Neurotransmitter Methods

266 Pages·1997·18.125 MB·English

by  Richard C. Rayne

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Preparation of Brain Slices
Ting Wang and Ira S. Kass
1. Introduction
The brain-slice technique has been utilized in electrophysiological, morph-
ological, biochemical, and pharmacological studies of almost all brain
structures. A search of the literature between 1991 and 1995 on the Ovid
Medline revealed 4387 entries that used the brain-slice technique; of these,
2038 are relevant to the study of receptors and neurotransmitters. This tech-
nique is widely used because it has many advantages over in vivo methods.
It provides precise control over experimental conditions, such as temperature,
pH, and drug concentration. It also allows the examination of metabolic
parameters and electrophysiological properties without contamination from
anesthetics, muscle relaxants, or intrinsic regulatory substances. The stability
of electrophysiological recordmg is greatly improved as the heart beat and
respiration of the experimental animal are eliminated. The cells being studied
can be located, identified, and accessede asily. Use of the brain slice has greatly
increased our knowledge of the mammalian central nervous system (CNS). This
technique is contmuously improving and will remain valuable for a long time.
Preparation of brain slices is an indispensable procedure for a variety of
experiments. The goal of the slice preparation is to obtain a thm piece of brain
tissue containing the cells of interest and to maintain the slice m a viable
(although artificial) condition that is similar to its in vivo environment. In this
chapter we describe procedures that are fundamental for brain slice prepara-
tion, Because the hippocampus is the most widely used tissue for brain slices,
Its isolation will be used here to illustrate the steps of the procedure. Two slic-
ing methods, using either a vibratome or a tissue chopper, are described. Our
discussion of these methods covers the steps from the decapitation of the ani-
mal to the storage of slices in artificial cerebrospinal fluid (aCSF). Some of the
From Methods m Molecular Bro/ogy, Vol 72 Neurotransmrtter Methods
Edlted by. R C Rayne Humana Press Inc , Totowa, NJ
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2 Wang and Kass
notions and dogma mentioned throughout are derived from the authors’ per-
sonal experience or anecdotal reports. There is no standard procedure for the
preparation and the existing technique is not perfect. Beginners are encouraged
to explore new approaches and test alternatives.
1.1, Preparation of Artificial Cerebrospinal Fluid
The aCSF mimics the extracellular environment of neurons and provides the
necessary ingredients that allow neurons to survive m vitro for at least several
hours. Because metabolic processes and the maintenance of the ionic gradient
across the cell membrane constantly consume ATP, glucose and oxygen must
be supplied to generate energy.
The common ingredients in aCSF are NaCl, KCl, IGI2PO4, NaHC03, glu-
cose, MgS04, and CaC12. Although the ideal composttton of aCSF mirrors that
of the cerebrospinal fluid in vivo, investigators often use an aCSF that differs
from CSF. The concentration of the rontc components of the aCSF vary slightly
from lab to lab to meet different experimental purposes. For example:
1, The response of the NMDA receptor to its agonist is reduced by raising the con-
centration of magnesium.
2. The excrtabihty of neurons is increased by raising the potassmm or lowering the
calcium or magnesium concentrations
3. Cells m slices survive longer and withstand anoxra better in solutions with higher
glucose concentrations (I) or lower potassium concentrations (2).
The alteration of the aCSF ingredients obviously affects the electrophysi-
ological properties of the neurons in slices. Our experiments showed dramatic
changes in the firing rate, the tiring pattern, and the shape of action potentials
of CA1 neurons when the ionic concentration of the aCSF was changed
(unpublished observation). Caution must be exercised to keep the osmolarity
and pH constant when one alters the composition of aCSF. It is important to
bear in mind that multiple biological effects may be produced by changing a
single component of the aCSF. For example, raising the concentration of
potassium not only increases the excitability of the neuron, but also increases
the activity of Na/K-ATPase (3).
