Fernandez et al. (2003) identified Eimeria species in samples containing from two to eight oocysts originated from experimental infections, using the same methodology. Yet, Haug et al. (2007) observed

that there is a variation in the detection of each Eimeria species, depending on the number of oocysts used and the technique adopted for DNA extraction. Compound C manufacturer In field samples, many factors may interfere in the success and effectiveness of diagnosis by PCR, especially in regards to the presence of contamination. According to Haug et al. (2007) the DNA extraction process in stool samples is influenced by the formation of inhibitors of Taq DNA polymerase that affect the reaction. At least three Eimeria species were simultaneously identified using a multiplex PCR. These data are consistent with literature that reveals the occurrence of mixed infections in animals, thus hindering the accurate diagnosis of the species applying techniques traditionally Nutlin-3a purchase used ( Long and Joyner, 1984, Shirley, 1995 and Williams, 2001). In Japan, Kawahara et al. (2008) identified E. brunette 65.6%, E. maxima and E. necatrix 50%, E. tenella 37.5%,

and E. acervulina at 25% in the properties evaluated, using real time PCR for the detection of ITS-1. In China Sun et al. (2009) identified E. tenella, E. praecox and E. acervulina in more than 70% of the properties. Yet in South Korea, Lee et al. (2010) identified a high prevalence of the seven species emphasizing E. acervulina, E. tenella and E. brunetti. These data reinforce the idea that several factors influence the presence and prevalence of Eimeria species in each region. The morphological evaluation of oocysts also resulted in the diagnosis of a wide variety of species in the farms. Luchese et al. (2007) also identified frequencies ranging from 45.52%, 18%, 14% and 12.32% for E. maxima, E. brunetti, E. tenella and E. acervulina, respectively, through the morphology.

Terra et al. (2001), evaluated 60 carcasses of slaughtered broiler chickens in the city of Monte Alegre do Sul, Brazil, and identified frequencies of 90% for E. maxima, 86.4% for E. tenella, 86% for E. mitis, and 25% for E. acervulina and E. necatrix, through the morphology. However, Santos et al. (2003) failed to identify the occurrence either of Eimeria species in farms of São Paulo state using morphology, considering the observed overlap in the measurements of oocysts among species and the occurrence of infections caused by mixed infections, preventing diagnosis. According to this data ( Table 1), morphology could be a sensitive method for the discrimination of Eimeria species in field trials. Nevertheless, morphology is a technique with A sort of limitations to be used as a single tool for diagnosis of Eimeria species. It means that results obtained with this method should be carefully interpreted ( Woods et al., 2000 and López et al., 2007).

, 2010 and Crocker et al., 2010). actin-Gal4 (#3954 and 4414) and tubulin-Gal4 (#5138) drivers were obtained from the Bloomington Stock Center; nsyb-Gal4 was a gift from J. Simpson; Mef2-Gal4 was a gift from R. Galindo; all were

backcrossed six to eight generations to the iso31 background. UAS-inc-RNAi.1, Galunisertib research buy UAS-inc-RNAi.2, and UAS-Nedd8-RNAi are in the iso31 background and correspond to VDRC stocks 18225, 18226, and 28444, respectively ( Dietzl et al., 2007). UAS-Cul2-RNAi, UAS-Cul3-RNAi, and UAS-Cul3 Testis-RNAi correspond to NIG-Fly stocks 1512R-3, 11861R-2, and 31829R-2, respectively. UAS-inc and inc-Gal4 stocks were generated in the iso31 background (Bestgene). UAS-inc.4 and UAS-inc.9 are third chromosome insertions. inc-Gal4.1 is an X chromosome insert; inc-Gal4.2 LDK378 and inc-Gal4.3 are second chromosome insertions. As noted in the text, mutants in the CS and w1118 iso31 backgrounds were compared to their respective matched genetic backgrounds. For crosses involving transgenes, control animals were obtained by crossing transgenes to the appropriate isogenic background (e.g., for elavC155-Gal4 x w1118; UAS-RNAi, control crosses of elavC155-Gal4 x w1118 were performed). For X-linked transgenes, progeny from reciprocal crosses provided an additional control. One- to

