Arabidopsis thaliana). Classifying these paralogous gene pairs into high, low, and no morphological diversification groups, based on knock-out data, we found that the divergence rate of both gene expression and protein sequences were significantly higher in either high or low morphological diversification groups compared with those in the no morphological diversification group. These results strongly suggest that the divergence of both expression and protein sequence are important sources for morphological diversification of duplicate genes. Although both mechanisms are not mutually exclusive, our analysis suggested that changes of expression pattern play the minor role (33%–41%) and that changes of protein sequence play the major role (59%–67%) in morphological diversification. Finally, we examined to what extent duplicate genes are associated with expression or protein divergence exerting morphological diversification at the whole-genome level. Interestingly, duplicate genes randomly chosen from A. thaliana had not experienced expression or protein divergence that resulted in morphological diversification. These results indicate that most duplicate genes have experienced minor functionalization.The differentiation of both gene expression and protein function is thought to be important as a mechanism of the functionalization of duplicate genes. However, it has not been addressed whether expression or protein divergence of duplicate genes is greater in those genes that have undergone functionalization compared with those that have not. We examined a total of 492 paralogous gene pairs associated with morphological diversification in a plant model organism ( Arabidopsis thaliana. Using these data, we found that the divergence of both expression and protein sequence were important sources for morphological diversification of duplicate genes. Although both mechanisms are not mutually exclusive, our analysis suggested that expression divergence is the minor contributor and protein divergence is the major contributor to morphological diversification. However, the expression or protein sequence of randomly chosen duplicate genes did not show significant divergence that resulted in morphological diversification. These results indicate that most duplicate genes experienced minor functionalization in the genome.The relationship between morphological and molecular evolution is a central issue to the understanding of eukaryote evolution. In particular, there is much interest in how duplicate genes have contributed to morphological diversification during evolution. As a mechanism of functionalization of duplicate genes, differentiation of both gene expression and protein function are believed to be important. Although it has been reported that both expression and protein divergence tend to increase as a duplication ages, it is unclear whether expression or protein divergence in duplicate genes is greater in those genes that have undergone functionalization compared with those that have not. Here, we studied 492 duplicate gene pairs associated with various degrees of morphological diversification in  Here, to address how duplicate genes have contributed to morphological evolution, we classified Arabidopsis duplicate genes into high, low and no morphological diversification groups based on knock-out data, and examined the divergence rates of both expression pattern and protein sequence among the three morphological diversification groups.From the literature and from our earlier work see  [25],[26R)/(1+R)), because the transformation is more sensitive for examining expression differences R)/(1+R)) values to paralogous gene pairs among the three morphological diversification groups, the log((1−R)/(1+R)) values increased as morphological diversification increased (R)/(1+R)) was divided by Ks. This is because expression divergence is expected to increase as duplication timing becomes earlier and Ks increases in a nearly linear fashion with duplication age Ed (log ((1−R)/(1+R))/Ks) is an indicator of the expression divergence rate between a paralogous gene pair: high and low Ed indicates high and low expression divergence at the same duplication age, respectively. When we calculated Ed between a paralogous gene pair in the three morphological diversification groups, Ed increased as morphological diversification increased  of the expression intensities among experimental conditions. Here, we transformed the value as log between paralogous gene pairs increased as morphological diversification increased , we examTo analyze the kinds of amino acid replacements that have occurred during morphological diversification, we classified all amino acid replacements as either ‘chemical radical’ or ‘conservative’ on the basis of an amino acid classification generated in an earlier report Ed and Ka/Ks ratios for paralogous gene pairs without morphological diversification were defined to be the threshold of higher divergence rate of expression pattern and protein sequences, respectively. We then counted the numbers of paralogous gene pairs with a higher divergence rate in each of the high and low morphological diversification groups  were obtained from TAIR (www.arabidopsis.org). Duplicate genes were defined as proteins that matched other proteins in a BLAST search with E<1×10−4http://micans.org/mcl/). In every pair of each family, we examined the amino acid identity and the coverage . We found 405 paralogous gene pairs with amino acid identity >0.3 and coverage >0.5. Since tandem duplicates have a higher chance of exhibiting similar expression due to leaky expression or conserved sequences by gene conversion than non-tandem duplicates We used data from the available literature and from our bank of previously generated T-DNA insertional mutants To examine the degree of morphological diversification between the genes of the paralogous gene pairs, we classified morphological changes into seed, vegetative and reproductive phenotypes, according to the definition of Meinke et al As a control set, we identified from the literature165 duplicate genes that did not show morphological diversification. Absence of morphological diversification was defined as the observation of morphological change only upon the deletion of multiple paralogs; deletion of each gene separately did not induce morphological change. After removing tandem paralogous gene pairs, we found 95 paralogous gene pairs with amino acid identity >0.3 and coverage >0.5 .http://www.arabidopsis.org/). We compiled 1280 microarray datasets under 634 conditions, consisting of 82 different developmental stages, 72 biotic treatments, 285 abiotic treatments, 11 nutrient treatments, 81 hormone treatments, 40 chemical treatments, 21 cell cycle stages and 42 different genotypes. The array intensities were processed with the Bioconductor (http://www.bioconductor.org) affy package in the R software environment (http://www.r-project.org). Specifically, the array intensities were adjusted to reduce background with the mas5 function, and the normalize quantiles function was used for between-array normalization. The background-corrected and background-normalized intensities were used for further analysis.We obtained Affymetrix ATH1 data from the AtGenExpress expression atlas at TAIR  A. thaliana genes because the methylation of 500 bp upstream regions is considered to be sensitive for gene expression We obtained the mapping data of known cis-regulatory elements in 1 kb promoter regions of all A. thaliana (TAIR7) were obtained from TAIR (www.arabidopsis.org). Pairwise alignment was performed with the program CLUSTALW to align coding regions Nucleotide sequences of A. thaliana genes using BLASTP We randomly chose genes from the total set of annotated A. thaliana genes (TAIR7). For a chosen gene, similarity searches were conducted against all annotated Figure S1Expression divergence, synonymous, and nonsynonymous distances among random paralogous gene pairs and among paralogous gene pairs with no, low, and high morphological diversification. (A) Relationship between expression divergence and morphological diversification (defined in the main text). Expression divergence is log ((1−R)/(1+R)), where R is the correlation coefficient of paralogous gene pairs among different experimental conditions. (B) Relationship between Ks and morphological diversification. (C) Relationship between Ka and morphological diversification. The random sample included 1000 pairs of paralogs. The distributions of expression divergence, Ks and Ka are shown as box plots with the solid horizontal line indicating the median value, the box representing the inter quartile range (25%–75%), and the dotted line indicating the first to the 99th percentile.(0.25 MB PDF)Click here for additional data file.Figure S2Divergence of cis-regulatory element and methylation of promoter regions among paralogous gene pairs with no, low, and high morphological diversification. (A) Relationship between proportion of overlapped cis-regulatory elements and morphological diversification. The proportion of overlapped cis-regulatory elements is the number of overlapped cis-regulatory elements over the number of observed cis-regulatory elements in promotor regions of two paralogous genes. (B) Relationship between proportional difference of methylation and morphological diversification. The proportional diffrerence of methylation is the difference of proportion of methylated cytosine in promoter regions of two paralogous genes. These distributions are shown as box plots with the solid horizontal line indicating the median value, the box representing the inter quartile range (25%–75%), and the dotted line indicating the first to the 99th percentile.(0.22 MB PDF)Click here for additional data file.Table S1Paralogous gene pairs with no, low, and high morphological diversification.(0.05 MB PDF)Click here for additional data file.Table S2Statistical difference  in (0.01 MB PDF)Click here for additional data file.Table S3Number of paralogous gene pairs with a high divergence rate of protein sequence  and/or expression  in the high and low morphological diversification groups.(0.03 MB PDF)Click here for additional data file.


