In a genetic screen, we found a zebrafish strain in which mitochondria fail to attach to the dynein retrograde motor. This strain carries a loss-of-function mutation in actr10, a member of the dynein-associated complex dynactin. The abnormal axon morphology and mitochondrial retrograde transport defects observed in actr10 mutants are distinct from dynein and dynactin mutant axonal phenotypes. In addition, Actr10 lacking the dynactin binding domain maintains its ability to bind mitochondria, arguing for a role for Actr10 in dynactin-mitochondria interaction. Finally, genetic interaction studies implicated Drp1 as a partner in Actr10-dependent mitochondrial retrograde transport. Together, this work identifies Actr10 as a factor necessary for dynactin-mitochondria interaction, enhancing our understanding of how mitochondria properly localize in axons.Mitochondrial transport in axons is critical for neural circuit health and function. While several proteins have been found that modulate bidirectional mitochondrial motility, factors that regulate DOI:http://dx.doi.org/10.7554/eLife.22234.001 Mitochondrial transport in axons is critical for the formation and function of the nervous system. This organelle generates the ATP necessary for energy demanding functions in all cells, but neurons are especially reliant on mitochondria to maintain their electrically polarized state. After depolarization, ATP-dependent ion pumps are employed to repolarize the cell and prepare it for another action potential. It is estimated that at rest alone, neurons use 4.7 billion molecules of ATP per second . BecauseIn addition to maintaining axon health, mitochondrial motility is also necessary to maintain mitochondrial health and function. Mitochondria undergo fission-fusion dynamics that facilitate both the replenishment of proteins in this organelle and the maintenance of mitochondrial DNA quantity and integrity  display axon terminal swellings in the central and peripheral nervous systems indicative of retrograde transport abnormalities. Analysis of cargo localization and movement in the actr10 mutant revealed clustering of mitochondria, but not other cargos analyzed, at microtubule plus ends due to failed retrograde mitochondrial movement. This phenotype was vastly different from loss of either Dynein heavy chain or p150, potentially indicating a unique function for Actr10 in mitochondrial retrograde movement. Furthermore, we demonstrated that abnormal mitochondrial movement in actr10 mutants is due to failed attachment of mitochondria to the dynein-dynactin complex in the absence of Actr10. Importantly, Actr10 engineered to lack the dynactin binding domain maintains mitochondrial interaction, hinting at a specific role for Actr10 in mediating dynactin-mitochondria interaction. Finally, we provide biochemical and genetic evidence that Drp1, a Dynamin-related protein previously implicated in microtubule minus end-directed mitochondrial movement  mediated disruption of the first exon of actr10, which phenocopied the mutant axon terminal swellings  system to identify novel mediators of retrograde cargo transport in axons . pLL axoulations . Using aulations . RNAmappulations  identifiwellings .10.7554/actr10 mutants, can arise due to a number of intracellular abnormalities, including disruptions in retrograde cargo transport . actr10 mutants displayed mitochondrial accumulation in pLL axon terminals  analyses of pLL axon terminals to determine if mitochondria are the predominant cargo mislocalized in actr10 mutants, mitochondria in single pLL axons were tagged with TagRFP using 5kbneurod:mito-TagRFP plasmid injection and organelle movement was visualized by confocal microscopy . In contrast, both the frequency of retrograde mitochondrial transport and the proportion of mitochondria moving in the retrograde direction were dramatically decreased in actr10 mutants  tagged Actr10 in individual pLL neurons using zygotic injection of a 5kbneurod:mRFP-actr10 DNA plasmid and assessed axon terminals. While mRFP-Actr10 expression in wildtype axons had no apparent effects  nor p150 were significantly mislocalized in actr10 mutants, though we did observe a trend of increased DHC in actr10 mutant axon terminals . These experiments demonstrated that loss of Actr10 does not impede dynein-dynactin complex localization or dynein retrograde movement in axons.Previous work on Actr10 function assayed mitotic spindle positioning during cell division in cultured fibroblasts and nuclear positioning in fungi to show that Actr10 depletion phenocopied dynein loss of function . In assoerminals . Next, tat 4 dpf . Additioin vitro synthesized mRNA. First, we confirmed that a GFP tagged version of dynein intermediate chain 2b (i2b), a core dynein protein, could integrate into the motor complex. mRNA encoding i2b-GFP was injected into zygotes and anti-GFP antibodies were used to immunoprecipitate the complex. Extracts were subjected to western blot analysis and probed with anti-DHC antibodies. i2b-GFP can integrate into the core dynein complex . Upon further investigation, we found that previous studies revealed a surprising relationship between mitochondria and peroxisomes, including a large number of shared membrane proteins, such as Drp1  and mature (4 dpf) neurons while p150a is enriched only in mature neurons 4 dpf . Loss of p150a alone has no discernable effect on axons or mitochondrial localization  from HEK293T cells. Whereas Actr10△7–△10 deletions showed reduced binding to dynactin, Actr10△7 most consistently failed to immunoprecipitate dynactin in all experiments ; n = 4. dynactin ; n = 3. dynactin ; n = 2, K38A variant  in pLL neurons using injection of a plasmid encoding the respective open reading frame under an inducible promotor. Mitochondria were labeled by 5kbneurod:mito-EGFP co-injection. In wild-type larvae, expression of Drp1K38A-mRFP caused mitochondrial clustering in the perinuclear region, phenotypically similar to the results observed in cultured fibroblasts  that is essential for interaction with the dynactin complex but inconsequential for interaction with mitochondria. This piece of data is critical as it demonstrates that Actr10 can interact with this organelle independently of dynactin, arguing for a direct role for this protein in mitochondria-dynactin interaction. Together, our data argue that Actr10 is necessary for dynactin-dependent, retrograde transport of mitochondria in axons.We provide multiple pieces of evidence supporting a function for Actr10 in mitochondrial transport. First, mitochondria are the only cargo analyzed whose localization or retrograde transport is significantly perturbed with loss of Actr10. If dynein-mediated cargo movement was generally disrupted, other cargos, including lysosomes and dynein itself, would fail to move in the retrograde direction as well. Second, the axonal phenotype of dynactin and dynein loss-of-function mutants is vastly different from actr10 mutants have slight deficits in the distance moved by mitochondria in both the anterograde and retrograde directions. While we cannot definitively ascertain the underlying mechanism of this disruption, at least three possibilities exist. First, bidirectional transport distance could be affected by the abnormal localization of mitochondria. Mitochondrial localization directly affects local levels of ATP, which in turn could compromise the processive activity of ATP-dependent motors necessary for long distance transport. Another possibility is local deficits in mitochondrial activity: if mitochondrial health is compromised, this could account for the bidirectional transport deficits of mitochondria. Finally, it is possible that Actr10 or its interacting proteins, such as p62, regulate motor engagement. It is largely thought that cargos typically have anterograde and retrograde motors attached simultaneously and direction of transport is attributed to regulation of motor engagement. More frequent oscillations in motor engagement due to loss of Actr10 or Actr10 interactors could account for the shortened distances moved by mitochondria in both directions. At this point, we do not have a full explanation for these transport parameter alterations but they are a topic of continued interest.Actr10 could directly link mitochondria to dynactin or potentially facilitate this interaction through other dynactin pointed end proteins, such as p62. p62 is of particular interest in this regard as it is predicted to rely on Actr10 for binding to the dynactin complex . Based oneurospora and aspergillus demonstrated that loss of Actr10 phenocopies the abnormal nuclear positioning observed with loss of dynein  mitochondrial retrograde, but not anterograde, transport is disrupted in actr10 mutants while all mitochondrial movement is inhibited in p150a/b mutants; and (3) localization and transport of all other cargos are not disrupted in actr10 mutants. Together, these results argue that loss of Actr10 does not impact all dynein-dynactin function. In addition, a variant of Actr10 lacking the dynactin binding domain retains its ability to interact with mitochondria, further substantiating a direct role for this protein in mitochondria-dynactin interaction. Together, our data argue that Actr10 participates in mitochondrial attachment to the retrograde motor protein complex in neurons in addition to potentially facilitating Arp1 capping and nuclear positioning in other contexts.Given the previous data implicating Actr10 in Arp1 capping and subsequent dynactin stability and dynein function , we wereThe interaction between Drp1 and Actr10 in mitochondrial retrograde transport may provide a link between mitochondrial movement and mitochondrial fission. Originally, we investigated the role of Drp1 in retrograde mitochondrial transport as previous studies revealed the unique ability of this GTPase to elicit mitochondrial localization to microtubule minus ends , 1998. UK38A could work. We cannot differentiate between these possibilities at this point. Nevertheless, given the intriguing data generated here, it is tempting to speculate that, upon the GTP-GDP transition, not only do mitochondria undergo fission but Drp1 also recruits the retrograde motor protein complex through interaction with Actr10 to facilitate separation of these daughter mitochondria. The precise nature of Drp1-Actr10 interaction and the role of this interaction in mitochondrial retrograde transport is a subject of current investigation.Similarly, our work in conjunction with other studies supports the argument that Drp1 participates in both mitochondrial fission and retrograde mitochondrial transport. Drp1 translocates to mitochondria where it can bind to receptors Fis1 and Mff . In its actr10-dync1h1 and actr10-p150a/b mutant phenotypes in axons. In addition, we identified Drp1, a GTPase implicated in mitochondrial fission, as an Actr10 interactor with a potential role in mitochondrial retrograde transport. Together with previous studies on mitochondrial motility, a complex picture is emerging in which mitochondrial transport and fission/fusion are dependent on a core group of proteins, with an independent set of proteins regulating mitochondrial docking. In conclusion, further work is crucial to understanding how mitochondrial transport and dynamics are orchestrated in axons, which will ultimately allow us to better understand how this critical organelle is positioned and maintained. As mitochondrial transport and dynamics have been implicated in neurological disorders, a more complete mechanistic understanding of their regulation will provide insight into disease pathology.The coordinated regulation of mitochondrial transport is necessary for the formation and maintenance of active neural circuits. Our work identifies Actr10 as a crucial protein for mitochondrial interaction with the dynein-dynactin complex, regulating retrograde transport. In addition, we argue that the role of Actr10 is specific and not due to a general loss of dynactin stability or dynein function. The strongest pieces of evidence supporting this assertion are the ability of Actr10 to bind mitochondria in the absence of dynactin and the disparate