Astrocyte reactivity and inflammation-induced depression-like behaviors are regulated by Orai1 calcium channels

Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca2+ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors including anhedonia and helplessness. These findings identify Orai1 as an important signaling hub controlling astrocyte reactivity and astrocyte-mediated brain inflammation that is commonly observed in many neurological disorders.

5. Overall, the amounts of released cytokines are low (~50 pg/ml for IL-6 after Tg/PDBu, Fig. 4B, compared to ~3200 pg/ml after LPS (Lu, X et al, J Neuroinflamm.) or 400 pg/ml after Il-1a/TNFa stimulation (Nakajima et al., 2022), in this later report, release of PGE2 was shown to be enhanced by KD of Orai2.In addition, Thrombin induced release is probably too low to detect by ELISA (only mRNA levels are shown in Fig. 4E and show a much lower induction compared to Fig. 4A).As all the behavioral assays are performed with LPS injection, the authors need to compare the effects of LPS on cytokine release from WT and KO astrocytes (see above).Importantly, how does acute inhibition of Orai1, i.e. with BTP-2 affect LPS induced release from WT or KO astrocytes? 6. Are the similar results on release of inflammatory cytokines from astrocytes observed when culturing reactive astrocytes without using the AWESOM protocol?LPS treatment in WT mice induces reactive astrocytes as shown in Fig. 5, is it possible that KO of Orai1 blocks the transition into reactive astrocytes also in cultures?7. Fig. 2 F-H: absent NFAT translocation is expected in the absence of Orai1, novelty? 8. Do mice with Orai1 deleted microglia show differences in LPS induced depression like behavior and vice versa, do Orai1 deficient astrocyte mice show altered neuropathic pain? 9. Since there are significant gender-specific differences seen in neuropathic pain (Tsujikawa et al., 2023) and in inflammation induced depression by LPS (this MS), from which gender was the RNA-Seq data derived?Are there gender specific differences in the RNAseq data isolated from male or female mice?Can gender-specific differences be seen in cytokine release of cultured astrocytes, potentially treated with BDNF? 10. Figure S1  This manuscript should be of interest to the broad readership of Nat communications.
Reviewer #3 (Remarks to the Author): Regulation of astrocyte-mediated brain inflammation by Orai1 channels.

Novakovic et al.
Astrocytes are a major CNS cell type and are increasingly recognized as important components of the CNS response to injury and disease, adopting what is commonly referred to as a 'reactive' phenotype.However, the molecular mechanisms underlying reactive astrogliosis are largely unknown.Ca<sup>2+</sup> release from internal stores, in an IP<sub>3</sub> dependent manner, is thought to be central to astrocyte function.Following depletion of internal Ca<sup>2+</sup> stores, ongoing signaling must be maintained by store-operated Ca<sup>2+</sup> entry (SOCE).Navakovic and colleagues have investigated the role of SOCE in regulating astrocyte reactivity by genetically ablating a key subunit of the Ca<sup>2+</sup> release activated Ca<sup>2+</sup> channel (CRAC) system, Orai1.Novakociv and colleagues report that deletion of Orai1 from astrocytes downregulates the expression of key glycolytic enzymes, metabolic intermediates and impairs ATP production.Furthermore, in their hands, Orai1 deletion reduces cytokine production in the hippocampus, reduces reactive astrogliosis and impacts inhibitory synaptic transmission in hippocampal CA1.Building on these observations, Novakovic et al., then report that mice with astrocyte-specific Ora1a are protected against inflammation induced depression.
Given the increasing recognition of astrocytes as key players in CNS disease, the manuscript is timely and the central theme of the manuscript appears novel.The manuscript itself contains a large volume of work, which has obviously required a large investment of time and effort on behalf of the authors.However, in my opinion, there are issues which need to be addressed before the manuscript can be considered for publication in Nature Communications.

