All tags agonists-activators-antagonists-and-inhibitors Brain slice electrophysiology video protocol

Brain slice electrophysiology video protocol

Brain slice electrophysiology allows the study of a synapse or neural circuit in isolation from the rest of the brain, in controlled physiological conditions. This protocol describes the preparation of hippocampal slice and electrophysiology recording.


Solutions and reagents

2 L of aCSF (artificial cerebrospinal fluid) solution

127 mM NaCl
1.0 mM KCl
1.2 mM KH2PO4
26 mM NaHCO3
10 mM D-glucose

Weigh out salts using a fine balance to make up 2 L of artificial cerebrospinal fluid (aCSF) solution. Top it up to with deionised H20 to around 1.9 L, leaving enough room to add other components later. Shake well and make sure all salts have dissolved. Bubble aCSF with carbogen 95% O2/5% CO2 for 15-20 minutes to stabilize the pH to 7.3-7.4. Afterwards add 4.8 ml of 1M CaCl2 and 2.6 ml of 1M MgCl2 using a suitable pipette to give a final concentration of:

2.4 mM CaCl2
1.3 mM MgCl2

Top it up with deionised H20 to 2 L. Bubble for a further 15 minutes with carbogen.

Intracellular recording solution

140 mM Potassium gluconate
10 mM KCl
1 mM EGTA-Na
10 mM HEPES
4 mM Na2ATP
0.3 mM Na2GTP

pH adjusted to 7.3 with 0.5M KOH and the osmolarity adjusted with 1M sucrose.

Test compounds

CGP 55845 hydrochloride
Tetrodotoxin citrate


Brain slice preparation

  1. Chill 1 L of aCSF over dry ice until < 4oC and add 250 ml of aCSF to slice holding chamber.
  2. Setup vibratome by filling the buffer chamber wtih cold aCSF solution, setting the desired thickness (2-500 µM for brain slices) and adjust the cutting speed to the desired setting (in the video protocol we used number 0.15 mm/min).
  3. 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.
  4. Mount the brain (cerebellum side) onto the vibrotome specimen disc using superglue, orienting the sample such that the cortex faces the razor blade. Add a supporting piece of agar behind the brain, away from the side of the vibratome, to provide structural support during the slicing.
  5. Set the limit stop positions of the microtome to define the start and stop positions of the slicing. Set the frequency of the vibrotome to maximum (100 Hz).
  6. Use the Up-rocker 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.
  7. 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.
  8. Carefully hemisect the brain down the midline and transfer the individual slices to a holding chamber pre-filled with aCSF.
  9. Store the slices for at least 1 hour at room temperature to allow the brain tissue to recover from the mechanical shock of slicing.

Tips: It is important to continually top up the chamber with cold aCSF solution to keep the brain tissue cool whilst slicing.

If unfamiliar with brain anatomy, use a reference such as Paxinos and Watson, The Rat Brain Atlas to ensure slicing of the correct brain area.

Image: Figure 35 from the Rat Brain Atlas



Electrophysiology recording preparation

  1. Prepare intracellular recording solution into a 1 ml microcentrifuge tube from pre-prepared stock solutions.
  2. Optional: Add Alexa Fluor® 633 to a final concentration of 50 µM to allow for further IHC experiments of the recorded brain neurones. See IHC protocol.
  3. Make an electrode filler by melting a 1 ml 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.
  4. Fill the electrode filler with the intracellular solution.
  5. Fabricate a glass recording pipette by using appropriate glass capillaries and pipette puller.
  6. Prepare test compounds and add it to aCSF to acheive the final working concentration that will be used in the experiment.

Tip: It is important to ensure the batch specific molecular weight is used to determine an accurate concentration of test compounds. Check out our FAQ on small molecules for more information.


Electrophysiology recording

  1. 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 5 ml/min using metal Hoffman clamps.
  2. Place brain slice into recording chamber using a small brush and secure the slice with reference electrode.
  3. 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.
  4. Attach the pipettes to the electrode holders of the patch-clamp amplifier headstages and turn into position.
  5. Using fine control micromanipulators, descend the recording pipettes to the region of interest within the brain slice. In the video, the CA1 region of the hippocampus.
  6. If required, use a coarse-manipulator to position an appropriate stimulating electrode again to the appropriate region of the brain slice to stimulate inputs to recorded neurones.
  7. Once pipette is in contact with a neurone within the brain slice, apply negative pressure to pipette via 1 ml syringe. Monitor resistance of seal formation on oscilloscope or computer.
  8. Once seal resistance has exceeded 1 GΩ, using amplifier and computer software to compensate transients and apply further negative pressure to rupture cell membrane gaining whole-cell access to neurone.
  9. Perform current-voltage relationship using computer controlled software to access neuronal health and to assess for presence of active membrane conductances.
  10. Once happy with quality of recording, perform set experiment applying test compounds via syringes connected in-line with the aCSF flow.
  11. Monitor response of test compound using computer controlled software and perform electrophysiological tests such as current voltage relationships, evoke excitatory or inhibitory post synaptic potentials.
  12. Optional: Once experiment has finished, remove brain slice from recording chamber and fix tissue overnight with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Continue with IHC to identify proteins of interest.

View our IHC protocol.


Acknowledgment

The video protocol was produced by Abcam in partnership with NeuroSolutions.

Reference

  • Paxinos, George, and Charles Watson. The rat brain in stereotaxic coordinates: hard cover edition. Access Online via Elsevier, 2006.

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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