Springer modern techniques in neuroscience research windhorst johansson 1999 springer 3540644601

Contents

Chapter 1
Cytological Staining Methods
Robert W. Banks
Introduction. . . . . . . . . . . . . . . . . .
Subprotocol 1: Fixation, Sectioning and Embedding
Subprotocol 2: Ultrastructure . . . . . . . . . .
Subprotocol 3: The Golgi Method . . . . . . . .
Subprotocol 4: Single-Cell Methods. . . . . . . .
References . . . . . . . . . . . . . . . . . .

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Chapter 2
Application of Differential Display and Serial Analysis

of Gene Expression in the Nervous System
Erno Vreugdenhil, Jeannette de Jong and Nicole Datson
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: Differential Display: Practical Approach . . . . . . . . . .
Subprotocol 2: Serial Analysis of Gene Expression (SAGE): Practical Approach
Subprotocol 3: Digestion of cDNA with Anchoring Enzyme . . . . . . . .
Subprotocol 4: Binding to Magnetic Beads. . . . . . . . . . . . . . . .
Subprotocol 5: Addition of Linkers . . . . . . . . . . . . . . . . . . .
Subprotocol 6: Tag Release by Digestion with Tagging Enzyme . . . . . . .
Subprotocol 7: Blunting Tags . . . . . . . . . . . . . . . . . . . . .
Subprotocol 8: Ligation to Ditags . . . . . . . . . . . . . . . . . . . .
Subprotocol 9: PCR Amplification of Ditags . . . . . . . . . . . . . . .
Subprotocol 10: Ditag Isolation . . . . . . . . . . . . . . . . . . . .
Subprotocol 11: Concatemerisation . . . . . . . . . . . . . . . . . . .
Subprotocol 12: Cloning Concatemers . . . . . . . . . . . . . . . . .
Subprotocol 13: Sequencing . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 3
Methods Towards Detection of Protein Synthesis in Dendrites and Axons
Jan van Minnen and R.E. van Kesteren
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: In Situ Hybridization of Cultured Neurons . . . . . . . .
Subprotocol 2: In Situ Hybridization at the Electron Microscopic Level . .
Subprotocol 3: Single-Cell Differential mRNA Display . . . . . . . . .
Subprotocol 4: Functional Implications of mRNAs in Dendrites and Axons:
Metabolic Labeling of Isolated Neurites. . . . . . . . . . . . . . .
Subprotocol 5: Intracellular Injection of mRNA . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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VIII

Contents

Chapter 4
Optical Recording from Individual Neurons in Culture
Andrew Bullen and Peter Saggau
Introduction . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . .

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102
102
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115
117
122
125

Chapter 5
Electrical Activity of Individual Neurons In Situ:
Extra- and Intracellular Recording
Peter M. Lalley, Adonis K. Moschovakis and Uwe Windhorst
Introduction . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: General Arrangement and Preparation for
Electrophysiological Recording and Data Acquisition . . . . .
Subprotocol 2: Extracellular Recording . . . . . . . . . . . . .
Subprotocol 3: Intracellular Recording with Sharp Electrodes . . .
Subprotocol 4: Intracellular Recording and Tracer Injection . . . .
Summary and Conclusions . . . . . . . . . . . . . . . . . .
Supplier List. . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .

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128
134
146
158
165
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168

Chapter 6
Electrical Activity of Individual Neurons: Patch-Clamp Techniques
Boris V. Safronov and Werner Vogel
Introduction . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . . . .
Applications. . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .

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173
176
180
187
188
189
191

Chapter 7
Microiontophoresis and Pressure Ejection
Peter M. Lalley
Introduction . . . . . . . . . . . .
Subprotocol 1: Microiontophoresis . . .
Subprotocol 2: Micropressure Ejection .
Comments . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . .
References . . . . . . . . . . . . .

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193
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207

209
209
209

Chapter 8
An Introduction to the Principles of Neuronal Modelling
Kenneth A. Lindsay, J.M. Odgen, David M. Halliday, Jay R. Rosenberg
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A Philosophy of Modelling . . . . . . . . . . . . . . . . . . . . . .

213
214

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Contents

Formulation of Dendritic Model . . . . . . . . . . . . . .
The Discrete Tree Equations . . . . . . . . . . . . . . . .
Formal Solution of Matrix Equations . . . . . . . . . . . .
Solution of the Discretised Cable Equations . . . . . . . . .
Generating Independent and Correlated Stochastic Spike Trains
Equivalent Cable Construction. . . . . . . . . . . . . . .
Generalized Compartmental Methods . . . . . . . . . . .
The Spectral Methodology . . . . . . . . . . . . . . . .
Spectral and Exact Solution of an Unbranched Tree. . . . . .
Spectral and Exact Solution of a Branched Tree . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
Notations and Definitions . . . . . . . . . . . . . . . . .
Appendix . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 9
In Vitro Preparations
Klaus Ballanyi
Introduction. . . . . . . . . . . . .
In Vitro Models . . . . . . . . . . .
En bloc Preparations . . . . . . . . .
Brain Slices . . . . . . . . . . . . .
Determinants of Ex Vivo Brain Function
Conclusions . . . . . . . . . . . . .
References . . . . . . . . . . . . .

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307
307
310
311
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324
325

Chapter 10
Culturing CNS Neurons: A Practical Approach to Cultured
Embryonic Chick Neurons
Åke Sellström and Stig Jacobsson
Introduction. . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . .

Troubleshooting . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . .

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327
329
329
330
333

334
334
337
337
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338

Chapter 11
Neural Stem Cell Isolation, Characterization and Transplantation
Jasodhara Ray and Fred H. Gage
Introduction. . . . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . . .

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340
341
343
351
352
354

IX


X

Contents

Applications.
References .
Suppliers . .
Abbreviations
Glossary . .

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357
357
359
360
360

Chapter12
In Vitro Reconstruction of Neuronal Circuits: A Simple Model System Approach
Naweed I. Syed, Hassan Zaidi and Peter Lovell
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

361
362
364
368
371
376

Chapter 13
Grafting of Peripheral Nerves and Schwann Cells into
the CNS to Support Axon Regeneration
Thomas J. Zwimpfer and James D. Guest
Introduction . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: Harvest and Implantation of PN Grafts into the CNS
Subprotocol 2: Schwann Cell Guidance Channels. . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .

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379
383
392
406

Chapter 14
Cell and Tissue Transplantation in the Rodent CNS
Klas Wictorin, Martin Olsson, Kenneth Campbell
and Rosemary Fricker
Introduction . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: Dissection of Embryonic/Fetal CNS Tissue.
Subprotocol 2: Preparation of Tissue/Cells . . . . . . .
Subprotocol 3: Transplantation into Adults . . . . . . .
Subprotocol 4: Transplantation into Neonates . . . . . .
Subprotocol 5: Transplantation into Embryos . . . . . .
References . . . . . . . . . . . . . . . . . . . . .

