Despite the availability of evidence based treatments, many people with major depression receive no or delayed professional treatment, which may put them at risk for adverse outcomes. The aim of this study was to examine which demographic and need factors distinguish early, delayed and no treatment use.N = 434). Mental health care use was assessed during this same period and at follow up (three years later). Multinomial regression analysis was used to distinguish early, delayed and no mental health care users with respect to demographic and need factors.Data were obtained from the Netherlands Mental Health Survey and Incidence Study-2 (NEMESIS-2). People with a diagnosis of major depression in the past 12 months were included (The majority of participants accessed treatment early (62%). Early treatment users were characterized by more severe and persistent symptoms and were more likely not to have a partner compared to no treatment users. The majority of those without treatment reached remission in three years (85%). Delayed treatment users were, compared to early users, characterized by relatively mild symptoms and a persistent or new major depressive disorder at follow up.Early access to treatment and the finding that need factors determine mental health care use among people with depression show that the filters along the pathway to treatment are not influenced by unfavorable determinants like education or age. Major depression is a highly prevalent mental disorder  and is aCross-sectional epidemiologic studies have shown that increased mental health care use is associated with having more severe symptoms and a comorbid mood or anxiety disorder , 18–21. In summary, based on earlier research findings from cross sectional studies, delayed treatment users may be distinguished from early treatment users by several demographic (gender or age) and need factors (severity of symptoms). However, most knowledge is derived from cross-sectional designs which preclude the investigation of long term associations. Large representative longitudinal population studies regarding mental health care use are rare , 30–32. The aim of the present study is to examine whether early, delayed and no treatment users differ regarding demographic  and need factors . For the present paper we used data from the Netherlands Mental Health Survey and incidence Study-2 (NEMESIS-2). This general population study has a longitudinal design which improves understanding of mental health care use over time.1 2010–2012; follow up T2 2013–2015). The methods employed have been described in greater detail elsewhere [1) three years after baseline (2010–2012), of which 5303 could be reinterviewed (80.4% response). All these 5303 participants were approached for second follow (T2) up three years after T1 (2013–2015), of which 4618 could be reinterviewed (87.8% response) [1 and T2 was not significantly associated with all individual 12-month mental disorders at baseline (controlled for sociodemographic factors) [The primary aim of NEMESIS-2 was to provide up- to- date information on the prevalence, incidence and course of DSM-IV mental disorders in the general population and its consequences in terms of service use and functioning. NEMESIS-2 is a longitudinal study which incorporated 3 waves  . Attritifactors) , 37.0) were selected, and we assessed whether they did or did not use primary care or mental health care for their psychological problems in this same period and at follow up (three years) (T1). Because of this relatively small subsample (n = 287) we also made the same selection of respondents in the second wave (T1 and T2): we selected people (who were not selected at baseline as described above) with a diagnosis of major depression 12 months prior to T1 and assessed whether they did or did not use primary care or mental health care for their psychological problems at T1 and follow up (T2). Since we used the same selection criteria, the data could be pooled and the analysis could be performed on the whole group. This resulted in 434 respondents .From the 6646 subjects that were included during the baseline measurement, people with a diagnosis of major depression in the preceding 12 months at the baseline measurement  3.0, which was developed in the World Mental Health Survey (WMHS) Initiative , 38–40. At baseline, mental health care use was measured with the following question: ‘In the previous 12 months, have you visited any of the following professionals or institutions for emotional problems or alcohol or drugs problems of your own?’ and at follow up: ‘Since the last interview, did you visit any of the following professionals or institutions because of emotional or alcohol or drugs problems of your own?’ Primary care included general medical professionals . Specialized mental health care included psychiatrists, psychologists, psychotherapists, mental health care or addiction institutes, part-time or full-time psychiatric treatment , 35.0). For the people that were selected at T1, mental health care use was assessed 12 months prior to T1. At follow up, we assessed if people report any mental health contact between the two successive measurements (T0-T1 or T1-T2).Mental health care use refers to at least one contact made in primary care and or specialized mental health care for emotional or addiction problems in the past 12 months prior to baseline measurement ;Delayed treatment users  but who reported contact at follow up ;Early treatment users ).Three groups of mental health care users were distinguished:0-T1 cohort and at T1 for the T1-T2 cohort. The predictor variables included the following:Potential predictors of mental health care use were recorded at baseline for the TDemographic factors.Gender categorized into male and female;Age categorized into 18–44 years old or 45–66 years old ;Education defined as primary education/lower secondary education; higher secondary education; higher professional education, university.Partner status defined as partner or no partner;Job status defined as having a job or no job;Need factors.Comorbid anxiety disorder: This variable relates to comorbidity with any anxiety disorder in the past 12 months before the To or T1 measurement with the CIDI 3.0.Disability days on work or normal daily activities based on the CIDI 3.0 question ‘About how many days out of 365 in the past 12 months were you totally unable to work or carry out your normal activities because of your (sadness/or/discouragement/or/lack of interest)’.We categorized this variable into 0 = ≤ 2 weeks and 1 = ≥ 2 weeks .Duration depressive episode in past 12 months based on the CIDI 3.0 question ‘About how many days out of the last 365 were you in an episode?’ We categorized this question in: 0 = ≤ 3 months and 1 = ≥ 3 months .Severity of the depressive symptoms was measured with the Inventory of Depressive Symptomatology, shortened version (QIDS-sr16) in the CIDI 3.0 [16 manual).CIDI 3.0 . The aimMajor depression at follow-up measurement this variable related to a persistent or new 12-month major depression at follow-up measurement (T1 or T2), measured with the CIDI 3.0.0−T1 and T1−T2) were merged into one dataset. In the final dataset , there were no duplicates in the selected respondent of T0 and T1. If respondents were both in the group at T0-T1 and T1-T2, they were excluded from the T1-T2 cohort.The data from the two waves . The dependent variable, primary care or mental health care use, consisted of three categories: early treatment use, delayed treatment use, no treatment use. Early treatment use was used as the reference category, because delayed and no treatment users were of specific interest.p < .05 was used in all analyses.We conducted a series of univariate analyses for each predictor separately (unadjusted odds ratios (OR)). Then, a multivariate analysis was performed to determine the effect of individual predictors in which all predictors were entered simultaneously (adjusted OR). The regression analyses were performed using SPSS 21 and a two sided significance level of 0 -T1 or T1-T2), we added an identification variable for the individual cohorts in all regression models (dummy variable study ‘cohort’). The identification variable was significant in the comparison between the no treatment users and early treatment users (p = .03), but not in the comparison between delayed and early treatment users (p = .11). Therefore, a sensitivity analysis was conducted were the analyses were performed separately on the T0 -T1 (N = 287) or T1-T2 (N = 147) study cohorts. These data were compared with the results of the total group (N = 434). This sensitivity analysis revealed no substantial differences between the two cohorts (results available on request). Furthermore, we performed the multinomial regression analyses with or without the identification variable. The results from these analyses revealed no substantial differences between the analyses as well (results available on request). Since the sensitivity analyses did not reveal substantial differences, but the variable was significant in the no treatment users versus early treatments users we performed the analyses on the dataset including the identification variable.In order to adjust for possible differences between the two waves (Tn = 47), duration depressive episode in past 12 months  and severity of depressive symptoms . In order to adjust for the missing data we imputed data for these variables in the dataset using a Fully Conditional Specification (MCMC) model with a Predictive Mean Matching (PMM) model type and 1E-012 as singularity tolerance and N = 10 imputations. Sensitivity analyses on the original dataset and imputed dataset yielded no significant differences (results available on request). Therefore, the analyses were performed on the imputed dataset.Missing values were present for three variables; disability days on work and/or normal daily activities . Respondent were, on average, 44 years old (range 18–67 year) at baseline. Most respondents (39%) completed higher secondary education or primary education/ lower secondary education (33%). About half (48%) reported living with a partner (Table n = 271) reported mental health care use in the same period (T0 or T1) (early treatment users). About a quarter  of the respondents reported no mental health care use at follow up (T0 -T1 or T1-T2) (no treatment users). Finally, 14% (n = 58) of the respondents did not report mental health care use at first measurement (T0 or T1), but did three years later (delayed treatment users)  and 24% (n = 14). Among the no treatment users 11% (n = 11) reported a depressive disorder at follow up.About a quarter of the early and delayed treatment users reported a major depressive disorder at follow up, respectively 23% (0-T1 or T1-T2 and less disability days (< 2 weeks), were more likely to be in the no treatment group. People without a partner were more likely to receive mental health care in the 12 months prior to the first measurement.Univariate analyses showed that no treatment users could be distinguished from early treatment users by several factors related to need for care Table . People 0-T1 or T1-T2 remained significant  and no persistent or new major depressive disorder between TUnivariate analyses showed that delayed treatment users could be distinguished from early treatment users by two need for care factors. People who reported <2 weeks of disability days and people who report none to mild symptoms (as compared to early treatment users) were more likely to be in the delayed treatment group compared to early treatment users Table .Table 3Dp = 0.06 for the univariate analyses p = 0.02 for the multivariate analyses)  Table .p = <.001) and a comorbid anxiety disorder . Furthermore, delayed treatment users were less likely to have a partner .By changing the reference category in the multinomial regression analyses, we examined demographic and need factors between delayed and no treatment users as well. Multivariate analyses showed that delayed treatment users, compared to no treatment users, were more likely to have a major depressive disorder between baseline and follow-up . Early treatment users were categorized by more severe and persistent depressive symptoms. The majority of the no treatment users reached remission after three years (85%), which suggests that they were able to solve their problems without professional help or that the depressive symptoms were self-limiting. This high remission rate among no treatment users is in line with earlier research findings in the Netherlands , 32, 33.2%. EarlyIn the present study the group of delayed treatment users was relative small (14%) and they were, compared to early treatment users, more likely to have mild depressive symptoms and to report a persistent or 12-month depression after three years , 20, 26.Except for having a partner in the group of no treatment users, no other demographic factors were associated with early, delayed or no treatment use. A possible explanation is that we focused our analyses on recent treatment use. Other studies examined life-time treatment contact or examined the time between the onset of symptoms and first treatment contact, which could explain possible differences , 11.The study results suggest that the accessibility of the mental health care system for major depression in the Netherlands is relatively good (>60% early users) and largely determined by clinical need for care related factors, such as severity of symptoms. Delayed or no treatment users are characterized by less severe and less complex symptoms compared to early treatment users. The fact that demographic factors (except for partner status in the no treatment users) were not associated with treatment use may be a positive result, since people should not receive help based on their level of education, gender or age. Not everyone from the no treatment users group reached remission after three years (11%) , 30. PosThere are several strengths and limitations of this study that need to be taken into account when interpreting the results. A strength of the study is the longitudinal design. To our knowledge, there is only one international published study that examined determinants of early, delayed and no treatment use in a large representative population study, this study focused on no treatment seeking among people with an alcohol use disorder . This deAnother limitation of this study is the definition of mental health care use. In the present study mental health care use refers to at least one contact made in primary care and or specialized mental health care for emotional or addiction problems. We were not able to examine the nature, intensity or the adequacy and perceived effectiveness of the received treatment. In addition, this study only examined demographic and need factors in relation to mental health care use. Barriers from patients’ perspective were not examined. However, several studies showed that attitudinal beliefs like the desire to handle problems by themselves , 48 or tMost people with a major depressive disorder in the past 12 months accessed mental health care early on (62%). When determining mental health care utilization among people with a major depressive disorder, need factors seem to be important in this process. An important finding is that not everyone from the no treatments users reached remission after three years (11%). In those patients, the mild stage of illness may provide a window of opportunity to prevent further disease progression. Therefore, further research should examine reasons for non-participating in professional care for this specific group.