five-day-old animals eclosing from LD-entrained cultures were loaded into glass tubes and assayed for 5–7 days at 25°C in LD cycles on cornmeal, agar, and molasses food using DAM5 monitors (Trikinetics). Animals were allowed to acclimate after loading for 1–2 days before data collection was initiated. For females, virgins were assayed. Locomotor data was collected

in 1 min bins, and Linifanib (ABT-869) a 5 min period of inactivity (Shaw et al., 2000 and Huber et al., 2004) was used to define sleep. Sleep parameters were analyzed with custom software written in MATLAB (Mathworks). Dead animals were excluded from analysis by a combination of automated filtering and visual inspection of locomotor traces. For statistical analysis of all sleep parameters that approximate normal distributions, unpaired Student’s t tests were used when comparing two genotypes; for comparisons of more than two genotypes, one-way ANOVA followed by Tukey-Kramer post hoc tests were used. For comparisons of sleep bout length, nonparametric Kruskal-Wallis tests followed by Bonferroni-corrected Mann-Whitney post hoc tests were used. For analysis in constant darkness, LD-entrained animals were placed in darkness and assayed otherwise as above. To assess rhythmicity and period length, data were binned at 30 min and analyzed with chi-square periodograms (p = .01); autocorrelation analysis yielded essentially identical results.

The reduction of the initial microbial load of the shredded carrots after singular and combined decontamination treatments are given in Table 2. As shown in Table 1 and Table 2, it was observed that the logarithmic reductions of 1.3 and 0.9 in precut treatments were determined for a single ultrasound treatment for TVC and YMC, respectively. In some decontamination outcome studies, the chlorine combined ultrasound treatments did not exceed the efficacy of the single ultrasound application, which is a very important result from the stand point of the antimicrobial effect of ultrasound. In both treatments with and without

chlorine the number of microorganisms was reduced by approx. 1 logarithmic unit in these experimental conditions which was applied for decontamination purposes. Huang et al. (2006) used the combination of chlorine check details dioxide c-Met inhibitor and ultrasound to kill the nalidixic acid resistant Salmonella enterica, serotypes Enteritidis, Typhimurium, and Mission and nalidixic-novobiocin resistant E. coli O157:H7 on apples and lettuce. The studies regarding the microbial reduction in these samples by chlorine dioxide at 0, 5, 10, 20, and 40 ppm with and without 170 kHz ultrasonic treatment for 10 min

are shown in Table 3. The results of Huang et al. (2006), demonstrate that chlorine dioxide can effectively reduce the numbers of test organisms from samples, and ultrasound application can promote the antimicrobial effect of chlorine dioxide on Salmonella and E. coli O157:H7 inoculated apples

and lettuce samples and a single treatment of ultrasound caused however an additional 1.2–1.9 log10 CFU/g reduction in the samples. The decontamination efficiency of chlorine dioxide when combined with ultrasonication and applied to both test organisms showed that the inoculated apple samples were higher than the inoculated lettuce. This result could be that the structural differences and irregular surfaces of lettuce may provide some protection for the microbial cells. As shown in Table 4, a 1.52 log10 CFU/g additional reduction was obtained with an ultrasound application on E. coli O157:H7 inoculated apples, in experiments which applied ultrasound with the chlorine dioxide, the reduction values were additionally increased in the range of 0.6–2.4 log10 CFU/g depending on the chlorine dioxide concentrations (5–40 ppm). In the lettuce experiments, it was determined that an additional reduction in Salmonella spp. was obtained between 0.3 and 0.65 log10 CFU/g using the ultrasound treatment. São José and Vanetti (2012) studied the effect of ultrasound (45 kHz, 10 min, 25 °C) in the presence of 5% hydrogen peroxide and 40 mg/L peracetic acid on cherry tomatoes. The reduction of the total viable count, yeast and mold count, and inoculated S. enterica typhimurium that adhered to the surface of the tomatoes was evaluated ( Table 5).

