Smith / Llinás / Kostyuk Commentaries in the Neurosciences

1. Auflage 2014
ISBN: 978-1-4831-4873-1
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 678 Seiten

Commentaries in the Neurosciences is a compilation of the commentaries segment featured in the Neuroscience journal. This book, however, does not provide a comprehensive account of all fields in neuroscience, but rather articles that highlight developments during the past years. The topics covered include chemistry and dynamics of neurotransmitter storage particles; release of central and peripheral neurotransmitters; and transmitters in the enteric nervous system. This book also discusses the properties of neuroreceptors; chemistry and connections of the cerebral cortex; and intracellular recording and ionic transmembrane currents. This text explains as well the invertebrate nervous systems; functions of the nervous system including neuro- and psychopharmacology; and the mind-body problem. This book will be of use not only to research workers interested in keeping updated with developments in different areas of the neurosciences, but also to advanced undergraduate and graduate students who are studying the nervous system.

Autoren/Hrsg.

Smith, A. D.

Llinás, R.

Kostyuk, P. G.

Leseproben

SYNAPTOSOMES AND CELL SEPARATION

IAN G. MORGAN,     Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601, Australia

Publisher Summary

When fractionating a complex tissue like brain, experiments should begin by separating it into its constituent cell classes. Any subcellular fraction prepared from brain is intrinsically heterogeneous as it is derived from several cell types. But even if it were possible to separate cell types satisfactorily, the fractions obtained would still be heterogeneous because of the fact that it is possible to subdivide the classes of neurons and glial cells into a series of subclasses. This chapter discusses the preparation of synaptosomes and synaptosomal subfractions and the related problem of distinguishing glial from neuronal material. Methods for preparing synaptosomal subfractions often begin with a crude mitochondrial fraction. However, it is preferable to begin with a synaptosomal fraction—best prepared on a Ficoll-sucrose gradient because of the greater ease of osmotic shock. As the synaptosomal fraction has lower levels of mitochondrial, myelin, and membranous contamination, a crude mitochondrial fraction should be used only if time is an absolutely crucial factor.

Preparation of synaptosomes

Preparation of subfractions from synaptosomes

Separation of neurons from glia

Conclusions

Logically, when fractionating a complex tissue like brain, experiments should begin by separating it into its constituent cell classes. Unfortunately, at the present time there are practical limitations on doing this, as will become clear later. As a result, any subcellular fraction prepared from brain is intrinsically heterogeneous since it is derived from several cell types. But even if it were possible to separate cell types satisfactorily, the fractions obtained would still be heterogeneous due to the fact that it is possible to subdivide the classes of neurons and glial cells into a series of subclasses. Moreover, even within a given cell there are different regions: an important problem for the neuron where dendritic, cell body, axonal and synaptic regions can be distinguished.

Any brain subcellular fraction suffers from some of these types of heterogeneity, although certain fractions, such as myelin, and nerve-endings and their subfractions, escape them to some extent. Very often, this heterogeneity is not important for the interpretation of results, but the finer the analysis, the more likely it is that it will be necessary to take it into account.

A crucial point in subcellular fractionation experiments, that of deciding on the aim of the experiment, applies to any tissue—not specifically to the nervous system. Basically there are two types of fractionation experiment: experiments aiming to draw a parallel between the distribution of markers, and the constituent under study, and experiments aiming to obtain as pure a fraction as possible for detailed studies of its composition or metabolism. While, ideally, the criteria for the techniques used in both types of experiment should be the same, in practice increases in the purity of a fraction are generally accompanied by decreases in yield, and vice versa. In practice, therefore, the optimum fractionation procedures for the two types of experiment are different.

In the classical analytical experiment—that of determining if a given tissue constituent is associated with a given subcellular fraction—a parallel is drawn between the distribution of the constituent and that of a known marker. In these experiments a standard procedure of high resolution is required, yet complete recovery of material is also necessary so that balance-sheets can be drawn up. The latter point is generally accepted, but the former is often neglected. Many experiments have reported localizations’ of enzymes in synaptosomal plasma membrane using procedures which do not effectively resolve them from glial, or even microsomal elements. This problem is compounded by the difficulty of choosing markers. Many are chosen by analogy with other tissues, particularly liver, which, given that few reference fractions can be isolated in pure form from brain, is unfortunately the best that can be done.

