Confocal Light Microscopy of Brain Cells and Tissue: Image Analysis & Quantitation
James N. Turner 1,2, William Shain 1,2, Donald H. Szarowski 1, Sharie Lasek 1, Beth Sipple 1, Christopher Pace 3, Khalid Al-Kafahi 4, Ali Can 4 and Badrinath Roysam 4
1 Wadsworth Center, and 2 School of Public Health, The University at Albany, Albany, New York, 12201, USA, 3 Department of Biology, University at Albany, SUNY, Albany, NY, 12222, USA and 4 Electrical Computer and Systems Engineering, Rensselaer Polytechnic Institute, Troy, New York, 12180, USA
Received for publication February 1, 1999
The brain is an inherently three-dimensional (3-D) structure with tissue regions that occupy large complex volumes and cells that have complicated morphologies, e.g. branching fields of dendrites and axons extending in all three dimensions. The linear dimensions range from micrometers to tens of millimeters. The confocal light microscope can image relatively large volumes of tissue, and is therefore an ideal imaging tool for studying the brain and its cells. We have used histochemical and immunohistochemical labels to delineate structure at the tissue, cellular, and subcellular levels. The 3-D images are analyzed by custom software that automatically segments and counts nuclei over large tissue regions, automatically montages any number of 3-D images, and traces branching structures such as neuronal processes or blood vessels. Examples of nuclear detection in the rat hippocampus, where the cell density is high and nuclear images are close together, and of tracings of the dendritic field of a dye-filled neuron from the visual cortex of the kitten are shown. Complete penetration of thick tissue sections by large immunolabeling molecular complexes is demonstrated in tissue doublelabeled for glial fibrillary protein and laminin. The ability to correlate multiple labels in 3-D space is another strong point for confocal light microscopy. The influence of deconvolution on the quality of 3-D images is also demonstrated.
Review Amyloid $\beta$ Peptides and Presenilins in the Pathogenesis of Alzheimer's Disease
Takeshi Iwatsubo
Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033
Received for publication January 26, 1999
Amyloid $\beta$ peptides, especially those ending at the 42nd residue (A$\beta$42), is the initially and predominantly deposited A$\beta$ species in the brains of patients with Alzheimer's disease and Down's syndrome. The pathogenetic significance of overproduction and/or deposition of A$\beta$42 is highlighted because mutations in multiple different genes responsible for early-onset familial Alzheimer's disease, i.e. those in $\beta$APP, presenilin 1 and 2 genes, led to an increase in the secretion and deposition of A$\beta$42.
Review Morphological Aspects of Superoxide Dismutase-1 Mutation in Amyotrophic Lateral Sclerosis and its Transgenic Mouse Model
Noriyuki Shibata 1, Makio Kobayashi 1, Asao Hirano 2, Kohtaro Asayama 3, Seiko Horiuchi 4, Mauro C. Dal Canto 5 and Mark E. Gurney 6
1 Department of Pathology, Tokyo Women's Medical University, Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, 2 Division of Neuropathology, Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467-2490, USA, 3 Department of Pediatrics, Yamanashi Medical University, Shimokato 1110, Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, 4 Department of Biochemistry, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, 5 Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago IL 60611-3008, USA and 6 CNS Diseases Research Unit, Pharmacia and Upjohn Inc., 301 Henrietta Street, Kalamazoo, MI 49001, USA
Received for publication December 1, 1998
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily involves the motor neuron system. Recent investigations have obtained evidence that mutations in the gene for superoxide dismutase-1 (SOD1) are detected in a subset of familial ALS patients, and that mutant SOD1-expressing transgenic mice develop ALS-like clinicopathological features. Several in vitro studies suggest that SOD1-mutated ALS is caused by a newly acquired neurotoxicity of mutant SOD1, but not by reduced enzyme activity. From the viewpoint of morphology, we analyzed the immunolocalization of SOD1 and some other substances in spinal cords from familial ALS patients with SOD1 Ala4$\rightarrow$Val mutation. The spinal cords of the ALS patients demonstrated the characteristic neuronal hyaline inclusions (NHIs) immunoreactive with antibodies to ubiquitin and phosphorylated neurofilament protein (NFP) in the lower motor neurons and cordlike swollen axons. The NHIs contained the epitopes of SOD1 and N$\varepsilon$-carboxymethyllysine (CML), one of the major advanced glycation endproducts (AGE), whereas there was no focal accumulation of SOD1 and CML in control individuals. Immunoelectron microscopy depicted the SOD1 and CML determinants on the granule-associated thick linear structures that mainly compose the NHIs. We also performed a similar study on mice carrying a transgene for Gly93$\rightarrow$Ala mutant SOD1. The spinal cords of the transgenic mice were characterized by the appearance of NHIs resembling those of familial ALS and by vacuolar degeneration. The mouse NHIs were immunoreactive for ubiquitin, phophorylated NFP, SOD1 and CML. Our findings of the coexistence of SOD1 and AGE in both the human and mouse NHIs indicate that certain substances are implicated in glycoxidation in the presence of oxidative stress originating from mutant SOD1 and finally deposited in the NHIs, suggesting a pathogenic role of the oxidative processes in motor neuron degeneration in vivo.
