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Freie Universität Berlin Charité University Medicine Berlin Humboldt University Berlin Max-Delbrück-Center for Molecular Medicine, Berlin-Buch |
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Freie Universität Berlin Charité University Medicine Berlin Humboldt University Berlin Max-Delbrück-Center for Molecular Medicine, Berlin-Buch |
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GRK 1123: |
Cellular Mechanisms of Learning and Memory Consolidation |
This Research Training Group is funded by the German Research Council DFG
| AG Ahnert-Hilger | AG Behr | AG Geiger | AG Haucke | AG Heinemann/ Kempter |
| AG Multhaup | AG Wulczyn | AG Rosenmund | AG Schmitz/ Brecht |
AG Sigrist |
Prof. Dr. rer. nat. Gudrun Ahnert-Hilger
Centrum für Anatomie, Institut für Integrative Neuroanatomie
Charité, Universitätsmedizin Berlin
Schumannstr. 20/21
10117 Berlin
gudrun.ahnert@charite.de
Topic
Contribution of synaptic vesicles to developmental and activity-dependent neuronal plasticity
Title
Regulation of vesicular transmitter transporters - impact of Go2, light-dark-cycles and enhanced stimulation
Question of the projectSynaptic vesicles do not represent a homogenous population even in a given synapse. How synaptic efficiency will be regulated at the level of the vesicle by its fusion competence, transmitter content and variations in the transmitter transporter equipment and/or due to the cycling between different vesicle pools is addressed in the project.
Current State of ResearchSynaptic vesicles concentrate neurotransmitters by means of specific vesicular transporters which they release following an action potential by forming a transmembrane SNARE complex between the vesicular and the plasma membrane. The SNARE complex involves the vesicular protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP 25 (Sutton et al., 1998; Jahn and Südhof, 1999; Lin and Scheller, 2000). Synaptic vesicles release their transmitter content as variable multimolecular packets into the synaptic cleft. Transmitter content and fusion capacity are regulated independently (Travis et al., 2000; van der Kloot et al., 2002). The availability of synaptic vesicles for fusion (Pieribone et al., 1995) and their individual transmitter content define the postsynaptic answer and add to synaptic strength.
Previous work of the group in the field
• Go2 and vesicular transmitter transporters
Go2 regulates the storage of VMAT containing vesicles. It was found that the vesicular content triggers G-protein activation and that VMAT2 by its first intravesicular loop works like a G-protein coupled receptor (Ahnert-Hilger et al., 2003; Brunk et al., 2006a,b). Deletion of Go2a disturbs the balance in the striatal dopaminergic system leading to a loss of cocaine-induced sensitization of locomotor activity (Brunk et al., 2008).
Go2 also regulates VGLUT activity by shifting the chloride dependence of the transporter to lower chloride concentrations while its deletion abolishes chloride dependence of VGLUT (Winter et al., 2005). The physiological relevance of these observations are still not clear and further complicated by the fact that the ionic basic for vesicular glutamate transport are far from being understood.
• Sorting of VGLUT to the plasma membrane depending on a day-night cycle
The amount of VGLUT in synaptic vesicular fractions undergoes a strong diurnal regulation (Yelamanchili et al., 2006). This regulation is mediated by a sorting out of VGLUT to the plasma membrane (Darna et al., in revision).
• Parallel occurrence of VGLUT and VGAT in the same terminal
Using immunoisolation we found that VGAT resides in considerable amounts on VGLUT2 containing vesicles and vice versa. Postembedding immunogold electron microscopy revealed that cerebellar and hippocampal mossy fiber terminals harbour VGLUT and VGAT containing vesicles.
Objectives
The projects will unravel basic mechanism on how synaptic vesicles contribute to synaptic plasticity. The regulation of transmitter uptake/storage by the G-protein Go2 and by diurnal influences on the amount of transporters per vesicle will be addressed. As a second approach we focus on the coexistence of different vesicular transmitter transporters in the same terminal and its dynamics following pathological stimulation.
Methods
Cell culture techniques (cell lines and sub area-specific primary nerve cell cultures of mouse hippocampus or raphe), transfection of cultured cells, permeabilization of cells, subcellular fractionation, immunoisolation and immoprecipitation, transmitter uptake and release, HPLC analysis, and immnucytochemical techniques including EM (in cooperation): Breading of deletion mutants of Gao1 Gao2 or both splice variants and of Gaq
Dissertation topics
Cooperation with other Members
AG Haucke : Characterization of the endocytotic route of VGLUT following stimulation and in context of a day night cycle
AG Schmitz : Characterization of physiological consequences of the G?o2-mediated down regulation of the activity of VGLUTs; development of autaptic cultures mainly from gyrus dentatus granule cells.
AG Heinemann/Kempter: Electron microscopic analysis of VGLUT and VGAT in hippocampal mossy fiber terminals under resting conditions and in kindled rats.
Scholarship Holders:
| AG Ahnert-Hilger | AG Behr | AG Braunewell | AG Haucke | AG Heinemann |
| AG Kempermann | AG Kempter/Schmitz | AG Kuhl | AG Multhaup | AG Nitsch |