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Chapter 15
Histological Staining Methods
Robert W. Banks
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: Architectonics . . . . . . . . . . . . . . . . . . . .
Subprotocol 2: Hodology . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 3: Histochemical Methods: Neurochemistry and
Functional Neurohistology, Including the Molecular Biology of Neurons
Subprotocol 4: Silver-Impregnation Methods in the
Peripheral Nervous System . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Contents

Chapter 16
Optical Recording from Populations of Neurons in Brain Slices
Saurabh R. Sinha and Peter Saggau
Introduction. . . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . .

Suppliers . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . .

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469
471
471
477
480
484
485
486
486

Chapter 17
Recording of Electrical Activity of Neuronal Populations
Hakan Johansson, Mikael Bergenheim, Jonas Pedersen and
Mats Djupsjöbacka
Introduction. . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: Multi-Unit Recording . . . . . . . . . . . . .
Subprotocol 2: Examples of Analysis and Results . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . .

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487
488
496
500
501
502

Chapter 18
Time and Frequency Domain Analysis of Spike Train and Time Series Data
David M. Halliday and Jay R. Rosenberg
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 1: Time Domain Analysis of Neuronal Spike Train Data . . . . . . . .
Part 2: Frequency Domain Analysis. . . . . . . . . . . . . . . . . . .

Part 3: Correlation Between Signals. . . . . . . . . . . . . . . . . . .
Part 4: Multivariate Analysis . . . . . . . . . . . . . . . . . . . . . .
Part 5: Extended Coherence Analysis – Pooled Spectra and Pooled Coherence
Part 6: A Maximum Likelihood Approach to Neuronal Interactions . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

503
505
510
516
527
530
533
539
539
541

Chapter 19
Information-Theoretical Analysis of Sensory Information
Yoav Tock and Gideon F. Inbar
Introduction. . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . .
The Neural Code . . . . . . . . . . . . . . . . . . .
Basics of Information Theory . . . . . . . . . . . . .
Random Continuous Time Signals . . . . . . . . . . .
Information Transmission with Continuous Time Signals .
Information Transmission – The Method . . . . . . . .
Summary – the Practical Procedure . . . . . . . . . .

Upper Bound to Information Rate and Coding Efficiency .
The Muscle Spindle: Experimental and Simulation Results

545
547
547
551
555
557
560
563
564
566

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XII

Contents

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chapter 20
Information-Theoretical Analysis of Small Neuronal Networks
Satoshi Yamada
Introduction . . . . . . . . . . . . . . . . . . . . .
Theory . . . . . . . . . . . . . . . . . . . . . . . .
Procedures and Results . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . .

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573
573
579
585
587
588

Chapter 21
Linear Systems Description
Amir Karniel and Gideon F. Inbar
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 1: Static Linear Systems. . . . . . . . . . . . . . . . . . . . . .
Part 2: Dynamic Linear Systems . . . . . . . . . . . . . . . . . . . .
Part 3: Physical Components of Linear Systems . . . . . . . . . . . . .
Part 4: Laplace and Z Transform . . . . . . . . . . . . . . . . . . . .
Part 5: System Identification and Parameter Estimation. . . . . . . . . .
Part 6: Modeling The Nervous System Control . . . . . . . . . . . . . .
Part 7: Modeling Nonlinear Systems with Linear Systems Description Tools .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

589

592
594
596
605
611
615
618
624
624

Chapter 22
Nonlinear Analysis of Neuronal Systems
Andrew S. French and Vasilis Z. Marmarelis
Introduction . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . .

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571

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627
628
629
637
639

Chapter 23
Dynamical Stability Analyses of Coordination Patterns
David R. Collins and Michael T. Turvey
Introduction . . . . . . . . . . . . . . . . . .
Part 1: Stationary Methods . . . . . . . . . . . .
Part 2: Nonstationary Analyses . . . . . . . . . .
Part 3: Phase Space Reconstruction . . . . . . . .
Postscript . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . .
Glossary . . . . . . . . . . . . . . . . . . . .

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641
641
654
660
663
665
666
667

Chapter 24
Detection of Chaos and Fractals from Experimental Time Series
Yoshiharu Yamamoto
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .

669


Contents

Part 1: Theoretical Backgrounds
Part 2: Procedure and Results .
Concluding Remarks . . . . .
References . . . . . . . . .

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669
675

685
686

Chapter 25
Neural Networks and Modeling of Neuronal Networks
Bagrat Amirikian
Introduction. . . . . . . . . . . . . . . . . . .
Network Architecture and Operation . . . . . . . .
Model Neurons, Connections and Network Dynamics
Learning and Generalization. . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .

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689
691
692
697
703

Chapter 26
Acquisition, Processing and Analysis of the Surface Electromyogram
Björn Gerdle, Stefan Karlsson, Scott Day and Mats Djupsjöbacka
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 1: Muscle Anatomy and Physiology. . . . . . . . . . . . . . . .
Part 2: Signal Acquisition and Materials . . . . . . . . . . . . . . . .
Part 3: Registration Procedures . . . . . . . . . . . . . . . . . . .
Part 4: Signal Processing . . . . . . . . . . . . . . . . . . . . . .
Part 5: Results . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 6: Noise, Artifacts and Cross-talk . . . . . . . . . . . . . . . .
Part 7: Special Applications . . . . . . . . . . . . . . . . . . . . .
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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705
706
716
721
722
728
733
735
743
745
752
753

Chapter 27
Decomposition and Analysis of Intramuscular Electromyographic Signals
Carlo J. De Luca and Alexander Adam
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . .

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759
760
760
767
772
774
775
776

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777
777
778

779
785
786
786

Chapter 28
Relating Muscle Activity to Movement in Animals
Gerald E. Loeb
Introduction. . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . .

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XIII


XIV

Contents

Chapter 29
Long-term Cuff Electrode Recordings from Peripheral Nerves
in Animals and Humans
Thomas Sinkjær, Morten Haugland, Johannes Struijk and Ronald Riso
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

787
788
798
799

Chapter 30
Microneurography in Humans
Mikael Bergenheim, Jean-Pierre Roll and Edith Ribot-Ciscar
Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comments . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . .
Supplier . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . .

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803
804
808
814
816
817
819
819

Chapter 31
Biomechanical Analysis of Human and Animal Movement
Walter Herzog
Introduction . . . . . . . . . . . . . . . . . . . . . .
Part 1: External Biomechanics . . . . . . . . . . . . . . .
External Force Measurements Using Force Platforms . . .

External Movement Measurements Using High-Speed Video
Surface Electromyography . . . . . . . . . . . . . . .
Part 2: Internal Biomechanics . . . . . . . . . . . . . . .
Muscle Force Measurements . . . . . . . . . . . . . .
Joint Contact Pressure Measurements . . . . . . . . . .
Movement Measurements . . . . . . . . . . . . . . .
Theoretical Determination of Internal Forces . . . . . . .
Future Considerations . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .

