Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.
),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:112-114.
THE BRAIN SLICE TECHNIQUE
Shankaranarayana Rao BS and Raju TR
The brain slice technique was first applied to
metabolic studies of small tissue sections as early
as 1920. With the advent of the microelectrode
technique at the beginning of the 1950s it was
possible to characterize the resting membrane
potentials of cells contained in freshly sliced tissue
and compare them to brain cells in situ. It was soon
realized that such slices provided new routes for
the study of synaptic phenomena. Towards the end
of the 1960s the use of surviving, metabolically
maintained tissues from the brain for
electrophysiological and pharmacological studies
was becoming an accepted, valued and widely
applied technique. Brain slice technique became
very popular after the advent of the whole-cell
recording technique in the mid 1980s and its
application to sections of brain tissue (Sticker 1997).
The Brain Slice preparation and maintenance for
electrophysiological recordings
The in vitro brain slice preparation often is used
to study processes involved in synaptic plasticity
and to evaluate the role of native receptor subtypes
in neurotransmission. Brain slices have been used
for physiological studies since the early 1970’s, and
recording from neurons in brain slices using patch
pipettes gained widespread use in the late 1980s,
often with the aid of video microscopy. Brain slices
allow recording from semi-intact neural circuits,
with the advantages of mechanical stability and
control over the extracellular environment. Whole-
cell recording in brain slices is often combined with
imaging techniques and indicator dyes to measure
intracellular pH, calcium concentration, etc. It can
also be combined with retrograde tracing
techniques to record responses from neurons that
project to a certain brain area. Brain slices are used
for a wide variety of studies including synaptic
plasticity and development, network oscillations,
intrinsic and synaptic properties of defined
neuronal populations, and many others
(Dingledine, 1984; Kettnmann and Grantyn, 1992).
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Depending on the experimental needs several
variants of the technique have been developed and
are being used. One distinction between the
variants relates to how thick the slices have been
cut. The thin slice technique was developed to allow
visualization of individual cells in slices less than
250µm while the thick slice technique is used in
experiments where connectivity and maintenance
of normal dendritic structure are crucial for the
study. Once prepared, slices can be kept alive in
various media for hours or days. Ultimately slices
can be kept in culture.
Best conditions for brain removal
Animal should be treated under the less stressful
conditions. Thus, animals should be housed in their
cages just till the beginning of the experiment. The
fastest and easiest way of animal execution is by
decapitation, although alternatively halothane (for
anesthesia), or CO2 (suffocation) could be
employed. These last two methods are less
recommendable, because they take more time, and
could produce some neuronal damage prior to the
brain removal. Anesthesia application is not
advisable, because of the possible interference of
the drug in the neural physiology. Once animal is
decapitated, skull should be removed with scissors
(scalpel is not recommended), cutting along the
sagital axis from caudal (posterior) to rostral
(anterior) part, and then open the two skull pieces
laterally. For shelling out the brain introduce the
forceps or the scissors in the caudal part, and
remove backwards the whole organ. Place it
rapidly (preferably less than 60 seconds) into
oxygenated ice-cold saline or artificial cerebrospinal
fluid. This solution should be so cold that it contains
a few ice crystals and to maintain this temperature
the container should be sitting on ice.
Faster dissection- better results
The process of brain removal should be gently
accomplished, but as fast as possible. The brain
should be placed in oxygenated ice-cold saline or
artificial cerebrospinal fluid. There, it should be
cooled for 3 minutes to 10 minutes. Next, the brain
is ready for slicing or dissection. To reduce damage
to the tissue, it is particularly important to keep
the brain immersed in oxygenated ice-cold saline
or artificial cerebrospinal fluid during the whole
process.
Slices should be kept at about 32-37°C in
oxygenated saline or artificial cerebrospinal fluid,
for at least 30 minutes before recording. Once this
time is over, the remaining slices are kept at room
temperature (25°C) before use, for a maximum of
5 - 10 hours.
Slicing procedure
Despite the many different procedures
employed, the main goal is to prepare a slice of
tissue where the neurons, fibers, synapses and glia
that are important to the experiment are in a viable
condition. The animals used in preparing slices are
most often small rodents; guinea pig, rat and
mouse. Young animals have some advantages for
the slice preparation. Their skulls are soft, and
therefore easier to remove. Since the brain is
smaller, cools more rapidly (3 minutes are usually
enough) when placed in ice-cold solution. About
the tissue, older animals are more susceptible to
anoxia, and the neuronal damage is higher as time
goes. More myelination and the presence of more
connective tissue may result in more damage to
the cells, and their processes during slicing, and in
a worse acquisition during the electrophysiological
recordings.