In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK).  Bacillus anthracis, is a binary toxin consisting of the 83 kDa Protective Antigen (PA) and 90 kDa Lethal Factor (LF). PA alone is non-toxic, but serves to translocate LF to the cytosol via the endosomal pathway  signaling pathway. Our search showed that anthrax lethal toxin (LeTx) had a similar activity profile on cancer cell lines as the mitogen-activated protein kinase kinase (MAPKK or MEK) inhibitor PD98059 in vitro and in vivoWe have used LeTx in xenograft studies to test the requirement for MKK signaling in tumor growth and angiogenesis To overcome limitations inherent in analysis of neovascularization in tumor models, and to gain further insight into the mechanism of LeTx action on tumor endothelium, we examined the effects of LeTx treatment on the growth of vascularization in the murine retina. Retinal vasculature forms postnatally, in a well-characterized and highly reproducible manner, providing a unique, convenient system to evaluate contributions to vascular formation and development. Since vascular growth develops primarily in two dimensions, the retina provides a convenient model to directly observe developmental angiogenesis, as well as a useful model to monitor and quantify changes in vascular growth.By analogy to the tumor model, we hypothesized that application of LeTx would disrupt MAPK signaling and alter normal retinal vascularization, specifically during the angiogenic phase of vascular development. Consistent with this, injection of LeTx at a time when active MAPK could be found in angiogenic buds resulted in the inhibition in branching morphogenesis of vasculature in the inner plexus. However, this was followed shortly afterwards by extensive focal neovascular growth into the outer retina that was driven by elevated vitreal VEGF. These results indicate that MAPK signaling plays a key role in retinal angiogenesis and that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.The mouse retina provides a highly regulated and reproducible system for the study of vascular development. It is a useful model for understanding normal as well as pathological angiogenesis, and has been extensively used to study vascular remodeling and maturation  and then increases through the first week, peaking around PN10 and remaining elevated through PN21 . By contTo determine where active ERK is expressed during retinal angiogenesis, we manually dissected retinas and examined them by immunofluorescence, staining them with an antibody to phosphorylated ERK. We observed that pERK staining was most pronounced in sprouting vessels, beginning at PN7 (data not shown), and especially at PN10 . These dPseudomonas exotoxin A in vitro, and found that, compared to SK-MEL-28, RECs were relatively insensitive to LeTx treatment, even after 72 hr incubation  and following the maturation of retinal vasculature (PN21). When retinas from treated eyes were manually isolated and stained with isolectin GS-IB4 from Griffonia simplicifolia (GSA) to identify endothelial cells, we observed that retinas treated with FP59 showed vascular destruction at both timepoints  into the vitreous and examined the retinal vasculature 4 and 8 days later. Vitreal injections of CI-1040 did not mimic the changes in retinal vascularization we observed following LeTx treatment  and S3. The observed transient inhibition of retinal vascular development followed by neovascularization that is caused by LeTx may be explained if the initial decrease in retinal vasculature in the inner plexus creates a hypoxic environment, causing elevated VEGF signaling that in turn triggers neovascularization. To test this we measured the level of soluble VEGF and other cytokines in the vitreous of eyes 1 and 4 days following LeTx treatment. We observed a significant increase in the levels of secreted VEGF into the vitreous at both timepoints compared to sham injected eyes . In contTo test whether increases in vitreal VEGF induce neovascular growth we coinjected LeTx with a VEGF neutralizing antibody (VEGF-NA) at both a low dose (1×) and high dose (10×) at PN10 and analyzed retinas 4 and 8 days following treatment . Using cIn this report, we have shown that LeTx can perturb developmental vascular growth in a murine model of retinal angiogenesis. LeTx treatment results in a transient delay in the formation of the inner plexus layers of retinal vasculature. This is accompanied by an elevation in the concentration of vitreal VEGF that subsequently drives extensive neovascular growth through the inner retina to the outer retinal region. To explain these observations we propose that LeTx acts directly on endothelial cells to prevent growth and remodeling of angiogenic sprouts as they extend into distal region of the retina. While it is possible that the immediate effects of LeTx are on another cell type that guides retinal angiogenesis, such as glial cells, Alfano et. al. in vivo. Oxygen levels are the primary regulator of blood vessel growth in the retina, and VEGF is a critical factor in endothelial cell growth, survival, and permeability, both during normal vascular development and during disease The retinal vasculature is an accessible and well characterized vascular system to study the development of blood vessels as the vasculature is restricted to two dimensions and can be visualized in vivo model system Our observation that active ERK was localized to angiogenic buds and neovascular outgrowths indicates that altered MKK signaling also may be a factor in retinal neovascularization. In support of this, increased ERK activity was detected in a rat model of ROP, and intravitreous injection of ERK inhibitors reduced retinal neovascularization in this Previously we observed that in mouse xenograft models LeTx causes a dramatic reduction in tumor volume that is preceded by an abrupt decrease in vascular perfusion and is accompanied by a decrease in tumor vascularization Several studies point to a role for MKK signaling in controlling the release of VEGF and other angioproliferative factors from tumor cells in vitro LeTx is considered the major virulence factor of anthrax and the mediator of host lethality. Vascular pathologies such as hemorrhage and septic shock are common features of anthrax and can be mimicked by the injection of LeTx alone In summary, to gain insight into the effects of anthrax LeTx on tumor blood vessels we turned to a mouse model of developmental retinal vascularization. We hypothesized that application of LeTx would disrupt MAPK signaling and alter normal retinal vascularization, specifically during the angiogenic phase of vascular development. Consistent with this, injection of LeTx at a time when active MAPK could be found in angiogenic buds resulted in the inhibition in branching morphogenesis of vasculature in the inner plexus. However, this was followed shortly afterwards by extensive focal neovascular growth into the outer retina that was driven by elevated vitreal VEGF. These results indicate that MAPK signaling plays a key role in retinal angiogenesis and that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.All experiments were done in compliance with the guiding principles of the “Guide for the Care and Use of Laboratory Animals.” All procedures were approved by the Van Andel Institute (VAI) Institutional Animal Care and Use Committee (IACUC).2 incubator.Retinal endothelial cells (RECs) were isolated and cultured essentially as described Bacillus anthracis (BH445) and purified essentially as described Pseudomonas exotoxin A Protective antigen (PA), lethal factor (LF) and LF-E687C (E687C), a mutant form of LF whereby a Glu to Cys substitution at the zinc-binding site results in elimination of enzymatic activity In vitro cell viability assays were performed as described 2O or 10 nM CI-1040 for 24 hr. To analyze the basal activity of the MAPK pathways during retinal vascular development, eyes were collected from C57BL/6 mice from postnatal day 0 to day 21, and retinas dissected. Retinal tissue was homogenized in RIPA buffer ) with a micropestle. Cells or homogenized tissue were then lysed in RIPA buffer, sonicated, and cleared by centrifugation. Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce Biotechnology). Five μg (for cell lysates) or 50 µg  were separated by denaturing SDS-PAGE and transferred to PVDF membranes (Millipore). Lysates of UV-treated SK-MEL-28 cells were used as a positive control for pERK, pp38, and pJNK activation. Membranes were immunostained with the following antibodies: MEK1  1∶2000 , MEK1  1∶200 (Santa Cruz #219), pERK 1∶1000 , ERK 1∶1000 , pp38 1∶1000 , p38 1∶1000 , pJNK 1∶1000 , JNK 1∶1000 . Membranes were probed with appropriate horseradish peroxidase-conjugated secondary antibodies at 1∶2000 . Specific proteins were visualized by chemiluminescence , followed by exposure to Kodak Biomax Light film (Eastman Kodak). Immunoblots were stripped and reprobed with β-actin  for a loading control.Immunoblotting was performed essentially as described All experiments were done in compliance with the guiding principles of the “Guide for the Care and Use of Laboratory Animals.” All procedures were approved prior to use by the Van Andel Institute (VAI) Institutional Animal Care and Use Committee (IACUC). C57BL/6 mice were obtained from Jackson Laboratories  and the VAI breeding colony. Mice were housed in the VAI animal facility with 12-hour light/dark cycle and free access to food and water. C57BL/6 mice at postnatal day 10 (PN10) were anesthetized by hypothermia as described Griffinia simplicificolia-IB4  and pERK  in 0.3% TritonX-100 in PBS. For pERK, retinas were further incubated with anti-rabbit Oregon Green  for one hour at room temperature. In order to identify the various retinal cell layers, retinas were incubated with 5 mg/mL DAPI (Invitrogen #D1306). Retinas were radially relaxed and flat-mounted on glass slides with Gel Mount Aqueous Media (Sigma #G0918).Eyes were enucleated 4 days or 8 days after intravitreal injection and fixed in 4% paraformaldehyde for 2 hours on ice. Retinas were dissected using standard procedures Epifluorescent images were acquired with a Nikon E600 microscope equipped with a Spot Insight QE camera. Identical illumination and camera settings were used to obtain all images. Optimization for color, brightness, and contrast was performed using Adobe Photoshop CS2; all optimization was performed identically for all images. For confocal microscopy, retinal whole mounts were visualized using a Zeiss LSM 510 META confocal microscope with Krypton-Argon , HeNe (543 nm) and Ti-sapphire (800 nm) pulse lasers. Three dimensional projections and depth coding images were produced from a series of z-axis images made up of 1 µm slices. All images were acquired and analyzed from peripheral regions of the retinal whole mounts to avoid region-specific differences within the retina.Vitreous fluid was collected as described Neovascular tufts were quantified using Imagine_0.16 software (developed in-house) and expressed as number of tufts per area as measured in square pixels. Threshold intensity was set to a high range to avoid false positives. A minimum of three retinas per treatment per collection timepoint was analyzed. Depth of vascular growth was calculated from analysis of immunofluorescently stained vasculature within confocal z-stack projections. Data are presented as a mean±standard deviation. Data were analyzed for significance by Student's t-Test. A p value<0.05 was considered significant.Figure S1LeTx induces neovascular growth into the outer retina. Retinas were isolated from mice treated on PN10 with either E687C/PA (left) or LeTx (right) and collected 8 days later. Retinas were stained with GSA (red) and DAPI (blue) and flatmounted for confocal microscopy. Retinal cell layers are indicated based on DAPI staining. Z-axis projections show that neovascular growth occurs through the inner retina and continue into the outer retina. GCL - ganglion cell layer; IPL - inner plexiform layer; INL - inner nuclear layer; OPL - outer plexiform layer; ONL - outer nuclear layer.(0.37 MB TIF)Click here for additional data file.Figure S2Intravitreal injection of the MEK1/2 inhibitor CI-1040 has no effect on retinal vascular growth. (A) Western blot analysis indicating activity of CI-1040 in SK-MEL-28 cells in vitro. SK-MEL-28 cells were either untreated (NT) or treated with vehicle (H2O) or 10 nM CI-1040 for 24 hr. CI-1040 treatment resulted in a loss of phosphorylated ERK (pERK) while having no effect on total ERK levels (ERK). Untreated (−) or UV-treated (+) SK-MEL-28 lysates were run as a positive control for pERK activation. (B) Retinas were isolated from mice treated on PN10 with either HBSS or 20 nM CI-1040, collected 4 days later, stained with GSA, and flatmounted for confocal microscopy. Z-axis projections (top) and depth coding images (bottom) indicate no effect of CI-1040 treatment on vascular growth into the inner plexus.(0.44 MB TIF)Click here for additional data file.Figure S3Intravitreal injection of the MEK1/2 inhibitor CI-1040 does not induce neovascular growth into the outer retina. Retinas were isolated from mice treated on PN10 with either HBSS or 20 nM CI-1040, collected 8 days later, stained with GSA, and flatmounted for confocal microscopy. (A) Z-axis projections (left) and depth coding images (right) show normal vascular development to the inner plexus and no formation of neovascular growth into the outer retina. (B) Epifluorescent images at low magnification of flatmounted, GSA stained retinas show no evidence of neovascular tuft formation 8 days after CI-1040 treatment by intravitreal injection.(0.59 MB TIF)Click here for additional data file.Table S1Measurement of secreted cytokines into the vitreous 1 day post treatment.(0.36 MB DOC)Click here for additional data file.Table S2Measurement of secreted cytokines into the vitreous 4 days post treatment.(0.40 MB DOC)Click here for additional data file.


