e., comparable to participants from the Down-group in

the asynchronous body condition). The present lesion data from a group of OBE-patients put previous anecdotal data about abnormal self-location INCB018424 research buy and first-person perspective on solid grounds. They also show that the detailed analysis of such clinical neuroanatomical data on self-consciousness translate to functional neuroimaging data on self-consciousness in healthy participants, highlighting collectively the significance of the TPJ as an important brain structure for self-consciousness related to self-location and the first-person perspective (Figure 5C). There are only a few carefully analyzed case studies in neurological patients with OBEs due Selleck Dinaciclib to focal brain damage or electrical brain stimulation. In addition, previous work has associated OBEs with many different brain structures, such as the right and left TPJ (Blanke et al., 2002, Blanke et al., 2004, Brandt et al., 2005 and Maillard et al., 2004), and several structures within the TPJ: posterior superior temporal gyrus (Blanke et al., 2004), angular gyrus (Blanke et al., 2002 and Brandt et al., 2005; Heydrich et al., 2011), and supramarginal gyrus (De Ridder et al., 2007 and Maillard et al., 2004), but also the precuneus (De Ridder

et al., 2007) and fronto-temporal cortex (Devinsky et al., 1989). Here we lateralized and localized brain damage in OBE-patients to the right TPJ. The right TPJ is the classical lesion site and side associated with visuo-spatial neglect (Halligan et al., 2003 and Karnath et al., 2001), a clinical condition shown to disturb the patient’s egocentric spatial relationship with extrapersonal space, visuo-spatial perspective taking (Farrell and Robertson, 2000), and own body perception such Phosphoprotein phosphatase as somatoparaphrenia (Vallar and Ronchi, 2009). A bilateral, but right lateralized, implication of the TPJ has also been observed during egocentric visuo-spatial

perspective taking (Maguire et al., 1998 and Ruby and Decety, 2001), multisensory integration, as well as imagined changes in self-location (Arzy et al., 2006, Blanke et al., 2005 and Schwabe et al., 2009) in healthy subjects. Despite the present strongly right-lateralized lesion data, our fMRI data reveal that self-location and first-person perspective likely depends on cortical processing in both TPJs. One of our patients suffered from OBEs due to left TPJ involvement. It may thus be that OBEs following interference with the left TPJ may be less reported by patients, potentially due to interference with the language cortex at the left TPJ. More data in larger patient samples in patients with OBEs will be necessary to clarify this. The TPJ is an excellent candidate for self-consciousness.

Subsequently, it has been established that high-affinity NMDARs are a common target for spillover-mediated signaling (i.e., Asztely et al., 1997; Isaacson, 1999; Overstreet

et al., 1999; Carter and Regehr, 2000; Scimemi et al., 2004). At PF-MLI synapses, NMDAR activation is only detected during high-frequency or high-intensity IOX1 datasheet molecular layer stimulation, indicating that NMDARs are located outside the postsynaptic density (Carter and Regehr, 2000; Clark and Cull-Candy, 2002). Such stimulation protocols produce synchronous activation of a high density of local fibers, generating extrasynaptic signaling that may be rare in vivo during physiological stimuli (Arnth-Jensen et al., 2002; Marcaggi and Attwell, 2005). We found that spillover from a single CF generates both AMPAR- and NMDAR-mediated depolarization of MLIs, suggesting that CF and PF stimulation activates different sets of receptors. In contrast to FFI mediated by PFs (Figure S3 and Mittmann et al.,

2005), CF stimulation generates a long-lasting (∼100 ms) component of inhibition to MLIs that contributes to the long-lasting buy PD-0332991 component of disinhibition to PCs (Figure 7). The persistent NMDAR-mediated component thus expands both inhibition and disinhibition to PCs, potentially enhancing the contrast between areas of active and inactive PCs. Typical FFI narrows the window for synaptic integration by providing a rapid increase in principal cell inhibition either that provides balanced regulation of excitation (Pouille and Scanziani, 2001; Wehr and Zador, 2003; Mittmann et al., 2005; House et al., 2011). Thus, we were surprised that blocking GABAARs had only small, variable effects on the number of CF-evoked