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821
822

822
824
825
830
831
834
836
838
844
845

Chapter 32
Detection and Classification of Synergies in Multijoint Movement
with Applications to Gait Analysis
Christopher D. Mah
Introduction . . . . . . . . . . . . . . . . . . . . . .
Dimensionality and Data Reduction . . . . . . . . . . . .
Principal Component Analysis Made Simple . . . . . . . .
Application to Gait Analysis . . . . . . . . . . . . . . . .
Force Fields and the Problem of Degrees of Freedom . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .

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849
850
850
854
864
866

Chapter 33
Magnetic Stimulation of the Nervous System
Peter H. Ellaway, Nicholas J. Davey and Milos Ljubisavljevic
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .

869


Contents

Subprotocol 1: Apparatus and Mechanisms . . . . .
Subprotocol 2: EMG Recording and Analysis Protocol
Applications . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .

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870
874
876
889

Chapter 34
In-vivo Optical Imaging of Cortical Architecture and Dynamics
Amiram Grinvald, D. Shoham, A. Shmuel, D. Glaser, I. Vanzetta,
E. Shtoyerman, H. Slovin, C.Wijnbergen, R. Hildesheim and A. Arieli
General Introduction . . . . . . . . . . . . . . . . . . . . . . . . .
Part 1: Optical Imaging Based on Intrinsic Signals . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 2: Voltage-sensitive Dye Imaging in the Neocortex . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Part 3: Combining Optical Imaging with Other Techniques . . . . . . . .
Targeted Injection of Tracers into Pre-Defined Functional Domains . . .
Electrical Recordings from Pre-Defined Functional Domains . . . . . .

Combining Micro-Stimulation and Optical Imaging . . . . . . . . . .
Part 4: Comparison of Intrinsic and Voltage-sensitive Dyes . . . . . . . .
Optical Imaging . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

893
896
896
901
930
930
942
957
957
958
959
960
960
960
961

Chapter 35
Electroencephalography
Alexey M. Ivanitsky, Andrey R. Nikolaev and George A. Ivanitsky
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . .
Subprotocol 1: EEG Recording . . . . . . . . . . . . . . . . . . .
Subprotocol 2: EEG Signal Analysis. . . . . . . . . . . . . . . . .
Subprotocol 3: Secondary EEG Analysis . . . . . . . . . . . . . . .
Subprotocol 4: Presentation of Results . . . . . . . . . . . . . . .

Advantages of the EEG in Comparison with High-Technology
Brain Imaging Methods . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 36
Modern Techniques in ERP Research
Daniel H. Lange and Gideon F. Inbar
General Introduction . . . . . . . . . . . . . . . . . . . .
Part 1: Review of EP Processing Methods . . . . . . . . . . . .
Processing methods . . . . . . . . . . . . . . . . . . . . .
Part 2: Extraction of Trial-Varying EPS . . . . . . . . . . . .
Layer 1 – Unsupervised Learning Structure . . . . . . . . .
Layer 2: Decomposition of EP Waveform . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .

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971
974
976
988
989

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991

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997
998
999
1001
1003
1012
1021
1021
1022

XV


XVI

Contents

Chapter 37
Magnetoencephalography
Volker Diekmann, Sergio N. Erné and Wolfgang Becker
Introduction . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . .
Applications. . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . . . . . .

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1025
1030
1034
1045
1048
1050
1051
1054

Chapter 38
Magnetic Resonance Imaging of Human Brain Function
Jens Frahm, Peter Fransson and Gunnar Krüger
Introduction . . . . . . . . . . . . . . . . . .
Technical Aspects of MRI Data Acquisition . . . . .
Data Evaluation and Visualization . . . . . . . . .
Physiologic Aspects of Brain Activation . . . . . . .
Paradigm Design . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .

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1055
1057
1063
1069
1077
1081

Chapter 39
Positron Emission Tomography of the Human Brain
Fabrice Crivello and Bernard Mazoyer
Introduction . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . .
Procedure . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . .
Applications. . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . .

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1083
1087
1088
1091
1093
1095
1096
1096
1097

Chapter 40
Magnetic Resonance Spectroscopy of the Human Brain

Stefan Blüml and Brian Ross
Introduction . . . . . . . . . . . . . . . . . .
Technical Requirements and Methods . . . . . . .
Applied MRS – Single-Voxel 1H MRS . . . . . . . .
Results: Neurospectroscopy . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . .
Glossary . . . . . . . . . . . . . . . . . . . .

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1099

1106
1115
1119
1137
1139
1142

Chapter 41
Monitoring Chemistry of Brain Microenvironment:
Biosensors, Microdialysis and Related Techniques
Jan Kehr
General Introduction . . . . . . . . . . . . . . . . . . . . . . . . .

1149

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Contents

Part 1: General Methods . . . . . . . . . . . . . . . . . . . . .
Stereotaxic Surgery on Small Rodents . . . . . . . . . . . . . .

Microdialysis Experiments on Awake Rats . . . . . . . . . . . .
Part 2: Implantable Sensors . . . . . . . . . . . . . . . . . . . .
Potentiometric Electrodes . . . . . . . . . . . . . . . . . . .
Measuring Extracellular K+ Ions by ISM . . . . . . . . . . . . .
Amperometric Electrodes. . . . . . . . . . . . . . . . . . . .
Measurement of Dopamine by Chronoamperometry . . . . . . . .
Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . .
Biosensor for Glucose . . . . . . . . . . . . . . . . . . . . .
Optical Sensors . . . . . . . . . . . . . . . . . . . . . . . .
Part 3: Continuous Sampling Devices . . . . . . . . . . . . . . . .
Cortical Cup Technique. . . . . . . . . . . . . . . . . . . . .
Push-pull Cannula . . . . . . . . . . . . . . . . . . . . . . .
Microdialysis . . . . . . . . . . . . . . . . . . . . . . . . .
Determination of Dopamine Release by Microcolumn Liquid
Chromatography with Electrochemical Detection (LCEC) . . . .
Determination of Serotonin in Microdialysis Samples by LCEC . . .
Determination of Aspartate and Glutamate in Microdialysis Samples
by HPLC with Fluorescence Detection . . . . . . . . . . . . .
Determination of GABA in Microdialysis Samples by HPLC
with Fluorescence (FL) and Electrochemical (EC) Detection . . .
Determination of Physiological Amino Acids in Microdialysis
Samples by Microcolumn HPLC with Gradient Elution and
Fluorescence (FL) Detection . . . . . . . . . . . . . . . . .
Determination of Acetylcholine in Microdialysis Samples by
Microbore Liquid Chromatography/Electrochemistry on
Peroxidase Redox Polymer Coated Electrodes. . . . . . . . . .
Microdialysis in the Human Brain . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Chapter 42
Invasive Techniques in Humans: Microelectrode Recordings and Microstimulation
Jonathan Dostrovsky
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1199
Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209
Suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1209
Chapter 43
Psychophysical Methods
Walter H. Ehrenstein and Addie Ehrenstein
Introduction. . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . .
Methods and Procedures . . . . . . . . . . .
Experimental Examples . . . . . . . . . . . .
Concluding Remarks . . . . . . . . . . . . .