When developing cortical neurons derived from siRNA-expressing progenitor cells were examined in E21 embryos, we found that neurons expressing the NP1-siRNA exhibited impaired radial migration to the cortical plate (CP) (Figures 6A and 6B), and displayed misorientation with respect to the CP (Figure 6B, arrows), as compared to neurons expressing control siRNA,

as previously demonstrated (Chen et al., 2008). This was quantified by the percentage of transfected cells located in the ventricular zone (VZ)/subventricular zone (SVZ), in the intermediate zone (IZ), and in the CP (Figure 6C), with much higher and lower fraction of NP1-siRNA transfected neurons located at the VZ/SVZ and CP, respectively, as compared to control cells. Further RG7204 nmr examination of cortical neurons Ipatasertib concentration expressing NP1-siRNA showed that a large fraction of them exhibited multiple neurites (multipolar) in the VZ/SVZ and IZ, whereas most neurons in these regions of control embryos had a single neurite (unipolar) or two neurites (bipolar), with only a small fraction of control cells in the VZ/SVZ exhibiting multipolar morphology (Figures 6A and 6B, arrowheads, and Figure 6D). Furthermore,

NP1-siRNA-expressing multipolar neurons located in the VZ/SVZ displayed a higher total neuritic length (Figure 6Fa) than control multipolar neurons in these regions, although the average neurite number per cell was not significantly different (Figure 6Fb). These polarization defects were illustrated by microscopic tracings of 20 randomly sampled control-siRNA and NP1-siRNA transfected neurons in various layers (Figure 6E). Our studies in cultured hippocampal neurons showed that Sema3A might regulate neuronal polarization by selectively promoting dendrite growth and suppressing axon growth (Figure 5). We thus have also examined the length of the leading process that becomes the

apical dendrite in control or NP1-siRNA transfected bipolar neurons. As shown in Figure 6G, NP1 downregulation resulted in significant reduction of the growth of the leading process in cells located at the IZ and CP, consistent with the promotion of dendrite growth by Sema3A signaling. In addition, the increased total neurite length of multipolar NP1-siRNA-expressing cells in the VZ/SVZ (Figure 6Fa) is also consistent with the Sema-3A-suppression on axon growth, although immunostaining of the abnormal processes in multipolar neurons for axon-specific (-)-p-Bromotetramisole Oxalate markers was not successful due to intense axon staining from nontransfected cells in these regions. We have also performed in utero electroporation using either one of the two NP1-siRNAs alone and found similar neuronal polarization results as to that described above for electroporation with both NP1-siRNAs together. Since neuronal polarization occurs in VZ/SVZ prior to radial migration, where downregulation of NP1 resulted in pronounced polarity defect, the failure of radial migration may be attributed in part to the polarization defect (see Discussion).

, 2007), and play a role in shaping the timing and dynamic range of cortical

activity (Cobb selleck products et al., 1995, Sohal et al., 2009, Cardin et al., 2009, Pouille and Scanziani, 2001, Gabernet et al., 2005, Cruikshank et al., 2007 and Pouille et al., 2009). Despite this wealth of knowledge, how PV cells contribute to the operations performed by the cortex during sensory stimulation is not known. Here we show that PV cells profoundly modulate the response of layer 2/3 Pyr cells to visual stimuli while having a remarkably small impact on their tuning properties. This modulation of cortical visual responses by PV cells is described by a linear transformation whose effects are visible in firing rate once above spike threshold and is well captured by a conductance-based model of the Pyr cell. These results indicate that PV cells are ideally suited to modulate response gain, an essential component of cortical computations that changes the response of a neuron SCR7 datasheet without impacting its receptive field properties. Gain control has been implicated, for example, in the modulation of visual responses by gaze direction (Brotchie et al., 1995 and Salinas and Thier, 2000) as well as by attention (Treue and Martinez-Trujillo, 1999 and McAdams and Maunsell, 1999). To control the activity of PV cells we conditionally expressed the light-sensitive proton pump Archeorhodopsin (Arch-GFP; to

