Reducing repressor activity allows re-activation of Notch in INPs to ectopically induce tll expression driving supernumerary neuroblast formation. Knocking-down hdac3 function prevents downregulation of tll during INP commitment. We propose that continual inactivation of stem cell identity genes allows intermediate progenitors to stably commit to generating diverse differentiated cells during indirect neurogenesis.Stem cells that indirectly generate differentiated cells through intermediate progenitors drives vertebrate brain evolution. Due to a lack of lineage information, how stem cell functionality, including the competency to generate intermediate progenitors, becomes extinguished during progenitor commitment remains unclear. Type II neuroblasts in fly larval brains divide asymmetrically to generate a neuroblast and a progeny that commits to an intermediate progenitor (INP) identity. We identified Tailless (Tll) as a master regulator of type II neuroblast functional identity, including the competency to generate INPs. Successive expression of transcriptional repressors functions through Hdac3 to silence  Indirect generation of differentiated cell types through intermediate progenitors allows tissue-specific stem cells to scale their progeny output to accommodate the development of appropriately sized organs in proportion to organism sizes. Indirect neurogenesis by the outer subventricular zone (OSVZ) neural stem cells drives the evolution of lissencephalic brains to gyrencephalic brains in vertebrates . The com-) immature INP. Three to four hrs after this transition, an Ase- immature INP upregulates Ase expression as it progresses through INP commitment. Once INP commitment is complete, an Ase+ immature INP transitions into an INP that undergoes six to eight rounds of asymmetric division. These molecularly defined intermediate stages during INP commitment provide critical landmarks to investigate the mechanisms that extinguish the activity of type II neuroblast functional identity genes.Two distinct neuroblast lineages function together to generate the number of neurons requisite for the development of adult fly brains . Similarearmuff (erm)  . During ff (erm) . brat-nuignaling . In paraignaling . This muignaling . Erm belignaling . Erm prefunction . Thus, Etailless (tll) as a master regulator of type II neuroblast functional identity. Tll is expressed in type II neuroblasts but not in INPs; moreover, Tll is necessary and sufficient for type II neuroblast functionality. Tll overexpression is sufficient to transform type I neuroblasts into type II neuroblasts, as indicated by changes in gene expression and an acquired competence for generating INPs. We identified hamlet (ham) as a new negative regulator of type II neuroblast maintenance. ham is expressed after erm in immature INP during INP commitment, and Erm and Ham function through Hdac3 to continually inactivate tll. Sequential inactivation during INP commitment suppresses tll activation by Notch signaling in INPs. We propose that silencing of the master regulator of stem cell functional identity allows intermediate progenitors to stably commit to the generation of differentiated cell types without reacquiring stem cell functionality.By comparing mRNAs enriched in type II neuroblasts or immature INPs, we identified - and Ase+ immature INPs in wild-type brains . We assessed the effect of this transient over-expression strategy on cell identity by quantifying the total type II neuroblasts and INPs per brain lobe based on Deadpan (Dpn) and Ase expression  Ase- immature INPs following 6–12 hr of Insb overexpression and (2) Ase+ immature INPs following 12–24 hr of Insb overexpression. We examined the expression of endogenous Erm in brat-null type II neuroblasts transiently overexpressing Insb to confirm their identity. We generated an erm::V5 allele by inserting a V5 epitope at the C-terminus of the erm reading frame, and showed that Erm::V5 recapitulates the published endogenous Erm expression pattern that we previously described (brat-null brains expressed cell identity markers indicative of Ase- immature INPs (Erm::V5+Ase-), whereas after 18 hr of Insb overexpression they expressed markers indicative of Ase+ immature INPs (Erm::V5+Ase+)  . These dbrat-null brains following 9 and 18 hr of Insb overexpression. By sequencing mRNA in triplicate following the transient over-expression strategy  transgene [tll::GFP(Bac)] where green fluorescent protein (GFP) is fused in frame with the tll reading frame. Consistent with previous study     . A close lineage . This real4(III) . Brains enotype) . Thus, Ttll is required to maintain type II neuroblasts in an undifferentiated state, direct assessment of its role in regulating the competency to generate INPs is not possible. As an alternative, we tested whether Tll overexpression is sufficient to induce ectopic type II neuroblast formation in the ventral brain region and in the ventral nerve cord that exclusively consist of type I neuroblasts. To unambiguously distinguish the two types of neuroblasts, we searched for robust protein markers of type II neuroblasts and their progeny. As Sp1 mRNA is uniquely detected in type II neuroblasts (Sp1::GFP(Bac) transgene. We found that Sp1::GFP marks type II neuroblasts and most of their progeny that are found exclusively in the dorsal brain region  in larval brains carrying the Sp1::GFP(Bac) transgene. While the ventral region of control brains contains 29 ± 2.1 type I neuroblasts, the same region of the brains overexpressing Tll contains 10.6 ± 2.5 type I neuroblasts and hundreds of ectopic type II neuroblasts (n = 10 brains per genotype)  . This reenotype) . Thus, etll can generate immature INPs that are marked by Erm::V5 expression , Ase, and Hamlet (Ham)  . Reverseing Insb . Thus, ttll expression during INP commitment by knocking-down their function in erm hypomorphic (hypoerm) brains. hypoerm brains display a low frequency of INP reversion to type II neuroblasts, and provides a sensitized genetic background for identifying genes required to prevent INP reversion to type II neuroblasts   . Thus, ham likely plays a role in preventing INPs from reverting to supernumerary type II neuroblasts.We tested if any of the candidate genes is required for inactivating roblasts . Because brains) . Althougansgene) . Consistham is the fly homolog of the vertebrate Prdm16 gene that encodes a transcription factor and has been shown to play a key role in regulating cell fate decisions in multiple stem cell lineages   as compared to control brains    . Second, brains) . To confSPR-Cas9 . We char brains) . Becauseall INPs , we concham knock-down drastically enhanced the supernumerary neuroblast phenotype in hypoerm brains, leading us to hypothesize that ham functions together with erm to suppress INPs from reverting to supernumerary type II neuroblasts. We tested this hypothesis by examining a potential genetic interaction between erm and ham during INP commitment. Although the heterozygosity of erm did not increase total type II neuroblasts , it enhanced the supernumerary neuroblast phenotype in ham deficiency heterozygous brains and in SK1ham homozygous brains  (enotype) . We concerm-null brains , overexpressing ham in INPs driven by Erm-Gal4(III) rescued the supernumerary neuroblast phenotype in erm,ham double heterozygous brains  (ham-null brains. We generated GFP-marked mosaic clones derived from single type II neuroblasts to confirm the origin of supernumerary type II neuroblasts in ham-null brains. In wild-type clones (n = 9 clones), the parental type II neuroblast was always surrounded by Ase- and Ase+ immature INPs  were always located far away from parental neuroblasts and surrounded by Ase+ cells that were most likely Ase+ immature INPs and ganglion mother cells  . This reure INPs . By conter cells . It is hSK1ham but not 1ham homozygous clones displayed a supernumerary neuroblast phenotype   (N-ZF) also rescued the supernumerary neuroblast phenotype in SK1ham homozygous brains   . We test at 25°C  and 4F. ctively) . Under i brains) . These rN-ZF)   . We foun brains) . Becausetll during INP commitment, preventing the re-activation of Notch signaling from triggering tll expression in INPs  tll consistently suppressed the supernumerary neuroblast phenotype in erm,ham double heterozygous brains   transgene mildly enhanced the supernumerary neuroblast phenotype in erm,ham double heterozygous brains  (tll during INP commitment. We tested this model by overexpressing erm or ham in type II neuroblasts that carry one copy of the tll::GFP(Bac) transgene. We found that Tll::GFP expression in type II neuroblasts overexpressing Erm was mildly reduced prior to upregulation of Ase expression, and declined further following upregulation of Ase expression (tll refractory to activation in INPs.Our findings strongly suggest that Erm and Ham inactivate  in INPs . Therefo brains) . This res brains . In thess brains . Furtherctively) . These dpression . By contpression . Togethetll is a putative target of Notch, we tested if inactivation of tll by Erm and Ham relinquishes the ability of Notch signaling to activate tll in INPs. We knocked-down Notch function in INPs in wild-type or SK1ham homozygous brains. Knock-down of Notch function in INPs had no effect on type II neuroblasts in wild-type brains    induced a drastically more severe supernumerary neuroblast phenotype in erm,ham double heterozygous brains  than in erm or ham single heterozygous brains   . Howeverenotype) . This rectively) . Thus, w in INPs .tll in INPs, Erm and Ham must function through chromatin-modifying proteins. We first tested if Erm and Ham inactivate tll by promoting sequential chromatin changes during INP commitment. We overexpressed full-length Ham or HamΔC-ZF driven by Erm-Gal4(III) in erm-null brains. erm-null brains alone contained 1824.6 ± 520.7 type II neuroblasts per lobe (n = 13 brains)   . Overexpctively) . This reham heterozygous brains. We knocked-down the function of genes known to contribute to the inactivation of gene transcription in ham heterozygous brains. Reducing the activity of PRC2 or heterochromatin protein 1, as well as decreasing the recruitment of Heterochromatin Protein 1, had no effect on the supernumerary neuroblast phenotype in ham heterozygous brains mγ, and Klumpfuss (Klu) induces the formation of supernumerary type II neuroblasts at the expense of generating immature INPs via the inhibition of erm activation mγ, and Klu in combination in INPs is sufficient to drive supernumerary type II neuroblast formation, Notchintra overexpression is not sufficient to transform a type I neuroblast into a type II neuroblast (data not presented). Thus, we propose that Notch functions as a general activator of genes expression in type II neuroblasts, and specific regulators of type II neuroblast functional identities must exist.Stem cell functional identities encompass the maintenance of an undifferentiated state and other unique functional features, such as the competency to generate intermediate progenitors. Because genetic manipulation of Notch signaling