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XVIII

Contents

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suppliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1238
1240

Chapter 44
Analysis of Behavior in Laboratory Rodents
Ian Q. Whishaw, Forrest Haun and Bryan Kolb
Introduction . . . . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . . . . . .
The Neurobehavioral Examination . . . . . . . .
Comments: Generalizing from Behavioral Analysis
References . . . . . . . . . . . . . . . . . .

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Chapter 45
Data Acquisition, Processing and Storage
M. Ljubisavljevic and M.B. Popovic
Introduction . . . . . . . . . . . . . . . . .
Outline . . . . . . . . . . . . . . . . . . . .
Part 1: Signal and Noise. . . . . . . . . . . . .
What is a Signal? . . . . . . . . . . . . . .
Noise . . . . . . . . . . . . . . . . . . .
Part 2: Signal Conditioning . . . . . . . . . . .
Amplification and Amplifiers . . . . . . . . .
Fundamentals of Filtering and Filters . . . . .
Part 3: Analog-to-Digital Conversion (Digitization)
Digital or Analog Processing? . . . . . . . . .
A/D Conversion . . . . . . . . . . . . . . .
Implementations . . . . . . . . . . . . . .
Part 4: Data Processing and Display . . . . . . .
Data Processing . . . . . . . . . . . . . . .
Data Display . . . . . . . . . . . . . . . .
Part 5: Storage and Backup . . . . . . . . . . .
Concluding Remarks . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . .
Glossary . . . . . . . . . . . . . . . . . . .

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1307
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Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1313

Chapter 1

Cytological Staining Methods
R.W. Banks

■

Introduction

General Introduction
Neurohistology is perhaps traditionally thought of as supplying information only about
the spatial or structural aspects of the neuron; however, it is my intention in the present
chapter and its companion (Chapter 15 in Sect.II) to present as far as possible a unified
view of neurohistology as a set of related problems centred on the relationship between
structure and function of nerve cells. Such problems are not unique to the subject, but
in the context of neurohistology they are uniquely complex. Neurons, as cells, are unrivalled in diversity of type, and other kinds of cells rarely match any neuron in the complexity of their spatio-temporal properties or in the range of genes expressed. The status
of neurohistology as a recognisable discipline is therefore dependent on these properties of nerve cells and nervous tissue, and its history is largely one of the development
of methods aimed at overcoming the difficulties presented by them. Of course, recognisable disciplines need not necessarily have sharp boundaries and it is perhaps already
apparent that I intend to take a fairly relaxed view as to what constitutes neurohistology.
The essential criteria are whether the investigation involves the nervous system and
whether it uses microscopy. Beyond those, it is a matter of taste where macroscopically
neuroanatomy and neuroimaging give way to neurohistology, and microscopically neurohistology gives way to cellular and molecular biology.
Within the discipline, boundaries must be arbitrary and harder to defend. The division into topics that can be described as cytological (this chapter) and histological sensu
stricto (Chap. 15) creates such a boundary that is more convenient than real; many of the
techniques covered will be applicable in either area. In a similarly cavalier fashion, I
shall gather several specific techniques under rather broad and by no means exclusive
headings so as to emphasize common purposes of the often disparate methods. It might
be argued that the overall purpose is to provide as close as possible a description of neurons and nervous systems in their living state. Clearly neurohistology alone is incapable
of reaching that end, but it is essential to its attainment. What is certain is that good neurohistology requires more than the mechanical application of various technical procedures aimed at a static description of the microscopic appearance of the nervous system.

I suggest that what is indeed essential is the intelligent and informed combination of
structural and functional elements, or at least of the interpretation of structure in functional terms. I hope to demonstrate the truth of this by placing several techniques in the
context of specific problems in neuroscience. Any protocols and practical advice given
in my chapters will be contained in these case-studies. Equally, if not more, important
will be the intervening sections in which the evolutionary development and theoretical
R. W. Banks, University of Durham, Department of Biological Sciences, South Road, Durham, DH1
3LE, UK (phone: +44-191-374-3354; fax: +44-191-374-2417; e-mail: )


2

R.W. Banks

backgrounds of various methods are briefly considered in order to highlight their possibilities and limitations.
The Beginnings of Neurohistology
“Often, and not without pleasure, I have observed the structure of the nerves to be composed of very slender vessels of an indescribable fineness, running lengthwise to form
the nerve” (Leeuwenhoek, 1717).
Leeuwenhoek's account of his observations on the spinal nerves of cows and sheep,
almost certainly the earliest histological description of a part of the vertebrate nervous
system, already carries an implicit functional interpretation, for there can be little doubt
that his use of the term 'vessels' is a reference to the hydraulic model of neural function
proposed by Descartes (1662). His observations are all the more remarkable in view of
the necessary limitation of his microtechnique to dissection with fine needles, freehand
sections made with a “little knife … so sharp that it could be used for shaving”, and
probably air-drying for mechanical stabilisation of tissue. Similar methods remained in
virtually exclusive use for the next hundred years or so until Purkinje, who was, significantly, professor of physiology at Wroclaw (Breslau), started hardening tissue in alcohol (spirits of wine), cutting sections with his home-made microtome and staining
them with various coloring agents including indigo, tincture of iodine and chrome salts
(Phillips, 1987).
These improvements enabled Purkinje to anticipate by two years Schwann's extension of the cell theory to animals by describing nucleated “corpuscles” from a variety of
tissues including brain and spinal cord (Hodgson, 1990). But new techniques rarely displace older ones entirely, and it was a combination of serial sectioning and microdissection with needles (teasing) of chromic-acid- or potassium-dichromate-fixed tissue that

allowed Deiters (1865) to demonstrate what had eluded Purkinje: the extension of the
nerve cell body in dendrites (“protoplasmic processes”) of progressively finer divisions,
and the continuity of the single axon with the cell body also.
The problem of how to study the contextual relationships of nerve cells and their
processes in situ was soon to be spectacularly solved by Golgi (1873) with “la reazione
nera”, in which the use of silver nitrate was inspired, no doubt, by contemporary experiments in photography. Cajal took those contextual relationships to their classical limits
in his magisterial exploitation of Golgi's technique (Cajal, 1995). He espoused Waldeyer's (1891) neuron doctrine in a modified and essentially modern form centred on his
concept of the dynamic polarization of the neuron (Cajal, 1906). Yet his insistence on the
separate identity of individual neurons had to await half a century and the development
of a new technology, electron microscopy, for its confirmation (Palade and Palay, 1954).

