Hepatitis E virus (HEV) causes adverse clinical outcomes in pregnant women, but the underlying mechanisms remain poorly understood. To delineate the mechanisms of pregnancy-associated adverse effects during HEV infection, we utilized a genotype 3 HEV from rabbit (HEV-3ra) and its cognate host (rabbits) to systematically investigate the clinical consequences, viral replication dynamics, and host immune and hormonal responses of HEV infection during pregnancy. We found a significant fetal loss of 23% in HEV-infected pregnant rabbits, indicating an early-stage miscarriage. HEV infection in pregnant rabbits was characterized by higher viral loads in feces, intestinal contents, liver, and spleen tissues, as well as a longer and earlier onset of viremia than in infected nonpregnant rabbits. HEV infection altered the pattern of cytokine gene expressions in the liver of pregnant rabbits and caused a transient increase of serum interferon gamma (IFN-γ) shortly after a notable increase in viral replication, which may contribute to early fetal loss. Histological lesions in the spleen were more pronounced in infected pregnant rabbits, although moderate liver lesions were seen in both infected pregnant and nonpregnant rabbits. Total bilirubin was elevated in infected pregnant rabbits. The serum levels of estradiol (E2) in HEV-infected pregnant rabbits were significantly higher than those in mock-infected pregnant rabbits at 14 days postinoculation (dpi) and correlated positively with higher viral loads in feces, liver, and spleen tissues at 28 dpi, suggesting that it may play a role in extrahepatic virus dissemination. The results have important implications for understanding the severe diseases associated with HEV infection during pregnancy. Hepatitis E virus (HEV), the causative agent of hepatitis E, is a leading cause of acute viral hepatitis worldwide and has emerged as a global public health concern in recent years , with an4–10–In pregnant women, adverse effects of HEV-1 infection include vertical virus transmission of >30%, preterm delivery, miscarriage, stillbirth , and fulminant hepatic failure (FHF), with a mortality rate of up to 30% , 13–17. 13–Hepeviridae, which contains two subfamilies, Orthohepevirinae and Parahepevirinae. The Orthohepevirinae subfamily consists of four distinct genera containing members that infect avian species (Avihepevirus); bats (Chirohepevirus); humans and other mammals (Paslahepevirus); and rodents, shrews, and carnivores (Rocahepevirus). The Parahepevirinae subfamily comprises only one genus (Piscihepevirus) of viruses that infect fish  –27, alth–28–ect fish , 34. Theountries , 36.Variant strains of HEV were isolated from rabbits in China, the United States, and several other countries –40. Sequ–44–48–52–In this study, we utilized an HEV-3ra infection of pregnant rabbit model to systematically investigate the adverse clinical outcomes associated with HEV infection during pregnancy and delineate the potential hormonal and immunological underlying mechanisms that may contribute to the severe diseases during pregnancy.n = 8) were either infected with HEV-3ra at 7 days of gestation or mock-inoculated with phosphate-buffered saline (PBS) (P = 0.0131) reduced number of kits born alive  compared to uninfected pregnant rabbits , indicating a fetal loss of 23.19% due to HEV-3ra infection  ; Fig. 1Anfection . Rabbit,nfection . None of10.1128/mbio.00418-23.3TABLE S1Table S1, DOCX file, 0.01 MB.Numbers of kits born alive and the numbers of stillbirths per litter in mock (PBS)-inoculated and HEV-3ra-inoculated pregnant rabbit groups. Download Copyright © 2023 Mahsoub et al.2023Mahsoub et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4 genome copies/g feces for HEV-P and 3.96 × 104 genome copies/g feces for HEV-NP) and remained positive until necropsy at 28 dpi, except for one HEV-NP rabbit, which tested negative at 7 dpi but was positive thereafter  than in HEV-infected nonpregnant rabbits (HEV-NP) , althougereafter . At 3 dpereafter  and B. Tereafter , but the10.1128/mbio.00418-23.4TABLE S210 copies of HEV RNA/gram) in fecal and large intestinal content (LIC) samples collected from HEV-3ra-infected pregnant (HEV-P) and nonpregnant (HEV-NP) rabbits. Download Table S2, DOCX file, 0.02 MB.Viral RNA load data . This indicates that fecal viral loads differed significantly among certain time points within the treatment group. Tukey’s post hoc analysis showed that, in the HEV-P group, fecal viral loads significantly increased during the 3- to 7-dpi and 7- to 10-dpi time intervals  . The increase in viral loads during subsequent intervals was not significant in both groups. Overall, the data show that HEV infection caused higher levels of virus replication in pregnant rabbits during the early stages of gestation than in nonpregnant rabbits. It is plausible that the increase in viral replication at the early stages of HEV infection in pregnant rabbits, along with other unfavorable virus-induced systemic and localized inflammatory responses, may contribute to the observed early fetal loss.We further analyzed and compared the fecal viral shedding data from 3 dpi to 28 dpi within each of the HEV-P and HEV-NP groups. The two-way analysis of variance (ANOVA) of the fecal viral RNA loads showed that the “infection × pregnancy” interaction was not significant; however, “time postinoculation” had a significant effect (ctively) . On the P < 0.001) than that of HEV-NP rabbits, which all tested negative (The amounts of HEV RNAs in sera from each rabbit were quantified in weekly serum samples by reverse transcriptase quantitative PCR (RT-qPCR). In HEV-P rabbits, viremia was detected as early as at 7 dpi in 2/8 rabbits, which remained positive at necropsy at 28 dpi. At 21 dpi, 4 more rabbits became viremic, bringing the total to 6/8 viremia-positive rabbits (75%). By 28 dpi, one rabbit became negative and one new rabbit tested positive, maintaining 6/8 pregnant rabbits with viremia . In HEV-negative . The vir10.1128/mbio.00418-23.5TABLE S310 copies of HEV RNA/mL or gram) in serum, bile, and tissue samples collected from HEV-3ra-infected pregnant (HEV-P) and nonpregnant (HEV-NP) rabbits. Download Table S3, DOCX file, 0.02 MB.Viral load data  due to higher individual rabbit variations in the HEV-P group , which prevented the differences from being statistically significant. Extrahepatically, HEV RNA was detected in spleen and ovary tissues, with substantially more positive samples in the HEV-P rabbits  than in the HEV-NP rabbits  (P < 0.05 and P < 0.001)  . Viral l HEV RNA .r = 0.70, P = 0.0510)  and LIC   and serum    to determine whether the magnitude of viral replication in liver affects extrahepatic viral dissemination. In HEV-P rabbits, viral loads in liver had a near-significant positive correlation with those in bile ( 0.0510)  and signFor viremia, in the HEV-NP group, one (33%) of the three viremic rabbits at 28 dpi tested positive for HEV RNA in the spleen, but all three were negative in the ovary. As for the HEV-P group, four (67%) of the six viremic rabbits at 28 dpi tested positive for HEV RNA in samples of both spleen and ovary tissues. These results indicate that viremia has a positive correlation with extrahepatic virus dissemination and that pregnancy is a risk factor.P < 0.05) (P < 0.05) than those in PBS-P rabbits (P < 0.05 and P < 0.01) (P < 0.05)  and liver enzyme alkaline phosphatase (ALP) in weekly serum samples from all animals using commercial enzyme-linked immunosorbent assay (ELISA) kits. We found that HEV-P rabbits had statistically significantly higher levels of TBB at 21 dpi and 28 dpi than those in HEV-NP rabbits ( < 0.05) . The ser rabbits . When an < 0.01) . PBS-P r < 0.05) . In all Since our pilot rabbit studies showed that there was no change in serum levels of the liver enzyme γ-glutamyl transferase (GGT) in HEV-infected pregnant and nonpregnant rabbits at 0 to 28 dpi , we deci10.1128/mbio.00418-23.2FIG S2FIG S2, PDF file, 0.2 MB.Pilot animal infection studies in pregnant and nonpregnant rabbits. To optimize the HEV infection study in pregnant rabbits and identify the appropriate virus strain, infection timing, and laboratory test parameters, we first conducted three pilot animal studies with a very small number of animals. The serum levels of γ-glutamyl transferase (GGT) in pregnant and nonpregnant rabbits experimentally infected with three different strains of genotype 3 rabbit HEV  were tested with a rabbit GGT ELISA kit . Results from pilot study 2 (0 to 28 dpi) and pilot study 3 (0 to 22 dpi) are shown here. There was no difference in serum level of GGT between infected and control and between pregnant rabbits and nonpregnant rabbits. Therefore, based on the pilot study result, GGT was not tested in this study. Also, based on collective virology and clinical data from the pilot studies, we chose to use HEV-3ra strain RC-39 for this study. P, pregnant rabbits; NP, nonpregnant rabbits. Download Copyright © 2023 Mahsoub et al.2023Mahsoub et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To determine the host systemic immune responses induced by HEV infection during pregnancy, we measured the weekly levels of proinflammatory cytokines interferon gamma (IFN-γ) and interleukin 6 (IL-6), as well as Th1 cytokines IL-2 and IL-12 in serum samples of both pregnant and nonpregnant rabbits using commercial ELISA kits. The cytokine levels among the four rabbit groups  were compared to determine the effects of pregnancy and HEV infection statuses on these cytokine responses. We found that the circulating levels of proinflammatory cytokines IFN-γ and IL-6, and Th1 cytokines IL-2 and IL-12, are similarly altered in HEV-3ra-infected rabbits regardless of pregnancy status.P < 0.05), while HEV-NP rabbits had significantly higher levels than PBS-NP rabbits at 7 and 14 dpi (P < 0.05) (P < 0.05) than the PBS-NP group (The levels of serum IFN-γ were significantly higher in both HEV-infected pregnant and nonpregnant rabbits throughout the course of infection. HEV-P rabbits had significantly higher levels of serum IFN-γ than PBS-P rabbits at 7 dpi ( < 0.05) . At all  < 0.05) . The aveNP group . At 7 dpThe average serum levels of Th1 cytokines (IL-2 and IL-12) at 0 to 28 dpi were not significantly different among the four groups, regardless of infection or pregnancy statuses  and D. TTo understand the host inflammatory responses to HEV infection in the liver microenvironment during pregnancy, we quantified the mRNA expression levels of a panel of proinflammatory and anti-inflammatory cytokines in the liver tissues collected at necropsy at 28 dpi from both pregnant and nonpregnant rabbits, uninfected or infected with HEV-3ra. As indicated for other parameters described above, in this study, we mainly focused on comparing the expression levels of cytokine genes in the HEV-P group with those in the HEV-NP group to examine the pregnancy factor and with those in the PBS-P group to examine the HEV infection factor.P < 0.05 for IFN-γ and P < 0.01 for TNF-α), irrespective of pregnancy status. The mRNA expression level of the IL-6 gene in the liver was higher in HEV-NP rabbits than in the other groups (P < 0.05) compared to HEV-P rabbits, where IL-6 expression was similar to the PBS controls. The IL-8 gene expression in the liver was higher in HEV-P rabbits than in the other groups (P < 0.05) compared to that of the HEV-NP rabbits. There was also an upregulation in mRNA expression of IL-18 and IL-10 genes in the liver of HEV-P rabbits compared to the other groups, but the differences were only significant compared to the PBS-P group (P < 0.05 and P < 0.01)  with liver IFN-γ mRNA levels compared to HEV-NP rabbits, which showed no such linear correlation   and B inr groups , but it r groups , but it  < 0.01) . The difrelation .10.1128/mbio.00418-23.6TABLE S4Table S4, DOCX file, 0.02 MB.Differences in fold change of gene expression levels of selected cytokines, chemokines, and pregnancy-related genes in the liver tissues among the four study groups of pregnant and nonpregnant female rabbits experimentally infected with HEV-3ra or mock-infected with PBS. Download Copyright © 2023 Mahsoub et