Internal solutions

Internal solutions are particularly important for slice electrophysiology. They are designed to mimic the internal osmolarity and ionic balance of cells when paired with ACSF. The type of internal solution you use depends on the experiment you want to do. However, the most common internal solution for current clamp recordings is a K-based internal and a Cs-based internal is typically used for voltage clamp recordings. There are a couple of somewhat common items you can add to your internal that help in visualizing cells. Alexa-fluor hydrazides (25 uM is usually enough) can be added to visualize cells acutely while you are patching. If you want to post-fix the tissue and label the neurons then you would use something like 0.1 uM biocytin. These can be added the day of the recording by making a stock solution and adding about 1 uL to your internal solution.

Some tips for making internal solutions: Use volumetric flasks if possible.

Always start with less water than you need, add the reagents and then top off the solution with water to the volume you need.
Always use high purity water, typically water made by a MilliQ machine or water that had ~18.2 MOhm of resistance (this is highly pure water).
Important: Always pH your solutions before and immediately after adding ATP/GTP. This is because ATP and GTP are acidic (especially ATP) and will lower the pH of your solution. ATP/GTP rapidly hydrolyze in acidic solutions so you do not want to lose all your ATP/GTP. Use KOH or CsOH (depending on the main ingredient in your internal solution) to adjust the pH to ~7.4.
Store the internal in the -20/80 C freezer. Your internal should last a long time.
Important: Always double check whether you are using the magnesium or sodium salts of ATP and GTP. These are important for setting the ionic gradients in your cells and depend on what else you are adding to your internal.

Osmolarity¶

The osmolarity of internal solution can be anywhere from 285-315 mOsmo. The internal solution omsolarity should be within 10 mOsmo of your external solution but realistically should be within 5 mOsmo. General consensus seems to be to start with balanced osmolarity (internal == external) and then adjust from there if you are starting from scratch. If you find your cells shrinking that means you internal solution osmolarity is too low compared to your external solution osmolarity. If you are checking the osmolarity of your solution at the end of making it and find it too low then add the main reagent which is usually KMeSO3, KGluconate, CsMeSO3 or CsGluconate. It is best to add these because they are already in excess in your internal so they won’t change your ionic gradients that much (think the Nernst equation). You can also add a sucrose crystal to the internal to raise the osmolarity on the fly. You can adjust your external solution by adding a little bit of water or reducing the glucose. If your cells are bloating and they are not unhealthy then you can raise the osmolarity of your external solution by adding some glucose. It is important that your internal is pretty isotonic. Otherwise this may change your capacitance and membrane time constant measures. These are particularly important for more advanced analysis of electrophysiology data. You can add water to your internal but there is a greater chance of diluting the internal too much thus changing the ionic gradient or diluting the ATP/GTP/phosphocreatine too much.

You should check the osmolarity of your internal solution at least once after making it. You can use a vapor pressure or freezing point depression osmometer. I have found the freezing point depression osmometers to be more accurate and consistent. Important: Make sure your sample is at room temperature before testing the osmolarity using either method.

Internal solution and pipette access resistance¶

The ionic composition of an internal solutions contributes to the pipette access resistance. If you think about the internal solution as a liquid wire then the more ions we have the lower the resistance since there are more charge carriers. Additionally, the smaller the ion species the lower the resistance since they can theoretically move faster (less friction) in the solution. The “friction” an ion experiences in a medium is measured as ionic mobility ( $\mu$ ). If you look at old sharp electrode recordings they used 2M NaCl for recordings outside the cell and 2M KCl for recordings inside the cell. The high concentration of a small salt helps reduce the pipette resistance to an acceptable value when using sharp high resistance pipettes. For “modern” internal solutions you generally have an excess of KMeSO3, CsMeSO3, K-Gluconate or Cs-Gluconate so we use them to modify the osmolarity since adding or removing so will not change the reversal potential of K+ that much. Theoretically internal solutions with lower molarity of these reagents will tend to have recordings with higher access resistance. We usually do not want to add large amounts of CsCl or KCl to our internal for whole cell patch clamp since it will change the Cl- reversal potential due to perfusion of the solution. We can look at the conductivity of a solution mathematically as $\sigma=F\sum_i |z_i| \mu_i c_i$ where $F$ is the Faraday’s constant, $|z_i|$ is the valence charge of an ion, $\mu_i$ is the ionic mobility and $c_i$ is the concentration. One last issue is that gluconate has a tendency to precipitate which will increase the access resistance.