nature of the nl1TgBAC(neurod:egfp) nl1 , actr10nc1h1mw20 , p150a  were crossed to WIKs to generate a mapping strain. Heterozygous F1 hybrids were identified using pair wise crosses. Eighty wildtypes/heterozygotes and 80 actr10 mutants were obtained from a single mapping pair. These larvae were lysed at 4 dpf in Trizol according to the manufacturer’s protocol. mRNA was extracted in phenol:chloroform:isoamyl alcohol and twice in chloroform before the pellet was redissolved in 20 µL of RNase/DNase free water. Sequencing was performed by the OHSU Massively parallel sequencing core on an Illumina HiSeq2000, generating approximately 30 million reads per condition. From this RNAseq data, RNAmapper identified a single nucleotide change in the start codon of the actr10 gene in the nl15actr10 mutant pool. The mutation  completely segregated with the mutant phenotype. For experiments, actr10 mutants were identified by genotyping according to the following protocol: PCR amplification of the region around exon 1 of the actr10 locus resulted in a 374 bp product  and actr10 mutant (195 bp) bands.RNAseq-based mapping was performed according to established protocols . actr10 actr10 was cloned from a 2 dpf zebrafish cDNA library and ligated into the pSC-A-amp/kan vector . All expression and mRNA synthesis vectors were constructed using Gateway compatible cloning as described  and a 2 dpf zebrafish cDNA library. The zebrafish Drp1 ortholog was cloned from 2 dpf cDNA, sequence verified, and cloned into the pDONR221 middle entry vector using BP cloning . To generate K38ADrp1, the middle entry vector was mutated to change Lysine 38 to Alanine . All expression plasmids were constructed by LR cloning using the 5kbneurod . TALENs targeting exon 1 of actr10 were designed using freeware  and synthesized as described  heterozygous outcrosses were injected with 10 pg guide RNA and 100 pg Cas9 mRNA at the one cell stage. Progeny were raised until adulthood and outcrossed to wildtypes to generate larvae. Larvae were genotyped using PCR amplification of the exon 3 locus  and cut with MwoI to identify insertions/deletions. Changes to the guide RNA target result in loss of the MwoI cut site in p150b mutants: wildtype (164 bp + 64 bp) from p150b mutants (224 bp).The escribed . Guide Rescribed . Cas9 mRescribed  was prodIn situ hybridization and antibody labeling were done according to established protocols ; α-p150 ; α-Lamp1 ; α-pJNK ; α-GFP ; α-DsRed ; α-Cytochrome c ; α-GFP ; α-c-myc ; and α-ATPβ .rotocols . The actDrp1-myc and K38A-mycDrp1 expression constructs were previously described  were cultured according to standard protocols in DMEM with 50 U/mL Pen-Strep. Cells tested mycoplasma free . This human cell line was used for its transfection ease and results were confirmed in vivo. Transfection was done in a 6-well plate using 2.5 µg total DNA and 5 µL of Lipofectamine2000 per well according to manufacturer’s protocols . The mammalian GFP-Actr10 clone was used previously  and obtaescribed . GFP-Actnl15actr10 mutant larvae were disrupted in 700 µL MB buffer  using a 26 gauge needle. After removal of the input fraction, lysates were subjected to increasing centrifugation speeds to fractionate mitochondria from a heavy and light fractions. The mitochondrial fraction was washed once in fresh MB buffer prior to addition of Laemmli buffer to the pellet and heat denaturation. Extracts were run on a 10% acrylamide gel, transferred to PVDF membrane, and incubated with antibodies prior to developing with the West Pico Substrate . After initial development, membranes were washed in water and PBS/0.1% Tween prior to incubation with the α-ATPβ antibody.Mitochondrial fractionation was performed according to established protocols . BrieflyAxonal transport analyses were done as described . BrieflyZebrafish larvae at 5 dpf were fixed and prepared for TEM analysis using standard protocols . Samplesneurod:egfp transgene in axon terminals and the mean fluorescence intensity was determined. Background from the same channel was subtracted prior to analysis. For quantification of western blots, mean intensity of was determined for signal and background using ImageJ. Statistical analyses were done in JMP. Prior to parametric analyses, data normality and variance were determined. Parametric data was analyzed using ANOVAs with Tukey-Kramer HSD contrasts for multiple comparisons. Non-parametric data was analyzed using Wilcoxon/Kruskal Wallis Tests. Image analyses and figure preparation was done using ImageJ, Adobe Photoshop, and Adobe Illustrator.Volumetric analyses to determine mean fluorescence intensity of immunolabeling was done using Imaris (Bitplane). For this analysis, a surface rendering was done using expression of the  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.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Tanya Whitfield as Reviewing Editor and Jonathan Cooper as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.actr10 mutants, and mutants in dynein and dynactin components have more severe and widespread phenotypes than actr10, supporting the notion that Actr10 functions specifically in transport of mitochondria. The authors suggest that Acrt10 interacts physically with the dynactin complex and brings it to mitochondria. Furthermore, the authors identify a region of the Actr10 protein that is required for physically interacting with the dynactin complex, but not mitochondria. Finally, biochemical and genetic experiments demonstrate that Actr10 interacts with the mitochondrial protein Drp1 and that a putative gain-of-function Drp1 mutant requires Actr10 to increase retrograde transport, supporting the idea that Drp1 recruits Actr10 to mitochondria.This interesting study describes a forward screen in zebrafish that identifies a component of the retrograde transport machinery. The authors propose that the Actr10 protein acts as a linker between mitochondria and the dynein/dynactin complex. Specifically, the authors set out to show that in Actr10 mutant zebrafish, retrograde transport of mitochondria, but not other cargo, is specifically compromised. Dynein transport is apparently normal in Essential revisions:1) The results showing that the trafficking effects are specific to mitochondria are not completely convincing, and require further experiments and controls. It is not clear that mitochondria accumulate more than other cargoes in axon swellings. The images should be analysed more quantitatively, avoiding potential effects of swelling size by normalising to area/volume. Anterograde and retrograde traffic should be quantified. 2) Critical controls are absent from several figures. Please see the specific comments from Reviewer 2.Please see the individual reviews, which are appended below, for further information.Reviewer #1:The conclusion about mitochondrial specificity would be even stronger if the authors analyzed more cargo using kymograph analysis-not just lysosomes and JNK. In a previous paper, the authors looked at a wide variety of cargo , so this should be technically feasible.actr10 and wt. Another good way to do it would be to normalize the intensity of each marker by taking a ratio to a volume-filling reporter, such as cytoplasmic GFP. If they are correct, the cyt10 to GFP ratio should be higher in actr10 mutants relative to wt, but the other markers should be the same in both genotypes.In addition to the kymograph analysis, the authors could quantify enrichment in an alternative, less biased way. For example, perhaps they could outline the entire neuromast and determine the average pixel intensity for each marker over the entire structure in One issue that the authors may wish to address a bit more, either through discussion or experiments, is the generalizability of these results. Is mitochondrial transport also affected in other neurons in the Actr10 mutant fish? What is the ultimate consequence of these defects to the neuron? Do they eventually degenerate? What is the ultimate fate of these fish? Is the Actr10 locus associated with any human diseases?Reviewer #2:I have raised a few queries into methodological and interpretative issues below. One particular issue is the "mitochondria specific" effect that is not supported by the Lamp1 and pJNK staining. I suggest the authors reconsider this.actr10 embryos .actr10 extracts performed at the same time as co-IP from i2b injected embryos.) This is in addition to the comment above.A&B: I am confused by the fact that the heavy fraction is strongly enriched for mitochondria. This suggests incomplete lysis of embryos. Probably just a technical issue, because 9F does not show the same enrichment?actr10 lysate. This in itself might affect transport and needs to be investigated.There appears to be less p150 in The loss of p150 from the mito fraction is striking, but the appearance in the supernatant cannot be correctly interpreted without using non-mitochondrial loading controls. I would suggest probing these blots with the following antibodies to ensure these fractions are "pure". (i) a nuclear protein that should label the heavy fraction (ii) another mito protein to confirm ATPβ. (iii) tubulin (DM1A) to confirm supernatant and mito fractions.actr10, particularly important in the case of the light fraction.These controls will also confirm equal loading for WT and C-D: control IPs are not presented to show how much p150 is immunoprecipitated using "beads only". This needs to be shown for each experiment. Also the level of GFP immunoprecipitated in Δ7 to Δ13 is much lower, so it is difficult to compare the efficiency of p150 IP.E: Again controls are missing. Is this specific?actr10. Beads only controls (no primary antibody) are also important here.actr10 axons it is less compelling to say that there is no effect of K38A because of a loss of interaction. i.e. if the effect of mutant actr10 on mitos is independent of the effect of DRP1 won't we still get the same result?eLife. Your revised article has been favorably evaluated by Jonathan Cooper (Senior editor), Tanya Whitfield (Reviewing editor), and two reviewers.Thank you for resubmitting your work entitled "Regulation of mitochondria-dynactin interaction and mitochondrial retrograde transport in axons" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below (see comments from Reviewer 2). In particular, it is essential that the immunoprecipitation experiments shown in Reviewer #1:actr10 mutants; second, they have re-analyzed image data with quantification methods that are more convincing and added new measures of transport that support their conclusions; third, they have added critical controls for biochemical experiments; finally, they have revised the text to emphasize alternative interpretations of key conclusions. This is now a thorough and convincing manuscript that identifies a key protein regulating the transport of specific axonal cargoes to maintain healthy axons, making this work of interest to both cell biologists and neuroscientists.The authors have been responsive to reviewers and improved the manuscript in several notable ways: First, they have performed electron microscopy to demonstrate that mitochondria accumulate in terminals of Reviewer #2:The authors have modified the manuscript, and it shows considerable improvement.I find 2 areas that require further explanation1) Reversals:Results section paragraph four, – Unclear- 0/100 micron in mutants?Needs to be only in moving mitochondria not per 100 micron, since there are less moving in mutants there is less chance for reversal?2) IP:actr10 extracts performed at the same time as co-IP from i2b injected embryos.)"My request: "it is critical to include the relevant control. i.e. western blot showing that the p150 signal is only present when i2b is pulled down.  