Saccharomyces cerevisiae has the ability to become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds. Among multistress resistance mechanisms is the one mediated by plasma membrane efflux pump proteins belonging to the ABC superfamily, questionably proposed to enhance the kinetics of extrusion of all these compounds. This study provides new insights into the biological role and impact in yeast response to acetic acid stress of the multistress resistance determinant Pdr18 proposed to mediate ergosterol incorporation in plasma membrane. The described coordinated activation of the transcription of PDR18 and of several ergosterol biosynthetic genes  during the period of adaptation to acetic acid inhibited growth provides further support to the involvement of Pdr18 in yeast response to maintain plasma membrane ergosterol content in stressed cells. Pdr18 role in ergosterol homeostasis helps the cell to counteract acetic acid-induced decrease of plasma membrane lipid order, increase of the non-specific membrane permeability and decrease of transmembrane electrochemical potential. Collectively, our results support the notion that Pdr18-mediated multistress resistance is closely linked to the status of plasma membrane lipid environment related with ergosterol content and the associated plasma membrane properties. Two of the described MDR/MXR transporter families occur in all classes of organisms and belong to the ATP binding cassette (ABC) superfamily, which uses the hydrolysis of ATP to translocate a variety of solutes across biological membranes3, or to the Major Facilitator Superfamily (MFS) which energetically drive their transport utilizing the transmembrane electrochemical gradient4. Although those proteins have been traditionally considered drug exporters, their physiological function and the exact mechanism of their involvement in resistance to cytotoxic compounds are still unclear and it is puzzling that a wide range of structurally and functionally unrelated substrates may be exported by a specific transporter. Moreover, numerous ATP-binding cassette (ABC) transporters implicated in MDR/MXR are present in yeast genomes, strongly suggesting that they might have important physiological roles even in the absence of drugs/xenobiotics3. In fact, recent studies support the concept that at least some MDR/MXR transporters exert their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds8. In particular, the role of ABC-MDR/MXR transporters in yeast plasma membrane lipid homeostasis is gaining increasing attention9 and can also explain the MDR/MXR phenotype exhibited by cells expressing these transporters. Indeed, the maintenance of plasma membrane integrity is crucial in yeast cell tolerance to stress and lipids play a critical role in determining membrane physical properties and regulating the function of membrane associated proteins15. The Saccharomyces cerevisiae ABC transporters demonstrated in the scientific literature as involved in lipid trafficking and membrane lipid homeostasis are shown in Fig. 18. Also, the ABC efflux pumps Pdr5, Yor1 and Ste6 are implicated in the translocation of phospholipids between the two plasma membrane monolayers, thus contributing to plasma membrane asymmetry20. Pdr10 is associated with detergent-resistant domains in the plasma membrane and affects lipid distribution and maintenance of membrane micro-environment for the adequate functioning of membrane embedded proteins including the ABC-MDR transporter Pdr1221. Pdr16 and Pdr17 do not localize to the plasma membrane and their expression was proposed to regulate lipid metabolic pathways24 but the underlying mechanisms were not detailed.The acquisition of multidrug/multixenobiotic resistance (MDR/MXR) is widespread in nature and has clinical, agricultural and biotechnological implications. MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a role in this phenomenon8, Pdr18 expression confers increased yeast resistance to the herbicides 2,4-dichlorophenoxyacetic acid , 2-methyl-4-chlorophenoxyaceticacid (MCPA), and barban, the agricultural fungicide mancozeb, the metal cations Zn2+, Mn2+, Cu2+ and Cd2+ 7, and to ethanol8. PDR18 expression was found to contribute to reduce [14C]-2,4-D intracellular accumulation but its action may be indirect since 2,4-D induces the decrease of plasma membrane ergosterol content, this deleterious effect being much stronger in the pdr18Δ background7.The present work focuses on the yeast ABC transporter Pdr18, previously described as a MDR/MXR determinant and proposed to mediate ergosterol incorporation in yeast plasma membrane25. Acetic acid tolerance may however be a highly desirable trait in industrial yeasts, since this weak acid is an industrial fermentation byproduct, its accumulation in the medium contributing to reduced fermentation performance27. Moreover, acetic acid is present in lignocellulosic biomass hydrolysates for the production of bioethanol and other bio-based chemicals29. Therefore, the understanding of the mechanisms underlying the toxic effects and adaptation and tolerance to acetic acid in the biotechnologically-relevant yeast species S. cerevisiae is essential to guide more efficient strategies either for food spoilage control or for the development of more robust industrial strains36.In this article, we describe Pdr18 role in yeast adaptation and tolerance to acetic acid stress. Acetic acid is widely used by the food industry as preservative of acidic food and beverages but there are spoilage yeasts that are able to cope with standard levels causing severe economic losses31 and restriction of the diffusional entry of the liposoluble acid form into the cell37. In particular, incorporation of a higher fraction of sphingolipids was found to improve membrane bilayer thickness and density, leading to the decrease of plasma membrane non-specific permeability in the highly acetic acid resistant species Zygosaccharomyces bailii37. Interestingly, the sphingolipid biosynthetic pathway was found to be up-regulated in response to acetic acid-induced stress38. Also, plasma membrane enrichment in saturated acyl chains arising from glycerophospholipids and complex sphingolipids was also found to occur in response to acetic acid stress and lead to reduced cell permeability and increased membrane order40. A chemogenomic study has identified in S. cerevisiae several acetic acid resistance determinant genes encoding ergosterol biosynthetic enzymes30 and the inclusion of a higher content of sterols in yeast plasma membrane demonstrated to counteract the deleterious effects of ethanol45 and heat shock47. The content of ergosterol is not only crucial for plasma membrane stability and adequate selective permeability barrier to avoid the passive diffusion of toxic compounds into the cell49, but is also related with the formation of lipid-raft domains that may modulate the activity of membrane-embedded pumps51.Yeast adaptation to weak acids is a complex multifactorial process considered to include mechanisms leading to the reduction of the intracellular concentration of the counter-ions through their increased efflux from the cell interiorPDR18 and of several ergosterol biosynthetic genes during the period of adaptation to acetic acid inhibited growth provides further support to the involvement of Pdr18 in yeast response to maintain ergosterol content in plasma membrane of yeast stressed cells. The observed impact that such biological role has in counteracting acetic acid-induced decrease of plasma membrane lipid order, increase of the non-specific permeability and decrease of the transmembrane electrochemical potential establishes a link between this ABC transporter biological activity and its involvement in multistress resistance.The present study provides new insights into the biological role and impact of Pdr18 in yeast cell response under stress, with a focus on acetic acid stress. The observed coordinated activation of the transcription of PDR18 was found to be required to partially overcome the effects of acetic acid induced stress in yeast growth   Fig. . While t mM Fig. . Moreoveant Fig. .Figure 2pdr18Δ mutant under acetic acid was rescued by the insertion of a centromeric plasmid expressing the PDR18 gene under the control of its natural promoter. Moreover, the expression of Pdr18 from the recombinant plasmid in the parental strain improved its resistance to acetic acid  confirmed the role of Pdr18 as an acetic acid resistance determinant Fig. . In factcid Fig. .pdr18Δ mutant . Under these conditions, growth curve of the parental strain in acetic acid supplemented MM4 medium exhibit a lag-phase of approximately 10 hours that was not detectable in the absence of acetic acid stress, while the duration of the acetic acid-induced lag-phase was extended to approximately 40 hours for the ant Fig. . Also, tant Fig. .PDR18 and selected ERG-genes in S. cerevisiae BY4741 cells were compared during cultivation of cells not previously adapted to acetic acid in MM4 or MM4 supplemented with 60 mM acetic acid at several time-points  after 5–8 hours, by the mid- to end of the phase of latency, while for ERG3 and ERG4 genes the activation levels are lower (2–5-fold) and were maintained throughout cultivation , compared to parental strain levels cells growing in the same conditions  factor from the fluorescence intensity of two spectral channels of Laurdan emission spectraPDR18 gene, when both strains were grown either in the absence (p = 0.845E−10) or in presence (p = 1.51E−24) of 60 mM acetic acid at pH 4.0  53% larger than the parental strain population, both grown in the absence of acetic acid, which indicates a higher cell heterogeneity for plasma membrane order within the pdr18Δ cell population  and a more heterogeneous profile for this trait (25% larger interquartile range), compared with the stressed parental strain population  values for each individual cell is shown in Fig. 4.0 Fig. . Parenta4.0 Fig. . Howevercid Fig. . Also noion Fig. . Adaptedion Fig. .Figure 5pdr18Δ deletion mutant assessed before was found to have a considerable impact on the physiological function of yeast plasma membrane as a selective barrier in the presence of acetic acid, with plasma membrane permeability