My new project involves electrophysiology on purkinje cells from the cerebellum. I got experience on other brain areas, such as locus coeruleus, so I am using the same aCSF as usual. However, it seems that something in my protocol is keeping healthy most of the cerebellum cells but purkinje cells, which seem all dead by the time I put them under the microscope.
My cutting solution is (in mM): 20 NaCl, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 85 sucrose, 25 D-glucose and 60 NaHCO3, saturated with 95% O2/5% CO2 at 4°C.
Cutting parameters: Sagittal slices, 250 – 300 um; Amplitud 750 um; Frequency: 80 Hz; Speed: 0.15 mm/s
Immediately upon cutting, slices are submerged in an normal aCSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.2 NaH2PO4, 11.1 D-glucose, 21.4 NaHCO3 and 0.1 ascorbic acid, saturated with 95% O2/5% CO2 at 35 °C, and are left to recover for at least 1 h.
My guess is that I should change the recovering temperature (colder) and/or the vibroslicer parameters but for the sake of time, I would appreciate any help on this.
It seems to me too that you have done everything right! But you may need to try an alternative protocol that is being used for cerebellar slicing and those beautiful Purkinje cell!
Also, in my opinion, you should use a colder buffer (ice cold). But try a warmer aCSF (even around 36 degrees Celsius) if you observe no success with the following (which is around 32 degrees C):
To improve slice quality, perfuse mice/rats transcardially with an ice-cold dissection buffer composed of (in mM): 110 Choline Cl, 7 MgCl2, 2.5 KCl, 1.25 NaH2 PO4, 0.5 CaCl2, 25 Glucose, 11.6 Na-ascorbate, 2.4 Na-pyruvate, and 25 NaHCO3 equilibrated with 95% O2 and 5% CO2. Dissect the cerebellum in ice-cold buffer as mentioned.
Then transfer slices to a submerged chamber with ACSF composed of (in mM): 125 NaCl, 26 NaHCO3, 1.25 NaH2 PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 25 glucose, equilibrated with 95% O2 and 5% CO2 at 32C for 20 min. Keep the slices at room temperature until recording for up to 6 hr.
Check the following well-established protocols too:
Article Electrophysiological properties of in vitro Purkinje cell so...
Article Ephaptic Coupling Promotes Synchronous Firing of Cerebellar ...
Thanks a lot for your help. I will try to recover my slices at room temperature. Let´s see what happens.
I would also appreciate if you guys know good (amplitude, frequency...) parameters for cutting the slice.
The fact that all the neurons except PCs were fine in the slice tells me that probably I should cut at a different speed/frequency...
I have been recording from Purkinje cells for years and this is what I do:
first of all, I do all the brain extraction and slicing at 4 degrees Celsius (for the surgical part, I use chambers that have been encased in ice to keep the brain cold which is a good protection against ischemia (e.g. it slows down the ischemic death of Purkinje cells which are very sensitive to ischemic conditions).
Secondly, surgery, slicing and slice storing is done in aCSF with 1 mM kynurenic acid to prevent excitotoxicity.
Third, directly after slicing, the slices are warmed to 32-34 degrees in a waterbath for an hour, that seems to get rid of dead surface material. After that time period, the slices are kept at room temperature.
Good luck.
Thanks a lot, Claudia. It´s been very helpful.
Do you remember by any chance the slicing parameters?
I slice at 225 um thickness, and an advance of the blade of 0.8 mm/s. The vibration on the Leica VT1200 S is set at 1
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