Cerebellar Neurodynamics Predict Decision Timing and Outcome on the Single-Trial Level.

Goal-directed behavior requires the interaction of multiple brain regions. How these regions and their interactions with brain-wide activity drive action selection is less understood. We have investigated this question by combining whole-brain volumetric calcium imaging using light-field microscopy and an operant-conditioning task in larval zebrafish. We find global, recurring dynamics of brain states to exhibit pre-motor bifurcations toward mutually exclusive decision outcomes. These dynamics arise from a distributed network displaying trial-by-trial functional connectivity changes, especially between cerebellum and habenula, which correlate with decision outcome. Within this network the cerebellum shows particularly strong and predictive pre-motor activity (>10 s before movement initiation), mainly within the granule cells. Turn directions are determined by the difference neuroactivity between the ipsilateral and contralateral hemispheres, while the rate of bi-hemispheric population ramping quantitatively predicts decision time on the trial-by-trial level. Our results highlight a cognitive role of the cerebellum and its importance in motor planning.

Insulin-like growth factor I (IGF-1) Ec/Mechano Growth factor--a splice variant of IGF-1 within the growth plate.

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.

Fibroblast growth factor 23 and Klotho are present in the growth plate.

INTRODUCTION: Regulation of phosphate homeostasis is essential for mineralization and enchondral ossification. Fibroblast growth factor 23 (FGF23) and its obligatory co-receptor Klotho (KL) play a key role in this process by influencing both renal phosphate reabsorption and vitamin D metabolism. In disease, excessive action of FGF23 leads to hypophosphatemic rickets, while its deficiency causes tumoral calcinosis. Although osteocytes and osteoblasts are widely seen as the primary source of FGF23 under physiological conditions, the origin of systemic FGF23 remains controversial. In this study, we investigated the expression of FGF23 and KL in porcine growth plate cartilage, adjacent tissues, and parenchymal tissues. MATERIALS AND METHODS: Tissue samples were obtained from 4- to 6-week-old piglets. mRNA expression was quantified by real-time PCR and normalized to 18S rRNA. Immunohistochemical staining was performed for FGF23, KL, collagen type X, and FGF receptor 1. Growth plate chondrocyte subpopulations were acquired by collagenase digestion of growth plate explants and subsequent density gradient centrifugation. RESULTS: We could detect both FGF23 and KL mRNA and protein in growth plate chondrocytes. FGF23 expression was mainly found in hypertrophic and resting chondrocytes. Furthermore, significant expression of both genes was observed in bone, liver, and spleen. CONCLUSION: These data challenge previous expression analyses, in particular theories of bone as the exclusive source of FGF23. Moreover, significant expression of FGF23 and KL within the growth plate and adjacent tissues imply a potential local role of FGF23 in chondrocyte differentiation and tissue mineralization.

Increasing expression of oxytocin and vasopressin receptors in the equine conceptus between Days 10 and 16 of pregnancy.

Oxytocin (OT) and arginine vasopressin (AVP) have been detected in the yolk sac of the pre-attachment equine conceptus. Therefore, we have assessed the presence of OT and AVP receptors in equine conceptuses between Days 10 and 16 of pregnancy by qualitative PCR, quantitative PCR and immunohistochemistry. Expression of OT receptor and of the AVP receptors V1aR and V2R could be verified after sequencing the RT-PCR products of the expected length. The size of conceptuses used for quantitative PCR significantly increased with day of pregnancy (P<0.01) as did their quantitative expression of OTR (P<0.01). Immunohistochemistry of OTR resulted in weak trophectodermal abundance on Day 10, increasing at Day 12. On Day 14, staining intensity increased in individual cells of the trophectoderm while it decreased in other cells; this trend became more apparent on Day 16. The endoderm of the trophoblast and surrounding subtrophoblastic compartments always showed moderate staining for OTR. On Day 10 immunoreactive V2R protein was localised in the trophectodermal apical membrane; on Day 12 it was also present in the basal membrane and weakly in the cytoplasm. On Day 14 only individual trophectodermal cells showed positive supranuclear cytoplasmic areas or V2R, whereas on Day 16 about one-third of the trophectodermal cells were stained entirely and intensely positive. These results suggest an involvement of OT and AVP action in the development and expansion of the early equine conceptus.

Significance of aquaporins and sodium potassium ATPase subunits for expansion of the early equine conceptus.