suppress activity; Chow et al., 2010) or the light-sensitive cation channel Channelrhodopsin-2 (ChR2-tdTomato; to increase activity; Boyden et al., 2005 and Nagel et al., 2003) in V1 using viral injection into PV-Cre mice ( Hippenmeyer et al., 2005). Targeted electrophysiological recordings were performed in anesthetized mice under the guidance of a two-photon laser-scanning microscope. We characterized PV cells in the adult PV-Cre mouse line immunohistochemically and electrophysiologically ( Figure 1; Figure S1, available online). We fluorescently labeled the cells expressing Cre by crossing PV-Cre mice with a tdTomato reporter line ( Madisen et al., 2010). tdTomato was present exclusively in neurons that were also Parvulin immunopositive for PV, confirming that cells

expressing Cre also expressed PV (97% ± 2%; mean ± standard deviation [SD]; n = 400 cells in 4 mice; Figure S1). Targeted loose-patch recordings from fluorescently labeled PV cells in layer 2/3 of the primary visual cortex in vivo (spontaneous rate: 2.1 ± 3.1 spikes/s; n = 79) showed that their spike-waveforms ( Figure S1) had faster kinetics than non-PV cells recorded using the same configuration, consistent with these cells being of the fast-spiking type ( McCormick et al., 1985, Connors and Kriegstein, 1986, Swadlow, 2003, Andermann et al., 2004 and Mitchell et al., 2007). Because the vast majority (∼90%) of the non-PV cells in layer 2/3 are Pyr cells ( Gonchar and Burkhalter, 1997), from here on we will refer to non-PV cells as Pyr cells.

, 2007). Collectively, these findings suggest that modest modulation of α- or β-secretase activity for extended time period can have a profound impact on Aβ pathology in aged brain. Beyond the level of plaque load, ADAM10 activity also affected the morphology of Aβ plaque. While Tg2576/DN Tyrosine Kinase Inhibitor Library price double-transgenic mice had more neuritic plaques with compact cores (versus Tg2576), most plaques found in the double-transgenic mice overexpressing WT or Q170H displayed irregular diffuse morphology. Neuritic plaques are known to be more tightly associated with AD pathogenesis than diffuse plaque. For example, fibrillar core-containing neuritic plaques are predominant in AD brains, whereas diffuse plaques

are more frequent in nondemented elderly (Selkoe, 2001). Furthermore, neuritic, but not diffuse, plaques are associated with pathological phenotypes of the disease, including dystrophic neurites, activated microglia, and reactive astrocytes (Figure 5). While further studies are warranted to delineate the mechanism underlying the observed differences in plaque morphology, our DAPT nmr findings suggest that enhanced ADAM10 activity may lessen Aβ pathology not only by decreasing plaque load but also by affecting plaque morphology. Currently, it is unclear how the two secreted APP ectodomains,

sAPPα and sAPPβ, engender different effects—neurotrophic versus neurodegenerative—on Bay 11-7085 neurons. Interestingly, a 35 kDa fragment derived from sAPPβ has been demonstrated to bind the cell surface death receptor DR6 and trigger axonal degeneration in neurons (Nikolaev et al., 2009). In addition to the extra 16 amino acids at the C terminus of sAPPα, the difference in where these ectodomains are generated, cell surface for sAPPα, and endosome for sAPPβ may play a key role in determining their distinct biological functions. At the cell surface, APP can be present as a dimer in cis or trans formation ( Wang and Ha, 2004). Structural and imaging studies have shown that liberated sAPPα