Female-biased dispersal (FBD) is predicted to occur in monogamous species due to local resource competition among females, but evidence for this association in mammals is scarce. The predicted relationship between FBD and monogamy may also be too simplistic, given that many pair-living mammals exhibit substantial extra-pair paternity.Tupaia tana) in Borneo, a behaviorally monogamous species with a genetic mating system characterized by high rates (50%) of extra-pair paternity. Genetic analyses provided evidence of FBD in this species. As predicted for FBD, I found lower mean values for the corrected assignment index for adult females than for males using seven microsatellite loci, indicating that female individuals were more likely to be immigrants. Adult female pairs were also less related than adult male pairs. Furthermore, comparison of Bayesian coalescent-based estimates of migration rates using maternally and bi-parentally inherited genetic markers suggested that gene flow is female-biased in T. tana. The effective number of migrants between populations estimated from mitochondrial DNA sequence was three times higher than the number estimated using autosomal microsatellites.I examined whether dispersal and gene flow are female-biased in the large treeshrew (These results provide the first evidence of FBD in a behaviorally monogamous species without mating fidelity. I argue that competition among females for feeding territories creates a sexual asymmetry in the costs and benefits of dispersal in treeshrews. Dispersal exerts an important influence on population genetics and demography, as well as on our ability to predict population-level responses to environmental disturbance Three non-mutually exclusive factors have been proposed to explain the association between mating systems and sex-biased dispersal: inbreeding avoidance, local resource competition (LRC), and local mate competition Monogamy in mammals is associated with female use of exclusive territories The predicted association between FBD and monogamy has rarely been examined in mammals, largely because most mammals are polygynous Marmota marmota) disperse more often than females Several polygynous mammals have been studied using these genetic techniques, and as predicted either no sex bias Predictions of a simple association between FBD and monogamy are complicated by increasingly common genetic results indicating substantial extra-pair paternity in putatively monogamous mammals Tupaia tana) in NE Borneo. Large treeshrews form behaviorally monogamous pairs that forage solitarily, potentially as an adaptation to intraspecific foraging competition T. tana is one of the highest ever recorded for a mammal (50%), but variance in reproductive success does not vary between males and females T. tana are not subject to intense sperm competition In this study I use multiple genetic methods to test for FBD and gene flow in the large treeshrew  females than among adult males in the population T. tana by comparing gene flow estimated from bi-parentally inherited microsatellite markers and a maternally inherited mitochondrial DNA (mtDNA) marker. Bayesian methods based on the coalescent T. tana populations. If gene flow is female-biased, then the migration rate for mtDNA should substantially exceed the migration rate for bi-parentally inherited microsatellites.I tested the prediction of FBD in T. tana individuals at two sites in Sabah, Malaysia (NE Borneo) from 2002–2004 during a larger study on mating systems in treeshrews. The first site (N = 39 samples) was located in the Danum Valley Conservation Area  and consisted of undisturbed primary lowland rainforest. The other site (N = 15 samples) was located 53 km away in the Malua Forest Reserve . This area was heavily logged in the early 1980's and has yet to recover the multiple closed canopies  and tall emergent trees (up to 70 m) that characterize lowland rainforests in SE Asia et al.Large treeshrews are small (200–250 g), diurnal, frugivore-insectivores that inhabit the lowland tropical rainforests of Borneo and Sumatra. I collected ear clips for genetic analyses from 54 I extracted genomic DNA from ear tissue samples using Qiagen DNEasy tissue extraction kits . Seven previously-described, unlinked microsatellite loci named JS22, JS132, JS183, JS188, JS196, SKTg19, and SKTg22 were amplified from DNA extracts using the PCR conditions in Munshi-South & Wilkinson Snn) implemented in DNASP. Snn measures how often the most similar sequences in a data set (“nearest neighbors”) are from the same population, and produces a powerful test of genetic differentiation for sequence data in nearly all situations To examine differences in genetic variability between the primary and logged forest populations, I calculated the number of alleles and allelic richness at each microsatellite locus for each population using FSTAT v. 2.9.3.2 mAIc) between adult males and females using the “biased dispersal” module in FSTAT. One-sided P values were calculated using 10,000 randomizations. The assignment index is the probability that an individual's genotype occurred by chance in a population cAI values of zero for each population. Negative cAI values characterize individuals with genotypes less likely than average to occur in a population sample, and thus lower mAIc values for one sex  implies sex-biased dispersal. This index was chosen because both simulations and real data sets have indicated that this test has high power at detecting moderately intense biases in dispersal N = 14 females and 20 males).To test for FBD, I compared mean corrected assignment indices  between the two populations using both the microsatellite and mtDNA sequence data. Bayesian inference may be more accurate and efficient at sampling genealogy space than maximum likelihood approaches for many datasets eμN, where μ = mutation rate) and M (m/μ) from microsatellite data, equaling 4emN when multiplied together. For mtDNA, this method estimates 2fmN . Assuming an equal sex ratio and equal variance in reproductive success among males and females, fN is equivalent to eN/2 calculated from microsatellites. Higher migration rates for mtDNA than for microsatellites should thus indicate female-biased gene flow.If gene flow among large treeshrews is female-biased, then migration rates calculated for maternally inherited mtDNA should be higher than migration rates calculated for bi-parentally inherited autosomal markers. To test this prediction, I used the Bayesian coalescence approach implemented in MIGRATE 2.1.3 M distribution: minimum = 0.000001, maximum = 1000, mean = 100), starting parameters based on stF calculations, burn-in equaling 10,000 trees, five long chains sampling 2,000,000 genealogies, and an adaptive heating scheme . The same analysis was then repeated using the estimates of Θ and M obtained from the first analysis as starting parameters. In this second analysis, a search window for the exponential prior distribution was set according to the distribution of parameter estimates from the first analysis . For the mtDNA dataset, I used the same analytical strategy with the F84 model of DNA sequence evolution instead of the stepwise microsatellite mutation model. However, I increased the number of sampled genealogies to 10,000,000 to achieve convergence, and used wider windows in the second run . These analyses produced values of ΘM  estimated in each direction between the two populations along with their approximate 95% confidence intervals emN) by summing ΘM in each direction and dividing by four for microsatellites and two for mtDNA.To estimate the effective number of migrants from microsatellite data, I ran 10 sequential iterations in MIGRATE using a stepwise mutation model with constant mutation rates, an exponential prior distribution  in the primary forest and from two to six alleles (mean = 4.0) in the logged forest (P<0.0001), as well as for four out of the seven loci .Microsatellite allelic diversity was moderate in both d forest . Allelicd forest . GenotypmAIc for adult females than for adult males  and positive for males (mean = 0.48), indicating that females are more likely to be immigrants than males. Two method-of-moment estimators of relatedness, Lynch and Ritland's r  from primary to logged forest and 2fmN = 3.35 (95th percentile = 0.07–13.46) from logged to primary forest. These two estimates produce an overall estimate of fmN = 5.77. Assuming an equal sex ratio and low variance in male reproductive success, this value is equivalent to emN = 11.54 effective migrants exchanged per generation between the two populations.Bayesian inference of migration rates produced an estimate for mtDNA of 2emN = 12.26 (95th percentile = 5.93–15.27) from primary to logged forest and 4emN = 2.04 (95th percentile = 1.05–3.40) from logged to primary forest. These estimates correspond to an overall effective number of migrants exchanged per generation of emN = 3.58, which is more than three times less than emN estimated for mtDNA.Microsatellite estimates of the effective number of migrants were substantially less than mtDNA estimates. Bayesian inference produced an estimate across all seven loci of 4cmAI and pairwise relatedness). These methods detect sex-biased dispersal only when adults have been thoroughly sampled and the sex bias is intense T. tana despite moderate sample sizes and genetic variability at seven microsatellite markers, suggesting that dispersal is substantially female-biased in this species. The magnitude of the difference between males and females in mAIc (1.18) for large treeshrews was similar to values for two other cases where a sex bias was also confirmed using trapping data Multiple genetic analyses presented here provide evidence of FBD in large treeshrews. As predicted for FBD, adult females had significantly lower mean values than males for two different tests  rather than male philopatry.Evidence of FBD in timators , but negT. tana samples for this study violate the assumptions of an equal sex ratio and equal variance in male and female reproductive success. However, variance in reproductive success was not different between males and females, and the sex ratio of offspring was equal in these populations T. tana.The prediction of greater migration (i.e. gene flow) rates for maternally inherited markers than bi-parentally inherited markers was also supported. The overall number of migrants per generation estimated using mtDNA was more than three times higher than the microsatellite estimate. The substantially higher migration rate for mtDNA suggests that historical gene flow in large treeshrews has been female-biased. Recent studies have raised concerns that migration rates and confidence intervals estimated from mtDNA using maximum likelihood coalescence techniques are often not accurate C. russula, which also exhibited lower cmAI values among females than males C. russula only persist for less than one breeding season, placing them at the short-term end of the continuum of pair duration in behaviorally monogamous mammals This study is only the second to find genetic evidence of FBD, and the first to report female-biased gene flow, in a behaviorally monogamous mammal. Evidence for FBD is more prevalent among polygynous mammals T. tana may also limit the ability of females to produce young on poor-quality territories, or during periods of resource scarcity. These physiological and behavioral limitations on reproduction are likely to produce intense competition between females for resources, and may be the main factor driving females to disperse away from their natal territory to settle on a high-quality territory for their own reproduction.Greenwood T. tana populations in primary forests and logged forests in Sabah, Malaysia. Southeast Asia has experienced greater rates of deforestation than other tropical regions The costs and benefits influencing the evolution of behavioral monogamy appear to influence dispersal patterns in large treeshrews. The fitness benefits that females gain from dispersal and the proximate factors influencing dispersal rates are fruitful areas for future research that could be addressed using provisioning experiments. Benefits males gain from philopatry, if any and if they are indeed philopatric, also deserve closer examination. The results from this study also indicate that gene flow is ongoing between