The results showing that the trafficking effects are specific to mitochondria are not completely convincing, and require further experiments and controls. It is not clear that mitochondria accumulate more than other cargoes in axon swellings. The images should be analysed more quantitatively, avoiding potential effects of swelling size by normalising to area/volume. Anterograde and retrograde traffic should be quantified. actr10 mutant axon terminals. While we cannot rule out a role for Actr10 in the movement of another cargo, these data indicate that not all cargo rely on Actr10 for retrograde transport.To address this issue, we reanalyzed our immunolabeling experiments to control for axonal volume, assayed the transport of additional cargos in axons, and perform TEM analyses of mutant axon terminals. Altogether, these data demonstrated that there is no identifiable change in the amount of other cargos in 2) Critical controls are absent from several figures. Please see the specific comments from Reviewer 2.To address this comment, we repeated a subset of these experiments with bead only controls as recommended. These additional controls confirmed our previous observations and are now included in the revised manuscript. Please find below an outline of our specific responses and where the corrections have been made in the text and figures. Please see the individual reviews, which are appended below, for further information.Reviewer #1:The conclusion about mitochondrial specificity would be even stronger if the authors analyzed more cargo using kymograph analysis-not just lysosomes and JNK. In a previous paper, the authors looked at a wide variety of cargo , so this should be technically feasible.We performed live imaging experiments to analyze the transport of two additional cargos in response to this review, peroxisomes and late endosomes/lysosomes labeled by Lamp1. JNK transport, as performed in our earlier work, was not feasible at 4 dpf, the time-point at which our other experiments were performed in this paper . The results of our experiments were very informative. First, we analyzed peroxisome transport. Surprisingly, there were small alterations in the distance of peroxisome retrograde transport and velocity of anterograde peroxisome movement . Upon fuIn addition to the kymograph analysis, the authors could quantify enrichment in an alternative, less biased way. For example, perhaps they could outline the entire neuromast and determine the average pixel intensity for each marker over the entire structure in actr10 and wt. Another good way to do it would be to normalize the intensity of each marker by taking a ratio to a volume-filling reporter, such as cytoplasmic GFP. If they are correct, the cyt10 to GFP ratio should be higher in actr10 mutants relative to wt, but the other markers should be the same in both genotypes.actr10 mutants. In addition, we used the same approach to analyze the amounts of Dynein heavy chain and p150 in actr10 mutants.We agree this type of analysis is indeed more suitable for signal quantification in axon terminals and thank the reviewer for this suggestion. As suggested by the Reviewer, we used volumetric analyses in Imaris software to determine the mean fluorescent intensity relative to the volume of axon terminals (determined using the cytoplasmic EGFP fill to create a “surface”) for Cytochrome c (mitochondria), Lamp1, and pJNK in One issue that the authors may wish to address a bit more, either through discussion or experiments, is the generalizability of these results. Is mitochondrial transport also affected in other neurons in the Actr10 mutant fish? What is the ultimate consequence of these defects to the neuron? Do they eventually degenerate? What is the ultimate fate of these fish? Is the Actr10 locus associated with any human diseases?actr10 mutants do not degenerate prior to larval death. In addition, the actr10 locus is not associated with any disease states save a short report demonstrating transcription of actr10 is increased in an in vitro model of prion disease .As Reviewer suggested, we elaborated briefly on the general mutant phenotype in the Results section. Specifically, we stated that the mutant is not homozygous viable and both CNS and PNS axons have these large swellings. Due to the limitations of imaging in other types of neurons (those positioned deeper within the embryo) with the current protocols we have in place, we did not visualize mitochondrial transport in other neuronal subtypes at this time. While we do not discuss it in the manuscript, the axons in the Reviewer #2:I have raised a few queries into methodological and interpretative issues below. One particular issue is the "mitochondria specific" effect that is not supported by the Lamp1 and pJNK staining. I suggest the authors reconsider this.Results section and The analysis shown in the graphs in In my opinion the swelling doesn't just contain mitochondria. This is true for other genetic mutations that give rise to axonal swelling in the literature.In relation to the specificity of actr10 mutation for mitochondria in comparison to pJNK and LAMP it would be necessary to demonstrate that transport of these cargoes is not affected in actr10 mutant axons. This could be achieved using kymograph analysis.EM could also be used to identify the contents of these swellings. Alternatively, the authors should remove the claim that these swellings are purely a consequence of mitochondrial accumulation.actr10 mutants. Based on our analyses, we can only say that the other cargos we analyzed are localized and transported normally and dynein is capable of moving normally in the retrograde direction. In addition, no other cargo appears significantly accumulated in actr10 mutant axon terminals based on our TEM analyses. However, it is possible that movement of other cargo, not analyzed in our studies, is also interrupted in actr10 mutants. We would like to clarify that our main motivation for the analysis of other cargos was to show that loss of Actr10 does not impact all dynein-mediated cargo transport. This point is now discussed throughout the manuscript.The points raised by this reviewer are critical to clarify and address for the submitted manuscript. Importantly, we do not to claim that mitochondria are the only cargo which is abnormally localized or transported in actr10 mutants, we reanalyzed the data using volumetric surface analyses in Imaris as described above. This type of analysis of fluorescence intensity within axon terminals defined by the presence of cytoplasmic EGFP  demonstrated that Cytochrome c immunofluorescence is significantly increased in actr10 mutants while Lamp1 and pJNK are not. This is now shown in In addition, to more clearly show that at least some non-mitochondrial cargos are localized normally in We also analyzed the transport of two additional cargos, peroxisomes and lysosomes, using our established protocols. The results are discussed above in regard to Reviewer 1 point 1 and depicted in actr10 mutants. These analyses clearly show a preponderance of mitochondria in the axon terminal swellings of lateral line axons. This is shown in Finally, we collaborated with Amy Herbert and Kelly Monk (now co-authors on the paper) to perform TEM analyses of the PNS axon terminals in “Our data does not preclude a function for Actr10 in the retrograde transport of as of yet unidentified cargo in axons or other roles for Actr10 in cells, which could vary based on a number of factors including cell type and developmental stage.”.Results section and actr10 mutants in what is now We have used a different kymograph to show mitochondrial transport in Results section and We have now specified the number of axon terminals analyzed in wildtype and mutant animals as well as the number of biological replicates in subsection “Actr10 functions autonomously in neurons to regulate axon morphology and mitochondrial localization “of the revised manuscript.Results section and actr10 mutant axon terminals relative to controls.We quantified the p150 fluorescence intensity using volumetric analyses in Imaris software. This quantification is shown in  in vivo (e.g. Gross JCB 2002).actr10 mutants in our imaging experiments. Rather, based on the population analyses shown in We appreciate the question and think the analysis of reversals adds to the value of the manuscript. To address this issue, we quantified the number of reversals in mitochondrial and dynein movement in the revised manuscript. The results are now reported in the text. We observe no change in reversal frequency of mitochondria or dynein-positive cargos in The need for bead only controls was duly noted. To address this concern, we repeated the specified immunoprecipitation experiments with no primary (bead only) controls. These experiments confirmed that i2b-GFP can interact with the dynein complex and mRFP-Actr10 but not mRFP-Δ7Actr10 can interact with dynactin. These new Western blots are now shown in A&B: I am confused by the fact that the heavy fraction is strongly enriched for mitochondria. This suggests incomplete lysis of embryos. Probably just a technical issue, because 9F does not show the same enrichment?The loss of p150 from the mito fraction is striking, but the appearance in the supernatant cannot be correctly interpreted without using non-mitochondrial loading controls. I would suggest probing these blots with the following antibodies to ensure these fractions are "pure". (i) a nuclear protein that should label the heavy fraction (ii) another mito protein to confirm ATPβ. (iii) tubulin (DM1A) to confirm supernatant and mito fractions.These controls will also confirm equal loading for WT and actr10, particularly important in the case of the light fraction.While the Reviewer raised a valid point, we believe that the observed variations in this experiment are due to the less stringent nature of this particular fractionation . This means that the mitochondrial fraction is only highly enriched for mitochondria relative to the other fractions as described by Prudent et al., 2013. The comparison to the mitochondrial loading control is therefore quite important as it shows that, relative to the amount of mitochondrial protein in this fraction specifically, the amount of p150 precipitated is dramatically reduced. To clarify this point, we have quantified the levels of p150 for each fraction relative to the input as well as mitochondrial loading in each fraction. This quantification is now shown as There appears to be less p150 in actr10 lysate. This in itself might affect transport and needs to be investigated.actr10 mutant fraction relative to wildtype siblings has now been quantified. In addition, the levels were normalized to the mitochondrial control (ATPβ) in the corresponding fractions to correct for any variation in loading. We felt it was important to normalize levels of p150 to mitochondria in each fraction, as the question we were addressing in this experiment was mitochondrial attachment to dynactin in the presence or absence of Actr10. This is now shown in The amount of p150 in the actr10 mutants, as detected by Western blot  controls are now shown for Drp1 functions with Actr10 in mitochondrial retrograde transport“.Because of time limitations, we were not able to repeat the Actr10-Drp1 coimmunoprecipitation with whole cell extracts probed for GFP; however this immunoprecipitation was replicated >3 times with consistent results. This is now stated in the text in subsection “K38A relocalizes mitochondria in a microtubule-dependent fashion to microtubule minus ends, implicating the retrograde motor in this process. 3) Our data also shows that loss of Actr10 causes accumulation of mitochondria in axon terminals due to failed transport while Drp1K38A causes accumulation of mitochondria in the cell body, likely due to increased retrograde movement. In contrast, if Drp1K38A functioned in a pathway parallel to Actr10, we would predict: 1) a further reduction in mitochondrial number in the axon; 2) a rescue  in mitochondrial density in the axon terminals; and 3) mitochondrial accumulation in the cell body of actr10 mutants, none of which are observed. Together, we use these data to argue that Drp1 in its GDP-bound form, is working with Actr10 to regulate mitochondrial localization in axons. This argument is now better articulated in the Discussion section.With regard to Drp1K38A’s impact on mitochondrial localization: we agree with the reviewer that the results of this experiment on their own are not entirely conclusive and should be interpreted with caution. This is now explicitly noted in the Discussion section of the revised manuscript. However, we also argue that there are multiple pieces of evidence to support our argument that Drp1 and Actr10 function together. 1) We show that the K38A mutation in Drp1 strengthens the interaction with Actr10. 2) In addition, work from others  demonstrates that expression of Drp1actr10 mutant axon terminals, leading to the reduction in number in the axon capable of being transported. Without a motile pool, Drp1K38A would not be able to decrease the number of mitochondria in the mutant axons to the same degree as in wildtype axons. This is now pointed out in the revised text. Future experiments using live imaging of mitochondrial transport would likely be able to discern between these possibilities.We also outlined an addition caveat related to this experiment. It is possible that the motile pool of mitochondria (~50% based on our work) is trapped in Unfortunately, these types of experiments are not possible without the generation of additional stable transgenic lines which would take approximately 6 months.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below (see comments from Reviewer 2). In particular, it is essential that the immunoprecipitation experiments shown in In the revised manuscript, we have addressed the three additional concerns raised by reviewer two in response to our resubmission. First, we changed the way we analyzed mitochondrial reversal frequency to make it relative to the number of mitochondria present in the axons. Second, we performed the necessary control experiments for


