Is the cause of diseases such as Alzheimer‘s, epilepsy and schizophrenia rooted in a deficiency or rise in chemical messengers? The „Functional Cell Biology" working group at the Charité University Hospital‘s Institute for Integrative Neuroanatomy in Berlin is looking into this and similar issues. The results are forming the basis of a fundamental understanding of the diseases and are intended to provide indications for therapeutic approaches. Researchers are employing the latest imaging and image analysis techniques and methods in the course of their investigations.
„Functional Cell Biology" Working Group
As Professor of the Centre for Anatomy at the Charité‘s Institute for Integrative Neuroanatomy, Dr. Gudrun Ahnert-Hilger leads the „Functional Cell Biology" working group, where her colleague Dr. Johannes-Friedrich Zander works. One of the research projects currently being worked on by Dr. Ahnert-Hilger and Dr. Zander is looking at the area of the brain which deals with memory, the hippocampus. „Our work is intended to contribute to a better understanding of the causes of epilepsy or Alzheimer‘s", says Dr. Zander, explaining the objective of the research project. This is because „in pathophysiological terms, the hippocampus plays a key role in the development of these diseases." The hippocampus is the area of the brain which, among other things, deals with the consolidation of memory. „In the hippocampus, memory content is transferred from the short-term to the long-term memory", says Dr. Zander. We now know that the central nervous system (CNS) is not a static organ, but is permanently changing as a function of activity and experience. „In this connection, the hippocampus is viewed as a ‚module for explicit memory‘", continues Dr. Zander - this means that patients with bilateral damage to the hippocampus are incapable of learning new facts and events.
The Focus of Interest: Mossy Fiber Synapses
Microscopic and functional differences divide the hippocampus into two corresponding areas called the dentate gyrus and the cornu ammonis. The cells of the dentate gyrus and the cornu ammonis communicate via synapses.
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Synapses are specialized links in the brain which allow communication between individual nerve cells, the neurons. „Each synapse has special anatomical characteristics. One of the hippocampal synapse types is extraordinary in its extension and referred to as a mossy fiber synapse", says Dr. Zander. „This synapse is involved in an important processing step in the hippocampal circuit, though its precise function has not yet been fully understood. That is why we are particularly interested in it", he continues.
It is the particular properties of mossy fiber synapses, such as their synaptic strength, synaptic facilitation and their strategic position in the hippocampus which make them so interesting to scientists. There is much to indicate that „mossy fiber synapses are responsible for a wide variety of processes in the brain", says Dr. Zander, adding that scientists are attempting gradually to get to the bottom of this.
Communication in the Cells
Information between the cells in the synapses is actually exchanged by means of neurotransmitters. These are biochemical messengers which pass on, amplify or modulate electrical stimuli from one nerve cell to another nerve cell or other cell. The „Functional Cell Biology" working group is interested in two main neurotransmitters: GABA, short for gamma-amino butyric acid, and glutamate.
„The development and function of the human brain depends essentially on the availability of gamma-amino butyric acid (GABA), an important neural messenger in the central nervous system", explains Dr. Zander. Diseases such as epilepsy and schizophrenia are directly linked to a deficiency of the GABA neurotransmitter. The glutamate messenger is also very importance. In healthy people, glutamate is responsible for memory performance, learning ability and concentration. It has now been scientifically proven that the concentration of this substance is higher in Alzheimer‘s patients and that this leads to continuous stimulation of nerve cells.
Dr. Zander and Dr. Münster-Wandowski, his colleague establishing the post-immunogold labelling technique in the lab and are using modern technology to get to the bottom of the transport mechanisms of vesicular glutamate and vesicular GABA. „Among other things, our research work consequently makes use of electron microscopes equipped with modern digital recording and analysis software", says Dr. Zander.
Gold Labels as Signposts
Dr. Zander‘s working group has come to appreciate above all the versatile potential of digital image evaluation. „We employ image analysis methods to evaluate gold particle labels of the vesicular glutamate transporters (VGLUT)1/2 and of the vesicular GABA transporter (VGAT) in relation to the surface area of the hippocampal mossy fiber (MF) terminals", is how Dr. Zander describes the method used.
More precisely, the brain region of interest was weakly fixed, dehydrated and embedded in acrylic resin (Lowicryl) at low temperature (freeze substitution). Results obtained with Lowicryl show good preservation of cellular contents and ultra structure. By means of the low temperature, protein denaturation was reduced as well as protein structural conformation was maintained. Embedded material was cut into 70 nm thin ultra slices, mounted on grids and immunolabelled using primary antibody against VGLUTs or VGAT, subsequently exposed to secondary antibody - coupled to 10 or 15 nm gold particles - recognizing the first antibody, (postembedding immunogold labelling). To visualize the ultrastructural localization of gold labelling, it is necessary to stain labelled thin sections with heavy metals.
The software used by the working group is iTEM from Olympus Soft Imaging Solutions (OSIS). „In addition to the imaging platform, we also use our TEM camera from OSIS", says Dr. Zander. Once the images have been recorded, the software rapidly guides a user to his or her goal: localizing and counting the transport proteins per vesicle and synapse.
The Ideal Assistant: Digital Image Analysis
Dr. Zander specifies the area around the mossy fiber terminal as his region of interest. At any point, explains Dr. Zander, you have the option of viewing the steps in question to check them. „I can even modify the parameters for detecting the gold particles if necessary", he says. Best of all: „We can always vary the parameters later without having to run the detection process again." Most images are recorded at 20,000 to 30,000x magnification and thus show only a section of the mossy fiber terminal. This is because mossy fibers have very large presynaptic terminals. „So we combine the pictures of partial areas of the mossy fiber terminal and the corresponding gold particle numbers", explains Dr. Zander „and standardize both parameters to 1 µm²." The measurements cover a total of 25 - 51 mossy fiber terminals per label (VGLUT1, VGLUT2 and VGAT) on at least two sections of rat brain.
The detailed results of the investigations can be found inter alia in Zander et al., J Neurosci. 2010 Jun 2; 30(22):7634-45. They have provided researchers at the Charité University Hospital with important indications. „This is gradually giving us a better idea of the links", says Dr. Zander. But each piece added to the jigsaw puzzle is only a tiny one. We are still a long way from a really complete understanding on synaptic function in health and disease. At the Charité University Hospital, they are pleased that digital imaging and image analysis features allow many of the complex evaluation steps to be automated. „This saves us a lot of time which we can use evaluating and preparing subsequent steps - time which we hope will help us achieve the real goal, which is to deal efficiently with diseases such as epilepsy or Alzheimer."
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Keywords: Digital Image Analysis GABA GABA Transporter Gamma-amino Butyric Acid Johannes-Friedrich Zander Memory Cells Mossy Fiber Synapses nerve cells Neuroanatomy Neuroimaging Neuroscience Neurotransmitter Olympus Soft Imaging Solutions TEM Transmission Electron Microcopy Transmission Electron Microscope VGLUTEmail request
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