Digital image analysis assists Berlin's Charité University Hospital in its research into understanding how memory content is stored
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.
The Charité University Hospital Berlin
The Charité in Berlin is one of Europe's largest university hospitals, and celebrated its 300th anniversary in 2010. Doctors and scientists research, cure and teach here to top international standards. Over half of Germany's Nobel prizewinners in medicine and physiology, including Robert Koch, Paul Ehrlich and Heinrich Wilhelm Waldeyer, come from the Charité. The university hospital is recognized as an outstanding training facility throughout the world. The campus is spread over four sites including over 100 hospitals and institutions. With over 13,000 staff, the Charité has an annual turnover of around one billion Euros, making it one of Berlin's largest employers. Research has a high priority at the Charité. Over 3,000 scientists are involved in over 1,000 research projects, working groups and collaborations; of these scientists, 1,500 are financed by the private sector. Since 2004, research has focused primarily on the neurosciences, oncology, cardiovascular and musculoskeletal issues, immunopathology and infectiology. However, subjects such as rheumatology, allergology, molecular medicine/genetics and transplant medicine also feature heavily.
Figure 1: With over 13,000 staff, the Charité University Hospital is Berlin's largest employer, where doctors and scientists research, cure and teach to top international standards.
"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. 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.
Figure 2: Dr. Zander records images of his immunogold-labeled samples on a high-resolution transmission electron microscope (TEM). He switches to a separate workstation to evaluate and analyze the images, allowing other working groups to use the TEM for their research projects.
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 labeling technique in the lab 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. To do this, each transporter is separately labeled with specific polyclonal antibodies from rabbits or guinea pigs. "Evidence of the antibody bond is provided by a gold particle-coupled antiserum", continues Dr. Zander. The particles of gold used in this immunogold labeling method are 10 nm and 15 nm in size. The software used by the working group is iTEM from Olympus Soft Imaging Solutions GmbH (OSIS). "In addition to the imaging platform, we also use our TEM camera from OSIS", says Dr. Zander. "The software and hardware interact perfectly." 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
Digital image analysis is performed with the aid of the high-resolution TEM camera. "The images are calibrated directly and also optimized in terms of contrast, so we have a perfect image immediately. This is a basic prerequisite for all the subsequent analysis steps", says Dr. Zander, highlighting some of the benefits of digital imaging. Dr. Zander now specifies the area around the mossy fiber terminal as his region of interest (ROI). "This is how I tell the software which area of the image I want to evaluate", says Dr. Zander, explaining the next step. The software then takes over and prepares the images for the actual analysis which is restricted to the selected ROI. 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. Thus he can specify, for example, how the software has to deal with particles at edges or within what limits errors are to be taken into account. "This system provides all the freedom we need", he continues. 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 software provides options to store the measured data in either Microsoft Excel or TXT format, allowing the analysis process to be automated still further.
Figure 3.1: The image recorded on the transmission electron microscope is loaded into the iTEM image analysis software (a). The next step is to select the area of a mossy fiber terminal which is of interest using a polygon function and to define this as one's region of interest (ROI) (b). The original image from (a) is processed further with shading correction and NxN filtering. The ROI from (b) is superimposed over the resulting image in the form of an overlay (c). The next step is to define the particle parameters (mean diameter, roundness) for subsequent analysis (d).
Figure 3.2: The measured data obtained are drawn into the overlay of the image and saved in MS Excel format (e). The data are evaluated further in MS Excel. Figure f consequently represents the intensity of the immunolabeling in the CA3-MFS, measured in gold particles/µm². VGLUT1, VGLUT2 and VGAT transporters in the CA3-MFS were detected by individual labels (gold labels = 10 nm) by the post-embedding method. The MFS of the hippocampi of two adult rats were measured (Animal 1: a,c,e and Animal 2: b,d,f), the embedding procedures differing slightly. In each case 25 - 51 presynaptic terminals with an area of 3.7 to 4.8 µm² were evaluated. Figure g shows a further result. This shows a comparison between Animal 1 and Animal 2 with regard to the individual labels for the neurotransmitter transporters VGLUT1, VGLUT2 and VGAT. The number of gold particles per presynaptic surface area (top part of the figure) and the number of gold particles due to non-specific labeling in the area of dendrites and cell nuclei (bottom part of the figure) were standardized to 1 µm².
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."
Acknowledgement This text was produced with the kind support of: Dr. rer. nat. Johannes-Friedrich Zander, Charité - Universitätsmedizin Berlin, Centre for Anatomy, Institute of Integrative Neuroanatomy, Functional Cell Biology, Philippstr. 12, 10115 Berlin, Germany