Although mGluR5-antagonists prevent fear and anxiety, little is known about how the same receptor in the amygdala gives rise to both. Combining in vitro and in vivo activation of mGluR5 in rats, we identify specific changes in intrinsic excitability and synaptic plasticity in basolateral amygdala neurons that give rise to temporally distinct and mutually exclusive effects on fear-related behaviors. The immediate impact of mGluR5 activation is to produce anxiety manifested as indiscriminate fear of both tone and context. Surprisingly, this state does not interfere with the proper encoding of tone-shock associations that eventually lead to enhanced cue-specific fear. These results provide a new framework for dissecting the functional impact of amygdalar mGluR-plasticity on fear versus anxiety in health and disease.DOI:http://dx.doi.org/10.7554/eLife.25665.001 But this line of investigation remains largely unexplored in the amygdala, as much of our current understanding is based primarily on studies that used systemic administration of mGluR5 antagonists to modulate these behaviors in rodents  impairs the acquisition of auditory fear conditioning . Further rodents .RS)−3,5-dihydroxyphenylglycine (DHPG)  . For inse (DHPG) , as welle (DHPG) . This rae (DHPG)  showed te (DHPG) , would aTo address these questions, we first examined the impact of bath application of DHPG on principal neurons of the LA using whole-cell current-clamp recordings in coronal brain slices prepared from adult male rats . As repo+) was paired with a foot shock (US) and the other was not (CS−) . Next, the same animals received in vivo infusions of DHPG into the BLA, followed by the same sequence of tone habituation and weak-US conditioning (Day 4). In contrast to saline, infusions of DHPG in the same animals caused a significant increase in freezing to both the CS+ and CS− during tone habituation  of awake, behaving rats . Followiot (CS−) . Using tn Day 4, . Moreoven Day 4, .10.7554/+ and CS− . Surprisingly, despite DHPG triggering an immediate and significant enhancement in freezing to both the CS− Day 4, , 24 hr l+ and CS−immediately after the infusion of DHPG indicative of fear generalization? To address this question, we quantified freezing exhibited immediately before the onset of any tones. Interestingly, these animals exhibited high levels of freezing (33.6 ± 10.7%) even before the onset of the first tone , thereby leading to distinct effects on specific versus indiscriminate fear. DHPG treatment alone enhanced LA neuronal excitability without eliciting any LTP in vitro . Consist in vivo . In othe in vivo . While t in vivo . Thus, uwithout any pre-exposure to tones during a habituation session – and fear memory was still enhanced. This suggests that it is the tone-shock pairing in the presence of DHPG that boosts the subsequent cue-specific fear learning, not the pre-exposure to tones. In other words, DHPG may persistently increase excitability of amygdalar neurons such that pairing CS+ with even a weak US in the presence of DHPG is sufficient to subsequently enhance fear learning. Interestingly, a similar effect of DHPG on neuronal excitability has been observed in the hippocampus  weighing 200–300 grams  were obtained using a Vibratome , transferred to a submerged holding chamber containing oxygenated ACSF and allowed to recover for 1 hr at room temperature. Individual slices were then transferred to a submerged recording chamber (28 ± 2°C) and neurons were visually identified using an upright differential interference contrast microscope .Rats were anaesthetized using halothane and decapitated. The brain was quickly dissected out and transferred to oxygenated, ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 115 NaCl, 25 glucose, 25.5 NaHCOSCR_000325), unless otherwise stated. Neurons were used for recording if the initial resting membrane potential (Vm) <-60 mV, series resistance (sR) was 15–25 MΩ and were rejected if the sR changed by >20% of its initial value. For all recordings, neurons were held at −70 mV.Patch pipettes  pulled from thick-walled Borosilicate glass using a P1000 Flaming/Brown micropipette puller  and filled with an internal solution containing (in mM) 120 K-gluconate, 20 KCl, 10 HEPES, 4 NaCl, 4 MgATP, 0.3 NaGTP, 0.2 EGTA and 10 phosphocreatine  were used to patch on to principal neurons in the Lateral Amygdala. For voltage clamp experiments, potassium was replaced with equimolar cesium in the internal solution. In experiments where evoked responses were recorded, a bipolar electrode  connected to an ISO-Flex stimulus isolator (A.M.P.I.) was used to stimulate the thalamic inputs to the amygdala. Data were recorded with an EPC-9 amplifier , filtered at 2.9 kHz and digitized at 20 kHz. Data acquisition and stimulus delivery were performed using Patchmaster software , while analysis of electrophysiological data was performed using custom-written programs in IGOR Pro  was used for analyzing the traces. A total of 7 cells  were used for the sEPSC experiments. 12 cells  were used for the mEPSC experiments.LA neurons were clamped at −70 mV and spontaneous excitatory postsynaptic currents (sEPSCs) were isolated with picrotoxin (75 µM). Miniature excitatory postsynaptic currents were isolated by clamping LA neurons at −70 mV in the presence of TTX (0.5 µM) and picrotoxin (75 µM). For both sets of experiments baseline current traces of 5 min duration (recorded at least 5 min after achieving whole-cell configuration) were recorded. 100 µM of 3,5-Dihydroxyphenylglycine  was applied in the bath solution for 10 min and then washed out for 15 min . Continuf-I) relationship, the number of action potentials fired during a 600 msec pulse of depolarizing current injections from 50 pA to 300 pA was recorded. Two pulses for each current magnitude were recorded and the average number of spikes was obtained for each current magnitude. After measuring baseline firing, the firing properties of the same neuron were measured during a 10 min bath application of 50 µM DHPG, starting 5 min from the onset  were recorded in response to stimulation of internal capsule fibers containing thalamic inputs to the LA (at 0.05 Hz). In the ‘DHPG’ group, 50 µM DHPG was bath applied to the slices for 10 min after achieving a stable whole-cell configuration followed by a 30 min washout. In the ‘Weak Pairing’ group, potentiation was induced at the thalamic inputs after recording a stable baseline for 5 min. LTP induction consisted of 10 stimuli delivered at 30 Hz and repeated 15 times with an interval of 30 s between trains . The posThe animals were handled for 2 days to habituate to the experimenter before the start of any experimental procedures. After this 2 day handling period, the animals were bilaterally implanted with stainless steel cannulae targeted at the amygdala. After the surgery, the animals were allowed to recover for 7–8 days. Then, the animals were habituated to the conditioning context on Day 1 and Day 2 for 25 min per day , left. OFor targeted infusion of DHPG into the BLA, rats were surgically implanted with bilateral, chronic, intracranial stainless steel guide-cannulae  aimed at the dorsal half of the BLA.Rats were subjected to anesthesia with 5% isoflurane  and then maintained under anesthesia with 1.5–2% isoflurane. The level of anesthesia was regularly monitored throughout the procedure using the pedal withdrawal reflex to toe pinch. The animal was placed and head fixed on a stereotaxic frame. Body temperature of rats was maintained with a heating pad. Burr holes were drilled at the stereotaxic coordinates of the BLA  and seven animals were used in the control group . The locations of the cannulae placement for the eight animals used in the experimental group study are shown in −1. The injection cannula was taken out 5 min after the end of infusion, to allow the drug to diffuse into the tissue. After the completion of experiments, cannulae placement was confirmed using standard histological methods . Conditioning was performed in a box with metal grids on the floor . Fear recall was performed in two contexts, one was a modified homecage , and the second was a hexagonal box made of plexiglass . Lighting conditions and floors and walls were different between the three contexts. All chambers were cleaned with 70% alcohol before and after each experiment.The behavior of the animals was recorded using a video camera mounted on the wall of the sound isolation box and a frame grabber (sampling at 30 Hz). The videos were analyzed offline for further quantification of freezing behavior . Infrare+: 5 kHz continuous tone) was paired  with a co-terminating 1 s scrambled foot shock , whereas the other was not (CS−: 15 kHz clicks). Thus, ten CS+-US pairings with ten randomly interleaved CS− presentations were given in random order during this differential conditioning procedure. In the testing session, the animals were introduced into either context B or context C. The animals were allowed to explore the context for 3 min and then presented with both CS− and CS+  in a pseudo-random order  to test recall of fear memory.During context habituation, the animals were allowed to explore context A for 25 min in each session. Next, in the tone habituation session  the animBehavioral response was hand scored offline using video recordings of tone habituation and all testing sessions. Response to the auditory stimuli was evaluated in the form of a freezing response. Freezing was defined as the absence of movement except due to respiration . The timAfter the experiment was concluded, rats were deeply anesthetized . Coated silver wires with a bare tip were inserted into the cannulae (1 mm projection) and electrolytic lesions  were made to mark the in vivo infusion sites. The animals were then perfused transcardially with ice-cold saline (0.9%) followed by 10% (vol/vol) formalin. The perfused brain was left in 10% (vol/vol) formalin overnight. Coronal sections (80 μm) were prepared using a vibratome  and mounted on gelatin-coated glass slides. Sections were stained with 0.2% (wt/vol) cresyl violet solution and mounted with DPX . The slides were imaged to identify and reconstruct infusion sites .