Conn Quantitative and Qualitative Microscopy

1. Auflage 2013
ISBN: 978-1-4832-6817-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 510 Seiten

Methods in Neurosciences, Volume 3: Quantitative and Qualitative Microscopy is a collection of papers that deals with microscopic techniques in statistical measures. This volume describes microscopy using sophisticated stains and dyes to advance observation of tests and experiments. Section I describes autoradiography including micro chemical methods, high-resolution autoradiography, and single- or double-label quantitative autoradiography for use in imaging of brain activity patterns or determining cerebral physiology. Section II discusses the quantification of structures through statistical and computational methods including dynamic video imaging technology. Section III explains the use of tracers, toxins, or dyes in tracing neuronal connections. One paper addresses the use of small injections of axonally transported fluorescent tracers. Section IV explains staining technology such as using the silver impregnation method for frozen sections of human nervous tissue that are gathered from tissues preserved in formalin. Section V addresses freezing techniques and those using freeze-fracture methods in neurobiology. The text also discusses cryoprotection and other freezing methods to control ice crystals found in fixed or unfixed brain tissues. Section VI presents the combined and high-resolution methods in polarization microscopy and microscopic investigations. Cellular biologists, micro-chemists, and scientific researchers in the field of micro- and cellular biology will appreciate this book.

Autoren/Hrsg.

Conn, P. Michael

Inhaltsverzeichnis

1;Front Cover;1
2;Quantitative and Qualitative Microscopy;4
3;Copyright Page;5
4;Table of Contents;6
5;Contributors to Volume;10
6;Preface;14
7;Volumes in Series;16
8;Section I: Autoradiography;18
8.1;Chapter 1. Autoradiographic and Microchemical Methods for Quantitation of Steroid Receptors;20
8.1.1;Introduction;20
8.1.2;Microchemical Measurement of Steroid Receptors in the Central Nervous System;21
8.1.3;Quantitative Autoradiography for Measurement of Steroid Receptor Levels in Central Nervous System;40
8.1.4;Conclusions and Prospectus;48
8.1.5;Acknowledgments;49
8.1.6;References;49
8.2;Chapter 2. High-Resolution Autoradiographic Mapping of Drug and Hormone Receptors;52
8.2.1;Thaw-Mount and Dry-Mount Autoradiography with Thin Frozen Sections;54
8.2.2;Apposition (Sandwich) Techniques;60
8.2.3;Conclusions;64
8.2.4;References;66
8.3;Chapter 3. High-Resolution Autoradiographic Imaging of Brain Activity Patterns with Radiolabeled 2-Deoxyglucose and Glucose;67
8.3.1;Preparation of Rats for Intravenous Injection of Deoxy[14C]glucose or [14C]glucose;68
8.3.2;High-Resolution Autoradiography at Regional Topographic Level;69
8.3.3;Comparison of Radiolabeled 2-Deoxyglucose and Glucose as Metabolic Indicators and Consideration of Time Course for 2-Deoxyglucose Studies;73
8.3.4;Dry-Mount Autoradiography for Cellular Resolution;75
8.3.5;Quantitative Considerations in 2-Deoxyglucose Studies;78
8.3.6;Summary and Conclusions;80
8.3.7;References;80
8.4;Chapter 4. Double- and Single-Label Quantitative Autoradiography for Cerebral Physiology;81
8.4.1;Quantitative Autoradiography: The Basics;81
8.4.2;Physiological Quantitative Autoradiography;87
8.4.3;Digital Image Analysis for Double-Label Quantitative Autoradiography;90
8.4.4;Double-Label Techniques;95
8.4.5;Summary and Conclusion;103
8.4.6;Acknowledgment;103
8.4.7;References;103
8.5;Chapter 5. Combination of Tritiated Thymidine Autoradiography and Neuropeptide Immunocytochemistry to Determine Birthdates and Migration Routes of Luteinizing Hormone-Releasing Hormone Neurons;107
8.5.1;Introduction;107
8.5.2;Preparation of Animals for Autoradiography;109
8.5.3;Preparation of Paraffin Sections;110
8.5.4;Immunocytochemical Procedures;110
8.5.5;Controls for Immunocytochemistry;112
8.5.6;Autoradiography;113
8.5.7;Controls for Autoradiography;115
8.5.8;Acknowledgment;122
8.5.9;References;122
9;Section II: Quantification of Structures: Statistical and Computational Methods;124
9.1;Chapter 6. Techniques and Technology for Dynamic Video Imaging of Cellular Fluorescence;126
9.1.1;Introduction;126
9.1.2;Chemical Probes for Cellular Function;127
9.1.3;Digital Video Imaging Technology;132
9.1.4;Biological Results of Dynamic Video Imaging;144
9.1.5;Comparison of Imaging with Photometric Systems;146
9.1.6;Limitations and Artifacts;151
9.1.7;Future Developments;151
9.1.8;References;152
9.2;Chapter 7. Three-Dimensional Computer