can bind as a ligand to APP at cell surface and disrupt APP dimer complex to exert its neuroprotective effect ( Gralle et al., 2009 and Wang and Ha, 2004). Therefore, it is interesting to speculate that ADAM10 cleavage of APP may shift the complex formation toward neurotrophic APP-sAPPα (or its cleavage derivatives) versus APP-APP dimerization at the cell surface. Accumulating evidence shows that elevated hippocampal neurogenesis improves memory function (Zhao et al., 2008) and that downregulation of hippocampal neurogenesis is associated with cognitive impairments in AD (Choi et al., 2008). Notably, adult neurogenesis has been reported to be affected by all three early-onset familial AD genes, APP, PSEN1, and PSEN2, and by Aβ in AD mouse models ( Mu and Gage, 2011), suggesting its tight link to the etiology and pathogenesis of the disease.

Second, we can

address the question of whether feature attention is dissociable from spatial attention by determining whether, for a given spatial attention state, fluctuations in feature attention affect behavior. Finally, fluctuations in attention can reveal the cortical extent of modulation by either form of attention. If distant groups of neurons are comodulated by attention, then the strength of their attentional modulation should be correlated on a trial-to-trial basis. These analyses require an estimate of the animal’s attentional state on a single trial. An instantaneous measure of spatial attention based on the responses of populations of V4 neurons can reliably predict an animal’s ability to perform a difficult psychophysical task several hundred milliseconds in the future (Cohen and Maunsell, 2010). We used this measure and an analogous measure of feature attention to predict behavior to examine spatial extents of the two types of GS-7340 cost attention. Our task had four attention conditions: each trial belonged to one of two spatial attention conditions (left or right) and one of two feature attention conditions (orientation or spatial frequency). Using similar methods to those in our previous study (Cohen and Maunsell, 2010), we quantified attention on a single trial as the similarity of the population response to the mean responses in each attention condition. This method is not an ideal decoder to distinguish

between correct and incorrect trials based on population responses. Instead, we tested the hypothesis that a single-trial extension of the traditional definition Anti-diabetic Compound Library of attention, which compares mean responses in different attention conditions (e.g., Figure 2) could predict behavior. We focused our analyses on trials with a single, difficult orientation change or a single, difficult spatial frequency change for which all trials had valid attentional cues. The average performance

on these trials was 34% correct across all data sets (total correct trials divided by total correct plus total missed trials), which is in a range where attention can be the difference between correct and incorrect trials. We first plotted the population response on each trial these in an n-dimensional space in which each of the n simultaneously recorded neurons represented one dimension. If we recorded 83 neurons in the two hemispheres combined, the population response on each trial would be a point in an 83-dimensional space. For ease of visualization, we have plotted these responses for two simultaneously recorded neurons in an example recording session (in a two-dimensional space; Figures 4A and 4C), but the actual analyses used all simultaneously recorded neurons in a high-dimensional space. We then projected each response onto a putative “spatial attention axis” and a putative “feature attention axis” using a process that is illustrated for the data from an example recording session in Figures 4A–4D.

After a 2 hr incubation, we observed significant increases in amounts of dCREB2-b compared to amounts in brains cultured in 20-mM-Mg2+ medium (Figure 7E). The increase in

dCREB2-b in Mg2+-free medium is strongly suppressed by the NMDAR antagonist MK801 and by dNR1 mutation, suggesting that in the absence of Mg2+ block, Ca2+ entry through dNMDARs increases dCREB2-b protein levels. We next wanted to determine whether the increase click here in dCREB2-b expression observed in elav/dNR1(N631Q) flies is sufficient to inhibit LTM formation. Thus, we plotted one-day memory scores after spaced training as a function of dCREB2-b protein levels in wild-type and hs-dCREB2-b flies heat shocked for various durations. As seen in Figure 8A, defects in LTM formation were highly correlated with dCREB2-b protein expression. Heat-shocked wild-type flies and non-heat-shocked hs-dCREB2-b flies had normal one-day memory and similar dCREB2-b expression. On the other hand,