APs in individual MLIs (Figure 4). We considered three potential factors that could produce variability in the effectiveness of CF-FFI, including the magnitude of FFI, the location of FFI relative to CF-mediated excitation, and the potential for a fraction of MLI inputs to promote MLI excitability (Chavas and Marty, 2003). Since CF-mediated inhibition of PF-evoked spiking was robust (Figure S6) and somatic inhibitory conductance injection effectively decreased CF excitation of MLIs, we predict that the locations of excitatory and inhibitory conductances could promote the transmission of somatic CF-mediated excitation (Brown et al., 2012) despite reciprocal inhibition. Although MLIs are generally thought to be electronically compact because of their high input resistance and short dendrites, their thin dendrites behave as passive cables that filter synaptic responses, resulting in sublinear integration (Abrahamsson et al., 2012). This suggests that shunting that depends on location (i.e., Gulledge and Stuart, 2003) may be important for MLI inhibition.

, 2011). Second, the hexanucleotide expansion was highly associated with disease in the same cohort of ALS cases Veliparib price and controls that was used to identify the chromosome 9p21 region within the Finnish population. Furthermore, the association signal based on the presence or absence of the expansion was many times greater than that indicated by the surrounding SNPs (p value based on expansion = 8.1 × 10−38 versus 9.11 × 10−11 based on the most associated SNP rs3849942 in the initial Finnish ALS GWAS) (Laaksovirta et al., 2010).

Third, the hexanucleotide repeat expansion was not found in 409 population-matched control subjects or in 300 diverse population samples screened in our laboratory. Fourth, we found that a large proportion of apparently unrelated familial ALS and FTD cases carried the same hexanucleotide repeat expansion within C9ORF72. Within this cohort of European-ancestry familial samples, we identified SAR405838 three additional multigenerational families within which the repeat expansion segregated perfectly with disease. Fifth, FISH analysis demonstrated that the repeat expansion

is large in size (at least 1.5 kb to be visualized by this technique, Figure 2C), and such long expansions are typically pathogenic ( Kobayashi et al., 2011). Finally, another group independently discovered the same genetic mutation to be the cause of chromosome 9p21-linked FTD/ALS ( DeJesus-Hernandez et al., 2011). Our data indicate that both ALS and FTD phenotypes are associated

with the C9ORF72 GGGGCC hexanucleotide repeat expansion. Several members of the GWENT#1 and DUTCH#1 pedigrees manifested clinical signs of isolated motor neuron dysfunction or isolated cognitive decline, whereas other affected members developed mixed ALS-FTD symptomatology over the course of their illness ( Pearson et al., 2011). It is interesting to note that the frequency of the repeat expansion was almost identical in our ALS and FTD case cohorts, suggesting that carriers of the mutant allele are equally at risk for both forms of neurodegeneration. Our data support the notion that the observed clinical and pathological overlap between ALS and FTD forms of neurodegeneration may be driven in large part by the C9ORF72 hexanucleotide repeat expansion. MTMR9 The identification of the cause of chromosome 9p21-linked neurodegeneration allows for future screening of population-based cohorts to further unravel the overlap between ALS and FTD and to identify additional genetic and environmental factors that push an individual’s symptoms toward one end of the ALS/FTD clinical spectrum. Some early observations may already be made: among our Finnish FTD cohort, we identified several patients carrying the pathogenic repeat expansion who presented with nonfluent progressive aphasia.

A comparison of non-selective with selective counts indicated that the proportion of injured cells (Eq. 

(2), data not shown) was not significantly influenced by temperature (p = 0.228), aw (p = 0.371) or water mobility (p = 0.411). Storage time just significantly influenced the proportion of injured cells (p = 0.044), as longer storage times led to check details increasing proportions of injured cells. These results do not support a hypothesis that the mechanism of inactivation changed from membrane damage at lower temperatures (≤ 50 °C) to ribosomal degradation at higher temperatures (> 50 °C) as suggested by Aljarallah and Adams (2007). Heating cells to temperatures just above their maximum growth temperature causes damage to the cytoplasmic membrane, which in enteric bacteria can be detected by plating the cells on non-selective media and media containing bile salts. If cells are treated at sufficiently high temperatures, death results from ribosome degradation, and there will be a small or no difference in the ability of the survivors to grow on selective and non-selective media. Aljarallah and Adams (2007) observed these effects using Salmonella treated at 53 °C and

Alpelisib manufacturer 60 °C at water activities of 0.99 and 0.94. Results in the present study indicated that there were no significant differences in the proportion of injured cells among those exposed to different water activities and temperatures. However, one major difference in our study is that we investigated lower water activities

