Hello all,
I have been working to set up a new extracellular recording (LFP)-type rig in the lab. The person who trained me on measuring LTP in in-vitro mouse hippocampal slices used a patch-clamp type rig to record. With this type of setup, magnification is excellent and the strong backlighting beautifully showcases the Schaffer collaterals, making electrode placement and subsequent recordings pretty easy. The slicing technique he used (and I am now using) is as follows: extract brain, cut off olfactory bulbs/frontal lobes and cerebellum, separate hemispheres, flip each hemisphere over to rest on its medial surface, turn so that the ventral surface is facing me and then make slightly angled cuts (towards me) on the dorsal surface of the brain (removing just enough cortex to make a flat surface to mount). Essentially this results in semi-transverse (?) slices. I mount both hemispheres next to one another on the cutting surface of a vibratome, dorsal side down, cutting 350 micron slices.
Now, on to my rig. I have figured out a method for backlighting with an LED strip that works OK (could be brighter, for sure, but cannot find one better), and the stereomicroscope provides less than optimal magnification. It's difficult to visualize the Schaffer collaterals with the precision that one could achieve using a patch-clamp rig. The variability is frustrating. Am I fighting a losing battle here? Is there a better way to be doing this on my type of rig? Better slicing plane, lighting, etc? I've read about people using coronal as well as sagittal slices. Any input would be much appreciated!
The visibility of slice will be better with the proper upright microscope equiped with special contrast options then with the simple stereomicroscope. However, recordings from the Schaffer collaterals are not that demanding. With some practice, one can use the stereomicroscope. For the extracellular recordings, you don't need to visualize the individual cells. The lighting could be achieved with the bright LEDs or halogen lamps. There are guide lights based on halogen lamps, they could provide quite a good illumination. Regarding the orientation of slicing, people use different planes - coronal, transverse, horizontal, etc. As long as the pathway is preserved, you should get good, high quality field EPSP responses. The method that you are using is also quite popular. As for the frustration, one should remember that not all slices are good. For example, using horizontal sections of tho hemispheres, I usually get only 3-4 good slices with proper fEPSP responses. In other slices, the cell processes are probably damaged due to inapropriate blade orientation.
As Anton said, you do not need to see individual cells for extracellular recordings, you just need to be able to see the stratum radiatum. I personally prefer a stereomicroscope for LFP. If you do struggle to see the radiatum, you may just need a little more contrast, the easiest is to have the light coming at a sharper angle or using two light sources. I have set up a rig using this:
https://www.amazon.co.uk/dp/B007LBELH4/ref=asc_df_B007LBELH457722537/?tag=googshopuk-21&creative=22146&creativeASIN=B007LBELH4&linkCode=df0&hvadid=310891327396&hvpos=1o1&hvnetw=g&hvrand=2358909252108849907&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9046183&hvtargid=pla-492132031899
They are electrically very noisy until you change the powersupply but otherwise fine.
Feel free to get in touch if you need more help.
Vincenzo Marra I'm not trying to see individual cells at all, I'm just trying to see the white-ish band of Schaffer collaterals when the slice is backlit. With using lighting from above (as I am guessing you are doing with the light you suggested) , do you still look for this white band, or other landmarks in the stratum radiatum? Thanks!
Hi Jacci Newel , sorry I misunderstood the way your microscope is set up. If your slice does not look at all like the one in the file attached, I think it may still be a contrast issue. It's hard to tell without seeing the rig but an easy way to improve your contrast would be to tilt your transmitted light source a bit to get some sort of 'oblique' effect. It could be as easy as putting a bit of tin foil to cover part of the light source. You could even 3D print a wedge in clear-ish plastic to get a gradient.
Hello Jacci,
You already got some nice answers from others. I agree with them entirely: you do not need a patch clamp setup for LFP, it should work.
I would just emphasize what Anton explained already: with this way of slicing, you may have only 1 or 2 slices with intact Schaffer collaterals. You can imagine the hippocampus as a banana: if you slice in a horizontal manner, only a few slices will be radial to the shape of it. Try many slices, and start by putting your stimulation electrode really close to your recording electrode. In this configuration, you can measure LFP even if the Schaffer collaterals are cut and really short. With your improving expertise, you will know just by the shape of the hippocampus if the slice is correctly radial or not and you can put your electrode further away.
Moreover, your slice could be plainly dead: your protocol is correct, butcheck your cutting and recording solutions. You can rehearse on young animals also: P14 slices should survive well.
Vincenzo Marra Here are some pictures of my rig and setup, as well as a pic of my brain slices as well as a slice under the stereomicroscope with the amount of contrast I am getting. Please let me know what you think, or if you have suggestions! Is it similar to your setup? Another question, do you use the accompanying heating element with your rig? What temperature do you set it at? Thanks in advance!
Jacci Newel I have recorded fEPSP with a similar setup. Your slices look fine but, as said before, some slices will be better than others. Hard to say at low mag but I think the 5 on the chuck should all give you a response. The contrast looks fine although that particular slice does not seem the best. When you have a relatively narrow stratum radiatum you may be better off having your electrode near the stratum pyramidalis and definitely not in the L-M. Regarding temperature controllers, I try to run experiments at 35+-1 C, if you are interested in plasticity or interneurons, temperature will be pretty important but if for now you are just trying to get your signal, I would not worry about the temperature.
If your stimulator, amplifier and respective electrodes are set correctly you should see an fEPSP in most of your slices.
PS: If you want a cheap diffuser for your light you can use a pringles crisps cap folded over a few times.
Vincenzo Marra Thanks so much! Sorry for the terrible pic of the slice, it was taken a few months ago when I first started doing all of this! And thanks for the Pringles cap tip. :-) And thank you @Sylvain Rama for your input as well!
Vincenzo Marra Sylvain Rama I had a few more questions for you, if you don't mind! I have been learning all of this in order to eventually run LTP experiments on mice for an Alzheimer's project. I have noticed that I cannot get above (150-160% of baseline) sustained LTP when stimulating at about 60 microamps (about 40% of max in my I/O curve, this is where I can get the most stable baseline). We've checked my slices for viability on the patch-clamp rig, and the cells are reliably alive. I'm using 1.3 mM of magnesium (rather than 2 mM in normal recording ACSF) for LTP recording. I am incubating my slices for 1-1.5 hours at room temperature in this LTP recording ACSF, and recording at room temperature. I mentioned temperature in my previous post and you said that it is important. My PI wants me to get 200-250% sustained LTP, which my predecessor was able to do on the patch-clamp rig, but I cannot on my rig. Do you think that temperature has anything to do with the fact that I can't get up to 200-250%? Or another factor? Thanks again in advance!
Hi Jacci Newel , yes temperature can definitely affect it. Are you measuring the slope of your fEPSPs?
In many ways it is easier to see a large effect with patch clamp for a number of reasons (one of them being that most people discard changes of 0%).
Hi Vincenzo Marra , yes, I am measuring the slope of my fEPSPs. My colleague wasn't actually using patch clamp to do the experiment, he was just using the rig one would use for patch clamp to do LFPs. This gave him superior visibility and precision when placing electrodes. Do you think I should incubate and record at a higher temperature then?
Hi Jacci Newel I would definitely record at physiological temperature if you can.
The recovery/incubation sounds good, I personally have a short 'warm recovery' period but I do not think it makes a big difference and having recovery at room temperature for a little longer (e.g. 60-90 minutes) always worked fine.
Vincenzo Marra Okay, sounds good! Thanks so much for all of your help! :-)
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