Review Aggression and Serotonin -Contribution of Inhibitory Mechanisms-
Shuichi Ueda 1, Atsuko Ishizuya-Oka 1, Akira Nishimura 2, Yoshihiro Takeuchi 2 and Kanji Yoshimoto 3
1 Department of Histology and Neurobiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293 and 2 Department of Pediatrics, and 3 Department of Legal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841
Received for publication December 8, 1998
Transplantation of fetal serotonergic neurons into the hypothalamus restored muricide (mouse killing behavior) in the rat with raphe lesion induced by 5,7-dihydroxytryptamine (selective serotonergic neurotoxine). Immunohistochemical and neurochemical studies indicated that recovery of serotonergic innervation in the lateral hypothalamic area by the graft brought about the inhibition of muricide. The grafted serotonergic neurons are strongly related to the inhibitory action on the muricide. Transplanted serotonergic neuron system can result in a reinnervation of the host, and leads to a reestablishment of the aggressive behavior.
Key words: Aggressive behavior, Rat, Transplantation, Serotonin, Immunohistochemistry
Review Three Dimensional Protein Localization Using High Voltage Electron Microscopy
Maryann E. Martone, Thomas J. Deerinck, Stephen J. Young and Mark H. Ellisman
National Center for Microscopy and Imaging Research, University of California, San Diego, San Diego CA 92093-0608, USA
Received for publication December 24, 1998
Three-dimensional (3D) protein localization at the electron microscopic level is of great benefit when determining the precise distribution of a given protein relative to complex subcellular structures. We have developed several 3D ultrastructural labeling approaches for use with higher voltage electron microscopy. High voltage electron microscopy allows for the use of much thicker sections than conventional transmission electron microscopy. Many of our staining protocols are based on the photooxidation of diaminobenzidine (DAB) by fluorescent markers. As a fluorescent dye is excited in the presence of DAB, the reactive oxygen generated gradually oxidizes DAB into an insoluble polymer which can be rendered electron dense. We previously demonstrated that photooxidation by the fluorophore eosin can be used for high resolution immunolocalization. Because of its small size, eosin-conjugated reagents penetrate well into tissue slices, providing labeling through a reasonable depth of tissue. When combined with high voltage electron microscopy using thick sections, the 3D distribution of a variety of proteins can be visualized. Analysis and interpretation of these 3D labeling patterns is further facilitated by the use of electron tomography, which allows for the derivation of 3D structure from a series of images taken at different orientations. In this review, several examples of 3D protein localization using these technologies are presented.
1 Second Department of Pathology, Kumamoto University School of Medicine, Kumamoto 860-0811, 2 Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo and 3 Exploratory Research Laboratory, Chugai Pharmaceutical Co. Ltd., Shizuoka
Received for publication December 8, 1998
Macrophage scavenger receptor (MSR) is one of the major receptors of macrophages. It plays an important role in the pathological deposition of cholesterol in macrophagederived foam cells during atherogenesis through receptor-mediated uptake of modified low density lipoproteins. MSR is also important for macrophages to recognize and eliminate pathogenic microorganisms. Targeted disruption of the MSR gene resulted in a reduction in the size of atherosclerotic lesions in atherosclerosis model mice. MSRknockout mice were more susceptible to infection. Hepatic granuloma formation induced by a single intravenous injection of heat-killed Corynebacterium parvum was significantly delayed in MSR-knockout mice. Using MSRknockout mice as immunization animals, five monoclonal antibodies against human MSR were successfully produced. Immunohistochemistry using these antibodies revealed a restricted distribution of MSR protein on tissue macrophages in various tissues and organs. These antibodies will provide a new approach to study the role of MSR in normal and various pathological conditions in humans.