(< 0.6) over a wider temperature range (21 °C–80 °C). Salmonella survival data at 21 °C during 168 days (6 months) of storage (results not shown) showed that populations were maintained under these conditions, with log reduction values of 0.001, 0.003, 0.002, 0.003 and 0.005 log CFU/day at aw levels of 0.16 ± 0.01, 0.26 ± 0.002, 0.34 ± 0.009, 0.41 ± 0.01 and 0.53 ± 0.05, respectively. These data indicated a significantly better survival of Salmonella at lower aw levels (0.16 Non-specific serine/threonine protein kinase and 0.26) as compared to higher ones (0.34 to 0.53) (p < 0.001). Significant differences in survival were also observed between the two highest aw levels (0.41 and 0.53). However, no significant differences in survival were found between aw levels of 0.16 and 0.26 (p = 0.541), 0.34 and 0.41 (p = 0.730) or 0.34 and 0.53 (p = 0.074). No influence of water mobility at the same aw level was observed (p = 0.917). Because the survival rates were essentially linear at 21 °C, the Geeraerd-tail model, the Weibull model (with β ≠ 1 in Eq.  (5)) and the biphasic-linear model were not suitable for describing the data. The Baranyi and the log-linear models were appropriate in describing the data for all conditions (ftest < Ftable) and showed similar statistical fit parameter values ( Table 2). Fig. 1 presents data on Salmonella survival at 36 °C during 168 days (6 months) of storage.

, 2009, Farkas et al., 2009, Chee et al., 2008, Guzman, 1984, Gonzales et al., 2004 and Alcaino et al., 2002). In addition, in areas where C. felis appears to be predominant, C. canis still represents a significant proportion of fleas in dog populations with a prevalence of 10–12.5% in Europe and up to 21% in the USA ( Franc et al., 1998, Gracia et al., 2007, Beck et al., 2006 and Durden

et al., 2005). The goal of a successful flea control program is to eliminate fleas quickly, continuously, and to prevent them from producing viable eggs Vismodegib purchase that contaminate the environment. The present study was conducted in order to determine the efficacy of a soft, beef-flavored chewable formulation of afoxolaner (Nexgard®, Merial) against the dog flea, C. canis, after a single oral administration to dogs. The study was designed to assess the long-acting efficacy of afoxolaner against adult fleas evaluated 12 or 24 h after weekly infestations and in the prevention of environmental contamination with flea eggs. Thirty-two beagle dogs (18 males and 14 females, 8–58 months of age, weighing 7.6–15.7 kg) were included in the study. Dogs had not been exposed to ectoparasiticides within 3 months prior to treatment. The protocol of the study was reviewed and approved by the www.selleckchem.com/products/BI6727-Volasertib.html Merial Institutional Animal Care and Use Committee. Dogs were handled with due regard for their welfare (USDA, 2008). All animals were housed individually. All dogs received

commercial food, once daily, in a sufficient amount to maintain body weight appropriate for the breed, and water was provided ad libitum. The dogs enrolled in the studies underwent a full physical examination by a veterinarian on Day −7 and were examined once daily for health observations. The study design was in accordance with the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of parasiticides for the treatment, prevention and control of flea and tick infestation on dogs and cats (Marchiondo et al., 2013), and was conducted in accordance with Good Clinical Practices as described in International Cooperation on Harmonisation

of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guideline GL9 ( EMEA, 2000). The dogs were acclimatized to study conditions 7 days prior to treatment and were observed for general health Adenosine conditions throughout the study. On Day −7, they were infested with 100 (±5) adult C. canis fleas from an Irish strain originate from Tipperary County (collected in the field on dogs some years ago and maintained as laboratory strain since). Dogs were ranked by decreasing live flea counts pre-treatment and allocated to 8 blocks of 4 animals each. Within each block, each dog was randomized to Groups 1, 2, 3 or 4 and weighed on Day −3 for dosage calculations using a calibrated balance. On Day 0, dogs were offered their normal ration prior to treatment. Dogs assigned to Groups 1 and 3 remained untreated and served as controls.