al.2023Mahsoub et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ESR1 and ESR2), progesterone receptors (PRMC1 and PRMC2), and progesterone-induced blocking factor (PIBF-1), were also quantified in the liver and placenta tissues collected at necropsy at 28 dpi. No significant changes in these gene expressions were found between HEV-infected or uninfected pregnant or nonpregnant rabbits  than those in their matching uninfected rabbit groups (P < 0.0001) than that in uninfected pregnant rabbits and numerically higher than that in HEV-infected nonpregnant rabbits. Splenic lesion scores were not different between HEV-infected and uninfected nonpregnant rabbits  and TNF-α    with serum IL-12 levels at 28 dpi and negatively  with serum IL-6 levels  was observed between spleen lesion scores and serum IL-12 levels at 28 dpi; other correlations for liver and spleen lesions were insignificant   and seru 0.0494) . Also, i6 levels . On the nificant , and G.At 7 days of gestation, i.e., the seventh day after copulation, rabbits in the two pregnant groups were inoculated with HEV or PBS , and this was the 0-dpi time point. The levels of pregnancy hormones (estrone [E1] and estradiol) were measured in the serum samples of all pregnant and nonpregnant rabbits at 0, 7, 14, 21, and 28 dpi .P < 0.05). The average serum E2 levels in HEV-P rabbits were slightly higher than those in HEV-NP rabbits, while PBS-P rabbits had lower serum E2 levels than those in PBS-NP rabbits  than those in PBS-P rabbits. The average E1 levels in HEV-P rabbits were also higher (0.38-fold to 0.95-fold) than those in HEV-NP rabbits, while no such differences were observed between the PBS-NP and PBS-P groups . The ave rabbits . Collectr = 0.75, P = 0.0322)      , spleen  0.0213) , fecal s 0.0219) , and LIC 0.0267) ; a posit 0.1760) . In the  0.1760)  to K. Thex vivo model utilizing the decidua basalis and fetal placenta was used to compare HEV-1- and HEV-3-induced pathogenic effects at the maternal-fetal interface. It was reported that HEV-1 replicated at significantly higher levels than HEV-3, causing dysregulation in the secretion of several cytokines, which contributed to severe decidual and placental tissue injuries  and shorter gestation period , in this present study, we aimed to utilize the pregnant rabbit model to systematically determine the roles and underlying mechanisms of pregnancy-associated sex hormone (estrogens), host immune responses, and viral replication kinetics in liver and extrahepatic tissues in the development of HEV-induced adverse clinical outcomes associated with pregnancy. The results from this study showed that HEV-3ra-infected rabbits had a significantly lower number of kits per litter than uninfected pregnant rabbits, with a fetal loss of ~23%; however, we were unable to reproduce the other pregnancy-associated severe outcomes such as maternal death or stillbirth reported by the other two groups , 53. Then = 9) compared to the other HEV-infected pregnant rabbits in the HEV-P group, implying a much lower burden of virus infection. Data from this individual rabbit may signify the importance of negative impact on clinical outcomes of enhanced virus replication during pregnancy and the importance of efficient antibody response to prevent viremia in pregnant rabbits and thus prevent severe diseases.In this study, we systematically quantified and compared viral loads in liver as well as in extrahepatic tissues, including serum, feces, intestinal contents, spleen, kidney, and ovary tissues in both HEV-infected pregnant and nonpregnant rabbits during the course of HEV-3ra infection. We found that viral loads in fecal samples at all time points and in LIC at necropsy were generally higher in infected pregnant rabbits than in nonpregnant rabbits, thus indicating an enhanced viral replication in pregnant rabbits. Furthermore, the onset of viremia was 1 week earlier with higher incidence and longer duration in HEV-infected pregnant rabbits than in infected nonpregnant rabbits. Importantly, we found that incidence of viral dissemination and viral loads in tissues and bile were either significantly or numerically higher in infected pregnant rabbits than in nonpregnant rabbits. Intriguingly, our results of viral loads, viremia incidence, and virus dissemination in HEV-infected pregnant rabbits are similar to what has been reported in an immunosuppressed rabbit model . Since w10.1128/mbio.00418-23.1FIG S1405) (8 rabbits per group) and cutoff values (dashed line) are plotted. (A) PBS-NP, PBS-inoculated nonpregnant rabbits; (B) PBS-P, PBS-inoculated pregnant rabbits; (C) HEV-NP, HEV-inoculated nonpregnant rabbits; (D) HEV-P, HEV-inoculated pregnant rabbits. Among the four groups, only two rabbits (rabbits 31 and 33) in the HEV-infected pregnant rabbit group (D) tested seropositive for anti-HEV IgG antibodies at 28 dpi. Rabbit 31 had an OD value that is slightly higher than the cutoff value, while rabbit 33 had a very high OD value above the cutoff. The lack of seroconversion in most of the infected rabbits is expected since the experiment lasted only 28 days. Anti-rabbit HEV IgG antibodies titers were assayed using an HEV ORF2 antigen-based ELISA. Briefly, serum samples were collected weekly from 0 to 28 days postinoculation (dpi). Serum samples were diluted at 1:200 in blocking buffer. Preimmune and hyperimmune anti-HEV positive rabbit sera were included as negative and positive controls, respectively. Download FIG S1, PDF file, 0.2 MB.Anti-HEV IgG antibody responses in HEV-3ra-inoculated or mock-inoculated pregnant and nonpregnant female rabbits. Individual values of optical density at 450 nm  cells and monocytes; therefore, reduction of the number of any of these cells may affect the overall level of IL-6 in the blood. It has been reported that HEV-positive pregnant FHF women had significantly lower CD4+ cell counts than HEV-negative pregnant FHF women or control patients , which had histological lesions in both HEV-infected pregnant and nonpregnant rabbits. We found that there were numerically higher expression levels of IL-18 and IL-10, significantly higher IL-8 mRNA levels, and significantly lower IL-6 mRNA levels in the liver of HEV-P rabbits than in the liver of HEV-NP rabbits. The significantly higher levels of the proinflammatory cytokine IL-18 and anti-inflammatory cytokine IL-10 in HEV-P rabbits than in PBS-P rabbits suggest that, in some infected pregnant rabbits, the host immune system may reach inflammatory homeostasis in the liver. In HEV-infected pregnant rabbits, the negative correlation of serum estradiol with IFN-γ gene expression in liver and its positive correlation with viral loads in liver may be one potential mechanism for how estrogen can promote viral replication at the site of infection. As indicated above, unlike in the HEV-NP group, the level of liver IL-6 mRNA expression in the HEV-P group was not upregulated, which may contribute to the increased viral titer (and histopathology) in the liver of pregnant rabbits. In addition to its protective role during liver injury , in a moIn this study, regardless of pregnancy status, we found liver damage in both HEV-infected groups during necropsy at 28 dpi and prior to the development of detectable serum anti-HEV IgG antibodies, with a median histological lesion score of 2.5 . Through regression analyses, we showed that liver IL-6 and TNF-α mRNA expression levels and serum IL-12 levels at 28 dpi may contribute to liver histological lesions in HEV-infected pregnant rabbits. Serum total bilirubin is a common marker of liver function, and a high level of blood TBB is indicative of liver injury . HEV-infIn summary, we showed that pregnant rabbits experimentally infected with HEV-3ra can recapitulate some , but not all, pregnancy-associated adverse clinical outcomes seen in HEV-infected pregnant women. Although liver histological lesions were similar in both HEV-infected pregnant and nonpregnant rabbits, the underlying factors  contributing to severe diseases are different and more pronounced in pregnant rabbits. The HEV-infected pregnant animals showed higher viral replication, worse viremia markers, higher extrahepatic virus dissemination, and altered inflammatory responses. The positive correlations between estradiol levels and both viral replication and cytokine responses in HEV-infected pregnant rabbits point out a significant role of pregnancy hormones in HEV pathogenesis during pregnancy.FJ906895), originally isolated from rabbits in China , while the rabbits in the two negative-control groups were each similarly mock-inoculated with PBS buffer.All rabbit studies were approved by the Institutional Animal Care and Use Committee at Virginia Tech (IACUC number 19-047). A total of 32 HEV-negative, 6.5-month-old, NZW female rabbits  were randomly divided into 4 groups of 8 rabbits per group, control nonpregnant (PBS-NP), control pregnant (PBS-P), infected nonpregnant (HEV-NP), and infected pregnant (HEV-P). Prior to arrival, serum samples were collected from all animals and tested to be free of anti-HEV IgG antibodies and HEV RNA using an ELISA and RT-qPCR or nested RT-PCR, respectively. Upon arrival, rabbits were housed separately in cages in an animal biosafety level 2 (ABSL-2) facility at Virginia Tech. At 7 days of gestation, the two HEV-3ra infection groups were intravenously inoculated with HEV-3ra infectious virus stock  or weekly (serum) thereafter until necropsy at 28 days postinoculation (dpi), which coincided with 2 to 3 days postdelivery for the pregnant rabbits. Pregnant rabbits were monitored daily for clinical signs, and the number of kits born  per litter was counted and recorded immediately after each rabbit’s delivery. When pregnant rabbits gave birth, samples of placenta tissues were collected, and during necropsy at 28 dpi, samples of liver, spleen, kidney, and ovary tissues were collected from all rabbits. One set of tissue samples was snap-frozen on dry ice and stored at −80°C for extracting RNA to quantify viral loads and various gene expression levels. A second set of tissue samples was fixed in 10% neutral buffered formalin and processed for routine histological evaluation. Bile and LIC samples were also collected at necropsy and stored at −80°C.Tissue slides were stained with hematoxylin and eosin and examined in a blinded fashion by a board-certified veterinary pathologist (T.L.). Liver microscopic lesions were scored from 0 to 4 based on the presence of phlebitis and intralobular inflammation . Spleen histological lesions were scored from 0 to 4 based on the presence of granular cell necrosis . Kidney, placenta, and ovary tissues were also evaluated for the presence or absence of histological lesions.Total RNA was extracted from 200-μL aliquots of 10% (wt/vol) fecal suspensions in PBS, 20% (wt/vol) LIC suspensions in PBS, and 50 μL of bile, using 1 mL of TRI reagent (Molecular Research Center), following the manufacturer’s instructions. Total RNA was extracted from 100 μL of serum samples using Quick-RNA viral kit (Zymo Research), following the manufacturer’s instructions. Samples of tissues  were prepared as 10% (wt/vol) tissue homogenates in TRI reagent using gentleMACS dissociator, following the “frozen tissue” RNA program instructions. Total RNAs were extracted from the respective 10% tissue homogenate of spleen, ovary, and kidney by following the TRI reagent protocol. For liver and placenta samples, after homogenization and centrifugation, the supernatants were transferred to genomic DNA (gDNA) eliminator spin columns in the RNeasy Plus minikit (Qiagen), and total RNA was subsequently extracted following the manufacturer’s instructions. The quality and concentration of extracted total RNAs from each tissue were determined using NanoDrop 1000 spectrophotometer (Thermo Scientific).In vitro-transcribed HEV-3ra RNAs were used to generate a standard curve in each RT-qPCR run, which covered a quantification range from 4 × 102 to 4 × 107 RNA copies per reaction. The total RNA extracted from each of the tissue samples was diluted at 250 ng/μL, thus allowing the use of 1 μg total RNA (4 μL)/reaction as recommended in the kit. The