I currently perform fEPSPs recordings in the stratum radiatum of dorsal CA1 while stimulating Schaffer collaterals in mouse hippocampal brain slices. I cut 300 micrometers thicks sections at 45 degrees angle from the coronal/sagittal plane, in order to preserve the fibers in the dorsal hippocampus. The cutting speed is 0.06 mm/s. I use cold choline for both cardiac perfusion and cutting and at 32 degrees for recovery (10 min). I then move them in normal aCSF.
Unfortunately, the amplitude of my fEPSPs is only as large as the fiber volley.
I am afraid that this is mainly due to the survival of my slices (from decapitation until the last slice it takes me more than 30 min) but I've been suggested also to use bicuculline to increase the excitatory events. Could this be helpfull?
Thank you!
Dear Vincenzo,
for the recording of fEPSP, i propose to read this paper: Theta burst stimulation-induced LTP: Differences and similarities between the dorsal and ventral CA1 hippocampal synapses.
This study is done in rats but i have tested in mice with success. I can propose you the following:
1) locate the dorsal part of hippocampus vertically against the blade (at the slices you can clearly see the pyramidal layer)
2) after the decapitation, remove as fast as you can the brain into a 0-4 oC acsf solution. (i usually use a petri with cold acsf where i locate the brain. And the petri into a box with ice in order to maintain the temperature in the right levels). Be careful: the acsf MUST be oxygenized at least for 30 min with O2/CO2. Tissue must not stay without O2 at all.
3) Leave the slices to rest for at least 1 hour at 35 oC. i left them over 1.5 hours.
4) Also, if you want to test the health of your hippocampal slices you could cut ventral slices that after 2 hours of rest in 35 oC they produce the synchronous spontaneous endogenous activity of SPWs. You can observe this activity in dorsal slices sometimes. This activity is organised by a small volume of cells. So if you see it then your slice is in perfect condition.
5) Be careful where you stimulate. At first i would locate the recording electrode on pyramidal layer and the stimulation electrode at radiatum. So, i would expect to see a small fEPSP and a large Population spike. After that you move toyw recording electrode down to radiatum. Your EPSP will be reversed and will get bigger.
I hope that all these will help you with your experiments.
Also, Bicuculline will cause epileptic events in your slice. You can try it after you get a good fEPSP response to see the difference of GABA blockade.
Hi Vincenzo,
Two things came to my mind are the location of your stimulation electrode and your recording electrode.
1) If you have a bipolar stimulation electrode, in order to stimulate as many fibers as possible it is better if the two feet of your stimulation electrode are not parallel to Schaffer Collaterals but span them as in the figure attached (from Power JM et al.,1997, J. Neurophysiology). Another reason would be that your stimulation electrode is too far away from your recording electrode.
2) As Stylianos mentioned in his 5th point, your recording electrode may not stand exactly on str. radiatum but lay more apically (close to the str. lacunosum moleculare). The 2nd attached figure (B) may give you an idea (from Kloosterman F. et al., 2001, J. Neurophysiology-though the one of the stimulation electrodes is on CA3b in this paper). You can also check the 3rd attached figure from Isomura Y et al.,2002, J. Neurophysiology
Hi Vincenzo
Dont use bicuculline, it will, as mentioned previously cause loss of GABAa and cause epileptiform activity. The main difference we find between mouse and rat slices is the fact that the temperature has to be as low as possible for mouse. We use ACSF that is "slushy" for initial chillling and cutting,(you can do this by putting the ACSF in a bag over dry ice. You also have to be careful as you dont want to "freeze" the brain. Temperature is most important. Also mouse slices take longer to recover after cutting (1.5 to 2 hours).
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