We are trying to prepare healthy striatal brain slices to do some whole cell patch-clamp electrophys recordings.
The brain slices that we get seem to have (selectively?) lost the MSNs - they look like fried-eggs and cannot be patched. We do see many other cell types in the slices, but these are also difficult to get good gigaseals and difficult to break through to whole-cell configuration. The cutting procedure is given below.
Slices health doesn't change when we get them under the microscope - so we don't think it is the recording solution (which we have been using for years).
We've tried "sodium-spike in procedure" (Ting et al 2018) and we don't think that was better.
Are there any special tricks for keeping MSNs alive in brain slices?
Experiment details:
CompressTome VF-310-0Z
NMDG-NAC perfusion/cutting solution 4-6 ºC (as per Ting et al 2018)
HEPES-ACSF incubation solution (as per Ting et al 2018)
Species: Rat Wistar
Age: 4-6 weeks
Assuming everything is well-bubbled with carbogen, this looks like an osmolarity issue. Check for steep osmolarity changes especially between incubating and recording. You may be able to gradually replace the solution with the recording one ( 1-2 dilution steps while incubating, depending on how big the difference is), to give the cells more time to adapt closer to the final osmolarity.
Try a slice that includes striatum-GP-SN or as best as can be achieved along those lines
Thank you Michael T. Craig
, Apostolos Mikroulis and John C Hedreen for your helpful advice. After much optimization we settled on the following:- Increasing osmolarity from 300 mOsm originally to ~310 mOsm helped reduce/prevent cell swelling
- Our slices are coronal orientation and through the tail striatum (caudolateral striatum) in which the fibers appear to be orientated such that we are not cutting through them
- Transcardiac perfusion, cutting and recovery were all done at room temperature - this improved slice quality
We settled on the following protocol:
NMDG-NAC perfusion/cutting solution @ RT, 310 mOsm
NMDG-NAC recovery solution @ 33 ºC, 30 min.
Sodium-spiking during the recovery period (1-3 month protocol from Table 3; Ting 2018)
HEPES-ACSF incubation solution @ RT, 310 mOsm
ACSF (standard) recording @ RT, 310 mOsm
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