Reconstruction of Perforated Synapses;153
9.2.1;Introduction;153
9.2.2;Computer-Assisted Three-Dimensional Reconstruction;154
9.2.3;Study of Perforated Synapses;160
9.2.4;Results and Conclusions;167
9.2.5;Acknowledgments;171
9.2.6;References;171
9.3;Chapter 8. Determination of Numerical Density of Perforated and Nonperforated Synapses;172
9.3.1;Introduction;172
9.3.2;Disector as Sampling Probe;176
9.3.3;Disector Method in Synaptic Studies;179
9.3.4;Numerical Density of Synapses and Perforated Synapses;182
9.3.5;Results and Conclusions;186
9.3.6;Acknowledgments;188
9.3.7;References;188
9.4;Chapter 9. Efficient and Unbiased Sampling of Nerve Fibers for Estimating Fiber Number and Size;189
9.4.1;Introduction;189
9.4.2;Efficiency, Bias, and Experimental Design;191
9.4.3;Sources of Random Error;191
9.4.4;Sources of Bias;192
9.4.5;Unbiased Sampling;193
9.4.6;Systematic Random Sampling;194
9.4.7;Practical Aspects of Sampling;197
9.4.8;Biological Examples;199
9.4.9;References;203
9.5;Chapter 10. Methods for Analyzing Neuronal Connections in Mammals;205
9.5.1;Introduction;205
9.5.2;Pathway Tracing with [3H]Proline;206
9.5.3;Pathway Tracing with Wheat Germ Agglutinin–Horseradish Peroxidase;213
9.5.4;Pathway Tracing with Carbocyanine Dyes;219
9.5.5;Acknowledgments;223
9.5.6;References;223
9.6;Chapter 11. Image Analytic Techniques for Quantification of Immunohistochemical Staining in the Nervous System;225
9.6.1;Introduction;225
9.6.2;Tissue Preparation for Image Analysis;225
9.6.3;Image Analysis;226
9.6.4;Recommended Procedure for Quantification of Immunohistochemical Staining;245
9.6.5;References;246
9.7;Chapter 12. Methods for Visualizing and Analyzing Individual Axon Arbors;247
9.7.1;Introduction;247
9.7.2;Labeling Axons with Extraaxonal Horseradish Peroxidase;247
9.7.3;Labeling Axons with Phaseolus vulgaris Leucoagglutinin;253
9.7.4;Analysis;257
9.7.5;Acknowledgments;260
9.7.6;References;260
10;Section III: Tracing Neuronal Connections:Tracers, Toxins, and Dyes;262
10.1;Chapter 13. Phaseolus vulgaris Leucoagglutinin Anterograde Axonal Transport Technique;264
10.1.1;Introduction;264
10.1.2;Protocols and Notes;265
10.1.3;Issues;271
10.1.4;Applications;274
10.1.5;Concluding Remarks;276
10.1.6;Acknowledgments;276
10.1.7;References;276
10.2;Chapter 14. Retrograde Axoplasmic Transport of Neurotoxins;278
10.2.1;Introduction;278
10.2.2;Retrograde Suicide Agents;279
10.2.3;Methodological Problems;280
10.2.4;Suicide Transport as a Neuroanatomical Method;281
10.2.5;Experimental Form of Motor Neuron Diseases;283
10.2.6;Theories of Motor Neuron Diseases;285
10.2.7;Pain Control by Retrograde Sensory Ganglionectomy;286
10.2.8;Eradication of Herpesvirus Persistence in Sensory Ganglia;289
10.2.9;Perspectives of Suicide Transport as a Research Method;289
10.2.10;Acknowledgment;290
10.2.11;References;290
10.3;Chapter 15. Tracing Neuronal Connections in the Periphery: Renal Nerves;292
10.3.1;Introduction;292
10.3.2;Fluorescent Dye Method;294
10.3.3;Combination of Fluorescent Tracers and Immunohistochemistry;299
10.3.4;Innovative Uses of Fluorescent Tracers;302
10.3.5;Problems of Interpretation;303
10.3.6;References;306
10.4;Chapter 16. Small Injections of Axonally Transported Fluorescent Tracers;308
10.4.1;Pressure Injections;309
10.4.2;Iontophoretic Injections;315
10.4.3;Implantation of Glass Micropipettes;320
10.4.4;Advantages and Disadvantages of Different Injection Techniques;323
10.4.5;General Considerations;326
10.4.6;Material Sources;329
10.4.7;Acknowledgments;330
10.4.8;References;330
11;Section IV: Staining Technology;332
11.1;Chapter 17. Fluoro-Gold and 4-Acetamido-4'-isothiocyanostilbene-2,2 ' -disulfonic Acid: Use of Substituted Stilbenes in Neuroanatomical Studies;334
11.1.1;4-Acetamido-4'-isothiocyanostilbene-2,2 '-disulfonic Acid;334
11.1.2;Fluoro-Gold;338
11.1.3;Acknowledgments;346
11.1.4;References;346
11.2;Chapter 18. Silver Impregnation Method for Frozen Sections of Human Nervous Tissue Using Ammoniacal Silver–Dichromate Solution;347