being inferred based on propidium iodide (PI) passive uptake  than those registered for unstressed cells  and this cell population was unable to recover from acetic acid-induced permeabilization during lag-phase to levels similar to unstressed cells , a dye sensitive to membrane potential. Since this diS-C3(3) assay was used to compare two isogenic strains , the activity of other MDR-pumps is considered identical and the differences in λmaxeq believed to reflect differences in membrane potential58. Results indicate that even in the absence of stress, parental strain cells exhibit a significantly higher (p = 0.038) plasma membrane potential than pdr18Δ cells , used in this assay as a positive control, confirmed the disruption of membrane potential under these conditions , consistent with the proposed role for Pdr18 at the end of the ergosterol transport process of the newly synthesized sterols from the endoplasmic reticulum (ER) to the plasma membrane. Moreover, the activation level of the late genes of the ergosterol pathway tested (ERG3 and ERG4) was more moderate and the activation profile less dependent of the growth phase. It would be important to know the sensors that perceive the fluctuations in the membrane environment of acetic acid stressed cells and transduce the signals to PDR18 gene resulting into the compensatory changes in the ergosterol profile and how these processes are controlled.The transcriptional activation under acetic acid stress of a number of genes of the ergosterol biosynthetic pathway has been revealed by transcriptomic analysis65. Ergosterol has then to be transported from the site of its synthesis to the plasma membrane and although this transport is known to be fast, the mechanism by which newly synthesized sterols are transported from the ER to the plasma membrane is not fully understood67. An essential role for vesicular transport pathways in transporting ergosterol, from its site of synthesis to the plasma membrane has been ruled out and ergosterol transport was proposed to occur by equilibration (t1/2 ∼ 10–15 min) of endoplasmic reticulum and plasma membrane ergosterol pools via a bidirectional, nonvesicular process66. To reconcile an equilibration process with the high ergosterol concentration present in plasma membrane, and based on the observation that a large fraction of ergosterol is found in the plasma membrane condensed with sphingolipids in membrane rafts that coexist with free sterol, Baumann et al.66 hypothesized that the concentration of free sterol is similar in the plasma membrane and endoplasmic reticulum and that only free (non-raft) sterol molecules have access to a nonvesicular transport pathway that connects the two organelles. Based on the results of the present study, we propose the involvement of the plasma membrane associated ABC transporter Pdr18 in the active ergosterol transport process at the plasma membrane level, allowing the high physiological ergosterol concentration present in this membrane.Yeast cells synthesize ergosterol in the membrane of the endoplasmic reticulum via a cascade of coupled enzymatic reactionsS. cerevisiae has the ability to acquire resistance to multiple drugs (MDR) or to multiple xenobiotic compounds (MXR), i.e. to become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds70. The acquisition of resistance to multiple stressing agents is due to a number of mechanisms, among them the mechanism mediated by efflux pump proteins localized at the plasma membrane and belonging to either the ABC (ATP-binding cassette) superfamily or the MFS (major facilitator superfamily) and proposed to enhance the kinetics of extrusion of these compounds71. The presence of a large number of ABC and MFS transporters homologous to MDR/MXR characterized transporters in the genomes of yeasts and other organism strongly suggests that they may play important physiological roles even in the absence of the cytotoxic compounds. Accumulating evidence has shown that ABC proteins perform endogenous activities extending beyond their accepted role as drug exporters, among them the transport of lipids73 as schematized in Fig. PDR18 has a paralogous gene in S. cerevisiae, SNQ274, no demonstration of the eventual involvement of Snq2 in lipid transport and homeostasis was so far reported. However, the affinity of Snq2 transporter for sterols was hypothesized due to its role in alleviating estradiol toxicity in S. cerevisiae75. Moreover, we were not able to identify any phenotype of resistance to acetic acid associated to SNQ2 expression. The coordinate control of plasma membrane ergosterol composition by Pdr18 and the so far well accepted role of Pleiotropic drug resistance (PDR) transporters in drug/xenobiotic efflux activity  is a possible model. However, based on the data gathered in this study, the involvement of Pdr18 in multistress resistance may merely result from its physiological role in ergosterol transport and proper distribution within plasma membrane proved to have a significant impact in plasma membrane properties, especially relevant under acetic acid stress. Apart from serving as substrates of certain ABC transporters, lipid molecules are also crucial molecular determinants that affect the trafficking and functioning of certain ABC transporter proteins78. Therefore, the role of Pdr18 in ergosterol homeostasis at the plasma membrane level is likely to affect the proper functioning of other efflux pumps of the ABC superfamily or the MFS, already proposed to be required for acetate efflux31, or the essential plasma membrane H+-ATPase activity79. Collectively, results support the notion that multistress resistance mediated by Pdr18 is closely linked to the status of plasma membrane lipid environment, specifically related with ergosterol content and the associated plasma membrane properties.Like other organisms, the yeast Saccharomyces cerevisiae parental strain BY4741  and the derived deletion mutant pdr18Δ were obtained from the EUROSCARF collection (http://web.uni-frankfurt.de/fb15/mikro/euroscarf), as well as plasmid pYCG_PDR18, expressing the PDR18 gene from its natural promoter, and the corresponding cloning vector, pRS416, used for phenotypic complementation assays.4)2SO4 , 20 mg/L L-methionine, 20 mg/L L-histidine , 60 mg/L L-leucine and 20 mg/L L-uracil , adjusted to pH 4.0 with HCl. Solid medium was prepared by the addition of 20 g/L agar  and the pH was set to 4.5 with HCl. Cells harboring the cloning vector pRS416 or derived plasmids were grown in the same medium lacking uracil supplementation (MM4-U medium) to maintain selective pressure.Yeast cells were cultivated at 30 °C with orbital agitation (250 rpm) in liquid minimal growth medium supplemented with the amino acids and the nucleotide to support growth of the auxotrophic strains (MM4). MM4 contained 1.7 g/L yeast nitrogen base without amino acids and ammonium sulphate , 20 g/L glucose , 2.65 g/L  at the desired concentration. The starting OD600nm of 0.05 (lane a in the figures), followed by two serial dilutions of 1:5 each (lanes b and c). These cell suspensions were plated as 4 µL spots onto the surface of MM4 pH 4.5 solid medium, supplemented or not with acetic acid. Complementation tests were perfomed with yeast cells harboring either the empty vector pRS416 or the recombinant plasmid pYCG_PDR18. For the complementation tests, spot assays were prepared as described before and cell suspensions were spotted onto MM4-U agar plates that were incubated at 30 °C for 72 hours.Yeast cell suspensions used for the spot assays were prepared from a mid-exponential cell culture grown in MM4 medium, diluted to an OD80. The real-time Reverse Transcription–PCR protocol used followed the manufacturer’s instructions  and the primers used for the amplification of each target cDNA were designed using the Primer Express software   under unstressed conditions was set as 1 and the remaining values are relative to that value.For gene transcription assays, BY4741 cells were harvested at adequate time points from liquid medium cultivations performed in the presence and absence of 60 mM acetic acid, as described above. Total RNA was extracted by the hot phenol methodpdr18Δ strains were cultivated in MM4 either in the presence or absence of acetic acid stress (60 mM at pH 4.0) as described above. Exponentially-growing cells were harvested by centrifugation , resuspended in homogenization buffer containing 50 mM Tris/HCl (pH 7.5), 2.5 mM EDTA and a protease inhibitor cocktail . Cells were broken by vortex-mixing with glass beads (Glaperlon 0.40–0.60 mm). The cell membranes were recovered by centrifugation  to remove unbroken cells and finally the cell membranes were pelleted by ultracentifugation at 25 000 rev./min for 1 hour . The pelleted cell membranes were resuspended in a buffer containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 20% glycerol and protease inhibitors at the concentrations mentioned above, stored frozen and freeze-dried until sterol extraction. Sterol extraction involved addition of 250 μL 5-α-cholestane  in dichloromethane and a lipid extraction using 600 μL methanol (Merck) and 300 μL dichloromethane (Merck). Samples were homogenized for 60 seconds at 2–4 °C in a homogenizer  at 10,000 rpm. Afterwards, 300 μL ultra-pure water and 300 μL dichloromethane were added. Samples were centrifuged . The lower phase was filtered through an anhydrous sodium sulfate (Merck) layer and solvent evaporated under N2 at 20 °C. Lipids were saponified with 500 μL of 2.5 N KOH (Merck) at 80 °C for 1 hour. Saponified lipids were extracted with 1 ml of saturated NaCl (Merck) and 5 ml hexane (Merck). Organic phase was evaporated under N2 at 20 °C. Derivatization was done with 100 μL N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (Sigma) at 80 °C for 12 hours. Derivatized sterols were analyzed using GC-MS  with a DB5 column . The carrier gas was helium (flow rate of 1 mL/min). Oven temperature was held at 120 °C for 1 min, increased to 300 °C at a rate of 2 °C/min, and held at 300 °C for 20 min. Injection and detector temperatures were 300 °C. Peak identification was based on retention time and comparison with external standards in the spectral database. Quantification was achieved through