1.2. Anesthesia, Decapitation,
and Removal of the Brain from the Skull
The animal should be deeply anesthetized before being sacrificed. Inhala-
tion anesthetics, such as ether, halothane, isofluorane, or methoxyfluorane, are
often used. However, some investigators prefer such anesthetics as ketamine
or chloral hydrate, which need to be injected. For some experiments, the use of
anesthetics may complicate the interpretation of the experimental results,
Preparation of Brain Slices 3
especially in studies of anesthetics themselves. Decapitation with a small guil-
lotine is one method of sacrificing animals without an anesthetic. In this case,
guidelines for the use of animals in experiments must be followed carefully.
After the animal is sacrificed, the skull should be opened quickly and the
brain removed into chilled aCSF (44°C). The low temperature slows down
the metabolic rate of cells and reduces their energy consumption. These effects
allow cells to survive the ischemic period during brain slice preparation. The
brain also becomes more firm when the temperature is lowered, facilitating the
dissection and slicing of the brain.
1.3. Oksection of the Brain Tissue
In order to facilitate slicing, it is best to prepare from the whole brain a
tissue block containing the cells of interest. This is achieved by cutting away
the excessive surrounding brain tissue with a sharp blade, but intentionally
leaving part of the surrounding tissue intact to protect and support the region of
interest when it is later sliced. The shape and size of the tissue block varies
according to the brain region targeted for slicing. At least one flat surface
should be created on the tissue block for mounting onto the slicing stage. Mak-
ing the dissection on a chilled platform and repeatedly pouring chilled aCSF
over the brain tissue will keep the temperature low and increase the viability of
the slices.
7.4. Mounting onto the S/icing Stage
After the tissue block is made, it is mounted onto a stage for slicing. For the
vibratome method, the tissue block must be glued to the slicing stage.
Cyanoacrylate (Krazy glue, Super glue) or Histoacryl is commonly used for
this purpose. Although a toxic effect of cyanoacrylate glue has been suspected
(4), no systematic study on its toxicity has been found in the literature.
To prevent the tissue block from leaning back because of the advancing
vibratome blade, the tissue block may be supported by an agar gel block. Some
authors use Plexiglas instead of agar, but the edge of the blade will be damaged
if it touches the Plexiglas. When a tissue chopper is used, a piece of filter paper
moistened with aCSF is placed between the tissue block and the slicing stage.
This will minimize the variation in the slice thickness and orientation, because
the filter paper reduces sliding of the tissue block during slicing.
1.5. S/icing
The vibratome and tissue chopper methods are two major techniques used to
slice tissue blocks. The vibratome seems to produce better slices and is gaining
popularity. The available commercial vibratomes include the Vibratome series
from Technical Products International (St. Louis, MO), and the Vibroslice
4 Wang and Kass
series from Campden Instruments (Loughborough, UK). One advantage of
vibratome slicing is that the whole tissue block is submerged in cold aCSF;
thus, the tissue temperature can be kept low and the slices gain immediate
accesst o the oxygen-saturated aCSF during slicing.
The tissue chopper, on the other hand, has the advantage of low cost and
faster slicing. The available commercial tissue choppers Include the Tissue
Slicer from Stoelting (Wood Dale, IL) and McIlwain Tissue Chopper (Mickle
Laboratories Engineering, Gromshall, Surrey, UK). Slicing is improved if
the subarachnoid membrane is removed.
Most commonly, slices of 300-500 pm are made. This thickness range pro-
vides good cell preservation without compromising the diffusion of oxygen
into the core of the slice. Thinner slices are preferred for optical experimental
techniques m order to visualize cells; however, the cells within about 50 pm
from the cut surface may be damaged. Commercial razor blades are used for
the slicing and are usually shipped covered with a thin film of oily material to
protect against oxidation. Blades should be cleared of the film with 75% alco-
hol and rinsed thoroughly with dlstilled water before use.