11.2.1;Introduction;347
11.2.2;Procedure;348
11.2.3;Results and Discussion;349
11.2.4;References;351
11.3;Chapter 19. Silver Impregnation Method for Neurons Using Synthetic Surfactants: A Contribution to Golgi Method;352
11.3.1;Introduction;352
11.3.2;Experimental Procedures;352
11.3.3;Results and Discussion;355
11.3.4;Acknowledgments;355
11.3.5;References;356
12;Section V: Freezing Techniques;358
12.1;Chapter 20. Use of Freeze-Fracture in Neurobiology;360
12.1.1;Introduction;360
12.1.2;Freezing Techniques;361
12.1.3;Specimen Preparation;362
12.1.4;Fracturing/Replicating;363
12.1.5;Replica Cleaning and Mounting;365
12.1.6;Replica Interpretation;366
12.1.7;Replica Artifacts;374
12.1.8;Conclusions;374
12.1.9;Acknowledgments;375
12.1.10;References;375
12.2;Chapter 21. Cryoprotection and Freezing Methods to Control Ice Crystal Artifact in Frozen Sections of Fixed and Unfixed Brain Tissue;377
12.2.1;Introduction;377
12.2.2;Variables Influencing Ice Crystal Formation;379
12.2.3;Methods;381
12.2.4;Observations on Freezing Damage in Fixed and Unfixed Tissue;382
12.2.5;Specific Cryoprotection Procedures;396
12.2.6;Conclusions;400
12.2.7;Acknowledgments;401
12.2.8;References;401
13;Section VI: Combined and High-Resolution Methods;404
13.1;Chapter 22. Double-Label [3H]2-Deoxyglucose and[14C]2-Deoxyglucose Method for Mapping Brain Activity Underlying Two Experimental Conditionsin the Same Animal;406
13.1.1;Introduction;406
13.1.2;Double-Label 2-Deoxyglucose Experimental Protocol;407
13.1.3;Obtaining Autoradiographs Showing [3H]2-Deoxyglucose and[14C]2-Deoxyglucose Uptake;409
13.1.4;Obtaining a Veridical Image of 2-Deoxy[3H]glucose Activity;412
13.1.5;Experimental Tests of Double-Label 2-Deoxyglucose Experiment;417
13.1.6;Data Analysis Procedures for Double-Label 2-Deoxyglucose Experimen;421
13.1.7;Optimizing Double-Label Experimental Protocol;424
13.1.8;Other Double-Label Methods;426
13.1.9;Acknowledgment;428
13.1.10;References;428
13.2;Chapter 23. Application of Incident Light Polarization Microscopy;430
13.2.1;Introduction;430
13.2.2;Incident Light Polarization Microscopy;430
13.2.3;Microscope;433
13.2.4;Application to Lateral Line Preparation;435
13.2.5;Application to Other Preparations;438
13.2.6;Improvement of Quality of Images;439
13.2.7;Comparison with Other Methods;440
13.2.8;Acknowledgments;441
13.2.9;References;441
13.3;Chapter 24. Light Microscopic Localization of Drug and Neurotransmitter Receptors in the Brain;442
13.3.1;Introduction;442
13.3.2;Methodology;444
13.3.3;Use of Receptor Autoradiography;452
13.3.4;Future of Receptor Localization;455
13.3.5;References;455
13.4;Chapter 25. Light and Electron Microscopic Investigation of Somatostatin-Containing Neurons in the Central Nervous System;457
13.4.1;Introduction;457
13.4.2;Methods for Light and Electron Microscopic Immunohistochemical Investigations;458
13.4.3;Types of Somatostatin-Containing Neurons in Cerebral Cortex,Striatum, and Spinal Cord;462
13.4.4;Ultrastructure of Somatostain-Containing Neurons in Rat Cerebral Cortex, Striatum, and Spinal Cord;464
13.4.5;Conclusion;471
13.4.6;Acknowledgment;472
13.4.7;References;472
13.5;Chapter 26. Reduced Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase Histochemistry: Light and Electron Microscopic Investigations;474
13.5.1;Introduction;474
13.5.2;Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase Histochemistry;475
13.5.3;Distribution and Characteristics of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase-Positive Neurons;478
13.5.4;Ultrastructural Localization of Nicotinamide Adenine Dinucleotide Phosphate–Diaphorase-Positive Neurons;484
13.5.5;Coexistence of Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase-Positive Neurons and Neuropeptides;486
13.5.6;Acknowledgments;487
13.5.7;References;488
14;Addendum;490
14.1;Addendum to Article [6];492
14.2;Acknowledgments;492
15;Index;494

