Werner Zuschratter

Assembly of Active Zone Precursor Vesicles OBLIGATORY TRAFFICKING OF PRESYNAPTIC CYTOMATRIX PROTEINS BASSOON AND PICCOLO VIA A TRANS-GOLGI COMPARTMENT

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.

Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

Spatial and sub-cellular localization of the membrane cytoskeleton-associated protein α-adducin in the rat brain

Studies on the identification and characterization of constituents of rat brain synaptic junctions have lead to the isolation of cDNA clones encoding segments of alpha-adducin. These and other studies suggest that adducin, a protein involved in promoting the assembly of actin and spectrin filaments at the plasma membrane, may play a role in dynamic assembly-disassembly processes underlying synaptic plasticity. In order to verify that brain alpha-adducin is indeed a constituent of synaptic structures, we have generated monoclonal antibodies against epitopes in the C-terminal region of alpha-adducin and have determined its spatial and sub-cellular distribution in postnatal day-30 rat brain. Alpha-adducin is found to be highly enriched in regions with high synapse densities of the hippocampus, corpus striatum, cerebral cortex and cerebellum. Immuno-electron microscopic analysis of peroxidase stained sections of the hippocampus and the cerebellum revealed that alpha-adducin is localized at distinct sub-cellular structures. In the CA1 and CA3 regions of the hippocampus alpha-adducin immunoreactivity is found in a distinct subset of dendrites and dendritic spines. In the molecular layer of the cerebellum, a distinct fraction of pre-synaptic terminals of parallel fiber terminals is labeled. In both cases the majority of synaptic structures does not contain adducin. Significant immunoreactivity is also detected in processes of glial cells both in the hippocampus and the cerebellum.

Interretinal transduction of injury signals after unilateral optic nerve crush

In this study, we report that partial unilateral optic nerve crush in the rat affects the number of retinal ganglion cells of the contralateral eye still in continuity with the ipsilateral superior colliculus. The reduction in cell number of the uncrossed retinal projection was accompanied by a microglia response and could be prevented by the local intravitreal application of the anti-inflammatory agent dexamethasone. Interestingly, the level of neuronal activity after optic nerve crush as evidenced by thallium autometallography was enhanced in the termination area of the uncrossed projection, the rostro-medial superior colliculus, suggesting that a dying-back mechanism is not involved. We propose that injury signals from the damaged optic nerve and retina are transduced to the unaffected eye.

Acquisition of multiple image stacks with a confocal laser scanning microscope

Image acquisition at high magnification is inevitably correlated with a limited view over the entire tissue section. To overcome this limitation we designed software for multiple image-stack acquisition (3D-MISA) in confocal laser scanning microscopy (CLSM). The system consists of a 4 channel Leica CLSM equipped with a high resolution z- scanning stage mounted on a xy-monitorized stage. The 3D- MISA software is implemented into the microscope scanning software and uses the microscope settings for the movements of the xy-stage. It allows storage and recall of 70 xyz- positions and the automatic 3D-scanning of image arrays between selected xyz-coordinates. The number of images within one array is limited only by the amount of disk space or memory available. Although for most applications the accuracy of the xy-scanning stage is sufficient for a precise alignment of tiled views, the software provides the possibility of an adjustable overlap between two image stacks by shifting the moving steps of the xy-scanning stage. After scanning a tiled image gallery of the extended focus-images of each channel will be displayed on a graphic monitor. In addition, a tiled image gallery of individual focal planes can be created. In summary, the 3D-MISA allows 3D-image acquisition of coherent regions in combination with high resolution of single images.

Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus)

Visinin-like protein (VILIP) is a neuronal EF-hand Ca(2+)-binding protein. In the chick brain, it is widely expressed, e.g. in neurons of the visual pathway and the cerebellum. In the cerebellum, a presynaptic localization of VILIP in glutamatergic parallel- and climbing-fiber terminals has been observed. Here, we describe the distribution of immunoreactivity (IR) detected by antibodies against chick VILIP in the gerbil hippocampus at the light and electron microscopic level. VILIP antibodies stain neurons in the whole hippocampal formation including pyramidal cells in the CA1 and CA3 region of the Ammons horn and granule cells of the dentate gyrus. In CA3 neurons, VILIP-IR is localized in dendrites and dendritic spines.

Detection of Dendritic Spines in 3-Dimensional Images

This paper demonstrates a method to analyse connections in biological neural networks. The signal flow from one neuron to another is established by an axon which carries the output of a neuron, a synapse which is divided into presynapse and postsynapse, and a dendrite which collects numerous signals from a large number of synapses and delivers them to the destination neuron. The postsynapse including its connection to the dendrite is denoted as a dendritic spine. Due to the observation that shape and size of dendritic spines as well as their distribution along a dendrite vary with behavioral experience and age, a relation between learning processes and the morphology of spines can be conjectured. This can be verified only by statistical analysis. Therefore, a pattern recognition system is required which automatically clusters the set of spines into classes in an unsupervised learning process and selects the features to discriminate them. Our approach is based on the idea that the skeletons of the spines including radius information will constitute all significant information.

Tracking on treelike structures in 3D confocal images

Confocal microscopy is qualified to perform volume scans of nerve cells with dendrites and spines. Length and diameter of the dendrite branches and the spines should be determined to analyze the influence of learning processes. A prerequisite for that is the recognition of the dendritic structure with branching off and spines. Because the microscope operates at the resolution limit the images are blurry, noisy and only poorly sampled. In contrast to other methods which are based on binary images and thinning algorithms, our method tracks and dendritic tree faster and in the gray-level domain using simple geometric models.An explicit segmentation is unnecessary and knowledge about shape and structure of the dendrite is included as a priori information. For large trees, first a low resolution scan is captured to crete a rough model. The algorithm allows to refine this model using higher resolution scans for interesting regions along the dendrite. The large unimportant areas between the dendrite branches are not scanned at high resolution to save time and disc space. In a second step, the parameters of the model are adapted to the microscope image by minim zing the deviation of the microscope image from the mode image convolved by the microscope point spread function. Features like number, diameter, length and position of the dendrite branches and spines can be easily calculated from the model. An interactive user intervention is possible at the model domain.