Once the tissue is removed, it is normally cooled
down by placing it in an ice-cold oxygenated
artificial cerebrospinal fluid (ACSF) to minimize
the metabolic activity. Cutting is done in ice-cold
ACSF using a vibratome. The angle and vibration
frequency (usually near the maximum) of the blade
should be adjusted to prevent the tissue being
pushed while cutting the slices. Slicing should be
accomplished in less than 10 minutes. Take
carefully the brain slices using a cut and fire
polished Pasteur pipette filled with the ice-cold
solution. A standard slice is cut at 300-400mm
thickness. Regions of the slices that are thicker than
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this exhibit centrally-located necrotic cells
suggestive of hypoxic damage. The slices are then
incubated at a temperature of around 360C for at
least 1 hour. Oxygenation and normal pH are
maintained by bubbling the ACSF with 95%O2 /
5%CO2. This allow the tissue to recover from the
damage imposed by the preparation and adjust to
the new extra cellular milieu as well as to changed
metabolic activity.
Composition of the artificial cerebrospinal fluid
(ACSF)
The artificial cerebrospinal fluid should be
freshly prepared for each experiment (quantities
in grams are given in brackets for 2 litre solution
preparation). It contains NaCl 126 mM (147.3 g),
KCl 2.5 mM (3.7 g), NaH 2PO4 1.25 mM (2.9 g),
MgCl 2 1.3 mM (2.48 g), CaCl 2 2.4 mM (7.1 g),
Glucose 11mM (4 g) and NaHCO3, 25 mM (4.2 g),
with a pH of 7.3 when gassed with 95% O2 and
5% CO2. If you want to create some stocks, you
can prepare the inorganic component (NaCl, KCl,
NaH2PO4, MgCl2 and CaCl2), and just before the
experiment add the Glucose and NaHCO 3 . To
adjust the pH correctly, solutions should be bubbled
for at least 30 minutes. This solution could be
employed as extracelullar solution in the patch-
clamp experiment.
The above mentioned composition of ACSF
reflects the basic requirements for maintaining
healthy slices. Depending on the experimental
needs, the composition might have to be adjusted
considerably.
Slice Chambers
For experimental use slices must be kept in an
environment providing appropriate oxygenation,
pH, osmolarity and temperature. In addition,
depending on the techniques used, it is necessary
to have excellent visual control, good mechanical
access and stability. Most commonly used
chambers allow superfusion of ACSF across the
slice. This imposes special demands on the
mechanical stability of the superfusion system.
Most of the slice chambers are temparature
controlled and the superfusion rate of the ACSF is
atleast 1ml/min. The flow rate determines the O2
and 5% CO2 escape by diffusion as the perfusate
travels along the tubing supplying the chamber.
Submersion chambers are normally used for whole-
cell patch recording, whereas interface chambers
are better suited for monitoring extracellular field
potentials (Sticker 1997).
Temperature
Although the body temperature of small rodents
is around 38oC, most investigators maintain the
slices at 30-35 oC in the experimental chamber.
There are two reasons for this, firstly, it has been
found that preparations survive longer and in a
healthier state at the lower temperature. Secondly,
the higher humidity resulting from warmer
solutions leads to the formation of droplets on
recording and stimulating electrodes. Many
researchers depending on their experimental design
tend to work at room temperature.
Cell visualization
The real advantage of the slice is its accessibility,
especially the visibility of structures such as cell
body layers. Thin slices of (250-300µm) allow a
Advantages and disadvantages of brain slice preparations
Advantages
1 Direct visualization
2 Technical accessibility
3 Mechanical Stability
4 Ease of use
5 Control of extracellular medium
6 Precise control over concentration
of drugs
References
1. Dingledine R (1984) Brain slices, Plenum Press, New York.
2. Kettnmann H and Grantyn R (1992) Practical
electrophysiological methods. Wiley-Liss Inc. New York.
greater optical resolution due to smaller effect from
light scattering. The slices from younger animals
have thinner myelin sheets, which also improves
visibility. The cells and parts of the dendrites can
be visualized using a 40 times high numerical
aperture, long working distance water immersion
objective. To further improve the contrast between
different cells, Normaski or Hoffman optical
arrangements are preferred.
Electrophysiological evaluation of brain slices
The assessment of electrical parameters of slices
depends on the characteristics of the particular cells
within the tissue and varies from brain region to
brain region. The basic indicators include resting
membrane potential, input resistance and
amplitude of the action potential. More sensitive
measures include the ability of the cells to produce
a regular, rhythmic train of action potentials after
the injection of a small current. Damaged neurons
will often respond with a single action potential at
the onset of the current pulse. Besides direct cellular
parameters, amplitudes of extracellular fields
reflect the synaptic action and are convenient for
assessment of the overall state of a slice or at least
of small regions within a slice (Sticker 1997).
Disadvantages
Loss of afferent and efferent connectivity
Shearing of dendritic processes and axons;
Damage to neurons and glia
Tissue debris around the cutting surface mixed
with healthy cells
Slow release of cellular enzymes and ions
from damaged cells
Altered metabolic state; the artificial conditions may
alter cellular metabolism in many ways
Loss of vascular and hormonal regulation
3. Sticker C (1997) Slices of brain tissue. In: Neuroscience
Methods, Martin R (ed.) Harwood academic publishers,
Australia. pp 3-10.
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