hs-dCREB2-b flies expressed increasing Kinase Inhibitor Library cell line amounts of dCREB2-b protein upon increasing heat-shock duration. This increase in dCREB repressor expression was correlated with a decrease in one-day memory in a linear fashion in the range tested. Significantly, we found that data from elav/dNR1(wt) and elav/dNR1(N631Q) flies plotted on the same graph fit the same regression line as our hs-dCREB2-b data; elav/dNR1(wt) flies expressed wild-type levels of dCREB2-b and had one-day memory scores comparable to wild-type and non-heat-shocked dCREB2-b flies, while elav/dNR1(N631Q) flies expressed similar amounts of dCREB2-b and had similar however memory to hs-dCREB-2b flies heat shocked for 30 min. In contrast, dNR1EP3511 flies did not show any increases in dCREB2-b mRNA or protein but had poor LTM scores (see also Figures 7B and 7C). These results indicate that the increase in expression of the dCREB2-b repressor in Mg2+ block mutants is

correlated with and sufficient to cause the decrease in LTM observed in these mutants, while memory defects in dNR1 hypomorphs likely occurs through a different mechanism. Although the mechanism through which Mg2+ block restricts NMDAR activity is well known, the cellular and behavioral functions of Mg2+ block have not been extensively studied. In this study, we used transgenic flies expressing dNR1(N631Q) to show that Mg2+ block is important for formation of LTM. Previous studies of hypomorphic mutants have shown that NMDARs are required for both learning and LTM. In contrast, our Mg2+ block mutants do not have learning defects. This suggests that although Ca2+ influx through NMDARs is important for learning, inhibition of influx during uncorrelated activity is not. Notably, elav/dNR1(N631Q) flies have slightly enhanced learning. Consistent with this result, NMDAR-dependent induction of hippocampal LTP is enhanced in the absence of external Mg2+ ( Mizuno et al.

Unlike in controls, 27% of Rh6-lacZ-positive R8 axons stalled at the medulla neuropil border, while 8% terminated incorrectly in the M1/M2 layers (265 axons, n = 13) ( Figures 8B–8F). Second, NetBcd8 was directed to a subset of ectopic layers using MH502-Gal4, a driver active in lamina Selleckchem MK0683 neurons L1/L2, ascending T1 medulla neurons and C2/C3 neurons throughout development. Transient expression in R cells during larval and pupal development was suppressed using ey3.5-Gal80 and lGMR-Gal80 transgenes ( Figures 8G and S7). Expression analysis at 55 hr confirmed that high levels of NetB are present in layers M1/M2 ( Figure 8H). Despite the presence of endogenous Netrins

in the M3 layer, 32% of Rh6-lacZ-positive R8 axons stalled at the medulla neuropil border, while 19% stopped in the M1/M2 layers (227 axons, n = 12) ( Figures 8I–8J). Using NP1086-Gal4 ( Rister

et al., 2007), we also expressed NetBcd8 in T1 neurons, which extend dendrites into layer M2 and axons into the lamina. Membrane-tethered ligand was not detected in the medulla, but in the lamina, and consistently, R8 axon targeting to layer M3 was unaffected ( Figures S7L–S7M′). This confirms that axons are the primary site of NetB release, and ectopic ligand expression using MH502-Gal4 can be mainly attributed to lamina neurons L1 and L2. The increased percentage of redirected axons to defined NetB-expressing layers with MH502-Gal4 compared to the effects of wide ectopic expression using ap-Gal4 supports the model FK228 in vivo that layer-specific localization of Netrins is sufficient for R8 axon targeting. Recent studies identified at least four molecular mechanisms that control layer-specific targeting in the nervous system by cell-cell interactions independently of neural activity. First, combinatorial expression of homophilic cell surface molecules promotes the recognition and stabilization of contacts between matching branches of pre- and postsynaptic neuron subsets. For instance, four members of the immunoglobulin MRIP superfamily of

