J Tokyo
(
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THE
APPLICATION OF SUBCELLULAR
TECHNIQUES TO THE STUDY
NERVOUS SYSTEM
FRACTIONATION
OF THE
Victor P. WHITTAKER
Arbeitsgruppe Neurochemie, Max-Planck-Institut ftir biophysikalische Chemie,
Postfach 2841, D-3400 G6ttingen, FR Germany
(Received December 4, 1987)
Abstract
The application of the technique of subcellular
fractionation to the central nervous system is
described. Nerve terminals are detached to form
sealed structures (synaptosomes) which retain
most synaptic functions including the
main-tenance of a membrane potential and depolariza-tion-induced, Ca2'-dependent transmitter release, nuclei and mitochondria are liberated fragments of myelin generated, and the endoplasmic
retic-ulum comminuted to form vesiculated membrane
fragments (microsomes). All these structures can be separated by differential and density-gradient
centrifugation from each other and from the
cytosol. Synaptosome preparations permit many aspects of synaptic function to be studied in isolation from the rest of the nervous system.
Applied to electric organ, modifications of the technique permit the isolation, from the purely
cholinergic electromotor synapses, of purified
syn-aptic vesicles and synaptosomes.
Milder methods of tissue comminution permit
the isolation from the central nervous system of
neurone cell bodies and various types of glial cell.
Biochemical and physiological analysis of such
preparations are leading to a greater understand-ing of nervous system function.
Substance of a lecture given to the 3rd students of the
Tokyo Women's Medical College on Nov 6 1987. Dr
Whittaker is grateful to the Japanese Society for the Promotion of Science for a short term fellowship for
travel to and within Japan.
The Emergence of Cell Biology
as a New Science
Cell biology emerged as a new science in the late 50's and early 60's as the result of the coming together of subcellular fractionation techniques
and electron microscopy, later supplemented by
tissue culture, immunochemistry and
immuno-cytochemistry.
Subcellular fractionation techniques were
orig-inally applied to liver, a soft tissue with oniy one
main cell type. The tissue is homogenized in iso-osmotic sucrose by the application of liquid shear forces generated between the stationaly wail of a glass "mortar" and the rotating wall of a glass or plastic "pestle" (Fig. Ia). The intensity of shear
may be varied by varying the clearance between
pestle and mortar and the spee.d of rotation of the
pestle. The cell's plasma membranes are broken,
the ceil organelles (e.g. nuclei, rpitochondria) are
liberated, the internal cell membranes (rough and
smooth endoplasmic reticuium) are comminuted
to small vesiculated membrane fragments (called microsomes) and the soluble cytoplasmic consti-tuents are released (Fig. Ib). The application of increasing centrifugal forces to the homogenate and the successive supernatants results in three particulate fractions, consisting respectively (in order of decreasing particle size and increasing
field intensity) of nuclei, mitochondria and
micro-somes, and a supernatant, consisting of soluble cytoplasmic constituents.
Enzymes and other functional components of
2
organelles and so can be used as "markers" for them. Thus the nuclei have a high DNA content,
mitochondria contain the enzymes required for
celi respiration (e.g. succinate dehydrogenase) and
the soluble cytoplasm, the enzymes of glycolysis (e.g. Iactate dehydrogenase). The concept of the
localization of cell functions to specific cell or-ganelles is one of the main generalizations of cell biology and qualifies it to be regarded as a disci-pline in its own right.
Application to Nervous Tissue
In the late 50's, I became interested in the phenomenon of bound acetylcholine. This
sub-stance is a transmitter at cholinergic nerve ter-minals and is one of some 20 transmitters used by
the brain. In homogenates it is not free, but bound
to membranous particles. In this state, it is immune to the action of cholinesterase and is pharmacologically inactive. Treatment of the
homogenate with detergents or denaturing agents
releases the acetylcholine from its bound state and
so renders it both pharmacologically active and
susceptible to hydrolysis by cholinesterase.
I found that this bound acetylchoiine was
sedimented in the mitochondrial fraction from brain tissue. This fraction is, however, much
more complex in composition than the
corre-sponding fraction from liver. Electron-microscopic
examinationi} showed that it contained particles
of three main types: myelin fragments derived from the breakup of myelinated nerves,
mito-chondria, and a complex type of particle consist-ing of a plasma membrane enclosconsist-ing a cytoplasm
rich in small vesicles and mitochondria (Figs. Ic, 2a, b). This type of particle could be identified as a
nerve ending which had survived homogenization but had been torn away from its axon and post-synaptic attachment and in the process had re-sealed. Such particles I named ``synaptosomes"2).
A simple type of density gradient centrifuging
sufficed to separate the three types of particle, and
bound acetylcholine was concentrated in the frac-tion of intermediate density containing the
syn-aptosomes.
Synaptosomes retain, to an astonishing degree,
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SYNAPTOSouESFig. 1 Scheme showing the fractionation of liver and
nervous tissue. (a) Homogenizer, (b) liver cell, (c) neurone, Glial cells break up in a similar way to liver cells and
contribute nuclei, mitochondria, microsomes and
natant to the primary fractions.