PCR quantification data were then used to calculate the viral RNA loads as genome copies per gram of tissue. Fold increase in viral loads in pregnant rabbits (HEV-P) compared to nonpregnant rabbits (HEV-NP) was calculated as follows: (HEV RNA copies in HEV-P − HEV RNA copies in HEV-NP)/HEV RNA copies in HEV-NP.HEV-3ra genomic RNA was quantified using SensiFAST probe No-ROX one-step kit (Bioline USA Inc.) with primers and probe listed in RPS18) was used as a housekeeping gene to calculate the normalized value of threshold cycle (ΔCT) for target genes in each rabbit. Cytokine gene mRNA expression levels were normalized in each infected rabbit as follows: ΔΔCT of HEV-P (or -NP) rabbit = ΔCT of HEV-P (or -NP) rabbit/mean ΔCT of PBS-P (or -NP) group. The same formula was also followed for negative-control rabbits as follows: ΔΔCT of PBS-P (or -NP) rabbit = ΔCT of PBS-P (or -NP) rabbit/mean ΔCT of PBS-P (or -NP) group. The 2−ΔΔCT value was then calculated, which reflects the fold change in gene expression in infected rabbit; this value in the control group is expected to be 1.0 but may become higher depending on individual variability within the control group. The fold change in cytokine gene expression after normalization between different groups was calculated as follows: /normalized cytokine gene expression level in group B = fold increase or decrease in group A compared to group B.Two-step RT-qPCR assays were developed and optimized to quantify the gene expression levels of a panel of inflammatory cytokines in liver tissues as well as the pregnancy hormone genes in liver and placenta tissues. Briefly, cDNA was synthesized from total RNA extracted from liver and placenta tissues, using high-capacity cDNA reverse transcription kit  and random primers according to the manufacturer’s instructions. The cDNAs were then used to quantify the mRNA expression levels of cytokine and pregnancy hormone genes by using SensiFAST SYBR No-ROX kit (Bioline USA Inc.) and rabbit gene-specific primers  designedThe levels of total bilirubin (TBB) and alkaline phosphatase (ALP) in weekly serum samples collected from each rabbit at different time points (0 to 28 dpi) were measured using commercial kits (MyBioSource), rabbit TBB competitive ELISA  and rabbit ALP ELISA  kits, respectively, according to the manufacturer’s instructions.A panel of cytokines were assayed in weekly serum samples of each rabbit by using the following rabbit-specific commercial ELISA kits: IL-2, IL-6, and IL-12  and IFN-γ , following the manufacturers’ instructions. The serum levels of pregnancy hormones estrone (E1) and estradiol (E2) in each rabbit were determined using ELISA kits . The fold change in serum cytokine level between different groups was calculated as follows: (serum cytokine concentration in group A − serum cytokine concentration in group B)/serum cytokine concentration in group B = fold increase or decrease in group A compared to group B.t test or ordinary two-way ANOVA followed by Tukey’s multiple-comparison test. Some serum samples at some time points had undetectable levels of cytokines, liver enzymes, or hormones and thus were not included in statistical analyses. In this case, data were analyzed by mixed-effects model (restricted maximum likelihood [REML]), followed by Tukey’s test. Statistical correlations were calculated with Pearson’s correlation coefficient test. A P value of <0.05 was considered statistically significant.All data were analyzed with GraphPad Prism 9.3.1 (GraphPad Software Inc.). The differences between the mean values of two treatment groups were analyzed by two-tailed unpaired Student's


Although Ca2+ depletion has been known to cause ER stress which in turn activates the unfolded protein response (UPR), how UPR sensors/transducers respond to excess Ca2+ when ER stores are overloaded remain largely unclear.Maintaining homeostasis of Ca2+ can directly sensitize the IRE1α-XBP1 axis. The overloaded ER Ca2+ in TMCO1-deficient cells can cause BiP dissociation from IRE1α, promote the dimerization and stability of the IRE1α protein, and boost IRE1α activation. Intriguingly, attenuation of the over-activated IRE1α-XBP1s signaling by a IRE1α inhibitor can cause a significant cell death in TMCO1-deficient cells.Here, we report for the first time that overloading of ER Ca2+ in ER stores and the selective activation of IRE1α-XBP1 axis, underscoring an unexpected role of overload of ER Ca2+ in IRE1α activation and in preventing cell death.Our data establish a causal link between excess CaThe online version contains supplementary material available at 10.1186/s13578-023-01062-y. EnvironR stress . To adapR stress –8. UnderR stress , 9.IRE1α is the most conserved and extensively studied UPR sensor protein , 11, whi2+ with the sarco-/endoplasmic-reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin (TG) can lead to ER dysfunction and to activate the UPR [2+ binding potential of many chaperones, such as BiP, GRP94, and calreticulin, which act as buffer proteins for maintaining the ER Ca2+ homeostasis, while Ca2+ in turn is also important for the activity of these chaperones [2+ depletion could significantly affect protein processing and then lead to the accumulation of unfolded proteins in the ER [2+ depletion-triggered accumulation of unfolded proteins can compete to bind BiP and thus to activate the UPR pathway [It is known that depletion of ER Ca the UPR . To main the UPR . Previouaperones , 21. ER n the ER –24. Mostn the ER , 26, whi pathway , 25.2+ overload happens in multiple diseases including Alzheimer’s disease [2+ stores may have therapeutic potential for treating these diseases. However, whether and how UPR sensors/transducers respond to the excess Ca2+ when ER stores are overloaded remains unclear. As an ER Ca2+ load-activated Ca2+ channel (CLAC), transmembrane and coiled-coil domains 1 (TMCO1) is important for maintaining ER Ca2+ homeostasis [2+ overload. We and others have reported that TMCO1 deficiency can cause cerebrofaciothoracic dysplasia (CFTD) [2+-dependent manner. Moreover, the ER Ca2+ overfilling signaling directly sensitizes the IRE1α-XBP1 axis, which is essential for modulating cell fate and helping cell survival.ER Ca disease –32, targeostasis , whose da (CFTD) , defectsa (CFTD)  and defea (CFTD) . In thisEdem1, a classical XBP1s target gene  of TMCO1 could increase the expression of IRE1α and promote IRE1α overactivation and XBP1 splicing  and KD HeLa cells with an IRE1α inhibitor KIRA6 . We founTo further support that the overactivation of IRE1α in TMCO1 KD cells is indeed caused by TMCO1 depletion, a TMCO1-IRES-mCherry construct was transfected back into the TMCO1 KD cells. We found that expressing TMCO1-IRES-mCherry but not mCherry in KD cells could attenuate the increase in IRE1α expression and its phosphorylation , TG or DTT, in the presence or absence of BAPTA-AM. We found that treatments with these ER stressors could significantly increase the mRNA  and carbachol (Cch), were used to treat WT and KD cells, and then the cytosolic Ca2+ was measured by the Ca2+ indicator fura-2. Significantly higher amplitudes of Ca2+ transients were detected in KD cells than in WT cells upon treatment with these Ca2+ inducers  [2+ store.Although TM, DTT and TG treatment could sensitize the IRE1α-XBP1 pathway in a Canal Ca2+ , but notnal Ca2+ , caused  (miGer)  in KD ce2+ overfilling in the activation of IRE1α. Based on our previous results [2+ permeation function was constructed. We found that the increased Ca2+ release after TG treatment in KD cells could be rescued by the expression of WT TMCO1 but not the D140A mutant, demonstrating that the regulation of Ca2+ signaling by TMCO1 depends on its Ca2+ permeability  of IRE1α is required for IRE1α activation [2+ binding [We next investigated how the overload of ER Cativation , 50. Contivation , deletio binding , 53 rema2+ overload could affect the interaction between BiP and IRE1α. Previous studies have reported that the association between BiP and IRE1α is mediated by the nucleotide binding domain in BiP and the LD of IRE1α in a nucleotides-independent manner [2+. As the estimated Ca2+ concentration in the ER lumen is around 0.5 to 0.8 mM in the resting state [2+, that is, 0.5, 0.8 and 1.2 mM, were used in the pull-down assay. Our result showed that the interaction between BiP-NBD and IRE1α-LD could only be disrupted by 1.2 mM Ca2+ but not by 0.8/0.5 mM Ca2+ , ER-resident Ca2+ transporters, and Ca2+ channels, including ryanodine receptors (RyRs), ER-resident inositol 1,4,5-trisphosphate receptor (IP3Rs), and SERCA [2+ depletion caused by SERCA inhibitors, such as TG, can trigger a robust activation of ER stress [2+ homeostasis include not only Ca2+ depletion but also Ca2+ overfilling, with the latter being also an important ER Ca2+ homeostatic feature. However, it is still unclear whether ER Ca2+ overload induces ER stress. We previously reported that deficiency of TMCO1 in mouse granulosa cells could activate ER stress mainly mediated by the activities of the IRE1α pathway [2+ overload. Here in this study, using in vitro and in vivo mouse model of ER stress, we have demonstrated that the overloading ER Ca2+ caused by TMCO1 depletion directly sensitizes the IRE1α-XBP1 axis. Therefore, our findings underscore an unexpected role of overload of ER Ca2+ in ER stress regulation.Maintaining homeostasis of Cand SERCA –75. It iR stress , 77. Of  pathway , it is s2+-binding sites, whose occupancy is likely decreased upon ER Ca2+ depletion within the physiological range [2+ depletion is expected to favor the dissociation of BiP from UPR sensors [2+ depletion activates UPR sensors through accumulation of unfolded proteins in the ER, activation of UPR sensors by ER Ca2+ overload is more related to its function to directly disrupt the BiP-IRE1α interaction  disrupts BiP interaction with IRE1α  was generated in Shanghai Model Organisms Center, Inc. , as described previously [Tmco1+/+ (C57BL/6 J) or Tmco1−/− mice aged 8 weeks were used for all experiments. Mice were housed in a temperature-controlled environment with a 12-h light/dark cycle and free access to water and feed. All animal experiments were approved by the Institute of Zoology Institutional Animal Care and Use Committee.The eviously . The mal2 environment. TMCO1 KD HeLa cells were described previously [HeLa cells, TMCO1 KD HeLa cells and HEK-293 T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C in a 5% COeviously .UPRE-mCherry reporter has been previously described, which occurred subsequent to endogenous XBP1 splicing . The UPRFor lentivirus production, HEK-293 T cells were co-transfected with lentivirus vector and packaging plasmids psPAX2 and pMD.2G using PEI. Following 48–72 h of transfection, lentivirus particles were collected for experiments.