− and CS+ were analyzed by repeated measures ANOVA as all the data passed the normality test. Further, post hoc Sidak’s test was used to analyze the differential freezing responses in different sessions and the differential freezing responses to CS− or CS+. The variances for all the datasets of CS− and CS+ were statistically similar except for the habituation session on Day 4. Repeated measures ANOVA was also used to analyze the pretone freezing across contexts/sessions followed by post hoc Sidak’s test where all the datasets were normal. The variance of pretone freezing during the habituation session on Day 4 was more than the other sessions. Two-way repeated measures ANOVA was used to analyze the percentage EPSP slopes across groups in the LTP experiments. This was followed by Tukey’s post-hoc test for comparison between groups for each minute. All the data sets passed the normality test. All statistical tests were performed using GraphPad Prism .All values are expressed as mean ± SEM. Each data set was evaluated for statistical outliers, which were defined as greater than twice the standard deviation away from the mean. According to this criterion, one neuron was excluded from mEPSC experiment in  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.eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom, Jennifer L Raymond (Reviewer #2), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Fabricio do Monte (Reviewer #1).Thank you for submitting your article "Activation of the same mGluR5 receptors in the amygdala causes divergent effects on specific versus indiscriminate fear" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:+, CS- and the context on the day of training. The manuscript is well written, and the study was generally well designed.The authors address the interesting question of the role of mGluR5 in fear learning and anxiety. The authors demonstrate that activation of mGluR5, with the agonist DHPG in the amygdala, promotes subsequent fear learning to a specific sound. Mechanistic insight into this phenomenon is provided by showing that coactivation of mGluR5 with a subthreshold LTP inducing stimulation leads to robust and persistent LTP, and that DHPG increases excitability of amygdala neurons, which likely underlies the enhancement of LTP. The authors also make a case that mGluR5 activation also leads to indiscriminate fear and anxiety by showing that DHPG injected in the amygdala during a "tone only" habituation period increases freezing behavior that is unrelated the tone. Perhaps most interesting is the observation that specific tone-shock associations can be learned in the presence of DHPG, even though the animals are exhibiting indiscriminate freezing in response to CSEssential revisions:The reviewers agreed that two key control experiments are necessary to support the authors' claims:+ and CS- observed on day 5 may be caused exclusively by the extra-conditioning session that animals received on the previous day. Therefore, to confirm that the observed increase in freezing response was due to intra-LA infusion of DHPG, a control group undergoing the same behavioral protocol, but receiving intra-LA infusion of saline on day 4, needs to be performed. The lack of a saline-control group becomes more problematic in 1) Most notably, in 2) The interpretation of the increase in sEPSCs in In addition, the reviewers raised several points that should be addressed with revisions to the text:a) Rudy and Matus-Amat (2009) have demonstrated that infusion of DHPG into the basolateral amygdala increases freezing induced by a weak foot shock conditioning. This work should be acknowledged in the Introduction, and a clear explanation of how the present manuscript differs from the previous study needs to be provided. More generally, the authors should be sure to emphasize what is novel in their findings, given that it known that mGluR5 is required for fear memory and LTP in the amygdala, and DHPG can prime hippocampal neurons for LTP.b) The subjects of the present study were 5-7 weeks old male Sprague-Dawley rats. Rats at this age are still considered adolescents. Considering that adolescent rats can differ from adults in learning and memory, as well as in fear responses, the authors should make it clear that they are using adolescent animals. In addition, they should cite previous studies on fear conditioning using adolescent rats in the Introduction and Discussion.c) Based on the map drawings of histological placement presented on d) The Abstract and the last sentence of the Discussion talk about a new "framework" for examining mGluR5-plasticity. What is that framework? Using agonist instead of antagonist? Thinking of fear and anxiety as independently modulated?+ and DHPG during the habituation period is required for subsequent fear association with the CS+, but they do not actually test this. Does DHPG injected in the amygdala in their home cage without a habituation period or pre-exposure to CS+ enhance subsequent fear learning? DHPG may persistently increase excitability of amygdalar neurons (as occurs in hippocampus) such that presentation of CS+ with the weak US after DHPG is sufficient to enhance learning.e) In the authors' schematic model they suggest that pairing of CSf) The authors also trained mice for fear learning to specific tones in different contexts. What is the role of the different contexts here? Does DHPG affect context specific fear learnings as well as to tones? This would strengthen the authors' conclusion that mGluR5 promotes discriminate learning.+ fear learning and LTP.g) The authors demonstrate that DHPG increases excitability of amygdalar neurons and so it is unclear if mGluR5 activity specifically is required to promote fear learning and anxiety or if simply increasing excitability will suffice. One way to test this would be to infuse a GABAA blocker into the amygdala during tone habituation, and test if this increases subsequent CS- start to go away when the associative conditioning begins?h) What is the behavior like during the conditioning session on Day 4, and especially towards the end of that session? Is there any evidence that the pretone freezing and the freezing to the CS Essential revisions:The reviewers agreed that two key control experiments are necessary to support the authors' claims:+ and CS- observed on day 5 may be caused exclusively by the extra-conditioning session that animals received on the previous day. Therefore, to confirm that the observed increase in freezing response was due to intra-LA infusion of DHPG, a control group undergoing the same behavioral protocol, but receiving intra-LA infusion of saline on day 4, needs to be performed. The lack of a saline-control group becomes more problematic in 1) Most notably, in We agree with the reviewers regarding the need for two key control experiments and we thank them for raising these issues. As summarized below, we have now performed both experiments and the new data are included in the revised manuscript.1) As suggested, to confirm that the observed increase in the freezing response was due to intra-LA infusion of DHPG, a control group undergoing the same behavioral protocol but receiving intra-LA infusion of saline on Day 4, were tested. To this end we infused saline twice (on Day 3 and Day 4) instead of saline (Day 3) followed by DHPG (Day 4).+ and not to CS-. Further, the animals showed cue-specific fear (significantly higher freezing in response to CS+ relative to CS-). Therefore, the indiscriminate fear observed in the DHPG-infusion group was not due to reintroduction to the same context where the animals were conditioned earlier, but a specific effect of DHPG infusion. Similarly, the saline-followed-by-saline infusion control group did not show enhancement of cue-specific fear on Day 5, thereby ruling out any effect of reconditioning. We also observed that the pretone freezing to the context is not enhanced in the saline-followed-by-saline group during habituation on Day 4, thereby confirming that the indiscriminate fear was caused by the DHPG infusion.We found that during habituation on Day 4, the saline infused rats showed enhanced freezing, compared to habituation on Day 3, only to CSWe have added a supplementary figure  depictin2) The interpretation of the increase in sEPSCs in We are grateful to the referee for raising this point. As advised, we have now carried out recordings of miniature excitatory postsynaptic currents (mEPSCs) in the presence of TTX. New data collected from these recordings show that even in the presence of 0.5µM TTX there is a significant increase in the frequency of mEPSCs elicited by bath application of 100µM DHPG, followed by a 15 washout (One-way repeated measures ANOVA followed by Holm-Sidak’s multiple comparison test). This clearly demonstrates the synaptic effects of DHPG, the point highlighted by the referee. We have reported this new result in the revised text and added the data to revised In addition, the reviewers raised several points that should be addressed with revisions to the text:a) Rudy and Matus-Amat (2009) have demonstrated that infusion of DHPG into the basolateral amygdala increases freezing induced by a weak foot shock conditioning. This work should be acknowledged in the Introduction, and a clear explanation of how the present manuscript differs from the previous study needs to be provided. More generally, the authors should be sure to emphasize what is novel in their findings, given that it known that mGluR5 is required for fear memory and LTP in the amygdala, and DHPG can prime hippocampal neurons for LTP.We thank the referee for raising these important points. We have made revisions in the relevant sections of both the Introduction and Discussion to include the specific points mentioned by the referee.a) The revised Introduction now clearly acknowledges the earlier study by Rudy and Matus-Amat , in which they showed that infusion of DHPG into the basolateral amygdala increases freezing induced by conditioning using a weak foot shock. Also, we have highlighted how the present study differs from the earlier study and how the strategy employed here attempts to address questions not examined by Rudy and Matus-Amat .b) Furthermore, we have also revised the Discussion to emphasize the novelty of our findings in light of earlier reports on mGluR5 and amygdalar LTP, fear memory and anxiety . Finally, we now mention findings on DHPG priming of hippocampal LTP and how they compares with our results in the amygdala , which is also related to points raised in part e) below.Together, we have made extensive revisions in the Introduction and Discussion to emphasize the novelty of our findings, and how they compare/contrast with earlier reports .b) The subjects of the present study were 5-7 weeks old male Sprague-Dawley rats. Rats at this age are still considered adolescents. Considering that adolescent rats can differ from adults in learning and memory, as well as in fear responses, the authors should make it clear that they are using adolescent animals. In addition, they should cite previous studies on fear conditioning using adolescent rats in the Introduction and Discussion.We have revised the manuscript to mention that adolescent rats were used . We have also added the relevant references as advised by the referee .c) Based on the map drawings of histological placement presented on As suggested by the reviewer, we have made corrections throughout the manuscript to mention that the infusions were targeted at the basolateral amygdala (BLA) instead of the lateral amygdala. We have also added a representative image of the site of infusion in d) The Abstract and the last sentence of the Discussion talk about a new "framework" for examining mGluR5-plasticity. What is that framework? Using agonist instead of antagonist? Thinking of fear and anxiety as independently modulated?We have revised the Discussion (first paragraph) to provide greater clarity on this important point raised by the referee. The findings reported here provide a framework for thinking about how fear and anxiety can be modulated independently by the same receptor in the BLA by using an mGluR5-agonist instead of an antagonist. Further, we find a similar separation at the cellular level between modulations of associative synaptic plasticity versus intrinsic plasticity. These represent a departure from earlier strategies using mGluR5-antagonists that did not explain how activation of the same receptor gives rise to both fear and anxiety. We have also made small modifications in the Abstract within the word limits allowed.