This sort of experiment generally gives some idea of whether the constituent is associated with the marker. But conclusions should always be confirmed by isolating the particular subcellular fraction absolutely pure. Whether the localization reported is exclusive can be answered by looking at the percentage distribution results, and the preparative enrichment of the constituent compared to the marker. The ultimate test would be to isolate all other subcellular fractions in pure form and test them for the presence of the constituent. In practice, our technology falls somewhat short of this, since all the fractions obtained are contaminated to some extent. Care needs to be exercised in adapting these criteria to reality.

In analytical experiments a method need only give a fraction in which the appropriate marker is highly enriched, and in which other markers are less enriched. This does not mean that the fraction is suitable for preparative work, since, to take the case of lysosomes, the specific activities of relevant enzymes in pure lysosomal fractions from liver may be 100 times those of the homogenate (Stahn, Maier & Hannig, 1970). Fractions enriched 10–20 times can be obtained from brain (Koenig, Gaines, Mcdonald, Gray & Scott, 1964), yet the lysosomes may only make up 10–20% of these fractions. It is obviously ludicrous to study the composition of lysosomes using such fractions, yet they are useful ‘reference fractions’ in the analytical approach.

For preparative work, in order to be able to judge the significance of the results, as complete an inventory as possible of the components of the fraction is necessary. It is not possible to put an arbitrary limit on the degree of purity necessary before results become meaningful. Generally, several minor components are less important than one major contaminant, even if the total percentage contamination is the same. Moreover, if the contaminant has itself been purified, it may be possible to allow for its presence. Particular care over contamination must be taken in experiments involving immunology, since biochemical contaminants may be highly antigenic and so give rise to major immunological components.

After these preliminary remarks, the main focus of this article will be upon the preparation of synaptosomes, and synaptosomal subfractions, and upon the related problem of distinguishing glial from neuronal material.

PREPARATION OF SYNAPTOSOMES

Preparation of synaptosomes has been in the past, and remains today, an empirical affair which has recently been reviewed by COTMAN (1974) in an admirably succinct article. Within certain limits, the precise conditions of homogenization do not appear to be crucial for isolating synaptosomes. Nor do the lower, and higher, centrifugal conditions of the first step, the preparation of the ‘crude mitochondrial’ fraction, appear to be absolutely crucial, although the levels of contamination with endoplasmic reticulum can be reduced by choosing the appropriate conditions (Cotman, Brown, Harrell & Anderson, 1970), and by washing the fraction exhaustively (Morgan, Wolfe, Mandel & Gombos, 1971; Gurd, Jones, Mahler & Moore, 1974).

Various gradients have been used for isolating synaptosomes, primarily made with sucrose or Ficoll. A synaptosomal peak can be easily defined by measuring occluded lactate dehydrogenase, but for more knowledge of the composition of the fractions, morphological studies are necessary. These have been performed on sucrose (Whittaker, 1968) and Ficoll (Joó & Karnushina, 1975) gradients. As a generalization it appears that synaptosomal fractions are never pure, but fractions containing up to 40–50% synaptosomes can be obtained. More importantly, by selecting dense fractions the contamination with membranous particles and fragments can be reduced, but at the expense of a corresponding increase in mitochondrial contamination. Conversely, less dense fractions are much less contaminated with mitochondria, but are more contaminated with membrane fragments. In practice, this means that the gradient used to isolate synaptosomes can only be chosen empirically once the aim of the experiments has been decided.

If the synaptosomes are to be used for subsequent fractionation, then, in general, denser fractions are preferable. As mentioned above, the denser fractions tend to be less contaminated with membranous particles, which therefore reduces the potential contamination with glial membranes and endoplasmic reticulum. If synaptosomal soluble components are required then denser fractions are also to be preferred since there is some evidence that there are less contaminating glial structures (Joó & Karnushina, 1975). When synaptosomal mitochondria are to be prepared, it is obviously preferable to start off from the...