In order to uncover the roles of the transporters, we are using state of the art imaging techniques (quantitative electron microscopy and confocal microscopy) to map the precise localizations of individual transporter protein types as well as their splice variants.
We combine imaging with stereological measurements and immunochemical measurements of the tissue content of the individual transporter proteins. This enables us to calculate the exact transporter densities at the various locations (number of transporter molecules per square micrometer cell membrane).
The studies require access to highly specific antibodies. We therefore produce a large number of different antibodies which we subject to extensive purification and rigorous testing. (For detailed discussions see: Danbolt et al., 1998, 2001; Holmseth et al., 2005, 2006, 2012)
"What a robot can do, a robot should do." We use robotic equipment to minimize manual work (fully automated ELISA assays, development of Western blots and immunolabeling of tissue sections).
A novel and user-friendly database system, which is based on an invention by KP Lehre, is being developed by Science Linker AS. The system handles documentation, authentication, and tracking of project data and materials (animals, samples, patients, mails, ideas, files, results, etc). A unique feature of this system is that data relations can be changed without affecting previously entered data. Thus, collaborators will know relevant project details, making it easier to document study protocols, use of reagents, etc. In addition to this, some datasets representing serial brain sections are being uploaded in an brain atlas database [Collaboration with Bjålie].
In addition, we produce animals with specific genetic modifications: e.g. the cell or organ selective deletion of one gene at a certain age allowing normal development of the animal until the time of study. This is required in order to be able to distinguish between the primary role of a transporter from those of secondary changes. Further, genetically modified animals are important controls in drug testing (e.g. Lehre et al., 2011) and immunocytochemistry (e.g. Holmseth et al., 2012). We have conditionally deleted four transporter genes: GAT2 (Zhou et al., 2012 ) , GAT3 (unpublished) , BGT1 (Lehre et al., 2011; Zhou et al., 2012 ) and EAAT2 (Zhou et al., 2014)
Transporter function and structure
Information on transporter localizations and numbers are important, but not sufficient. We also need to know more about the properties of the molecules themselves. How they operate, how they are regulated, and so on. In particular, the demonstration of high glutamate uptake in synaptic terminals in spite of low levels of transporters (Furness et al., 2008) raised the question if nerve terminal EAAT2 is behaving differently than astroglial EAAT2 or if this is an artifact due to a higher rate of heteroexchange than net uptake [Collaboration with Peter Larsson].
Brain metabolisms and the roles of EAAT2 in nerve terminals
Conditional EAAT2 knockouts are being used to uncover the physiological importance of glial versus nerve terminal uptake. Changes in brain metabolism is being measured using NMR [Collaboration with Ursula Sonnewald].
Brain ultrastructure and computer simulations
Because of the fine structure of the nervous system (e.g. the sharpest electrodes used for electrophysiological recording being more than 10 times thicker than synaptic clefts), and because of the dynamic regulation of virtually all tissue components and proteins, the nervous system cannot be measured without changing it. To overcome this problem, we are using the above obtained data to perform computer simulations. We generate 3D-models of brain tissue based on electron micrographs of serial ultrathin sections (e.g. Mathiisen et al., 2010) using state of the art serial electron microscopy [Collaboration with G Knott, DA Rusakov and NH Risebro].
Apart from being interesting in itself, data generated from the above studies are being combined with data from our collaborators specifically to improve our understanding of memory formation, epilepsy, amyotrophic lateral sclerosis (ALS), bacterial meningitis, stroke and Huntington's and Alzheimer's diseases.