Major issues:
(i) For the central claims of the manuscript to be valid, Orai1 deletion must be cell autonomous and exclude potential off-target effects.However, in my opinion, the authors provide no such evidence and we are left to believe the specificity of the Aldh1l1-CreER<sup>T2</sup> and GFAP-Cre lines.While these lines have been widely used, it is my opinion, that the authors should still demonstrate specificity in their hands.Otherwise, isn't it suspicious that expression of IBA1 (a Ca<sup>2+</sup> binding protein) is reduced in Orai1 cKO mice compared to wild types?Or is this juust coincidental?Along similar lines, I do not see any indication of how pure their astrocyte cultures are.Surely, it is important to exclude microglial contamination when measuring cytokine levels (either RNA or protein)?Finally, and a more nuanced point, even if Cre-mediated recombination is/was limited to astrocytes, the time course of protein turnover and how this relates to the experimental paradigms is not fully evident (as it was only assessed in cultured cells: see below).Perhaps, the authors could also comment on why tamoxifen was added to cultures: I thought the active metabolite 4-hydroxy tamoxifen needed to be added.How does this impact their results?
(ii) The authors make extensive use of cultured astrocytes in their work.Whether cultured astrocytes fully recapitulate <i>in vivo</i> astrocytes is hotly debated (for example, see Foo et al., Neuron, 2011).In an attempt to offset this criticism, the authors use "AWESAM" astrocytes which they claim are "stellate astrocytes with complex morphology, long processes and a more <i>in vivo</i> like transcriptome".The original paper describing "AWESAM" astrocytes showed that these cells express high levels of proteins associated with vesicle trafficking (Wolfes et al., J Gen Physiol, 2017), which is not the situation for <i>in vivo</i> hippocampal astrocytes (Chai et al., Neuron, 2017).Furthermore, the author's own data showing cell morphology (Fig. 4G) does not correspond to a "stellate" structure.Coupled with the extreme treatments used (e.g.prolonged PDBu exposure) the authors should, in my opinion, be much more circumspect with the conclusions they draw.While this could be offset by appropriate <i>in vivo</i> measurements, this type of experiment is generally lacking.
(iii) Personally, I am not sure if the data presented really offer a mechanistic explanation for what is observed.The authors refer several times to Ora1 being a "key checkpoint" for pro-inflammatory cytokine production.However, this protein is involved in refilling ER Ca<sup>2+</sup> stores, as emphasized by the authors, so what exactly is the link to increased transcription and, perhaps more importantly, cytokine release?In this respect, the paper feels slightly superficial.
(iv) The lack of observed depressive effects induced by inflammation in female wild type mice impacts the global significance of the study, and should be more thoroughly addressed by the authors.