Therefore, at present, no firm conclusions seem to be possible on the degree to which envelope ICMs or phase ICMs may differentiate between different disorders. At a very general level, it may be hypothesized that disorders with a high degree of structural alteration may be associated with strong changes in both envelope and phase ICMs (e.g., AD), whereas disorders with less prominent connectomic changes (e.g., PD) may primarily show altered phase ICMs (Table 1). The current data point to a preferential pathophysiological involvement of certain ICMs, which may be altered in specific subnetworks in the respective disorders. However, more neurophysiological investigations of envelope

Selleckchem Navitoclax ICMs and phase ICMs are required, which ideally should be combined with source space analyses (Hipp et al., 2012, Brookes et al., 2012 and Marzetti et al., 2013). This might allow the identification of ICMs that reflect GSK1120212 in vitro network pathologies with high specificity and sufficient sensitivity to monitor longitudinal change during disease progression or recovery. Computational modeling has taken up the challenge of investigating the mechanisms

underlying ICMs. One central motivation of such simulations has been to explore the dynamic implications of structural brain connectivity (Bullmore and Sporns, 2012). In addition to incorporating information about anatomical connections (Hagmann et al., 2008), these models also include a generalized description of the dynamics of regional neural populations (Figure 6A). Typically, the models assume largely uniform features for the dynamics of the nodes or the interconnections (Deco and Corbetta, 2011 and Deco et al., 2011). The results of several such modeling approaches (Zhou et al., 2006, Honey et al., 2007, Deco et al., 2009 and Haimovici et al., 2013) converge on a number of central MTMR9 findings. In particular, the models reproduce empirically observed correspondences between structural connectivity and envelope ICMs (Honey et al., 2009). As a result, envelope ICMs found in the models

typically reflect topological features of the underlying connectome, such as modules and hubs (Honey et al., 2007). Models further suggest that structural modularity can endow ICMs with dynamics on different temporal scales (Figure 6B). Intramodular links may provide a substrate for fast interactions, while intermodular connections allow the integration of nodes across modalities at longer timescales (Pan and Sinha, 2009). It is currently unclear to what extent this difference between topological scales may contribute to the physiological distinction between envelope and phase ICMs. Interestingly, similar results were found in models differing strongly in their local node dynamics (Figure 6A), which may be represented by chaotic oscillators (Honey et al., 2007), phase oscillators (Cabral et al., 2011), neural mass models (Deco et al., 2009), or simple discrete excitable nodes (Haimovici et al., 2013).

, 1999 and Orimo et al., 2008). Cortical deposits of synuclein that occur late in the disease presumably contribute to cognitive problems. Certain nonmotor manifestations of PD can respond to dopamine replacement, raising questions about the significance

of synuclein deposition outside the nigrostriatal projection. However, many symptoms do not respond, and the widespread accumulation of synuclein presumably accounts for many of the dopamine-resistant symptoms. It is important to note that the relationship between α-synuclein deposition and neuronal dysfunction remains unclear. In the substantia nigra, substantial cell loss occurs before symptoms develop, suggesting that protein deposition is not as important as cell loss. However, cell loss may not accompany synuclein deposition elsewhere. In the enteric nervous system, Lewy pathology www.selleckchem.com/products/VX-770.html is indeed not associated with cell loss (Annerino et al., CB-839 in vitro 2012), raising the possibility of a functional rather than anatomic disturbance. On the other hand, synuclein deposition itself may not even produce dysfunction, and pathologic investigation of many older individuals (up to 30% of centenarians) reveals extensive synucleinopathy (incidental Lewy body disease) with no clear neurological symptoms (Ding et al., 2006 and Markesbery et al., 2009). Indeed, synuclein aggregation may represent a neuroprotective response,

with a different species of synuclein responsible for dysfunction. Although synuclein deposition has thus superseded cell loss as evidence of degeneration, its actual role in the degenerative process remains unknown. α-synuclein has also been

implicated in at least two other disorders, multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Interestingly, these conditions also produce clinical parkinsonism but involve the deposition of α-synuclein until in different cells from those affected by typical PD. MSA can begin with parkinsonism, autonomic failure, or cerebellar ataxia but usually progresses to involve one or both of the other components, resulting in the recognition that these initially disparate conditions reflect a common disorder. However, the parkinsonism observed in MSA does not generally respond well to dopamine replacement, presumably because the pathology affects many cell populations in addition to dopamine-producing cells of the substantia nigra, including postsynaptic medium spiny neurons in the striatum (Papp and Lantos, 1994 and Sato et al., 2007). In contrast, PD affects preferentially the dopamine neurons, with spared postsynaptic cells still responsive to dopamine replacement. In MSA, α-synuclein deposits in glial (generally oligodendroglial) cytoplasmic inclusions (GCIs) (Spillantini et al., 1998a and Tu et al., 1998) rather than in the neuronal Lewy bodies or dystrophic neurites more characteristic of PD.