™ Reverse Transcription System . Quantitative real-time PCR reactions for target genes were performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix . Gapdh was used as a normalization control for mRNA. The primer sequences are shown in Reagents and Tools Table.Total RNA from cells or tissues was extracted by TRIzol Reagent . RNA (1 μg) was reverse-transcribed with GoStriptCells or tissues were harvested, washed with phosphate-buffered saline (PBS) and lysed on ice with RIPA buffer , and 0.5% sodium deoxycholate) containing 50 mM DTT, 1 mM EDTA, 1 mM PMSF, 1 mM NaF and protease inhibitor cocktail (Roche). Then, lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. The total protein was separated by SDS‒PAGE and incubated with primary antibodies for 1 h at room temperature and then at 4 °C overnight. Finally, secondary antibodies conjugated with horseradish peroxidase were incubated for 2 h at room temperature. The bands were visualized by chemiluminescence (Millipore). Density measurements of bands were performed in ImageJ.Cells were treated with 25 μg/ml CHX and harvested at the indicated time points. Lysates were analyzed by 10% SDS‒PAGE followed by immunoblotting.SUnSET assay was performed as described previously . Cells wCells were lysed in NETN buffer  containing protease inhibitor cocktail (Roche). After incubation for 1 h at 4 °C, the total cell lysates were centrifuged at 4 °C. For pulldown of FLAG-tagged proteins, the cell supernatant was incubated with M2 beads (Sigma‒Aldrich) at 4 °C overnight with a rotating mixer. The next day, the beads were washed with NETN buffer three times. Samples were separated by 10% SDS‒PAGE and analyzed by immunoblotting.For IRE1α-LD-FLAG protein purification, a total of three 10-cm dishes of HEK-293 T cells expressing IRE1α-LD-FLAG were lysed in NETN buffer containing protease inhibitor cocktail for 45 min at 4 °C. The total cell lysates were centrifuged at 4 °C, and the cell supernatants were incubated with M2 beads (Sigma‒Aldrich) overnight at 4 °C with a rotating mixer. The beads were washed with NETN buffer, and then the IRE1α-LD-FLAG proteins were eluted with Flag peptide in buffer B . The eluents were concentrated and purified with a centrifugal ultrafiltration tube . Finally, the concentration of purified protein was determined by NanoDrop Spectrophotometer (Thermo-Fisher Scientific).2+ at room temperature for 2 h. After that, the beads were washed with buffer B. And then, IRE1α-LD-FLAG was analyzed by anti-FLAG immunoblotting. Bacterially purified BiP-NBD proteins were visualized by Ponceau S staining.For the pulldown assay between GST-BiP-NBD (residues 28–405)  and IRE12+ measurement was performed as described previously [2+ content in the ER, cells were loaded with 4 µM Fura-2 AM  and 0.02% pluronic F-127  in ECS  for 40 min at room temperature in the dark. After three washes with ECS, the cells were incubated for another 10 min in ECS buffer for de-esterification. TG (1 μM) was applied to release Ca2+ from the ER in Ca2+-free Hank's balanced salt solutions: 140 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, and 1 mM MgCl2, pH 7.4. Different Ca2+ release inducers were applied in the same manner as TG. Ca2+ imaging was taken every 3 s with a Nikon inverted microscope (Eclipse TiE) with a 40 × magnification oil-immersion objective by alternatively excitation at 340 nm and 380 nm. Single-cell Fura-2 images were captured and processed by Metamorph software (version 7.0). The Ca2+ level was determined by the 340/380 fluorescence ratio after the Ca2+ image background was subtracted. The ER Ca2+ content was calculated by the area under the curve induced by the indicated reagents that induce Ca2+ release.Intracellular Caeviously . To meast test and one-way ANOVA followed by Tukey’s multiple comparison test were used for statistical significance analysis. All statistical tests were performed by GraphPad Prism 8.Data are shown as the mean ± SEM, and two tailed unpaired Student’s Additional file 1. TMCO1 KD leads to the over-activation of IRE1α and regulates RIDD. Related to Fig. P < 0.01, n.s., no significance. (b-d) qRT-PCR analysis of Bip (b), Chop (c) and SPARC (d) mRNA levels in WT or TMCO1 KD cells. Data are shown as mean ± SEM from three independent experiments. n.s., no significance, *** P < 0.001. (e) qRT-PCR analysis of XBP1 mRNA splicing in WT or TMCO1 KD cells treated with 1 μM TG for the indicated times. Bar graphs represent the mean ± SEM from three independent assays. *P < 0.05; **P < 0.01, n.s., no significance. (f) Western blotting analysis of mCherry and XBP1s levels in the HEK-293 T expressing UPRE-mCherry treated with 1 μM TG along with/without 50 μM 4μ8C for 6 h. GAPDH was used as a loading control. Quantification of the relative protein levels are shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001, n.s., no significance. (g) Western blotting analysis of TMCO1 levels after TMCO1 KD in the HEK-293 T. GAPDH was used as a loading control. Relative quantification of each protein was shown in the down panel. Bar graphs represent the mean ± SEM from three independent experiments. *P < 0.05; ***P < 0.001. (h) 1 μM TG-triggered Ca2+ transients in WT  and TMCO1 KD  HEK-293 T cells. Each trace line in H is an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of TG-triggered Ca2+ mobilization curves. ***P < 0.001. (i) IRE1α, XBP1s, TMCO1 levels were analyzed by immunoblotting in the WT or TMCO1 KD HEK-293 T cells. GAPDH was used as a loading control. Relative quantification of each protein was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. **P < 0.01; ***P < 0.001.Additional file 2. Effects of TM and DTT on the ER Ca2+ transients. Related to Fig. P < 0.01. (b) 3 μg/ml TM or 1 μM TG-triggered Ca2+ transients in WT  and TMCO1 KD  cells. Each trace line in B is an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of TG-triggered Ca2+ mobilization curves. ***P < 0.001. (c) 5 mM DTT or 1 μM TG-triggered Ca2+ transients in WT  and TMCO1 KD  cells. Each trace line in C is an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of TG-triggered Ca2+ mobilization curves. ***P < 0.001.Additional file 3. TMCO1 knockdown leads to the overload of ER Ca2+ store. Related to Fig. 2+ transients in WT  and TMCO1 KD  HeLa cells. Each trace line is an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of Cch-triggered Ca2+ mobilization curves. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. (b) 1 μM iono-triggered Ca2+ transients in WT  and TMCO1 KD  HeLa cells. Each trace line is an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of iono-triggered Ca2+ mobilization curves. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. (c) 100 μM ATP-triggered Ca2+ transients in WT , TMCO1 KD  cells. Each trace line was an average of Ca2+ responses in each group. Right panel, statistical analysis of the average peak area of ATP-triggered Ca2+ mobilization curves. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. (d) The resting G-CEPIA1er fluorescence ratio signals were detected in WT (n = 454) or TMCO1 KD (n = 540) cells. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001.Additional file 4. TMCO1 KD activates IRE1α to prevent cell death. Related to Fig. P < 0.01, ***P < 0.001. (b) Western blotting analysis and quantification of proteins in WT mice injected with TM for the indicated times. GAPDH was used as a loading control. Data represent as the mean protein intensity normalized to GAPDH ± SEM from 3 independent experiments. Each point represents independent animal. *P < 0.05, n.s., no significance. (c) Picrosirius red staining of liver tissues from Tmco1+/+and Tmco1−/− mice injected with TM (magnification × 200). Scale bar, 100 μm. Three animals per group were analyzed. (d) Western blotting analyses of proteins extracted from liver tissues of Tmco1−/− mice and Tmco1+/+ mice at 8 months of age (three mice per group). GAPDH is used as a loading control. Data represent as the mean protein intensity normalized to GAPDH ± SEM from 3 independent experiments. **P < 0.01.Additional file 5: Table S1. The sources of antibodies used in this study. Table S2. The sources of plasmids used in this study. Table S3. The sources of primers used in this study. Table S4. The sources of chemicals used in this study.


Here, we critically examine the biophysical properties of jGCaMP8f and report development by rational design of two novel variants of jGCaMP8f.Genetically encoded biosensors for monitoring intracellular calcium changes have advanced our understanding of cell signaling and neuronal activity patterns in health and disease. Successful application of GCaMP biosensors to a wide range of biological questions requires that sensor properties such as brightness and dynamic range, ligand affinity and response kinetics be tuned to the specific conditions or phenomena to be investigated. Random as well as rational targeted mutations of such sensor molecules have led to a number of important breakthroughs in this field, including the calcium sensors GCaMP6f and GCaMP6fin vitro biophysical properties of jGCaMP8f and selected variants by fluorescence spectroscopies and compared their performance monitoring intracellular Ca2+ transients with previously developed fast and bright GCaMP sensors by live cell imaging.We determined the 2+ not only majorly affects the kinetic responses of GCaMPs but also their brightness and fluorescence dynamic range. We developed novel variants jGCaMP8f L27A which has threefold faster off-kinetics and jGCaMP8f F366H which shows a ∼3-fold greater dynamic range than jGCaMP8f, in vitro as well as in HEK293T cells and endothelial cell line HUVEC in response to ATP stimulation.We demonstrate that the physiologically highly relevant Mgin vivo imaging outcomes with increased signal-to-noise ratio. The L27A variant with faster kinetics than jGCaMP8f has larger cellular responses than previous fast GCaMP variants. The jGCaMP8f generation and novel improved variants presented here will further increase the application potential of GECIs in health and disease.We discuss the importance of optimization of biosensors for studying neurobiology in the context of the novel variants of jGCaMP8f. The jGCaMP8f F366H variant with a large dynamic range has the potential to improve  Fluorescence intensity increases as a result of a conformational change in the CaM-RS20-cpEGFP complex (2+ binding)  firing rate at the presynaptic terminals where GCaMPs were initially introduced  . The restroduced . Faster  peptide . The taroff-kinetics than GCaMP6f. We measured the biophysical properties of jGCaMP8f in order to determine how targeted mutations affect them. We report novel variants L27A and F366H with further increased off-rate and fluorescence dynamic range, respectively, demonstrated in vitro and in cellular environments.jGCaMP8f  is a memE. coli XL10-Gold and BL21 (DE3) Gold cells were purchased from Invitrogen. Subsequently, jGCaMP8f was cloned into a bacterial expression vector (jGCaMP8f pET30b) using DNA assembly .pGP-AAV-syn-FLEX-jGCaMP8f-WPRE was purchased from Addgene (Plasmid #162379). pET30b vectors were obtained from Novagen. ® Site-Directed Mutagenesis Kit (New England Biolabs), a range of mutations of jGCaMP8f pET30b were generated. Primers (5′-3′) used for mutagenesis were:Using Q5A18S: CAAGGAGGTGAGCACCGCCGTGL27A: CATCGCCCGCGCAATGGGCCTGAAGD294A (EF-1): CTCCCTATTTGCCAAGGACGGGGD330A (EF-2): AATGAAGTAGCTGCCGACGGTGF366A: GTTCGGTGTGGCTGATAAGGATGF366H: GTTCGGTGTGCATGATAAGGATGD367A (EF-3): GGTGTGTTTGCTAAGGATGGCD403A (EF-4): AGGGAAGCAGCCATCGATGGGAfter mutagenesis, DNA sequences were sent to Genewiz for checking.E. coli B21 Gold cells using 0.5 mM isopropyl thio-β-D-galactoside (IPTG) then purified on a NiNTA column  at 4 °C as described previously .Protein concentration was calculated from the absorption spectra of the proteins at wavelength 280 nm using the molar extinction coefficient of 26,360 M+, 100 mM KCl and 5 mM EGTA in the presence or absence of 2 mM MgCl2, pH 7.5) at 20°C were recorded using a Fluorolog-3 spectrofluorometer (Horiba©) at 492 nm excitation and 500 – 550 nm emission wavelengths. Subsequently, a saturated amount of CaCl2 was added to obtain the maximum fluorescence emission intensity. Data were normalized and plotted using GraphPad Prism 9 software.Fluorescence emission spectra of 0.5–1 μM jGCaMP8f or variant proteins in assay buffer  and the fluorescence spectra were recorded on a Fluorolog-3 (Horiba©). GCaMP6f quantum yield measured in Ca2+ -saturated buffer was used as a reference (ΦCa+2+ = 0.59) (protein = ΦGCaMP6f × (Sprotein/SGCaMP6f).The concentration of jGCaMP8f proteins was adjusted such that the absorbance at the excitation wavelength (492 nm) was between 0.001 and 0.1. A series of dilutions was prepared in a buffered solution  . Data weKa for each jGCaMP8f protein, a series of buffers were prepared. Depending on their respective pH buffering range, an appropriate buffer was used for the measurements . The pH titrations were performed by recording fluorescence spectra in Ca2+ -free  or Ca2+-saturated  measuring 1 μM protein at 0.5 pH unit intervals at 20°C . Excitation wavelength was set at 492 nm and emission in the 500–600 nm range was recorded. BAPTA was chosen as Ca2+ chelator because of its stable affinity for Ca2+ over the pH range.To determine the apparent p2+ association and dissociation rates were measured using a TgK Scientific KinetAsyst™ double-mixing stopped-flow apparatus in single mixing mode at 20°C. The fluorescence was excited