595th MEETING, MANCHESTER
595th Meeting
Held at the University of Manchester on 22, 23 and 24 July 198 1
The Biochemistry of the Single Cell
Techniques Group Colloquium organized and edited by R. W. A. Oliver (Salford)
Techniques for the production and purification of single nerve cells
NEVILLE N. OSBORNE
Nufield Laboratory of Ophthalmology, University of Oxford,
Walton Street, Oxford OX2 6 A W , U.K.
Rationale for single-cell analysis
The human brain has approx. IO’O nerve cells and the vast
population of neurons presents a formidable challenge to the
biologist attempting to understand how the nervous system
works. Three approaches have generally been used. The first is
to try to localize the tissue constituent by making it in some way
visible in histological sections, e.g. by staining, by using
fluorescent dyes or by radioactive labelling followed by
autoradiography. Procedures of this sort have become
increasingly sophisticated, as exemplified by the development of
the whole field of immunohistology, where specific monoclonal
antibodies may be used.
The second major approach is to separate component parts,
e.g. cell bodies, glia, myelin, nuclei, synaptosomes, synaptic
vesicles etc., and study them in isolation. While studies of this
kind have many advantages, they can suffer from certain
drawbacks, such as the possibility of changes occurring in the
constituents caused by the elaborate separation of fractionation
procedures employed.
The third, and possibly the oldest approach, is the subject of
this contribution. It consists of the direct quantitative microchemical analysis of identified samples obtained under controlled conditions from specific sites in the nervous system. The
originators of direct quantitative histochemistry were Kai
Linderstrtam-Lang and Heinz Holter in the early 1930s. Their
classical studies involved producing serial frozen sections from
the gastric mucosa (Holter & Linderstrtam-Lang, 1935), staining
and chemically analysing alternate sections and then making a
correlation between the distribution of pepsin and several
specific cell types. David Glick (Glick, 1938) and Alfred Pope
(Pope, 1952) were among the first to apply the original
Lang-Holter methodology to the nervous system, and it became
evident that original procedure had to be modified if it was to be
applied generally, because adjacent systems in the nervous
system can be very dissimilar in cellular composition.
In recent years many studies have attempted to characterize
biochemically the individual cells within nervous tissue, the most
prominent developers being Lowry, Giacobini and Hyden (see
Osborne, 1974). There are various procedures for isolating
defined components of the nervous system by dissection and
analysing them with suitable biochemical methods, some of
which I shall deal with briefly. Detailed information on
microbiochemical procedures and their application are described
elsewhere (Lowry, 1963: Lowry & Passonneau, 1972: Osborne,
1974; Giacobini, 1975; Glick, 1977: Wu, 1978).
Isolation of sample
The nervous system functions rapidly on receiving stimuli and
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injury and therefore what happens to the sample before
analysis may be at least as important as the analysis itself. In
practice, everything should be done to maintain the functional
integrity of the nerve cell after dissection, or the metabolism of
the neuron should be stopped abruptly by means of rapid
freezing. Two further considerations are necessary in studies on
isolated neurons. Firstly. how much contamination of glia, blood
vessels etc. is associated with the neuron? and secondly, can the
isolated neuron’s be regarded as ‘normal’ neurons insofar as
they are free of significant damage and alteration to their
characteristics? The answer to both of these questions is
determined to a large extent by the efficiency of the experimenter, although in absolute terms it is impossible to dissect a
neuron totally free of glia without damaging it considerably (see
Johnston & Roots, 1972; Osborne, 1977).
The most general method used for isolating single nerve cells
is as follows. A suitable dissecting microscope is a prerequisite.