Subprotocol 1
Fixation, Sectioning and Embedding
Part 1: General
Histological and
Cytological Methods

■■

Introduction

“The principles of biological microtechnique may perhaps be reduced to one – the principle that when we make a microscopical preparation of any sort, we ought to try to understand what we are doing…” (Baker, 1958).
The physico-chemical, as well as the spatio-temporal, properties of living nervous
tissue are not amenable to much histological work so it is generally necessary to modify


1 Cytological Staining Methods

them in various ways in order to produce a usable specimen. In this section we shall
look at some preparative techniques that are basic to much histological study and that

may be conveniently grouped under the heading of fixation, sectioning and embedding.
Since they are not specific to neurohistology, the treatment of these techniques will be
brief. It is particularly instructive, however, to consider them in the context of their historical development, which, together with that of the various methods of dyeing and
staining, is typically a continuing story of progressive problem-solving by eclectic use
of technologies derived from contemporary advances in other fields, principally chemistry and physics.
The natural products ethanol, in the form of spirits of wine, and acetic acid, in the
form of vinegar, have always been used in the preservation of organic material, but only
the first was commonly used in early microtechnique. This is because what was sought
was hardening of the tissue, enabling it to be cut into thin sections, and of the two agents
only ethanol had the desired effect (Baker, 1958). Hardening by the purely physical
method of freezing was also possible and was used by Stilling in 1842 (cited by Cajal,
1995) to prepare sections of brain and spinal cord. With the development of inorganic
chemistry in the late 18th and early 19th centuries several substances were found to
harden animal tissues sufficiently to allow them to be sectioned, and their particular effects were exploited either as single hardening agents or in various mixtures, many of
which continue in use to the present day. The most important are
– mercuric chloride,
– osmium tetroxide,
– chromium trioxide and
– potassium dichromate,
all of which were in use in microtechnique by about 1860. The subsequent rise of organic
chemistry led to the introduction of the remaining classical 'hardening agents'
– picric acid (2,4,6-trinitrophenol) and
– formaldehyde (methanal),
the latter as late as 1893 and only after its previous use as a disinfectant (Baker, 1958).
As infiltration and embedding of tissue in solid media became standard practice (see
below), the hardening property of these substances lost its relevance and attention could
then centre on their role in fixation of the non-aqueous components of the cell. A cell
that has been killed or rendered non-viable by chemical action is necessarily artefactual
to a greater or lesser extent when compared to the living cell. The amount of artefactual
distortion of some feature of interest in the living state can be taken as a measure of the

quality of fixation in that respect, whether it be fine structure, enzyme activity, lipid extraction, or whatever. Moreover, in view of the physico-chemical complexity of the cell,
it is not surprising that any single substance combines both good and poor fixative qualities when assessed on different criteria. To some extent the deficiencies of one fixative
can be counteracted by the complementary benefits of another when used in combination, either sequentially or together. This is necessarily an empirical process, the results
of which are in general unpredictable, but it is an approach that has led to the introduction of many important fixatives and fixation procedures.
As an example, we shall follow the development of one of the most widely used procedures, involving a combination of aldehydes with osmium tetroxide, the version in
current use in Durham being given in example 2 below. Although osmium tetroxide
rapidly destroys enzyme action, Strangeways and Canti (1927) found that it very faithfully preserves the fine structure of the cell as revealed by dark-ground microscopy.
Fine-structure preservation is critically important for most electron microscopy because of the very high spatial resolution that it provides, so in the first two or three decades of electron microscopy osmium tetroxide was widely used as the only fixative, typ-

3


4

R.W. Banks

ically as a 1 % solution in 0.1 M phosphate or cacodylate buffer at about pH 7.3 (Glauert,
1975). It had the additional advantage of imparting electron density to those components of the specimen that reacted with the osmium tetroxide, and thus increasing image contrast. But the consequent loss of cytochemical information, especially about the
localisation of enzyme activity which was preserved by formalin fixation (Holt and
Hicks, 1961), prompted Sabatini, Bensch and Barrnett (1963) to assess various aldehydes for their ability to preserve cellular fine structure better than formalin while retaining high levels of enzymic action. Of the nine aldehydes assessed, including formaldehyde and acrolein, the best results were obtained with glutaraldehyde (pentane 1,5dial, C5H8O2), which was used as a 4–6.5 % solution in 0.1M phosphate or cacodylate
buffer at pH 7.2. Its superior performance is usually attributed to its relatively small size,
enabling rapid penetration, and its two aldehyde groups, which are thought to allow glutaraldehyde to form stable cross-linkages between various molecules, especially proteins. Moreover, when combined with a second fixation with osmium tetroxide, fine
structural preservation was as good as with osmium tetroxide alone even if the blocks
had been stored 'for several months' before the second step. In an early modification of
the procedure Karnovsky (1965) advocated the inclusion of 4 % formaldehyde in the
primary fixative, on the basis that formaldehyde, being much smaller than glutaraldehyde and with only a single aldehyde group, would penetrate tissue more rapidly, stabilizing it sufficiently long for glutaraldehyde to act and thus permit the fixation of larger
blocks. Whether or not this is a correct explanation for the action of the aldehyde mixture, the fixative has become probably the most widely used for electron microscopy,
though the strength is usually reduced by half, apparently prompted by considerations
of the osmotic potential of the fresh solution.
Ever since Leeuwenhoek wielded his “little knife” the importance of sectioning in microtechnique has been clear and, as we have seen, fixation, whether chemical or physical, was initially developed to harden tissue sufficiently for it to be sectioned. Sectioning

is necessary not only to make specimens suitably transparent to photons or electrons,
but also to reduce the spatial complexity of a specimen to convenient limits. Analysis
may be greatly facilitated, and frequently is only made possible at all, by selecting section thickness and orientation appropriate to the scale of spatial structure required of
the specimen. The 3-dimensional structure of components larger than the section
thickness can then be recovered by reconstruction from serial sections. But in neurohistology, until the discovery of the Golgi method, the complex shapes of complete nerve
cells could not easily be traced in sections, and microdissection with needles of the fixed
material remained in widespread use throughout much of the latter half of the 19th century. Perhaps because it is incompatible with microdissection, embedding tissue in a
medium that could itself be hardened to give mechanical support during sectioning appears to have been adopted relatively late into neurohistology. Embedding, when first
used, was just that; the tissue was scarcely, if at all, infiltrated by the medium, but merely
surrounded by it in order to retain the relative positions of separate components. Large,
gel-forming molecules such as collodion (nitro-cellulose) and gelatine have been used
since the earliest days of embedding when, it is no coincidence, both of these substances
were also being used in the production of the first photographic emulsions. A low viscosity form of nitro-cellulose (“celloidin”) eventually became widely used in neurohistology, particularly when sections greater than about 20 µm in thickness were required.
According to Galigher and Kozloff (1964), paraffin wax, a product of the then emergent
petroleum industry, was first introduced as a purely embedding medium by Klebs in
1869 but almost immediately (1871) an infiltration method, essentially similar to that in
current use, was devised by Born and Strickler. Neurohistologists do not appear to have
taken up paraffin embedding immediately, but certainly by the end of the last decade of
the 19th century it was being routinely used by them both for thin (2 µm) and serial sec-


1 Cytological Staining Methods

tions. Biological electron microscopy necessitated the use of new embedding media, opportunely provided by the plastics industry from the 1940s onwards. Glauert (1975)
gives a very full account of them: the most widely used are the epoxy resins. Although
glutaraldehyde fixation and resin embedding were developed to meet the needs of electron microscopy, the quality of their histological product is such that light microscopy
has also benefited, as the following case study will show.

