How do you identify 'healthy' neurons in slices (brain, spinal cord) for Patch-Clamp recording?

I'm not sure about the dorsal horn but in the brain I would make sure to choose cells that don't have irregularities in the appearance such as darker and lighter spots. Under DIC optics, cells with dark areas or a dark outline around the cell tend not to be as healthy. Good luck.

Yes, it is very important to patch just healthy neurons because just with these ones you will get stable and true response. You can get patch more easily with damaged ones but from these it is impossilbe to get stable recording. First of all, you have to prepare very healthy slices with good airation. Second one, you have to patch cells which are located more deep in the slice (about 50 -100 mkm) but not superficial cells. Ussually superficial ones are dead ones. It is true that unhealthy cells have dark border and very rigid membrane (it is impossible to see any invagination of the cell membrane caused by flow of solution from patch pipette). Also healthy cells have volume. And the last thing I can advise you try to patch slices from very young mice (about 7 - 15 days) to get some experience. A reason to do this is that percent of healthy neurons is higher than in old ones even if you have bad preparation of slices.   

Training ones eyes to identify healthy neurons or rather patchable neurons takes experience and varies from area to area. As Vitaliy mentioned, usually neurons located deeper in a slice tend to be better. However, I would recomend to have a high positive pressure in your pipette which can clean the cell surface well. This helps a to identify a better cell. Moreover, a better cell tends to be better embeded in the extracellular matrix and so, a higher pressure does not make it fly away from the pippete.

However, the amount of pressure which is high depends on the size of the cell. A 50 micron hippocampal cell can withstand a pressure of 100-150 psi while a 20 micron granule cell or an astrocyte might be able to take up only upto 60-80 psi.

good luck!

I have experience with acute brain slices prepared from different regions of the brain, but not spinal cord. If the slices are overall healthy, the good healthy cells appear full of volume and have smooth membranes visible through DIC. The irregularities and spotty appearance of cells is a bad sign. The "fullness" of the cells can be seen as the cell surface appearing concave or convex under DIC. This depends on the polarization, in most cases you can change the direction of polarization on your microscope such that a membrane that was appearing concave will look convex after changing the polarization. If the cell is nice and healthy and smooth, when you lower your patch pipette near the cell surface using a little bit of positive pressure, you should be able to see a tiny depression of the cell surface, this is exactly the time to apply suction. If the cell is healthy this works well and you can get a good seal with high resistance. 

I want to add some remarks regarding spinal cord slices. You already mentioned the necessity for good optics. I think this is especially or even more important in spinal cord slices, because the tissue can be more dense and the details can be less clear. Therefore I recommend patching with a 60x objective, which improves the visibility of the patch formation in comparison to a 40x or 20x magnifications.  Another strategy to better find the healthy cells in the spinal cord is to use fluorescently labeled cells. When we tried to patch motoneurons in adult mouse spinal cord slices, it was very helpful to use ChAT-GFP mice. Healthy cells had nice fluorescent labeling, whereas unhealthy cells were dim. The fluorescence signal also helps to go for cells deeper in the tissue which also increases the chance of getting a nice and healthy cell which is not impaired by the slicing procedure.  Are you using neonatal or adult mice?

Thanks all. Andreas: I agree with what you said. I was using 40x this summer and it was hard to see the definition on the cells, especially the 'dimple' that forms when you approach with positive pressure. I also had a GFP line that helped me see certain neurons. So you found that the healthy cells had a lot of GFP? I was told that high GFP expressing cells may be the ones that you should avoid, since the expression of GFP may be harmful to the cells. What do you think? I'm using young adult mice, 3-6 weeks. 

It depends a lot on preparation, area, eg. the cell that you'd avoid in acute slices are 'healthiest' in cultured slices. As somebody mentioned about GFP loaded cells, the rule of thumb that i and some people i know follow is, 'brighter the cell weaker it is'. Sometimes in fluorescence you see really bright cells, but you don't find any neuron in DIC. If you are patching on cells expressing viruses or beads, best bet is to go for cells with less expression.The duration of expression and animal age are very critical. I would suggest you to judge yourself based on how your preparation is, try finding the best ones in the field of interest. It also depends what kind of experiments you are doing, eg. recording for short duration,long term, or pharmacology.   

Hi Alexander, I agree, the cells that are much brighter than the average shouldn't be the first choice. Those look often additionally suspicious under infrared light, with  higher contrast areas, which has already been identified by previous comments as indications for unhealthy cells. I was also wondering how much the expression of GFP could impair the physiology of a neuron, but in my experience cells are typically not unhealthy because of sufficient GFP expression.

One point many slice physiologists agree on: the healthier the slices, the easier the patching.  Take good care during slice preparation, and the ratio of healthy vs unhealthy cells will improve and finding the healthy cells won't be that challenging anymore.