Minor issues:
(i) In general, the manuscript would benefit from tidying.There were several instances of incomplete text (e.g.pSIRV-NFAT-eGFP and was a gift: page 35), figures were cited out of order in the text and some were missing (Fig. 6J: page 17), references were incorrectly cited (there is no Ref 79 listed: Supp Figure 2).Are mouse genotypes really correctly cited with appropriate nomenclature?(ii) In general, Ca<sup>2+</sup> signaling in astrocytes is much more complex than presented by the authors in the 'Introduction' -see for example, the various types of Ca<sup>2+</sup> measured in hippocampal and striatal astrocytes (see Chai et al., Neuron, 2017).
(iii) My personal opinion is that some of the authors claims are not substantiated by the data.mRNA levels do not reflect protein levels, and deletion of one channel subunit could affect the stability of other subunits at the protein level (Page 6).Likewise, does the <i>in vitro</i> calibration for fura-2 accuratrely reflect the <i>in vivo</i> situation, or is this an approximation?(see Helmchen, CSH Protocols, 2011).
(iv) Images of GFAP, IBA1 and C3 levels in hippocampal slices are not convincing.Would larger images work better?Even assuming that the GFAP and IBA1 responses were supporting extensive gliosis, why is the C3 signal so low (Figure 5 and Supp Fig 6).
(v) The electrophysiological measurements show an interesting effect on excitatory and inhibitory transmission but the measurements appear superficial.Why was the analysis limited to mini-analysis? (vi) Could the authors speculate in the "Discussion" about the potential therapeutic aspects to their work?
We thank the reviewer for their constructive comments which we believe have significantly improved the manuscript.
1. Is the reduced ATP release seen in Orai1 deleted astrocytes (Toth et al)  3. Release of many pro inflammatory cytokines usually entails activation of P2RX7 channels and inflammatory cytokines usually entails activation of P2RX7 channels and indeed, acute stress induces depressive like behavior has been shown to be meditated by astrocytic meditated by astrocytic and microglial P2X7 channels (Zhao YF et al., 2022, see also 51 citations on astrocytes, P2X7 and microglial P2X7 channels (Zhao YF et al., 2022, see also 51 citations on astrocytes, P2X7 and inflammatory cytokines).These data need to be discussed and using the available RNA Seq Seq data, the authors need to show in immunohistochemical staining of adult astrocytes from tamoxifen ochemical staining of adult astrocytes from tamoxifen induced Orai1KO (or GFAP-Cre) if levels of P2X7, and some selected components such as Cre) if levels of P2X7, and some selected components such as pannexins, connexins, TLR receptors (or other pathways of inflammation (Fig. 2B)) are altered in pannexins, connexins, TLR receptors (or other pathways of inflammation (Fig. 2B)) are altered in vivo.If downregulated, mediators of the classic LPS triggered NLRP3 inflammasome, potentially ted, mediators of the classic LPS triggered NLRP3 inflammasome, potentially indirectly protect mice from inflammation induced anhedonia, helplessness and despair.In spinal indirectly protect mice from inflammation induced anhedonia, helplessness and despair.In spinal slices, astrocytic connexin43 hemichannels respond to LPS (Panattoni et al., Mol Brain 2021).We should note that these effects are not likely relevant for the whole-animal peripheral LPS injection studies we carried out in Figures 5, 6, 7, and 8 of the manuscript because LPS is not known to cross the blood brain barrier (BBB).   5. Overall, the amounts of released cytokines are low (~50 pg/ml for IL-6 after Tg/PDBu, Fig. 4B, compared to ~3200 pg/ml after LPS (Lu, X et al, J Neuroinflamm.) or 400 pg/ml after Il-1a/TNFa stimulation (Nakajima et al., 2022), in this later report, release of PGE2 was shown to be enhanced by KD of Orai2.In addition, Thrombin induced release is probably too low to detect by ELISA (only mRNA levels are shown in Fig. 4E and show a much lower induction compared to Fig. 4A).As all the behavioral assays are performed with LPS injection, the authors need to compare the effects of LPS on cytokine release from WT and KO astrocytes (see above 6.Are the similar results on release of inflammatory cytokines from astrocytes observed when culturing reactive astrocytes without using the AWESOM protocol?LPS treatment in WT mice induces reactive astrocytes as shown in Fig. 5, is it possible that KO of Orai1 blocks the transition into reactive astrocytes also in cultures?Seq data derived?Are there gender specific differences in the RNAseq data isolated from male or female mice?Can gender-specific differences be seen in cytokine release of cultured specific differences be seen in cytokine release of cultured astrocytes, potentially treated with BDNF? astrocytes, potentially treated with BDNF?While it is excellent that data can be reproduced, also in a different mouse model, all data with GFAP-Cre should be moved to the supplemental figures.   