the internal standard peak area. Sterol concentration was normalized taking into account phosphate content in the freeze-dried samples.For quantification of ergosterol in yeast cell membranes, the parental and pdr18Δ strains either in the absence or presence of acetic acid, by the normalized ratio of Laurdan fluorescence emission measured in two channels. Growth curves were performed as described above and cells at mid-exponential phase (OD600nm = 0.5) were centrifuged , washed three times and resuspended in 200 µL PBS buffer . These cell suspensions were deposited in a glass 8-wells chamber  treated with poly-L-lysine  for better adherence of living yeast cells. The incubation with 5 µM Laurdan  was performed at room temperature for 1 hour followed by two washing steps with PBS. Images were obtained by two-photon excitation fluorescence microscopy on a Leica TCS SP5  inverted confocal microscope (DMI600), using a Ti:sapphire laser  as the excitation source, and an apochromatic water immersion objective . The excitation wavelength was set to 780 nm and the fluorescence emission of Laurdan was collected at 400–460 nm and 470–530 nm. Laurdan exhibits shifts in its fluorescence emission maximum in response to changes in membrane hydration and solvent relaxation and this shift can be estimated through a normalized ratiometric measurement of the fluorescence intensity recorded in the two spectral channels, the generalized polarization (GP) value81. GP images were obtained and analysed using homemade software developed in a MATLAB environment , with the GP value defined as GP = (I400–460 − G·I470–530)/(I400–460 + G·I470–530). Both channel intensities are corrected for background contributions, and the calibration factor G was obtained from imaging Laurdan in DMSO using the same experimental conditions as those set for the samples under study82.Membrane lipid order was estimated in exponentially-growing cells of parental and pdr18Δ grown in the presence or absence of acetic acid were estimated based on the shift in the maximum emission wavelength of the fluorescent probe DiS-C3(3) (3-3′-Dipropylthiacarbocyanine iodide), as described before83. For the timepoints tested, yeast cells were harvested as described above for other assessments and resuspended in citrate-phosphate (CP) buffer to a final OD600nm of 0.5. 5-mL aliquots of cell suspensions were labelled with DiS-C3(3) (Sigma-Aldrich) at a final concentration of 10−7 M, for 30 minutes at room temperature with orbital agitation of 70 rpm. Carbonyl cyanide p-chlorophenylhydrazone (CCCP) was added to an exponentially-growing culture of parental strain cells, to a final concentration of 5 µM, and this condition was used as a positive control for full membrane depolarization84. Fluorescence emission spectra of the cell suspensions were collected from 555 to 790 nm (at wavelength excitation of 531 nm) in 0.5 cm × 0.5 cm quartz cuvettes using a SLM-Aminco 8110 Series 2 spectrofluorometer .Differences in the plasma membrane potential of the parental strain BY4741 and the derived deletion mutant pdr18Δ cells either in the presence of absence of 60 mM acetic acid. PI was added to 1 mL of 4 × 107 cells/mL to a final concentration of 20 µM, cell suspensions and incubated in the dark with orbital agitation . Cells exposed to PI were centrifuged , washed twice and ressuspended in PBS buffer to a final 107 cells/mL aliquots. PI-fluorescence emission spectrum  were obtained using a SLM-Aminco 8100 series 2 spectrofluorometer with double excitation and emission monochromators MC400 , in a right-angle geometry. The light source was a 450 W Xe arc lamp and the reference a Rhodamine B quantum counter solution. All measurements were performed in 0.5 cm × 0.5 cm quartz cuvettes and under a constant temperature of 30 °C by using a thermostated sample holder with a Julabo circulating water bath . Gain adjustment of the apparatus was performed using a positive control for full permeabilization . The spectra obtained were integrated and the area under the curve was calculated.Plasma membrane permeability was assessed by the passive uptake of propidium iodide , from cells harvested at adequate time points of the cultivation of parental strain and To evaluate the population heterogeneity for the cell permeability trait, at adequate time points the cell population strained with PI was observed using an Axioplan microscope equipped with adequate epifluorescence interface filters . Fluorescence images were obtained with a cooled charge-coupled device camera , and the images were analyzed with MetaMorph, version 3.5. Cell-to-cell fluorescence intensity was defined as the average of pixel by pixel intensity in the selected region of interest and a minimum of 50 cells/experiment were used. The fluorescence images were background corrected by using dark-current images.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Material


Microcebus murinus) is a primate model of cerebral aging which can also present with amyloid plaques and whose Aβ is highly homologous to humans'. In an attempt to characterize this primate model and to evaluate the potential of plasma Aβ as a biomarker for brain alterations, we measured plasma Aβ40 concentration in 21 animals aged from 5 to 9.5 years. We observed an age-related increase in plasma Aβ40 levels. We then evaluated the relationships between plasma Aβ40 levels and cerebral atrophy in these mouse lemurs. Voxel-based analysis of cerebral MR images , showed that low Aβ40 levels are associated with atrophy of several white matter and subcortical brain regions. These results suggest that low Aβ40 levels in middle-aged/old animals are associated with brain deterioration. One special feature of mouse lemurs is that their metabolic and physiological parameters follow seasonal changes strictly controlled by illumination. We evaluated seasonal-related variations of plasma Aβ40 levels and found a strong effect, with higher plasma Aβ40 concentrations in winter conditions compared to summer. This question of seasonal modulation of Aβ plasma levels should be addressed in clinical studies. We also focused on the amplitude of the difference between plasma Aβ40 levels during the two seasons and found that this amplitude increases with age. Possible mechanisms leading to these seasonal changes are discussed.Accumulation of amyloid-β (Aβ) peptides in the brain is a critical early event in the pathogenesis of Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. There is increasing interest in measuring levels of plasma Aβ since this could help in diagnosis of brain pathology. However, the value of plasma Aβ in such a diagnosis is still controversial and factors modulating its levels are still poorly understood. The mouse lemur ( After t42 and/or Aβ40 levels and white matter lesions  and lacunar infarcts , a small (70–150 g) primate model of cerebral aging. It has a short lifespan of ~12 years in captivity  and confirmed previous studies demonstrating higher plasma Aβ levels in the oldest animals. MR images were recorded for each animal in order to evaluate relationships between plasma Aβ40 levels and cerebral pathology. A voxel-based morphometry (VBM) study revealed a relationship between lower plasma Aβ40 levels and atrophy of white matter and subcortical brain regions. We also investigated the seasonal rhythmicity of plasma Aβ40 in mouse lemurs by measuring its concentration during two consecutive seasons. Mouse lemurs displayed strong seasonal variations of plasma Aβ40 levels with an increase in winter. We examined the amplitude of the difference between plasma Aβ40 levels in the two seasons and found that this amplitude increased with aging.In the present study, we assessed plasma AβThis study was carried out in accordance with the recommendations of the European Communities Council directive (2010/63/EU). The protocol was approved by the local ethics committees CEtEA-CEA DSV IdF . All mouse lemurs were born in a laboratory breeding colony , and maintained at steady ambient temperature (24–26°C) and relative humidity (55%). Their seasonal and daily rhythms were controlled by an artificial photoperiod regimen of alternating 6 month seasons of short day lengths  and long day lengths . The shift from one season to the other was made without progressive transition. Animals were fed with fresh fruits, milky mixture  and meal worms.Twenty-one mouse lemurs (5–9.5 years old) were involved in this study. They were split into two groups according to age at first blood sampling: middle-aged  and old . Two blood samples were collected from each animal: the first one was taken during winter, 1 month after the shift in the season, and the second one was obtained 6 months later during summer. MRI images were recorded for each animal.40 was measured as previously described  tests  following the manufacturer's protocol for non-diluted plasma samples. With this kit, 50 μl of plasma is required to perform one measure. Antibodies used in these kits are coated with monoclonal antibodies against the N-terminal part of human Aβ and the secondary antibodies are rabbit antibodies specific for the C-terminal part of the human Aβ40 sequence. The calibration standards (synthetic Aβ40 peptides) were provided with the kits. The calibration standards and negative controls always performed within the manufacturer's range and samples' Aβ concentrations always stood within the calibration range.As mouse lemurs constitutively produce Aβ peptides, their plasma Aβ concentrations are much lower than those of transgenic mice overexpressing mutated forms of APP, so mouse lemur Aβ2, matrix = 128 × 128, slice thickness = 230 μm, acquisition time = 32 min).Cerebral imaging was performed by MRI in all the animals involved in the study. Brain images were recorded on a 7.0 Tesla spectrometer  using a four channel phased-array surface coil  actively decoupled from the transmitting birdcage probe . Briefly, animals were anesthetized by isoflurane (4% for induction and 1–1.5% for maintenance). Respiratory rate was monitored to ensure animal stability until the end of the experiment. Body temperature was maintained by an air heating system. Two-dimensional fast spin echo images were recorded with an isotropic nominal