To measure, quantify and compare Ocular Aberrations due to nuclear cataracts.Department of ophthalmology and school for ophthalmic technicians, college of medicine and health sciences, Sultan Qaboos University, Muscat, Oman.Retrospective case controlled study.113 eyes of 77 patients with nuclear cataract (NC) were recruited from outpatient clinic of a major tertiary referral center for Ophthalmology. Patients having NC with no co-existing ocular pathologies were selected. All patients were subjected to wavefront aberrometry (make) using Hartmann-Shack (HS) aberrometer. Consents were taken from all patients. Higher order Aberrations (HOA) were calculated with Zernike polynomials up to the fourth order. For comparison 28 eyes of 15 subjects with no lenticular opacities (control group) were recruited and evaluated in an identical manner. No pupillary mydriasis was done in both groups.20) and spherical aberration (Z42) were found to be significantly greater amongst NC group, compared to normal control group.Total aberrations were almost six times higher in NC group compared to control  subjects. The HOA were 21 times higher in NC group, and coma was significantly higher in NC eyes compared to normal (control) group. The pupillary diameter was significantly larger in control group  compared to NC (3.05mm ± 1.9145) subjects (probably due to younger control age group). Amongst Zernike coefficients up to fourth order, two polynomials, defocus (ZNuclear cataracts predominantly produce increased defocus and spherical aberrations. This could explain visual symptoms like image deterioration in spite of normal Visual acuity. Cataract is the leading cause of preventable blindness worldwide.Aberrometry does not provide direct imaging of the eye; it provides a picture of the eye's optical quality. Corneal refractive surgery is one of the fields where this technology has contributed a lot.5This study was undertaken to determine whether wavefront aberrometry can be used as a tool in eyes with nuclear cataracts to assess their visual function, and thereby, help cataract surgeons as well as patients in predicting quality of visual outcome.Aberration means a deviation from the proper or expected course, a defect of focus such as blurring or distortion in an image. It may be a physical defect in the optical element, as in a lens, that causes such imperfection. Scattering of light,Spheres and cylinders are not the only refractive errors of the eye. Additional refractive errors known as higher order aberrations (HOA) also exist. These additional refractive errors are normally so small that they have little effect on routine vision. In summary HOA are the refractive errors beyond spheres and cylinders. They are important because patients with such aberrations may experience uncomfortable visual symptoms even when they have six-by-six vision.While cortical opacification is measured objectively by determining the percentage of lens that is opaque, nuclear opacification is determined by measuring the optical density  system analysis is a class 1 laser device. Written consent was obtained from all patients. Being a non-interventional study, the approval from Medical Research Ethics Committee was not required. Patients were selected on the basis of clinically observable nuclear cataract with no co-existing ocular pathology. A standard ophthalmic assessment was performed in all patients. This included best corrected visual acuity (BCVA) using Snellen chart at six meters, Slit-lamp biomicroscopy , and intraocular pressure measurement (Goldmann). Aberrometry was performed by a single trained senior technician (HB) using HS Aberrometer (Zeiss Asclepion Germany). Picture acquisition was done by dimming the room light and target light so that maximum diameter of the patient pupil could be achieved without using a mydriatic. The fellow eye was provided with a target at a distance of 4 meters to avoid accommodation. Where auto-refraction failed to converge (as happened in few young patients with good accommodation), a manual refraction in the aberrometer was performed. The measurements were repeated at least three times or till a well focused image of the eye was obtained. All corneal contact procedures that might have affected the corneal epithelium or tear film (like applanation tonometry) were done after the aberrometry. The root mean square (RMS) used for analysis was computed by Zywave.For comparison 28 eyes of 15 subjects with no clinical detectable cataract or any other ocular pathology, except refractive errors, were recruited and served as a control group. These subjects were mainly selected from the staff members of the hospital, or relatively young relatives of the patients. All control eyes were subjected to similar psycho-optical tests and procedures as eyes with NC, except A-scan and applanation tonometry which were not done in control eyes.The exclusion criteria were: (1). A Hartmann image that could not be analysed automatically. (2). Eyes with associated corneal, vitreous and retinal pathologies. Normal eyes in control group did not have any ocular disorder.2≤). The subscript 2 denotes the Zernike order  while the superscript (≤) means the mode or the aberration within that order.Zernike modes or aberrations are labeled as double index scheme (Z2), cartesian astigmatism (Z22), trefoil of cosine phase (Z33), Coma like aberrations (third order component (Z3), tetrafoil of cosine (Z44), tetrafoil of sine phase (Z4−4), and spherical aberrations (Z4). Corneal topography was measured simultaneously using placido disk attached to the HS wavefront sensor.The HOA were measured up to fourth order Zernike polynomials without dilating the pupil. From the Zernike coefficients, we calculated defocus  was applied as the distribution did not follow a normal pattern.Mean Total aberrations (HOA plus Lower order aberrations) were six times larger (11.6011 SD ±24.2214) in NC eyes than in control eyes (1.7364 SD ±1.0752), however, this difference was not found to be significant, probably due to younger age group of the control eyes. Mean HOA for NC group (n=113) was 6.7900 SD ±15.7432) compared to.3150 (SD ±.1451) for control group (n=28). This difference was significant (p=.003). Coma in NC group was almost 13 times higher (20.2052 SD ±54.3304) than in control group (1.5018 SD ±1.1315). This too was statistically significant (p<.001) .20 p<.001, mean for NC group being - 28.4146 SD ± 54.9631) and spherical aberration . Aberrations like Cartesian astigmatism (Z22), triangular astigmatism (Z3−3), coma of sine phase (Z3−1), spherical aberration (Z4−2) were found to be higher in NC group but not significantly.2−2) coma of cosine phase (Z31), trefoil of cosine phase (Z33), spherical aberration (Z40), tetrafoil of cosine phase (Z44) and tetrafoil of sine phase (Z4−4) were found to be higher in control group compared to NC group; however, the measurements were not statistically significant  versus only −.025 SD ±.203 for control group. Negative polarity in spherical aberration in NC has also been reported by Kuroda et al.NC Group: Amongst 12 Zernike coefficients, six aberrations  showed positive polarity while other six  showed negative polarity. The negative polarity means the wavefront in the papillary area is relatively delayed, indicating a myopic shift  out of 12 Zernike coefficients showed positive polarity, while other seven aberrations  showed negative polarity  and 6th order component (S6) in a single case of nuclear cataract.40) component in our patients with NC, though our series had larger number of cases. In a latter study by the same authors, they found that nuclear opacities always induced negative spherical aberration which is confirmed by our study as well,42 polynomial than eyes in control group (p<.045). Although increase in other spherical aberration components  was not statistically significant, we believe the trend followed due to pupil not being dilated in our cases.Kuroda et al reported negative spherical like aberration, defined as 4Study by Nisha Sachdev et al has also reported statistically significant spherical aberration in nuclear opacification compared to normal human eyes.Also since our cataract grading was not based on pentacam or LOCS scale, the difference in the density of NC between two groups could be the reason for such a large difference. The increase in total aberrations (TA) and coma in our series is well documented by Nisha Sachdev et al.40) in nuclear cataracts in our patients is strongly corroborated by similar observation by the same authors . This suggests delay of wavefront when the ray travels inside a hard nucleus with increased refractive index. Such negative polarity of spherical aberrations (wavefront delay) is well consistent with the slower velocity measurements of extracted senile nuclear cataractous lenses using ultrasound, than in normal lenses.40) in our control group could be due to relatively younger age group (mean 44.46 years). For the sake of interest, polarity is usually positive in cortical cataracts and negative in NC.Total aberrations (11.601 in NC group versus 1.736 in control group in our study) increase in eyes with cataract due to local refractive changes in the lens. This has been observed by Kuroda et alThe limitations of our study were that LOCS III system for cataract grading was not possible, and the use of Pentacam Scheimpflug photography for in-vivo measurements and quantification of lens density would have been more scientific; however, a recent study has shown that LOCS III criterion as an economic cataract grading system provides data that are in satisfactory concordance with the results obtained by Pentacam Scheimpflug system.16Until early nineties there was no way to clinically measure such aberrations, or even if HOA were significant and measurable, there was no practical way to correct them. HS aberrometer with Scheimpflug cameraIn conclusion, wavefront technology is a unique tool for measuring and quantifying aberrations produced by the eye as a whole optical system. HS aberrometer measures the deterioration of image quality objectively and quantitatively in eyes with cataract. The results suggest that nuclear cataracts induce high spherical aberrations and varying amounts of tetrafoil.


