions on this subject, it seems that the onus is on the authors to provide this information.
We concur that this is an important point and have devoted a year-long effort to address it. Note, however, that the strategy employed by Stogsdill et al. does not actually allow conclusions about their recombination efficiency. As described above, it only allows the conclusion that some recombination took place. The Stogsdill et al. Nature paper (2017) is a bit confusing on this point. This approach is thus not appropriate to address the question raised by the reviewer.
We have performed two experiments to address the issue raised by the reviewer.
First, we used a viral (i.e. AAV2/5) approach to express Rpl22 with a triple HA-tag, also known as Ribotag, which allows us to purify ribosome-bound mRNA from targeted cells for downstream gene expression analysis. The novel construct is driven by the GfaABC1D promoter and includes two additional features which make it particularly useful. First, upstream of Ribotag is a membrane-targeted, Lck-mVenus followed by a self-cleaving P2A sequence. This allows easy visualization of targeted astrocytes. Second, we have incorporated a cassette of four copies of six miRNA targeting sequences (4x6T) for mIR-124 as was recently published (Gleichman et al., 2023) to eliminate off-target expression in neurons. Based on qPCR analysis, the updated construct allowed >95% de-enrichment of neuronal mRNA and slightly improved observed recombination rates (~10% per gene) relative to an earlier version without 4x6T. Mice that were injected with tamoxifen at P1, similar to other experiments in the paper, were then stereotactically injected at ~P35-40 within the dorsal hippocampus with AAV2/5-GfaABC1D-Lck-mVenus-P2A-Rpl22-HA-4x6T. Approximately 3 weeks later, acute slices were prepared, visualized for fluorescence, and both CA1 and nearby cortex that was partially targeted were isolated for downstream ribosome affinity purification with HA antibodies. Total RNA was saved as input. qPCR was performed using assays that are sensitive to the exons that are floxed in the Nlgn123 cKO mice, so that our quantifications are not confounded by potential differences in non-sense mediated decay. Our control data reveals a striking enrichment of an astrocyte marker gene (e.g. aquaporin-4) and de-enrichment of genes for other cell types. In the CA1, we observed robust loss of Nlgn3 (~96%), Nlgn2 (~86%), and Nlgn1 (65%) gene expression. Similarly, in the cortex, we observed a similarly robust loss of Nlgn3 (93%), Nlgn2 (83%), and Nlgn1 (72%) expression. Given that our targeting of astrocytes based on Ai14 Cre-reporter mice was ~90-99%, these reductions are striking and definitive. The existence of some residual transcript reflects the presence of a small population of astrocytes heterozygous for Nlgn2 and Nlgn3. In contrast, Nlgn1 appears more difficult to recombine and it is likely that some astrocytes are either heterozygous or homozygous knockout cells. Although it is thus possible that Nlgn1 could provide some compensation in our experiments, it is worth noting that Stogsdill et al. found that only Nlgn2 and Nlgn3 knockdown with shRNAs resulted in impaired astrocyte morphology by P21. Moreover, they found that Nlgn2 cKO in astrocytes with PALE of a Cre-containing pDNA impaired astrocyte morphology in a gene-dosage dependent manner and suppressed excitatory synapse formation at P21. Thus, our inability to delete all of Nlgn1 doesn’t readily explain contradictions between our findings and theirs.
Second, in an independent approach we have cultured glia from mouse quadruple conditional Nlgn1234 KO mice and infected the glia with lentiviruses expressing inactive (DCre, control) or active Cre-recombinase. We confirmed complete recombination by PCR. We then cultured human neurons forming excitatory synapses on the glia expressing or lacking neuroligins and measured the frequency and amplitude of mEPSCs as a proxy for synapse numbers and synaptic function. As shown in the new Figure 9, we detected no significant changes in mEPSCs, demonstrating in this independent system that the glial neuroligins do not detectably influence excitatory synapse formation.
(2) Along the same lines, the authors should be careful not to overstate their findings in this direction. For example, the figure caption for Figure 2 reads 'Nlgn1-3 are efficiently and selectively deleted in astrocytes by crossing triple Nlgn1-3 conditional KO mice with Adh1l1-CreERT2 driver mice and inducing Cre-activity with tamoxifen early during postnatal development'. This is not technically correct and should be modified to reflect that the authors are not in fact assessing deletion of Nlgn1-3, but only expression of a tdTomato reporter.
We agree – this is essentially the same criticism as comment #1.
(3) In general, the animal numbers used for the experiments are rather low. With an n = 4 for most experiments, only large abnormalities would be detected anyway, while smaller alterations would not reach statistical significance due to the inherent biological and technical variance. For the most part, this is not a concern, since there really is no difference between WTs and Nlgn1-3 cKOs. However, trends are observed in some cases, and it is conceivable that these would become significant changes with larger n's, e.g. Figure 3H (Vglut2); Figure 4E (VGlut2 S.P., D.G.); Figure 6D (Vglut2). Increasing the numbers to n = 6 here would greatly strengthen the claims that no differences are observed.
We concur that small differences would not have been detected in our experiments but feel that given the very large phenotypes of the neuroligin deletions in neurons and of the phenotypes reported by Stogsdill et al. (2017), which also did not employ a large number of animals, a very small phenotype in astrocytes would not have been very informative.
Minor points:
(1) Please state the exact genetic background for the mouse lines used.