Figure 6.Comparison of cytokine induction in astrocytes from male and female mice.Astrocytes were separately cultured from individual as described in the paper.Regardless of.The sex of the mouse, cell stimulation with Tg/PdBu caused strong induction of numerous cytokine genes which was suppressed to similar extents in male and female astrocytes.This result indicates that astrocyte Orai1 channels make equal contributions to inflammatory cytokine synthesis in both sexes.We thank the reviewer for thoughtful comments and recommendations. 1.For the central claims of the manuscript to be valid, Orai1 deletion must be cell autonomous and exclude potential off-target effects.However, in my opinion, the authors provide no such evidence and we are left to believe the specificity of the Aldh1l1-Cr CreERT2 and GFAP-Cre lines.While these lines have been widely used, it is my opinion, that the authors should still demonstrate specificity in their hands.Otherwise, isn't it suspicious that expression of IBA1 (a Ca2+ binding protein) is reduced in Orai1 cKO mice compared to wild types?Or is this juust coincidental?Along similar lines, I do not see any indication of how pure their astrocyte cultures are.Surely, it is important to exclude microglial contamination when measuring cytokine levels (either RNA or protein)?Finally, and a more nuanced point, even if Cre-mediated recombination is/was limited to astrocytes, the time course of protein turnover and how this relates to the experimental paradigms is not fully evident (as it was only assessed in cultured cells: see below).Perhaps, the authors could also comment on why tamoxifen was added to cultures: I thought the active metabolite 4-hydroxy tamoxifen needed to be added.) The authors make extensive use of cultured astrocytes in their work.Whether cultured astrocytes astrocytes fully recapitulate fully recapitulate in vivo astrocytes is hotly debated (for example, see Foo et al., Neuron, 2011).In an attempt to In an attempt to offset this criticism, the authors use "AWESAM" astrocytes which they claim are offset this criticism, the authors use "AWESAM" astrocytes which they claim are "stellate astrocytes with complex morphology, long processes and a more in vivo like like transcriptome".The original paper describing "AWESAM" astrocytes showed that these cells transcriptome".The original paper describing "AWESAM" astrocytes showed that these cells express high levels of proteins associated with vesicle trafficking (Wolfes et al., J Gen Physiol, 2017), which is not the situation for in vivo hippocampal astrocytes (Chai et al., Neuron, 2017).Furthermore, the author's own data showing cell morphology (Fig. 4G) does not correspond to a "stellate" structure.Coupled with the extreme treatments used (e.g.prolonged PDBu exposure) the authors should, in my opinion, be more circumspect with the conclusions they draw.While this could be offset by appropriate in vivo measurements, this type of experiment is generally lacking.
In general, the manuscript would benefit from tidying.There were several instances of incomplete text (e.g.pSIRV-NFAT-eGFP and was a gift: page 35), figures were cited out of order in the text and some were missing (Fig. 6J: page 17), references were incorrectly cited (there is no Ref 79 listed: Supp Figure 2).Are mouse genotypes really correctly cited with appropriate nomenclature?        (ii) In general, Ca 2+ signaling in astrocytes is much more complex than presented by the authors in the 'Introduction' -see for example, the various types of Ca 2+ measured in hippocampal and striatal astrocytes (see Chai et al., Neuron, 2017). in-situ 2+   2+    2+   (iii) My personal opinion is that some of the authors claims are not substantiated by the data.mRNA levels do not reflect protein levels, and deletion of one channel subunit could affect the stability of other subunits at the protein level (Page 6).Likewise, does the in vitro calibration for fura-2 accurately reflect the in vivo situation, or is this an approximation?(see Helmchen, CSH Protocols, 2011).
in vitro in vitro  2+                 (iv) Images of GFAP, IBA1 and C3 levels in hippocampal slices are not convincing.Would larger images work better?Even assuming that the GFAP and IBA1 responses were supporting extensive gliosis, why is the C3 signal so low (Figure 5