perturbs the regulation of differentiation during asymmetric stem cell division , the roltivation . These rtll functions as a master regulator of type II neuroblast functional identities   . Tll ovedpn, E(spl)mγ and klu loci in embryos, suggesting that Tll likely regulates their expression. The phenotypic effects of loss- and gain-of-function of tll on type II neuroblasts mimic those of dpn, E(spl)mγ and klu. The vertebrate homolog of Tll, Tlx, has been shown to function as a transcriptional activator during neurogenesis mγ and klu expression. Tll also binds pntP1 and btd loci in embryos. Similar to Tll overexpression, mis-expression of PntP1 or Btd can transform type I neuroblasts in the ventral brain region into type II neuroblasts  transgenic protein identified hundreds of putative Tll target genes that include all previously characterized regulators of type II neuroblast functional identities . Tll binogenesis . Thus, Troblasts . Thus, iham as a putative regulator of INP commitment was unexpected given that a previously published study concluded that Ham functions to limit INP proliferation . After removing the fix solution, samples were incubated with primary antibodies for 3 hr at room temperature. Three hours later, samples were washed with PBST and then incubated with secondary antibodies overnight at 4°C. On the next day, samples were washed with PBST and then equilibrated in ProLong Gold antifade mountant (ThermoFisher Scientific). The confocal images were acquired on a Leica SP5 scanning confocal microscope . More than 10 brains per genotype were used to obtain data in each experiment.Larval brains were dissected in PBS and fixed in 100 mM PIPES (pH 6.9), 1 mM EGTA, 0.3% Triton X-100, and 1 mM MgSO11/Df(2L)Exel8040brat larvae carrying ts2xTub-Gal80, Wor-Gal4 and UAS-insb transgenes using TRIzol (ThermoFisher Scientific) and mRNA was purified using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol. First strand cDNA was synthesized using a 1st Strand cDNA Synthesis Kit for RT-PCR [AMV] (Roche) according to the manufacturer’s protocol. cDNA is amplified by using gene-specific primers. qRT-PCR was performed using ABsolute QPCR SYBR Green ROX Mix (ThermoFisher Sientific). Data were analyzed by comparative CT method, and the relative mRNA expression was presented.Total RNA was extracted from control or treated 11/Df(2L)Exel8040brat larvae carrying ts2xTub-Gal80, Wor-Gal4 and UAS-insb transgenes were cultured in corn meal caps. One third of the larvae (18 hr Insb OE sample) were allowed to grow at 25°C for 102 hr and then transferred to 33°C to induce transient Insb overexpression for 18 hr. One-third of the larvae (9 hr Insb OE sample) were allowed to grow at 25°C for 111 hr and then transferred to 33°C to induce transient Insb overexpression for 9 hr. The remaining third of the larvae (0 hr Insb OE) were allowed to grow at 25°C for 120 hr. Fifty brains from each group were isolated on the same day, and immediately processed for RNA extraction. Biological triplicate brain samples were collected. Paired-end Poly-A-mRNA libraries were generated to be assayed by Illumina HiSeq 4000. Differential genes expression was based on absolute linear fold change >or equal 1.5.Control or treated http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). We align the reads to the dm6 reference genome including mRNAs (http://www.genome.ucsu.edu/) with HISAT2 (version 2.1.0) and default parameters. Alignment results were validated through a second round of FASTQ followed by quantification and analysis. For differential expression analysis, Stringtie (version 1.3.4) quantifies expression at the gene and isoforms levels and DESeq2 (version 1.20.0) performs differential expression testing. We compared transcriptional profile between 0 hr vs 9 hr, 0 hr vs 18 hr, and 9 hr vs 18 hr. We identified genes and transcripts as being differentially expressed based on absolute linear fold change >or equal 1.5.Biological triplicate brain samples for each time point were sequenced using Illumina HiSeq 4000. The samples had around 46, 45 and 44 million reads. Quality of the raw reads for each sample was checked using FastQC , (**) and (***), respectively in figures. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your demonstration that Tailless is a master regulator of type II neuroblast identity is an important concept and this confirms recent work by Hakes and Brand, 2020. The demonstration that overexpression of Tailless in INPs causes dedifferentiation to type II neuroblasts, while in type I neuroblasts, it causes ectopic type II neuroblasts is of importance. The genetic manipulations performed in the paper showed that Hamlet and Earmuff in the INPs inhibit Tailless, an effect that appears to be mediated by HDac3,This work therefore brings new sights into how type II neuroblast lineages are specified.Decision letter after peer review:eLife. Your article has been reviewed by Utpal Banerjee as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Inactivation of stem cell functionality genes by histone deacetylation enables robust progenitor commitment" for consideration by The reviewers have extensively discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Your demonstration that tailless is a master regulator of type II identity is an important new concept, although this was also recently shown by Hakes and Brand, 2020. It was also important to show that overexpression of Tailless in INPs causes dedifferentiation to type II neuroblasts, while in type I neuroblasts, it causes ectopic type II neuroblasts. Your genetic manipulations allowed you to show that Hamlet and Earmuff in the INPs inhibit Tailless. They also appreciated that the function of Erm and Ham is modified by HDac3, although the reviewers thought that too much attention was given to this effect and suggested to remove it from the title of the paper as of less significance. The reviewers found the work to be novel and convincingly, bringing new sights into how type II neuroblast lineages are specified.Below is a series of experiments that would correct the weaknesses of the manuscript as well as a very long series of editorial changes that should be very helpful for you to fix small problems.I should add that the absence of a Materials and methods section was quite disturbing and should not have happened.Essential revisions:Below is a series of suggested revisions:– To confirm if ectopic type II neuroblasts generated by Tll overexpression generate INPs, clones should be done/followed . Or use the NBGal4 that is expressed in type I NBs to drive UAS-Tll, and focus on a brain region where there is normally no type II NBs, and quantify how many ectopic type II NBs are generating erm-V5+ INPs. A control should be provided to show that there is normally no erm-v5 expression in this region.– Figure 2C and 2D would benefit from single split channel images, type II lineage should be labelled with a RFP or GFP, to see clearly that type II NBs are ectopically expressing Ase, and they are not type I NBs.– Figure 3F-H should show split images, very difficult to see which cells are Dpn+ Ase- from these pictures. Also, is there an independent validation that can be done to convince us that these cells are really type II NBs? How about using a Tll antibody to definitively mark these cells, this would also strengthen the point that erm and ham inhibits Tll.tll should be explored. What happens to the expression of Tll when Hdac3 is manipulated?– Figure 6 section – the connection between Hdac3 and – The authors suggests that Ham and Erm inhibits Tailess, is this specifically in the INPs, or everywhere? An over expression experiment involving UAS-Ham and UAS-Erm in type II NBs would clarify this point.– The Material and methods section were not available for review which made the interpretation of some experiments difficult. The authors must include the Solexa sequencing raw data in a repository publicly accessible.Reviewer #1:Drosophila type II neuroblasts(NBs) stably commit to the INP identity and do not revert back to stem cell fate. There are several novel and significant findings reported. First, Tailless was shown to be necessary and sufficient for the type II neuroblast functional identity; second, Hamlet was identified to be a novel regulator of INP commitment after Erm, and it functions as a transcription repressor, and is required to suppress Tll re-activation by Notch signaling in INPs; Third, Ham and Erm may function through HDAC3. These novel findings illustrate robust mechanisms to down-regulate "stemness" in intermediate progenitors, and will be of great interest to readers of eLife.This manuscript by Rives-Quinto et al., is addressing the question how intermediate progenitors (INPs) generated by 1) The authors showed modest increase in TllGFP in trans-heterozygous mutant of erm and hamlet that is likely corresponding to the modest increase in the number of type II NBs per lobe in such mutants. I also would like to see how Tll expression changes in hamSK1 homozygous mutant brains. I understand the reason for lacking of this data might be the difficulty to put hamsk1 homozygous mutant and tllGFP together, but Tll antibody is available that will make this experiment feasible.tll locus to silence its expression in INPs, but further experiments will be needed to confirm this model.2) It was shown that erm and ham repress Tll expression in INPs, but whether this is direct or indirect is not known. The interaction between ham, erm and Hdac3 are only shown by genetic interactions. It should be noted that the data presented here suggested an attractive model that erm and ham recruit Hdac3 to Reviewer #2:Drosophila neuroblasts as a lineage model since it nicely allows them to track cell fates and progenitor competency.In this manuscript the authors explore the mechanism by which stem cells have the competency to generate intermediate progenitors. The authors use tll) as a type II neuroblast identity gene, earmuff (erm) and Hamlet (Ham) as putative repressors of neuroblast genes in INPs.The authors used a type II neuroblast tumor model where they then express in a time-controlled manner insb. Insb causes neuroblasts to differentiate into intermediate progenitors. To identify the genes necessary for type II neuroblast functional identity they sequence the tumors+insb at several time points and look for genes that are downregulated in the differentiating tumor. On the other hand, genes that are upregulated during type II neuroblast differentiation allowed the authors to identify candidate genes important for repressing neuroblast functional identity genes in INPs. This was a very nice strategy and allowed the authors to identify tailless  and this should be discussed in this manuscript.The authors show that RNAi of tll in INPs.By a series of epigenetic experiments, the authors find that erm and ham genetically interact and are both required to maintain INP commitment, preventing their reversion to neuroblasts. They further show that reducing the levels of both erm and ham (double heterozygous) is sufficient to de-repress By using a series of ham mutants and domain analysis the authors claim that the N-terminus zinc finger of Ham is required to repress neuroblast genes in INPs. These experiments and result interpretation are however not clear.tll was previously described as a putative target of Notch, the authors explore how Notch regulates INP commitment. The authors show that reducing Notch levels rescues the ham mutant phenotype, and that Notch constitutive activation enhances the phenotype of erm, ham double heterozygous. The connection between Notch and Tll was not experimentally explored.Because To test the hypothesis that Erm and Ham required chromatin repressing complexes to repress neuroblast genes in INPs the authors tested the role of several candidate chromatin regulators. The RNAi of Hdac3 in type II neuroblasts, or in the background of either ham or erm heterozygotes causes an increase in type II neuroblast number. Since reducing Hdac3 levels by itself does not completely mimic ham or erm mutant phenotypes, this might indicate that Hdac3 is not the only chromatin regulator acting downstream of these genes.tll and Notch or tll and erm/Ham is not as clear.Overall, the authors characterize the function of several genes responsible for either neuroblast or progenitor fate, but the connection between – The Material and methods section were not available for review which made the interpretation of some experiments difficult. The authors should include the Solexa sequencing raw data in a repository publicly accessible.