+ and DHPG during the habituation period is required for subsequent fear association with the CS+, but they do not actually test this. Does DHPG injected in the amygdala in their home cage without a habituation period or pre-exposure to CS+ enhance subsequent fear learning? DHPG may persistently increase excitability of amygdalar neurons (as occurs in hippocampus) such that presentation of CS+ with the weak US after DHPG is sufficient to enhance learning.e) In the authors' schematic model they suggest that pairing of CSWe apologize for the lack of clarity on this important point. Assuming that the “schematic model” the reviewer is referring to is + and CS-) caused an indiscriminate and immediate increase in freezing to both tones, as well as the context (left panel). However, subsequent fear association only enhanced freezing to the CS+, i.e. cue-specific fear, without increasing fear of the CS-.1) Presentation of the two tones (CS+ with a weak US failed to form a strong fear memory (middle panel).2) In the absence of DHPG, the association of the CS+, but not the CS-) was formed by the same weak conditioning in the presence of DHPG (right panel).3) However, a significantly stronger fear memory . This indicates that it is the tone-shock pairing in the presence of DHPG  in the hippocampus .“DHPG may persistently increase excitability of amygdalar neurons such that pairing CSf) The authors also trained mice for fear learning to specific tones in different contexts. What is the role of the different contexts here? Does DHPG affect context specific fear learnings as well as to tones? This would strengthen the authors' conclusion that mGluR5 promotes discriminate learning.In this study we focused only on an auditory version of discriminative fear conditioning in rats. Hence, we used distinct tones in different contexts to minimize the potential contributions of context in fear generalization. This strategy was guided by our earlier finding that amygdalar neurons encode the switch from cue-specific to generalized fear, behavioral measures we use in the present study to examine the effects of in vivo activation of mGluR5 in the same brain area .We agree with the referee that a similar effect may exert its influence contextual fear learning too. Based on our findings, it is quite likely that DHPG infusion would lead to immediate increase in indiscriminate fear of contexts, but still enhance context-specific fear 24 hours later. We have revised the Discussion to mention this point based on the referee’s comment (second paragraph).+ fear learning and LTP.g) The authors demonstrate that DHPG increases excitability of amygdalar neurons and so it is unclear if mGluR5 activity specifically is required to promote fear learning and anxiety or if simply increasing excitability will suffice. One way to test this would be to infuse a GABAA blocker into the amygdala during tone habituation, and test if this increases subsequent CS- information in a manner that subsequently increased freezing to the CS- as well , not just to the CS+ alone (i.e. specific fear). Thus, these findings provide a separation between the effects of DHPG on anxiety versus specific fear, as well as neuronal excitability versus associative synaptic plasticity.In the present study, the DHPG-induced increase in excitability in amygdalar neurons was manifested is two ways – enhancement in both intrinsic and synaptic excitability. The in vivo impact of DHPG was an indiscriminate and immediate increase in freezing to both context and tones, without affecting subsequent formation cue-specific fear. In other words, though the synaptic plasticity triggered by CS-US association took place in the backdrop of heightened excitability through enhanced action potential firing and enhanced spontaneous synaptic activity, it did not interfere with the effective encoding of cue-specific fear memory. An alternative outcome could have been that the DHPG-induced increase in neuronal excitability also affected the inputs receiving CSAR activity can lead to similar effects. Indeed, enhancement of excitability of amygdalar neurons by reducing GABAAR activity also has similar effects  on anxiety and generalization of fear. Further, enhanced excitability of amygdalar neurons also plays a role in enhancement of cue-specific fear memory . Therefore, the suggestion from the reviewer that modulating GABAA activity may also have similar effects is possible. Based on this point raised by the referee, we have revised the Discussion to mention earlier work on the role of amygdalar GABAAR in fear and anxiety (third paragraph).We agree that mGluR5 activity is not the only way to mediate such effects. Modulation of GABA- start to go away when the associative conditioning begins?h) What is the behavior like during the conditioning session on Day 4, and especially towards the end of that session? Is there any evidence that the pretone freezing and the freezing to the CS- does not go away when the associative conditioning begins. Interestingly, there is higher indiscriminate freezing by the end of the conditioning session. We have now summarized these data in We thank the referee for raising this interesting question. We have carefully reviewed the data based on this query. The animals show indiscriminate freezing throughout the conditioning session on Day 4. The pretone freezing to CS


In recent reports, re-irradiation with stereotactic body radiotherapy for lung tumors in patients previously treated with thoracic radiation therapy resulted in several serious toxicities. Serious non-lung toxicities were observed mostly in patients with central tumors, but we experienced a case of fatal gastric perforation after a second stereotactic body radiotherapy in a patient with a peripheral lung tumor.An 83-year-old Asian man was diagnosed with T2N0M0 lung cancer in the form of squamous cell carcinoma in the lower lobe of his left lung. He was treated with stereotactic body radiotherapy of 40 Gy in 4 fractions and the tumor decreased in size in partial response. The local tumor recurred 8 months after the first stereotactic body radiotherapy, and he was re-irradiated with a second stereotactic body radiotherapy of 50 Gy in 4 fractions. A Sengstaken–Blakemore tube was inserted below his diaphragm by laparoscopic surgery before the second stereotactic body radiotherapy in order to reduce the stomach dose by keeping his stomach apart from the tumor. Two months after the second stereotactic body radiotherapy, he developed fatal gastric perforation and gastropleural fistula penetrating his diaphragm.To the best of our knowledge, this is the first report about a gastric perforation after stereotactic body radiotherapy for lung tumors and it warns of serious complication of stereotactic body radiotherapy in not only centrally located but also peripherally located tumors like in this case. Stereotactic body radiotherapy (SBRT) plays a major role in the treatment of early-stage non-small cell lung cancer and oligometastatic lung tumors . In receAn 83-year-old Asian man with a left lung tumor was referred to our hospital for SBRT in 2008. He had no clinical symptoms. Chest computed tomography (CT) showed a 3.5 cm-sized tumor located close to his left diaphragm. Magnetic resonance imaging of his head and fluorodeoxyglucose-positron emission tomography (FDG-PET)/CT showed no metastatic lesions. The maximum standardized uptake value (SUVmax) of the tumor on FDG-PET/CT was 6.9. The serum cytokeratin-19 fragment (CYFRA 21-1) level was elevated to 8.7 ng/mL. A transbronchial lung biopsy revealed squamous cell carcinoma. The lung cancer was staged at T2N0M0 based on the sixth TNM classification of malignant tumors. He had undergone upper lobectomy for stage I squamous cell lung cancer in 1993, and his medical history also included hypertension, atrial fibrillation, renal sclerosis, and chronic bronchitis with low respiratory function. Radical left lower lobectomy was not applicable because of his advanced age, history of left upper lobectomy, and low respiratory function; therefore, he selected SBRT.SBRT was performed with the self-breath-holding technique using a respiratory monitoring system. Image guidance was performed using a CT-on-rails system for all sessions. A total dose of 40 Gy in 4 fractions was delivered to 95% of the planning target volume. The details of irradiation are shown in Table Immediately after the completion of SBRT, he developed vomiting and upper abdominal pain, and he was consequently treated with a histamine H2-receptor antagonist. Two months after SBRT, CT revealed a partial response of the lung tumor, and the serum CYFRA 21-1 level had decreased to the normal range. However, approximately 3 months after SBRT, he developed hematemesis. Endoscopy showed a deep ulcer on the fornix of his stomach Fig. , and he The second SBRT was performed 11 months after the first SBRT. A Sengstaken–Blakemore tube was inserted below his diaphragm by laparoscopic surgery before the second SBRT in order to keep his stomach away from the tumor. The balloon was expanded with 200 ml of normal saline. A total dose of 50 Gy in 4 fractions was delivered to the isocenter. The Dmax of the stomach was 48.0 Gy, but we considered that the high-dose region differed from the previous one treated with the first SBRT, based on the change of the stomach shape by the Sengstaken–Blakemore tube. The dose distribution and dose volume histogram of the stomach are shown in Figs. One month after the second SBRT, he was hospitalized with melena and anemia. An endoscopy revealed a deep ulcer in the same region as after the first SBRT, and he was treated with a proton pump inhibitor Fig. . Subsequet al. reported the following serious toxicities after re-irradiation with SBRT for central tumors: three cases of grade 5 hemoptysis and one case of grade 4 vena cava superior stenosis and grade 4 fistula between the trachea and gastric tube [et al. reported a case of grade 5 aorta-esophageal fistula after re-irradiation with SBRT for a central tumor previously treated with chemoradiotherapy [et al. reported a case of fatal hemoptysis after re-irradiation with SBRT for central disease [In recent reports, re-irradiation with SBRT for lung tumors previously treated with thoracic radiation therapy resulted in several serious toxicities , 11, 17.otherapy , while TIn our case, the patient had a peripheral tumor that was treated twice with SBRT, and consequently developed serious gastric perforation. Referring to the endoscopic images, we suspected that this resulted from the high dose of the second SBRT delivered to the same gastric region as the first SBRT. Although we tried to extend the distance between the stomach and the tumor and to change the high-dose region in the stomach by inserting a spacer to reduce the stomach dose, we could not sufficiently extend the distance because of coalescent resulting from the first SBRT.et al. analyzed the predictors for severe gastroduodenal toxicity in patients treated with SBRT using 33 to 60 Gy  in 3 fractions for abdominopelvic malignancies [et al. [TM . Rigid registration was used to create fusion CT images focusing on the fornix of the stomach. The Dmax of the stomach in the summed plan was 83.5 Gy, which was considerably higher than the predictive doses.Bae gnancies . Forty p [et al.  were simFinally, although a detailed case presentation on serious gastric perforation after SBRT for lung tumors has not yet been reported, based on the present case, we conclude that we should carefully evaluate the stomach doses in both the first and second SBRT. Moreover, we should also be cautious of the toxicity after the first SBRT, and should observe the stomach with endoscopy immediately before the second SBRT.To the best of our knowledge, this is the first report about a gastric perforation after SBRT for lung tumors. Our case of re-irradiation with SBRT for a lung tumor suggests that it is necessary to pay attention to not only the tumor location in the lung but also to the doses to the normal tissue and any toxicity after initial irradiation.