Leseproben

[1]

Autoradiographic and Microchemical Methods for Quantitation of Steroid Receptors

N.J. MacLusky, T.J. Brown, E. Jones, C. Leranth and R.B. Hochberg

Publisher Summary

During the past few years, considerable progress has been made toward the development of specific new methods for the analysis of steroid receptor concentrations in discrete, anatomically defined regions of the brain. These methods are based on two experimental approaches. The first method includes a variety of modifications of earlier subcellular fractionation-based steroid receptor assay procedures combined with the Palkovits microdissection method to achieve the necessary anatomical resolution. The second approach is based on quantitation of the uptake of radiolabeled steroids by the tissue through densitometric analysis of autoradiograms prepared by the exposure of tissue sections against film. This chapter outlines the principal features of these two methodological strategies: microchemical measurement of steroid receptors in the central nervous system (CNS) and quantitative autoradiography for the measurement of steroid receptor levels in the central nervous system. Measurement of steroid receptor levels in individual microdissected cell groups from the CNS, however, presents special technical problems that restrict the range of methodology that can be successfully applied. For more than two decades, the primary method for high-resolution localization of steroid binding sites in the brain has been autoradiography. Perhaps the most exciting potential application of these methods reviewed in the chapter is the possibility for integrating high-resolution measurements of steroid receptor levels with other biochemical and anatomical techniques. Using combinations of experimental approaches, it may be possible to achieve a far greater understanding of the mechanisms of steroid hormone action on the brain.

Introduction

Over the past two decades, biochemical methods have been established for measurement of all five major classes of steroid hormone receptor in the central nervous system (CNS). Many of these procedures also allow selective estimation of the extent to which the receptors are occupied by endogenous circulating hormones. Despite the wide range of methodology available, however, progress toward defining the relationship between steroid–receptor interactions in different regions of the brain and responsiveness to changes in circulating steroid levels has been relatively slow. A major problem is that the majority of the available steroid receptor assay methods are relatively insensitive, which necessitates use of large tissue samples and/or pooling of tissue from several animals for each determination. This limitation seriously constrains attempts to correlate receptor binding with physiological responses. Studies on the effects of intracerebral hormone implants strongly suggest that neuroendocrine and behavioral responses to gonadal steroids may require exposure of only one, or a few, highly localized target structures to estrogen (1–3). Moreover, several reports have suggested that steroid receptor concentrations in different regions of the brain may be selectively influenced by such factors as reproductive status and afferent neural input (4–7). Thus, overall measurements of receptor concentrations in crudely dissected brain samples may give a misleading picture of the relationship between receptor binding and responses to circulating steroids.

The CNS presents a unique challenge with respect to the methodology for steroid receptor assays. Not only are overall receptor concentrations relatively low, in comparison to steroid target organs such as the liver and reproductive tract, but the target cells in the brain are distributed heterogeneously, dispersed in groups among much larger numbers of nontarget neurons and glia (8,9). This makes measurements of steroid receptors in the brain particularly difficult, since the assay methods must combine levels of sensitivity and specificity that are considerably greater than those required for most other steroid–sensitivity structures.