An activity-dependent long-lasting asynchronous release of GABA from identified fast-spiking inhibitory neurons in the neocortex can impair the reliability and temporal precision of activity in a cortical network. 2+ sensitivity, suggesting that they rely on different Ca2+ sensors and/or involve distinct pools of vesicles. In addition, asynchronous release is modulated by the endogenous Ca2+ buffer parvalbumin. Functionally, asynchronous release decreases FS-cell spike reliability and reduces the ability of P neurons to integrate incoming stimuli into precise firing. Since each FS cell contacts many P neurons, asynchronous release from a single interneuron may desynchronize a large portion of the local network and disrupt cortical information processing.Networks of specific inhibitory interneurons regulate principal cell firing in several forms of neocortical activity. Fast-spiking (FS) interneurons are potently self-inhibited by GABAergic autaptic transmission, allowing them to precisely control their own firing dynamics and timing. Here we show that in FS interneurons, high-frequency trains of action potentials can generate a delayed and prolonged GABAergic self-inhibition due to sustained asynchronous release at FS-cell autapses. Asynchronous release of GABA is simultaneously recorded in connected pyramidal (P) neurons. Asynchronous and synchronous autaptic release show differential presynaptic Ca In the cerebral cortex (neocortex) of the brain, fast-spiking (FS) inhibitory cells contact many principal pyramidal (P) neurons on their cell bodies, which allows the FS cells to control the generation of action potentials . FS-cell-mediated rhythmic and synchronous inhibition drives coherent network oscillations of large ensembles of P neurons, indicating that FS interneurons are needed for the precise timing of cortical circuits. Interestingly, FS cells are self-innervated by GABAergic autaptic contacts, whose synchronous activation regulates FS-cell precise firing. Here we report that high-frequency firing in FS interneurons results in a massive (>10-fold), delayed, and prolonged (for seconds) increase in inhibitory events, occurring at both autaptic (FS–FS) and synaptic (FS–P) sites. This increased inhibition is due to asynchronous release of GABA from presynaptic FS cells. Delayed and disorganized asynchronous inhibitory responses significantly affected the input–output properties of both FS and P neurons, suggesting that asynchronous release of GABA might promote network desynchronization. FS interneurons can fire at high frequency (>100 Hz) in vitro and in vivo, and are known for their reliable and precise signaling. Our results show an unprecedented action of these cells, by which their tight temporal control of cortical circuits can be broken when they are driven to fire above certain frequencies. In the cerebral cortex, the control of neuronal population discharge pattern and timing is of fundamental importance for information processing and cognitive operations Distinct cortical interneuron classes have a wide range of preferred firing patterns 2+-binding protein parvalbumin (PV) in these cells; and the characteristic firing of APs at high constant rate with no adaptation. Another factor that distinguishes FS cells from other interneuron types is their hypothesized rigid (non-plastic) action as precision devices during cortical operations, leading to the view that they represent a dedicated system for regulating the timing of activity in neocortical circuits Despite the large heterogeneity of cortical inhibitory neurons, the main population of perisomatic-targeting fast-spiking (FS) interneurons is relatively homogeneous throughout the cerebral cortex. Several factors likely contribute to making these interneurons highly specialized for the control of P spike precision, including their short membrane time constants; intrinsic excitability Presynaptic terminals at several synapses respond to trains of APs with a delayed and long-lasting asynchronous release of synaptic vesicles FS cells are self-innervated to a great extent by GABAergic autapses Here we show that trains of APs in FS cells elicit asynchronous GABA release that can persist for several seconds at both autaptic and FS–P synapses and can be blocked by increasing slow intracellular calcium buffering. Although asynchronous release was proposed to change postsynaptic gain for a relatively prolonged period after presynaptic activity has subsided We recorded from layer V FS interneurons in acute slices obtained from rat somatosensory cortex. FS interneurons were visually identified under infrared videomicroscopy as multipolar neurons lacking clear apical dendrites. During electrophysiological recordings, these cells typically showed FS firing behavior, namely brief APs and abrupt high-frequency firing (>40 Hz) in response to depolarizing direct current steps , inset  of the drop in spike reliability was due to GABAergic autaptic asynchronous release. Again in GBZ recordings, nonspecific drifts of recording conditions were ruled out, as repeated control experiments performed after delivering the high-frequency trains yielded values for spike reliability that were indistinguishable from those obtained in the first control sequence. Importantly, these experiments were performed using a local perfusion system that efficiently removed ambient GABA, as evidenced by the lack of significant change in the total FS-cell membrane resistance when switching the flow from control to GBZ, and back to control solution (p>0.05), ruling out a role for tonic inhibition in these effects. Values of membrane resistance (Rm) in the presence of local perfusion are higher than those previously reported for FS interneurons Rm values for FS cells were in line with previously published results in all other experiments that did not use a local perfusion . In addition, there were no appreciable differences in asynchronous release in experiments with or without local perfusion, ruling out the possibility that asynchronous release was due to increased impact of small GABAergic events.To directly test the actual contribution of asynchronous GABA release to the spiking reliability reduction seen in FS cells, we repeated the frozen noise stimulations in the presence of 10 µM GBZ and compared the response of FS cells before and after blocking GABA+ channels.In P neurons, GABAergic contacts from FS cells are mostly located at the soma, contributing to the electrotonic shunt responsible for synchronization during rhythmic cortical activity. We have shown that asynchronous release from FS cells affects both the FS cells via autapses and the postsynaptic P cells . We ther– reversal potential close to the physiological level reported for cortical P cells . The effect of asynchronous release on spike reliability and precision in P neurons was greater in the first 200 and 300 ms following presynaptic spike trains, respectively, and recovered within a few hundred milliseconds later (data not shown). Interestingly, the immediate large effect of asynchronous release on P neurons was reduced spike reliability. Later, during the decaying phase of asynchronous release, spikes could be evoked more reliably, but with a much reduced precision  polysynaptic activation\recruitment of other GABAergic cells within the circuit, (2) retrograde signaling to presynaptic GABAergic terminals, or (3) asynchronous GABA release from FS-cell autaptic terminals.Polysynaptic activation would imply that stimulation of a FS neuron is capable of triggering spikes in some synaptically connected cells, which would then activate other interneurons, leading to a feedback inhibition. This type of mechanism has been described to explain delayed disynaptic inhibition between P cells 2+], which rapidly trigger the synthesis or release of the retrograde molecule. This rapid mode of action is incompatible with our observation that high concentrations of either BAPTA or EGTA in the recording pipette blocked delayed self-inhibition in FS cells slowly (tens of minutes), which is inconsistent with a rapid somatodendritic diffusion of these compounds. The time course of this effect is more consistent with the diffusion of these compounds to presynaptic terminals. Indeed, the time required for BAPTA to block delayed inhibition closely matches the time course of its effect on synchronous autaptic responses. This concurrence in the time course of the two effects indicates that both mechanisms are located remotely at the same autaptic sites. Moreover, the time course of the BAPTA effect in our experiments was similar to the one previously observed with connected FS–P cell pairs and is consistent with simulations estimating the diffusion speed of internally infused Ca2+ chelators from the soma to presynaptic terminals As the increase in sIPSC frequency follows high-frequency trains of spikes, it is possible that this intense activity triggers the activation of a retrograde signaling pathway increasing GABA release from presynaptic neurons. A similar, albeit more persistent, phenomenon described in the cerebellum was shown to involve postsynaptically released glutamate acting on presynaptic NMDA glutamate receptors The delayed increase of inhibition in response to spike trains cannot be accounted for by electrical coupling. Indeed, gap-junction coupling functions as a low-pass filter: ∼100-mV spikes are sensed as ∼1-mV spikelets in electrically coupled cells Two other lines of evidence support the hypothesis that the delayed sIPSCs result from asynchronous autaptic release. First, this phenomenon occurred only in FS cells showing functional autaptic transmission, and the size of the autaptic response correlated with the increase of delayed sIPSCs. Secondly, asynchronous GABAergic events following a spike train in FS interneurons were also observed in connected P neurons and other postsynaptic FS interneurons, which did not fire APs and were held at hyperpolarized potentials. This increase of GABAergic events in P cells was very similar in amplitude and time course to that observed in parallel in FS cells. Altogether, these results indicate that retrograde signaling from FS cells to presynaptic GABAergic terminals can be ruled out. We therefore conclude that intense spike trains in FS interneurons induce delayed and prolonged asynchronous GABA release onto themselves and other postsynaptic targets.Our results show that asynchronous autaptic release from FS interneurons is large (>10-fold increase in sIPSC rate) and could be reliably induced for as long as the cell integrity was maintained. Most of our recordings were made in preparations from animals at an age (18–25 d old) where neuronal maturation of the FS cell connectivity pattern is well advanced Asynchronous autaptic release required trains of spikes at relatively high frequencies (>100 Hz). Although this is consistent with stimulation protocols used to evoke asynchronous release in many other studies 2+-buffering action of EGTA, is fully consistent with published data indicating that temporal precision of hippocampal basket PV-positive interneurons may be enhanced by the tight coupling of P/Q-type Ca2+ channels and Ca2+ sensors that trigger vesicle release at their active zones 2+ channels Given the prevailing view that transmitter release at PV-positive basket cell terminals is rapid, highly efficient, and temporally precise 2+ buffer EGTA, even at 10 mM, the delayed and long-lasting asynchronous autaptic release was blocked by high concentrations of EGTA. As suggested previously for hippocampal CCK-positive basket cells, the effect of the slow Ca2+ buffer may result from a reduction of the long-lasting presynaptic Ca2+ transient following repeated stimulations 2+ channels, which are more loosely coupled to exocytosis 2+ channels, which tightly couple Ca2+ entry and synaptic vesicle exocytosis at their terminals. However FS cells can fire at rates at least one order of magnitude higher than the low-range cutoff frequency (>50 Hz), and therefore these neurons are more likely to induce long-lasting residual Ca2+ signals at their terminals. The results of our experiments indicate that residual Ca2+ plays a major role in asynchronous release. Whether the two modes of release (synchronous and asynchronous) involve the same vesicle pools, release from the same active zones, and/or