at 492 nm and the emission was collected using a 530 nm cut-off filter. For Ca2+ association, 500 nM jGCaMP8f or variant proteins in 50 mM HEPES-K+, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, pH 7.5 at 20°C were rapidly mixed with increasing concentrations of Ca2+ , calculated free [Ca2+] ranged from 108 nM to 252 μM in the mixing chamber). For Ca2+ dissociation, 500 nM jGCaMP8f or variant proteins in 50 mM HEPES-K+, 100 mM KCl, 2 mM MgCl2, 200 μM CaCl2, pH 7.5 at 20°C were rapidly mixed with 50 mM HEPES-K+, 100 mM KCl, 2 mM MgCl2, 10 mM EGTA, pH 7.5 at 20°C. Experiments were repeated at least three times. Kinetic records shown are the average of at least four traces. Averaged data were fitted to a single or double exponential using KinetAsyst software (TgK Scientific) to obtain association or dissociation rates. The error quoted is the standard error of the fit. For the rate plots, the mean values obtained from at least three experiments are shown, the error bars represent S.E.M.Ca2+ concentration [Ca2+] was calculated using the two-chelators Maxchelator program  was used to verify the calculated [Ca2+].Free Ca program . Fluo-3 ©) at specific excitation and emission wavelengths, 20°C. When bound to Ca2+, the Fluo-3 excitation peak is at 506 nm and the emission peak is at 526 nm; the respective wavelengths for jGCaMP8f and all variant proteins are 492 nm and 512 nm. One μM jGCaMP8f or variant proteins in assay buffer  were titrated by stepwise addition of a solution of 500 mM CaCl2 until no further changes in fluorescence emission intensity were observed. Experiments were done in triplicates. Data was corrected for dilution, normalized and expressed as mean ± S.E.M. Kd and Hill coefficient for indication of cooperativity (n) were obtained by fitting the data to the Hill equation using GraphPad Prism 9 software.Fluorescence emission spectra were recorded using a Fluorolog-3 spectrofluorometer  at 300,000 cells per dish for 24 h. Cryopreserved pooled primary human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell GmbH (Heidelberg Germany). HUVEC were grown in a human growth medium (HGM) which comprised media M199 supplemented with 20% FCS. A total of 50 μg/mL of the antibiotic gentamicin along with ECGS  and heparin (10 U/mL) were added to the media. HUVEC were grown on 24 well trays or 9 mm glass coverslips that had been pre-treated with porcine skin gelatine (1% w/v) for 30 min at 37°C. Cells were cultured for 4 days in a 5% CO2 incubator at 37 °C until fully confluent as assessed by visual examination by light microscopy.HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Media  supplemented with 10% fetal bovine serum and 4 mM L-glutamine, in 75 cmTransfection of HEK293T was accomplished using Lipofectamine™ 2000 (Invitrogen): 2.5 μg of plasmid DNA was combined with 5 μL Lipofectamine™ 2000 reagent in Opti-MEM, this was then added to the 2 mL cell culture medium per dish. Transfection of HUVEC was by Nucleofection using an Amaxa Nucleofector 2b device and the HUVEC old transfection reagent  according to manufactures. Experiments were performed 24 h after transfection.2, 1 mM MgCl2, 1 mM NaH2PO4, 10 mM glucose, 20 mM Na+-HEPES pH 7.4) and placed on the stage of an inverted Olympus IX71 fluorescence microscope. The microscope was equipped with an Olympus UPLSAPO × 100 1.40 NA objective and an OptoLED light source  for EGFP (470 ± 20 nm) fluorescence. Excitation light was directed to the specimen via a 500DCXR dichroic mirror  and emitted light collected through a 535 ± 30 nm emission filter onto an Ixon3 EMCCD camera  operated in frame transfer mode at full gain and cooled to −68 °C. Images were acquired at 20 frames/s using Winfluor software.1 Intracellular Ca2+ release in HEK293T cells was induced by ATP. ATP was administered as a bolus of 50 μL diluted in imaging buffer to give a final concentration of 50 μM in HEK293T cells and 2 μM for HUVEC. The higher sensitivity of HUVEC to ATP is reflected in the different ATP concentrations used. Full dynamic range was measured following the addition of 10 μM ionomycin. The fluorescence changes during ATP stimulation were analyzed using Winflour software. A square region of interest (ROI) was placed over a peripheral cytoplasmic region of each cell. The mean background fluorescence was measured by placing an ROI with the same dimensions at a point at which no cell was present. GraphPad Prism 9 was used to plot and fit data. Time-to-peak was determined by the time in which the rise amplitude increased from 10 to 90%. Decay t1/2 was determined from an exponential function fitted for individual records when possible, or by determining the time required for the signal dropping to half the amplitude. Data are expressed as mean ± S.E.M. For further statistical analysis one-way ANOVA tests were carried out with post-hoc Dunnett’s multiple comparison test. Different number of data points could be extracted for the different parameters due to the variability of individual cell responses. For example, following a clear rising phase, the decay phase may include oscillations or plateaus that complicate fitting and analysis. In some cells when ionomycin was added, no clear response was seen.Transfected HEK293T cells or HUVEC were transferred to a physiological saline solution   . The relD: 7ST4) .We generated variants by introducing mutations in the CaM EF-hands and selected sites in the eNOS peptide . Mutatio2+ binding sites of CaM and/or in the eNOS peptide of jGCaMP8f will weaken the binding affinity resulting in faster calcium off-kinetics  ] . In desi binding , was exp2+/CaM – eNOS peptide complex may increase the brightness and fluorescence dynamic range . The F366 residue in CaM is adjacent to EF-3 and is involved in hydrophobic interactions with the peptide. We designed two mutations, F366H and F366A, with the view of reducing hydrophobicity and size to increase dynamic range.In turn, mutations that stabilize the Ca2+ affinity and kinetic responses of the variants in comparison with jGCaMP8f. We furthermore tested the response of jGCaMP8f, its novel variants and previously generated fast GCaMP variants to ATP stimulation in HEK293T cells and HUVEC.We were interested to see how these mutations affected the brightness and dynamic range, apparent Ca2+ binding and an off-rate of 37 s–1 at 20°C (2+ and at pH 7.2 as opposed to our buffer that contains 2 mM Mg2+ and is adjusted to pH 7.5. Remeasuring the dynamic range of jGCaMP8f in the absence of Mg2+ (pH 7.5), we got a remarkably different result: the fluorescence of jGCaMP8f increased 31-fold upon Ca2+ binding. We then compared fluorescence intensities with or without added Mg2+ in the presence of EGTA and in the presence of Ca2+ , we obtained a 42-fold and a 37-fold increase upon Ca2+ binding, respectively, compared to 31-fold for jGCaMP8f ] in saturating Ca2+ of jGCaMP8f, 54,226 M–1cm–1 and a quantum yield of 0.52 gave a brightness value of 28.3 mM–1cm–1. This compares well with 29.7 mM–1cm–1 measured for GCaMP6f in the absence of Mg2+  48,107 M–1cm–1 (quantum yield 0.54) and F366H, εo(492 nm) 21,203 M–1s–1 (quantum yield 0.53) suggest that lower brightness of the apo-form rather than increased brightness of the Ca2+-bound form of the F366H variant underlies its increased fluorescence dynamic range compared to jGCaMP8f. In contrast, for the L27A variant, the Mg2+ induced large increase in fluorescence intensity in the apo form accounts for the loss in dynamic range. Neither fluorescence intensity nor εo(492 nm) is affected by Mg2+ in saturating Ca2+. The only available data for comparison of the apo form with and without Mg2+ is for jGCaMP8f (2+ εo(492 nm) is 8,413 M–1cm–1, in the absence of Mg2+it is reduced to 1,930 M–1cm–1, consistent with the 4-fold increase in fluorescence intensity by Mg2+ in the absence of Ca2+  present revealed that for jGCaMP8f and the L27A and F366H variant, the intensities increased for both the Ca2+-bound and apo-forms, the dynamic ranges decreased with increasing pH, peaking at pH 7 , R1 , R1 . With anas 22 ms .–1) initial on-rate (R1) (off-rate of the L27A variant was 84.4 s–1 (decay t1/2 of 8.2 ms)  . The off 8.2 ms)  threefol–1 initial on-rates (R1) measurable in the μM [Ca2+] range  and D403A (EF-4) mutations had very little effect on either the kinetic or fluorescence responses of jGCaMP8f (off-rate (25 s–1) than jGCaMP8f , D330A (EF-2), and F366H variants were studied.Measurement of the Ca2+ dependence of the four phases of the on-fluorescence response of jGCaMP8f and its L27A, D294A (EF-1), D330A (EF-2), and F366H variants, revealing that rates (R1 ↑) of the fast, rising phase  are highest at around 1–2.5 μM [Ca2+] for all five proteins  of 5 s–1 which increases to ∼30 s–1 in the 1–2.5 μM [Ca2+] range for the F366H variant, while only at high [Ca2+] for the other four proteins (kobs for the fourth phase (R4 ↑) is slow ∼1 s–1  shows that for the jGCaMP8f, the L27A and F366H variants the rapid first phase represents the major fraction of the amplitude . In cont ∼200 ms . Therefo 3 and 4 . The lowKd for Ca2+ in the low 1–4 μM range with Hill coefficient (n) values of 2–5 . The F366H variant responded with a ΔF/Fo of 9 (ΔF/Fo measured in response to 2 μM ionomycin was 12), while for L27A the ΔF/Fo was 1 (ΔF/Fo measured in response to 2 μM ionomycin was 2.8). Time to peak values were 1.4 s, 1.8 s and 1 s for jGCaMP8f and its F366H and L27A variants, respectively. Thus, the on-kinetic responses of the three proteins were similar, however, the decay t1/2 for the L27A variant of 4.6 s was 2.3-fold smaller than for jGCaMP8f (10 s) and F366H variant (11 s). Moreover, the amplitude of fluorescence increase for jGCaMP8f F366H was 3.75-fold greater than for jGCaMP8f . Previously, GCaMP3fast and GCaMP6fu were developed which had in vitro decay t1/2 of 11 and 8 ms, respectively, at 20°C  for GCaMP3fast and 0.44 (p = 0.003) for GCaMP6fu (p = 0.0009) and 3.5-fold (p < 0.0001) greater ΔF/Fo than jGCaMP8f. The full dynamic ranges (ΔF/Fo measured in response to 10 μM ionomycin) of the sensors were similar with, interestingly, GCaMP6f exceeding even that of jGCaMP8f F366H (p = 0.002)  ; Table 32+ binding to CaM and hence its interaction with target peptides and proteins is affected by Mg2+ and Mg2+ is also present in the cells, our measurements are carried out in a buffer containing 2 mM MgCl2 and found striking differences in the dynamic ranges when comparing results obtained in the absence of Mg2+. jGCaMP8f is reported to have a significantly greater fluorescence dynamic range than the GCaMP6 generation, in comparable conditions, in the absence of Mg2+ at pH 7.2  of ≥500 s–1 likely represents Ca2+ binding to the CaM EF-1 and EF-2 sites of the N-lobe, the transient drop of fluorescence in the second phase (R2) then corresponds to Ca2+dissociation from the N-lobe as the [Ca2+] is not sufficient for saturating N-lobe Ca2+ binding. Slow Ca2+ binding to the C-lobe follows in phase 3 (R3) and the binding is stabilized in phase 4 (R4). At [Ca2+] > 2.5 μM, the N-lobe can hold onto Ca2+ better and the second phase is no longer detectable. Thus, fluorescence rapidly increases in a more straightforward manner. Our data with the D294A (EF-1) and D330A) (EF-2) variants, which only show the slow 3rd and 4th phases, support this explanation.The complex on-rates and amplitudes is favorable for the L27A and F366H variants with fast initial on-rates (R1) of ≥500 s–1 in the physiologically relevant 1–2.5 μM intracellular [Ca2+], together with jGCaMP8f. The on-rate of jGCaMP8f and its L27A and F366H variants is 3.4 and 1.5-fold faster than that the fast decay GCaMP3fast and GCaMP6fu, respectively, at 20°C  involved in peptide binding to the CaM C-lobe. In contrast, in jGCaMP8f EF-3 or the EF-4 mutation that disabled Ca2+ binding did not significantly affect the properties of jGCaMP8f, while mutating EF-1 and EF-2 resulted in very fast off-rates but drastically slowed-down on-response times, implicating their important role in binding the eNOS peptide which is essential for restoring fluorescence of the cp-EGFP β barrel.jGCaMP8f and the F366H variant have a 13-fold faster GCaMP6fu , 