Pieces of tissue approx. 1 mm square are placed on a glass slide
containing a few drops of cold physiological solution. A drop of
0.1% Toluidine Blue or Methylene Blue is added and left in
contact with the tissue until the nerve cells stain weakly before
washing away. Steel wires, glass needles and microscalpels can
now be used to tease the individual cells away from the
surrounding tissues. Single cells can then be lifted free by using
fine wire needles. The dendrites of the nerve cells are transferred
to the appropriate medium. An experienced worker is able to
isolate 2-6 neurons a minute, depending on the dendritic
complex of the neurons. To make scalpels fine enough for
microdissection, halved razor blades of untempered steel are
broken transversely in the teeth of a pair of good quality pliers
and then fixed on to wooden handles. Needles of steel or glass
can also be easily produced to suit the experimenter.
A number of methods exist for isolating specific neurons or
neuronal parts from nervous tissues. The choice of method will
depend on the problem investigated. For example, anoxia or
anaesthetics affect ATP or lactate profoundly. so when
analysing these parameters it is necessary to freeze the tissue
rapidly from the living state. In contrast, if RNA and DNA are
to be analysed, it is possible to preserve the tissue which
formalin treatment, which destroys most enzymes. The best
known method, dating back to the studies by Anfinsen et al.
(1 942) on the retina, is first to produce freeze-dried sections and
then, under microscopic vision, tease out specific components
freehand. The freeze-dried samples can then be weighed, by
using a quartz-fibre balance, and analysed (see Lowry &
Passonneau, 1972). Another method is to isolate cells from
paraffin-was-embedded or from ‘deparaffinized’ fixed sections. It
is possible to use micromanipulators to dissect the components under an oil-immersion objective, free of contamination.
This procedure has been used particularly for RNA analysis
(see Edstrom & Neuhoff, 1974). Another alternative is to isolate
cells directly from fresh material. This approach has been very
583
BIOCHEMICAL SOCIETY TRANSACTIONS
584
successful in the isolation of specific invertebrate neurons,
because they are generally much larger than vertebrate neurons
and survive the dissection process much better. It is possible to
isolate neurons by simple hand dissection directly from fresh
tissue (Osborne, 1974; McCaman & Dewhurst, 1970; Hyden.
1959; Giacobini, 1956) or from tissues slightly pretreated with
digestive enzymes (Chen et al., 1971) or from tissues stabilized
with an ethylene glycol solution to facilitate the cell dissection
(filler & Schwartz, 1971). Another method for obtaining cells is
Table 1. Introduction of microchemical methods in neurobiology: a chronological review (from Giacobini, 1978).
First measurement of enzyme activity
with the Cartesian diver in nervous
tissue
Measurement of RNA in a single neuron
Histological sampling methods for single
neurons including weight determination of single neuron
Ribonucleotide analysis of individual
neurons
Quantitative determination of enzyme
activity in single neurons
First determination of enzyme activity
with the Cartesian diver in a single
neuron and its components
Determination with X-ray microspectrography of dry organic mass of
single neurons, lipids and proteins
Quantitative chemical analysis of
isolated glia cells
Linderstrem-Lang (1937):
Click (1938)
Hyden (1943)
Lowry (1953)
Edstrem (1953)
Lowry el al. (1956): Lowry
(1957): Robins el al.
(1957): Giacobini &
Zajicek (1956)
Giacobini & Zajicek (1956):
Giacobini (1957. 1959)
Brattgard & Hyden (1952)
Cycling technique for substrate
determination
Measurement of intracellular oxidationreduction states in single neurons
Measurement of substrates in an
isolated neuron
Introduction of radiochemical micro
methods for determination of enzyme
activity in single cells
Measurement of Na+ and K f in single
neurons with micro flame photometry
Protein analysis at the cellular level by
micro disc electrophoresis
Microchromatography of dansylated
derivatives
Assay of serotonin and amino acids in
single giant cells of the snail with
dansyl-t.1.c. micro chromatography
Assay of norepinephrine and dopamine
with gas chromatography-mass spectrometry in single sympathetic cells
Radiometric assay of acetylcholine,
hydroxytryptamine and dopamine in
single invertebrate nerve cells
Lowry (1957): Hyden el al.
(1958): Hyden (1959):
Giacobini (1959)
Lowry ef al. (1961)
Terzuolo el al. ( 1964.
