I'm studying hippocampal pyramidal and granule cells in coronal mouse brain slices using patch-clamp. The mice are 2-3 months old. I've been able to record from CA1 pyramidal cells and DG granule cells, and both cell types can look fairly good in my slices. However, most of the CA3 pyramidal cells look dead and I fail to patch them, and the CA3 field itself looks fuzzy at low magnification. I'm not sure why that happens. Can this be due to the slicing plane (coronal) I'm using? Should I switch to horizontal or sagittal slices?
I'm using mixed NaCl/sucrose ACSF for cutting (ice-cold) and recovery (30 min at 35-37°C) and standard ACSF for storage and recording.
Mixed NaCl/sucrose ACSF: NaH2PO4 1.25 mM, NaCl 87 mM, KCl 2.5 mM, MgCl2 7 mM, CaCl2 0.5 mM, glucose 25 mM, NaHCO3 25 mM, sucrose 75 mM. Osmolarity: 330-335 mOsm/kg.
Standard ACSF: NaH2PO4 1.25 mM, NaCl 126 mM, KCl 3 mM, MgSO4 1.25 mM, CaCl2 1.25 mM, glucose 10 mM, NaHCO3 26 mM. Osmolarity: 300 mOsm/kg.
Hi Alexey!
I doubt that direction of the slicing will affect the survival of the cells. But you can cut more or less number of axons depending on the orientation of the slicing.
In my case, the key for cell survival was ultra-fast cooling and transition of the brain into oxygenated ice-cold ACSF solution in a vibratome chamber.
Specifically, after decapitation the head was immediately dropped into 200 mL glass beaker with ACSF that was about 50% in ice form and 50% in liquid form. After cooling of the head for about 30 sec, I quickly removed the brain, cut the area that should be sliced and glued it (with Super Glue) to a metal disk, which was already set up in a dry and precooled chamber (at -80C or at -20C, if -80C is not available) of a vibratome. Then, the chamber was filled with pre-oxygenated and precooled ASCF (with chunks of ice, ~30%) and again oxygenated during the slicing.
Thus, the time between decapitation and beginning of the slices should be minimized to less than 1 min, and during almost all time the brain should be in ice cold ACSF.
I agree with most what Anatoli said but this sentence got my attention: "time between decapitation and beginning of the slices should be minimized to less than 1 min", If "After cooling of the head for about 30 sec", that leaves you only 30 sec to remove the brain and glue it. I wish I was that quick!!
Alexey, my advice to you would be to use Juvenile mice (2 - 3 weeks) to start with and then perfect your slicing with these, then you can try the old 2-3 months mice you mentioned. Good luck!
I also doubt that the cutting plane is responsible, CA3 pyramidal cells are delicate, I also do something similar to Anatoli's protocol, the speed and low temperature are crucial. You can also try other cutting solutions, this is the one I use: 210 sucrose, 2.8 KCl, 2 MgSO4, 1.25 Na2HPO4, 25 NaHCO3, 1 MgCl2, 1 CaCl2, and 10 D-glucose. I've seen people who also add kynurenic acid while cutting, but I haven't tried that.
With this protocol, we've been able to obtain recordings from CA3 pyramidal neurons from 1 to 2-year-old rats as well as mice the age you are trying to record.
Good luck!
Hello, I'm Abby, Chief Scientific Officer at Precisionary Instruments, where we've been crafting Compresstome vibratomes and microtomes for nearly two decades. With a wealth of experience in tissue sectioning, I'm here to assist you.
When it comes to cutting acute mouse slices from older (mature) mice, there are typically two critical factors to consider: cutting speed and ACSF or cutting solution optimization.
For cutting speed, faster is often better. Our Compresstome vibratome is designed to allow you to section mouse brains significantly faster than other vibratomes, thanks to the agarose-embedding process, which stabilizes your tissue. To understand this concept better, you can check out how it works here: https://precisionary.com/tissue-sectioning-help/how-is-compresstome-different/
Regarding ACSF cutting solution, we've collaborated closely with Dr. Jonathan Ting of the Allen Institute, who has published an optimized recipe for cutting acute mouse brain slices from older specimens. You can find his recommended solution here: https://pubmed.ncbi.nlm.nih.gov/29553547/
Furthermore, Dr. Ting hosted a webinar in partnership with Precisionary Instruments, providing an excellent overview of optimizing ACSF solutions. You can access the recorded webinar through this link: https://precisionary.com/support/product-support-menu/webinars/
If you have any more questions or need further assistance, please don't hesitate to reach out, and I'll be happy to respond here on ResearchGate.
Best regards,
Abby Chu, MD-PhD
CSO
Precisionary Instruments
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