Expansion of the equine conceptus can be divided into blastocoel and yolk sac phases. The endodermal layer transforming the blastocoel into the yolk sac is completed around day 8 of pregnancy. From that time, the size of the spherical conceptus increases tremendously due mainly to the accumulation of fluid rather than cell multiplication. In this study, we have investigated the abundance and localisation of Na(+)/K(+)-ATPases and aquaporins (AQP) in the equine conceptus on days 8, 10, 12, 14 and 16 by multiplex reverse transcriptase PCR, Western blot and immunohistochemistry. During conceptus expansion, the ectoderm of the yolk sac exhibited basolateral abundance of alpha1ATPase, apical localisation of AQP5, and membrane and cytoplasmic expression of AQP3. With increasing conceptus size its cells showed an extensive enlargement of the apical membrane surface by microvilli. From day 14 onwards, the yolk sac endoderm forms arc-like structures with attaching sites to the ectodermal layer and shows intensive staining for alpha1ATPase, AQP5 and AQP3 in the membrane as well as in the cytoplasm. In the yolk sac ectoderm, the arrangement of these proteins is comparable with the collecting ducts of kidney with AQP2 being replaced by the closely related AQP5. The detection of phosphorylation sites for protein kinase A suggests a similar AQP5 traffic and regulation as known for AQP2 in the collecting ducts of the kidney. The arrangement of these proteins in equine embryos indicates at least partially the mechanism of conceptus expansion.

Tyk2 and signal transducer and activator of transcription 1 contribute to intestinal I/R injury.

Previously, we have shown that the Jak-signal transducer and activator of transcription signaling constituents Tyk2 and STAT1 play a role in the development of multiple organ failure during endotoxin shock. Here, we report that Tyk2 and STAT1 contribute to death caused by intestinal I/R injury. Tyk2- and STAT1-deficient mice showed increased survival to I/R because their intestines were protected from gross histomorphological tissue destruction and neutrophil infiltration. On the molecular level, the reduced ischemia induced inflammatory response in mutant versus wild-type mice was accompanied by an impaired up-regulation of the adhesion molecules P-selectin and intercellular adhesion molecule 1 and of the matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-14 in the reperfused intestine. In conclusion, this study demonstrates for the first time that Tyk2 or STAT1 promote intestinal I/R-induced shock based on a deregulated local inflammatory response and a destruction of the gut intestinal barrier.

Organ-specific and differential requirement of TYK2 and IFNAR1 for LPS-induced iNOS expression in vivo.

Lipopolysaccharide (LPS) is an integral structural component of the outer membrane of Gram-negative bacteria and the principal active agent in the pathogenesis of endotoxin shock. LPS is a potent inducer of a variety of cytokines and inflammatory agents that lead to a profound alteration of gene expression patterns in cells and organs. The gene coding for the inducible nitric oxide synthase (iNOS) is highly responsive to LPS in vitro and in vivo and accounts for the production of nitric oxide (NO). The Janus kinase (JAK) family member tyrosine kinase 2 (TYK2) is a constituent of the interferon (IFN) type I response pathway and an important effector in the progression of endotoxin shock. Macrophages deficient for IFNalphabeta receptor chain 1 (IFNAR1) or TYK2 were shown to have an impaired LPS-induced iNOS expression. Here we determined the contribution of IFNAR1 and TYK2 to iNOS expression in vivo in a lethal LPS challenge model. TYK2 and IFNAR1 were found to be crucial for the LPS-induced iNOS mRNA and protein expression in spleen and lung that could be attributed to the Mac3-positive population. In liver LPS-induced iNOS mRNA expression was only partially impaired in TYK2-deficient mice and was unimpaired in IFNAR1-deficient mice, indicating organ specificity. TYK2(-/-) and IFNAR1(-/-) mice also differ with respect to IFNgamma production upon LPS challenge in that TYK2(-/-) mice show a defect while IFNAR1(-/-) mice do not. Our data suggest that iNOS is induced through IFNAR1 and TYK2 in Mac3-positive cells which are the main source of iNOS in spleen and lung. The LPS-induced iNOS expression in liver is independent of IFNAR1 and partially dependent on TYK2, which is most likely due to the lack of IFNgamma production in the absence of TYK2.

Immunohistochemical localization of androgen and oestrogen receptors in canine hair follicles.

Skin biopsies from seven different body sites were obtained from 21 dogs of different breeds (short, normal, long hair), which were presented for euthanasia. Commercially available polyclonal antibodies were used for the immunohistochemical detection of androgen and oestrogen receptors. Both receptors showed a similar distribution in canine skin, with specific intranuclear staining. In the epidermis, the percentage of androgen receptor (AR)-positive cells, but not that of oestrogen receptor (ER)-positive cells, was significantly higher in samples from the thorax and the flank. In the dermal papilla, the percentage of ER-positive (but not AR-positive) cells was significantly lower in biopsies from the flank. No significant difference was found for both receptors between the locations in the outer root sheath, among the three different hair types, between sex and between intact and castrated dogs.

Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts.

Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.