During the past few years, considerable progress has been made toward the development of specific new methods for the analysis of steroid receptor concentrations in discrete, anatomically defined regions of the brain. These methods are based on two experimental approaches. The first includes a variety of modifications of earlier subcellular fractionation-based steroid receptor assay procedures combined with the Palkovits microdissection method (10) to achieve the necessary anatomical resolution. The second approach is based on quantitation of the uptake of radiolabeled steroids by the tissue through densitometric analysis of autoradiograms prepared by exposure of tissue sections against film. This article briefly outlines the principal features of these two methodological strategies.

Microchemical Measurement of Steroid Receptors in the Central Nervous System

General Principles

In common with nonneural target tissues, steroids appear to act on the brain through receptor proteins that are recovered in the soluble cytoplasmic (cytosol) fraction of the cell after subcellular fractionation. Binding of the steroid to its receptor site results in transformation of the receptor to a form that binds tightly within the cell nucleus, from which it can be extracted only by disruption of the chromatin using DNase or high-ionic-strength buffers (11–14). The different properties of the native and hormone-bound forms of steroid receptors necessitate use of somewhat different methodologies for assay of occupied and unoccupied receptor sites. Since the majority of unoccupied receptors are extracted into the cytosol after tissue homogenization, these receptors can be assayed relatively easily by equilibrating tissue cytosol fractions with appropriate stereospecific labeled ligands, followed by separation of the receptor-bound and free components. Nuclear bound steroid–receptor complexes are separated by preparation of a cell nuclear fraction from which the majority of unbound receptors are eliminated by washing with low-ionic-strength buffer. The remaining receptors are then labeled by exchanging a radioactive ligand for the unlabeled steroid in the nuclear-bound receptor complex by incubating either the intact nuclei or a soluble nuclear extract with the labeled ligand (for a general review of steroid receptor assay procedures, see Ref. 11).

Cytosol Steroid Receptor Assays

The major differences between cytosol steroid receptor assay methodologies involve the technique used for separation of bound and unbound ligand. For studies of the steroid receptor content of relatively large regions of the brain (e.g., the entire hypothalamus and preoptic area) a wide variety of such techniques have been employed. Measurement of steroid receptor levels in individual microdissected cell groups from the CNS, however, presents special technical problems which greatly restrict the range of methodology that can be successfully applied. There are three main problems. The first concerns the process for dissection of the tissue, which must be accomplished without significant degradation of the relatively sensitive steroid receptor molecules. Second, the extremely small tissue samples must be homogenized under conditions which will ensure reproducible extraction of the nonnuclear-bound receptor sites. Finally, separation of the receptor-bound hormone must be accomplished with maximum efficiency to allow detection of the small number of receptors present in individual microdissected tissue samples.

The procedure currently in use in our laboratories for measurement of cytosol steroid receptors is as follows.

General Cytosol Steroid Receptor Assay Procedure

For cytosol steroid receptor assays in microdissected regions of the brain, the tissue must initially be frozen and sectioned to allow dissection using the Palkovits “punch” technique (10). In the rat brain, no special precautions are necessary for preservation of the receptors during the microdissection procedure. However, perfusion of the animals via the left ventricle with ice-cold aqueous 10% (v/v) dimethyl sulfoxide prior to removal of the brain may offer some advantages for progestin receptor assays (15); this point will be discussed further below. The brain is rapidly removed, blocked with a razor blade, mounted in the required orientation on a cryostat chuck, and frozen on dry ice. Serial 300-µm-thick coronal sections are cut and thaw-mounted on glass slides. Regions of interest are then removed from the frozen sections under a dissecting microscope using stainless steel needles ranging in diameter from 0.3 to 1 mm. Figure 1 illustrates the scheme that we currently use for studies of the regulation of estrogen and progestin receptor levels in the adult rat brain (PVPOA, periventricular preoptic area; mPOA, medial preoptic area; BNST, bed nucleus of the stria terminalis; ARC, arcuate nucleus; ME, median eminence; VMN, ventromedial nucleus of the hypothalamus; AMYG, pooled cortical and medial nuclei of the amygdala).

Fig. 1 Microdissection scheme for the hypothalamus and preoptic area. Twelve consecutive 300-µm thick sections are cut from each brain and mounted...