Example 1: The Primary Ending of the Mammalian Muscle Spindle – A Case Study
of the Use of 1 µm Thick Serial Sections in Light Microscopy

■■

Materials

Muscle spindles partially exposed by removal of overlying extrafusal muscle fibres for
direct observation in the tenuissimus muscle of the anaesthetized cat.

■■

Procedure

Fixation
1. 5% glutaraldehyde in 0.1M sodium cacodylate buffer pH 7.2 for 5 min. in situ. [Glutaraldehyde is usually obtained as a 25% solution. It polymerizes easily and so should
be kept below 4 °C until required.]
2. The same fixative for 4–14 days after excision of portions of muscle each about 10
mm long containing one spindle. [Variation in total fixation time was due to postal
despatch between laboratories. There was no obvious difference in the quality of fixation of muscles fixed for different times.]
3. Washed in the buffer for 30 min.
4. 1 % osmium tetroxide, buffered, for 4 hours. [Osmium tetroxide penetrates tissue
very slowly, but the tenuissimus muscle is typically less than 1 mm thick and could
be adequately fixed in this time. OsO4 is made up as a 2 % stock solution and kept
refrigerated in a sealed bottle. The working strength fixative is made by diluting the
stock solution with an equal quantity of 0.2M sodium cacodylate buffer.]

Dehydration and Embedding
1. Dehydrated in a graded series of ethanol – 70 %, 95 %, 100 % (twice) – for 10 min each
at ambient temperature.
2. 50:50 mixture of ethanol and propylene oxide (1,2-epoxy propane) for 15 min. [Propylene oxide is usually included as an intermediate solvent and is analogous to the
use of “clearing agents” in paraffin embedding procedures. The refractive index of
most clearing agents is similar to that of dehydrated proteins and other cellular components; they were originally used to make fixed tissue transparent, hence the name

which has persisted even though they rarely have that function today. For alternative
dehydration methods see Glauert (1975).]
3. Propylene oxide for 15 min.
4. 50:50 mixture of propylene oxide and Epon (complete except for the accelerator) left
overnight in an unstoppered container in a fume cupboard. [Evaporation of the propylene oxide results in a very well infiltrated block.]

5


6

R.W. Banks

5. Drained excess infiltration medium blotted; transfered to fresh complete Epon.
6. Flat-embedded in an aluminium foil mould; polymerized for 12 hr at 45 °C and 24 hr
at 60 °C.
Sectioning and Staining
1. Sections cut manually at 1 µm thickness in groups of 10 on an ultramicrotome with
conventional glass knives. [If necessary, the sections can be spread on the water surface using chloroform vapor from a brush held close to them, or by radiant heat from
an electrically heated filament. Glass knives need to be replaced regularly; use of a
mechanical knife-breaker ensures close similarity of shape in successive knives. Accurate positioning to within a few µm of a new knife with respect to the block face
can be achieved by lighting the back of the knife, such that the gap between knife
edge and block face appears as a bright line.]
2. Coverslips [50x22 mm is a convenient size] scored with a diamond marker and broken into strips about 3 mm wide were used to collect the sections directly from the
water trough of the knife by immersing one end of the strip under the surface of the
water (Fig. 1.A). [The sections, either as a ribbon or individually, are easily guided
with a toothpick-mounted eyelash onto the strip, which is held in watchmakers’ forceps. A simple technique to ensure adequate adhesion of the sections is to draw one
face of the strip of coverslip over the tip of the tongue and allow it to dry.]
3. The back of each strip was dried with a soft tissue, leaving the sections free-floating
on a small drop of water on the front of the strip.

4. The sections were thoroughly dried onto the strip using a hot plate at about 70° C.
[Best done by keeping a glass slide permanently on the hot plate and placing the
strips onto the slide (Fig. 1.B).]
5. Stained with toluidine blue (Fig. 2.A) and pyronine (Fig. 2.B) at high pH by placing a
drop of the stain on the sections and heating until the stain starts to dry at the edge

Fig. 1. Stages in the preparation, staining and mounting of serial, 1 µm thick, epoxy resin-embedded sections. A: A sort ribbon of sections is guided onto a strip of glass cut from a coverslip, using
an eyelash mounted on a toothpick. The strip of coverslip is held in watchmakers’ forceps. B: The
back of the coverslip is dried using a soft tissue, leaving the ribbon of sections free-floating on a
drop of water on the front of the coverslip, which is then placed on a glass slide on a hot-plate to
flatten the sections and dry them. The same arrangement is used to stain the sections as described
in the text. C: Several strips are mounted under a single large coverslip and the slide is labelled to
indicate the order of the sections.


1 Cytological Staining Methods

Fig. 2. Structural formulae of various dyes and chromogens mentioned in the text. A: Toluidine
blue. B: Pyridine. C: Lucifer yellow. D: JPW1114. E: Calcium Green-1. F: FM1–43. G: DiA.

of the drop. Washed with water and differentiated with 95 % ethanol. [Staining solution is made by dissolving 0.1g toluidine blue + 0.05g pyronine + 0.1g borax (sodium
tetraborate) in 60 ml distilled water, and should be filtered periodically.]
6. Dried on the hot plate and mounted using DPX (Distrene-Plasticizer-Xylene). [5
strips each with, say, 10 sections can be conveniently mounted under a single 50x22
mm coverslip (Fig. 1.C). Of course, the strips should be mounted with the sections
uppermost.]

■■

Results

The primary ending of a tenuissimus muscle spindle in the cat occupies about 350 µm of
the mid portion of the spindle and typically requires some 50 serial, 1 µm, longitudinal
sections for its complete examination. The ending is generally considered to comprise
the expanded sensory terminals of a single group Ia afferent nerve fibre, together with
the system of preterminal branches, both myelinated and unmyelinated, that serve to
distribute the terminals among the several intrafusal muscle fibres. There are commonly six intrafusal fibres of three different kinds. Figure 3 shows a selection of micrographs
taken with a x100 oil-immersion plan achromat objective (N.A. 1.25); structures consid-

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Fig. 3. Examples of results of serial-section analysis using 1 µm epoxy resin-embedded material.
A-F: Longitudinal sections taken in the primary sensory region of a mammalian muscle spindle.
This spindle contained 5 intrafusal muscle fibres, part of only one of which (a bag1 fibre) is shown.
The sections are serial except that one section has been omitted between A and B, and one between D and E. Scale bar = 10 µm. G: Contour reconstruction of the sensory terminals on the bag
fibre shown in A-F. Scale bar = 50 µm. mpt, myelinated preterminal branch; n, nucleus; pt, unmyelinated preterminal branch; t, sensory terminal.