1 Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, 2 Third Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511 and 3 First Department of Surgery, Gunma University School of Medicine, Maebashi, Gunma 371-8511
Received for publication February 12, 1999
Sugar transporters are integral membrane proteins responsible for the transport of sugars across the cellular membranes. They play important roles in the transfer of sugars across different compartments separated by epithelia in the body. Preferential localization of them in specific plasma membrane domains of epithelial cells is crucial in the vectorial transfer of sugars. Modification of the transporter genes with the molecular biology techniques and visualization of the expressed gene products by immunohistochemical methods have shed light on the molecular mechanism underlying the specific localization of the transporter molecules.
Application of Real-Time Confocal Microscopy to Observations of ATP-Induced Ca2+-Oscillatory Fluctuations in Intact Corneal Epithelial Cells
Katsura Kimura, Hiroshi Yamashita, Tomoko Nishimura, Shiho Mori and Yoh-ichi Satoh
Department of Histology, Iwate Medical University School of Medicine, Uchimaru 19-1, Morioka 020-8505
Received for publication December 16, 1998 and in revised form January 16, 1999
Digital imaging of intracellular calcium ion concentration ([Ca2+]i) dynamics has been a fundamental technique in cell biology. The present study examines the possibility of using the confocal microscope for [Ca2+]i imaging of living tissue. We analyzed the ATP-induced [Ca2+]i dynamics of rabbit corneal epithelium as an experimental model. After loading the cells with Indo-1, a realtime confocal microscope (Nikon RCM/Ab) revealed individual images. ATP (100 $\mu$M) in the perfusate elicited an increase in [Ca2+]i. Superficial cells showed a biphasic change in [Ca2+]i composed of an initial spike phase followed by a persistent plateau phase. On the other hand, oscillatory fluctuations were evident in the mid-wing cell layer, and they were often synchronized, indicating intercellular communication. The spikes in the wing cells did not affect the [Ca2+]i dynamics of the superficial cells. In conclusion, the optical slicing effect of confocal microscopy is suitable for observing thick specimens, such as living tissues composed of various cell types. This is the first report to describe [Ca2+]i dynamics in intact epithelial tissue specimens, and to reveal heterogeneous responses of different cell layers.
Key words: Cornea, Confocal microscopy, Intracellular Ca2+, ATP
Localization and Interaction of Hyaluronic Acid and Inter-$\alpha$-Trypsin Inhibitor in Stimulated Preovulatory Rat Ovaries
Guang Wei Sun, Hiroshi Kobayashi and Toshihiko Terao
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka, 431-3124
Received for publication May 29, 1998 and in revised form November 24, 1998 and re-revised form February 1, 1999
Proteins of the inter-$\alpha$-trypsin inhibitor (I$\alpha$I) family are reported to stabilize the expanding cumulus extracellular matrix by direct binding with hyaluronic acid in mouse. The aim of this study is to compare the expression and distribution of hyaluronic acid and I$\alpha$I in the rat ovaries during gonadotropin-induced follicle development. Immunoand affinity histochemical staining was performed in rat ovary using a polyclonal antibody raised against purified human I$\alpha$I and a hyaluronic acid-specific probe (biotinylated hyaluronic acid-binding protein). During a preovulatory stage, relatively large amounts of hyaluronic acid were present in the follicular fluid as well as on the surface of expanding cumulus cells and theca/stromal cells. The area of I$\alpha$I reactivity in stimulated preovulatory follicles was almost the same as that of diffuse deposition of hyaluronic acid. In an in vivo rat model, inhibition of hyaluronic acid synthesis resulted in inhibition of cumulus oocyte complex (COC) expansion accompanied by reduced staining for I$\alpha$I and hyaluronic acid. Western blot analysis of I$\alpha$I and immunoassay for hyaluronic acid in tissue extracts from hCG-stimulated ovaries indicated marked increase in I$\alpha$I and hyaluronic acid expression. These findings support the hypothesis that the co-distribution of I$\alpha$I and hyaluronic acid may serve to organize the formation of the cumulus extracellular matrix in the rat ovary.