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): The reviewer appreciates the careful and thorough revision of the manuscript and the detailed comments and additional experiments which have improved the manuscript.The minor points that remain are listed below.The new Figure 6 is indeed very exciting and adds significant information.which would be near zero) and also to include the AUC of SOCE as a bar graph.In addition, Fig. 1F is confusing: Why is the relative Orai1 expression not reduced in the Cre+ tam+ induced cells? Figure 1B is not mentioned in the text on page 7 and line 164, page 7 is confusing as the Tg peak is analyzed and the mRNA expression not altered.Please check figures versus text.Analysis of expression of inflammasome related genes was carefully carried out and presented in the comments to reviewers (Fig. 1).Is there a mention of these important results within the manuscript?P2RX7 channels are not mentioned in the text, despite extensive literature on their role in inflammation, also in astrocytes.Please cite Zhao YF et al., 2022, for role of P2RX7 in depressive like behavior and add to discussion.Same applies to the replies to additional points that yielded important and interesting results.The reviewer is aware of the already extensive supplementary results and figures, but the manuscript would benefit from addition of Figures 3 and 5  Astrocytes are a major CNS cell type and are increasingly recognized as important components of the CNS response to injury and disease, adopting what is commonly referred to as a 'reactive' phenotype.However, the molecular mechanisms underlying reactive astrogliosis are largely unknown.Ca<sub>2+</sup> release from internal stores, in an IP<sub>3</sub> dependent manner, is thought to be central to astrocyte function.Following depletion of internal Ca<sup>2+</sup> stores, ongoing signaling must be maintained by store-operated Ca<sup>2+</sup> entry (SOCE).Navakovic and colleagues have investigated the role of SOCE in regulating astrocyte reactivity by genetically ablating a key subunit of the Ca<sup>2+</sup> release activated Ca<sup>2+</sup> channel (CRAC) system, Orai1.Novakociv and colleagues report that deletion of Orai1 from astrocytes downregulates the expression of key glycolytic enzymes, metabolic intermediates and impairs ATP production.Furthermore, in their hands, Orai1 deletion reduces cytokine production in the hippocampus, reduces reactive astrogliosis and impacts inhibitory synaptic transmission in hippocampal CA1.Building on these observations, Novakovic et al., then report that mice with astrocyte-specific Ora1a are protected against inflammation induced depression.
Given the increasing recognition of astrocytes as key players in CNS disease, the manuscript is timely and the central theme of the manuscript appears novel.This is a revision of a previously submitted manuscript.
Major issues: In general, the authors answered all comments raised in review.However, while I acknowledge the effort put into the new Ca<sup>2+</sup> imaging data, I do not feel as though they fully answer the question of what is the mechanistic connection of Orai1-mediated Ca<sup>2+</sup> signaling and the observed effects on synaptic transmission.At least one plausible explanation put forward by the authors is an effect on Ca<sup>2+</sup>-evoked gliotransmitter release.As the evidence points towards the gliotransmitter involved being ATP it would be relatively easy to pharmacologically tests the link between Ca<sup>2+</sup> signaling, ATP release and synaptic transmission.
of the current manuscript is a repeat of data as shown in Toth et al; Fig. 1D indeed is nearly identical to Fig. 1D in Toth et al., and the Thrombin response (Fig. 1I) a repeat of Fig. 2C in Toth et al.Supplementary Figure 7A-D a repeat of recordings done in Toth et al.While it is excellent that data can be reproduced, also in a different mouse model, all data with GFAP-Cre should be moved to the supplemental figures.Reviewer #2 (Remarks to the Author): In this work Murali Prakriya et al. identify Orai1 calcium channels regulate astrocyte metabolic reprogramming and pathogenic function in the context of neuroinflammation and inflammationinduced depression.Authors represent astrocytes lacking Orai1 shows significant decrease of pro-inflammatory signals (IL-               observations, astrocyte Orai1 KO mice reduces CNS inflammation in LPS-evoked neuroinflammation model.Furthermore, Astrocyte Orai1 channels have been shown to regulate inflammation-induced depression behaviors.