erably less than 0.5 µm in size are easily resolved. Each field of view covers a distance of
a little over 100 µm in the long axis of the spindle. The most prominent structure visible
in the micrographs is the central portion of one of the intrafusal fibres, specifically the
bag1. In the region of the primary ending, the sarcomeres of the intrafusal fibres are almost entirely replaced by a collection of nuclei (Fig. 3C, n). Projecting from the surface
of the fibre are the sensory terminals (Fig. 3F, t). These can be traced between the sections as can portions of the myelinated (Fig. 3B, mpt) and unmyelinated (Fig. 3E, pt)
preterminal branches. The dark structures within the terminals are mostly mitochondria. Several accessory, fibroblast-like cells are also visible forming a sheath around the
bag1 fibre. A contour line reconstruction of this part of the sensory ending on the bag1
fibre, based on these and other intervening sections, is shown in Figure 3G. A 3-dimensional reconstruction of the complete ending was published by Banks in 1986. A similar

1 Cytological Staining Methods

serial-section analysis was recently used by Banks et al. (1997) in a correlative histophysiological study of multiple encoding sites and pacemaker interactions in the primary ending.

Subprotocol 2
Ultrastructure
■■

Introduction

“The dimensions of the [synaptic] cleft are now known and its detection has led many,
perhaps rather hastily, to consider the neuron (discontinuity) versus the reticular controversy (transynaptic cytoplasmic continuity) to be ended.” (Gray, 1964).
The advent of the electron microscope removed the barrier to the study of so-called
ultrastructure, or spatial organisation, on a finer scale than the resolution of the light
microscope. It permitted not only synaptic clefts but also structures one or two orders
of magnitude smaller to be made visible in sections of biological material. The effect on
microtechnique was, however, more evolutionary than revolutionary except that observation of living cells and tissues is scarcely possible with the electron microscope. It
might be supposed that without the possibility of direct comparison with living cells the
quality of ultrastructural fixation could only be assessed subjectively, but physical fixation by rapid freezing is entirely feasible (see, for example, Verna, 1983), thus providing
an objective standard for chemical methods. Freezing is not generally applicable mainly
because of its limitation to very small thicknesses of tissue in order to prevent ice crystal
formation (see, for example, Heuser et al., 1979), but it can be important or even essential in some studies and, with sufficient ingenuity, can be applied to relatively inaccessible structures within the brain (Van Harreveld and Fifkova, 1975). Despite the necessity
for freezing in some special applications, ultrastructural neurohistology depends overwhelmingly on chemical fixation, the techniques being derived directly from practices
and principles originally developed for light microscopy, as has been outlined above. In
this section we will look briefly at the role that fixation played in the functional interpretation of synaptic structure. Of primary importance here was the fixation of lipids
by OsO4, so preserving membrane structural integrity. This revealed not only the discontinuity of neurons at the synaptic cleft, but the presence of characteristic round vesicles of 30–50 nm diameter in the presynaptic terminals of synapses with chemically
mediated transmission (Gray, 1964). The vesicles were, of course, immediately recognised as being correlated with, or structurally equivalent to, the neurotransmitter quanta. The dynamic nature of vesicle recycling during transmission was clearly established,
among others, by Heuser and Reese (1973) who used immersion fixation of frog sartorius muscles, in a Karnovsky-type fixative, after various durations of nerve stimulation

and post-stimulation recovery.
Immersion fixation was initially used in ultrastructural studies on the CNS, but it was
necessary to cut the tissue finely in order to obtain high quality results, so the spatial
relationships of structures greater than about 1 mm in size were lost. Nevertheless, using this technique, Gray (see 1964 review) was able to identify two major types of central
synaptic structure and to recognize that they were differentially distributed on the dendrites and somata of the post-synaptic neurons. They were characterised by electrondense material associated with the post-synaptic membranes that were of greater (type
1) or lesser (type 2) thickness and extent, and their locations led Eccles (1964) to suggest
that they might correspond to excitatory and inhibitory synapses, respectively. Despite

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this and other important advances made using immersion fixation, the advantages of
perfusion in maintaining high quality fixation while retaining larger scale structural relationships in the CNS are such that it very soon became the method of first choice (Peters, 1970). At first veronal-acetate-buffered OsO4 was used (Palay et al., 1962) and subsequently aldehydes, with or without subsequent treatment with OsO4 (Karlsson and
Schultz, 1965; Schultz and Karlsson, 1965; Westrum and Lund, 1966). Immediately, and
virtually simultaneously, several authors described the occurrence of flattened presynaptic vesicles in some synapses. Uchizono (1965) was able to correlate round vesicles
with Gray type 1 and flattened vesicles with Gray type 2 synapses; utilizing the known
interneuronal origins and functional effects of certain synapses in the cerebellar cortex,
he further concluded that the first were excitatory and the second inhibitory. The identification was criticised on several grounds, not least that the flattening depended on aldehyde fixation which, if prolonged, would induce even the normally round vesicles to
flatten (Lund and Westrum, 1966; Walberg, 1966; Paula-Barbosa, 1975). However, many
later observations have substantially confirmed Uchizono’s conclusion so that what is
perhaps most interesting and instructive in this case is the usefulness of an incidental
product of fixation, an artefact that without the functional correlation would otherwise
be regarded as undesirable.

Example 2: Synapses of the Cerebellar Cortex
■■

Materials

Cerebellar cortex of the adult rat, anaesthetised with an intraperitoneal injection of sodium pentobarbitone.

■■

Procedure

Fixation
1. Systemic perfusion with a Karnovsky fixative, made up as follows (proportions given
for 100 ml)
– Solution A: 2g paraformaldehyde dissolved in 40 ml water at 60 °C, 1N NaOH added dropwise (2–6 drops) until the solution clears.
– Solution B: 10 ml of 25 % glutaraldehyde mixed with 50 ml of 0.2M sodium cacodylate buffer, pH 7.3.
Solutions are kept at 4 °C until required, then mixed to give 100 ml complete fixative. Techniques of perfusion vary considerably in their elaboration; the method I
have adopted is simple and seemingly reliable: it aims to minimise the time between induction of anaesthesia and effective fixation. A peristaltic pump [WatsonMarlow MHRE 200] is used to provide the driving force [many authors use hydrostatic pressure] and the fixative is introduced immediately the cannula is in place,
beginning at a relatively low speed until signs of onset of fixation are evident (limb
and tail extension), and progressively increasing the speed over the first few minutes. Fixation is continued for about 10 minutes, consuming about 500 ml fixative
for an adult rat. Pressure is not monitored.
The cannula is fashioned from a 21G hypodermic needle, angled at its mid-point
and ground transversely at the tip. A blob of epoxy resin applied to the tip before


1 Cytological Staining Methods

grinding facilitates introduction of the cannula into the ascending aorta via an incision in the apex of the left ventricle, and the cannula can then be clamped in
place using an artery clamp. During surgery and insertion of the cannula, the
pump is kept running at a very slow speed to prevent the introduction of air bubbles into the vasculature. As soon as the cannula is clamped in place, the wall of
the right atrium is cut and the pump speed is increased to initiate fixation.
2. After perfusion the brain is removed and placed in fresh fixative until required.