cell adhesion molecules, Sidekick 1 and 2 and Dscam and DscamL, are expressed and required in subsets of bipolar, amacrine, and retinal ganglion cells for targeting to different inner plexiform sublayers (IPLs) in the chick retina (Yamagata and Sanes, 2008). In Drosophila, the leucine-rich repeat protein Caps may play an analogous role, as it is specifically expressed in R8 cells and target layers M1–M4 and, thus, could promote homophilic interactions to stabilize connections within correct columns and layers ( Shinza-Kameda et al., 2006). Second, concise temporal transcriptional control is used to regulate the levels of ubiquitous cell surface molecules and, thus, adhesiveness of afferent and target neurons to balance branch growth and targeting.

Zach Hall, and original LRP4 constructs were gifts from Dr. Tatsuo Suzuki. Flag-MuSK was generated as previously described (Zhang et al., 2008). To generate Flag-LRP4 and Flag-ecto-LRP4, we amplified full-length LRP4 and ecto-LRP4 cDNA by PCR from original LRP4 construct and subcloned into HindIII/BglII sites in pFlag-CMV1 downstream of this website an artificial signal peptide sequence and a Flag epitope. LRP4-miRNA construct

miLRP4-1062 was generated using the BLOCK-It Pol II miR RNAi Expression Vector Kit (K4936-00, Invitrogen), which has been previously described and verified to be most potent in inhibiting LRP4 expression (Zhang et al., 2008). For all the constructs, the authenticity was verified by DNA sequencing in Eurofins MWG Operon. LRP4loxP mice, in which the exon 1 of the LRP4 gene was flanked by loxP sites, were generated as described in Supplemental Experimental Procedures. They were crossed with CP-673451 chemical structure HSA-Cre and HB9-Cre transgenic mice to generate muscle or motoneuron specific knockout (KO) as well as double knockout (dKO) LRP4 mutant mice. The mutant mice were generated on a BL6/129 mixed background and backcrossed into C57BL/5J mice.

Crosses generated the expected Mendelian numbers of each genotype and genotyping procedures were described in Supplemental Experimental Procedures. Mice were housed in a room with a 12 hr light/dark cycle with ad libitum access to water and rodent chow diet (Diet 1/4” 7097, Harlan Teklad). Experiments with animals were approved by Institutional Animal Care and Use Committee of the Georgia Health Sciences University. Whole-mount staining of diaphragms, quantitative analysis of NMJs, single muscle fiber assay, and electrophysiological recording were performed as previously described with modification (Dong et al., 2006-2007 and Li et al., 2008) (see Supplemental Experimental Procedures for details). Electron microscopic secondly studies were carried as described previously (Wu et al., 2012). Briefly, entire diaphragms (P0 and P15) were isolated and fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer for 1 hr at 25°C and 4°C overnight. Synaptic

segments of muscles were isolated and fixed in sodium cacodylate-buffered (pH 7.3) 1% osmium tetroxide for 1 hr at 25°C. After washing three times with phosphate buffer, 10 min each, synaptic segments were dehydrated through a series of ethanol steps (30%, 50%, 70%, 80%, 90%, and 100%), rinsed with 100% propylene oxide three times, embedded in plastic resin (EM-bed 812, EM Sciences), and subjected to serial thick sectioning (1–2 μm). Some sections were stained with 1% toluidine blue for light microscopic identification of phrenic nerves. Adjacent sections were cut into ultra-thin sections, mounted on 200 mesh unsupported copper grids, and stained with uranyl acetate (3% in 50% methanol) and lead citrate (2.6% lead nitrate and 3.5% sodium citrate [pH 12.0]). Electron micrographs were taken by a JEOL 100CXII operated at 80KeV.