Critical Electrolyte Concentration of Chicken Erythrocyte Chromatin
Jos\'{e} Ricardo P. Falco 1, 2, Maria Luiza S. Mello 1, Silvya S. Maria 1 and Neiva A. Grazziotin 1, 3
1 Department of Cell Biology, Institute of Biology, UNICAMP, 13083-970 Campinas, SP, Brazil, 2 Department of Cell Biology and Genetics, CCB/UEM, 87020-020 Maring\'{a}, PR, Brazil and 3 University of Passo Fundo, 99001-970 Passo Fundo, RS, Brazil
Received for publication September 28, 1998 and in revised form January 5, 1999
This study examined whether chicken erythrocyte chromatin, whose great physicochemical stability is promoted by histone H5, has a critical electrolyte concentration (CEC) value comparable to that of other chromatin types with special condensed packing states but devoid of H5. The affinity for toluidine blue molecules under Mg2+ competitive binding action was thus investigated. The CEC value was found to be 0.12 M, which is close to that of heterochromatin from mouse liver cells and to sperm chromatin with histone H1 variants. Acetic ethanol fixative was found to affect the CEC, possibly by influencing the chromatin packing state and DNA phosphate availability through dissociation of proteins from the DNA. The phenomenon of programmed cell death was considered to contribute to high CEC values by inducing special states of chromatin condensation.
The Relationship between Apoptosis and Keratinization in Human Epidermis
Kuniko Nakamura 1, Yuko Ito 2, Kenji Matsumoto 2, Eriko Daikoku 2, Kimihiro Kiyokane 1 and Yoshinori Otsuki 2
1 Department of Dermatology, and 2 Department of Anatomy and Biology, Osaka Medical College, Takatsuki, Osaka 569-8686
Received for publication September 29, 1998 and in revised form January 23, 1999
The relationship between keratinization in human epidermis and apoptosis is studied using the technique of in situ end labeling of DNA strand breaks combined with transmission electron microscopy. Moreover, quantitative analysis of free 3'-OH DNA ends in individual keratinocytes was performed on a computer using the NIH Image program. The number of free 3'-OH DNA ends in keratinocyte nuclei was significantly higher in cells from the lower spinous layer than in the basal layer, although chromatin condensation was not detected in the keratinocytes of the lower spinous layer. As chromatin condensation progressed from the upper spinous cells to the granular cells, the number of 3'-OH DNA ends increased and was highest in granular cell nuclei. However, no other characteristic of apoptosis, such as decreased cell volume, membrane blebbing or apoptotic bodies was observed in keratinocytes in any layer of the epidermis. Moreover, horny cells lost their nuclei, together with the abrupt decrease in the number of 3'-OH ends. Therefore, keratinization does not thoroughly satisfy all the criteria of apoptosis, although cells undergoing keratinization show both chromatin condensation and DNA fragmentation. It is noteworthy that DNA fragmentation has already occurred in keratinocytes of the lower spinous layer which show no apoptotic changes.
Key words: Apoptosis, Keratinization, Epidermis, In situ DNA nick end labeling, Transmission electron microscopy
1 Department of Anesthesiology and 2 Department of Anatomy & Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566
Received for publication December 9, 1998 and in revised form January 21, 1999
The induction and functional involvement of macrophages and postganglionic sympathetic fibers have been reported in rat neuropathic pain models. In the present study, we examined the morphology and structural relationship among macrophages, postganglionic sympathetic fibers, the dorsal root ganglion (DRG) neurons, and the satellite cells by light and electron microscopic examination. We used the rat spinal nerve ligation model reported by Kim & Chung. On postoperative day (POD) 1, when sympathetically maintained pain was confirmed, macrophages invaded from the nerve ligation site and reached the DRG neuronal area. Macrophages might have induced the demyelination of the spinal nerve and the degeneration of the satellite cells, and then the DRG neurons, from which the satellite cells were detached, lost the insulation from other neurons or nerve fibers. After the invasion of macrophages, postganglionic sympathetic fibers with TH-like immunoreactivity migrated into the same area, and occupied sites around the neuronal soma which were surrounded by satellite cells under normal conditions. At POD 180, the sympathetically maintained pain had disappeared, and the DRG neurons and spinal nerve showed normal structure without any invasive macrophages or postganglionic sympathetic fibers. The above changes in the morphological arrangements of macrophages and postganglionic sympathetic fibers in the DRG were thus elucidated and we suggest that this process may be part of the mechanisms underlying the development of the sympathetically maintained pain.