Hippocampal slices for patch clamp and field potential, are prepared from mice using coronal, horizontal, sagital, parasagittal or transverse section. A few papers show that slices orientation can affect synaptic plasticity. (Bartlett et al., Mol Brain. 2011 Nov 15;4:41. doi: 10.1186/1756-6606-4-41.,Sherwood et al., Neurochem Int. 2012 Sep;61(4):482-9. doi: 10.1016/j.neuint.2012.04.021. Epub 2012 Apr 27.)
It is convenient and quick to get hippoampal slices from coronal, horizontal, sagital, parasagittal method. Some researchers said that cutting in a parallel way of the apical dendrites growth,transverse section, make slices health. But transverse section method is a little tricky and need more practice.
1. Which orientation are used in your lab to get hippocampus slices? And why?
2. I found that I got a smaller field EPSP response in horizontal and parasagittal slice than transvers slice.
For us, we find that there is a difference between rats and mice.
In rats, one the hippocampi are laying flat on a piece of wet filter paper, we like to have the dorsal hippocampi parrallel to each other before cutting. We tend to straighten the dorsal hippocampus and let the ventral part get more and more separated from each other as we move towards the tip (ventral).
For mice, we don't straighten the dorsal hippocampus as much as we do in rat. You end up with 2 C-shaped pieces of tissue rather than 2 L-shaped pieces of tissue (rat).
Check this site out too:
http://www.functionalneurogenesis.com/blog/2011/04/dorsoventral-vs-septotemporal-hippocampus/
We used both coronal and horizontal sections for recording fEPSPs in CA1, and actually found that horizontal sections are somewhat better in our hands. However, the CA1 area tends to be smaller in horizontal sections and it is perhaps more difficult to find the optimal position of stimulating and recording electrodes.
Small EPSPs could be due also to how you treat the slices before recordings. We had the best results by incubating them in an interface-like chamber, in the aCSF used for recording, for 1 hour before starting the experiments.
We have found that transverse sections seem to preserve the CA3-CA1 pathway to a greater extent than coronal sections. We can obtain good fEPSPs in either preparation, but the transverse allows us to place the stimulating electrode further back within CA3 and still measure good fEPSPs (0.5-1 mV) in CA1. With coronal sections, the stimulator typically has to be placed somewhat closer to the recording electrode.
Slice health can depend on many factors, as suggested. Another factor that can suppress responses is higher adenosine levels, which can be evaluated easily by applying an antagonist (DPCPX or caffeine, for example). If your responses are greatly enhanced by the antagonist, it suggests a higher basal level of endogenous adenosine in your slices.
I also find that CA1 pyramidal neurons in adult mouse slices are healthier when cut by approximating a transverse slicing angle as opposed to just coronal sectioning of dorsal hippocampus. This can be achieved by blocking the brain a number of ways but probably the most straightforward way is after the description of Bischofberger et al 2006 Nature Protocols, the so called magic cut angle.
Just an interesting note in follow up to Alexander's comment...I also observed that there is a lot of adenosine released during the slicing trauma, and this particularly notable in CA3-CA1 Schaffer collateral pathway in mouse slices. The high adenosine levels produce a very strong pop spike and fEPSP suppression, and this is mediated by A1ARs and can be blocked by antagonists like DPCPX. The relaxation of this high adenosine phase back to 'normal' takes time and not coincidentally is fully relieved in about the hour time period conventionally allowed for slice recovery before staring your serious recording sessions. You can know this is true if you try evoking responses immediately after slice preparation (which you can't) but if you then apply DPCPX you will find you actually can get the usual amplitude responses just minutes after slicing. I found that very interesting and had previously always wondered why everyone used 1 hour for slice recovery.
If you have slices that are degrading quickly or a static slice holding chamber that allows accumulation of metabolites in a small volume, I have seen that adenosine can build up in the slices and make evoked responses from CA1 either highly variable or notably small. This can be partly relieved by using solution flow for slices in a holding bucket or by simply refreshing the static aCSF solution in the holding bucket frequently.
As Jonathan notes, mouse slices may indeed have higher endogenous adenosine levels, as we reported a couple of years ago:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855550/
As for the reasons people wait for 1 hr, I think there were some earlier studies that suggested ATP/adenosine levels were highest in this post-recovery time period. There was also an interesting discussion some years back on general slice methods that makes for fun reading:
http://www.ncbi.nlm.nih.gov/pubmed/7475244
Alexander, I remember when your paper first came out on the difference between mouse and rat for adenosine tone in brain slices...it was extremely helpful to me at the time when I was seeing some interesting alterations in eCB mediated short term plasticity in one of our mouse models. There was definitely some issue with obscured measurements due to high adenosine levels. It is impressive how powerful the effects of adenosine can be on synaptic function and I don't think it gets a fair share of attention.
Hi Mason,
Here's a JoVE article that demonstrates getting transverse sections.
http://www.jove.com/video/2330/preparation-acute-hippocampal-slices-from-rats-transgenic-mice-for
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