resolution of 230 μm  with the SPMmouse toolbox (http://spmmouse.org) dedicated to animal brain morphometry  corresponding to tissues with cortical gray matter (GM), white matter and subcortical nuclei (WM-SC), and cerebrospinal fluid (CSF) characteristics, using locally developed priors. The intensity of the pixel in each probability map represents the probability of the pixel to be GM, WM, or CSF. Then brain images and tpm were spatially transformed to the standard space, defined by Sawiak et al., using a GM mouse lemur template  was designed to evaluate relative changes in GM and WM-SC tpm values, a parameter reflecting atrophy, as a function of age. The sex of the animals and total intracranial volumes (TIV) were considered in the design matrix and were treated as covariates of no interest. More specifically, with the GLM, if the brain of one animal is defined by the number “xj,1 corresponds to the age of each animal j. Sj,1 and Sj,2 correspond to the sex of the animals. Sj,1 = 1 if the animal j is a male and = 0 otherwise; Sj,2 = 1 if the animal j is a female and = 0 otherwise.With 40 levels. Winter and summer plasma Aβ40 levels were added in the model. For this study, age, sex and TIV were considered in the design matrix and were treated as covariates of no interest. More specifically, with this second GLM, the signal within a pixel ; 40 levels; 40 levels; xj,1, xj,2 and xj,3 correspond to the age, winter plasma Aβ levels and summer plasma Aβ levels for each animal j. Sj,1 and Sj,2 correspond to the sex of the animals. Sj,1 = 1 if the animal j is a male and = 0 otherwise; Sj,2 = 1 if the animal j is a female and = 0 otherwise.With 40 levels = 75.2 and 35.5 pg/ml with a TIV of 2150 mm3):As an example, on the basis of the GLM-2 model, for the first animal for the second animal , with extent threshold values of 500 voxels, meaning that clusters required 500 contiguous voxels to be selected as relevant . P < 0.05 was set as the level of statistical significance for each test.Paired Student's 40 was evaluated in 21 middle-aged or old mouse lemurs in winter and summer  and 64% (p = 0.024) in winter and summer, respectively  and summer  periods  was found in old animals  or plasma Aβ40 levels  and cerebral atrophy [assessed from reduction in tissue probability maps (tpm)]. We did not detect any significant relationship between the age of the animals and gray matter, white matter or subcortical nuclei atrophy . We found a negative relationship between plasma Aβ40 levels and white matter and subcortical nuclei atrophy when both summer and winter Aβ40 were included in the model  increases with age. Finally, evaluation of cerebral atrophy showed that aging was not associated with cerebral atrophy in the middle aged/old animals from this study. Interestingly, when data were adjusted for age, low Aβ40 levels were associated with white matter and subcortical nuclei atrophy. Thus, at a given age, animals with lower plasma Aβ40 levels were more atrophied than animals with higher Aβ40 values.First, we found an age-related increase in plasma Aβ40 levels than middle-aged ones confirms a trend reported in a previous study in lemurs . ΔAβ40 increased with age. This result was not expected as the amplitude of most seasonal cycles  tends to decrease with aging in mouse lemurs . Aging did not modulate cerebral atrophy in our study either. Thus, the relationship between plasma Aβ40 and atrophy was independent of aging. However, our study suggests that plasma Aβ40 measures alone should not be taken as a surrogate marker for cerebral atrophy as it evolves with the age of the animals. In other words, plasma Aβ40 increases with aging and at a given age lower Aβ40 levels are associated with cerebral atrophy. In a previous study, we showed that low plasma Aβ40 levels in mouse lemurs are associated with high cerebral intracellular labeling with antibodies specific to amyloid and APP  due to vascular alterations. The authors suggested that the association of low plasma Aβ40 levels with increased WMH may reflect increased deposition of Aβ40 in cerebral vessel walls after its clearance, resulting in impaired cerebral blood flow (Kaffashian et al., 40 lowering in the atrophied animals, new investigations are now required. Developing techniques for perfusion measurements combined with brain vessel histopathology should be an elegant way to link plasma Aβ and brain morphology variations and to determine the potential of plasma Aβ40 as a biomarker of brain pathology.Our result showing that low Aβ40 is modulated by seasonal variations. Seasonal effects should thus be taken into account when studying plasma Aβ, in preclinical as well as in clinical studies. Since seasonal changes are amplified in mouse lemurs, they appear to be a useful model for evaluating the impact of seasons on amyloid metabolism. Further mechanistic studies should now be initiated to understand the origin of the link between plasma Aβ40 concentrations and cerebral atrophy as well as of the plasma Aβ changes associated with seasonal variations. We also observed that in normal conditions and after adjustment for age, low plasma Aβ40 is correlated with brain tissue atrophy in several white matter and subcortical regions including the corpus callosum, internal capsule, putamen, globus pallidus, thalamus and geniculate nucleus. The origin of these changes remains to be evaluated but supports a strong link between plasma Aβ40 concentrations and brain morphometry.To conclude, we confirmed an age-related increase of plasma Aβ and showed for the first time that plasma AβCG and MD designed the study; CG and ZH performed the blood sampling; CG and A-SH performed the biochemical analysis; CG, A-SH, and MD were responsible for statistical analyses and wrote the manuscript; A-SH and MD performed the MRI voxel wise analysis; ZH revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cortical surface recording techniques such as EEG and ECoG are widely used for measuring brain activity. The prevailing assumption is that surface potentials primarily reflect synaptic activity, although non-synaptic events may also contribute. Here we show that dendritic calcium spikes occurring in pyramidal neurons (that we showed previously are cognitively relevant) are clearly detectable in cortical surface potentials. To show this we developed an optogenetic, non-synaptic approach to evoke dendritic calcium spikes in vivo. We found that optogenetically evoked calcium spikes were easily detectable and had an unexpected waveform near the cortical surface. Sensory-evoked dendritic calcium spikes were also clearly detectable with amplitudes that matched the contribution of synaptic input. These results reveal how dendritic calcium spikes appear at the cortical surface and their significant impact on surface potentials, suggesting that long-standing surface recording data may contain information about dendritic activity that is relevant to behavior and cognitive function. Surface EEG recordings are thought to primarily detect synaptic activity. Here the authors devise an optogenetic method to evoke dendritic calcium spikes in layer 5 pyramidal cells of the rat somatosensory cortex, and report that optogenetically evoked, as well as sensory-evoked dendritic calcium spikes make a significant contribution to surface EEG recordings. The standard textbook explanation of surface potentials is that they primarily reflect cooperative postsynaptic activity2 because synaptic currents are assumed to be the dominant electrical current source5 (but see ref. 6 as an example of non-synaptic EEG component). However, other current sources, such as the active properties of cortical pyramidal cell dendrites could potentially contribute7. For instance, voltage-gated calcium (Ca2+) channels around the main bifurcation point of apical dendrites (550–900 μm from the soma in rats8) give rise to characteristic depolarizing plateau potentials (dendritic Ca2+ spikes)9. These dendritic Ca2+ spikes can be generated spontaneously or by sensory inputs10, last for 50 ms in anesthetized animals8 and up to several seconds in awake behaving rats11, and represent a large current source12. A recent study further demonstrated the correlation between dendritic Ca2+ spikes and animal behavior13. Despite their magnitude, functional significance, and proximity to the cortical surface, it remains unclear how dendritic spikes contribute to surface potentials, although it has been observed that dendritic spikes can influence the local field potential (LFP)15. We sought to determine the extent to which dendritic Ca2+ spikes contribute to the surface potentials through combined use of multi-channel extracellular recordings, optogenetics, pharmacology, and a “micro-periscope” that allows layer-specific light delivery.For nearly a century, brain surface recording techniques such as electroencephalogram (EEG) and electrocorticogram (ECoG) have been the most widely used technique for recording brain activity16. The fluorescence image of the cortical slices taken from the recorded rats showed the confined viral expression in L5 , we recorded the extracellular local field potentials (LFPs) in the hindlimb area of the primary somatosensory cortex in the anesthetized rats, while optogenetically stimulating layer 5 (L5) of the cortex Fig. . Layer-s L5 Fig. .Fig. 1Op2+ spikes can be evoked in L5 pyramidal neurons by depolarizing the apical dendritic initiation zone17. Light pulses delivered to L2/3 caused a sink in these layers, however we chose not to use this approach because it was difficult to separate the effect of the ChR2 current from activation of voltage-sensitive postsynaptic currents. We therefore chose a second method for evoking dendritic Ca2+ spikes based on the “critical frequency” approach that uses high-frequency backpropagating action potentials (bAPs) generated at the soma19. This method has been shown to activate the same dendritic Ca2+ channels as direct dendritic depolarization in vitro20 and has been shown to be effective in vivo21. By activating ChR2 in L5, we avoided the confounding influence on the LFP that would otherwise have occurred by direct activation of ChR2 in the dendrites . In other words, we could remotely activate the