– The title does not represent what has been clearly shown in this manuscript and should be altered. The authors should not emphasize the chromatin regulation aspect.– Abstract should be modified as well according to new title.– Tll::GFP expression pattern in type 1 neuroblasts should be shown. This is essential to corroborate that Tll is a type II neuroblast specific functional gene . Please show an image of an entire central brain lobe  and an image of a ventral nerve cord .– To confirm if ectopic type II neuroblasts generated by Tll overexpression generate INPs, clones should be done/followed .tll to analyzing erm needs to be re-written as it is hard to follow.– Subsection “Ham is a new regulator of INP commitment” – the authors need to say what are the candidate transcription factors identified. Could be included in a table and text. The transition from analysing – Every time erm, ham double heterozygotes are used the exact alleles used should be included in the text and figures (e.g. subsection “Ham promotes stable INP commitment by repressing gene transcription”).– All figures – uniformize genotypes shown in graphs and labels. Homozygous mutants should either be represented as e.g. Ham sk1/sk1 or HamSk1 -/-.– All figures should say the stages being analyzed.– Figure 3 – legend is very incomplete. Legend should be reviewed and information about the Ham alleles provided. It is not clear what the SK mutants really are molecularly.– In the legend of Figure 3 it is mentioned that Ham is expressed in iINPs and INPs. This is hard to see in the figure which is small. Is Ham expressed in the NB? It would be helpful to know to distinguish role in INP formation vs. commitment.– In Figure 3C – since the information about the Ham antibody is not available, it is not clear if the antibody should recognize HamSk1 and SK4 since the majority of the protein should be produced. Assuming the authors used the previously published Ham Ab, which recognizes the N-terminus of Ham, the authors should discuss why there is no staining in SK1 mutants.– Section regarding Figure 4 (subsection “Ham promotes stable INP commitment by repressing gene transcription”) – It is concluded that the N-terminus ZF domain of Ham is the domain responsible for repressing neuroblast genes in INP. However, in both Ham1 and in HamSK1, the N-ZF domain are present and wild-type. Since in HamSk, where this N-ZF is present there is a phenotype, how can it be concluded that this is the domain responsible? Unless this SK mutant causes Ham degradation and, in the end, re-expressing Ham, either full-length or just the functional domain rescues the KO phenotype. The authors should clarify this experimentally and in the text.tll::GFP in wt background.– Figure 5 – 5C include tll in INPs” – rephrase this sentence. The connection between tll and Notch is not demonstrated, pose as an hypothesis.– Subsection “Inactivation during INP commitment relinquishes Notch’s ability to activate tll in INPs” – rephrase to say that "Ham overexpression can replace endogenous Erm function". In the wild-type situation this is not true as both erm or ham mutants have a phenotype.– Subsection “Inactivation by Hdac3 relinquishes Notch’s ability to activate tll should be explored. What happens to the expression of Tll when Hdac3 is manipulated?– Figure 6 section – the connection between Hdac3 and tll off in the erm or ham loss of function? This was not shown, and in Figure 5C and D, double heterozygotes erm, ham cause tll expression in INPs, the opposite phenotype of what is in the model.– Figure 7 – why is The model should be re-drawn as it is hard to interpret and contains too many speculations.Reviewer #3:eLife.Lee and Colleagues have previously shown that the nuclear protein Insensible inhibits the activity of notch downstream proteins such as dpn through culling mediated proteolysis and Insb over expression triggers premature differentiation in type II NBs. Utilising this tool, they show that in Brat mutant brains, over expression of Insb can promote differentiation, and performing RNAseq, they found that tailess is a master regulator of type II identity. Consistent with what was recently shown by Hakes and Brand, 2020, Tailess over expression in INPs cause dedifferentiation to type II NBs, and its over expression in type I NBs causes ectopic formation of type II NBs. Using some very nice genetics, the authors show that Hamlet and Erm in the INPs are required to inhibit Tailess. Furthermore, Tailess has previously been shown to be activated by Notch. In the INPs, they show that Notch intra is less capable of inducing INP to Type II NB reversion in the absence of Erm and Ham. Finally, the repressive function of Erm and Ham is modified by HDac3. The work is novel, and convincingly shows that ham and erm are required for Tll inactivation, and brings new sights into how type II NB lineages are specified. This work is suitable for publication in  Essential revisions:Below is a series of suggested revisions:– To confirm if ectopic type II neuroblasts generated by Tll overexpression generate INPs, clones should be done/followed . Or use the NBGal4 that is expressed in type I NBs to drive UAS-Tll, and focus on a brain region where there is normally no type II NBs, and quantify how many ectopic type II NBs are generating erm-V5+ INPs. A control should be provided to show that there is normally no erm-v5 expression in this region.We thank reviewers for the suggestion.tll driven by GAL4 drivers of various strength, and found that highly levels of Tll overexpression are required to convert type I neuroblasts in the ventral brain region into type II neuroblasts.We overexpressed We have added a new panel of images  showing transformed type II neuroblasts in the ventral-medial region of the brain generate immature INPs marked by Erm::V5 expression.UAS-tll driven by a pan-neuroblast Gal4 . This result shows that Tll overexpression transforms greater than 60% of ventral type I neuroblasts (Dpn+Sp-1::GFP-Ase+) into type II neuroblasts (Dpn+Sp-1::GFP+Ase-).We added quantification of total type I neuroblasts per ventral brain lobe that overexpressed a We also added a new paragraph (Discussion) to discuss how brain.– Figure 2C and 2D would benefit from single split channel images, type II lineage should be labelled with a RFP or GFP, to see clearly that type II NBs are ectopically expressing Ase, and they are not type I NBs.We thank reviewers for the suggestion.We have now included single-channel, higher-magnification images of brain samples shown in Figure 2C and 2D. We encircled type II neuroblast lineages with yellow dotted lines, and used colored arrows and arrowheads to label distinct cell types in the lineage. A key to colored arrows and arrowheads is provided in the legend for every figure.tll-mutant type II neuroblasts indicated by white arrows ectopically express Ase. First, the UAS-tllRNAi transgene was driven by a type II neuroblast-specific Gal4 driver , which has been well-characterized and widely used in many studies. Second, tll-mutant type II neuroblasts  are surrounded by immature INPs and INPs, identical to type II neuroblasts in wild-type brains . Third, the diameter of tll-mutant type II neuroblasts is significantly smaller than type I and type II neuroblasts in wild-type brains, and reduced cell diameter is a typical diagnostic marker of neuroblast differentiation. Ectopic Ase expression is only detected in the late stage of type II neuroblast differentiation. One of the type II neuroblasts in tll knock-down brains clearly show strong ectopic Ase expression, but the neighboring type II neuroblast has yet to upregulate Ase expression. These reasons led us to conclude that tll-mutant type II neuroblasts shown in Figure 2D ectopically express Ase due to premature differentiation.Three lines of evidence led us to conclude that – Figure 3 F-H should show split images, very difficult to see which cells are Dpn+ Ase- from these pictures. Also, is there an independent validation that can be done to convince us that these cells are really type II NBs? How about using a Tll antibody to definitively mark these cells, this would also strengthen the point that erm and ham inhibits Tll.We thank reviewers for the suggestion.We have included single-channel images of brain samples shown in Figure 3F-H to allow for clear visualization of type II neuroblasts (Dpn+Ase-).We obtained a commercially available Tll antibody used in a previously published study, but this antibody was unable to detect specific signals in larva brains after trying multiple immunofluorescent staining protocols.– Figure 6 section – the connection between Hdac3 and tll should be explored. What happens to the expression of Tll when Hdac3 is manipulated?We thank reviewers for the suggestion.tll in INPs”) describing findings from this experiment.We have now included a panel of images and quantification of Tll::GFP expression relative to Dpn expression in INPs in Figures 6E-G. We have also added a paragraph  describing findings from this experiment.We have now included a panel of images and quantification of Tll::GFP expression relative to Dpn expression in neuroblasts in Figures 5F-I. We added text (subsection “Inactivation during INP commitment relinquishes Notch’s ability to activate – The Material and methods section were not available for review which made the interpretation of some experiments difficult. The authors must include the Solexa sequencing raw data in a repository publicly accessible.We apologize for our mistake. We added the Materials and methods section.