depend on the same Ca2+-sensitive elements of synaptic vesicle release machinery is yet to be clarified.In contrast to synchronous release, which was unaffected by the slow Ca2+ buffer PV in PV−\− mice was a small, but significant, increase in autaptic asynchronous release from FS autaptic terminals, suggesting a role of PV in limiting the buildup of residual Ca2+  was significantly larger than in WT animals −/− mice Consistent with this model, we found that the effect of deleting the endogenous CaCa2+ see . Similar2+ buffer PV, raises the question of whether it results from dialysis of endogenous buffers and/or other important metabolites that are lost during invasive whole-cell recordings. It is noteworthy that large and robust asynchronous release (both autaptic and synaptic) was always detectable immediately after patch rupture (<1 min) in all of our whole-cell experiments. A presynaptic washout of metabolic compounds in the intra-terminal cytoplasm during the first minutes of whole-cell recording is highly unlikely, as indicated by the timing (tens of minutes) required for 10 mM BAPTA and 10 mM EGTA to reach presynaptic sites and block asynchronous release. Moreover, our experiments in the presence of 1 mM EGTA in the whole-cell pipette showed that the magnitude of asynchronous release remained constant for long recordings (>50 min) in all tested neurons. Possible effects related to whole-cell dialysis in the first minutes of recordings would have resulted in changes of asynchronous release properties soon after whole-cell establishment. Finally, asynchronous release was detectable in few perforated-patch experiments (data not shown), directly ruling out the possibility that it is due to imbalanced ion homeostasis and/or dialysis due to whole-cell recording.The specific sensitivity of asynchronous release to high concentrations of EGTA, together with its modulation by the endogenous CaFS interneurons are fast and precise elements of cortical circuits, instrumental in synchronizing large populations of neurons in a network . Indeed,Our results suggest that the function of FS cells may not be limited to the phasic nature of fast cortical inhibition but might, under some conditions, be involved in more sustained modulation of network properties.Previous studies described asynchronous release at many synapses in various central nervous system areas, hypothesizing its potential role in controlling the gain or excitability of postsynaptic cells by generating a smooth excitatory or inhibitory tone . This is probably due to the intrinsic and synaptic properties of FS cells that are tuned to act as very efficient coincidence detectors Interestingly, asynchronous synaptic release from FS interneurons had an even more pronounced effect on postsynaptic P neurons than on autaptically connected FS cells . Each FS cell contacts several P neurons, actively contributing to their synchronous firing during network oscillations. The strong reduction of spike reliability and precision of P cells response to frozen noise stimulations produced by asynchronous GABA release from a presynaptic FS cell implies that P neurons are more sensitive to disorganized inhibitory events than are basket cells. This enhanced effect of asynchronous GABA release in P cells might be explained partly by the slower kinetics of their intrinsic and synaptic properties, and may act as an effective shunt of cortical excitation after intense sensory activation. Our experiments were performed in relatively quiescent cortical slices and in the constant presence of glutamate receptor antagonists. In vivo, P neurons are known to be constantly bombarded by excitatory and inhibitory synaptic activity yielding a high conductance state that has been proposed to be important for the correct integration properties and computation capabilities of cortical neurons To process incoming information, cortical neurons embedded in a network utilize coding strategies relying either on the average rate of AP firing or on the exact (millisecond range) spike timing of several neurons firing synchronously By switching between two modes of GABA release at their autaptic and synaptic nerve terminals, FS interneurons can effectively filter network activity, behaving as a precise coordinator when activity is normal, but quickly breaking the synchrony pattern when activity levels overshoot a certain threshold during physiological states in order to prevent/decrease generalized synchronous activity observed during epileptic discharges.−/−3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM MgSO4, and mM 0.5 CaCl2 (equilibrated with 95% O2–5% CO2). Coronal slices (300 µm) were cut with a vibratome  from a block of brain containing sensorimotor cortex. Slices were then incubated in oxygenated artificial cerebrospinal fluid containing the following: 126 mM NaCl, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2 and 10 mM glucose (pH 7.4), initially at 32°C for 30 min, and subsequently at room temperature, before being transferred to the recording chamber. Recordings were obtained at a temperature of 30–32°C from layer V interneurons visually identified using infrared video microscopy. Basket cell interneurons were identified in current-clamp by their fast firing behavior and the absence of a large emerging apical dendrite. In all experiments, GABAA-receptor-mediated currents were isolated by adding 6,7-dinitroquinoxaline-2,3-dione  to the bath perfusion. For most whole-cell recordings, patch-clamp electrodes  were filled with a “high Cl–” intracellular solution containing the following: 70 mM Kgluconate, 70 mM KCl, 2 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 2 mM MgATP, and 0.3 mM Na2GTP (pH 7.3) corrected with KOH (290 mOsm). The estimated ECl was approximately –16 mV based on the Nernst equation, without correction for gluconate-generated liquid junction potential. Under these recording conditions, activation of GABAA receptors resulted in inward currents at a holding potential (Vh) of –70 mV. For experiments testing the effects of Ca2+ chelators at high concentrations, patch pipettes were filled with a similar solution: 60 mM Kgluconate, 70 mM KCl, 2 mM NaCl, 10 mM HEPES, 2 mM MgATP, and 0.3 mM Na2GTP, but containing either 10 mM K4BAPTA or 10 mM EGTA. For experiments with WT and PV−/− mice, we used exactly the same low Mg2+ solution described in Caillard et al. ECl of approximately −16 mV. In order to minimize washout of PV from WT cells in the whole-cell configuration, recordings were carried out right after breaking in.All procedures were performed in accordance with Italian , European, and United States guidelines. For electrophysiological recordings, Sprague Dawley rats or C57BL/6J mice  were injected into the recorded cell, in order to elicit unclamped, presumed axonal APs, producing typical GABAergic synchronous responses. These were characterized by fixed latency, high peak-amplitude fluctuation and short-term depression Autaptic responses were detected using the high-chloride intracellular solution as previously described t-test, and differences were considered significant if p<0.05.Signals were amplified, using a Multiclamp 700B patch-clamp amplifier , sampled at 20 kHz, filtered at 10 kHz, and stored on a PC. Data were analyzed using pClamp (Axon Instruments) and Origin  software. Custom written software (J. R. Huguenard) was used for analyzing spontaneous GABAergic events . Results−/− experiments, train stimulations  were repeated 20 times in each cell, and we compared a 3-s baseline period  with the time course of asynchronous response after the train (in bins of 200 ms). In general, direct detection of synaptic events was preferred to exclude any additional slower post-train effect such as AHP and, possibly, GABAB autaptic responses or axonal GABAA-autoreceptor-mediated currents In most cases, we used locally written software (Detector [J.R.H.]) to detect and sort sIPSCs 2+ buffers on asynchronous response . For all experiments, measurements of spike reliability and SD of the mean spike timing (spike jitter) were performed with Spike2 software . Spikes were first threshold detected, and then raster plots were constructed, and since most spikes were reliable in control conditions, we delimited areas to analyze around each peak of the peri-spike time histogram, manually adjusting the time limits in order to compare only spikes that were reliably and precisely induced in the control condition. “Spike clusters” were defined as time periods in which spikes occurred repeatedly with a reliability of at least 20% in the control condition. For example, a period in which a spike occurred only once in 30 trials (reliability = 3.3%) was removed from the analysis. We then compared the reliability and precision of APs across each noise condition to those AP clusters occurring within the same periods under control conditions. Reliability was measured as the total number of spikes occurring within single AP clusters divided by the number of trials, and for jitter analysis, spike clusters were required to be at least 20% reliable in all tested conditions (because jitter cannot be accurately measured with low numbers of spikes). Spike precision and reliability were determined for each spike cluster and then averaged across all spike clusters within selected time bins .Input current stimuli were generated using a custom-written Octave script, by convolving traces of 3-s-long Gaussian noise with a synaptic function Figure S1Automatized method for detection and sorting of sIPSCs. (A) Voltage-clamp recording from a FS interneuron (top trace) in the continuous presence of the glutamate receptor antagonist DNQX to isolate spontaneous inhibitory GABAergic synaptic currents. Individual events were detected  with a threshold-triggered process from a differentiated copy of the recorded trace (bottom) and sorted as type 1, 2, or 3 events. The stimulation train artifacts were digitally removed. Detected synaptic events are color-coded. Type 1 (green) events are completely isolated events. Type 2 (blue) events are those arising from a flat baseline, but with an event on their decay phase. Type 3 (red) events are those arising on the decay of a previous event. (B and C) Same traces of (A) at faster time scales before (B) and after (C) inducing asynchronous autaptic release. Note that this approach allows accurate detection of quantal synaptic events occurring at very high frequency.(3.57 MB TIF)Click here for additional data file.Figure S2The total number of APs determines the magnitude of asynchronous autaptic release. Increasing the number of APs in the train caused proportional increase in asynchronous release (measured as SD of the mean post-train noise) regardless of their imposed frequency.(0.64 MB TIF)Click here for additional data file.Figure S3Asynchronous release from FS interneurons is not due to firing from other FS cells embedded in the network. (A) Simultaneous whole-cell  and cell-attached  recordings (in the continuous presence of 10 µM DNQX) from two closely spaced FS interneurons (see schematic shown in the inset at left). Single APs in the FS cell held in whole-cell induced prominent autaptic responses . This protocol never evoked spikes in the second FS interneurons recorded in cell-attached mode. (B) Same pair of FS interneurons as in (A). No spikes were detected in the FS neuron held in cell-attached  when a 0.5-s-long train at 200 Hz was induced in the FS neuron recorded in whole-cell . Note the presence of asynchronous autaptic release (black arrowheads). (C and D) Same experiment as (A) and (B) following patch rupture in the FS interneuron previously held in cell-attached. The two cells were connected by GABAergic synapses as shown by the presence of unitary synaptic responses (C). Identical high-frequency trains in the presynaptic cell induced both autaptic and synaptic asynchronous release . AP firing in FS interneurons (both single and trains of spikes) never induced firing in nearby FS cells whether they were synaptically connected (n = 4) or not (n = 12), ruling out the possibility that increased sIPSC frequency in response to high-frequency trains is due to depolarizing GABAergic responses that would induce firing in neighboring FS interneurons. These experiments were performed using GAD67-GFP mice (1.55 MB TIF)Click here for additional data file.