1966)
Giacobini & Grasso (1966):
Giacobini & Marchisio
(1966)
Buckley el al. ( 1 967):
Consolo el al. ( 1 968)
Carlsson el al. ( 1 966,
1967)
Hyden et al. (1966)
Neuhoff el al. (1969):
Neuhoff & Weise (1 970)
Osborne ( 1 972)
Koslow el al. ( 1 972)
McCaman er al. (1973a.b)
simply to isolate them from tissue cultures. although it is
arguable whether cultured neurons can really be compared with
the same cells in the intact state.
Chemical analvsis
An arsenal of microprocedures exists for analysing a whole
spectrum of substances in isolated neurons (Table 1). Because of
their undoubted importance in neurobiology, a number of
scientists have been attracted to the analysis of transmitter
substances in single neurons. It has been estimated by
Giacobini (1975) that a single sympathetic cell body, which
measures about 30,um across its diameter. contains about
mol of noradrenaline. One would therefore require a procedure with at least this sensitivity to detect noradrenaline in a
sympathetic neuron. Only fluorimetry-cycling procedures (see
Lowry & Possonneau, 1972) have a sensitivity in this range. but
have not been applied to transmitter analysis. By using large
neurons which exist in certain invertebrates, however. the
sensitivity could be reduced by at least 3-fold. since these cell
bodies have diameters of about 120,um. Consequently, techlike gas
niques with a sensitivity of at least 10-I2~.
chromatography, fluorimetry. radiometry, gas chromatography-mass spectrometry, t.1.c.-mass spectrometry or micro
t.l.c.-dansyl(5-dimethylaminonaphthalene-1 -sulphonyl) chloride
chromatography have been used with some success in identifying
transmitter substances in isolated giant neurons (see Table 2).
Most of the examples shown in Table 2 are from invertebrate
neurons, though certain vertebrate cells have also proved
large enough for analysis.
General comments
As illustrated in this brief article, important data have already
been established through the use ui suitable techniques for the
analysis of single neurons. One limitation is the degree of
accuracy required. Working with small quantities of material
creates the problem of error caused by contamination, dirty
glassware, inefficiency of procedure or experimenter. etc., as a
micro error in the micro scale is, in effect, a macro error in the
macro scale. While qualitative methods for analysing isolated
neurons are always to be preferred, they are often very difficult
to apply with success, simply because of the scale of the system.
Nevertheless, in compensation for this deficiency, chemical
analysis of isolated neurons has already supplied us with several
kinds of information that other methods have not yet achieved,
e.g. visualization procedure.
I thank the Volkswagenwerk Stiftung for financial support.
Anfinsen, C. B., Lowry, 0. H. & Hastings. A. B. (1942) J . Cell. Comp.
Physiol. 20,23 1-237
Brattgard, S . - 0 . & Hyden. H. (1952) Acfa Radiol. Suppl. 94. 1-48
Buckley, G., Consolo, S., Giacobini. E. & McCaman, R. (1967) Acra
Physiol. Scand. 7 1.34 1-347
Carlsson, B., Giacobini, E. & Hovmark, S. (1966) Acla Phvsiol. Scand.
68, Suppl. 277
Carlsson, B., Giacobini, E. & Hovmark, S. (1967) Acla Physiol. Scand.
71,379-390
Table 2. Measurement of known transmitters in single neurons
Transmitter
Acetylcholine
y-Aminobutyric acid
Noradrenaline
Serotonin
Dopamine
Histamine
Amino acids
Amount
Method
per cell
Neuron
Aplysia giant neurons
10-12mol Enzymic
Motor neurons, Deiter’s cells 3 x 10-14mol Enzymic cycling
10-15 m o ~Micro spectrofluorimetry
L-7 sympathetic-ganglion cell
4 x 10-12mol Dansyl-t.1.c.
Giant cell of slug
5 x 10-12mol Micro t.1.c.
