The neuronal organization of the hippocampus is extensively studied. However, the synoptic connections between neurons of the hilus and neurons of the granule cell layer are still debate. In the present study, we utilized automated micromanipulation technique combined with visual image processing to analyze the synaptic connections between hilar mossy cells or mossy fibers, and granule cells or basket cells in the granule cell layer. The stereoscopic microscope image of the dentate gyms of coronal slice in adult rat brain was converted into a binary image. The area of the dentate gyms is detected by genetic algorithms. This was matched with the template image made from a microscopic photo of cresyl violet-stained coronal section of the rat brain by parallel and rotation matching. A glass microelectrode inserted into the apex of the dentate gyrus by the micromanipulation system. Lipophilic fluorescent tracer DiI or DiD was injected into the hilus by a nanoinjector. </p

Sano, Tetsuya

Yamamoto, Hideki

Okayama University Digital Information Repository

Micromanipulation with Stereoscopic Imaging

Okayama University Scientific Achievement Repository

EngineeringIndustrial & Management Engineering fieldsOkayama University Year 2004Micromanipulation with StereoscopicImagingTetsuya Sano Hideki YamamotoOkayama University Okayama UniversityThis paper is posted at eScholarship@OUDIR : Okayama University Digital InformationRepository.http://escholarship.lib.okayama-u.ac.jp/industrial engineering/22IEEE IST 2004 ~ lntcmalional Workshop on Imaging Systcms and Tcchniqucs Strcsa, Italy, 14 May 2004 Micromanipulation with Stereoscopic Imaging Tetsuya San0 and Hideki Yamamoto Faculty of Education, Okayama University Tsushima 3-1-1, Okayama, 700-8530, Japan Phone: +8 1-86-25 1-7723 FAX: C8 1-86-25 1-7723 e-mail:yamamoto@cc.okayama-u.ac.jp Abstract - The neuronal orgunizution ofthe hippocumpus is exten- sively studied However, the synaptic connections between neurons ofthe hilus and neurons of the grunule cell Inyer ure still debate. In the present mi+, we utilized automated micromunipulution tech- nique combined with visual imugeprocessing to unalyze the synup- tic connections between hilar mossy cells or mossy fibers. and granule cells or basket cells in the grunule =el/ layer. The stereo- scopic microscope imuge of the dentate gynrs of coronul slice in udult rut bruin was converted into U binary imuge. The urea of the dentate arm is detected by genetic ulgorithms. This was matched with the template image mode Pam U microscopic photo of cresyl violet-stuined coronal section ofthe rut bruin by parullel und rotu- tion mutching. A glass microelectrode inserted into the apex of the dentate gvnrs by the micromunipulution system. Lipophilicfluores- cent tracer DiI or DiD was injected into the hilus by U nanoinjector. Keywords - h u g e  Processing , Micromunipulution, Neuronal Or- gunkdon, Microinjection I. INTRODUCTION The hippocampus is a prominent brain structure which plays an important role in memory and leaming. Its cytoar- chitecture is relatively simple compared to that of cerebral cortex, and its intrinsic and extrinsic neuronal connections are well known. However, its intrinsic synaptic connections, especially that between dentate granule cells and hilar in- terneurons, are still debate. To examine neuronal reorganization induced by brain in- sults, a precise anatomical study comhiniig immunofluores- cent tracing and visual image processing are required. Vis- ual image processing is widely utilized in experimental neu- roanatomy, and computer-based morphological analysis is an indispensable technique in neuroscience. Although the injection of fluorescent tracers are generally done manually under microscopic observations, an automated micromanipulation technique combined with computer-based visual image processing may he helpful to reduce labor. In addition, an analysis of fluorescently traced neurites by visual image processing may be useful to elucidate the structure of normal and reorganized neuronal circuits because the hippo- campus has highly branched, three-dimensional neuronal organization. In the present study, lipophilic fluorescent dyes DiI and DiD were injected into small regions of the hilus of 0-7803-8591-8/04/$20.00 2004 IEEE 48 the rat brain slice by an automated micromanipulation to ana- lyze synaptic connections of the hilar neurons with granule cells and interneurons in the dentate granule cell layer. We selected the hilar region near to the apex of the granule cell layer as the target because it was easy to determine distances from the apex to the target from its characteristic triangular morphology. 11. METHODS A Defection method of injection area Figure1 shows the microinjection system. The system con- sists of a stereoscopic microscope, two CCD cameras, a micromanipulator and a personal computer. A target is set on the stage of the microscope. The cameras are mounted to the eye pieces of the microscope. The micromanipulator has three degrees of freedom. The microelectode is set to the micromanipulator. The (X, Y ,  Z) coordinate is set as shown in Fig. 1. The Y axis is perpendicular to the stage of the mi- croscope. The X Z  plane is parallel to the stage. The left im- age and the right image inputted to the personal computer that performs image processings to direct the microelectrode and the target in the images. The distance between the micro- electrode and the target is measured three dimensionally by the stereovision method. The microinjection system guides the microelectrode to reach the injected point witbin the tar- get. The measurement of the distance between the tip position of the microelectrode and the injected point, and the move- ment of the microelectrode are carried out alternately to re- duce the distance to 0. Fig.1 Microinjection system. Fig. 2 Dcntalo gym of rat brain. Fig. 3 Dcntale syrus aftcr Ggion scgmenwtian. I ........... ~~ ~ ~ Fig. 4 Exhactcd arca afthc dcntatc gyrus. A coronal slice of adult Sprague-Dawley rat brain of (Smm in thickness) was placed in a recording chamber filled with phosphate-buffered saline. The stereoscopic