2016. Nfast and GCaMP6fu variants, in each case reduced compared to the parent GCaMP. In contrast, jGCaMP8f F366H shows a 25-fold fluorescence enhancement upon Ca2+ binding, 3-fold greater than jGCaMP8f in our hands (in the presence of Mg2+), but similar off-rate to jGCaMP8f. Thus, simultaneously increasing brightness, dynamic range and off-rate of GCaMPs remains elusive.The fluorescence dynamic range of ∼5 is similar for the fast-decay jGCaMP8f L27A, GCaMPfin vitro. However, response to cell [Ca2+] transients is not always entirely consistent with what the in vitro experiments predict. There are many possible reasons for this, for example there can be affinity differences in the cells compared to what is measured in solution, pH changes can affect sensor responses.The greater amplitude (3.75-fold in HEK293T and 1.9-fold in HUVEC) of the response to ATP stimulation by our F366H variant compared to jGCaMP8f is consistent with the 3-fold difference we measured 2+ transients of different magnitude and time course. Our cell data show the expected range of response times and amplitudes, similar to other reports of GCaMP cell responses , both in vitro and in cellular environments. The off-kinetics of L27A is as fast as that of GCaMP3fast and GCaMP6fu, however, the jGCaMP8f L27A variant may be more advantageous in imaging applications as it has a greater fluorescence dynamic range in the cellular environment than the earlier fast variants. The jGCaMP8f F366H variant gives a 2–3.75-fold greater fluorescence increase in cells than jGCaMP8f whilst preserving its fast kinetics unless saturation distorts its response kinetics.In summary, we present a thorough characterization of jGCaMP8f and two newly developed variants, the faster decay L27A and F366H with an increased dynamic range, through which we highlight the complexities of the kinetics of this sensor generation. We demonstrate how Mg2+ indicators are highly applicable for studying cell signaling involving transient rises of intracellular [Ca2+] to understand physiological or disease conditions. An important consideration for the application for GECIs for imaging neural activity is the kinetics of the intracellular [Ca2+] transient generated by AP-evoked Ca2+ influx and terminated by Ca2+ pumps, which is slower than the ion channel activities driving APs. In cells, the millisecond response times of GECIs translate into transients over seconds. Nevertheless, increasing the off-rate of GCaMPs is important for faithful reporting of intracellular Ca2+ transients and for increasing the resolution of oscillating Ca2+ transients. However, there are still limitations to the sensitivity and fidelity of detection. It is important to understand how closely the fluorescence changes map the neural activity under investigation. We note that many in vivo or in cell studies cannot give detailed kinetic data of Ca2+ signals. This may be explained by the low expression level and/or low brightness of GECIs in vivo, their action as a Ca2+ buffer, slow kinetics, the complexity arising from the non-linearity of the data , respectively, therefore have potential applications to study physiological or disease conditions. For example, simultaneous recording of APs and GCaMP6s or GCaMP6f fluorescence in vivo in the primary visual cortex of transgenic mice revealed that in typical population imaging conditions only ∼10% of single AP events were detected , D330A (EF-2), A18S as well as designed, generated the F366A and F366H variants by SDM, and carried out all This work was funded by BBSRC grant BB/M02556X/1 to KT.


Demonstrably, auxin-activated proton pumps, AGPs, Ca2+ channels, and auxin efflux “PIN” proteins, embedded in the plasma membrane, combine to generate cytosolic Ca2+ oscillations that ultimately regulate oscillatory growth: Hechtian adhesion of the plasma membrane to the cell wall and auxin-activated proton pumps trigger the release of dynamic Ca2+ stored in periplasmic AGP monolayers. These four major components represent a molecular PINball machine a strong visual metaphor that also recognises auxin efflux “PIN” proteins as an essential component. Proton “pinballs” dissociate Ca2+ ions bound by paired glucuronic acid residues of AGP glycomodules, hence reassessing the role of proton pumps. It shifts the prevalent paradigm away from the recalcitrant “acid growth” theory that proposes direct action on cell wall properties, with an alternative explanation that connects proton pumps to Ca2+ signalling with dynamic Ca2+ storage by AGPs, auxin transport by auxin-efflux PIN proteins and Ca2+ channels. The extensive Ca2+ signalling literature of plants ignores arabinogalactan proteins (AGPs). Such scepticism leads us to reconsider the validity of the universal growth oscillator proposed here with some exceptions that involve marine plants and perhaps the most complex stress test, stomatal regulation.Since Darwin’s “Power of Movement in Plants” the precise mechanism of oscillatory plant growth remains elusive. Hence the search continues for the hypothetical growth oscillator that regulates a huge range of growth phenomena ranging from circumnutation to pollen tube tip growth and stomatal movements. Oscillators are essentially simple devices with few components. A universal growth oscillator with only four major components became apparent recently with the discovery of a missing component, notably arabinogalactan glycoproteins (AGPs) that store dynamic Ca Pairs of kidney-shaped guard cells form stomatal pores that respond to ambient conditions by opening and closing and thus allow a trade-off between water retention and CO2 entry. A critical balance depends on the osmotic generation of turgor pressure and the elasticity of guard cell walls with anisotropic reinforcement. Guard cell turgor depends on the regulated influx and efflux of specific osmolytes. Opening and closing are distinct processes; both depend on Ca2+ signalling. However, a simple model remains elusive. Even after 50 years, the extensive literature shows that major problems remain stubbornly unresolved. This implies that a major piece of the puzzle is missing. Certainly, the glaring omission of AGPs from the current discussion is a strong hint of their possible relevance, especially considering their location and Ca2+ binding properties. The following discussion outlines how AGPs may contribute to the Ca2+ homeostasis that underlies the mechanical dynamics of stomatal function. This may resolve the apparent paradox of Ca2+ required for both opening and closing stomata [About four hundred million years ago, bryophytes evolved an upright sporophyte with a terminal sporogenous capsule that enhanced aerial spore dissemination but created the opposing demands of water retention while permitting carbon dioxide entry. The development of a waterproof cuticle saved water but restricted CO stomata ,2,3,4.2+ homeostasis, which was quite unanticipated. The Plant Kingdom has recruited reverse proton gradients; a novel extrapolation of Mitchell’s proton pump integrates auxin-controlled growth with cell surface glycoproteins and Ca2+ signalling as a major regulator of plant growth , and protein X-Ray diffraction by Perutz and Kendrew. Peter Mitchell’s discovery of chemiosmosis and the proton motif force generated by proton gradients across a membrane is one of the great Nobel Prize winners [t growth . This co2+. The Venn diagram emphasises common attributes shared by the four membrane components that are uniquely linked by their common function in Ca2+ homeostasis.Auxin efflux PIN proteins transport auxin.Auxin activates proton pump to generate protons.2+ from AGP-Ca2+.Protons release Ca2+ channels supply cytosolic Ca2+.Open CaA Venn diagram: four components embedded in the plasma membrane generate cytosolic Ca2+-binding sites [2+ stores of animal cells, plants locate their dynamic Ca2+ storage at the most strategic point of entry, the plasma membrane. The release of dynamic Ca2+ then identifies a novel function of proton pump activity.Cell surface arabinogalactan glycoproteins (AGPs) were for many years with only modest evidence  considerng sites . Further2+-dependent Ca2+ oscillator [2+ gradient; this suggested Ca2+ influx could originate from external stores in the cell wall [2+ and the cell surface Ca2+ oscillator explained its crucial role in pollen tube growth. The wide ramifications include not only tip growth of pollen tubes but numerous other plant processes such as phyllotaxis and even stomatal movements [Another centre of activity involved National Service at Number Two Radio School, RAF Yatesbury, where two years as an instructor in radio theory, with a focus on oscillators, provided the essential backdrop to biological oscillators. This knowledge was later critical in identifying the AGP-Cacillator . Over 25ell wall . Howeverell wall  became sovements .2+ homeostasis that regulates plant growth and differentiation thus exemplifying the parsimony of nature  released to the cytosol by inositol triphosphate IP3 of the Ca2+-signalling phosphoinositide (PI) pathway [2+ homeostasis: Indeed, it is now clear that plants store dynamic Ca2+ externally as periplasmic AGP-Ca2+ rather than in the ER. One hardly expects immediate acceptance of this model. Time sifts. Paradigms shift. Indeed our inference extrapolates Peter Mitchell’s chemiosmotic model that couples proton gradients across mitochondrial membranes with ATP generation [2+ homeostasis and growth. Somehow, plants have bypassed the enormous complexities of hormonal regulation in animals with an elegant strategy that regulates plant growth by unifying an auxin-activated proton pump with AGPs and Ca2+ homeostasis. Sydney Ringer’s serendipitous discovery of Catraction  was follmination . These d pathway . Many co pathway  reminiscneration   of the Arabidopsis genome create significant genetic redundancy and therefore a challenge to functional analysis.2+ hypothesis by two groups with substantial contributions to AGP structural chemistry generated multiple mutants that lack AGP glucuronic acid, the Dupree group at Cambridge UK, and the Showalter group at Athens Ohio. The genetic redundancy of glucuronosyl transferases that add GLcA to AGP polysaccharides involved complementary approaches to the generation of multiple GlcAT gene knockouts: Firstly, T-DNA insertion lines involving Ti plasmid transformation mediated by Agrobacterium tumefaciens generated Arabidopsis mutants in four glucuronosyl transferases  [A critical test of the AGP-Ca and -E) . Secondly, similar Arabidopsis mutants were generated by the CRISPRCas9 multiplexing approach for GlcAT14A, GlcAT14B, and GlcAT14C . 