Gluconate vs methanesulfonate¶

Gluconate and methanesulfonate are biologically neutral anions that are carriers for K+ or Cs+. Gluconate (HOCH2(CHOH)4CO2H) is an organic anion which is just an oxidized version of glucose. Gluconate chelates K+ and Ca2+ ions. Gluconate can be used as a substitute for Cl- to maintain osmotic balance without contributing to Cl- currents. Methanesulfonate (CH3SO3H) is a mesylate or organosulfuric compound and a strong acid. Unlike gluconate, methanesulfonate is not known to chelate or bind to other metal ions in water. You may see recipes with methanesulfuric acid (CH3SO4H), however I believe this is mostly likely a mistake. Some older papers use methanesulfuric acid but the recipes are bit different from todays recipes so it can be used but is not typical. I am not aware of a situation where gluconate or methanesulfonate would be better than the other. I have used both and found both to work well. What you use will probably depend on what your labs uses.

Other chemicals¶

HEPES: Anion buffering agent that maintains pH despite changes in carbon dioxide and does not bind to metal ions.
EGTA: buffers Ca++ prevents rundown/inactivation of Ca++ currents, prevents Ca++-dependent plasticity, prevents the membrane from reforming in whole-cell recordings.
Spermine: blocks Ca++ channels and Kir channels (normal function).
QX-314: blocks voltage-gated Na+ channels. Use the bromide version.
TEA-Cl: blocks K+ channels.
ATP: provides energy to the cells.
GTP: provide energy to the cells.
Phosphocreatine: reserve of phosphate to recycle ATP.
Mg++: Need a small amount, usually supplied with ATP or by adding MgCl2, involved in Kir ion channel function and as a cofactor for enzymes.
K+: The main positively charged ion in potassium-based internals.
Cs+: The main positively charged ion in cesium-based internal. Blocks K+ channels (particularly voltage-gated) and used to improve space-clamp.
Cl-: Chloride levels are extremely important. When studying Cl- currents you can manipulate the levels to change the reversal potential which allows you to record Cl- currents at -70 or at 0 mV. To study normal cell physiology, like rheobase, spiking behavior, etc, there is generally just a little bit of Cl- compared to the ACSF.
Na+: Sodium levels are extremely important. Generally there is a little bit of Na+ so that Na+ currents go into the cell. Too much Na+ can also cause excitotoxicity so unlike Cl-, which we can manipulate to change the reversal potential, Na+ levels are left as is.

Potassium-based internal¶

Potassium internals are used for recordings where you do not want to block an channels. This is very important for current clamp where you want to the neuron to spike as naturally as possible. You can also use potassium internals for recording specific ionic current such as Ih currents in voltage-clamp.

Potassium gluconate¶

Potassium methanesulfonate¶

Cesium internal¶

Cesium internal solutions are used for voltage-clamp recordings. Cesium blocks many inwardly-rectifying potassium channels as well as all 4 HCN channels. By blocking potassium and HCN channels, cesium internal makes it easier to voltage-clamp a cell by increasing the resistance of the cell, thus reducing the current needed to conteract changes in membrane potential. By increasing the resistance you also reduce space-clamp issues since it becomes easier to clamp to the distal parts of a cell. Space-clamp occurs when the effectiveness of voltage-clamp deceases the farther you get from the point of access to the cell (higher resistance means less current needed to adjust voltage). Space-clamp means that dendrites may not be effectively clamped which can impact your ability to record mEPSCs from more distal dendrites. Cesium internal needs about 2-4 min to perfuse throughout the cell for it to be effective.

I have found cesium internals to come in several varieties. There is the standard variety which is generally used to record AMPAR mEPSCs at -70 mV. There is a high chloride version that allows you to record GABAA currents at -70 mV as long as you block AMPA and NMDA currents. There is a low chloride version for recording AMPA currents at -70 mV and Cl- currents at 0 mV.

Recipe notes:

QX-314 (bromide) is used to block voltage-gated sodium channels. Be sure to use the bromide version so you do not change the chloride ionic gradient.

Standard Cesium methanesulfonate¶

A standard cesium interal used to measure AMPAR currents at -70 mV. You can record NMDAR currents at +40 mV as long as GABAA and AMPA receptors are blocked. One caveat to measuring NMDAR currents is that you can actually measure evoked NMDAR without blocking AMPAR currents however you the peak amplitude must be measured later than the actual peak because there are still AMPAR currents but they decay quickly. IPSCs can be recorded at 0 mV as long as NMDA and AMPA receptors are blocked.

Low chloride cesium methanesulfonate¶

This low chloride version of the cesium internal that allows you to measure AMPAR currents at -70 mV and GABAA currents at +0 mV as long as you block NMDA currents. The reversal potential of AMPA receptors is at 0 mV and the reversal potential of GABAA receptors is at -70 mV.

Cesium methanesulfonate with Ca2+ channel block¶

This internal is very similar to the standard cesium methansulfonate internal except that is has spermine added to block Ca2+ channels.