Background: Cancer pain involves nervous system damage and pathological neurogenesis. Neuropathic pain arises from damage to the nervous system and is driven by ectopic signaling. Both progesterone and pregabalin are neuroprotective in animal models, and there is evidence that both drugs bind to and inhibit voltage-gated calcium channels.Aims: This study was designed to characterize the effects of progesterone and pregabalin in preclinical models of cancer and neuropathic pain in both sexes.Methods: We measured peripheral sensory signaling by intracellular in vivo electrophysiology and behavioral indicators of pain in rat models of cancer-induced bone pain and neuropathic pain.Results: Female but not male models of cancer pain showed a behavioral response to treatment and pregabalin reduced excitability in C and A high-threshold but not low-threshold sensory neurons of both sexes. Male models of neuropathic pain treated with pregabalin demonstrated higher signaling thresholds only in A high-threshold neurons, and behavioral data indicated a clear recovery to baseline mechanical withdrawal thresholds in all treatment groups. Female rat treatment groups did not show excitability changes in sensory neurons, but all demonstrated higher mechanical withdrawal thresholds than vehicle-treated females, although not to baseline levels. Athymic female rat models of neuropathic pain showed no behavioral or electrophysiological responses to treatment.Conclusions: Both pregabalin and progesterone showed evidence of efficacy in male models of neuropathic pain. These results add to the evidence demonstrating differential effects of treatments for pain in male and female animals and widely differing responses in models of cancer and neuropathic pain. These include nociceptive mechanical and chemical stimuli that result from the cancer growth and metastasis to the bone, as well as damage to and pathology of the nervous system itself. As a result, cancer pain is described as a unique pain state that includes aspects of nociceptive, neuropathic, and inflammatory pain.4Neuropathic pain is a prevalent and often intractable state of pain arising from pathology of the peripheral or central nervous system and driven by ectopic signaling from damaged or pathological neurons.5 spinal cord injury,6 and chemotherapy-induced NEP.7 In a rat model of sciatic cuff–induced NEP, PRO treatment starting immediately after model induction and lasting for 10 days significantly limited the development of mechanical allodynia.8 Early clinical reporting describes treatment with PRO sharply reducing or abolishing pain in patients with late-stage metastatic breast cancer including bone metastases.9 However, PRO has yet to be investigated in animal models of cancer pain.In models of neuropathic pain (NEP), treatment with progesterone (PRO) has produced beneficial results, including restoring myelination of damaged neurons and ameliorating mechanical and thermal withdrawal thresholds in animal models of nerve crush,10 all of which are involved in the generation and maintenance of NEP and which progesterone treatment has been experimentally demonstrated to reduce. In cultured rat striatal neurons, supraphysiological doses of PRO have been demonstrated to inhibit excitotoxic neuronal cell death by direct inhibition of L-type voltage-gated calcium channel (VGCC) action, without effect on glutamate-mediated ion channels.11Calcium signaling plays a well-established role in neuronal inflammation, demyelination, and excitotoxic cell death,12 PRE is utilized to treat neuropathic cancer pain in the clinic. Some clinical studies demonstrate its utility in comparison to other drugs,14 whereas other studies find no beneficial effects.15 Despite these discrepancies, PRE has not been tested in animal models of cancer pain excluding chemotherapy-induced neuropathies. Similarly to PRO, PRE has been shown to act as a VGCC antagonist by binding at the α2-δ auxiliary subunits of P/Q-, N-, and L-type VGCCs.16 This VGCC inhibition and the resulting reduction of Ca2+-mediated excitatory glutamate release at neuronal synapses confers a neuroprotective benefit17 and is thought to be responsible for the effectiveness of PRE in treating NEP.12Pregabalin (PRE) is a well-established anticonvulsant and analgesic drug approved for the management of NEP associated with diabetic peripheral neuropathy and postherpetic neuralgia. PRE also demonstrates clinical utility across many other NEP conditions, including chemotherapy-induced peripheral neuropathy, trigeminal neuralgia, fibromyalgia, and postsurgical pain.Due to the evidence of neuropathic involvement in cancer-induced bone pain (CIBP) and the widespread evidence of efficacy of the VGCC antagonists PRO and PRE in conditions of NEP, this investigation was designed to characterize the behavioral and electrophysiological effects of these drugs in a rat model of CIBP. Our findings are indicative of a limited and possibly sexually divergent response in CIBP and prompted further investigation in our well-established rat model of sciatic cuff–induced neuropathy.2 in growth medium supplemented with 10% fetal bovine serum (FBS) and antibiotics . MRMT-1 cells were grown in RPMI 1640 (Thermo Fisher Scientific) and tested for mycoplasma contamination prior to experimental use. Cell numbers were quantified in 96-well plates using crystal violet staining, measuring absorbance at λ = 570 nm with an optical plate reader . Cells treated with PRE were plated with dialyzed FBS, and cells treated with PRO were plated with charcoal-stripped FBS (Thermo Fisher Scientific) for all measurements. All were plated at 8000 cells/well and measured 24 h posttreatment. Cell numbers are indicated relative to their respective vehicle-only control for each dose. Cell harvesting for in vivo implantation was performed on subconfluent cultures; adherent cells were suspended and kept lightly agitated in sterile Hank’s Balanced Salt Solution on ice.The mammary rat metastasis tumor (MRMT-1) rat mammary carcinoma cell line  was used in all in vitro and in vivo work. Cells were maintained in a humidified incubator at 37°C with 5% CO12 Vehicle-treated controls  were tested in parallel with experimental animals. Drug solutions were freshly prepared and administered by daily intraperitoneal injection.Progesterone  was administered in vivo at 30 mg/kg, suspended in sesame oil. Pregabalin was administered at 4 mg/kg dissolved in 0.9% saline. Earlier experimentation has demonstrated the doses included here to be relevant in animal models of pain and within published safe dosing ranges.18 and guidelines established by the Canadian Council on Animal Care with ethical approval from the McMaster University Animal Research Ethics Board. All experimental animals were housed in pairs with access to food and water ad libitum in a temperature-controlled room under a 12-h light:dark cycle.All procedures were conducted according to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of PainMale and female Sprague-Dawley (SD) rats  weighing 170–200 g were utilized for all cancer models. Rats were randomly assigned to cancer or sham surgery groups. MRMT-1 cells (3.0 × 104) resuspended in 20 μL Hank’s Balanced Salt Solution were implanted in the distal femur of each cancer pain model rat. Cells for sham surgical controls were suspended at the same concentration and inactivated by three heat:freeze cycles prior to implantation.2) and oriented in a supine position with their right hind limb fixed to a stationary convex support to maintain the limb in a flexed position. A small incision was made on the medial side to expose the quadriceps femoris and the vastus lateralis was incised to expose the medial epicondyle of the femur. A small cavity was drilled between the medial epicondyle and the adductor tubercle with a 0.8 A stereotaxic drill equipped with a 1.75-mm burr. A 25-gauge needle was inserted into this cavity to penetrate the intramedullary canal. The needle was removed and replaced with a blunted 25-gauge needle attached to a Hamilton syringe containing the live MRMT-1 or heat/freeze-inactivated MRMT-1 (sham) cell suspension. The suspension was dispensed slowly into the canal and the syringe was left in place for 1 min to prevent leakage. The cavity was then sealed with dental amalgam and fixed using a curing light. The wound was flushed with sterile deionized water, and muscle, fascia, and skin were sutured. Cancer cell implantation to the distal femur was performed as described in detail in previously published methods.20Rats were anaesthetized with inhaled isoflurane , xylazine , and acepromazine  given intraperitoneally, and the right sciatic nerve was exposed at the mid-thigh level. One cuff of 0.5-mm polyethylene (PE-90) tubing  was slit longitudinally and fitted around the exposed nerve. The muscle and skin of the wound were then sutured separately.Male and female SD rats and female RNURats were exposed to handling and behavioral testing equipment for a 1- to 2-week acclimation period and assigned individual identification prior to model induction. All behavioral testing was repeatedly performed by the same operators, who were blinded to group assignment throughout the duration of the study. Behavioral testing was performed prior to model induction to obtain baseline data and weekly beginning on day 7 following model induction, continuing until endpoints, which were week 3 post model induction for all CIBP models and week 6 post model induction for all NEP models. With the exception of week 1 acute testing, all behavior measurements were performed prior to daily drug administration.23 Postural disequilibrium of the animal could indicate an allodynic response to normal ambulation, so a reduction in weight borne by the tumor-afflicted limb of the animal was accepted as evidence of an inability or aversion to utilize that limb, providing indirect evidence of nociception.Weight, area, and time distribution between all points of pressure of freely moving animals were recorded with the Dynamic Weight Bearing test 2.0 (DWB) . Each animal was recorded in the DWB apparatus for 5 min/test and recordings were manually validated with DWB software version 2.0.59 (BioSeb). Results were exported as mean weight and time for each point of pressure across the validated experiment time. DWB has been validated as a useful test for animal models of CIBP.no use, 1 = severe limp, 2 = moderate limp, 3 = slight limp, 4 = normal use). This scale has been validated in mouse models of cancer-induced bone pain.25The open field observational limb use scale is an operator-derived numerical representation of the use of the animal’s ipsilateral limb, scored over a 5-min period of free ambulation  were applied to the plantar surface of the ipsilateral hind paw to determine mechanical withdrawal thresholds using the up–down method of Dixon27 as applied to rodents by Chaplan et al.28 A von Frey filament was applied a maximum of five times for 3–4 s each, at 3-s intervals, to different spots on the plantar surface of the ipsilateral hind paw in ascending order of force until a clear withdrawal response was observed, starting with the 2 g filament. When a withdrawal occurred, the next lightest filament was reapplied, and the process continued until a 50% withdrawal response threshold was derived. Brisk foot withdrawal in response to the mechanical stimulus was interpreted as a valid response. A reduction in 50% mechanical withdrawal threshold by the tumor- or cuff-afflicted limb was indicative of allodynia.To quantify mechanical sensitivity, brisk foot withdrawal in response to normally innocuous mechanical stimuli with von Frey filaments was measured. Rats were placed in a 30 × 30 × 30 cm Plexiglas box designed for von Frey testing with a clear floor containing 0.5-cm-diameter holes spaced 1.5 cm apart for access to the paws.29 and cancer pain.31 Briefly, action