2+ homeostasis. This was consistent with the greatly decreased glucuronidation and Ca2+-binding capacity of AGPs isolated from leaves and roots of glcat14a/b/d mutants [2+ transients in roots with attenuated Ca2+ wave propagation. Significantly, exogenous Ca2+ suppressed/rescued these AGP glucuronidation mutant phenotypes.A comprehensive and detailed study of plant growth by the Dupree group showed that multiple glucuronosyl knockouts severely impaired Ca mutants , while tA parallel study by the Showalter group focussed on sexual reproduction and the role of multiple glucuronosyl transferase (glcat) mutants in Arabidopsis. Over many years significant contributions to AGP biology by the Showalter group have demonstrated the widespread involvement of AGPs encoded by 85 genes and 11 GLCAT genes. Their current work demonstrates that multiple glcat mutants significantly impair sexual reproduction, particularly pollen development, polytube block, and successful fertilisation that requires cytoplasmic calcium oscillations in synergid cells after physical contact with the pollen tube tip followed by normal embryo development in Arabidopsis.Taken together, the Dupree and Showalter groups corroborate the pinball model and support a unified theory of plant growth based on the properties of membrane glycoproteins.2+ homeostasis, therefore Glucuronic-bound Ca2+ is the essential source of cytosolic Ca2+. The novel idea of a Ca2+ store strategically located at the surface of the plant plasma membrane was previously overlooked because Mother Nature keeps some of her best secrets well hidden.Multiple glucuronosyl transferase knockouts severely impair Ca2+-binding centre.In a conventional pinball machine, pinball trajectories are largely random. Thus, although a strong visual metaphor a molecular pinball machine  has limi2+ has been apparent for many years [2+. While Vanneste and Friml concluded [2+ signalling is poorly understood”, Dindas and colleagues confirmed [2+-mediated metabolic regulation. An alternative explanation postulates complex auxin-activated Ca2+ channels [2+ channel. While they demonstrate Ca2+ influx within seconds of IAA application, it completely ignores the source of dynamic Ca2+ bound by cell surface AGP-Ca2+ stores, hence overlooking the direct role of the proton pump. Although a connection between auxin and Cany years , both auny years  with a concluded  that “thonfirmed  a close channels : Auxin  is of pThe proton pump acts as a central hub regulated by virtually all factors known to regulate plant growth, hormones, and classical plant hormones  generati2+ from AGP-Ca2+ and also generates the membrane potential. Thus AGP-Ca2+ dissociation initiates cytosolic Ca2+ oscillations while a large membrane potential enables K+ influx increasing cytosolic osmolyte levels including malate derived from starch. These Ca2+ oscillations provide the cytosolic Ca2+ that triggers vesicle exocytosis [2+ is evidenced by the over 200 EF-hand Ca2+-binding proteins as key transducers mediating Ca2+ action encoded in the Arabidopsis thaliana genome [In turgid guard cells, the proton pump activated by auxin releases Caocytosis ,40. Indea genome . + osmolyte via the specific K+ efflux channel GORK [2+ oscillations that regulate osmolyte influx and efflux respectively; increased cytosolic Ca2+ opens stomata [+ efflux channels and convert malate to starch. Such low demands presumably reflect the much weaker Ca2+ oscillations hence a low level of metabolic activity. This resolves the paradox, depicted in In flaccid cells, osmotic stress and dehydration generate abscisic acid, which triggers stomatal closure  by inactnel GORK  and recy stomata ,45. On tThe coastal town of Brighton afforded access to a wide range of ecological habitats including chalk downland, freshwater, estuarine, marine, and saltmarsh environments. These included the Lewes Brooks where salt ingress from the adjacent tidal river Ouse formed a clear ecological gradient. 2+ binding role of classical AGPs [2+ homeostasis regulated by a molecular pinball machine in the plasma membrane [2+ binding were of great interest, particularly in seawater species of flowering plants where high Na+ could disrupt Ca2+ binding. This was an evolutionary conundrum faced by flowering plants seeking to escape drought through the return to an oceanic abundance. Indeed, evolutionary adaptation to high salt has occurred in a family of seagrasses represented by Zostera marina [Ulva lactuca is also adapted to a marine environment but 90% of Chlorophyta are freshwater species; thus Ulva probably migrated from fresh to seawater very much earlier than Zostera [2+-binding [These early influences, which included summers living with a farm family and working on a mixed dairy/arable farm, led to a curiosity about natural processes and a career in biochemistry. On retirement joining the faculty at Sussex University as a visiting professor, Dean Timothy Flower’s interest in saltmarsh ecology stimulated my approach to salt stress and led to the discovery of upregulated AGP secretion in cultured cells . Elucidacal AGPs  that ledmembrane ,47,48,49a marina ,22. It c Zostera , yet qui-binding . Thus itWhile highly glycosylated classical AGPs are the most abundant in cell extracts, a small minority of AGPs with just a few arabinogalactan glycomodules are designated as non-classical; such minimal glycosylation directs them into the Golgi secretion pathway targeted to the plasma membrane . Their f2+ signalling [This review recapitulates recent significant progress in AGP biology represented by the molecular pinball machine of the plasma membrane that unifies proton pumps, AGPs, and Cagnalling .2+ can both open and close stomata, with AGPs as the missing link and source of cytosolic Ca2+. Arguably, the proton motive force controls both the opening and closing of stomata. Thus positive and negative regulation of the proton pump involves an auxin accelerator and an abscisic acid brake. That simple analogy explains how low Ca2+ levels close stomata, emphasising elegant control of both water loss and carbon dioxide gain.Here, we presented a critical test of the pinball hypothesis and suggested how it resolves the paradox that Ca2+ homeostasis and its related tropisms. It has taken many years to appreciate the versatility of these cell surface glycoproteins.Recent work validates the pinball hypothesis and is a suitable closure to a research career extending over 60 years initiated by the discovery of hydroxyproline-rich cell wall proteins  in 1960.


Here, by using two-photon microscopy and whole-cell recordings, we show two distinct astrocytic Ca2+ signals in the murine barrel cortex: a small, long-lasting Ca2+ increase during sleep and a large, widespread but short-lasting Ca2+ spike when aroused. The large Ca2+ wave in aroused mice was inositol trisphosphate (IP3)-dependent, evoked by the locus coeruleus-norepinephrine system, and enhanced sensory input, contributing to reliable sensory transmission. However, the small Ca2+ transient was IP3-independent and contributed to decreased extracellular K+, hyperpolarization of the neurons, and suppression of sensory transmission. These events respond to different pharmacological inputs and contribute to distinct sleep and arousal functions by modulating the efficacy of sensory transmission. Together, our data demonstrate an important function for astrocytes in sleep and arousal states via astrocytic Ca2+ waves.Despite extensive research on astrocytic Ca 2+ signaling to the modulation of sensory transmission in different brain states remains largely unknown. Here, the authors show two types of Ca2+ signals in the mouse barrel cortex with distinct function in sensory transmission during sleep and arousal states.The contribution of astrocytic Ca More than a century ago, Cajal indicated that astrocytes might play a critical role in sleep by extending their processes into the synapse to block sensory transmission6. Later electron microscopy studies demonstrated that sleep deprivation led to extensions of astrocytic processes towards the synaptic cleft7, supporting Cajal’s idea. In recent decades, many more studies have examined the functional role of astrocytes in cognition during sleep and waking states18. In addition, astrocytes have also been suggested to affect circadian rhythm11 and control slow oscillation sleep19, as well as neural circuits within distinct behavioral states21, which suggests a direct functional role for astrocytes in sleep and arousal. However, the direct functional astrocytic role in emotional arousal states is not clear.It is increasingly appreciated that rather than being mere supporting cells as purely nonfunctional glues for neurons, glial cells may play active roles in sleep, arousal, and cognitive processing22. During periods of high arousal states, brain electroencephalogram (EEG) recordings show desynchronized, high-frequency, low-amplitude patterns, which are modulated by short bursts of high-frequency neuromodulatory activity23. The sleep-wake cycle and emotional arousal states are primarily regulated by neuromodulators12. Astrocytes have a unique capability to amplify and extend the effects of neuromodulators over a large population of synapses, making them a particularly intriguing component of the brain’s signaling system12. Previous findings, including our own, also showed that sensory input can induce local astrocytic Ca2+ waves24 and can trigger widespread Ca2+ signals in the cerebral cortex mediated by α1 adrenergic receptor activation in waking states25. The locus coeruleus-norepinephrine (LC-NE) projection is recognized to play a major role in regulating the behavioral state and state-dependent processing of sensory information27. However, the functional role of astrocytes in sleep, arousal, and their relationship with monoamine neuromodulators are not clear.Sleep, wakefulness, and arousal represent distinct alert states characterized by unique emotional activity profiles and different cognitive functions2+ indicators (GCaMP6f) into this circuitry, enabling the identification of unique astrocytic Ca2+ signals during sleep, arousal, and sensory transmission25. We conduct functional studies in live mice, incorporating dual whole-cell recordings in single neurons and local field potential (LFP) recordings in layer II/III of the barrel cortex. We show that astrocytic signaling is differentially regulated by the previously described function of norepinephrine in modulating sleep and arousal, as well as sensory input28, demonstrating a fundamental role for astrocytes in sensory processing and emotional arousal.In this study, we incorporate genetically encoded Ca2+ signaling during different behavioral states, a genetically encoded calcium indicator (GCaMP6f) using a GfaABC1D promoter was delivered selectively into astrocytes using adeno-associated virus (AAV) vectors without detectable astrogliosis29, enabling detection of Ca2+ transients2  for groups of neurons in layer II/III of the barrel cortex  recordings that demonstrated slow waves of 0.5–4 Hz for EEG recording31  in 6 mice, Fig. 2+ transients had a faster latency, 3.5 ± 2.3 s compared with 7.8 ± 1.8 s in the sleep state  from the LC to enable system priming32. To confirm these findings, we stimulated the LC and compared the two kinds of Ca2+ transients and found that LC stimulation changed the small Ca2+ transients during sleep to larger Ca2+ transients and caused powerful arousal in the mice  , similar to the data reported above for LC stimulation with whisker stimulation.Previous studies have shown that astrocytic Ca01) Fig. . To conf1-AR) antagonist terazosin (100 µM), and this drug significantly blocked LC stimulation-induced Ca2+ signaling  antagonist metoprolol (10 µM) was not effective  at significantly blocking LC-induced Ca2+ signaling. Thus, α1-AR but not α2-AR appears to be involved in the LC/NE-mediated induction of barrel cortex signaling.Because these findings appear to show the involvement of NE signaling in this effect, subsequent experiments were designed to identify specific NE receptors involved in the LC/NE-mediated induction of barrel cortex signaling using receptor antagonists. First, we introduced the alpha-1 adrenergic receptor  before and after LC stimulation and revealed that LC stimulation significantly increased the amplitude of whisker stimulation-induced EPSPs -induced astrocytic Ca2+ transients, from 16.2 ± 1.4 mV to 21.5 ± 1.5 mV , while LFP EPSPs occurred with a latency of 20.2 ± 1.3 ms after whisker stimulation  to specifically stimulate the transfected astrocytes. Administration of CNO significantly induced Ca2+ in astrocytes, which was 68.5 ± 3.4% with a duration of 21.3 ± 2.6 s , and whisker stimulation induced EPSPs, which increased from 14.1 ± 1.5 mV before UTP to 18.5 ± 2.2 mV  promoter; the MrgA1 receptor can be selectively stimulated with Phe-Met-Arg-Phe-NH2 (FMRF) amide. The results showed that administration of FMRF (100 µM) significantly induced Ca2+ in astrocytes  = 4.7, **P < 0.01, n = 27 ROIs in 7 mice). Most importantly, EPSP-induced neuronal firing  also increased by 55.6 ± 26.3%  = 27.34, *P < 0.01, n = 27 ROIs in 7 mice, Fig. 2+ waves appear to enhance sensory responsiveness, suggesting that astrocytes act as a gain to mediate the salience or impact of sensory flow into the neocortex by modulating their Ca2+ signals to induce a brain arousal state.In addition, UTP (100 µM) induced Ca2+ signals induced by whisker stimulation were examined in the presence of two antagonists: mGlu5 receptor antagonist MPEP (50 µM) and mGlu2,3 receptor antagonist LY341495 (10 µM). The resulting Ca2+ signal, ΔF/F0, decreased