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all the main features of the synapse,
morpholog-ical, physiologicai and biochemical.
Morphologically, we see the typical synaptic
fine structure: a cytoplasm packed with synaptic vesicles and small mitochondria, and surrounded
by a sealed plasma membrane with (under
appriate staining conditions) presynaptic dense
pro-jections and an adhering patch of postsynaptic
membrane.
Physiologically, the synaptosome, if incubated
briefly with glucose in a balanced saline solution,
behaves like a miniature cell, taking up K' and
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Fig. 2 (a) the crude mitochondrial
showing its three main constituents: myelin fragments (My), mitochondria (Mi) and detached nerve terminals (synaptosomes, S).
(b) An isolated synaptosome and a typical cortical
axodendritic synapse juxtaposed so as to show the
faithful retention of synaptic morphology by the
aptosome.
Detached terminat
(synaptosome)
fraction from brain
extruding Na' and thereby acquiring a membrane
potential. It may then be depolarized and so
caused to release transmitter.
Biochemically, the metabolizing synaptosome
takes up energy metabolites and transmitters (or their precursors) including several amino acids;
the kinetics of the uptake show that it is carrier mediated and specific for a given substrate and its
close chemical analagues. Drugs affecting syn-aptic function may be studied in such a system and are often found to interfere with transmitter
uptake. The synaptosomal enzymes are in an
occluded state unless released by osmotic
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aptosomes into separate fractions consisting of (O) soluble cytoplasm, (D) monodisperse synaptic vesicles, (E) microsomes, (F, G) synaptosomal plasma membranes, (H) disrupted synaptosomes, (I) intraterminal chondria.
tion and synaptosomes behave like simple os-mometers over a limited range of osmotic
per-turbation.
Further work (Fig. 3) using a more complex
density gradient onto which osmotically disrupted
synaptosomes were loaded enabled the various
component parts of the synaptosome to be
separated2). These included: the small vesicles
(synaptic vesicles), the synaptosomal plasma membrane, the intraterminal mitochondria and the synaptosomal cytoplasm. Most of the bound
acetylcholine was 1ocalized in the synaptic ves-icles, which were thus shown to be the ultimate
storage sites of the transmitter in the nerve
terminal. The 1ocalization of transmitters in ves-icles was consistent with the electrophysiological
finding that transmitter is released from nerve
terminals in packets, called quanta. However,
transmitters are usually synthesized in the
cyto-plasm, and the cytoplasmic (non-vesicular) pool of
im-portant.
Synaptosomes preserve, as we have seen, most of the properties of the nerve terminals3)4) and
eriable synaptic function to be studied in the
test-tube, away from other parts of the nervous system
such as axons, cell bodies or glial cells. However, a
typical synaptosome preparation from
mam-malian brain is derived from all types of neurone utilizing many different transmitters. For some purposes this is an advantage -thus the recovery of a pharmacologically active substance mainly in
synaptosomes is strong evidence that it has a
transmitter role- but for further work on cho-linergic function we needed a source of purely cholinergic nerve terminals abundant enough for subcellular fractionation and further biochemical
work.
The Electromotor System of the Electric
Ray: A Model Cholinergic Synapse
I found such a source in the electric organ of the
electric ray, Tbrpedo marmorala. This consists of
stacks of flattened cells, the electrocytes, which
are derived from muscle, and like muscle, are
innervated by cholinergic motor neurones. Unlike muscle, the whole ventral surface of each elec-trocyte is covered with nerve terminals. Thus the tissue contains 500 to 1000 times more synaptic material than the equivalent weight of rnuscle (Fig. 4a). I had to modify my fractionation
tech-niques since this organ is full of collagen, which
makes conventional homogenization difficult5). I discovered that freezing in liquid nitrogen makes
the tissue brittle. It can then be smashed up into
small fragments. During the process of commi-nution, the nerve terminals are ripped open. Extraction with iso-osmotic NaCl extracts the synaptic vesicles together with soluble cyto-plasmic proteins and some membrane fragments. A very pure synaptic vesicle fraction can be
isolated by means of continuous density gradient
centrifuging in a zonal rotor. My colleagues and I
have intensively studied this preparation and now
understand quite well how it is able to accumulate acetylcholine in high concentration. It does this by
generating a proton gradient. Protons are then
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of comminution.
exchanged for acetylcholine via a carrier in the vesicle membrane6).
Vesicle recycling -the process whereby
ves-icles discharge transmitter and refill at the
ex-pense of the small but metabolically important
cytoplasmic transmitter pool- can be studied by isolating vesicles from terminals that have been
prestimulated to generate a pool of recycling
vesicles7}. Such vesicles can be separated from the
reserve pool by making use of small differences in size and density. They can be identified by their ability to take up newly synthesized transmitter: this in turn is identified by supplying the tissue
with isotopically labelled transmitter precursors.
Another method of comminution involves
minc-ing the tissue and then passminc-ing it through wire meshes of decreasing diameter8}9}. This generates
synaptosomes which are then purified by
frac-tionation on a step gradient (Fig. 4b). Such
syn-aptosomes can be lysed by exposure to
hypo-osmotic media and the plasma membranes
sep-arated out by a second step-gradient fractionation.
These plasma membranes may be used to study
the transporters responsible for taking up
cholineiO) and other metabolites into the terminal
-374-and also for surface markers which label the
terminal as "cholinergic"ii}.
We have successfully reconstituted the choline transporter in an artificial membrane and labelled iti2) by means of the choline analogue, choline
aziridium mustard. This compound has a three
membered N-C-C ring and can act as an alkylating agent. It preferentially labels the choline trans-porter. This has been tentatively identified as a protein of molecular mass 45 to 50 Kilodaltons and
isoelectric point of 5.1.
We have also identified two minor gangliosides (Chol-la and B) as cholinergic-specific surface markersii)i3). Antisera to these gangliosides in-duce the complement-mediated lysis of the
choi-inergic subpopulation of mammalian
synapto-somes. Such synaptosomes, if pretreated with
sheep anti-Chol-1 antiserum, may be isolated by
adsorbing them onto a column of immobilized
anti-sheep-IgG monoclonal antibodyi4}. We believe
that Chol-1 regulates the formation of mature cholinergic synapses and may therefore have a
role in synaptic plasticity in the cortical system involved in arousal, attentiveness and thus
learn-ing and memory.
The Isolation of Neural Cells
Subcellular fractionation techniques have been refined and extended to permit the isolation of multicellular structures (e.g. brain capillaries, renal glomerulae) and single cell types (e.g.
neu-rones, astrocytes, oligodendrocytes)i5). If prepared
from embryonic tissue, such cells may survive in culture and even develop. For this reason we have replaced the term "subcellular" by "tissue"
frac-tionation techniques when running advanced
teaching courses in these methods. In the other
direction, polysomes, ribosomes and glycogen
granules are examples of very smali subcellular
structures that can be isolated.
These techniques are enabling us to get a better insight into the way the nervous system functions
at the molecular level.
References
1) Gray EG, Whittaker VP: The isolation of nerve
endings from brain: an electromicroscopic study of cell
fragments derived by homogenization and
tion.J Anat (Lond.) 96: 79-88, 1962
2) Whittaker VP, Michaelson IA, Kirkland RJA: The
separation of synaptic vesicles, from nerve-ending ticles (``Synaptosomes''). Biochem J 90: 293-303, 1964
3) Whittaker VP: Chapter 1; The synaptosome and Chapter 2; The synaptic vesicle, In Handbook of
Neurochemistry (Lajtha A ed) 2nd ed, vol 7, pp 1-69, Plenum, New York (1984)
4) Whittaker VP: Synaptosome. In Encyclopedia of
Neuroscience (Adelman G ed) vol 2, pp 1179-1181, Birkhauser, Boston (1987)
5) wnittaker VP, Essman WB, Dowe GHC: The
isolation of pure cholinergic synaptic vesicles from theelectric organs of elasmobranch fish of the family torpedinidae. BiochemJ 128: 833-846, 1972
6) Whittaker VP: The structure and function of inergic synaptic vesicles. Biochem Soc Trans 12: 561-576, 1984
7) Agoston DV, Dowe GHC, Fiedler W et al: A kinetic
study of stimulus-induced vesicle recycling in motor nerve terminals using labile and stable vesicle
markers.J Neurochem 47: 1584-1592, 1986
8) Dowdall MJ, Zimmermann H: The isolation of pure
cholinergic nerve terminal sacs (T-sacs) from the tric organ of juvenile Torpedo. Neuroscience 2: 405-421,
1977
9) IsraEl M, Manarache R, Mastour-Franchon P et
al: Isolation of pure cholinergic nerve endigns from the
electric organ of Torpedo marmovata. Biochem J 160: 113-115, 1976
10) Ducis I, Whittaker VP: High-affinity,
gradient-dependent transport of choline inte vesiculated
presynaptic plasma membrane fragments from the
organ of IIbrpedo marmorala and reconstitution of the solubilized transporter into liposomes. Biochem Biophys
Acta 815: 109-127, 1985
11) Richardson PJ, Walker JH, Jones RT et al:
tification of a cholinergic-specific antigen chol-1 as a
ganglioside. J Neurochem 38: 1605-1614, 1982
12) Rylett RJ, Whittaker VP: Identification of the affinity choline transporter of Torpedo electromotor nerve terminals using a 3H-choline mustard ligand. J
Neurochem 48: S66A, 1987
13) Ferretti P, Borroni E: Putative cholinergic-specific ganglioside in guinea pig forebrain. J Neurochem 46:
1888-1894, 1986
14) RichardsonPJ,SiddleH,LuzioJP:Immunoaffinity
purification of intact, metabolically active, cholinergic
nerve terminals from mamma}ian brain. Biechem J 219: 647-654, 1984
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