External solutions¶

External solutions are very important for getting good recordings. External solutions for slice electrophysoilogy are generally referred to as artificial cerebral spinal fluid (ACSF). There are many variaties of ACSF. Some recordings cannot be done without specific types of ACSF. Some protocols use different ACSF for cutting, incubation and recording while other protocols use one solution for everything. ACSF was designed to be a physiological buffer that matches external ionic balance, osmolarity and pH that neurons experience in-vivo.

A bicarbonate-based buffer¶

A majority of ACSF recipes are bicarbonate-buffer based solutions that need CO2 to maintain a pH of around 7.4. Bicarbonate is actually a terrible pH buffer because it is very volatile, especially compared to other to pH buffers like HEPES and degrades quickly in the presence of H+. However, bicarbonate is a very physiological buffer in that it is the main way the O2/CO2 balance is managed in our bodies. While bicarbonate is volatile, our bodies are constantly producing bicarbonate but this is not so in solution so the levels of bicarbonate are carefully chosen to ensure that the pH remains close to 7.4 while bubbling with carbogen without having to add an acid or base. There is a little bit of sodium phosphate buffer in ACSF, bicarbonate is by far the biggest contributor to pH buffering.

Osmolarity¶

Osmolality of ACSF is generally anywhere from 280-310 with the vast majority of papers reporting osmolality between 295-305. The ACSF osmolarity should be balanced with your internal osmolarity. It is general recommended that the difference in osmolarity between the ACSF be between -5 (higher osmolarity in the internal) to 10. In my hands, an osmolarity difference greater than 10 almost always causes problems with the access resistance where the access resistance is either high or rapidly climbs over the recording. There are two ways I have heard of to adjust the osmolarity of the the ACSF. One is to just add or remove water (this seems controversial). Adding/removing water will change your overall ionic concentrations. The other way is to add or remove glucose. For smaller changes you could potentially adjust the amount of NaCl/choline/sucrose/NMDG in the solution.

Common reagents¶

• Na+: Neurons need a lot of Na+ to spike since there are large amounts of Na+ channnels in neurons. You also have to have Na+ to get AMPA currents.

• Bicarbonate: Used as a pH buffer when the ACSF solution is bubbled with carbogen (95%O2/5%CO2). Your body has an endogenous bicarbonate buffer system so the bicarbonate is also an attempt to replicate this system. Bicarbonate is also used to prevent cell swelling however I have also been told that higher concentrations can make the membrane “stiffer”. Bicarbonate is provided as sodium bicarbonate or choline bicarbonate. If your experiments include manipulating the cholinergic system you probably want to use sodium bicarbonate.

• Sodium phosphate (monobasic monohydrate): Helps maintain pH and phosphate can technically be used for energy generation (the ATP).

• K+: Neurons need K+ for spiking in particular the afterhyperpolarization potential and maintaining the ionic balance. I have found that if you forget to K+ to the ACSF all the cells in the slice disapeer. Typically added as KCl.

• Mg: Mg+ blocks NMDARs. High levels inhibit activity and low or absent levels can induce spontaneous activity in slices. Mg+ is usually used in two different forms: MgCl2 6H2O or MgSO4 7H2O. The choice probably depends on how much Cl- **you are adding with other reagents (NaCl, CaCl2, KCl, etc).

• Ca++: Ca++ is need for Ca++ gated voltage channels as well as NMDAR currents. There are even some configurations of AMPARs that are permeable to Ca++. Ca++ is typically added using CaCl2 2*H2O.

• HEPES: HEPES is used to prevent edema in tissue slices MacGregor et al., 2001. HEPES is also a pH buffer that is resistant to changes in carbon dioxide levels.

• Choline: Methylated organic cation that replaces Na+ to prevent excitotoxicity (rapid influx of Na+) while maintaining the nature ionic gradient.

• NMDG: Methylated organic cation that replaces Na+ to prevent excitotoxicity (rapid influx of Na+) while maintaining the nature ionic gradient.

• Glucose: Provides an energy source to the cells and is used to adjust the osmolarity.

• Sucrose: Used increase the osmolarity of a solution and also to replace Na+ when trying to prevent excitotoxicity. One interesting thing about sucrose it that because it has more -OH groups than glucose it is more likely to draw water with it than glucoseEbrahimi & Sadeghi, 2016. So if you want to prevent water from going into the cell membrane then adding sucrose to the extracellular solution could help which is probably why it was orginally used in cutting solutions.

• Pyruvate: Energy source used in the citric acid cycle. Can also be used as an antioxidant. This helps keep cells healthy.

• Ascorbate: Also known as vitamin C or citric acid, can be used as an antioxidant and is involved in the citric acid cycle. This helps keep cells healthy.

