Dynamics of glycine receptor insertion in the neuronal plasma membrane

The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses

Evaluation of Adaptive Changes by Non-Invasive Imaging in Hepatic Vein Outflow Obstruction

Hepatic vein outflow obstruction induces remarkable changes of intra–hepatic blood circulation; the significance of these changes remains uncertain. Six patients with obstruction of the hepatic veins were evaluated by duplex Doppler ultrasound and computed tomography. The adaptive changes secondary to obstruction were analyzed and their significance was correlated with the clinical findings. Four patients presenting unilateral hepatic vein occlusion had unilateral reversed portal flow. Two of them, with lobar liver atrophy and contralateral compensatory hypertrophy required operation; the other two, with normal appearance of the liver, benefitted from conservative treatment. Two patients with bilateral hepatic vein occlusion, intra-hepatic bypasses, bilateral lobar atrophy and caudate lobe hypertrophy, received operations. Intrahepatic unilateral portal flow reversal compensates for unilateral hepatic outflow obstruction. The combination of complete or subtotal hepatic vein obstruction and atrophy–hypertrophy complex predicates advanced disease despite flow reversal or spontaneous shunt

Unresectable Malignant Biliary Obstruction: Treatment by Self-Expandable Biliary Endoprostheses

The primary goal in the treatment of malignant obstruction is the relief of jaundice. Although operative biliary bypass is a reliable method of palliation, nonoperative palliation may be desirable in selected patients

Wound healing and hyper-hydration - a counter intuitive model

Winters seminal work in the 1960s relating to providing an optimal level of moisture to aid wound healing (granulation and re-epithelialisation) has been the single most effective advance in wound care over many decades. As such the development of advanced wound dressings that manage the fluidic wound environment have provided significant benefits in terms of healing to both patient and clinician. Although moist wound healing provides the guiding management principle confusion may arise between what is deemed to be an adequate level of tissue hydration and the risk of developing maceration. In addition, the counter-intuitive model ‘hyper-hydration’ of tissue appears to frustrate the moist wound healing approach and advocate a course of intervention whereby tissue is hydrated beyond what is a normally acceptable therapeutic level. This paper discusses tissue hydration, the cause and effect of maceration and distinguishes these from hyper-hydration of tissue. The rationale is to provide the clinician with a knowledge base that allows optimisation of treatment and outcomes and explains the reasoning behind wound healing using hyper-hydration

In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous

Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation

Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution

Determining the neurotransmitter concentration profile at active synapses

Establishing the temporal and concentration profiles of neurotransmitters during synaptic release is an essential step towards understanding the basic properties of inter-neuronal communication in the central nervous system. A variety of ingenious attempts has been made to gain insights into this process, but the general inaccessibility of central synapses, intrinsic limitations of the techniques used, and natural variety of different synaptic environments have hindered a comprehensive description of this fundamental phenomenon. Here, we describe a number of experimental and theoretical findings that has been instrumental for advancing our knowledge of various features of neurotransmitter release, as well as newly developed tools that could overcome some limits of traditional pharmacological approaches and bring new impetus to the description of the complex mechanisms of synaptic transmission

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/1/emboj7601065.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102104/2/emboj7601065-sup-0001.pd