Question
Asked 27th Aug, 2017

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

I'm new to electrophysiology. I'm trying to record from dorsal horn neurons in the spinal cord. One of the hard things for me is identifying the neurons that are healthy and suitable for patching. It seems like that is something that comes with experience to make that judgement, and also good optics. I know which ones to stay away from: big, swollen cells with visible nuclei. But what are the specific features that one should look for that indicate a suitable cell?

Most recent answer

15th Mar, 2018
Yao Xue
Yale University
Hi Alexander. I don't know much about dorsal horn neurons. I'm working in hippocampus slice. Here are some of my personal experience:
1. As mentioned by many, superficial cells are usually not healthy. Cells that are not regular in shape are unhealthy.
2. When pipettes deep into the slice with positive pressure(which will create liquid flowing out of the tip), those seemingly healthy cells aren't able to endure the flow and will be scattered.  Only those healthy cells can survive the flow. You can test it by moving your pipettes around and see whether cell bodies are "resilient" enough.
3. When pipettes' tips touch the cells, the point of touching should be surrounded by a little ring of light under DIC.
4. Cells difficult to form gigaseal are unhealthy.
That's all. Hope some of them will help.
Best wishes
1 Recommendation

Popular Answers (1)

29th Aug, 2017
Vitaliy Reznikov
Universitat Politècnica de València
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.   
5 Recommendations

All Answers (10)

28th Aug, 2017
Puja Parekh
Weill Cornell Medical College
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.
3 Recommendations
29th Aug, 2017
Vitaliy Reznikov
Universitat Politècnica de València
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.   
5 Recommendations
30th Aug, 2017
Debanjan Dasgupta
The Francis Crick Institute
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!
3 Recommendations
31st Aug, 2017
Avani Shukla
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. 
3 Recommendations
4th Sep, 2017
Andreas Husch
Deutsches Zentrum für Neurodegenerative Erkrankungen
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?
3 Recommendations
4th Sep, 2017
Alexander Chamessian
Duke University Medical Center
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. 
4th Sep, 2017
Anurag Pandey
Cardiff University
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.   
2 Recommendations
4th Sep, 2017
Andreas Husch
Deutsches Zentrum für Neurodegenerative Erkrankungen
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.
3 Recommendations

Similar questions and discussions

How to find out what is killing our brain slices?
Question
16 answers
  • Roberto De PasqualeRoberto De Pasquale
We have been done visual cortical brain slices of mice (300 μm, 20-25 days) for electrophysiology. We´ve been doing it successfully for years and never had this kind of problem. Since the last three weeks, the slices are always completely dead. Not even a single cell survived. For dead I mean that we can clearly see the dead cells (large cell body and clearly visible nucleus). The slice is already dead even if we put it under the microscope immediately after cut.
 We´re using the following:
-       Cooled (0°C) oxygenated (5% CO2-95% O2) cutting solution (in mM): 206 sucrose, 25 NaHCO3, 2.5 KCl, 10 MgSO4, 1.25 NaH2PO4, 0.5 CaCl2, and 11 D-glucose.
-       ACSF; in mM: 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 25 D-glucose).
-       Leica vibratome 1100S
We´ve already tried the following:
We checked osmolarity and ph of both solutions many times with different machines and they are ok (300 mOsm-7,3).
We took distilled water from other laboratories.
We replaced all salts with brand new products.
We autoclaved all glass and then cleaned it with hydrochloric acid, then with alcohol and then with tons of distilled water.
We verified with weights samples that the balance is calibrated.
We thought that the problem could be the vibratome. However, it seems to be working normally. It vibrates horizontally and shows no visible sign of malfunction. The slices don´t look to have passed throught any kind of unusual mechanical damage.
We made a test cutting a brain with solutions from another laboratory, and the neurons were healty, so we suspect it is a contamination problem occurring during the preparation of the solutions in our lab. If this is the most likely hypothesis, we are afraid to do tests involving other laboratories and risk to contaminate their stuff.
We are continuing to replace components to isolate the problem but with no luck. As far as I know, bacteria can kill slices, but that requires at least few hours. I cannot think of a chemical specie capable of killing everything this way and persist after several washing processes. If someone has an idea of what can be going on, we would be grateful to receive an opinion.
What might cause increased access resistance over the course of whole-cell current clamp recording?
Question
4 answers
  • Matt RussoMatt Russo
Hello Everyone,
Recently, I've started experiencing an issue that I haven't encountered over the past couple years of doing whole-cell current clamp recording. I typically record from cells for about 30-40 minutes at a time, and this has not really presented an issue in the past. However, I've noticed over the past few months that my access resistance is dramatically increasing at a certain point during the recording session, and I'm not sure what's causing it. Here are some details pertinent to these experiments:
  • I use the following internal pipette solution: 130 mM K-Gluconate, 10 mM KCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 10 mM HEPES, 10 mM Na-phosphocreatine, and 0.5% Biocytin (for post-hoc streptavidin labeling). Usually have an osmolarity of ~295 mOsm
  • The external solution (aCSF) is constantly perfused through the recording chamber at a temperature between 32-34 degrees C. The osmolarity is usually somewhere around 305 mOsm.
  • Pipette resistance is typically ~2.5 MOhms.
  • I typically inject a slightly negative current (between -20 and -45 pA) to maintain a constant RMP around -68 mV. This varies based on the true RMP of the neuron, but on average, the current injection tends to fall somewhere in that range.
  • I inject a -80 pA test pulse to perform bridge balance and capacitance compensation. This is done about a minute after break-in, and the test pulse is immediately turned off once compensation is complete.
  • Because the issue only began recently, I thought it may be the batch of internal solution I was using, so I made a fresh batch but had the same problem.
I typically attribute this to membrane closure, but I feel that's unlikely to be causing this issue just by how frequently I've observed it recently. Just curious if anyone has any tips for how to alleviate this problem. Thank you all in advance for your help!

Related Publications

Article
We used a retrograde marker, rhodamine-labeled fluorescent latex microspheres and the enzyme papain to isolate a group of identified, dorsal horn projection neurons from rat spinal cord. This method has allowed us to study the electrical properties of these acutely dissociated cells with patch-clamp techniques. It will also provide us the opportuni...
Article
Full-text available
Dorsal spinal cord neurons receive and integrate somatosensory information provided by neurons located in dorsal root ganglia. Here we demonstrate that dorsal spinal neurons require the Krüppel-C(2)H(2) zinc-finger transcription factor Bcl11a for terminal differentiation and morphogenesis. The disrupted differentiation of dorsal spinal neurons obse...
Article
Whole-cell patch-clamp recording technique is a powerful tool to study intrinsic membrane properties and synaptic interactions in the spinal cord. Spinal cord slice is an idea preparation for electrophysiological studies under physiological and pharmacological manipulation that is difficult to perform in an in vivo preparation. Depending on experim...
Got a technical question?
Get high-quality answers from experts.