Our lab generally uses hybrid CD1/Bl6 mice to avoid artifacts produced by inbred genetic mutations in so-called ‘pure’ lines, especially Bl6 mice. This standard protocol was followed in the present study. Thus, the mice are on a mixed CD1/Bl6 hybrid background.
Reviewer #3 (Recommendations For The Authors):
(1) Figure 4 demonstrates that neuroligin 1-3 deletions restricted to astrocytes do not affect the number of excitatory and inhibitory synapses in layer IV of the primary visual cortex. This conclusion could be further strengthened if the authors could provide electrophysiological evidence such as mE/IPSCs.
We agree but have chosen a different avenue to further test our conclusions because slice electrophysiological experiments are time-consuming, labor intensive, and difficult to quantitate, especially in cortex.
Specifically, we have co-cultured human neurons with astrocytes that either contain or lack neuroligins (new Fig. 9). With this experimental design, we have total control over ALL neuroligins in astrocytes. Electrophysiological recordings then demonstrated that the complete deletion of all glial neuroligins has no effect on mEPSC frequencies and amplitudes. Although clearly much more needs to be done, the new results confirm in an independent system that glial neuroligins have no effect on synapse formation in the neurons, even though neurons depend on astrocytes for synaptogenic factors as Ben Barres brilliantly showed a decade ago. However, it is important to note that dissociated glia in culture, while synaptogenic, are reactive and may not faithfully recapitulate all roles of astrocytes in synaptogenesis.
(2) It would help readers if the images showing the punctate double marker stainings of excitatory/inhibitory synapses are presented in merged colors (i.e., yellow colors for red and green puncta colors).
We have tried to improve the visualization of the rather voluminous studies we performed and illustrate in the figures as best as we could.
(3) The resolutions of the images in the figures are not good, although I guess it is because the images are for review processes.
We apologize and would like to assure the reviewer that we are supplying high-resolution images to the journal.
(4) Typos in lines 82 and 274.
We have corrected these errors.
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eLife assessment
This important study examines whether synaptic cell adhesion molecules neuroligin 1-3 resident on astrocytes, rather than neurons, exert effect on synaptic structure and function. With convincing evidence, the authors report that deletion of neuroligins 1-3 specifically in astrocytes does not alter synapse formation or astrocyte morphology in the hippocampus or visual cortex. This study highlights the specific role of neuronal neuroligins rather than their astrocytic counterparts in synaptogenesis.
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Reviewer #1 (Public Review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative …
Reviewer #1 (Public Review):
Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative western blot analyses, imaging and electrophysiology. They also characterize the specificity of the cre line they used. Overall, this is a very clear and strong paper that is supported by rigorous experiments. The discussion considers the findings carefully in relation to past work. This paper is of high importance, because it now raises the fundamental question of exactly what neuroligins 1-3 are actually doing in astrocytes. In addition, it enriches our understanding of the mechanisms by which astrocytes participate in synapse formation and function. The paper is very clear, well written and well illustrated with raw and average data.
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Reviewer #2 (Public Review):
In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic …
Reviewer #2 (Public Review):
In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapses, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Neuroligins 1, 2 and 3 specifically from astrocytes, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.
Overall, this is a strong study that addresses an important and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, no alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes, are observed.
One caveat to this study is that the authors do not directly provide evidence that their Tamoxifen-inducible conditional deletion paradigm does indeed result in efficient deletion of all three Neuroligins from astrocytes. Using a Cre-dependent tdTomato reporter line, they show that tdTomato expression is efficiently induced by the current paradigm, and they refer to a prior study showing efficient deletion of Neuroligins from neurons using the same conditional Nlgn1-3 mouse lines but a different Cre driver strategy. However, neither of these approaches directly provide evidence that all three Neuroligins are indeed deleted from astrocytes in the current study. In contrast, Stogsdill et al. employed FACS and qPCR to directly quantify the loss of Nlgn2 mRNA from astrocytes. This leaves the current Golf et al. study somewhat vulnerable to the criticism, however unlikely, that their lack of synaptic effects may be a consequence of incomplete Neuroligin deletion, rather than a true lack of effect of astrocytic Neuroligins.
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Reviewer #3 (Public Review):
This study investigates the roles of astrocytes in the regulation of synapse development and astrocyte morphology using conditional KO mice carrying mutations of three neuroligins1-3 in astrocytes with the deletion starting at two different time points (P1 and P10/11). The authors use morphological, electrophysiological, and cell-biological approaches and find that there are no differences in synapse formation and astrocyte cytoarchitecture in the mutant hippocampus and visual cortex. These results differ from the previous results (Stogsdill et al., 2017), although the authors make several discussion points on how the differences could have been induced. This study provides important information on how astrocytes and neurons interact with each other to coordinate neural development and function. The …
Reviewer #3 (Public Review):
This study investigates the roles of astrocytes in the regulation of synapse development and astrocyte morphology using conditional KO mice carrying mutations of three neuroligins1-3 in astrocytes with the deletion starting at two different time points (P1 and P10/11). The authors use morphological, electrophysiological, and cell-biological approaches and find that there are no differences in synapse formation and astrocyte cytoarchitecture in the mutant hippocampus and visual cortex. These results differ from the previous results (Stogsdill et al., 2017), although the authors make several discussion points on how the differences could have been induced. This study provides important information on how astrocytes and neurons interact with each other to coordinate neural development and function. The experiments were well-designed, and the data are of high quality.
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