Figure 1 :
Figure 1: RNA Seq analysis of select molecules involved in the LPS inflammatory response.(A) Expression of key genes involved in LPS induced inflammatory responses and inflammasome pathway (TLR4, Nlrp3, P2RX7, P2RX4.)Expression was not altered in Orai1 cKO astrocytes.(B) By contrast, stimulated astrocytes showed marked downregulation of several genes including P2 X7, TLR4, and Panx2 in both WT and Orai1 cKO astrocytes.

Figure 2 :
Figure 2: Immunohistochemistry of P2X7, TLR4, and Connexin 43 in the hippocampus in .Immunostaining of hippocampal brain slices for P2RX7, TLR4, and Connexin43.We saw broad labelling of these proteins in the CA1 region of the hippocampus and this was not affected by ablation of Orai1.Coronal cut brain slices (30 µm thick) were labelled with antibodies for the indicated proteins.

Figure 4 :
Figure 4: Cytokine analysis of astrocytes grown in the presence of FBS.(A) Morphology of astrocyte grown in the absence (AWESAM) or presence of serum (FBS).Cells were stained for GFAP with a monoclonal Ab. (B) Cytokine induction in the astrocytes grown in the presence of serum (FBS).Thapsigargin/PdBu treatment stimulates induction of IL-6, MIP-1, and TNF-.

Figure 5 :
Figure 5: Comparison of A1 vs A2 markers.Comparison of A1 vs A2 markers.Astrocytes were stimulated with TG/PdBu to evoke Orai1 activation and the A1 and A2 evoke Orai1 activation and the A1 and A2 markers described by Liddelow were analyzed in WT and Orai1 cKO cells.analyzed in WT and Orai1 cKO cells.

Figure 7 :
Figure 7: Specificity of Orai1 deletion.(A) To assess whether the (A) To assess whether the Orai1 fl/fl Aldh1l1-Cre line affects Orai1 expression in microglia, we cultured line affects Orai1 expression in microglia, we cultured microglia from Orai1 fl/fl Aldh1l1-Cre mice treated with tamoxifen and mice treated with tamoxifen and examined Orai1 expression by real-time PCR.Orai1 expression istime PCR.Orai1 expression is unaffected in microglia in this line .(B) Purity of astrocytes.We stained unaffected in microglia in this line .(B) Purity of astrocytes.We stained our primary astrocyte cultures for GFAP (astrocyte marker) and IBA1 our primary astrocyte cultures for GFAP (astrocyte marker) and IBA1 (microgial marker).No IBA1 staining was visible in our cultures.The right panel shows microglia isolated from the same mouse stained for IBA1.

Figure 8 :
Figure 8: Analysis of Orai1 protein expression by IHC.(A) Orai1 is expressed in the CA1 hippocampus, particularly near the pyramidal layer (PL), but also in the stratum oriens (SO SO) and stratum radiatum (SR SR). ).(B) Higher magnification image shows astrocytes in the SR SR region co labeled with Orai1 in WT mice.cKO mice have less Orai1-gfap colocalization than WT mice.Scale bar = 25 µm.(C (C) Cells co-labeled with GFAP and Orai1 were quantified in the SO and SR layers using a 5 µm thick stacked image.Nuclei of GFAP+ cells were identified and cells with Orai1 staining within 5 µm of the nuclei were considered Orai1+ astrocytes.(Data are mean +/-SEM.n=4 mice/group; >100 cells/mouse.p=8.9x10 4 ).).

Figure 9 :
Figure 9: PKC activation alone (with PDBu) without concomitant Orai1 activation fails to induce inflammatory cytokine production.Astrocytes were treated with PDBu for 6 hours and cytokine mRNA levels were assessed by real-time PCR.

Figure 10 :
Figure 10: Orai1 channels regulate LPS evoked increases in astrocyte Ca 2+ signaling.(A) Schematic illustrating experimental protocol.GCaMP6f was expressed in astrocytes of the hippocampal CA1 using stereotaxic injections of AAV5 virus with an astrocyte-specific gfaAB1D promoter.After 2 to 3 weeks to allow for expression, mice were injected with either 1 mg/mL LPS or equivalent volume of saline.24 hours following intraperitoneal LPS injection, Ca 2+ 2+ fluctuations in CA1 astrocytes expressing GCaMP6 were imaged using 2PLSM.(B) Examples of Max ax-IP images of IP images of astrocytes transfected with gCAMP6f in the CA1 region of the hippocampus.(C) Sample traces from the soma, primary branches, and distal branches of astrocyte Ca 2+ fluctuations in brain slices from WT+saline, WT+LPS, cKO+saline, and cKO+LPS mice.(D) Summary graphs for frequency of calcium oscillations calculated over three minutes of imaging.Each dot represents one ROI (amplitude is the average of all peaks in one ROI, frequency is total # of events/3 minutes).WT saline: n=3 mice, 16 cells; WT+LPS: n=3 mice, 15 cells; cKO+saline: n=3 mice, 11 cells; cKO+LPS: n=3 mice; 13 cells.* = p<0.05,** = p<0.01,*** = p<0.005.

