Microchemical Analysis of Nervous Tissue: Methods in Life Sciences

By Neville N. Osborne

Microchemical Analysis of Nervous Tissue focuses on the use of microbiochemical methods in the analysis of nervous tissue, with emphasis on those related to the study of amines, amino acids, phospholipids, and proteins. Special attention is paid to the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. Comprised of 10 chapters, this volume begins with an overview of microprocedures used in neurochemistry, followed by a discussion on the importance of choosing the biological material for microanalysis. The isolation of nervous tissue for analysis is then considered, with particular reference to invertebrate neurons; cell components from fresh, "fixed," freeze-dried, and frozen impregnated tissue; and discrete areas of nervous tissue. Subsequent chapters describe some instruments and glassware used in microprocedures, along with the applications of such procedures; general techniques used in microprocedures; microdetermination of phospholipids as well as amines and amino acids as dansyl derivatives; and microelectrophoresis of proteins. This book will be of interest to molecular biologists, microbiologists, physiologists, and neurochemists.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483187389

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development.

CHAPTER 1

General Introduction

Publisher Summary

The physiology, morphology, functional role, and biochemistry of individual neurons are studied and the neurons in the nervous system are related to one another before a real insight is gained into the intricate mechanism of the nervous system. However, progress has been slow basically for two main reasons. First, the majority of neurons are difficult to characterize and study as entities because of their small size, and, second, there is a lack of suitable microprocedures that would permit the study of different biochemical parameters in individual neurons. This chapter describes some microprocedures recently developed in the laboratory. It focuses on the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. The microbiochemical methods are described related especially to the study of amines, amino acids, phospholipids, and proteins. The chapter also describes sensitive microprocedures and their applications are explained for studying small amounts of tissue, for example, isolated cells, discrete areas of brain, and biopsy material.

WHY a monograph on microprocedures in neurochemistry? This is not difficult to justify when one considers that the human brain has approximately 10¹⁰ nerve cells, while the tiny brain of the ant (Formica lugubris) has about 100,000. These vast populations of neurons present a formidable challenge to the biologist trying to understand how the nervous system works. From the mass of electrophysiological and electron microscopical data which has accumulated it can now generally be concluded that nerve cells are independent units (see e.g. Bullock, 1967; Bullock and Horridge, 1965; Eccles, 1964; Segundo, 1970; Horridge, 1968). Furthermore, each neuron has several parts: (1) receptive loci specialized in transducing the dozens of imputs which impinge on them in several ways; (2) pacemaker loci which inject spontaneous rhythms; (3) mixing and integrating loci; (4) threshold loci for initiating all-or-none nerve impulses in bursts and trains from 1 to 1000 per second; and (5) transmitter loci at each of the far ends of the nerve cell, where they influence up to several dozen others. Clearly, biochemical information to be gained from classical studies using relatively large amounts of nervous tissue (and therefore large numbers of cells which may have very different properties) is of limited value. This problem is complicated by the existence of vast numbers of glial cells which form a close and integrated association with the neurons. Obviously the physiology, morphology, functional role and biochemistry of individual neurons have to be studied and the neurons in the nervous system related to one another before a real insight is gained into the intricate mechanism of the nervous system. However, progress has been slow, basically for two main reasons. Firstly, the majority of neurons are difficult to characterise and study as entities because of their small size, and, secondly, there is a lack of suitable microprocedures which would permit the study of different biochemical parameters in individual neurons.

One way of circumventing these difficulties is either to separate disaggregated nervous tissue, thus obtaining populations of neurons and glial (Rose, 1968), or to fractionate homogenates of nervous tissue and secure relatively pure fractions of a constituent part of the different neurons, e.g. the nerve endings (Whittaker, 1973). Studies of this kind have many advantages, but they, too, suffer from certain drawbacks, such as the possibility that changes could occur in the constituents, caused by the elaborate separation or fractionation procedures employed; moreover, any differences there may be in the properties of similar structures obtained from the brain cannot be observed. Another approach is to analyse small defined areas of the nervous system or individual neurons where possible. This presupposes the presence of suitable microchemical procedures. Perhaps a distinction should be made here between macro- (normal), micro- and ultra-procedures, though one might think that such a distinction is meaningless, since they represent a scale continuum.