As stated above, adult forebrain GluN2B (protein and mRNA) levels are unaltered in GluN2B+/+ versus GluN2B2A(CTR)/2A(CTR) mice ( Figure 3A). We also specifically studied GluN2B levels in isolated protein GSK126 mouse fractions enriched in synaptic and peri/extrasynaptic

NMDARs, following an established protocol ( Milnerwood et al., 2010). Briefly, a synaptosomal preparation was made from the hippocampi of adult GluN2B+/+ and GluN2B2A(CTR)/2A(CTR) mice. This prep was then split into a Triton-soluble “non-PSD enriched” fraction including extrasynaptic NMDARs, plus a Triton-insoluble (but SDS-soluble) “PSD-enriched” fraction containing synaptic NMDARs. We found no differences in the levels of GluN2B between GluN2B+/+ and GluN2B2A(CTR)/2A(CTR) Hydroxychloroquine order hippocampi with regard to either total homogenate, “Non-PSD enriched” fraction, or “PSD-enriched” fraction ( Figure 3B). This biochemical

data is in agreement with observations that the NMDAR:AMPAR current ratios in evoked EPSCs measured at holding potentials of −80 and +40 mV are not altered in adult CA1 pyramidal cells of GluN2B2A(CTR)/2A(CTR) mutants compared to GluN2B+/+ controls (Thomas O’Dell, personal communication). Moreover, the decay time constant of NMDAR-mediated EPSCs recorded at +40 mV in GluN2B2A(CRT)/2A(CTR) mutants was found to be indistinguishable from GluN2B+/+ controls (Thomas O’Dell, personal communication), indicative of a similar GluN2 subunit composition. To promote excitotoxic neuronal loss, we stereotaxically

injected a small (15 nmol) dose of NMDA into the hippocampus (just below the dorsal region of the CA1 layer) and quantified the resulting lesion volume 24 hr later. Consistent Resveratrol with the position of the injection site, the lesions were centered on the CA1 subregion, an effect potentially enhanced by the known vulnerability of this subregion to excitotoxic insults (Stanika et al., 2010). However the lesion also spread to other hippocampal subregions (CA3, dentate gyrus) as well as a small intrusion into the thalamus. Importantly, analysis revealed that GluN2B2A(CTR)/2A(CTR) mice exhibited smaller lesion volumes in the hippocampus and the thalamic region (and smaller overall lesion volumes) than GluN2B+/+ mice ( Figures 3C–3F). Thus, the GluN2 CTD subtype also influences NMDAR-mediated excitotoxicity in vivo. We next investigated the mechanistic basis for the observed GluN2 CTD subtype-dependent differences in vulnerability to excitotoxicity. NMDAR-dependent activation of CREB-dependent gene expression protects against excitotoxicity (Lee et al., 2005) and can act as a protective response to excitotoxic insults (Mabuchi et al., 2001). We found that basal levels of CREB (serine-133) phosphorylation (normalized to total CREB) were unaltered in GluN2B2A(CTR)/2A(CTR) neurons (118% ± 12% compared to GluN2B+/+ neurons, p = 0.2).

Each encoding run was followed by a non-scanned recognition test. Participants were tested on the directly learned (16 AB, 16 BC) and inference (16 AC) associations for each triad type (Figure 1C). On each self-paced test trial, a cue was presented on the top of the screen (e.g., an A stimulus)

and two choice probes were presented at the bottom of the screen (e.g., two B stimuli from different triads). Participants indicated which of the two choice stimuli was associated with the cue. Participants were instructed that on inference trials, the association between the cue click here (A) and the correct choice (C) was indirect, mediated through a third stimulus (B) that shared an association with both the cue and the correct choice during encoding. To control for familiarity, the incorrect choice was a familiar item, but one that was not [directly or indirectly] associated with the cue. The order of test trials was pseudorandom, with the constraint that individual inference trials were tested before the corresponding AB and BC associations to ensure that an AC association was not formed during the test. Because of the repeated study-test nature of the design, participants were instructed prior to scanning that they would be tested on the directly learned associations as well as the indirect relationships.