potentials evoked by stimulation of the dorsal root and measured at the L4 DRG soma are used to compare the configuration parameters and conduction velocity of each neuron. Recorded neurons were classified as C-type high-threshold mechanosensitive fibers (CHTM), Aβ-type high-threshold mechanosensitive fibers (AHTM), or Aβ-low-threshold mechanosensitive fibers (ALTM) based on their action potential configuration, conduction velocity, and receptive field properties as determined by utilizing handheld mechanical stimulators.33 Other major factors, including the rate of adaptation and the tissue location of the receptive field, were used to further classify ALTM neurons as either cutaneous neurons (CUT) or muscle spindle (MS) neurons. MS neurons were classified as slowly adapting neurons with deep subcutaneous receptive fields activated by deep tissue manipulation of the muscle belly but not by cutaneous stimulation.Details of intracellular electrophysiological recording techniques have been reported previously in animal models of NEPSoma excitability thresholds were measured by evoking action potentials in the somata of DRG neurons by direct injection of depolarizing current. To quantify soma excitability, current injections of 100 ms each were injected into the soma, at amplitudes between 0.5 to 4 nA in increments of 0.5 nA. The thresholds of depolarizing current pulses were determined with the “Protocol Editor” function in the pClamp 9.2 software program (Molecular Devices). All animals were tested at model endpoint. CIBP model rats were recorded following week 3 behavioral testing and all NEP model rats were recorded following week 6 behavioral testing.n = 3 biological replicates plus or minus the standard error of the mean (SEM). Data are expressed as fold change relative to dose-matched vehicle controls. Differences within treatment groups are compared to untreated control (0 µM) by one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple comparisons test. In vivo behavioral data represent the mean ± SEM from male SD CIBP rats  and female SD CIBP rats . Only animals with verified tumor development are included in results for CIBP model animals. Data were collected from male SD NEP rats , female SD NEP rats , and female RNU−/− NEP rats . All behavioral results are compared between and within groups across the duration of the experiment, and groups are compared independently at endpoint. Differences between treatment groups over time are compared to vehicle control by repeated measures two-way ANOVA with post hoc Dunnett’s multiple comparisons test. Differences within treatment groups relative to respective baseline measurements are compared by repeated measures two-way ANOVA with post hoc Dunnett’s multiple comparisons test. Differences between treatment groups at endpoint are compared by one-way ANOVA with post hoc Tukey’s multiple comparisons test or Kruskal-Wallis test with post hoc Dunn’s multiple comparisons test for nonparametric limb use scale data. Acute response differences within treatment groups are compared by multiple t-tests. Electrophysiological data represent the mean ± SEM from independently recorded neurons from n ≥ 3 rats of each group. Differences between treatment groups are compared by Kruskal-Wallis test with post hoc Dunn’s multiple comparison test. All results were considered significant at P < 0.05. Analyses and charts were generated using GraphPad Prism 7 software .In vitro data represent the mean of Prior to use in animal models, the growth of MRMT-1 cancer cells treated with PRE and PRO was investigated in vitro to determine whether administration of these test compounds could affect cell growth and therefore tumor size and nociceptive outcomes in in vivo animal models. Cell number was measured in vitro in the presence of a range of PRE and PRO doses between 1 nM and 50 µM. Crystal violet staining showed no differences between vehicle-treated MRMT-1 cells and PRE- and PRO-treated cells at any dose . Due to Male CIBP model rats did not show sustained differences indicative of reduced nociception by any behavioral measures in PRE- or PRO-treated groups when compared to vehicle. There were no differences in 50% mechanical paw withdrawal threshold in the ipsilateral limb between PRO, PRE, and vehicle treatment groups of male CIBP model rats at any time point post model induction as measured by testing with von Frey filaments . All malFemale CIBP model rats showed evidence of a delay of onset of a reduced mechanical withdrawal threshold in both PRO- and PRE-treated groups relative to vehicle , where PThere were no differences in ipsilateral limb use scoring  as measured by observational scoring over a 5-min period of free ambulation between PRO, PRE, and vehicle treatment groups of  male andThere were no differences in ipsilateral limb weight bearing as a percentage of animal body weight as measured by dynamic weight bearing between PRO, PRE, and vehicle treatment groups of male  and femaAction potential responses to intracellular depolarizing current pulse injection were tested in vivo to determine the soma excitability thresholds in sensory neurons at the DRG of model animals. Representative recording images show the multiple injected current stimuli  of 100 mCurrent activation thresholds measured in nanoamps of CHTM neurons  were sigIn female CIBP model rats, the activation thresholds of CHTM neurons  in the vAll treatment groups of male NEP model rats  showed aIn male NEP model rats, there were no differences between any groups in the current activation thresholds of CHTM neurons . Activat−/− sciatic cuff–induced neuropathic pain model rats at any time point post model induction excluding week 3 as measured by testing with von Frey filaments (There were no differences in 50% mechanical paw withdrawal threshold in the ipsilateral limb between PRO, PRE, and vehicle treatment groups of female RNUilaments . At weekAll model groups showed reduced thresholds relative to naïve control animals in all postsurgical weeks (not marked on chart; −/− models of NEP, the current activation threshold of CHTM neurons (−/− female rats. There were no significant differences in thresholds of CHTM neurons between both PRE- and PRO-treated groups and either vehicle or naïve rats. Activation thresholds of AHTM (In athymic female RNU neurons  was decr of AHTM  and ALTM of AHTM  neurons  of AHTM  were sig−/− neuropathic pain model animals (All NEP model animals were measured at week 1 pretreatment and 1 h posttreatments. Male SD rat models of sciatic cuff–induced neuropathic pain showed an acute response to treatment with PRE, PRO, and PRE+PRO combination as an increase in 50% mechanical paw withdrawal threshold as measured by testing with von Frey filaments. Withdrawal thresholds of vehicle-treated animals did not change following treatment. No acute responses to treatment were seen in any groups of female immunocompetent SD rats  or immun animals .Figure This study was designed to assess the effects of PRE and PRO on the development of chronic hypersensitivity in CIBP and NEP animal models in both male and female groups. We present for the first time that these treatments, administered as repeated injections during the early phases of NEP and CIBP development, promoted the robust recovery of mechanical hypersensitivity in male NEP rats, partial recovery of female NEP and female CIBP rats, and no apparent effects on both male CIBP and female immunocompromised NEP models. These results add to the evidence indicative of differing mechanisms of pain generation in CIBP and NEP states and to the evidence of sexual divergence in both the mechanisms of chronic pain itself and in the response to pain therapeutics.34 In this study, both CHTM and AHTM nociceptive neurons and ALTM-CUT and ALTM-MS nonnociceptive neurons showed significant changes in CIBP models, and treatment with PRE induced the recovery of nociceptive neuron excitation thresholds to sham model levels in both sexes. There were no differences in treated ALTM neurons from vehicle control. In NEP male and female model animals, we have observed that CHTM and ALTM-CUT neurons did not change in any groups relative to naïve and that reductions in the thresholds of AHTM and ALTM-MS neurons were prevented or reversed by treatment with PRE in male NEP animals and were not significantly changed in any groups of female NEP animals. Taken together, these results indicate that in contrast to the strong behavioral responses to treatment in NEP model rats, which showed normally nonnociceptive ALTM thresholds equivalent to naïve, the absent and muted behavioral response to treatment in in male and female CIBP rats, respectively, may be associated with decreased signaling thresholds in ALTM-CUT and ALTM-MS neurons.The behavioral results of this study suggest that PRE and PRO have efficacy in treating male and female NEP rats, and electrophysiological data support this for PRE-treated animals, showing excitation thresholds in nociceptive neurons equivalent to naïve. However, CIBP model male animals show no behavioral response to PRE or PRO, and female CIBP rats show only limited evidence of a delay in the onset of hypersensitivity with both treatments. Our previous investigation of the intracellular electrophysiological characteristics of sensory neurons in this CIBP model indicated that there are both nociceptive and neuropathic components of the cancer-induced pain state. These include reduced signaling thresholds of ALTM-MS neurons in both vehicle-treated NEP and CIBP model rats relative to control and morphological changes at the spinal cord indicative of a possible role for ALTM fibers in the generation or maintenance of the neuropathic component of CIBP.35 and has demonstrated some utility in treating chronic NEP in rat models in male rats.8 PRE has demonstrated effectiveness for NEP in animal models and in humans, leading to its use as a first-line clinical therapy for NEP; however, its effectiveness is inconsistent.36 Both PRO and PRE inhibit VGCCs, and though the exact mechanisms of the PRO and L-type VGCC interaction have not yet been elucidated, PRE, like gabapentin, binds at the α2-δ subunits of P/Q-, N-, and L-type Ca2+ channels.16 T- and N-type VGCCs are expressed on sensory afferent neurons,38 and it has been reported that the α2δ-1 subunit is upregulated in DRG neurons in several animal models of pain, and this is causally related to the onset of pain behavior.39–41 It also has been reported that pregabalin reduces the depolarization-induced calcium influx at nerve terminals, resulting in a reduction of the presynaptic release of excitatory neurotransmitters, including glutamate, substance P, and CGRP.43 VGCCs are also expressed on glial cells44; however, their expression and function are less well understood.In this study, normally nociceptive CHTM and AHTM neurons in both pain models showed decreased excitability after treatment with PRE. Treatment with PRO produced partial reductions in neuronal excitability thresholds in CIBP animals, although none were significantly different from either vehicle or sham controls. There is preclinical and clinical evidence of the efficacy of both drugs for conditions involving neuronal damage and neuropathy; however, the exact mechanisms of action are unclear. PRO has been shown to have neuroprotective properties, including protection from excitotoxic cell death, demyelination, and reduction of neuronal inflammation and edema, all of which can contribute to the generation of ectopic signaling,46 Electrophysiological measurements from week 3 endpoint either may not reflect a delay in the onset of this pain state or may not have allowed for an adequate and comparable duration of recovery.A limitation of this study is that our animal models developed over different durations, where our endpoint electrophysiological data were recorded at week 6 in