HFE-related hereditary hemochromatosis (HH) is characterized by abnormally low expression of the iron-regulatory hormone, hepcidin, which results in increased iron absorption. The liver is crucial for iron homeostasis as it is the main production site of hepcidin. The aim of this study was to explore and compare the genome-wide transcriptome response to Hfe deficiency and dietary iron overload in murine liver and duodenum. Illumina™ arrays containing over 47,000 probes were used to study global transcriptional changes. Quantitative RT-PCR (Q-RT-PCR) was used to validate the microarray results. In the liver, the expression of 151 genes was altered in −/−Hfe mice while dietary iron overload changed the expression of 218 genes. There were 173 and 108 differentially expressed genes in the duodenum of −/−Hfe mice and mice with dietary iron overload, respectively. There was 93.5% concordance between the results obtained by microarray analysis and Q-RT-PCR. Overexpression of genes for acute phase reactants in the liver and a strong induction of digestive enzyme genes in the duodenum were characteristic of the Hfe-deficient genotype. In contrast, dietary iron overload caused a more pronounced change of gene expression responsive to oxidative stress. In conclusion, Hfe deficiency caused a previously unrecognized increase in gene expression of hepatic acute phase proteins and duodenal digestive enzymes.Iron is an essential trace element whose absorption is usually tightly regulated in the duodenum.  HFE-related hereditary hemochromatosis  is an autosomal recessive disorder in which absorption of iron is inappropriately high Iron plays crucial roles in cellular metabolism but, in excess, it can catalyze the formation of free radicals leading to oxidative stress and cell damage HH is characterized by inappropriately low expression of the iron-regulatory hormone hepcidin Hfe (−/−Hfe) provide a genetic animal model of HH Hfe−/− mice and carbonyl iron-fed mice develop iron overload, there are important differences between these two models. Hfe−/− mice lack Hfe protein and therefore have decreased expression of hepcidin Mice homozygous for a null allele of Hfe gene disruption on mRNA expression in the liver and duodenum, two organs with crucial roles in iron metabolism Hfe−/− mice and wild-type mice, with or without dietary iron overload. This allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein.Current RNA microarray technology allows expression profiling of the whole transcriptome. This methodology has been used to explore the effects of Hfe−/− mice and carbonyl iron loaded mice, and comparing it with that of wild-type mice fed a standard diet. This approach allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein. All the mice used were males and all had the same genetic background (C57BL/6).We used global microarray analysis to study gene expression in the liver and duodenum of −/−Hfe mice and 2 wild-type mice was subjected to microarray analysis. The Pearson correlation coefficient between the knock out mice and between the controls was in both cases 0.989. The results revealed 86 induced genes and 65 repressed genes, using a cutoff value of ±1.4-fold  with upregulation in −/−Hfe mice and downregulation by dietary iron overload.There were 11 upregulated and 7 downregulated genes that were affected by both he liver . In some−/−Hfe mice. The expression of Hamp1 and Tfrc was decreased and that of Lcn2 was induced. We confirmed these results using Q-RT-PCR, and also tested the expression of Hamp2, which was downregulated  of them showed concordant results by these two methods  showed concordant results between microarray analysis and Q-RT-PCR  of expression.Q-RT-PCR analyses were done on duodenal RNA samples from 5 Q-RT-PCR  and 4. THfe deficiency and dietary iron overload in murine liver and duodenum. This approach allowed the identification of genes whose expression is changed during iron overload and those genes whose expression is differentially influenced by lack of Hfe protein. The global transcriptional response to Hfe deficiency has been explored previously in the liver and duodenum of two mouse strains −/−Hfe mice and in mice with secondary iron overload produced by intraperitoneal injection of iron-dextran −/−Hfe mice have no concordance with ours. Regulation of hepatic gene expression, on the other hand, is similar for several genes, such as Hamp1, Tfrc and Mt1. The second report focused on gene expression in the duodenum The goal of this study was to explore and compare the genome-wide transcriptome response to −/−Hfe mice has upregulated expression of APPs such as serum amyloids, lipocalins and orosomucoids. Notably, the expression of serum amyloid genes  was upregulated in the −/−Hfe mice compared to being downregulated in dietary iron overload, suggesting that Hfe deficiency induces this gene expression by an iron-independent mechanism. However, hepatic interleukin-6 mRNA expression was not significantly changed by Hfe deficiency, so the potential involvement of this cytokine in the observed upregulation of APPs remains uncertain.The hepatic expression of acute phase proteins (APPs) can be induced by inflammatory mediators such as interleukin-6. Interestingly, the liver of Lcn2 expression is increased in the liver of both −/−Hfe mice and those with dietary iron overload, suggesting that this induction is iron-related.Lipocalin2  is an APP with antimicrobial properties through a mechanism of iron deprivation by siderophore binding Hamp1, Hamp2) as previously reported Hfe−/− mice, Hamp1 expression was downregulated as expected Hamp2 mRNA by Q-RT-PCR and found a -1.92-fold change. The low expression of hepatic Hamp1 in Hfe−/− mice is likely responsible for the increased iron absorption and low microphage iron content in these mice Dietary iron overload of the liver led to increased expression of both hepcidin genes  in the same experimental mice. Recent work demonstrates that Bmp6 is a key player in the signalling pathway that controls hepcidin expression Bmp6 mRNA expression by dietary iron overload was not evident in the current study.Inhibitor of DNA-binding/differentiation proteins, also known as Id proteins, comprise a family of proteins that heterodimerize with basic-helix-loop-helix (bHLH) transcription factors to inhibit their binding to DNA. Several studies have reported that Id proteins have important roles in differentiation, cell cycle and angiogenesis in various cell types Hfe−/− mice. Although these genes are induced under certain stress conditions, such as heat shock and ischemia–reperfusion, their expression is decreased by iron overload The gene expression of several heat shock proteins was downregulated in the liver and duodenum by both Hfe deficiency and dietary iron overload, with a considerably greater number of these genes downregulated in the liver of Hfe gene induces the expression of many genes in the duodenum coding for digestive enzymes, such as elastases, carboxypeptidases, trypsins, chymotrypsins, amylases, and lipases. In contrast, feeding mice with an iron-supplemented diet did not affect the expression of any of these genes. The upregulation of gene expression for digestive enzymes in Hfe−/− mice is surprising because overexpression of these enzymes has not been associated with HH.Our results indicate that disruption of the Hfe deficiency and dietary iron overload was the transcriptional repression of genes involved in antimicrobial activities, such as cryptdins. In mice fed an iron-supplemented diet, there was also a decrease in mRNA expression for genes involved in antigen processing and presentation, such as some genes of the MHC class II family.A common feature of the duodenal response to both Hfe deficiency or dietary iron overload. In the duodenum, the expression of the sodium-coupled neutral amino acid transporter Slc38a5 was induced in Hfe−/− mice and repressed in mice fed an iron-supplemented diet. In the liver, the expression of Slc46a3 was upregulated in Hfe−/− mice. This gene belongs to the Slc46 subfamily of heme transporters. It is thus a close relative of Slc46a1 , a recently identified, although controversial, heme transporter Slc11a2, contains an iron-responsive element (IRE) in the 3′UTR of its mRNA. This permits the regulation of Dmt1 mRNA levels according to the cellular labile iron pool by mediation of the iron regulatory proteins, IRP1 and IRP2. Under iron-replete conditions, IRP activity is reduced rendering the Dmt1 mRNA vulnerable to degradation. The opposite is true under iron-deficient conditions, which is believed to be the situation inside the enterocytes of HH patients and Hfe−/− mice Dmt1 has been observed in the duodenum of HH patients Hfe−/− mice Dmt1 in the Hfe−/− duodenum. This may be explained by the inability of our microarray probes and PCR primers to discriminate between IRE-positive and IRE–negative transcripts.The solute carrier molecules constitute a large family of proteins involved in membrane transport of diverse molecules. The gene expression of many family members was affected by Tfrc (transferrin receptor 1) by iron is also mediated through the IRE/IRP system Tfrc expression in the duodenum −/−Hfe mice Tfrc was not significantly changed in the duodenum of Hfe−/− mice, a result that agrees with a previous report The post-transcriptional regulation of Hfe deficiency, especially in the regulation of enzymes involved in glutathione-related detoxification of reactive intermediates.Excess free iron increases oxidant production Hfe deficiency results in increased gene expression of hepatic APPs and duodenal digestive enzymes. In contrast, dietary iron overload causes a more pronounced change of gene expression responsive to oxidative stress.In conclusion, The animal protocols were approved by the Animal Care and Use Committees of Saint Louis University and the University of Oulu (permission No 102/05).Hfe gene and 4 male wild-type control mice were fed a standard rodent diet (250 ppm of iron) and sacrificed at approximately 10 weeks of age. The generation of the −/−Hfe mice has been described elsewhere −/−Hfe mice. The duodenum and liver samples were immediately collected from anesthetized mice and immersed in RNAlater solution .Five male C57BL/6 mice homozygous for a disruption of the Total RNA extraction and quality control have been described previously −/−Hfe mice and 3 mice with dietary iron overload were used. As controls, RNA samples from the duodenum and liver of 4 wild-type mice (2 controls of the −/−Hfe mice and 2 controls of the mice with dietary iron overload) were used. All 10 samples were analyzed individually. The amplification of total RNA (300 ng), in vitro transcription, hybridization and scanning have been described before All microarray data reported in the present article are described in accordance with MIAME guidelines, have been deposited in NCBI's Gene Expression Omnibus public repository −/−Hfe mice were highly divergent from the other two. Thus, this sample was excluded from further analyses. The data were then filtered according to the SD of the probes. The percentage of data that did not pass through the filter was adjusted to 99.4%, implicating a SD value of almost 3. At this point, statistical analysis was performed using the empirical Bayes t-test for the comparison of 2 groups. Due to the small number of samples, the statistical results were considered as orientative and thus no filtering was applied to the data according to p-values. The remaining 280 probes were further filtered according to fold-change with ±1.4 as cut-off values for up- and down-regulated expression, respectively. The functional annotation tool DAVID  Array data were normalized with Chipster (v1.1.1) using the quantile normalization method. Quality control of the data included non-metric multidimensional scaling, dendrograms, hierarchical clustering, and 2-way clustering (heat maps). These analyses showed that data from one of the three duodenal samples from −/−Hfe and dietary iron overload) and 4 mice from each control group  were used. Exceptionally, for the analysis of mRNA expression in the duodenum of −/−Hfe mice, only 4 samples were used. RNA samples (5 µg) were converted into first strand cDNA with a First Strand cDNA Synthesis kit  using random hexamer primers. The relative expression levels of target genes in the duodenum and liver were assessed by Q-RT-PCR using the LightCycler detection system . The reaction setup, cycling program, standard curve method and primer pairs for Angptl4, Dnajb1 and Tfrc have been described before Hamp1 and Hamp2 primers have also been characterized previously For this analysis, duodenal and liver RNA samples from 5 mice of each experimental group Click here for additional data file.Dataset S2List of genes differentially expressed in the liver of mice fed an iron-supplemented diet(0.05 MB XLS)Click here for additional data file.Dataset S3Genes whose expression was altered in the duodenum of Hfe knockout mice(0.05 MB XLS)Click here for additional data file.Dataset S4Genes whose expression was affected in the duodenum of mice fed an iron-supplemented diet(0.04 MB XLS)Click here for additional data file.Dataset S5Sequences of the primers used in the Q-RT-PCR experiments performed in this study(0.06 MB DOC)Click here for additional data file.