L5 dendrites without directly stimulating the apical dendrites either optically or synaptically.Can = 17 animals) % of trials with maximum light amplitudes, we observed an additional sink in the upper and middle layers at distances corresponding to the typical location of dendritic Ca2+ spikes8 ~ 20 ms after the offset of the light stimulation18. Interestingly, the current sink in L2/3 was reliably accompanied by a current source in L1 . In the remaining cases, there was no discernable late sink at all  delivered to L5 reliably caused a current sink in L5 in all cases Fig.  but also L1 Fig. , right aall Fig. . The sin0 ms deli2+ spike including location, timing and a sharp dependence on the strength of somatic activation. To investigate this possibility, we performed a number of tests. First, we repeated the optogenetic stimulation experiments combined with a second micro-periscope for recording Ca2+ fluorescence in L2/3  with a long excitation wavelength (550 nm) to avoid direct activation of ChR2 was injected to L5. The light through this micro-periscope was shone tonically throughout the experiment so that any residual effect was not time locked to the ChR2 stimulation in L5. Here, optogenetic activation of L5 caused an all-or-none fluorescence transient in L2/3 (at higher stimulus strengths) similar to the current sink seen earlier  into L2/3 and L5. In these experiments, there was no change in the amplitude of the current sink in L2/3 . We therefore conclude that the late current sink in L2/3 was produced by currents in the dendrites of L5 pyramidal neurons.The sink in L2/3 therefore bore all the hallmarks of a dendritic Ca2/3 Fig. , left. Fier Fig. , right. ier Fig. . This is2+ spikes are effectively blocked by the GABAB receptor agonist baclofen25 due to down-regulation of Cav1 (L-type) channels12. Here, we found that the dendritic current sinks significantly decreased by local injection of 50 μM baclofen to L2/3  30 that is spontaneously open, and previous in vitro studies showed that Ih interacts with dendritic Ca2+ spikes33. This spontaneous leak current may account for the pronounced source of passive current. We tested this possibility by local application of Ih blocker ZD7288 (500 μM) on the cortical surface  the evoked surface potentials were clearly detected at multiple electrodes that were 500 μm apart. The surface potentials were also baclofen-sensitive  with similar characteristics . We conclude that apical dendritic Ca2+ spikes are detectable as positive potentials at the cortical surface in both anesthetized and awake brain.The appearance of a passive current source in the superficial layers correlating with the Caace Fig. . After Zray Fig. , we detepia Fig. . In all 2+ spikes on surface potentials in the absence of synaptic input to the dendrite (i.e. evoked by optogenetic stimulation of L5), we turned our attention to the effect of sensory-evoked Ca2+ spikes on surface potentials. We recorded LFPs, surface potentials, and Ca2+ fluorescence in the same cortical area—the hindlimb area of the primary somatosensory cortex—while electrically stimulating the contralateral hindlimb  this second current sink was abolished by local injection of baclofen in L2/3 . Moreover, the second surface spike was significantly wider than the first ; then we tested the classifier with the remaining half , the classifier predicts that the surface potentials correspond to Ca2+ spikes with 73.9% (34/46) accuracy  accuracy  implying that the late synaptic input is more or less invisible in the LFP unless it reaches threshold for a postsynaptic event like a dendritic Ca2+ spike. On the other hand, the fact that the late sink we observed, when visible, had the same amplitude as the first sink, implies that the Ca2+ spike produces extracellular signals of similar magnitude to very synchronous synaptic inputs.Both EPSPs and single or low-frequency bAPs have a comparably small effect on intracellular Ca44 whereas the late sink we recorded spread from depths that correspond to the location of the Ca2+ spike initiation zone in L5 pyramidal neurons8. Although the data suggest that the late extracellular sink is primarily due to dendritic Ca2+ spikes which represent a high conductance across a large area of membrane, it is still possible that the initiation of these spikes requires some combination of EPSPs, bAPs and/or NMDA spikes41 that are each likely to have a smaller impact on the extracellular potential.NMDA spikes are another form of postsynaptic dendritic spikes. However, they are normally confined to individual tuft branches4. Our study demonstrates that dendritic Ca2+ spikes in L5 pyramidal neurons can also evoke surface positive potentials that are as large as those evoked by EPSPs . In this case, the predictive power of this approach would be improved by first characterizing the surface potential resulting from evoked potentials.The conventional view is that the surface positive potential primarily reflects a current source passively generated by a current sink corresponding to EPSPs in pyramidal neurons arriving in L2/3 Fig. , left4. SPs Fig. , right. 2+ spikes while recording LFPs, surface potentials, and Ca2+ fluorescence. We showed that these optogenetically evoked dendritic Ca2+ spikes can be detected at the cortical surface as positive potentials and that sensory-evoked potentials can also exhibit the same characteristics. We conclude that L5 pyramidal neurons are not simple passive dipoles but exhibit a more complex, dynamically changing laminar profile of current sinks and sources due to dendritic Ca2+ spikes. These results have strong implications for the interpretation of surface potentials that till now have not taken dendritic Ca2+ spikes into consideration.In summary, we have developed a new method for optogenetically evoking dendritic CaAll experiments and procedures were approved and conducted in accordance with the guidelines given by the veterinary office of Landesamt für Gesundheit und Soziales Berlin.2, vol/vol, Abbott) before ketamine/xylazine anesthesia  was administered intraperitoneally and lidocaine  was injected around the surgical site. Body temperature was maintained at ~ 36 °C by a heating pad and the depth of anesthesia was monitored throughout virus injection. Once anesthetized, the head was stabilized in a stereotaxic instrument . The skull was exposed by a skin incision and a small hole (~ 0.5 × 0.5 mm2) was made above the hindlimb area of the primary somatosensory cortex (1.5 mm posterior to bregma and 2.2 mm from midline). AAV1.CamKIIa.hChR2(H134R)-eYFP.WPRE.hGH (Addgene 26969 P) or AAV1.CamKII.GCaMP6f.WPRE.SV40 purchased from the University of Pennsylvania Viral Vector Core was backloaded into a micropipette (Drummond) and was slowly injected  to L5. The pipette remained there for another 5 min after injection. The skin was sutured after retracting the pipette.Female Wistar rats (P21–23) were initially anesthetized with isoflurane (~ 2% in O2 micro right-angled prism (Edmund Optics), a 100 μm core multi-mode optical fiber , a custom-designed Grin lens , were assembled in-house using a UV curable adhesive (Noland). The other end of the optical fiber was coupled with a blue LED . The fiber was held and positioned by a stereotaxic micromanipulator .As shown in Supplementary Fig. 2+ spikes occurred. The resulting probability with the highest light intensity (12 mW/mm2) ranged from ~ 30 to ~ 100%.Optical stimulation of L5 through the micro-periscope was controlled by Power1401 and Spike 2 software (CED) and synchronized with the neural recording system via TTL signals. The duration of optical stimulation started at 10 ms and, if necessary, increased up to 30 ms to maximize the probability that dendritic Ca2, vol/vol, Abbott) before urethane anesthesia (0.05 mg per kg of body weight) in anesthetized experiments or ketamine/xylazine anesthesia  in awake experiments was administered intraperitoneally and lidocaine  was injected around the surgical site. Body temperature was maintained at ~ 36 °C by a heating pad and the depth of anesthesia was monitored throughout experiment. Once anesthetized, the head was stabilized in the stereotaxic instrument and the skull was exposed by a skin incision. A ~ 1.5 × 1.5 mm2 craniotomy was made above the hindlimb area of the primary somatosensory cortex and the dura matter was removed. The exact location of the hindlimb area was mapped using the intrinsic optical imaging45 or flavoprotein autofluorescence imaging technique46. The area was kept moist with rat ringer for the entire experiment . In LFP recordings a linear array of 16 electrodes  was perpendicularly inserted into the area such that the uppermost electrode was positioned at 100 μm below the pia. In surface potential recordings a 4 by 4 surface electrode array  was placed above the cortical area. In awake experiments the craniotomy was covered by silicone , and the micro-periscope and the surface electrode were fixed to the skull with dental cement. After recovery the animal was placed in a rectangular arena (60 cm × 40 cm) and was free to move thereafter. Electrical activity was bandpass-filtered at 1–9 K Hz, digitized at 10 K Hz, amplified by ERP-27 system and Cheetah software . LFPs were obtained by low-pass filtering the activity with the cutoff frequency of 300 Hz. Multi-unit activity was measured with a single tungsten electrode (~ 0.5 MOhm) and extracted through a bandpass filter (300–3 KHz).Animals were initially anesthetized by isoflurane /F0, where F is the fluorescence intensity at any time point and F0 is the average intensity over the prestimulus period for 100 ms. The eYFP fluorescence upon excitation was measured before loading the calcium indicator and was subtracted from ΔF/F.As shown in Supplementary Fig. 2+ fluorescence was measured using a custom-built two-photon microscope with B-scope , a water immersion objective  and a titanium sapphire laser . Animals were either transfected with AAV1.CamKII.GCaMP6f.WPRE.SV40 or injected with Cal-520 AM (AAT Bioquest) in L5 as described in the previous sections. A craniotomy (diameter ~ 3 mm) was made under Urethane-anesthesia over the hindlimb area of the primary somatosensory cortex and the dura matter was removed. To