Figure 1 :
Figure 1: The authors moved previously published mouse model data to the supplementary figures and added two bar graphs to the analysis of the new inducible Orai1KO experiments.I would recommend to indicate Tg-release in Fig 1E (as one would expect that the SOCE Area is analyzed,which would be near zero) and also to include the AUC of SOCE as a bar graph.In addition, Fig.1Fis confusing: Why is the relative Orai1 expression not reduced in the Cre+ tam+ induced cells?Figure1Bis not mentioned in the text on page 7 and line 164, page 7 is confusing as the Tg peak is analyzed and the mRNA expression not altered.Please check figures versus text.Analysis of expression of inflammasome related genes was carefully carried out and presented in the comments to reviewers (Fig.1).Is there a mention of these important results within the manuscript?P2RX7 channels are not mentioned in the text, despite extensive literature on their role in inflammation, also in astrocytes.Please cite Zhao YF et al., 2022, for role of P2RX7 in depressive like behavior and add to discussion.Same applies to the replies to additional points that yielded important and interesting results.The reviewer is aware of the already extensive supplementary results and figures, but the manuscript would benefit from addition of Figures3 and 5to the supplements and to the discussion.The dual activation protocol entails pretreatment with LPS (Signal 1) for several hours and then addition of ATP.How were the experiments in Reviewer Fig.3performed?
Figure 1: The authors moved previously published mouse model data to the supplementary figures and added two bar graphs to the analysis of the new inducible Orai1KO experiments.I would recommend to indicate Tg-release in Fig 1E (as one would expect that the SOCE Area is analyzed,which would be near zero) and also to include the AUC of SOCE as a bar graph.In addition, Fig.1Fis confusing: Why is the relative Orai1 expression not reduced in the Cre+ tam+ induced cells?Figure1Bis not mentioned in the text on page 7 and line 164, page 7 is confusing as the Tg peak is analyzed and the mRNA expression not altered.Please check figures versus text.Analysis of expression of inflammasome related genes was carefully carried out and presented in the comments to reviewers (Fig.1).Is there a mention of these important results within the manuscript?P2RX7 channels are not mentioned in the text, despite extensive literature on their role in inflammation, also in astrocytes.Please cite Zhao YF et al., 2022, for role of P2RX7 in depressive like behavior and add to discussion.Same applies to the replies to additional points that yielded important and interesting results.The reviewer is aware of the already extensive supplementary results and figures, but the manuscript would benefit from addition of Figures3 and 5to the supplements and to the discussion.The dual activation protocol entails pretreatment with LPS (Signal 1) for several hours and then addition of ATP.How were the experiments in Reviewer Fig.3performed?
due to reduced release (Toth et al) or due to an overall increase in AMP/ATP ratios and reduced ATP (Fig 3 F,G, current MS) and thereby leading to a reduced vesicular ATP concentration?Same applies for the reduced glutamate release reported in Toth et al. which may be due to altered glutamine metabolism.In Toth et al., a direct role of SOCE in release of vesicles was postulated.Is this result still valid?
). Importantly, how does acute inhibition of Orai1, i.e. with BTP-2 affect LPS induced release from WT or KO astrocytes?                                              2+               Lack of depression type behaviors in female mice.                     ) and Supp Fig 6).             2+         J Gen Physiol                      Journal of visualized experiments : JoVE 