Although randomized trials have established the clinical efficacy of treating all persons living with HIV (PLWHs), expanding treatment eligibility in the real world may have additional behavioral effects  or lead to unintended consequences . Using a regression discontinuity design, we sought to assess the effects of a previous change to Zambia’s HIV treatment guidelines increasing the threshold for treatment eligibility from 350 to 500 cells/μL to anticipate effects of current global efforts to treat all PLWHs.We analyzed antiretroviral therapy (ART)-naïve adults who newly enrolled in HIV care in a network of 64 clinics operated by the Zambian Ministry of Health and supported by the Centre for Infectious Disease Research in Zambia (CIDRZ). Patients were restricted to those enrolling in a narrow window around the April 1, 2014 change to Zambian HIV treatment guidelines that raised the CD4 threshold for treatment from 350 to 500 cells/μL . Clinical and sociodemographic data were obtained from an electronic medical record system used in routine care. We used a regression discontinuity design to estimate the effects of this change in treatment eligibility on ART initiation within 3 months of enrollment, retention in care at 6 months (defined as clinic attendance between 3 and 9 months after enrollment), and a composite of both ART initiation by 3 months and retention in care at 6 months in all new enrollees. We also performed an instrumental variable (IV) analysis to quantify the effect of actually initiating ART because of this guideline change on retention. Overall, 34,857 ART-naïve patients  newly enrolled in HIV care during this period; 23,036 were analyzed after excluding patients around the threshold to allow for clinic-to-clinic variations in actual guideline uptake. In all newly enrolling patients, expanding the CD4 threshold for treatment from 350 to 500 cells/μL was associated with a 13.6% absolute increase in ART initiation within 3 months of enrollment , a 4.1% absolute increase in retention at 6 months , and a 10.8% absolute increase in the percentage of patients who initiated ART by 3 months and were retained at six months . These effects were greatest in patients who would have become newly eligible for ART with the change in guidelines: a 43.7% increase in ART initiation by 3 months , 13.6% increase in retention at six months , and a 35.5% increase in the percentage of patients on ART at 3 months and still in care at 6 months [95% CI, 29.2%–41.9%). We did not observe decreases in ART initiation or retention in patients not directly targeted by the guideline change. An IV analysis found that initiating ART in response to the guideline change led to a 37.9%  absolute increase in retention in care. Limitations of this study include uncertain generalizability under newer models of care, lack of laboratory data , inability to account for earlier stages in the HIV care cascade , and potential for misclassification of eligibility status or outcome.In this study, guidelines raising the CD4 threshold for treatment from 350 to 500 cells/μL were associated with a rapid rise in ART initiation as well as enhanced retention among newly treatment-eligible patients, without negatively impacting patients with lower CD4 levels. These data suggest that health systems in Zambia and other high-prevalence settings could substantially enhance engagement even among those with high CD4 levels  by expanding treatment without undermining existing care standards. In a regression discontinuity analysis, Aaloke Mody and colleagues assess the implications for treatment initiation and retention of expanded eligibility for antiretroviral therapy in Zambia. Universal treatment with ART regardless of CD4 count  is based on randomized controlled trials showing the biologic efficacy of ART, but the public impact of expanding ART eligibility also depends on the behavior of patients and the capacity of health systems to absorb an influx of new patients.Direct effects of expanding ART eligibility include increased ART initiation, but it may also indirectly affect patient behavior  or create negative spillover effects through increased clinic congestion and crowding out of patients already eligible for ART.We leveraged a 2014 change to Zambia’s national ART guidelines that expanded ART eligibility to those with a CD4 between 350 and 500 cells/μL and pregnant or breastfeeding women to anticipate the effects of expanding treatment to those with CD4 above 500 cells/μL under treat-all.We used a regression discontinuity design to compare patients enrolling just before and after this 2014 change to Zambia’s national ART guidelines expanding the CD4 treatment threshold from 350 to 500 cells/μL to estimate the effects of the guideline change on ART initiation and retention in care in the entire patient population.Among 34,587 patients who newly enrolled in HIV care between August 1, 2013, and November 1, 2014, Zambia’s 2014 guideline change was associated with a 13.6% increase in the proportion initiating treatment within 3 months of enrollment, a 4.1% increase in retention in care at 6 months, and a 10.8% increase in the percentage of patients in care and on ART at 6 months in the entire clinic population.These effects were most pronounced in those patients who would have become newly eligible with the guideline, but ART initiation also increased without evidence of decreased retention in those who remained eligible as well as those who were not yet eligible for ART.We estimated that initiating ART in response to the change in guidelines led to a 37.9% increase in retention in care, indicating that 2.6 patients would need to be initiated on ART to prevent one episode of LTFU.Expanding ART eligibility to patients with higher CD4 counts in a real-world setting was associated with marked changes in patient behavior that were not observed in the more tightly controlled settings of randomized trials.Expanding treatment eligibility was associated with modest increases in ART initiation, overall retention in care, and the percentage of patients in care and on ART, without increasing clinic congestion or negatively impacting those who were already eligible for treatment before the 2014 guideline change .Adopting treat-all may lead to similar improvements in ART initiation and retention without associated negative effects; further research regarding the real-world effects of implementing treat-all is needed.Although expanding treatment eligibility improved ART initiation and retention in care, the overall HIV care cascade remained suboptimal. Further innovations in health systems are needed to increase early diagnosis and linkage and improve overall ART initiation and retention in care. Although randomized controlled trials (RCTs) have demonstrated that antiretroviral therapy (ART) in persons living with HIV (PLWHs), irrespective of CD4 levels, has broad clinical benefits, policies to expand HIV eligibility in the real world may have important additional effects that are not captured in the context of controlled trials. HPTN 052 first demonstrated in 2010 that treating the positive partner in a serodiscordant relationship reduces HIV transmission by as much as 96%. This was followed in 2015 by the INSIGHT START and TEMPRANO trials, which showed that immediate treatment reduced the incidence of serious AIDS-related events by 50%, even in patients with CD4 levels greater than 500 cells/μL . Based oThe current clinical evidence base for offering treatment to all persons with HIV provides limited insights on its anticipated effects in routine care settings. Broadly speaking, implementation science is motivated by the observation that guidelines rarely result in all intended effects and often lead to at least some unintended effects. Typically, individual-level RCTs establish clinical efficacy by only enrolling participants already willing to be treated, closely monitoring both treated and untreated patients, and delivering care in regulated environments to confidently capture the biological effect of treatment –9. In INOn April 1, 2014, Zambia updated its HIV treatment guidelines to expand ART eligibility to asymptomatic patients by increasing the CD4 threshold for treatment from 350 to 500 cells/μL and instituting treatment for all pregnant and breastfeeding women under Option B+ . We use The study was approved by the University of Zambia Biomedical Research Ethics Committee (UNZABREC) and institutional review boards at the University of California, San Francisco, and the University of Alabama, Birmingham, School of Medicine. The research in this paper was not prespecified in the original study protocol and consists of secondary analyses that were conceptualized to take advantage of the change to Zambian national guidelines that occurred during the study period. This manuscript was prepared according to STROBE guidelines .We analyzed ART-naïve, HIV-infected adults (greater than 15 years old) who newly enrolled in HIV care before and after the April 1, 2014 change in Zambia’s HIV treatment guidelines, from August 1, 2013, to November 1, 2014. Patients were from one of 64 clinics operated by the Zambian Ministry of Health that received technical support from the Centre for Infectious Disease Research in Zambia (CIDRZ), a Zambian nongovernmental organization that supports implementation of HIV care delivery and research across 4 of the 10 provinces in Zambia. According to Zambian national guidelines at the time of this study, patients eligible for ART first underwent three evaluation and counselling sessions prior to being initiated on ART about 3–6 weeks later. They were then followed up monthly for at least the first 6 months of treatment, after which they might be eligible to have their visits spaced out to 3-month intervals if considered stable. Patients ineligible for ART at the time of enrollment were followed up with clinical evaluations and CD4 counts every 6 months until becoming eligible for ART.Measurements were extracted from the national electronic medical record system used in routine HIV care in Zambia, SmartCare. This system involves providers manually filling out paper clinical forms during patient encounters in routine practice with data clerks, then entering this information into the electronic database. We used patient sociodemographic characteristics , clinical characteristics , and visit history  for our analyses.First, we describe in a multistate analysis the longitudinal experience of patients enrolling in HIV care, stratified by pre- and post-guideline change, by estimating the probability of a patient being in a particular care state at a given time point after enrollment. We defined four mutually exclusive and exhaustive states: (1) in care and not initiated on ART, (2) in care and on ART, (3) lost to follow-up  or died prior to initiating ART, and (4) LTFU or died after initiating ART. Death was combined with LTFU because of known inaccuracies in ascertainment of mortality from routine programmatic data . We thenSecond, we used a regression discontinuity design to estimate the effect of the change in treatment eligibility criteria on three outcomes in the entire population enrolling in care: (1) timely ART initiation (defined as within 3 months of enrollment), (2) retention in care at 6 months (defined as having made at least one visit between 3 and 9 months post-enrollment), and (3) being retained and on ART at 6 months (a composite outcome of having timely ART initiation and being in care at 6 months). The regression discontinuity approach potentially permits causal inferences by comparing patients just above or below an arbitrary threshold value in a small window or bandwidth around the threshold . This approach makes the assumption that patients immediately on either side will be similar on both observed and unobserved characteristics , thus making assignment to exposure “as if” random –27. In tt test or chi-squared test, as appropriate, (2) graphically confirming that there were no gross aberrations in the trends of new enrollees and overall clinic visits per week to suggest changes in other external factors that could affect patient behaviors, and (3) formally testing for continuity in the density of new enrollees at the threshold using the McCrary test (p = 0.86 for discontinuity at the threshold) ; “Always Eligible” RD + 6.2% ) and the percentage of patients in care and on ART at 6 months  but had a limited effect on retention in care  increase in retention in care; the effect was similar when restricted only to asymptomatic patients with a CD4 count above 350 cells/μL. This result indicates that 2.6 patients  would need to be started on ART to prevent one from becoming LTFU , the persisting benefit of counseling and disclosure that occurs in preparation for ART initiation, or the additional services that patients receive once they have started on ART . AdditioIt is important to note that we also did not observe any negative spillover effects associated with expanding ART eligibility. In fact, we noted a slight increase in ART initiation (RD + 6.2%) without any changes to retention in the patients who were eligible for ART even prior to the guideline change, which may have been due to an overall expansion of the ART supply. We also saw increased ART initiation in those who were not yet eligible for ART (RD +12.5%). We posit that this may have also been related to an informal loosening of eligibility criteria for those patients not yet officially eligible for ART, as there were no other changes made to the guidelines at that time that would have been expected to affect patient care in such a way . Lastly,Despite the overall benefits of expanding ART eligibility, with more patients initiated on treatment and retained in care, significant gaps are likely to remain in the care cascade even with adoption of treat-all. First of all, in our population, about 70% of patients still presented with more advanced disease and would have been eligible by the 2010 guidelines, highlighting the need for stronger efforts to diagnose patients and link them to care earlier. Furthermore, despite enrolling in care and being ART eligible, a significant proportion of patients still never initiate ART; a majority of these patients are LTFU after only one visit. Among those who do start ART, there is an additional subset that becomes LTFU shortly afterward, for whom the effect of expanding eligibility is in essence simply to shift attrition from pre-ART initiation to post-ART initiation. Currently, there is little evidence as to how this shift in attrition from pre- to post-ART initiation—also seen in the same-day ART initiation studies—affects subsequent re-engagement and drug resistance ,45,46. TRegression discontinuity and IV study designs can, provided that the central underlying assumptions can be met, quantify both the intended and unintended effects of interventions implemented in routine care and are thus a powerful, though underused, tool in the implementation science armamentarium. Traditional RCTs have high internal validity, but their external generalizability—imperative for informing policy decisions—is often limited –9. For eThere are several limitations of our study. First, it is important to recognize that our results, strictly speaking, are only applicable to the current care setting, under the core assumptions of the regression discontinuity and IV analyses. This makes generalizability to populations that are diagnosed, linked to care, or initiated on ART under different circumstances—such as with home-based testing, enhanced linkage procedures, same-day ART initiation, or hospitalization—uncertain. As we only assessed patients already presenting to clinic, we were also unable to assess whether expanding eligibility had more global effects, such as on patients’ decision to go for HIV testing or overall rates of linkage to care, although the similar number of patients enrolling in care prior to and after the guideline change would suggest minimal effects on earlier stages of the HIV care cascade. Second, we excluded patients enrolling closest to the implementation date to minimize bias both from “cross over” and also the transition period during gradual guideline rollout, precluding us from comparing patients enrolling immediately before and after the guideline change. Nevertheless, calendar time was not a significant predictor in our models, making it unlikely that secular trends over this time period meaningfully affected our results in the context of our patient restriction. Additionally, our results remained robust to sensitivity analyses varying criteria for patient restriction and also when adjusted for baseline patient covariates, despite a slight change in patient composition before and after the guidelines. Third, we were unable to assess virologic outcomes, as viral loads were not routinely collected in Zambia during our study period. Still, recent results from the Zambia Population-Based HIV Impact Assessment (ZAMPHIA) suggest that just under 90% of patients in care and on ART will be virally suppressed . Lastly,In conclusion, we found that the 2014 change in Zambia’s HIV treatment guidelines was associated with increased ART initiation, retention in care, and the percentage of patients on ART and still in care at 6 months without evidence of associated negative spillover effects, providing important new data on the effects of expanding ART eligibility to asymptomatic patients in sub-Saharan Africa. Furthermore, we quantify the behavioral effect of actually initiating ART on retention in care and also show that a substantial subset of patients will not see the benefits of ART despite being eligible for treatment, largely due to attrition. Although these results are highly relevant to Zambia’s transition to universal treatment in December 2016, further study is still needed into the actual experience of treat-all—particularly with regard to viral suppression and in the setting of new care models meant to improve the HIV care cascade . MoreoveS1 STROBE Checklist(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Research says that diabetes may develop in over 10% of non-diabetic pregnant women. Diabetes which generally occurs late in second trimester and third trimester of pregnancy, it is called gestational diabetes. Overweight or suffering from obesity before pregnancy is type 2 diabetes risk factor. In most cases, diabetic symptoms disappear after delivery. HLA-G has an important role both in mother and fetus tolerance during pregnancy, it may also be effective in the protection of pancreatic islet cells. In this study, concentration of these molecules in pregnant women with gestational diabetes in comparison with normal pregnant women was investigated.In this case-control study, we measured serum HLA-G levels in 24 pregnant women with gestational diabetes compared with 30 normal pregnant women using sandwich ELISA.HLA-G levels were significantly low in pregnant women with gestational diabetes in contrast to normal pregnant women (P=0.001).  In this study, we found that HLA-G levels were reduced in women with gestational diabetes compared with control group. Therefore, it is suggested that measurement of HLA-G in pregnant women can be considered as an indicator in prognosis of gestational diabetes. Hyperglycemia of a woman during pregnancy is a special type of diabetes that is called gestational diabetes mellitus (GDM), which is defined as different severities of carbohydrate intolerance first started or diagnosed during pregnancy and is considered as the most common medical complication during pregnancy. Patients can be divided into two categories: those whose diabetes is diagnosed before pregnancy (diabetes before pregnancy or diabetes mellitus) and those whose disease is diagnosed during pregnancy  . Blood sGestational diabetes and impaired glucose tolerance test during pregnancy will lead to an increased risk of prenatal death and progression to diabetes mellitus in 30-50% of women in the future : Two milliliter of blood was collected and after centrifugation serum was extracted. We measured serum soluble HLA-G by commercial ELISA kit  according to kit protocol. This kit uses double-antibody sandwich technique to measure serum concentrations of soluble HLA-G in case and control group. Briefly, we prepared five standard solutions with 2400ng / L, 1200ng / L, 600ng / L, 300ng / L, 150ng / L, 75ng / L concentrations by diluting stock 2400ng /L concentration. The first well was filled by blank and none of the samples and biotinylated HLA-G antibody were added. Then, we added 50 microliters of standard and 50 micro liters of streptavidin HRP in wells for standards, and in wells related to samples, 40 micro liters of samples plus 10 micro liters of biotinylated HLA-G antibody and 50 micro liters streptavidin-HRP were added to sample wells. Next, the plate was shaken and incubated for 60 min at 37°C. Distilled water was used to dilute 30X washing solution 30 times. Then we washed the plate five times. After washing, 50 microliters of chromogen A and 50 micro liters of chromogen B were added to each well, the plate was a little shaken and was incubated at 37°C for 10 min in the dark and then 50 microliters stop solution was added to each well. Finally, blank was considered zero and optical density (OD) was measured by ELISA reader under 450 nm wavelength. Concentrations of the samples were obtained according to standard concentration and a standard curve that was plotted by the device. We used SPSS Version 19 with Mann-Whitney test to analyze the data.Totally, 54 women participated in this study in which 24 of these women are suffering from gestational diabetes with no history of diabetes and also without taking any medications (type A1) as the case group, and 30 normal pregnant women the control group. Gestational age and age of women in control group were matched with case group. The age range of the patients in case group was 24 to 39 years with a mean and standard deviation of 29.8±4.10 and in the women in control group was 23 to 38 years with a mean and standard deviation of 28.9±3.62. Gestational age of pregnant women with GDM was between 27 and 34 weeks and 2 of these pregnant women were under 20 weeks of pregnancy. Demographic characteristics of the study population are shown in Statistical analysis showed no significant differences in age and weight between the case group and control group.Soluble HLA-G levels decreased in pregnant women with gestational diabetes compared with control group. As shown in In this study, the concentration of soluble HLA-G in pregnant women with gestational diabetes compared with normal pregnant women was studied and a significant decrease in the concentration of these molecules in pregnant women with gestational diabetes compared with normal pregnant group was observed (P=0.001). Many studies have examined the HLA-G soluble in a variety of disorders such as preeclampsia , tumors immune tolerance molecule, recommendations have been made (In different studies in regard to gestational diabetes and the role of HLA-G een made , 8, but een made . Hunt eteen made . Vautert et al. examined the intracellular interactions and showed that HLA-G molecules in addition to maternal-fetus tolerance, also play a role in regulating the immune response . In the In this study, we have shown that HLA-G in normal pregnant women is higher than pregnant women with diabetes, this could be related to the activity of these molecules in regulating the immune system. Higher concentrations of these molecules in normal pregnant women could be indicative of inhibition of the immune response and the regulatory role of this molecule in pregnancy and lower values in pregnant women with gestational diabetes may be an indicative of removal of this inhibition and regulation. It is suggested that this assumption can be the reason for developing gestational diabetes in these women comparing to normal control group. The results of this study showed significant reduction of HLA-G levels in pregnant women with gestational diabetes compared with normal pregnant women. So we can conclude that HLA-G molecule is among the factors for regulation and control of the immune response and the induction of tolerance and can be an important indicator in determining gestational diabetes. Therefore, it is recommended that HLA-G in pregnant women is tested as an effective molecule so as to take an important step in the prediction of gestational diabetes to prevent problems and consequences of this disease.