simultaneously record surface potential, a glass electrode (0.5–1 MOhm) was placed under the cover glass (diameter 3 mm) such that the tip was at the location where the maximum sensory response was observed in the intrinsic or flavoprotein autofluorescence imaging. The cover glass and the recording pipette were glued to the skull. Ca2+ fluorescence was measured at a frequency of 100.30 Hz (142 × 512 pixels) by a GaAsP photomultiplier tube . ΔF/F was calculated as (F-F0)/F0, where F is the fluorescence intensity at any time point and F0 is the average intensity over the prestimulus three data points (duration 29.91 ms).Car > 0.8) paired measurements of two-photon dendritic ΔF/F and surface potential averaged over 50–60 ms were halved into the training and the validation data (90 measurements for each). Fluorescence noise was defined as M0 + SD0 where M0 and SD0 are, respectively, the mean and the standard deviation of the fluorescence change when surface potentials were zero. A lower threshold θ1 was derived from the intersection of the horizontal line (M0 + SD0) with the line fitted to the training data. A higher threshold θ3 was derived from the intersection of the line (M0 + 3SD0) with the fitted line. The performance of the classifier was measured using the validation data set.Correlated  and slowly injected  to their target cortical layer. The pipette was angled to avoid damaging the dendrites under study.Sensory stimulation was provided by a single short electrical pulse  given to the contralateral hindpaw through a pair of conductive adhesive strips (Skintact).48, inverse CSD method50 was used due to its significant advantages. Principal component analysis was performed on all LFP waveforms recorded at 600 μm below the pia and only the first and second principal components were used for cluster analysis.Analyses were conducted using Matlab (Mathworks). In optogenetic experiments current source density (CSD) analysis was performed on the average of all 100 evoked potentials at 16 cortical depths. In sensory stimulation experiments 100 measurements were divided into two groups, with or without the late sink; the late sink was considered present if the peak exceeded 3 × s.d. of the prestimulus activity. Instead of the original CSD methodt tests or Wilcoxon tests at a significance level of 0.05. Each statistical test was chosen based on the data distribution using histograms. No statistical method was used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field. The variance was generally similar between groups under comparison. No blinding/randomization was performed.Unless otherwise stated all values are indicated as mean ± s.e.m. and significance was determined by two-tailed, paired The data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


This study provides bench marks for the expected abortion/absorption rates following the early ultra-sonographic diagnosis of twin pregnancies in comparable populations and supports earlier observations that the expected  sex ratio for twinning approximates to1♂♂:2♂♀:1♀♀.The purpose of this study was to investigate the proportion of twins carried to term and the sex ratio of twin calves at birth in Holstein-Friesian dairy cattle kept on commercial farms in Devon and Cornwall, England. Ten farms were used in the study. Fifty four cows with twin pregnancies were identified using trans-rectal ultra-sonographic examination between 30 and 70 days of gestation. The farm records were subsequently used to derive the number of calves born. Farm records of 66 additional sets of twin births with the sex of the calves recorded were also identified. Of the 54 cows diagnosed with twin pregnancies, 16 cows (29.6%) aborted or absorbed both fetuses, 11 cows (20.4%) carried one calf to term and 27 cows (50%) carried both calves to term. In the calf sex analysis of the additional 66 sets of twins: 13♂♂ calves (19.7%), 18 ♀♀ calves (27.3%) and 35 ♂♀ calves (53.0%). There was no statistically significant difference from an expected ratio of 1♂♂:2♂♀:1♀♀ ( Twinning in dairy cattle is associated with higher milk yields , an incrThe purpose of this study was to investigate the proportion of twins carried to term and the calf sex ratio of twin calves in Holstein-Friesian dairy cattle kept on 10 commercial farms in Devon and Cornwall, England.Ten Friesian-Holstein dairy farms in Devon and Cornwall were used in this study. Herd size ranged from 70 to 310 animals with a mean of 168. None of the farms instigated a specifically designed management protocol to deal with cows identified as carrying twins. Fifty four cows with twin pregnancies were identified during routine trans-rectal ultra-sonographic examination for pregnancy. A 7.5 MHz linear probe was used. The operator was a large animal veterinarian experienced in ultra-sonographic pregnancy diagnosis. Twins were identified between 30 and 70 days of gestation according to the last recorded service date and a diagnosis of twinning was made on the observation of two embryos/fetuses. The farm records were subsequently used to derive the number of calves born following the ultra-sonographic diagnosis. Farm records of an additional 66 sets of twin births with the sex of the calves recorded were identified. A chi-squared test was used to analyse the data for a random distribution giving a gender ratio of 1♂♂:2♂♀:1♀♀.p = 0.61). Of the 54 cows diagnosed with twin pregnancies between 30 and 70 days of gestation, 16 cows (29.6%) aborted or reabsorbed both fetuses, 11 cows (20.4%) carried one calf to term and 27 cows (50%) carried both to term. In the calf sex ratio analysis of the additional 66 sets of twins there were 13♂♂ calves (19.7%), 18 ♀♀ calves (27.3%) and 35 ♂♀ calves (53.0%). There was no statistically significant difference when the numbers of calves within each category were compared to an expected ratio of 1♂♂:2♂♀:1♀♀  [A comparative study of gestation losses in beef cattle at the US Meat Animal Research Centre found that 12.2% of cows with twin pregnancies aborted or reabsorbed both fetuses and 4.6% aborted or reabsorbed one fetus . López-G. (2009)  reportedet al. (2008) reported a calf sex ratio in 4,045 twin births in Holstein cows in Iran of 28.2%♂♂:48.8%♂♀:22.9%♀♀ [A sex ratio of 1♂♂:2♂♀:1♀♀ was reported in a large population of Holstein cows . The cal:22.9%♀♀ .Within the power of this study the distribution of the sexes of the twins were consistent with this random distribution of the sexes. In a review of the literature of twinning in cattle 92.6% were classified as fraternal or dizygotic twins and 7.4% classified as monozygotic or identical twins ,14. TherThis study provides bench marks for the expected abortion/absorption rates following the early ultra-sonographic diagnosis of twin pregnancies in comparable populations and supports earlier observations that the expected sex ratio for twinning approximates to 1♂♂:2♂♀:1♀♀.


However, O2 gas annealing converts these superhydrophobic ZnO NWs into a superhydrophilic state. For switching from superhydrophobic to superhydrophilic state and vice versa in cyclic manner, H2 and O2 gas annealing treatment was used, respectively. A model based on density functional theory indicates that the oxygen-related defects are responsible for CA switching. The water resistant properties of the ZnO NWs coating is found to be durable and can be applied to a variety of substrates including glass, metals, semiconductors, paper and even flexible polymers.Here, we demonstrate a facile approach for the preparation of ZnO nanowires (NWs) with tunable surface wettability that can be manipulated reversibly in a controlled manner from a superhydrophilic state to a superhydrophobic state. The as-synthesized ZnO NWs obtained by a chemical vapor deposition method are superhydrophilic with a contact angle (CA) value of ~0°. After H Recently, smart surfaces with controlled reversible and switchable wettability by external stimuli have attracted significant attention due to their potential applications including self-cleaning surfaces, inkjet printing, microfluidic channels, and controllable drug delivery1245679101114151618192021In the past few decades, the wetting properties of metal-oxide-nanostructured surfaces have been widely studied. As a wide band gap semiconductor, ZnO possesses good transparency and high electron mobility, which make it suitable for a wide range of technological applications. Lee and co-workers have shown that electronic devices based on superhydrophobic ZnO nanorods overcome water vulnerability effects2428292/O2 gas annealing treatment, and reversible wettability switching is also possible. These superhydrophobic coatings of ZnO NWs can be applied to various types of substrates on which direct ZnO NWs growth via chemical vapor deposition (CVD) is not possible due to high growth temperature.This work presents a novel approach for fabricating superhydrophobic ZnO nanowires (NWs) and their reversible water wettability switching from a superhydrophilic to a superhydrophobic state. The surface wettability is shown to be controlled by simply tuning the oxygen-related defects. The ZnO NWs can be made superhydrophobic/superhydrophilic by Ha = 0.325 nm and c = 0.521 nm. The scanning electron microscopy (SEM) image of as-grown ZnO samples is shown in d = 0.26 nm corresponds to the (002) plane, which indicates the nanowires grow along the <001> direction. The point energy dispersive X-ray (EDX) pattern .  