Giant cell of snail
lo-” mol Enzymic
Giant cell of Aplysia
1 0 - 1 3 m o ~G a s chromatography-mass spectrometry
Retzius cell of leech
I O-” mol Fluorimetry
Giant cell in snail
10-’2mol Radiometric
Aplvsia neuron
10-12mol Dansyl-t.1.c.
Giant snail neuron
Authors
McCaman ef al. (l973a)
Otsukaeral. (1971)
Giacobini er al. (1 970)
Osbcsne & Cottrell (1972)
Osborne (1971)
McCaman ef al. (3973b)
McAdoo (1978)
Powell & Cottrell (1974)
Weinreich el al. (1975)
Osborne (1977)
1981
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Techniques for the microinjection of single cells
C. MULLER, A. G R A S S M A N N and M. G R A S S M A N N
Institut f u r Molekularbiologie und Biochemie d e r Freien
Universitat Berlin, Arnimallee 22, 1000 Berlin 33, West Berlin,
a micromanipulator and connected t o a sturdy syringe as a
source of positive or negative pressure, enabling the investigator
t o fill the pipette through the tip or t o deliver material into a cell.
Germany
A volume of 10-lO-lO-llml can be placed either into the
nucleus or into the cytoplasm of a single cell. In order t o
facilitate the reidentification of injected cells, recipient cells are
grown on slides subdivided into numbered squares of I m m 2 .
Suspension-culture cells are prepared for microinjection by
binding them t o a substrate via a suitable linker, e.g., antibodies,
concanavalin A (Graessmann et al., 19806). U p t o 1000 cells
may be injected serially per hour and, on condition that the
injected material had n o cytopathic effects, recipient cells
respond efficiently and remain as viable as their uninjected
neighbours. Every tissue-culture cell line tested so far proved
suitable for microinjection, and virtually no limitations exist
regarding the material t o be transferred: intact virions, D N A
and R N A species, proteins and small metabolites a s well as
substances unrelated t o cellular metabolism have been microinjected successfully.
Standard techniques are employed to assay events in injected
cells: fluorescence microscopy to follow the fate of fluorescently
labelled injected materials: immunofluorescent staining techniques t o identify newly made gene products: autoradiography
to detect altered metabolic activities: virus-plaque assays t o
observe the synthesis of infective virions; cell-cloning techniques
t o identify cells with altered growth properties. Analysis of
newly synthesized polypeptides by polyacrylamide-gel electrophoresis is feasible with some hundred injected cells by
combining the techniques of immunoprecipitation and
fluorogrsphy (Celis et al., 19806: Richardson et al., 1980), and
biochemical studies on the fate of injected materials is further
facilitated by using a s recipients multinucleated cells I fused by
Most of the techniques currently in use for the transfer of
biological macromolecules into tissue-culture cells employ
carrier systems (e.g., liposomes, erythrocyte ghosts, calcium
phosphate/DNA co-precipitates) and/or fusion agents le.g.,
poly(ethy1ene glycol), Sendai virus1 t o mediate their uptake.
Since whole-cell cultures are processed with these techniques,
they are especially suited for biochemical analyses of the fate of
the material transferred. However, since an individual target cell
cannot be predetermined, these approaches are inadequate for
the study of events in single cells (for reviews, see Celis et al.,
1980a).
Direct microinjection of single tissue-culture cells with glass
micro-pipettes, developed independently in o u r laboratory
(Graessmann, 1968; Graessmann & Graessmann, 1971) and by
Diacumakos’ group (Diacumakos et al., 1970), allows the
introduction of biomolecules in purified form without the
involvement of helper macromolecules or chemical treatment.
T h e technical aspects of microinjection and the accessories
required have been described in detail fairly recently
(Graessmann et al., 1980a). In brief, micropipettes are drawn
from commercial glass capillaries to a tip diameter of about
0.5,um with the aid of a mechanical puller. Filling of the pipette
and microinjection are performed under a phase-contrast
microscope: the pipette is inserted into the instrument holder of
* Abbreviations: SV40, Simian virus 40; cRNA, complementary
RNA.
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