#LabHacks: Tips for successful patching of immature embryonic and young postnatal neurons

#LabHacks: Tips for successful patching of immature embryonic and young postnatal neurons

By Deepanjali Dwivedi

The invention of patch clamp recordings has revolutionised the field of neuroscience. The journey that started with Hodgin and Huxley poking electrodes in a squid axon has progressed to include optogenetics and two-photon patching. Making healthy tissue slices is an essential first step for successful patching. While it takes some level of practice to get those healthy slices going, a lot still depends on diverse components of the slicing and patching process. It becomes more challenging if you are patching from young pups or embryos when cells are immature and hence unhealthy. In general, you need to be extra careful at every step of patching from immature cells. Here are some steps which can assure successful patching of immature embryonic and young postnatal neurons.

1. Solutions are the key

For any electrophysiology experiment, quality of acsf solutions is crucial. The same holds true for patching from embryos and postnatal pups. Acsf solutions should be made in clean MQ water and preferably fresh every day before the experiment. The osmolarity of the solution should be 10-20mOsm more than the osmolarity of the internal. Check the pH of the acsf to make sure it’s in the physiological range (7.2-7.4) before proceeding with the experiment. For older animals acsf with special components like sucrose, NMDG or choline should be used when necessary to maintain healthy slices. However, for embryos and young pups, no special acsf is a prerequisite for healthy slices.

Frozen acsf should be used during dissection and slicing, but make sure that the solution is not over frozen. As embryo and pups brain are more delicate than adult brains, big ice crystals can be extremely damaging.

2. Dissection and Slicing

After dissection, the whole embryo should be immersed in cold acsf for 1-2 mins. Brain dissection should be completed in cold acsf, in as little time as possible. Thereafter the brain should be fixed against the agar block before slicing. For adult brains, it’s reasonable to do mounting and slicing of the full brain without the agar support. However, embryos and pups brains are soft,hence slicing them without agar support can damage the cells. For slicing of young brains, lower speeds are prudent on the vibratome (than the ones used for slicing brains of adult animals) to preserve slice health.

After slicing, the slices can be incubated for 0.5-1 hr in a standard slice holding chamber with carbogen (95% O2 and 5% CO2) bubbling. Be extra vigilant that bubbling is not exceptionally high as it can lead to mechanical damage to the slices.

3. Ideal pipettes for ideal patching

Pipettes are one of the most crucial components of a successful patch for young immature neurons of embryo and pups brains. As these cells are very small, we typically need higher resistance pipettes to patch them. However, higher pipette resistance can also lead to high series resistance, which will interfere with recorded data. Thus, an ideal pipette resistance for patching immature cells is between 4-5 MOhms. Additionally, pipettes should not be of very high taper, or they will further interfere with series resistance. Also, use a good puller, which gives stable pipettes across multiple pulls. Variability in pipette program can be more taxing for patching young, immature cells of embryos and pups than for mature cells of adult animals.

4. Good cells are the way to go

Any slice has both good and bad cells. You must know how to differentiate between them for best results. Ensure you have good DIC optics for differentiation. Rules for differentiating good and bad cells for embryo/pups slices are the same as that for adult slices. Bad cells look shrivelled, have high contrast borders and fail to form a good seal, while healthy cells look luscious, have more uniform shape and low contrast borders. Also, target cells at least 50-100uM below the top of the slice, but not deeper than 250uM, as the best slices are in the middle of the cells. While the cells at the top layer might have damaged dendrites and axons, cells below 250um deep into the slice are unhealthy because of limited oxygen supply.

5. The wind of positive pressure

It's common practice to put positive pressure in the pipette before entering the tissue. This prevents the pipette from getting clogged with the tissue before it can approach the cell. However, too much positive pressure might hinder the patching process of young cells as they might get blown away. This is because they are not very firmly tethered to their extracellular matrix. Thus, bare minimum, essential positive pressure should be used while approaching the cell. Also, try approaching the cells in gradual steps to keep the cell in its position.

6. Seal to record

As soon as a slight dimple appears on the surface of the cell (because of approaching positive pressure from the pipette), the pipette should be opened to the atmosphere to facilitate patching. A slight negative suction can be advantageous occasionally, but a negative suction too extreme might lead the cells to become sucked up into the pipette. As these cells are fragile, negative pressure should be given gently and kept to the bare minimum.

7. Gentleness at its best while opening

This is the most crucial step. You need to be extra careful when trying to open the immature cells, as they are more prone to lose the seal than mature cells. Using electrical pulse to open these cells might not work very well, as these cells have very high Input Resistance. Thus, even a small electrical pulse can cause great depolarisation in these cells, which can cause you to lose the patch. In all cases, try using gentle suction with your mouth with short pulses of “kissing” sound. Don’t increase the suction too much in any circumstance. You can substitute this with short, multiple, gentle suctions to achieve the same result.

8. One go or let it go

Patching is more of an all-or-none process. However, while patching mature cells of adult animals, if the cell’s series resistance is not ideal, a slight suction can improve it. However, the success rate of this process is lower for these immature cells. If series resistance is increasing and you want to improve it, it’s not a good idea to do that as you may lose the cell. If series resistance crosses the allowed range, a better idea is to just discard the cells and try again with a fresh pipette.

Some other tips

  • Before starting your experiments, it is advisable to sterilise your instruments either using ethanol or by giving them UV treatment for half an hour.
  • Use a fresh blade for slicing each day. Old blades might get microscopic rust or get blunt, both of which can damage the slices.
  • Use a harp with wide-spaced nylon threads to hold the slice in the slice chamber when working with slices made from embryonic and young pups to minimise damage.

About the author

Deepanjali Dwivedi is a fifth-year PhD student studying neuroscience at the National Centre for Biological Sciences (NCBS), India. She has extensive experience in whole-cell patch-clamp electrophysiology and data analysis using MATLAB. She is interested in ion channel physiology, neurological disorders, microglia and astrocyte biology, and interneuron functioning.

Banner image credit: Brain slice methods

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