all NEP models and at week 3 in all CIBP models. In addition, unlike the one-time nervous system damage of the cuff NEP model, our CIBP model reflects the progressive and often intractable nature of cancer pain in humans. CIBP is a conglomerate of multiple initiating factors, including a wide range of nociceptive and inflammatory stimuli, including mechanical distortion and pressure on host tissues, secreted inflammatory and nociceptive mediators, and neuropathy from tumor-initiated destruction and damage of nervous tissue and pathological growth of new and dysregulated sensory neurons.47–49 There is evidence in cancer pain states, however, that microglia do play a role in the maintenance of pain in female rats.50 To investigate whether the sex differences observed in our NEP models involved the T cell–dependent signaling systems in the spinal cord, we applied the sciatic cuff model to female RNU−/− athymic rats. Our hypothesis was that the lack of mature T cells would result in a chronic pain state with behavioral responses similar to those of immunocompetent male rats, as has been shown in other animal models of pain.47 It has also been demonstrated that T cell–deficient nude rats develop significantly reduced mechanical allodynia following chronic constriction injury compared with their heterozygous littermates.51 Our findings showed a behavioral difference between PRE- and vehicle-treated rats at only one time point, and that difference was not sustained. Female athymic NEP model rats in fact demonstrated more severe mechanical hyperalgesia in treatment groups than immunocompetent females, counter to our expectations, although this could reflect strain differences. Correspondingly, CHTM and ALTM-CUT neurons in vehicle-treated immunocompromised female NEP models showed increased excitability relative to naïve controls and were not recovered by treatment.Female CIBP rats treated with PRE and PRO showed a delay to the onset of mechanical hypersensitivity as measured by von Frey fibers. This delay was not reflected by measures of either weight bearing or limb use. Male CIBP model rats did not show behavioral responses to treatment. In NEP model rats, males demonstrated a strong response to drug treatment where PRE, PRO, and PRE+PRO treatment groups recovered to 50% mechanical thresholds equivalent to their baseline measurements by week 2. Although all NEP model female treatment groups remained at significantly higher withdrawal thresholds than vehicle-treated female controls throughout the experiment, no treatment groups recovered to baseline levels, in contrast to male rats. These sexually divergent effects in response to treatment are suggestive of mechanistic differences between male and female CIBP and NEP models. Substantial evidence indicates that sex differences in the behavioral responses to peripheral neuropathy in animal models may involve distinct hormonal and immune system pathways. After peripheral nerve injury, microglial–neuronal signaling in the spinal cord appears to mediate hypersensitivity in male mice, whereas in females, despite concurrent microglial proliferation, T cells infiltrate the spinal cord and maintain a hypersensitive state.Sex differences were also apparent in the acute behavioral response measurements performed at week 1 in all NEP model animals. Male NEP rats of all treatment groups responded with an increase in mechanical withdrawal threshold measured 1 h after treatment. These increases were not apparent in vehicle-treated controls, and no groups were significantly different in female immunocompetent and immunocompromised animals.52 and complete ablation of macrophages can result in severely impaired axon regeneration.53 It is possible that a partially beneficial immune response to chronic injury and NEP, as is the case in the sciatic cuff model, could be limited by the absence of T cells and by therapies that target them.These discrepancies in the response to treatment with PRE and PRO between sexes and immunocompromised animals indicate that the cellular immune response in the spinal cord of animal models of chronic pain is not a simple answer in this case. Inflammatory responses to injury also have adaptive functions enabling nerve repair involving interleukin-1 and tumor necrosis factor-α expression,In conclusion, we show that the analgesic effects of PRE and PRO can promote recovery of tactile hypersensitivity in response to treatment initiated during the early phase of NEP development. These effects are sex dependent and strongest in male rat models of NEP. Female rat models of NEP show a limited response to PRE and PRO treatment. Female CIBP models also show a limited response to treatment, and males do not respond. These results indicate that sex may be an important consideration for the therapeutic utility of both PRE and PRO.


T and Foxa2, results in changes to accessibility in DNA sequences containing their respective DNA-binding motifs and identify preferential motif arrangements that influence accessibility.A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Transcription factors interact with DNA to alter the accessibility of chromatin, which enables changes to gene expression during development or disease or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of transcription factors is difficult to study in the context of the native genome because every genomic site is distinct in multiple ways. Here we introduce the multiplexed integrated accessibility assay (MIAA), an assay that measures chromatin accessibility of synthetic oligonucleotide sequence libraries integrated into a controlled genomic context with low native accessibility. We apply MIAA to measure the effects of sequence motifs on cell type–specific accessibility between mouse embryonic stem cells and embryonic stem cell–derived definitive endoderm cells, screening 7905 distinct DNA sequences. MIAA recapitulates differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying E-box motifs common to epithelial–mesenchymal transition driver transcription factors in stem cell–specific accessible regions that become repressed in endoderm. We show that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell–specific, endoderm-specific, and shared accessibility-modifying transcription factor motifs. We also show that overexpression of two definitive endoderm transcription factors,  Genomic DNA acts as an instruction book for the cellular machinery to carry out functional processes such as RNA production  and DNA Transcription factors that interact with closed chromatin are thought to establish the accessibility of cell type–specific regions and initiate cell state change in differentiation , cancer Previous work has indicated specific transcription factor motifs and logic governing chromatin accessibility , but sucEscherichia coli adenine DNA adenine methylase (Dam) to the locus, given the high efficiency and stability of Dam methylation in cells  A. For inSupplemental Fig. S1). First, DNA sequences are amplified by primers outside of the homology arms to ensure only correctly integrated DNA sequences are amplified. Only undigested DNA sequences will be amplified at this step owing to the site of the GATC site of restriction enzyme cleavage between the PCR primers. Then, two additional PCR steps are used to further amplify DNA sequences and add Illumina sequencing adapters for high-throughput sequencing. If a DNA sequence is more accessible, it will have fewer read counts in the DpnI digested pool and more read counts in the DpnII digested pool  cells using a well-established differentiation protocol shown to yield >90% DE  before Rk-mer model trained on DNase-seq . We found in comparing control sequences with GC-content in the range of 30%–50%, MIAA replicates had 96%–100% of positive control DNA sequences yielding higher methylation than the average negative control DNA sequence, whereas SLOT had 4.5%–13.6% of positive control DNA sequences yielding higher methylation than the average negative control sequence , suggesting that MIAA provides a marked improvement over SLOT in the measurement of accessibility differences of single DNA sequences in the context of large libraries. Biological replicates of MIAA were also well correlated (Pearson's r = 0.5–0.79) .We tested a library of 5978 DNA sequences in eight biological replicates  for stem cells (ESCs) and four biological replicates  for DE cells. To gauge the reliability of MIAA, we included sets of positive and negative control DNA sequences used in our previous work that maximally pack 100-nt variable sequences with DNA sequence motifs shown to have an opening or neutral effect on chromatin by a Nase-seq . From MINase-seq C,D, withSupplemental Fig. S2). We do not know if this means that RAR-Dam can methylate ∼20% of inaccessible chromatin while it is tightly wound or if the methylation is happening during cell cycle phases when chromatin is accessible. We also found that retinoic acid binding sites within our sequence appeared to have no impact on MIAA results , suggesting that linking RAR to Dam is unlikely to confound our aim of measuring chromatin accessibility.We note that negative control  DNA sequences are still methylated at a rate of 20%–50%. In line with this result, we found ∼20% RAR-Dam methylation in two known native genomic inaccessible chromatin loci as measured by qPCR, compared with 85%–95% methylation at known RAR binding sites .We separately designed a pilot experiment of 2000 DNA sequences to determine whether MIAA could measure differential chromatin accessibility. First, we ran KMAC, a method for de novo motif enrichment , on diffSupplemental Fig. S6). We tested 213 native DNA sequences that DeepAccess predicted would be differentially accessible between ESCs and DE cells with MIAA, and found that as a group these DNA sequences showed differential accessibility between ESCs and DE cells   F. Althou< 0.001) F.Supplemental Fig. S7). We hypothesized that these DNA sequences contain motifs that result in decreases in accessibility in DE cells. We performed motif enrichment  on these DNA sequences and found that 98% (compared with 0% of endoderm native sequences) contained a match to the ZEB2 motif , a known transcriptional repressor that has been implicated in early gastrulation by repression of CDH1   , suggestadherin) , which wSupplemental Fig. S7). ZEB2 binding sites were also enriched in ESC-specific genomic accessible regions with 14% containing a ZEB2 motif relative to 9% in endoderm-specific accessible regions (P < 0.001 by hypergeometric test). In comparison, 12% of genomic ESC-specific accessible regions contained a SOX2 motif, 6% contained a POU5F1 motif, and 6% contained a KLF4 motif. KEGG biological pathway analysis of ZEB2 motif sites in ESC-accessible regions showed an enrichment of motif sites proximal to genes regulating pluripotency of ESCs (P < 0.001), including the key pluripotency regulators KLF4, SOX2, and NANOG, a finding that is consistent with a model of ZEB2 repression of pluripotency during DE differentiation . Unlike KMAC's pure enrichment approach, DeepAccess is able to learn nonlinear relationships between sequence motifs for predicting accessibility. From our set of motif hypotheses from both methods, we designed synthetic DNA sequences with either seven instances of one motif . We chose to pack each DNA sequence with the maximum number of motifs (54%–84% of the positions in each DNA sequence are part of a motif) while leaving space for sequence variation. For each DNA sequence, we also included a control in which the nucleotides are randomly shuffled to observe the influence of nucleotide content alone.We then hypothesized that we could identify and confirm with MIAA motifs that control chromatin accessibility in a cell type–specific manner through a set of synthetic, designed DNA sequences. By using cell type–specific DNase-seq data, we