In theory they do; however, in practice there is a change from macroscale (i.e. brain homogenates) to micro (i.e. one very large neuron, or microquantities of nervous tissue), and over this range many macroprocedures can be modified and scaled down. The next step, the ultra-microprocedure (parts of a single (20 μ) minute nerve cell), is a ‘quantum jump’ and often requires elaborate apparatus and new approaches.

The purpose of this monograph is to describe some microprocedures recently developed in this laboratory. Special attention will be paid to the choice of biological material and the various procedures used for the isolation by dissection of defined components of the nervous system. The microbiochemical methods described will be those related especially to the study of amines, amino acids, phospholipids and proteins. Many other extremely sensitive microprocedures (plus-ultra-microprocedures) have been developed within the last thirty years (see Chap. 5) and though their description is beyond the scope of the monograph, a brief review of some of these methods and their applications is presented. Perhaps it should be pointed out that emphasis is often laid only on the applicability of microprocedures for studying small amounts of tissue, e.g. isolated cells, discrete areas of brain, biopsy material, etc., whereas they also have other important virtues. Some micromethods, for example, are less time-consuming than normal procedures, and are for this reason employed even when the material available is unlimited. Moreover, the cost of analysing material by micromethods can often be very much less than that of similar normal scale studies.

References

BULLOCK, T. H. Signals and neuronal coding. In: QUARTON G.C., MELNECHUK T., SCHMITT F.O., eds. The Neurosciences: a Study Program. New York: The Rockefeller University Press; 1967:347–452.

BULLOCK, T. H., HORRIDGE, G. A.Structure and Function in the Nervous Systems of Invertebrates. San Francisco: W. H. Freeman, 1965.

ECCLES, J. C.The Physiology of Synapses. New York: Academic Press, 1964.

HORRIDGE, G. A.Interneurons. San Francisco: W. H. Freeman, 1968.

ROSE, S. P. R. (1968) The biochemistry of neurones and glia. In: Applied Neurochemistry (Eds. A. N. Davison and J. Dobbing), pp. 332–355.

SEGUNDO, J. P. Functional possibilities of nerve cells for communication and for coding. Acta Neurol. Latinoamer. 1970; 14:340–344.

WHITTAKER, V. P. The biochemistry of synaptic transmission. Naturwissenschaften. 1973; 60:281–289.

CHAPTER 2

Choice of Biological Material for Microanalysis

Publisher Summary

This chapter discusses the fact that the mammalian brain contains a great number of neurons, which presents a problem. The difficulty lies in the choice of appropriate experimental objects. In this respect, certain invertebrate nervous systems offer a number of advantages, in that they are organized in an orderly manner, have fewer nerve cells than the vertebrates, have specialized giant neurons, and can be individually characterized. There are certain vertebrate preparations, which contain populations of giant neurons, though they are difficult to characterize individually. Another important advantage of the invertebrate neurons is that they can retain their functional activity after dissection and survive for several hours or even days. This makes it possible to perform in vitro experiments on invertebrate nervous systems, monitoring the activity of individual neurons by means of intra- or extracellular recording while the environment of the cell can be controlled or changed by adding or substituting ions and inhibitors. The chapter also explains that there is not only an enormous variety of invertebrate cell preparations, but also of invertebrate preparations of giant synapses and giant axons, which are suitable for biochemical analysis.

