Dynamic Regulation of Synaptopodin and the Axon Initial Segment in Retinal Ganglion Cells During Postnatal Development.

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+-store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, ßIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons.

Axons are subdivided into functionally organized microdomains, which are required for generation and propagation of action potentials (APs). In the central nervous system (CNS), APs are generated near the soma in the axon initial segment (AIS) and propagated by nodes of Ranvier (noR). The crucial role of the membrane adapter proteins ankyrin-B and ankyrin-G as organizers of AIS and noR is now well established. By comparison, little is known on the localization and function of these proteins in sensory axon terminals of the peripheral nervous systems (PNS). Here, we tested the hypothesis that somatosensory PNS terminals are organized by distinct members of the ankyrin protein family. We discovered a specific distribution of ankyrin-B in somatosensory axon terminals of skin and muscle. Specifically, ankyrin-B was localized along the membrane of axons innervating Meissner corpuscles, Pacinian corpuscles and hair follicle receptors. Likewise, proprioceptive terminals of muscle spindles exhibited prominent ankyrin-B expression. Furthermore, ankyrin-B expression extended into nociceptive and thermoceptive intraepidermal nerve fibers. Interestingly, all studied somatosensory terminals were largely devoid of ankyrin-G, indicating that this scaffolding protein does not contribute to organization of mechanoelectric transduction zones in peripheral somatosensory neurons. Instead, we propose that ankyrin-B serves as a major membrane organizer in mechanoreceptive and nociceptive terminals of the PNS.

Effects of strength training with eccentric overload on muscle adaptation in male athletes.

In classic concentric/eccentric exercise, the same absolute load is applied in concentric and eccentric actions, which infers a smaller relative eccentric load. We compared the effects of 6 weeks of classic concentric/eccentric quadriceps strength training (CON/ECC, 11 subjects) to eccentric overload training (CON/ECC+, 14 subjects) in athletes accustomed to regular strength training. The parameters determined included functional tests, quadriceps and fibre cross-sectional area (CSA), fibre type distribution by ATPase staining, localisation of myosin heavy chain (MHC) isoform mRNAs by situ hybridization and the steady-state levels of 48 marker mRNAs (RT-PCR) in vastus lateralis biopsies taken before and after training. Both training forms had anabolic effects with significant increases in quadriceps CSA, maximal strength, ribosomal RNA content and the levels of mRNAs involved in growth and regeneration. Only the CON/ECC+ training led to significantly increased height in a squat jump test. This was accompanied by significant increases in IIX fibre CSA, in the percentage of type IIA fibres expressing MHC IIx mRNA, in the level of mRNAs preferentially expressed in fast, glycolytic fibres, and in post-exercise capillary lactate. The enhanced eccentric load apparently led to a subtly faster gene expression pattern and induced a shift towards a faster muscle phenotype plus associated adaptations that make a muscle better suited for fast, explosive movements.

Transfer of the sFLT-1 gene in Morris hepatoma results in decreased growth and perfusion and induction of genes associated with stress response.

PURPOSE: Inhibition of tumor angiogenesis is emerging as a promising target in the treatment of malignancies. Therefore, monitoring of antiangiogenic approaches with functional imaging and histomorphometrical analyses are desirable to evaluate the biological effects caused by this treatment modality. EXPERIMENTAL DESIGN: Using a bicistronic retroviral vector for transfer of the soluble receptor for the vascular endothelial growth factor (sFLT) hepatoma (MH3924A) cell lines with sFLT expression were generated. In human umbilical vein endothelial cells cultured with conditioned medium of sFLT-expressing hepatoma cells, the inhibitory action of secreted sFLT was determined using a Coulter counter and a thymidine incorporation assay. Furthermore, in vivo experiments were done to measure the effects on tumor growth and perfusion. Finally, the tumors were examined by immunohistochemistry (including computer-assisted morphometry) and DNA chip analysis. RESULTS: Stable sFLT-expressing hepatoma cells inhibited endothelial cell proliferation in vitro. In vivo, growth and perfusion, as measured by H(2)(15)O positron emission tomography, were reduced in genetically modified tumors. However, the immunohistochemically quantified microvascularization and macrovascularization, as indicated by CD31- and alpha-actin-positive area, revealed no significant changes, whereas the number of apoptotic cells was increased in sFLT-expressing tumors, although not significantly. DNA chip analysis of tumors with gene transfer showed an increase of genes related to apoptosis, signal transduction, and oxidative stress. CONCLUSION: Our results suggest that sFLT expression inhibits tumor growth and perfusion and enhances expression of apoptosis-related genes in this model. Enhanced expression of genes for signal transduction, stress, and metabolism indicates tumor defense reactions.