Konfokales Laserscanning-Mikroskop mit integrierter Elektrophysiologie
Zusammenfassung der Projektergebnisse
Project 1: Spatiotemporal organization of synaptic vesicles at the active zone. The influence of presynaptic proteins on synaptic vesicle clusters of active zones at the calyx of Held synapse was studied. Viral gene transfer into the cochlear nucleus allowed us to overexpress presynaptic proteins or to label the presynaptic compartment with membrane-bound GFP or to tag presynaptic proteins and study their distribution. We first identified the distribution of the matrix proteins Bassoon and Piccolo in calyces of different maturation stages. The number of active zones per calyx increased from 405±35 at P9 to 601±45 at P21 at unchanged density because of a concurrent inrease in calyx size. The active zones segregated into three classes: Bassoon-only, Piccolo-only, and mixed. Furthermore, we studied the role of the exocyst complex in presynaptic function. The dominant-negative Exo70dC decreased the structural complexity of the calyx while leaving synaptic transmission unchanged, suggesting that the exocyst complex does not act as a tethering complex in local synaptic vesicle recyling but rather is needed for membrane incorporation during postnatal maturation. Finally, we studied the role of synapsin proteins in synaptic vesicle organization and synaptic transmission. Synaptic vesicle distribution was examined after synapsin depletion (triple KO mice) and after overexpression of synapsin. We found that in wild-type synapses, the synapsin-dependent reserve pool contributes to the replenishment of the readily releasable pool (RRP), although accounting only for a small fraction of the SVs that enter the RRP. Furthermore we found that the amount of synapsin controls the volume of synaptic vesicles and size of the active zones: lack of synapsin proteins resulted in large synaptic vesicels and active zones while overexpression had the opposite effect. This suggests that synapsins have a regulatory role in the biogenesis of synaptic vesicles. Project 2: Synaptic mechanisms of odor discrimination. In this project we manipulated the postsynaptic composition of ionotropic glutamate receptors in granule cells of the olfactory bulb and found that deletion of GluA2 increased inhibition and accellerated odor discrimination time, while deletion of GluN1 had the opposite effects. Using confocal microscopy of entire single-standing granule cells we could rule out structural effects of the deletion of glutamate receptors on granule cell morphology. Furthermore, we study the role of sodium channels, T-type calcium channels and GABAA receptors in granule cell function. We used confocal microscopy to visualize the distribution of these proteins in identified granule cells. Project 3: Synaptic transmission in thalamic giant synapses of the mouse. The goal of this project is to characterize the structure and function of giant synapses contacting relay neurons in thalamic nuclei. We study giant synapses formed by projection neurons of the principal trigeminal nucleus onto relay neurons of the ventroposteromedial nucleus and synapses formed by cortical layer 5B pyramidal neurons onto relay neurons of the posteromedial nucleus. We use a novel approach to electrically stimulate identified presynaptic terminals. Briefly, the giant synapses are labeled by viral expression of synaptophysin-GFP and can be readily identified using the resonant scanner of the SP5 confocal. Whole-cell recordings are established from the relay neurons situated in the vicinity of the terminals and the neurons are filled with a red dye via the patch pipette. Using real-time video-rate imaging and overlay of two fluorescence channels we screen for labeled giant terminals in contact with the relay neurons. Subsequently, we use scanning gradient contrast imaging for the first time adapted to the confocal microscope (using the internal 700nm laser in conjunction with a Dodt tube and an additional external PMT) for the visualization of slice morphology. This is required to position a double barrel stimulation pipette precisely within 1 um of the labeled giant terminal for electrical stimulation. All three channels are displayed separately and superimposed in real time, a unique feature of our custom configured SP5 confocal microscope. Several projects employing this technique and studying thalamic giant synapses are close to completion.
Projektbezogene Publikationen (Auswahl)
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(2010). Acute genetic perturbation of exocyst function in the rat calyx of Held impedes structural maturation, but spares synaptic transmission. European Journal of Neuroscience 32:974-984
Schwenger, D. and Kuner, T.
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(2010). Synaptic inhibition in the olfactory bulb accelerates odor discrimination in mice. Neuron 65, 399-411
Abraham, N.M., Egger, V., Shimshek, D.R., Renden, R., Fukunaga,. I., Sprengel, R., Seeburg, P.H., Klugmann, M., Margrie, T.W., Schaefer, A.T., Kuner, T.
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(2010). Targeted three-dimensional immunohistochemistry reveals localization of presynaptic proteins bassoon and piccolo in the rat calyx of Held before and after the onset of hearing. J. Comp. Neurol. 518, 1008-1029
Dondzillo, A., Sätzler, K., Horstmann, H., Altrock, W.A., Gundelfinger, E.D., Kuner, T.
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(2012). Synapsin-dependent reserve pool of synaptic vesicles supports replenishment of the readily releasable pool under intense synaptic transmission. European Journal of Neuroscience 36:3005-3020
Vasileva, M., Horstmann, H., Geumann, C., Gitler, D., Kuner, T.
