Frequently Asked Questions about the 'protective recovery' method:
Q: Can I make 10X stock solutions for NMDG aCSF?
Stock solutions are possible if certain components are omitted (thiourea, ascorbate, pyruvate, etc) and added fresh. However, because of the inherent difficulty in preparing healthy adult brain slices and the multiple components that can't go into concentrated stocks, we favor making all solutions fresh and using them within a few days (one week at most).
Q: Does the protective recovery method work for other animal species?
A: We believe this method can work for any species. Several different labs have had success with with this method for adult brain slice preparation from mice, rats, birds, fish, bats, monkey, etc. We have also demonstrated excellent success in preparing brain slices from adult human brain biopsy (such as for damaged epileptic brain tissue that is surgically removed).
Q: Is the protective recovery method effective for all brain regions and cell types?
A: This method has been successfully implemented to study diverse neuronal subtypes throughout the nervous system; however, there is not enough evidence at this time to make a claim that the method works for all cell types. It is likely that slight modifications to the procedure will be necessary to be compatible with particular cell types in adult brain slices. In some cases a shorter protective recovery duration may improve the outcome for these difficult populations.
Q: Is it essential to perform a transcardial perfusion?
A: It may not be essential but is highly recommended for adult brain slice preparation. Transcardial perfusion represents the highest standard and will help take some of the voodoo out of the procedure. Once the NMDG aCSF is perfused throughout the brain, the neuronal activity and metabolic activity is largely shut-down, so it is no longer as important exactly how fast the brain is extracted and mounted for slicing. It is still a good idea to work quickly. Note that transcardial perfusion requires use of anesthetics. We have not systematically explored different anesthetics. We currently use intraperitoneal injections of Avertin (tribromoethanol) due to the large safety margin.
Q: Should I anticipate that all of my indices will match earlier data collected with standard slice preparation methods?
A: Although we would like to provide evidence that this is true for simplicity sake, there are several indications that this is not the case owing to the dramatically improved neuronal preservation with this protective recovery method. We have noted much higher mini frequencies in our whole cell recordings (mEPSCs and mIPSCs) in virtually all cell types and brain regions investigated. Hippocampal CA3-CA1 (Schaffer collateral) population spike amplitudes do not seem to be dramatically affected, but the smoothness of the responses are improved and the occurrences of secondary population spikes are reduced. This may indicate improved preservation of inhibition in our adult slices, consistent with improved functional integrity. Such differences may lead to notable differences in physiology, but which may in fact be a better approximation of the in vivo state. Further detailed investigations are necessary to determine the validity of this statement, but we note that similar observations of better-preserved inhibition were previously described for the sucrose aCSF cutting method compared to standard aCSF cutting method, and this resulted in an inability to induce standard hippocampal LTP in the absence of a GABA-A receptor antagonist [Kuenzi et al, 2000 J Neurosci Methods]. It is generally accepted that inhibition normally constrains LTP induction in vivo, thus lending some support to our claim. We urge those of you testing this particular adult brain slice method to keep an open mind and to help to continually refine the methodology.
Q: How do I make NMDG aCSF?
A: Following the formulation in the excel spreadsheet (see 'Recipes' tab), add all ingredients except for the divalents (Ca2+ and Mg2+) and the hydrochloric acid. At this point the pH will be 12 as NMDG is highly basic. Titrate the pH to 7.3-7.4 by addition of 10N HCl (~8 mL). Once the pH is in the correct range, add the Ca2+ and Mg2+ from concentrated stocks and top off the volume to 1 Liter with purified water (MilliQ water system recommended). Measure and adjust the osmolarity to 300-310 mOsm. The osmolarity should already be at or near this range with no adjustment, and this is a good indication of whether all the ingredients were added and in the correct amounts.
Note that an internet blog for patch clamping has indicated that NMDG is not stable and goes bad after ~3 weeks. We have never experienced any such problems and we routinely order large amounts of NMDG from Sigma (M2004-1KG) and store the containers at room temperature for many months.
Q: Is the temperature of the slicing solution important?
A: No - not particularly. NMDG aCSF (or Choline aCSF) is so effective in this protocol that it is possible to perform the transcardial perfusion and slicing procedure with room temperature NMDG aCSF. This may seem like a ridiculous idea given the conventional wisdom, but we have been preparing adult brain slices this way for 2 years and it works just as well as chilled solutions. This may have some advantages for those studying plasticity mechanisms, particularly since it is known that chilling and re-warming of the brain can cause extensive proliferation of dendritic spines, and presumably this is largely obviated by eliminating the chilling step. We have not tested warmed NMDG aCSF at this time. In general the slices deteriorate faster at higher temperatures, so we try to limit the duration that the slices are exposed to temperatures above room temperature in order to prolong slice health.
Q: Is sucrose aCSF effective in the protective recovery method?
A: We have attempted to use standard sucrose aCSF (as well as sucrose-HEPES aCSF) in the context of the protective recovery method but with poor results. The main finding is that neuronal membranes appear heavily damaged/dimpled when slices are exposed to sucrose aCSF recovery at elevated temperature of 32-34*C. This may suggest that sucrose becomes cell permeable, or alternatively that its permeability-blocking role is compromised at higher temperatures. The exact mechanism of this effect is not known at this time. This effect is also observed to a lesser extent for the Tris aCSF formulation, but is NOT observed with NMDG aCSF or Choline aCSF formulations, even for extended recovery incubation times at 32-34*C. It is still possible to see clear improvements compared to the standard protective cutting approach when performing the protective recovery step in sucrose aCSF or Tris aCSF, but it is essential to perform the protective recovery step at room temperature instead of elevated temperatures. It appears that Tris aCSF recovery is slightly more effective than sucrose aCSF recovery in young mice (3-5 weeks old) in our hands.
Q: Is the protective recovery method effective for all animal ages?
A: This method provides an alternative to the protective cutting method and is highly effective for preservation of neurons in brain slices from adult and aging animals. The method is not designed for use with juvenile or adolescent animals, and our own testing reveals that the procedure does not allow for full recovery of electrophysiological properties in young tissue when using NMDG aCSF or choline aCSF formulas. Modifications such as reducing the protective recovery duration (e.g. <5 min) or use of Tris aCSF appear to be effective in young mice. In general we recommend trying the protective recovery method in cases where the standard sucrose cutting method is not highly effective. We advocate for use of the NMDG protective recovery method in mice aged 6 weeks and older. In very old mice the choline aCSF formula may be required to achieve adequate preservation when examining certain cell types, such as hippocampal pyramidal neurons. Users should keep in mind the relative age equivalencies across species.