AS previously mentioned, the fact that the mammalian brain contains a great number of neurons presents a problem. The difficulty lies in the choice of appropriate experimental objects. Ideally one needs a nervous system that produces a reasonably complex repertoire of behaviour and has only a few cells, each of which can be recognised so that suitable experiments can be carried out on them. In this respect certain invertebrate nervous systems offer a number of advantages, in that they are organised in an orderly manner, have fewer nerve cells than the vertebrates, have specialised giant neurons and can be individually characterised. There are certain vertebrate preparations which do contain populations of giant neurons, though they are difficult to characterise individually. Another important advantage of the invertebrate neurons is that they can retain their functional activity after dissection and survive for several hours or even days (see e.g. Strumwasser, 1967). This therefore makes it possible to perform in vitro experiments on invertebrate nervous systems, monitoring the activity of individual neurons by means of intra- or extracellular recording while the environment of the cell can be controlled or changed by adding or substituting ions, inhibitors, or drugs, etc. These and the many other advantages in using invertebrate neurons in the analysis of individual cells are summarised in Table 1.

TABLE 1

ADVANTAGES OF INVERTEBRATE OVER VERTEBRATE CELL PREPARATIONS FOR MICROCHEMICAL ANALYSIS

There is not only an enormous variety of invertebrate cell preparations (see Table 2), but also of invertebrate preparations of giant synapses and giant axons (see Table 3) which are suitable for biochemical analysis. In addition, glial cells can often be dissected from invertebrates (see Kuffler and Nicholls, 1966) and analysed by biochemical procedures, thus allowing the study of the relationship between specific glial and nerve cells. Despite all these advantages, some scientists hesitate to compare the properties of neurons of the vertebrate central nervous systems with those of the invertebrate nervous system. Physical, biochemical and pharmacological differences do exist between vertebrate and invertebrate neurons, though they seem to consist mainly of differences in pharmacological sensitivity and some chemical characteristics rather than fundamental functional mechanisms and the mode of response of the cells to transmitters and drugs. Although electrophysiological properties of all neurons appear to be identical and in general comparable with those of electrogenetic cells, there are detailed electrophysiological differences between invertebrate and vertebrate nerve cells. Furthermore, the anatomy of invertebrate neurons differs in many ways from that of the vertebrates (Cohen, 1970). It is for these reasons that the biochemical analysis of mammalian cell preparations is of particular importance.

TABLE 2

INVERTEBRATE AND VERTEBRATE CELL BODIES SUITABLE FOR MICROANAYLSIS

TABLE 3

INVERTEBRATE GIANT AXONS AND GIANT SYNAPSES SUITABLE FOR MICROANALYSIS

The autonomic ganglia, which comprise a few thousand nerve cells organised in physiological units, have proved suitable material for identifying neurons with different pharmacological and biochemical properties. Single neurons isolated by microdissection and subcellular fractions of the ganglia have been analysed by microprocedures so as to follow changes involved in the process of synaptic plasticity (e.g. Giacobini, 1970). Populations of large nerve cell bodies also exist in the following mammalian tissues: cerebellum, hippocampus, anterior horn, spinal ganglion, nucleus supraopticus, cortex and Deiters’ nucleus (Table 2), and can be hand-dissected in excellent morphological condition (Fig. 1) from pieces of nervous tissue. However, care must be taken in order not to damage the chemical integrity of the neurons, since dyes (e.g. methylene blue) often have to be used to help in the identification process and also because vertebrate neurons are exceedingly susceptible to minute environmental changes. It may be worthwhile noting that the elegant experiments of Hydén showing changes in brain protein during learning were carried out on isolated hippocampus cells (Hydén, 1967; Hydén and Lange, 1970). In addition to Hydén (1972), Lowry and Passoneau (1972) and Giacobini (1970) have all been pioneers in the chemical analysis of isolated vertebrate neurons, and their work has contributed a great deal to our understanding of a number of important aspects in neurobiology. The point should be made, nevertheless, that studies on vertebrate nervous tissues, though exceedingly elegant, suffer from the drawback that the individuality of the cells cannot be exploited for technical reasons.

FIGS. 1 and 2 Free-hand dissected nerve cells isolated from the spinal nucleus of the trigeminal nerve, slightly stained with methylene blue solution. Photographed under Normarski optics. (Photographs by courtesy of V. Neuhoff.) Fig. 1, × 1800. Fig. 2, × 1000.

References

COHEN, M. J. A comparison of invertebrate and vertebrate central neurons. In: SCHMITT F.O., ed. The Neurosciences. New York: The Rockefeller University Press; 1970:798–812.