1.6. Storage of the Slices
Brain slices can be kept viable at 20-37°C for several hours in an aCSF-filled
storage chamber aerated with 95% O2 and 5% CO*. We routinely use slices for
intracellular recording after up to 6 h of storage. Another lab has reported
viability for 24 h (5). The following factors may contribute to the viability of
the slices:
1. The brain tissue should be handled delicately to reduce physical damage; it should
not be squeezed or twisted.
2. The preparation time should be short, a prolonged lschemic period 1s associated
with a fall in intracellular ATP and reduction in the recovery of population spike
(6-8) Although most investigators think that the brain slice preparation should
be accomplished in ~5 min, another group found that there was no significant
change in the amplitude of the population spike even with 30 mm postmortem
delay at room temperature (9).
3. Lowering the brain tissue temperature during the preparation 1s effective in
reducing the sensitivity of neurons to ischemla.
4. In order to reduce the neurotoxicity of the glutamate that may be massively
released during slice preparation, some authors use an aCSF containing 10 mM
magnesium and 0 5 m.M calcium for slicing and storage (10,ll)
Because the slices survive for a long period in the aCSF, and more than one
slice can be obtained from most brain regions, multiple experiments can be
performed with slices from one animal. This is especially useful in pharmaco-
logical studies, because the slices can be moved individually from the storage to
Preparation of Brain Slices 5
the recording chamber and then discarded after each drug test. Each slice can,
therefore, be examined without contamination by the previously tested drugs.
An incubation period of 1 h is necessary for cells to recover from the
ischemia and physical trauma of preparation. The transfer of slices from
the storage chamber to the experimental chamber should be made by using
a Pasteur pipet with its narrow opening attached to the rubber bulb (see
Section 3.15.). If the temperature or a component of aCSF in the experi-
mental chamber is different from that in the storage chamber, 20 min should
be allowed for the slice to adapt to the new environment in the experimen-
tal chamber.
The brain slices may be further treated to isolate indrvidual neurons or may
themselves be maintained in culture media for several weeks. For detailed
descriptions of slice culture and neuronal isolation, please see Chapters 2 and 3.
1.7. Shortcomings of the Brain Slice Technique
No matter how healthy the slice may appear, and how closely the compo-
nents of the aCSF mimic the m vivo cellular mdieu, the slice is surrounded by
an artificial environment, Many substancest hat are present in vivo and impor-
tant in regulating neuronal function, such as trophic factors and amino acids, are
not included in the aCSE Although local neuronal circuits may be preserved,
the synaptic connections of the slice to and from other brain regions are inter-
rupted. The effect of the artificial extracellular environment and the mterrup-
tion of synaptic input is illustrated by the change of the firing pattern of midbrain
dopamine neurons recorded in slices. In contrast to the irregular firing pattern
recorded in vivo, an extremely regular pacemaker-like firing pattern is a charac-
teristic feature of the dopamine neurons recorded in vitro (22,13). This pace-
maker-like firing pattern has never been observed in vivo. The change of firing
pattern may reflect a liberation of the cells from synaptic innervation, revealing
their intrinsrc activity. Alternatively, this may represent abnormal electrophysi-
ological activity because of the altered cellular environment.
2. Materials
2.1. The Vibratome Method
2.1.1. Preparation of the aCSF
The artificial cerebrospinal fluid (aCSF) contains 124 mMNaC1,2 mMKC1,
1.25 mM KH2P04, 26 mA4 NaHC03, 10 mM o-glucose, 2 m&f MgS04, and
2 mA4 CaQ. Dissolve the following in ultrapurified water and bring the final
volume to 1000 mL,: 7.25 g NaCl, 0.149 g KCl, 0.17 g KHzPO,+2, .18 g NaHC4,
1.8 g glucose, 0.24 g MgS04, and 0.294 g CaC12. Note that CaC12 should be
6 Wang and Kass
added last and as a dissolved solution. The final pH of the aCSF is 7.4 after it is
saturated with a 5% CO2 and 95% 02 gas mixture (see Notes 1,2, and 3).
2.1.2. Anesthesia, Decapitation,
and Removal of the Brain from the Skull
1. Halothane
2. Small animal guillotme.
3. One pair of straight surgical scissors (sharp/blunt, 16 cm)
4. One bone cutter (16 cm).
5. Spatula (20 cm)
6. Beaker (20 mL).
2.1.3. Preparation of the Brain Tissue Block
1. One Petri dish (60 x 10 mm).
2. Filter paper (55 mm, diameter).
3. Razor blade (Gillette, superstainless).
2.1.4. Mounting onto the Slicing Stage
1. Cyanoacrylate glue (Krazy glue)
2 Agar gel block (see Note 4).
3. Two flat-end spatulas (20 cm).
2.15. Slicing
1. Vrbratome (Vtbratome Series 1000, Techmcal Products Intemattonal).
2. Razor blade (Gillette, superstainless)
3. Glass Pasteur pipet with a rubber bulb
4. Alcohol.
2.1.6. Storage of the Slices
I. Beaker (200 mL).
2 95% O2 and 5% CO2 gas mixture
2.2. The Tissue Chopper Method
2.2.1. Preparation of the aCSF
Identical to the vibratome method (see Section 2.1.1.).
2.2.2. Anesthesia, Decapitation, and Removal
of the Brain from the Skull
Identical to the vibratome method (see Section 2.1.2.).
2.2.3. Preparation of the Brain Tissue Block
1. Petri dish (60 x 10 mm).
2. Filter paper (55 mm diameter).
3. Flat-end spatula (20 cm).
Preparation of Brain Slices 7
22.4. Mounting onto the Slicing Stage
1. Flat-end spatula (20 cm).
2. Filter paper (55 mm diameter).
2.2.5. Slicing
1. Tissue chopper apparatus (Stoelting Tissue Slicer)
2. Razor blade (Gillette, superstainless).
3. Sable #2 (fine) artist’s brush.
2.2.6. Storage of the Slices
Identical to the vibratome method (see Section 2.1.6.).
3. Methods
3.1. Vibratome Method
3.1.1. Anesthesia, Decapitation,
and Removal of the Brain from the Skull
1. Mount an agar gel block (see Note 4) on the vibratome stage with Krazy glue. A
tissue block, when prepared, will be placed next to rt (see Section 3.1 3 ).
2. Anesthetize the rat with halothane in a glass desiccator (see Note 5)
3. When the rat falls asleep, decapitate it using a small animal guillotme (see Note 6).
4. Expose the skull and cut along the sagittal suture from the foramen magnum to
the forehead with a pair of surgical scissors (see Section 2.1.2.); the sharp pointed
blade of the scissors should be placed on the inner side of the skull.
5. Using a bone cutter, make one cut at the foramen magnum on each temporal side
of the skull and expose the brain by carefully prying the skull open.
6. Hold the skull upside down and, using a spatula to sever the cranial nerves that
hold the brain to the skull, allow the brain to fall mto a 20 mL beaker containing
chilled aCSF.
3.1.2. Preparation of a Brain Tissue Block
that Contains the Hippocampus
1 Place the cooled brain with its dorsal side up and cerebellum toward you on a
piece of filter paper (see Note 7) in a Petri dish. Place ice under the Petri dish to
keep the brain cool.
2. Pour chilled aCSF into the Petri dish until the brain is half submerged
3. The two hippocampal formattons in the brain resemble an inverted V as viewed
from above (see Fig. 1A). Each hippocampus 1s a U-shaped structure with the top
opening of the letter U facing forward and medially (see Fig. 1 B). If the hippoc-
ampus on the right side of the brain is used, the blade should be placed 45’ across
the longitudinal fissure at a point one-third from the posterior end (see Fig. 1A)
and rotated 15” back from the vertical plane (see Fig. 1B).
4. The surface of the first cut is roughly perpendicular to the longitudinal axis of the
dorsal portion of the right hippocampus (stippled area m Fig. IB), and will be
used for mountmg onto the shcmg stage.
Wang and Kass
The 2:d Cut
The 3rd Cut \ HipP-mp-
Cerebral
Cortex
C
Fig. 1. Preparation of a tissue block containing a portion of htppocampus. (A) View
of the rat brain from above. The hrppocampus, represented by the dashed lines, IS
covered by the neocortex and IS not visible from above. The two solid lines across the
brain indicate the positions where the first and the second cuts are made Note that the
blade should be rotated 15” back from the vertical plane as shown m B (B) Lateral
view of the brain showing the right hippocampus. The strarght dashed lme IS
perpendrcular to the surface of the neocortex. The solid lines across the brain are 15”
offset from the dashed lme and indicate where the first and second cuts are made. Five
to six slices can be obtained from the stippled area that IS perpendicular to the longitu-
dmal axis of hippocampus. (C) Vrew of the second cut surface. Make the third cut
perpendicular to the surface of the second cut along the lme shown in the figure.
5. The second cut is parallel to the first one, and crosses the midpoint of the poste-
rior border of the right cerebral cortex (see Ftg. 1A). The tissue block between
the two cuts contains the dorsal portion of the hippocampus The cutting angles
can be adjusted to obtain other portions of the hippocampus.
6. The thrrd cut should be made perpendicular to the surface of the second cut and
along the line between the cerebellum and the cerebrum (see Fig. 1 C). Make the
third cut l-2 mm away from the cerebrum to avoid damaging the hrppocampus.
The surface of the third cut will be placed against the agar block on the slicing
stage (see Fig. 2). Some authors use different methods to make the hrppocampal
tissue block (see Note 8).
Preparation of Brain Slices 9
Cerebral Cortex
Slicing Stage
Fig. 2. Arrangement of the agar and tissue blocks on the slicing stage. Note that the
surface of the third cut of the tissue block is placed against the agar block.
3.1.3. Mounting onto the Slicing Stage
1. Spread a thm layer of cyanoacrylate glue on the slicing stage (see Notes 4 and 9)
to which an agar block has already been mounted (see Fig. 2).
2. Shovel the tissue block off the Petri dish with a spatula and place it on a piece of
dry filter paper with the mounting surface down. Wait for about 2-3 s and allow
the fluid on the mounting surface to be drained away by the filter paper. While
waning, dry the spatula by rubbing it on the filter paper
3. Using the spatula, shovel the tissue block from the filter paper onto the slicing
stage and place it next to the agar block (see Fig. 2). Withdraw the spatula while
using another spatula to gently hold the tissue block m place.
4. Clamp the slicing stage onto the vtbratome specimen vise with the agar block
furthermost from the blade (see Fig. 2) The tissue block and the blade should be
totally submerged in cold aCSF (4-8”C) that had been poured into the vibratome
and aerated with 95% 0, and 5% CO2 before sacnticmg the animal
3.1.4. Slicing
1. Adjust the vibratome section thickness to 400 pm and the amplitude of vibration
to maximum (Vibratome series 1000, Technical Products International).
2. Position the sectionmg blade at a 20° angle and advance tt forward, mto the tis-
sue block, at a speed that does not distort the tissue while slicing
3. Discard the first few shces until a cutting plane that is roughly perpendicular to
the longitudinal axis of the hippocampus is reached (shaded area m Fig. 1B). The
10 Wang and Kass
Fig. 3. Storage of brain slices. The shces are incubated in aCSF aerated with a 95%
0s and 5% COs gas mixture. The crosshatched area represents the nylon net on which
the slices are placed. A Pasteur pipet with its narrow opening attached to the rubber
bulb is used for transferring slices
hippocampus is sliced along with part of its surrounding tissues. Separate the latter
from the hippocampal slices using forceps and fine scissors after all the slices
have been made.
3.1.5. Storage of the Slices
1. Immediately after slicing, move each slice to a storage chamber using a Pasteur
pipet with the bulb placed over the narrow opening (see Fig. 3).
2. Incubate the slices in the storage chamber at room temperature (see Note 10) for
60 mm before use. A 200-n& beaker containing aCSF bubbled with 95% O2 and