extracted short (8- to 12-nt) DNA sequence motifs that we hypothesized would cause differential accessibility using two methods A. First,ne motif B, which t motifs C, which r = 0.62; P < 0.001) with MIAA-measured differential accessibility  , which we hypothesize is the result of overconfidence of neural networks on out-of-distribution inputs . Out of 38 tested motif sequences, 20 induced differential accessibility, and out of 38 motif pair sequences, 26 induced differential accessibility. We also found these results to be largely consistent across a secondary closed integration locus . Thus, MIAA was able to confirm that motifs identified using DeepAccess are able to result in observable changes to accessibility both between cell types and compared with shuffled control sequences . In comparing results from DeepAccess to KMAC, we found only 32% (8/25) of our KMAC motifs or motif pairs were differentially accessible compared with 74.5% (38/51) of DeepAccess , indicating our DeepAccess approach was successful in identifying motifs driving differential accessibility.Out of the 46 motif or motif pair sequences that induced differential accessibility across cell types and were compared with shuffled control sequences as measured by MIAA, DeepAccess predicted the correct direction of differential accessibility between the two cell types in 76% (35/46) of cases (P < 0.001 by Wilcoxon rank-sum test), whereas in ESCs, only the low GC-content bin had differential accessibility between negative and positive controls (P < 0.001 by Wilcoxon rank-sum test) , suggesting that GC-content is a contributor to MIAA-measured accessibility alongside motif composition. DeepAccess-predicted accessibility was consistent with MIAA, indicating these effects were to be expected from observations on DNase-seq .We noticed previously that the positive control DNA sequences from the um test) A becauseP < 0.001 by one-tailed Wilcoxon signed-rank test) in ESCs compared with endoderm cells across all GC-content bins, except in DNA sequences with <35% GC-content (N = 372). Altogether, these results indicate that GC-content alone is a sufficient DNA signal to drive accessibility in both ESCs and endoderm as measured by MIAA and also to drive accessibility differences between these two cell contexts through its heightened impact in ESCs.Because this result could be an effect of sequence motifs included in the high-GC-content negative control DNA sequences, we then examined the nucleotide-shuffled DNA sequences that we designed to act as controls for motif activity to see if the effect of GC-content on MIAA accessibility held in random DNA. We found that the GC-content of randomly shuffled sequences correlated with MIAA accessibility in both cell types B. We alsSupplemental Fig. S10).Consistent with previous research that suggests a relationship between GC-rich regions and accessibility , we founr = 0.6335) and held-out test data (Pearson's r = 0.5841)  and significantly improved from regression models that did not include motif effects   D, reinfoSupplemental Methods). We identified motifs for known transcription factors such as Pou and Sox motifs as ESC-enriched and motifs for T-box and Fox factors as enriched in DE cells. The regression weights for these differential accessibility–driving motifs were robust, showing high consistency between models trained on biological replicates (Pearson's r = 0.963) , indicating that although MIAA correlation at the level of individual DNA sequences is modest, our estimation of motif-level effects is highly reproducible. We also identified motif pair sequences that show interesting nonlinear activity with respect to differential accessibility compared to their motif sequence effects alone . We then selected the motifs with the greatest positive difference in regression weights between the overexpressed T (ESC + T) and the ESC conditions. We found that T overexpression increases MIAA accessibility most strongly in DNA sequences with a motif pair that partially matches the motif of a T homodimer with two motifs in a minus/plus orientation and is significantly enriched over other dimer orientations in T ChIP-seq peaks (P < 0.001 by χ2 test) . The second strongest motif is also significantly enriched in T binding in mouse DE as measured by ChIP-seq (P < 0.05 under Benjamini–Hochberg multiple hypothesis correction) , supporting that T binding is capable of increasing accessibility specifically at motif-containing DNA sequences in a fixed chromatin context.We then hypothesized we could connect our discovered motifs to transcription factors driving differential accessibility by ectopically expressing transcription factors known to bind to certain enriched motifs. We overexpressed the transcription factors ith MIAA A. We trarection) B. OveralFoxa2 overexpression and found that the third and fourth top motifs were enriched in sequences from FOXA2 ChIP-seq peaks (P < 0.05 under Benjamini–Hochberg multiple hypothesis correction) , which is consistent with data showing that Foxa2 overexpression also results in more changes to gene expression , and therefore may lead to secondary chromatin accessibility changes unrelated to the FOXA2 motif. Both T and Foxa2 overexpression resulted in increased accessibility at a TGTCAACATT motif, which is likely because it contains sequences capable of binding both factors and is consequently enriched in both T and FOXA2 ChIP-seq. We also found that both Foxa2 and T overexpression resulted in chromatin accessibility changes that brought cells closer to the MIAA profile of DE cells . Thus, overexpression of individual transcription factors is capable of increasing the chromatin accessibility of a specific cohort of motif-containing sequences in a controlled chromatin context, providing evidence that binding of these factors leads to increased chromatin accessibility.Similarly, we examined the motifs with the greatest increase in accessibility upon rection) C. Foxa2 Finally, we used MIAA to explore interactions between motifs that are difficult to measure from observational approaches such as DNase-seq because of the lack of suitably controlled genomic motif arrangements. To probe interaction effects over a constrained set of known transcription factors, we designed a new library from the consensus binding motifs of the ESC lineage-defining transcription factors POU5F1, SOX2, and KLF4 A and theSupplemental Fig. S16). We note that the consensus motifs for SOX17 and SOX2 are highly similar, sharing a common sequence (CATTGTTT), so it is likely that both Sox factors and possibly others bind to both motifs tested. In contrast, in our DNA sequences containing two motif instances, 17/18 significantly increased accessibility compared with shuffled DNA sequences in at least one cell type , indicating that MIAA is capable of reliably detecting accessibility changes resulting from a minimum of two motif instances and that all six motifs open chromatin in at least one cell type. We then tested for differential accessibility with 6-nt versus 20-nt distance between motifs, which we selected based on literature supporting preferential distances between SOX2 and POU5F1 and between KLF4 and POU5F1 .We found that single motif instances were able to significantly increase accessibility compared with shuffled DNA sequences for 2/6 transcription factors (SOX17 and GATA4) but were rarely able to make DNA significantly differentially accessible  and FOXA2-GATA4 and SOX17-GATA4 combinations driving endoderm accessibility .We then examined all homotypic and heterotypic conformations with one, two, or three motif instances for induction of accessibility and differential accessibility. Overall, we found that 35/42 conformations significantly increased accessibility compared with shuffled versions in at least one cell type, and 15 out of 42 motif conformations were statistically significant for differential accessibility induction after multiple hypothesis correction E. Of theIn several cases, homotypic motif arrays showed accessibility patterns inconsistent with the expression of their expected transcription factors. For example, homotypic SOX17 motifs drive ESC-enriched accessibility, and homotypic FOXA2 motifs drive accessibility equivalently in ESCs and endoderm in contrast to the endoderm-specific expression of both transcription factors. Though we chose canonical motifs for factors well known in the literature to be associated with ESCs and endoderm, motifs are often shared by multiple members of a transcription factor family. In fact, it has been shown that FOXD3 binds in ESCs to motifs that will eventually become occupied by FOXA2 in endoderm . This saIn addition, we observed several instances of heterotypic combinations of transcription factor motifs in which order (whether a transcription factor motif was closer to the 5′ or 3′ end of the designed ssDNA sequence) had an impact on accessibility. For ESC factor binding motifs, we found that one ordering of POU5F1 and KLF4 more strongly differentially opens chromatin, whereas the other opens chromatin equivalently in both cell types E. We alsSupplemental Fig. S20). Because such native genomic instances are rare and confounded by other differences, MIAA provides a more controlled approach to identifying motif arrangements with differential activity.Among endoderm factor motif combinations, we found that particular FOXA2 and GATA4 and SOX17 and GATA4 E orientaThe MIAA is a new assay for measuring changes in chromatin accessibility caused by short DNA sequences integrated into a fixed locus in the genome. Most prior approaches to understanding the control of chromatin accessibility have used correlative approaches that identify genomic DNA sequences that tend to coincide with accessible chromatin in a particular cell type  or leverr = 0.53; P < 0.001), which we believe can be partially attributed to the use of DeepAccess to scan for highly differential native sequences that are more likely to be causal for specifying differential chromatin accessibility. Through examination of randomly shuffled control DNA sequences, we identify a distinction between how a set of natively ESC-specific and endoderm-specific sequences achieved differential accessibility. The natively endoderm-accessible sequences opened chromatin more in endoderm than in ESCs and more than their shuffled versions on average, suggesting the presence of binding sites for endoderm-specific accessibility-promoting transcription factors. On an individual level, only a subset of sequences act in this way, suggesting that a 100-nt DNA sequence does not always fully recapitulate the chromatin accessibility status of native regulatory elements, which often span over a kilobase. This may be caused by the absence in MIAA of specific sequence elements outside the 100-nt sequence that either contribute to or interact with the 100-nt sequence in its native locus.We applied MIAA to study the effects of motifs on differential accessibility between ESC and DE cell states using a number of distinct experimental designs. Through the use of native genomic 100-nt DNA sequences transplanted to a fixed locus, we were able to recapitulate the differential accessibility from native DNase-seq , highlighting the power of MIAA to identify accessibility-altering motifs.In spite of its importance, predicting MIAA chromatin accessibility of held-out DNA sequences purely based on GC-content yields poor results, whereas much better results are achieved by accounting for binding motifs. Of the motifs that can be confidently matched to known transcription factor families, our results are consistent with the action of known tissue-specific pioneer factors including SOX2 and POU5F1 in ESCs and GATA4 and FOXA2 in the endoderm . We confWe then designed a library using consensus motifs of several key transcription factors in all possible combinations and orderings, from which