Blocks or slices are cut sufficiently thin (about 1 mm maximum) to allow penetration
of OsO4. The second fixation with OsO4, dehydration and embedding are as in example 1 above.

Sectioning
1. 1 µm thick sections for survey and alignment stained with toluidine blue and pyronine as in example 1.
2. Approx. 70–90 nm (silver-pale gold interference color) sections collected on formvar-coated grids and stained with lead citrate and uranyl acetate.

■■

Results

Several different kinds of synaptic association have been described in the cerebellar cortex, most synapses belonging to various kinds of axo-dendritic association. We shall
look briefly at three examples, one from the molecular layer and two from the synaptic
glomeruli of the granular layer. Synaptically, the molecular layer is dominated by the
parallel fibre-dendritic spine synapses between the granule and Purkinje cells. Figure
4A shows the outermost part of the molecular layer with the parallel fibres (pf) cut
transversely. Several parallel fibre-dendritic spine synapses (s) may be seen; note that
they are usually in close association with glial-cell processes whereas the parallel fibres
(the axons of granule cells) are clustered together and lack individual glial-cell sheaths.
A similar synapse is shown enlarged in Fig. 4B; it conforms to Gray's type 1, in particular there is a post-synaptic thickening and a sheet of extracellular material lies between
the pre- and post-synaptic membranes. The presynaptic vesicles (rv) are round in profile. Figure 4C shows a synaptic glomerulus of the granular layer. This is a complex
structure consisting of a central mossy-fibre rosette (mf) surrounded mainly by numerous profiles of granule-cell dendrites (gcd) and Golgi-cell axons. Mossy fibres and Golgi-cell axons both form axo-dendritic synapses with the granule-cell dendrites, but they
are of Gray types 1 and 2, respectively. A Golgi cell-granule cell synapse is shown in
greater detail in Fig. 4D. The post-synaptic thickening is much less well developed than
in a type 1 structure, and there is no obvious extracellular material between the pre- and
post-synaptic membranes. Many of the presynaptic vesicles are flattened. As is well
known, of course, both parallel and mossy fibres are excitatory, whereas Golgi cells are
inhibitory.

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Fig. 4. Examples of electron microscopy of mammalian CNS fixed by perfusion using a mixture of
aldehydes. Cerebellar cortex of the rat. A: Outermost part of molecular layer, cut transversely to
the parallel fibres. Scale bar = 1 µm. Bgc, Bergmann glial cell process; pf, parallel fibres; pm, pia
mater; s, synapse. B: Gray type 1 synapse between a parallel fibre varicosity and a Purkinje cell
dendritic spine. Scale bar = 0.5 µm. ds, Purkinje cell dendritic spine; gc, glial cell process; pf, parallel fibre containing microtubules (neurotubules); pfv, presynaptic varicosity of parallel fibre; rv,
round vesicles. C: Synaptic glomerulus in the granular layer. Scale bar = 1 µm. mf, mossy fibre rosette filled with round vesicles and forming numerous synaptic contacts with different granule
cell dendrites; gcd, granule cell dendrites. D: Gray type 1 (mossy fibre to granule cell dendrites)
and type 2 (Golgi cell axon to granule cell dendrites) synapses. Scale bar = 0.5 µm. Gca, Golgi cell
axon terminal, with flattened vesicles; gcd, granule cell dendrite; mf, mossy fibre rosette.


1 Cytological Staining Methods

Subprotocol 3
The Golgi Method
■■

Introduction

“Golgi is responsible for a method that renders anatomical analysis both a joy and a
pleasure.” (Cajal, 1995).
The Golgi method is central to neurohistology, almost serving to define the discipline. Here is a technique that by its ability to select single cells, more or less at random,
and fill them with a near-black precipitate while leaving the surrounding cells unstained
provided a straightforward means to solve the technical problem presented by the complex shapes and interrelationships of cells of the nervous system. These same staining

properties render it virtually useless in any other area of histology. We are told that the
method was discovered by accident, but insofar as it consisted simply of “prolonged immersion of the pieces [of brain], previously hardened with potassium or ammonium bichromate [sic], in a 0.50 or 1.0 % solution of silver nitrate” (Golgi, 1873), it was probably
only a matter of time before someone found it. It is worth recalling that Mueller had introduced potassium dichromate as a hardening agent as recently as 1860 (Baker, 1958).
From its earliest days the method has had its critics, but by acknowledging the criticisms at the outset we can perhaps best appreciate its limitations (and therefore its possibilities). Essentially, the criticisms can be expressed as two questions: i) Does the
method provide a representative sample of cells (especially neurons)? ii) When a neuron
stains, are all its neurites fully shown? The respective answers – probably not; and perhaps sometimes, but certainly not always – highlight the limitations which, it may be
seen, imply that we should be particularly cautious with quantitative results obtained by
means of the Golgi method. However, any method that selectively marks individual
neurons is liable to suffer the same criticisms and its results will require some sort of
complementary control. The Golgi method has continued to be important, even after
the introduction of electron microscopy and intracellular staining techniques, due to its
particular advantages: economical generation of information on different types of neuron and their interrelationships, and simplicity in execution.
It is not surprising, in view of its importance and long history, that the Golgi method
has spawned several variants, though the two principal ones were introduced by Golgi
himself. We shall refer to them as the rapid Golgi and the Golgi-Cox methods. Once
again it is instructive to consider briefly how these might have arisen; in the absence of
a rational physico-chemical basis for the variants (see below), one suspects them to be
due to a process of selection following empirical, if not to say playful, experimentation.
Could this be how Golgi came, in the last year or two of the 1870s, to substitute mercuric
chloride for silver nitrate after the initial fixation in Mueller's fluid? Mercuric chloride
(HgCl2) had only just been popularized as a fixative by Lang, writing in 1878, although
it was first used in microtechnique around the middle of the 19th century (Baker, 1958).
However, unlike silver nitrate, mercuric chloride led to individual cells being marked by
a white precipitate, which needed to be darkened by treatment with alkali. Cox made a
relatively minor modification to the method in 1891 by including the mercuric chloride
in the primary fixative, and it has remained essentially the same since.
Both the original and, especially, the Golgi-Cox methods suffer from being very prolonged procedures, sometimes up to several months in total. Golgi it was who found that
the addition of a small amount of osmium tetroxide to the primary dichromate fixative,
originally about 0.33 %, resulted in a great reduction in the amount of time needed for
the subsequent silver nitrate exposure. It seems unlikely that Golgi can have predicted

this effect of osmium tetroxide, but rather that it was a fortunate side-effect of an at-

Part 2:
The Differentiation of
Single Cells

13