pattern  acquiredThe surface wettability of the samples was analyzed by measuring the average static contact angle (CA) for a sessile water droplet of 5 μl. The water droplet spreads completely on the as-synthesized ZnO NWs samples and the surface displays superhydrophilic behavior with a CA value of 0°. When these ZnO NWs samples were stored in the dark for about 50 days, the surface wettability changes drastically and the samples become superhydrophobic with a CA value of 155.7°. For reversible wettability switching, many reports in the literature have focused particularly on the light illumination method31323132TMiSTM50 system. These spectra were recorded in transmittance mode, in which ZnO NWs were scratched from the Si substrate and mixed with KBr to form a pellet. A series of absorption peaks from 600 to 4000 cm−1 are found corresponding to vibrations of various impurities such as hydroxyl (-OH), carboxylate (COO−) and alkane (C-H) present on the ZnO NWs surfaces. The FTIR spectra of these three samples are shown in −1 assigned to hydroxyls for both dissociated and molecularly adsorbed water. Peaks between 2830 and 3000 cm−1 are due to the C-H stretching vibration of alkane groups. The peaks observed at 1630 cm−1and 1384 cm−1 are due to asymmetrical and symmetrical stretching of carboxylate groups (COO−), respectively343537To develop a smart approach for preparing superhydrophobic ZnO NWs rapidly and economically without using any chemical coating, firstly we have to understand the water wetting mechanism on ZnO NWs. The water wetting of metal oxide surfaces is generally governed by the surface topography and the surface defects. In the present case, a nanowire-like morphology was used for water wetting measurements and the same type of samples show both superhydrophilic and superhydrophobic behavior under UV light illumination and dark storage. Hence, the surface morphology doesn’t play significant role in the observed wide variation of CA on ZnO NWs samples. A comparative defect analysis of three types of ZnO nanowire samples have been performed to analyze the defects responsible for the variation in the water wetting behavior with dark storage and UV treatments. The ZnO NWs samples used for this analysis were as-synthesized ZnO NWs (superhydrophilic), dark storage (50 days) ZnO NWs (superhydrophobic) and UV-treated (15 min) ZnO NWs (superhydrophilic). Fourier transform infrared spectroscopy (FTIR) was used to examine the defects radicals attached on the surface of the ZnO NWs. The FTIR measurements were conducted using a Thermo Scientific NicoletPhotoluminescence (PL) and Raman measurements were done on these samples to analyze the possible defects present in these ZnO NWs samples. For PL measurements, a Horiba Jobin Yvon Lab RAM (HR 800 Evolution) system was used with a He–Cd laser of 325 nm wavelength and 30 mW power as an excitation source. The room temperature PL spectra is shown in 40−1 corresponds to the second order Raman spectrum arising due to the E2 (high)-E1 (low) multiple scattering process of ZnO, 380 cm−1 corresponds to A1 (TO) and the main peak at 437 cm−1 is assigned to the E2 (high frequency) optical phonon mode of ZnO which is associated to oxygen atoms present in the ZnO1 (LO) mode at 583 cm−1 is associated with lattice defects as oxygen vacancies and zinc interstitials45−1 peak and the absence of the 583 cm−1 peak indicates the oxygen rich nature of the ZnO NWs in the as-synthesized and UV treated ZnO NWs samples, whereas the enhanced 583 cm−1 peak in dark storage sample indicates its oxygen deficient nature. The defects observed in the Raman spectra are in consistent with the PL results and suggest that the dark storage ZnO samples are relatively oxygen deficient compared to the as-synthesized and UV-light illuminated ZnO NWs samples. A comparative analysis of CA and oxygen-related defects on the ZnO NWs reveal that the ZnO NWs surface with rare oxygen vacancy defects is more favorable for water wetting as compared to oxygen deficient ZnO NWs surface.The Raman measurements were acquired by using a Horiba Jobin Yvon Lab RAM (HR 800 Evolution) system with an Ar-ion laser of 514 nm wavelength and 50 mW power. The Raman spectra of the as-synthesized, dark stored and UV-treated ZnO NWs samples are shown in k-mesh was generated by the Monkhorst−Pack method, and all results were tested for convergence with respect to the mesh size (6 × 6 × 1). In all calculations, self-consistency was achieved with a 0.1 meV convergence of the total energy and the force was minimized using a BFGS scheme up to 0.01 meV/Å tolerance value. Van der Waals corrections were taken into account as per the Tkatchenko−Scheffler schemeA first-principles based density functional theory (DFT) calculation was performed to understand the water wetting phenomenon on different types of ZnO NWs surfaces. The nanowires are randomly oriented, and hence, it is quite possible that different types of planes come into contact with the water droplet during contact angle measurements. The ZnO nanowires show the wurtzite crystal structure where generally four low miller index surfaces dominates, i.e., the nonpolar  and is perfectly stable at 300 K. We have tried to understand the interaction of the surface with water molecules. It is noted that for clean and O-deficient surfaces the water molecule was adsorbed on top of the Zn-site , there is a competition of sharing excess electron to the nearby-attached electropositive H-atoms between the ZnO surface O-atoms and the O-atom in H2O  and reducing (H2 gas) ambient conditions during the annealing treatment of the ZnO nanowires. The ZnO NWs sample are treated at 300 °C in the presence of H2 or O2 gases. A series of experiments were done to calibrate the annealing parameters like H2/O2 gas flow rate and annealing time to obtain wettability switching of the ZnO NWs. The annealing temperature was kept constant at 300 °C to avoid the effect of the change in surface roughness at higher temperature2 gas at 300 °C temperature for 90 min is sufficient to make the ZnO NWs superhydrophobic with a CA of about 153.5°. The superhydrophobic ZnO NWs are highly water repellent as shown by 2 gas at 300 °C for 60 min. Different flow rates of O2 gas were used for the annealing treatment and again a 50 sccm flow rate was found to be sufficient to regain the ZnO NWs superhydrophilic nature. The change in CA on the ZnO NWs with different annealing treatments is shown in 2 gas annealing treatment creates oxygen vacancy defects in metal oxides whereas the O2 gas annealing treatment passivates the oxygen vacancy defects525354The temperature-dependent stability of hydroxyl defects on the surface of the O-terminated  for 60 and 90 min. The CA values found on 60 and 90 min Ar annealed ZnO NWs samples were 29° and 35°, respectively. The Ar gas was used to create an inert atmosphere, which should eliminate the effect of oxidizing (O2) and reducing (H2) gases present in the atmosphere. These observations suggest that water wetting on the ZnO NWs is strongly influenced by the presence of the H2/O2 gases during the annealing treatment.The heating treatment on the metal oxide nanostructures affects the contact angle322/O2 gas annealing treatments are not only suitable for tuning the contact angle but reversing the wettability of the ZnO NWs is also possible (2 gas (50 sccm) annealing treatment at 300 °C for 90 min makes the samples superhydrophobic with average CA value of 153.5 °C and an O2 gas (50 sccm) annealing treatment at 300 °C for 60 min was observed to be sufficient to regain the superhydrophilic nature. Alternating the annealing treatment of the H2 and O2 gases produces superhydrophobic and superhydrophilic states of the ZnO nanowires, respectively. Up to 10 cycles, the annealing treatment seems to be suitable for completely reversible switching of the ZnO NWs between the superhydrophilic and superhydrophobic states. It is important to notice that the contact angle on superhydrophilic ZnO NWs samples is not stable. It increases with time and becomes hydrophobic (CA=123°) and superhydrophobic (CA=155.7°) when the samples are stored in daylight and under dark conditions, respectively  annealing treatment the ZnO NWs become superhydrophobic. However, the O2 gas (50 sccm) annealing treatment was observed to be suitable to revert the superhydrophilic nature of ZnO NWs. The controlled reversible wettability switching is demonstrated by the alternating annealing treatments of H2 and O2 gases. Also, it has been shown that the superhydrophobic ZnO NWs can be utilized for hydrophobization of different types of substrates. Furthermore, this novel approach might help to prepare superhydrophobic nanostructures of different metal oxides and their controlled reversible wettability switching for future applications.In conclusion, we have accomplished the goal of preparing superhydrophobic ZnO NWs and controlling their reversible wettability switching from superhydrophilic to superhydrophobic states, by a fast and cost-effective approach. The ZnO NWs were grown by CVD. The as-synthesized ZnO NWs show a superhydrophilic nature and by an HZnO powder (99.99%) was purchased from Sigma-Aldrich. Activated charcoal powder and ethanol were purchased from Merck Company.The ZnO NWs were synthesized using horizontal a chemical vapor deposition (CVD) system. A mixture of ZnO and charcoal powder in a ratio of (1:1) was taken as a precursor and loaded into the alumina boat at the center of the tube furnace. Si substrates coated with ~5 nm gold were placed downstream in the tube furnace at a distance of 1 inch from the precursor boat. The furnace was then heated up to 1050 °C under a constant flow (200 sccm) of Ar gas. The system was at 1 atmospheric pressure.TMiSTM50 system. UV-Vis transmittance measurements were done using a Shimadzu UV-3600. The wetting properties of the samples were studied by measuring the contact angle (CA) of a 5 μl water droplet on the samples at room temperature using an optical CA meter.The samples were characterized using ZEISS EVO 50 scanning electron microscope (SEM), glancing angle X-ray diffraction (GAXRD) using phillips X’Pert, PRO-PW 3040 diffractometer and Technai G20-stwin high resolution transmission electron microscope (HRTEM) at 200 K. Photoluminescence (PL) and Raman measurements were taken using a Horiba Jobin Yvon Lab RAM (HR 800 Evolution) system. For PL measurements, 30 mW He–Cd laser of 325 nm wavelength was used as an excitation source. For the Raman measurements, an Ar-Ion laser of 514 nm wavelength and 50 mW power was used. The Fourier transform infrared (FTIR) measurements were done using a Thermo Scientific NicoletAlicat Scientific mass flow controller (MFC). A heater was used to heat the sample up to 300 °C during the annealing treatment.A small chamber of dimensions 3 × 2.5 × 1.5 inchHow to cite this article: Yadav, K. et al. A fast and effective approach for reversible wetting-dewetting transitions on ZnO nanowires. Sci. Rep. 6, 35073; doi: 10.1038/srep35073 (2016).