we provide evidence that a single binding site is sufficient to increase chromatin accessibility and as few as two binding sites are sufficient to induce differential accessibility between two cell types. These results suggest for the first time that individual transcription factor binding events in the absence of DNA-binding cofactors are capable of altering chromatin accessibility in mammalian cells.We also found that for motifs known to bind to both ESC and DE transcription factors, motif order has a subtle effect on accessibility, which provides support for specific transcription factor interactions driving accessibility change. This result illustrates the complexity of differential accessibility induction, which cannot simply be distilled to the presence of consensus motifs for differentially expressed transcription factors. In addition to the reuse of genomic motifs by different members of the same transcription factor family in different cell states , certainT was overexpressed, suggesting that MIAA has the capacity to measure the effects of transcription factor interactions on accessibility. Predicting differential accessibility from DNA sequence has been a much more difficult task than predicting cell type–consistent accessibility  are flanked by 25-nt primer sequences containing a GATC site and homology arms for CRISPR integration. We identified six native genomic sequences of size 100 nt from a pilot experiment that did not drive differential accessibility with MIAA but varied in GC-content. We randomly perturbed these native sequences three times each to obtain a total of 24 neutral sequence backgrounds. For our first experiment, we took each background and inserted either one motif seven times  or two motifs in which motif 1 is inserted four times  and motif 2 is inserted three times . For our second experiment, we limited ourselves to nine backgrounds that we expected to have high reproducibility to the set of 24. In this experiment, we tested sequences of size 70 nt. By using the consensus sequences of known ES key TFs  or DE key TFs , we inserted one, two, or three motifs into each sequence. We tested homotypic DNA sequences consisting of one unique motif, as well as heterotypic DNA sequences enumerating all possible motif orders. Consensus motifs for key developmental transcription factors are listed in Supplemental Methods). Cells were grown for 5–8 d after electroporation to obtain adequate quantities for doxycycline treatment. When indicated, cells were differentiated to DE before doxycycline treatment.Electroporations were performed in two to four biological replicates into p2L RAR-DamA126 ESCs . The second PCR step and third PCR steps further amplify sequences and add adaptors for Illumina sequencing. For primer information and further details, see Supplemental Code). Reads were normalized to reads per million over the total number of reads in the digest. DNA sequences were kept if they had a threshold number of total normalized reads over all replicates, based on the observation of high standard deviation at low total read counts. The threshold was selected based on visual inspection and can be found in the Supplemental Code. Once reads were normalized and high variability DNA sequences filtered, MIAA accessibility was computed as a proportion of DpnI/II read counts DpnII/(DpnI + DpnII).Reads were mapped to library DNA sequences by taking the reverse complement to the raw read, in which the first N nucleotides (between 70 and 100 based on the size of the designed sequence) are the designed variable DNA sequence. Perfect matches were counted using a custom R script  open in endoderm cells and closed in ESCs, (2) open in ESCs and closed in endoderm cells, (3) open in both cell types, or (4) closed in both cell types. A test set of 22,357 sequences is held out for performance evaluation.We obtain DNase-seq regions using the 100 nt centered at the MACS2 narrow peak call. Accessibility prediction is treated as a multitask classification problem, in which each genomic sequence (100 bp) is associated with a two-dimensional bit vector representing whether the sequence is open in each cell type (ESC and DE cell). We trained an ensemble of 10 convolutional neural networks. For specific details on network architecture, see We extracted motifs from DeepAccess by applying smoothed gradient ascent to score each nucleotide in the 100-nt DNA sequence by its importance for predicting the output  and multhttps://www.ncbi.nlm.nih.gov/geo/) under accession number GSE145920. Prefiltered unnormalized MIAA read counts are available as Supplemental Data. Accession numbers for previously published DNase-seq, ChIP-seq, and RNA-seq data that were used in this study are listed in Supplemental Table S2. Code for DeepAccess accessibility prediction and motif extraction is available at GitHub (https://github.com/gifford-lab/DeepAccess) and as Supplemental Code. Code for MIAA library processing and producing manuscript figures is available at GitHub  and as Supplemental Code.All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; The authors declare no competing interests.


Astrocytes, once thought to be passive cells merely filling the space between neurons in the nervous system, are receiving attention as active modulators of the brain and spinal cord physiology by providing nutrients, maintaining homeostasis, and modulating synaptic transmission. Accumulating evidence indicates that astrocytes are critically involved in chronic pain regulation. Injury induces astrocytes to become reactive, and recent studies suggest that reactive astrocytes can have either neuroprotective or neurodegenerative effects. While the exact mechanisms underlying the transition from resting astrocytes to reactive astrocytes remain unknown, astrocytic calcium increase, coordinated by inflammatory molecules, has been suggested to trigger this transition. In this mini review article, we will discuss the roles of astrocytic calcium, channels contributing to calcium dynamics in astrocytes, astrocyte activations along the pain pathway, and possible relationships between astrocytic calcium dynamics and chronic pain. Under physiological conditions, astrocytes occupy non-overlapping regions and function in discrete areas  and inhibitory γ-aminobutyric acid (GABA). The majority of Glu in the extracellular space is absorbed (80–90%) by excitatory amino-acid transporter (EAAT)-1 and 2, which are predominantly expressed in astrocytes  activity, on the other hand, is directly regulated by astrocytic Ca2+. Transient receptor potential (TRP) channel subfamily A (TRPA), a cation channel shown to be important in maintaining basal astrocytic Ca2+ concentration in astrocytes, controls the expression of GAT in a Ca2+-dependent manner , a structure of fine astrocytic processes wrapped around neuronal synapses, affect astrocytic buffering ability and provide spine stability in an astrocytic Ca2+ affects astrocytic lactate secretion is unknown, but the control of vascular smooth muscle tone, which is another important way of providing nutrients and oxygen, is directly regulated by astrocytic Ca2+  between synapses , but they could be more appropriate for providing sustained signaling in cases such as protecting tissues after an injury during recovery. While some alterations in astrocytic Ca2+ signals have been associated with dysfunctions in the CNS such as chronic pain, what kind of astrocytic Ca2+ changes lead to pathological conditions is still unclear. Since astrocytic Ca2+ signals triggered by different sources seem to have different functions, different sources of astrocytic Ca2+ are discussed in the next section.As described above, astrocytics Ca2+ sources contribute to astrocytic Ca2+ dynamics. Ca2+ releases from intracellular stores and influx from the extracellular space both contribute to astrocytic Ca2+ changes, albeit with different patterns. Intracellular sources of astrocytic Ca2+ include inositol triphosphate receptor (IP3R), ryanodine receptor (RyR), and mitochondrial permeability transition pore (mPTP). Ca2+ Channels that contribute to Ca2+ influx into astrocytes from the extracellular space include Orai channels, ligand-gated ion channels , TRP channels, sodium/calcium exchangers (NCX), and voltage-gated Ca2+ channels.Several Ca2+ increase occurs by activation of IP3R expressed on the endoplasmic reticulum (ER). Activation of IP3R leads to a robust Ca2+ increase in the cell body. Ca2+ dynamics become nearly absent in the cell body of astrocytes with inhibition of IP3R or knockout of IP3R2, a major type of IP3R in astrocytes, but some Ca2+ activity in astrocytic processes remains  generated by mitochondria and the increase in Ca2+ released from the ER, the mitochondrial membrane depolarizes via the opening of the mPTP, which ultimately leads to the necrotic death of astrocytes  on the ER, which sense Ca2+, cluster to activate Orai channels  could also contribute to astrocytic Ca2+ elevation. Several types of voltage-gated Ca2+ channels—L, N, R, and T types—are expressed in astrocytes. Although astrocytes are not electrically excitable, cultured astrocytes responded with an increase in intracellular Ca2+ to electrical stimulation  in astrocytes prevented astrocyte activation, reduced inflammation in the brain, and promoted myelin regeneration  and affective  aspects, which are separately processed by discriminatory and the affective pain pathways. Interestingly, in chronic pain models, reactive astrocytes are found not only at the site of noxious stimulation or injury but also along both pain pathways . Astrocya et al.  found tha et al. .Numerous studies have suggested that reactive astrocytes in the spinal cord are associated with chronic pain. Astrocyte activation, indicated by GFAP upregulation, is reported in various pain models , suggesting that reactive astrocytes in the thalamus may be the underlying cause  resulting from nerve injury , the region important for identifying the intensity and location of pain, is also enhanced by nerve injury. GFAP expression and frequency of astrocytic CaAstrocytes in brain regions known to process the cognitive-affective aspect of pain are also activated with chronic pain. Sciatic nerve ligation significantly enhanced GFAP expression in the cingulate cortex  subtype Cx43  or diffusion of extracellular ATP .While some studies suggest that aberrant changes in astrocytic Ca2+ increased in the S1 and the ACC following nerve injury  release and synapse formation, indicating that rewired circuits could cause neuropathic pain , which remodel synapses. Altered connectivity between neurons and associated hyperactivity may be the underlying cause of chronic pain. Since reactive astrocyte exhibits prominent Ca2+-dependent gliotransmitter release, enhanced Glu released by reactive astrocytes that excessively excite neurons may also contribute to chronic pain. Changes in neurotransmitter buffering activity could also contribute to the changes in neuron–astrocyte interaction ensuing chronic pain. Buffering of the inhibitory transmitter—GABA—by astrocytes via GAT is directly affected by astrocytic Ca2+, with more GABA taken up with increasing Ca2+. Although GAT activity was shown to enhance the synaptic efficacy of an inhibitory synapse by reducing ambient GABA that desensitize the inhibitory synapse  and the A2 (neuroprotective; Liddelow and Barres, 2+ is unknown. It is possible that astrocytic Ca2+ induced by different channels may drive reactive astrocytes to be one type or the other. Therefore, investigating how different astrocytic Ca2+ channels contribute to different types of reactive astrocytes in regard to chronic pain development will provide a deeper understanding of the roles of astrocyte in pain and may offer better targets for pain control.Astrocytic CaJC and YH contributed equally to the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.