microscope color image of the hippocampus was converted into a black and white image and the noises were eliminated by a median filter. Noises of the microscopic image were largely elimi- nated by this procedure (Fig.2). The threshold for binary im- age was determined by the mean concentration and the stan- dard deviation calculated i?om the dentate gyrus. By the re- gion segmentation, the area of the dentate gyrus is extracted. was 92.93 and the standard deviation was 5.39 respectively. Figure 3 shows the dentate gyrus after region segmentation. The small rigions in Fig. 3 is eliminated and the area of the dentate gyrus is detected as shown Fig.4. In this image, noises were eliminated. The triangular-shaped apex of gran- ule cell layer was clearly visualized because the dentate dispersed in hilus and molecular layer of the dentate gyrus, and CA3. B Detection of the apwc of the dentate granule cell luyer by ~ I I In the brain slice shown in Fig. 2, the mean concentration ~ I j I I granule cell layer was large enough compared to small noises genetic algorithms. For successful automated micromanipulation, the apex region of the dentate gyrus shown in Fig. 4 should be matched with the template image. A binary image of the template is shown in Fig. t first, the temr te image was made from microscop% photos of cresyl violet-stained coronal section of the rat brain (Fig.6). The area of the dentate gyrus was selected from microscopic photo image. A clearly defmed image of the dentate gyrus was obtained by this processing hecause the granule cells were strongly stained with cresyl violet compared to adjacent molecular layer and hilar region. It is suitable for the template because the images which reflected morphological characteristics of the dentate gyrus. The reference pattern is positioned in the binary image as shown in Fig.7. A correlation value between the reference pattem and the binary image that is shown in Fig4 is estimated. When the reference pattem and the pattem of the black pixels in the binary image is equals to each other, the correlation value is near to 1. If the reference pattem and the binary image is not match up each other the correlation value becomes small. Therefore, if the correlation value is small, the reference pattern is moved and the correlation value is estimated again. Finally, the position of the reference pattern where the correlation value becomes near to 1 can he detected. Afier the detection of the position, an angle of the reference pattem is detected in the same way. Additionally, it is needed to reduce the detection process time to perform the microinjection at a realistic rate. 49 However, these detection process requires a long time because the enormous amount of calculation is necessary. Therefore, we developed the detection algorithm using the genetic algorithm. position in row, the position in column is indicated by 6 hits And the angle ofthe reference pattem is indicated by 3 hits of gene. The position and the angle of the template was detected as A search area is 64x 64 pixels in the center of the image. Angle is selected 6om O", 45", 90", 135", 18U6, 225", 270'and 3 15'. shown in Fig.9. The maximum correlation coefficient was 0.330375 when the center of the template image was located at i=3 1 I j=174. Fig. 8 shows the structure of a gene. The size of the gene is set at 15 bits, and each bit is the value of 0 or 1.The Fig. 5 Tcmplate for the apcx of ulc dcntatc gyms. 128 pixel Fig. 6 Cresyl violct-stained coronal section of thc rat brain Fig.7 Detectioo mcthod of thc dentate p. Fig. 9 Rcsult of tcmplatc matching. di:Position in row dj:Position in colomn k:Number of angles 00 63 0 0  63 ( U 7  1 2  3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5  Figuro 8 Structllrc of a gcnc, whcrc lipophilic tncm M to be injccted 111. RESULTS The tip of microelectrode was successfully inserted into the target region and DiI or DID was slowly injected by a nanotransjector (Fig. 10, Fig. 11). In the present study, we injected lipophilic tracer into 4% paraformaldehyde-fmd brain slices because lipophilic trac- ers were incorporated into plasma membrane are slowly dis- persed along the outer layer of plasma membrane and provide sharp morphological image. Moreover, lipophilic tracers can be injected into 5-mm brain slice was processed as de- scribed in the methods. By using an automated microma- nipulation device, the tip of microelectrode was successfully inserted into the apex of dentate gyrus by 6 to 8 steps of movements within 2 to 3 minutes, and 0.5 to 1 pI of lipo- philic tracer was injected into granule cell layer (Fig. IO,  Fig. 11). The injected slices were retnrned into fixative and Fig I ?  Coniacd mtcrmcopic umgc uimon, af ,he hilar ncurvn, lrr ird with  UtU. Thhs RJCCJ ncunlr.> m mdicotcd by mow stocked 31 riiom temperoture or 4 1 under light-protested condiliun. Murpholugical properties o i  neurons in granular ccll layer. molecular layer, and hilus wiu  analyLed by conro- c d  laser scan microssope (Lciss) more than two months ;It?er Ihs injection. The hilar nsurons were beautifully mccd and the apex of the dcntltc gynrs. The injccted sitc is indicatcd by mow. their processes extending into the granule cell layer was clearly identified (Fig. 12) 1V. CONCLUSIONS We have shown that image processing and micromanipula- tion could be utilized for lipophilic tracer injection into the hilus of the dentate gyrus of rat brain. Local injection of trac- ers has been done by microscopic visual inspection and man- ual control of microelectrode. However its general use is lim- ited because sophisticated techniques and much burdens are required to operate micromanipulator. Especially when trac- ers, chemoatractants or neurotrophic factors must be injected into the areas with exact distance i?om the implanted neurons or regions to where growth cones of mons or dendrites are introduced, it is difficult to inject them in unfixed, unstained brain slice. Our system may be useful in these experimental studies. REFERENCES T.L. Babb, W.J. Brown, J. Pretoriu. C. Davcnport, J. Lieb and P.H. 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