Monoamines are implicated in the modulation of adult hippocampal neurogenesis in depression models and following chronic antidepressant treatment. Given the key role of Sonic hedgehog (Shh) in adult neurogenesis, we examined whether monoaminergic perturbations regulate the expression of Shh or its co-receptors Smoothened (Smo) and Patched (Ptc). Combined depletion of both serotonin and norepinephrine with para-chlorophenylalanine (PCPA) resulted in a significant decrease in Smo and Ptc mRNA within the dentate gyrus subfield of the hippocampus. However, selective depletion of serotonin, using the serotonergic neurotoxin 5,7-dihyrdroxytryptamine , or norepinephrine, using the noradrenergic neurotoxin DSP-4, did not alter expression of Shh and its co-receptors, Smo and Ptc. Acute treatment with the monoamine releasing agent, para-chloroamphetamine (PCA) significantly upregulated Smo mRNA within the dentate gyrus. However, acute or chronic treatment with pharmacological antidepressants that modulate monoaminergic neurotransmission did not regulate Shh cascade expression. These results indicate that robust changes in monoamine levels can regulate the expression of the Shh signaling cascade in the adult rodent brain. Animal models of depression exhibit decreased adult hippocampal neurogenesis Sonic hedgehog (Shh) is a powerful regulator of adult hippocampal neurogenesis and is essential for the maintenance of the adult stem cell niches ad libitum. All animal procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the TIFR Institutional Animal Ethics Committee.Male Sprague–Dawley rats  were group-housed and maintained on a controlled 12 h light/dark cycle with access to food and water in situ hybridization analysis.To induce a combined depletion of norepinephrine (NE) and serotonin (5-HT), animals were treated with p-chlorophenylalanine  as previously described In situ hybridization was carried out as previously described 35S-labeled UTP (Amersham) from plasmids provided by Dr. Martial Ruat . Slides were incubated with radiolabeled riboprobes (1 × 106 cpm/150 μl per slide) in hybridization buffer overnight and then subjected to stringent post-hybridization washes. Slides were exposed to Hyperfilm β-max (Amersham) for 2–3 weeks. Shh, Ptc or Smo sense riboprobes did not yield significant hybridization (data not shown) confirming the specificity of the signal observed with the antisense riboprobes. Shh, Ptc and Smo mRNA levels were quantitated with the Macintosh-based Scion Image software  using 14C standards  for calibration and to correct for non-linearity. For Shh mRNA, expression in the horizontal and vertical limbs of the diagonal band (VDB) and in the neocortex was quantitated. For Ptc and Smo mRNA, the expression in the dentate gyrus was determined. The optical density was determined by outlining an equivalent area in each section, and the means were determined from measurements taken on both sides of 4 individual sections/animal.n = 5/group) were treated with PCA or vehicle and sacrificed as described earlier. Hippocampi were subdissected in cold saline and frozen in liquid nitrogen. Total RNA was isolated from hippocampal tissue using Tri Reagent (Sigma), according to the manufacturer's protocol. RNA was quantified using Nanodrop  and RNA quality determined by resolving on 1% formaldehyde agarose gel. cDNA was synthesized from 200 ng of total RNA per sample using Quantitect reverse transcription kit , as per the manufacturer's protocol. cDNA was amplified and visualized using a SYBR Green kit  in a Realplex mastercycler (Eppendorf). Hypoxanthine phopshoribosyl transferase (HPRT) was used as the endogenous control. To compare HPRT and target gene, relative quantification was performed using comparative CT method CT method involved averaging duplicate samples of each target and endogenous control in both control and treatment samples [i.e. ΔCT = absolute CT value − endogenous control CT value and ΔΔCT = ΔCT treatment − ΔCT control]. The fold change was calculated according to the formula Quantitative real-time PCR (qPCR) was also performed for acute PCA treatment. Animals  with significance determined at p values < 0.05.Combined, but not selective, depletion of norepinephrine and serotonin levels downregulates Smo and Ptc mRNA levels in the dentate gyrus.Experiments were analyzed for differences using the unpaired Student's Treatment with the monoamine releasing agent, PCA, increases Smo mRNA in the dentate gyrus, however pharmacological antidepressants do not regulate the Shh signaling cascade.The expression of Smo, the signaling component of the Shh receptor complex, was found to be significantly decreased (∼30%) in the hippocampal dentate gyrus subfield following depletion of both NE and 5-HT by PCPA treatment. In addition, there was a small but significant decrease (∼20%) in the level of Ptc mRNA in the DG of PCPA treated animals as compared to the vehicle group . In striin situ regulation of Smo and Ptc mRNA were further validated by qPCR for acute PCA treatment. Acute PCA resulted in a 70% increase in Smo mRNA in the hippocampus as compared to vehicle treated controls . Hippocampal Ptc mRNA levels were found to be unaltered following acute PCA treatment . Further, Shh mRNA levels in the VDB and the neocortex, and Smo and Ptc mRNA levels in the DG were not altered by either acute or chronic treatment with the pharmacological antidepressants DMI, FLX or TCP (Acute PCA treatment resulted in a robust and significant upregulation of Smo mRNA (∼65%) within the DG subfield of the hippocampus . PCA treX or TCP .The present study indicates that robust changes in levels of both 5-HT and NE modulate the expression of the Shh co-receptors, Smo and Ptc, in the hippocampal dentate gyrus subfield. While depletion of both 5-HT and NE (PCPA) decreased Smo and Ptc hippocampal mRNA levels, selective depletion of either 5-HT  or NE (DSP-4) did not alter their expression. Further, acute treatment with the monoamine releasing agent PCA significantly increased Smo mRNA levels. In contrast, acute or chronic antidepressant treatment with the serotonin selective reuptake inhibitor, fluoxetine, the norepinephrine selective reuptake inhibitor, desipramine, and the monoamine oxidase inhibitor, tranylcypromine did not regulate the expression of the Shh cascade. Our study provides novel evidence of an influence of NE and 5-HT on the expression of the Shh signaling cascade, a major developmental pathway that continues to be expressed in the adult brain.The effects of Shh have been best studied during development, where it regulates the proliferation, survival and fate determination of embryonic neuronal and glial precursors In this regard, it is particularly interesting to note that selective depletion of NE (DSP-4) or 5-HT  does not alter expression of the Shh cascade whereas combined depletion of both monoamines (PCPA) results in a significant decline in hippocampal Smo and Ptc mRNA levels. While DSP-4 and 5,7-DHT selectively induce a decrease in the proliferation of hippocampal progenitors It is intriguing that ECS and PCA differ in their effects on hippocampal Smo mRNA expression, as ECS would also be expected to enhance monoamine levels within the hippocampus At present the mechanism by which NE and 5-HT may influence expression of the Shh signaling cascade is unclear. However, the cAMP-Protein kinase A (PKA) pathway, is an attractive candidate. Activation of the cAMP cascade by the β adrenoceptor agonist isoproterenol enhances Ptc expression in the pineal gland Unlike previous results In conclusion, our results provide novel evidence that monoaminergic neurotransmission modulates the Shh signaling cascade expression in the adult mammalian brain, and highlight the need for future studies to understand the mechanism of this regulation and the role of Shh signaling in the effects of monoamines on adult structural plasticity.