Participants practiced the encoding and test phases prior to scanning using stimuli different from those Ku-0059436 mw used during fMRI data collection. In a separate scanning session (separated by 1–7 days), an and object/scene encoding localizer and guided recall task was collected for multivoxel pattern classifier training and validation (see Supplemental Experimental Procedures). Whole-brain imaging data were acquired on a 3.0T GE Signa MRI system (GE Medical Systems). During each session, structural images were acquired using

a T2-weighted flow-compensated spin-echo pulse sequence (TR = 3 s; TE = 68 ms; 256 × 256 matrix, 1 × 1 mm in-plane resolution) with thirty-one 3-mm-thick oblique axial slices (0.6 mm gap), approximately 20° off the AC-PC line. Functional images were acquired using a GRAPPA parallel echo-planar imaging (EPI) sequence using the same slice prescription as the structural images (TR = 2 s; TE = 30 ms; flip angle = 90°; 64 × 64 matrix; 3.75 × 3.75 mm in-plane resolution, interleaved slice acquisition). For each functional scan, the first six EPI volumes were discarded to allow for T1 stabilization. An additional high-resolution T1-weighted SPGR scan (sagittal plane, 1.3 mm slice thickness, 1 mm2 in-plane resolution) was acquired during the first scanning session. Head movement was minimized using foam padding. Data were preprocessed and analyzed using SPM5 (Wellcome Department of Cognitive Neurology) and custom MATLAB routines.

, Wood Dale, IL, USA, cat# 51386). The

brain was sliced using standard razor blades (GEM, Personna American Safety Razor Co., Cedar Knolls, NJ, USA; cat# 62–0165) into 2 mm slices. PVN were either quickly frozen for quantitative PCR or immediately fixed in 2.2% formaldehyde for the ChIP analyses. The PVN was punched using a microdissecting needle of an appropriate size. In situ hybridization and immunostaining were performed as we previously described (Blechman et al., 2007). Total RNA was prepared from fish larvae and mouse PVN tissues using the TRI-REAGENT (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. RNA preparations were treated with DNase recombinant I, RNase free (Roche, Basel, Switzerland) to avoid contamination of genomic DNA. First-strand cDNA was synthesized from 0.5–1 μg of total RNA PI3K inhibitor using an oligo(dT)15 primer and SuperScript II reverse transcriptase (RT) (Invitrogen, Blackrock, Co. Dublin, Ireland). The PCR was performed

in 96 plates using a 7300 real time PCR system (Applied Biosystem, Foster City, CA, USA). The total cDNAs were amplified using a SYBR Green qPCR kit Abiraterone clinical trial (Finnzymes, Espoo, Finland) according to the manufacturer’s instructions. The relative quantity (RQ) was calculated using the Relative Quantification program (Sequence Detection System software version 1.2.2) according to the comparative method. Samples were normalized according an endogenous gene (β-actin for zebrafish very fish or hprt for mouse) and to a control time zero sample, which served as a calibrator. ChIP analysis was performed on 6-day-old larvae (pools of 50 larvae per treatment) or adult mouse PVNs (pools of five punches per treatment) as described in Blechman et al. (2011). For IP, we used 4 μl of affinity-purified anti-Otp, 1 μl of anti-Pol II, or control Rabbit IgG. Sequential ChIP-reChIP analysis was performed using a previously described

protocol (Furlan-Magaril et al., 2009). Proteins for IP were extracted from a pool of 100 fish larvae per treatment as we previously described (Blechman et al., 2007). IP was performed using affinity-purified anti-Otp antibody, which was covalently bound to an Affi-Gel 10 beads (Bio-Rad, Hercules, CA, USA) and incubated with the protein extracts for 3 hr at 4°C. Thereafter, beads were extensively washed and protein complexes were eluted (200 mM Glycine-HCl pH 2.7, 150 mM NaCl) for 15 min at room temperature. The resulting protein complexes were subjected to 12% SDS-PAGE under nonreducing conditions, followed by immunoblotting with an antibody directed to phospho-CREB(Ser133). Nitrocellulose membranes were stripped and reblotted with an anti-Otp antibody. Pools of 6-day-old zebrafish (ten larvae) were used for cortisol measurements.