to 19.2 ± 6.6% during waking states (56 ROIs in 5 mice), as shown in Fig. 0 was not significant . In mice with genetic deletion of the IP3 receptor , whisker stimulation-induced Ca2+ signals were significantly decreased to 21.6 ± 5.8%  blockers SN-6 (SN-6:2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester, 50 µM) and SEA-0400 methoxy]-phenoxy]-5-ethoxyaniline, 50 µM) had a modest effect on whisker stimulation-induced Ca2+ transients, with an average peak fluorescence of 58.6 ± 7.7% .The characteristics of astroglial Caant Fig. . This fi2+ transients during arousal was elucidated, we next assessed the functional effects of the small Ca2+ transients on neuronal activity and attempted to determine whether the smaller Ca2+ transients can induce deeper sleep . The results showed that ATP-induced Ca2+ transients decreased whisker stimulation-induced EPSPs . Similarly, the EPSPs recorded in LFPs also decreased from 0.82 ± 0.18 mV to 0.38 ± 0.14 mV  evoked [Ca2+]i transients with an average increase of 19.7 ± 4.1% during the sleep period. Furthermore, FMRF-induced [Ca2+]i transients decreased whisker stimulation-induced EPSPs, with an amplitude decrease from 14.4 ± 2.5 mV to 7.1 ± 1.2 mV with whole-cell recording  receptor antagonist 6-cyano-7-nitroquinoxaoine-2,3-dione  had no significant effect on the small Ca2+ transients . The N-methyl-D-aspartate receptor (NMDA) antagonist DL-2-amino-5-phosphono-pentanoic acid  also did not significantly alter the small transients . However, the mGlu5 receptor antagonist MPEP (50 µM) and mGlu2,3 receptor antagonist LY341495 (10 µM) significantly decreased Ca2+ transients , suggesting a role for mGLuR receptor-induced Ca2+ release from the ER. We then used the traditional agonists ATP  and UTP  to activate Gq receptors to determine their effects on sleeping Ca2+ transients in astrocytes. To exclude the involvement of neurons, tetrodotoxin TTX (1 µM) was applied together with these drugs. The results suggested that these agonists could induce small astrocytic Ca2+ transients  in mice with genetic deletion of the IP3 receptor (Type 2). In the absence of the IP3 receptor, whisker stimulation still induced Ca2+ transients in these mice 43 or through NCX35. To test this hypothesis, we administered the TRPA1 channel blocker HC030031 (100 µM) and found that whisker stimulation-induced Ca2+ transients were significantly decreased to 15.03 ± 3.1%  and SEA0400 (50 µM) on the surface of the brain, whisker stimulation-induced Ca2+ transients decreased . Thus, we used agonists  to induce Ca2+ transients in the presence of TTX (1 µM) to block neuronal activity and found that these agonists induced similar hyperpolarization  knockout mice, whose glymphatic system had been suggested to be impaired, and found a reduction in the extent of decrease/clearance of K+. Thus, it appears that the small Ca2+ transients that are associated with sleep may serve to inhibit sensory information from causing arousal, and this appears to be mediated by NE released by the LC. Moreover, during this state, it appears that the astrocytic glymphatic system is enabled to perform the essential function of clearance. Such findings raise the provocative possibility that during sleep, astrocytes suppress sensory information from reaching the neocortex to enable the necessary removal of metabolic waste from the brain.The data presented above provide compelling evidence for a direct role of astrocytic Ca2+ in regulating these processes, which are regulated by noradrenergic input to the barrel cortex that serves this role in modulating different brain states. The interesting findings demonstrated that astrocytes in the barrel cortex mediate these effects by distinct large Ca2+ transients to enhance arousal, while small Ca2+ transients induce deeper sleep and squelch sensory input in this circuit  gain.The results from our study confirm a fundamental role for astrocytes as major players in the modulation of neurons under physiological conditions2+ signaling during sleep to greater Ca2+ signaling in the arousal state. The latter LC/NE mechanism was previously suggested to play a role in modulating the brain arousal state50. The observations from the present study show that astrocytes are fundamentally and functionally involved in this modulation by enhancing sensory input and responsiveness during arousal and inhibiting sensory input during sleep. These findings expanded our understanding of the control of behavioral states.Another important finding from these experiments is the fact that the LC and its neurotransmitter NE play a major role in modulating these behaviors by switching between small Ca51. Our results further reveal that astrocytic calcium waves play a key role in these processes. Sleep, wakefulness, and arousal are distinct patterns in the brain, each with its own unique neuromodulator profile and variations in brain wave synchronization, frequency, and amplitude52. These findings broadened our understanding of the unique contributions of astrocytes to the regulation of these behavioral states. Our study provided evidence that astrocytes not only promote sleep by silencing sensory transmission but also have the ability to enhance sensory transmission during wakefulness and arousal by increasing their activity, providing insights into the complex interplay between astrocytes and neurons and their impact on brain function.In conclusion, by using integrative dual neuron and astrocyte recordings for local field potentials after NE or LC stimulation, we identified distinct roles of astrocytes in regulating sleep and arousal states, as demonstrated through the use of dual neuron and astrocyte recordings, as well as local field potentials and NE or LC stimulationAll animal procedures followed the National Institute of Health guidelines and were approved by the Institution of Animal Care and Use Committee at the University of Rochester (A3292-01) and the Institution of Animal Care and Use Committee at Sichuan Normal University (2021LS031).2), head restrained with a custom-made mini-frame and habituated to the restraint over one week in multiple sessions, with a total training duration of 3–4 h. A 1.5 mm craniotomy was then opened over the somatosensory cortex , the dura was carefully removed, and the mice were allowed 60 min of recovery prior to conducting the experiments. The craniotomy procedure lasted <20 min to minimize anesthesia exposure on the recording day. Mice were then head-strained, placed in a behavioral tube to minimize movement, and relocated to the imaging room, which was kept dark and asleep. The body temperature was maintained with a heating pad. For cortical drug surface application, artificial cerebrospinal fluid (aCSF) was perfused across the cortex of awake mice at a rate of 2 mL/min into a custom-made well with an ~200 μL volume through tubing with an ~100 μL volume, meaning the entire volume bathing the brain was exchanged every ~9 s. The aCSF solution contained 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 26 mM NaHCO3, pH 7.4. For imaging, the calcium indicator rhod-2 AM (Invitrogen) was loaded onto the exposed cortex for 30–40 min before applying agarose  and a coverslip. All mice were trained several times a day for three days before the experiments. The mice were anesthetized with 1.5% isoflurane during the surgery, and then after being mounted on the equipment, the isoflurane was stopped and the mouse was left to run freely on the wheel during sleep/wake states in the dark room, except that the heads were held tightly on a frame.Adult (10 weeks old) C57Bl/6 wild-type mice were used . Mice were housed in a facility with a light/dark cycle of 12/12 h and provided food and water ad libitum. The preparation for mouse experiments was modified from published protocols. Briefly, mice were anesthetized using isoflurane , clozapine-N-oxide (CNO) (Enzo Life Sciences), APV , MPEP (6-(phenylethynyl)-pyridine, Tocris), terazosin (Tocris), metoprolol (Tocris), SN-6 (2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester, Tocris), SEA0400 methoxy]-phenoxy]-5-ethoxyaniline, CM-4620 (Tocris), synthesized by Taisyo Pharmaceutical Co. Ltd), UTP .1D-cyto-GCaMP6f-SV40 , which is a genetically encoded Ca2+ indicator driven by the astrocyte-specific GfaABC1D promoter, was injected into the barrel cortex. The mice were anesthetized with 1–2% isoflurane with oxygen supply and were placed in a stereotactic head frame with a heating pad underneath. A small vertical incision was made on the skin, and craniotomy (0.5 mm × 0.5 mm) was performed with a drill. The virus was injected at a volume of 500 µL/site without dilution, and 3 injection sites were utilized. A glass micropipette with a tip of 10 µm was used for injection with microinjection control.pAAV5-GfaABC17. Images of astrocytic Ca2+ signaling were recorded every 2–3 s, which was sufficient to capture evoked responses while limiting laser-induced photodamage at a laser power of <30 mW. Prior to whisker stimulation experiments, anesthesia 0.5 mg/kg D-tubocurarine was injected to prevent small reflex movements that could distort imaging. Direct LC stimulation was applied using a bipolar concentric electrode. Stimulation consisted of a single train of 20–100 pulses . ROIs were extracted from the above-threshold pixels with the fluorescence of the GCaMP or Rhod-2 imaging, and ΔF/F signals were calculated to detect the periods that had multiple ΔF/F peaks above baseline.A custom-built microscope attached to a Tsunami/Millenium laser  and scan box  was used for 2-photon imaging through a 203 objective . The excitation wavelength was in the range of 800–820 nm. Emission wavelengths were split to detect fluo-4 and AlexaFluor 594 signals as previously described2, 10 mM HEPES, 4 mM Mg-ATP, and 0.3 mM Na-GTP (pH adjusted to 7.2 with KOH). The junction potential between the patch pipette and the bath solution was zeroed before forming a gigaseal. Patches with seal resistances of less than 1 gigohm were rejected. Data were low pass–filtered at 2 kHz and digitized at 10 kHz with a Digidata 1440 interface controlled by pClamp Software (Molecular Devices). Whisker stimulation was delivered using a picospritzer III (Parken Instrumentation) and Master 8 (A.M.P.I.). The amplifier bandwidths were normally 0.5 Hz to 100 Hz. EEG recording was digitized at 100 Hz and then subjected to spectral analysis using a complex demodulation procedure.Recordings were obtained from the layer II barrel cortex using glass microelectrodes. LFP signals were externally filtered at 6 Hz , bandpass filtered at 1–100 Hz and digitized (Digidata 1440 A by Axon Instruments). Recordings were analyzed offline using pClamp 10.2. Whole-cell recordings were performed with blind patching by observing the pipette resistance. Patch electrodes were fabricated from filament thin-wall glass (World Precision Instruments) on a vertical puller; the resistance of the pipette was approximately 6 to 9 megohms with intracellular pipette solution added. The pipette solution contained 140 mM K-gluconate, 5 mM Na-phosphocreatine, 2 mM MgClAcquired EEG/EMG signals were amplified  at a sampling frequency of 1 kHz. The EEG signal was filtered with high-pass: 0.5 Hz, low pass: 30 Hz, and EMG signal high-pass filtered at 10 Hz. Wakefulness was subdivided into quiet wakefulness (QW) or arousal wakefulness (AW) using the EMG peak-to-peak amplitude of all wake epochs across the 12-h recording. QW was defined as the 33rd percentile or less and AW as the 66th percentile or higher of all wake EMG peak-to-peak amplitude values. Concurrently with EEG recordings, spectral analysis of EEG recordings showed that most of its power resides in 4–6 Hz, which was interrupted intermittently with slow waves of 0.5–4 Hz.α = 0.01 level. The data are shown as the mean ± S.D. (standard deviation). For independent samples, a t-test (≤2 variables) or one-way ANOVA (>2 variables) was used; for paired samples, a paired t-test was used.All analyses were performed using SPSS 19 software (IBM), and all tests were two-tailed where significance was achieved at the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary