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- Weigh out salts using a fine balance to make up
two liters of artificial cerebrospinal fluid
solution, ACSF.
Top it up with deionized water to around one point nine
liters, leaving enough room to add other components later.
Shake well and make sure all salts have dissolved.
Bubble ACSF with carbogen, 95% oxygen, 5% carbon dioxide,
for 15 to 20 minutes to stabilize the ph to seven point
three to seven point four.
Afterwards add four point eight mils of one molar
calcium chloride,
and two point six mils of one molar magnesium chloride
using a suitable pipette to give a final concentration
of two point four mini molar and one point three mini molar.
Top it up with deionized water to two liters.
Bubble for a further 15 minutes with carbogen.
Then chill one liter of ACSF over ice until it is cold,
at or below four degrees C.
Mount a double edge razor blade onto the knife holder,
screw to tighten into place.
Set the microtome to the desired thickness, two to 500
microns for brain slices, and adjust the cutting speed
to the desired setting.
In this case number three or nought point one five
millimeters per minute.
Place the brain into pre chilled ACSF solution.
For hippocampal sections, trim the brain by cutting off
the cerebellum, which provides a flat surface to mount
the brain with,
and a small part of the prefrontal cortex.
Mount the brain, cerebellum side, onto the microtome
specimen disk using superglue.
Orienting the sample such that the cortex faces
the razor blade.
Add a supporting piece of a guard behind the brain
away from the slide of the vivo tome to provide structural
support during the slicing.
Set the limit stop positions of the microtome to define
the start and stop positions of the slicing.
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- Set the frequency of the microtome to maximum, 100 hertz.
Use the uprocker button to move the buffer tray and brain
to a position where the exposed surface is just below
the razor blade edge and press start to begin
the brain slicing.
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It is important to continually top up the chamber with
cold ACSF solution to keep the brain tissue cool
while slicing.
If unfamiliar with brain anatomy, use a reference such
as Paxinos and Watson, the rat brain atlas, to ensure
correct slicing of the brain area.
Using a transfer pipette, transfer each individual brain
slice containing the region of interest from the buffer tray
to a clean Petri dish prefilled with chilled ACSF.
Carefully hemisect the brain down the midline
and transfer the individual slices to a holding chamber
prefilled with ACSF.
Store the slices for at least one hour at room temperature
to allow the brain tissue to recover from the mechanical
shock of slicing.
Prepare intra cellular recording solution into a one mil
eppendorf tube from preprepared stock solutions.
Here we added alexa six three three dye to a final
concentration of 50 micro molar to allow for further IHC
experiments of the recorded brain neurons.
Make an electrode filler by melting a one mil plastic
Pasteur pipette over a bunsen burner flame.
Once the Pasteur pipette has turned opaque within the flame
pull each end apart to stretch the plastic and once cooled
down, cut allowing enough length to reach the bottom
of the recording pipette.
Fill the electrode filler with the intracellular solution.
Fabricate a glass recording pipette by using appropriate
glass capillaries and pipette puller.
It is essential to ensure that the batch specific
molecular weight is used to determine an accurate
concentration of test compounds.
On all our cam compounds, the batch specific molecular
weight is clearly displayed on the vial.
Prepare the test compound and make the stock solution
by weighing out the compound using a fine balance.
Stabilization instructions can be found on the certificate
of authenticity accompanying your product or on the website.
Some products may be difficult to solubilize and you may
find that rapid stirring, warming in a water bath
or saunication of the solution may help.
Solubility is temperature dependent, as such cooling or
freezing solutions may lead to precipitation of a product
out of solution.
It is therefore important to ensure that your product is
completely redissolved before use.
After making up the stock solution, alacort at the desired
volume and freeze until use.
On the day of experimentation, defrost a single tube and
add it to the ACSF to achieve the final working
concentration that will be used in the experiment.
Now the solutions have all been produced, the recording
can start.
Fill up a bottle with ACSF solution and testing
compound solution.
Bubble both with carbogen and adjust the flow rate of the
ACSF solution to approximately five mils per minute
using metal Hoffman clamps.
After one hour incubation at room temperature,
carefully place the brain slice into the recording chamber
using a transfer pipette or small brush.
Top up the chamber with ACSF solution.
Move the slice into place using a small brush,
and secure the slice with reference electrode.
Fill the glass recording pipettes with intracellular
solution using the pipette filler,
making sure the solution is all the way down at the tip
of the pipette.
Flick to get rid of any air bubbles.
Attach the pipettes to the electrode holders of a
packed clamp amplifier head stages and turn into position.
Using fine control micro manipulators descend the recording
pipettes to the region of interest within the brain slice.
Here the ca one region of the hippocampus.
If required use a course manipulator to position an
appropriate stimulating electrode again to the appropriate
region of the brain slice to stimulate inputs to
recorded neurons.
Here this is within the Schaffer collaterals of
the hippocampus.
Use the control dials of the micro manipulator to descend
the recording pipette into the ACSF solution covering
the brain slice.
Open the seal test function of the computer controlled
acquisition software and monitor the resistance of
the recording pipette.
The ideal resistance when filled with intracellular
recording solution is between five and eight mega ohms.
Using the pipette, offset or track functions of the
recording amplifier, set the offset current to zero prior
to advancing the electrode into the brain slices with
a micro manipulator.
At the same time apply pressure to the recording electrode
via a syringe connected to the electrode holder.
When a neuron is encountered, the conductivity through
the recording electrode is increasingly reduced as the tip
of the recording pipette gets close to the
neuronal membrane.
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Gentle positive pressure is applied via the syringe
to clear the tip of the recording pipette of any debris
that may occlude it as the recording electrode is
lowered through the brain slice.
Once close to a neuron, the height of the injected current
step will have fallen to approximately 20% of its
starting value.
Negative pressure is then applied via the syringe to
obtain a giga ohm seal and negative current applied to
the electrode via the acquisition software.
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After allowing the giga ohm seal to improve over time
we apply further negative pressure via the syringe to
rupture the membrane and gain direct access to the cytoplasm
of the neuron while maintaining a tight seal to prevent
current leakage.
We now have wholesale access to the recorded neuron,
as defined by the increased capacitance transients
visible in our current step.
Using the packed clamp amplifier the capacitance transients
are progressively canceled and electrophysiological
experiments can begin on the recorded neuron.
Using the computer controlled acquisition software
the health and electrophysiological properties of the
recorded neuron are assessed.
A current voltage relationship is constructed by injecting
square wave negative and positive rectangular current
injection of constant increment into the neuron.
The smooth membrane trace and action potential firing on
the positive current steps indicates the health of the
recorded neuron.
In the current clamp recording mode of the whole cell
packed clamp technique the neuronal membrane responses
to applied test compounds can be assessed.
In order to test the affects of test compounds,
switch the recording solution from ACSF in the main
ACSF bottle to the pre prepared test compound contained
within 50ml syringes connected to the recording chamber
by three way taps.
In this example we are applying a one micro molar
tetrodotoxin, a potent sodium channel blocker,
to a recorded neuron in the current clamp recording mode
of the whole cell packed clamp technique.
The cessation of spontaneous action potential firing can
clearly be observed.
Using the stimulating electrode placed within the
Schaffer collateral pathway of the hippocampus,
the responses to electrically evoked post synaptic
potentials can also be monitored.
Here a mixed excitatory glutamatergic EPSP and inhibitory
GABAergic IPSP can be seen in response to
electrical stimulation.
Once the experiment has finished, the brain slice is removed
from the recording chamber using a paintbrush and carefully
placed within a suitable glass container.
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A pipette is used to fill the container with four percent
paraformaldehyde in nought point one molar phosphate
buffer, ph seven point four, and stored in the fridge
until used for immunohistochemical processing.
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