GIACOBINI, E., Biochemistry of single neuronal modelsCOSTA, E., GIACOBINI, E., eds. Biochemical Psychopharmacology; 2. Raven Press, 1970:9–64.

HYDÉN, H. RNA in brain cells. In: QUARTON G.C., MELNECHIK T., SCHMITT F.O., eds. The Neurosciences. New York: The Rockefeller University Press; 1967:248–266.

HYDÉN, H. Macromolecules and behaviour. In: ANSELL G.B., BRADLEY P.B., eds. Arthur Thomson Lectures. London: Macmillan; 1972:3–75.

HYDÉN, H., LANGE, P. W. Protein changes in nerve cells related to learning and conditioning. In: SCHMITT F.O., ed. The Neurosciences. New York: The Rockefeller University Press; 1970:278–289.

KUFFLER, S. W., NICHOLLS, J. G. The physiology of neuroglial cells. Ergeb. Physiol. 1966; 57:1–90.

LOWRY, S. W., PASSONNEAU, J. V.A Flexible System in Enzymatic Analysis. New York: Academic Press, 1972.

STRUMWASSER, F. Types of information stored in single neurons. In: WIERSMA C.A.G., ed. Invertebrate Nervous Systems. Chicago: University of Chicago Press; 1967:291–319.

CHAPTER 3

Isolation of Nervous Tissue for Analysis

Publisher Summary

This chapter discusses that the isolation of characterized neurons, nervous tissue parts, and populations of cells is the most critical step in the biochemical analysis of the nervous component. When working with vertebrate tissue, the most hazardous period is usually between the moment the blood supply is cut off and the dissection. It cannot be overstressed that either the functional integrity of the nerve cell should be maintained after dissection, or the metabolism of the neuron should be stopped abruptly by means of rapid freezing. The chapter explains that the first alternative is essential for direct neurophysiological and neuropharmacological correlations and it can be achieved comparatively easily when using invertebrate neurons. The second alternative is often necessary when analyzing vertebrate nervous tissue, especially labile metabolites. A couple of pairs of fine forceps together with a microscalpel are all that is required to free a characterized cell from the surrounding nervous tissue.

THE isolation of characterised neurons, nervous tissue parts and populations of cells is often the most critical step in the biochemical analysis of the nervous component. When working with vertebrate tissue, the most hazardous period is usually between the moment the blood supply is cut off and the dissection. It cannot be overstressed that either the functional integrity of the nerve cell should be maintained after dissection, or the metabolism of the neuron should be stopped abruptly by means of rapid freezing. The first alternative is essential for direct neurophysiological and neuropharmacological correlations and it can be achieved comparatively easily when using invertebrate neurons for the reasons stated earlier. The second alternative is often necessary when analysing vertebrate nervous tissue, especially labile metabolites.

1 Isolation of Characterised Invertebrate Neurons

The problems encountered in dissecting individual neurons from the various isolated ganglia are generally caused by the illumination of the ganglion, the identification of the neuron involved and its removal, intact, from the ganglion. An essential first step is to pin the ganglion down in a relatively stretched out position. It should not be stretched too much, otherwise the cells will tend to ‘pop out’ when the connective tissue layers are removed. In the instance of the snail (Helix pomatia) circumoesophageal ganglia, a small bath (volume 0.7 ml) containing a nylon sheet at the bottom and filled with snail saline is most suitable. This stiff plastic sheet is necessary to retain the insect pins used for holding the ganglia down. Either transillumination, reflected light, or dark-field illumination can then be used for the identification and dissection of individual neurons, which is carried out under microscopic vision. The most useful method by far is dark-field illumination. One simple approach is to focus a pencil of light on to the cell concerned. This can be done by attaching a tapered glass rod to a microscope lamp, and bringing the glass tip (about 3 mm diameter) very close to the preparation, thus illuminating the cell alone. An alternative to this is the Dark-field Dissecting Stand obtainable from Brinkmann Instruments Company (Westbury, New York). Proper illumination is essential for identifying individual neurons within the