Some basic tips for making external solutions:

• Do not make stock solutions with sodium/choline bicarbonate. In the absence of CO2, bicarbonate quickly makes the solution alkaline. Once the solution is alkaline you can bubble with carbogen but it can take a while, if ever, for the solution to return to 7.4 pH. If you add an acid like HCl the bicarbonate turns in to NaCl, H2O and CO2 so avoid adding HCl to titrate the pH.

• Important: Bubble the ACSF with carbogen before adding MgSO4, MgCl2 or CaCl2 as these can precipitate out when the pH is above ~7.6.

• Suggested: Bubble CaCl2 with carbogen before adding to the ACSF.

• 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).

• Magnesium comes in a SO4 or a Cl2 form. Both forms are hydrated which means that the molecular weight include water. You will need to caculate the pure molecular weight of MgSO4 or MgCl2 to calculate the concentration and adjust the amount of reagent used to account for the hydration of the magnesium reagent.

• It is important to note that the NaCl and glucose/dextrose concentrations can be adjusted and are often different from lab to lab. These differences may be due to the osmolarity and ionic balance of the internal used.

External solution recipes¶

Standard/Recording ACSF¶

Standard ACSF is the primary solution for electrophysiology recording. It generally contains Na+, K+, Ca++, Mg++, sodium phosphate (typically monosodium monophosphate monohydrate), sodium bicarbonate and glucose. Most recording ACSF recipes I have seen have higher Ca++ than Mg++. The Ca++ and Mg++ concentrations will vary between labs. Additionally if you lower both Ca++ and Mg++ to 1.0 and 0.25 mOsmo respectively (which is sometimes called Ringer’s Solution) you can induce spontaneous activity in pyramidal cells. Recipes are handed down from lab personnel to lab personnel so the original considerations for using the recipe are typically lost to time. Below is a protocol that you can use. For the KCl, MgCl2, and CaCl2 you can make 10x stock solutions.

Notes:

• You can make a 10x stock solution of NaCl, KCl, MgCl2, CaCl2 and sodium phosphate.

• Once you add sodium bicarbonate you need you need to bubble the ACSF with carbogen before adding MgCl2 or CaCl2 as these can precipitate out when the pH above ~7.6.

Choline ACSF¶

Choline ACSF is like standard ACSF except that choline chloride is used instead of sodium chloride. Choline is a methylated organic cation that replaces NaCl to prevent excitotoxicity (rapid influx of Na+) while maintaining the ionic gradient. Unlike other Na+ substitution methods choline chloride maintains Cl- levels close to what is in ACSF. Choline ACSF can be used as a perfusion, cutting and short duration (~10 min) recovery solution. When used as a cutting solution it is generally chilled and when used as a recovery solution it is generally heated to ~32 C. Choline ACSF also contains thiourea (occasionally), Na-pyruvate and Na-ascorbate. Choline ACSF can be chilled before use or even frozen to a slush. However, I have found that slushy choline ACSF during the slicing step seems to be bad for adult tissue and leads to longer recovery time. Other work has shown the even ice cold solutions can have an adverse effect on the tissueKirov et al., 2004Eguchi et al., 2020Fiala et al., 2003. I advise using either room temperature or chilled choline ACSF. Choline is an agonist at acetylcholine receptors, particularly the $\alpha7$ containing receptorsAlkondon et al., 1997 and at concentrations far below (micromolar) what is used in the choline ACSF (millimolar). The question is whether there are long term effects of stimulating $\alpha7$ containing receptors and how long those receptors stay desenstized. A few types of neurons express high levels of receptors with the $\alpha7$ subunit (layer 1 interneurons, VIP interneurons, and some hippocampal neurons). One huge potential benefit of using choline ACSF is that choline is used to synthesize the lipid components of the membrane. Neurons undergo extreme membrane stress when the tissue is cut so it is possible that excessive choline provides a pool or choline used to stabilize membranes through synthesis of new membrane lipid components. When choline ACFS is used as a recovery solution do not leave the slices in choline ACSF for more than 10-15 min. In my hands prolonged incubation in choline ACSF increases access resistance to the point where it is impossible to get usable cells.

Recipe notes:

• You can make a stock solution of KCl, MgCl2, CaCl2 and sodium phosphate.

• Once you add sodium bicarbonate you need you need to bubble the ACSF with carbogen before adding MgCl2 or CaCl2 as these can precipitate out when the pH above ~7.6.

• Choline is solid as a dehydrated powder and hydrates fairly quickly. It should be stored in a desiccant box or under vacuum. When choline hydrates it seems to go bad quickly. Choline hydration can change how much choline you are adding since there is water mixed in with the choline.

NMDG ACSF¶

NMDG ACSF is similar to standard ACSF except that NMDG replaces the Na+. NMDG is a methylated organic cation that replaces Na+ to prevent excitotoxicity (rapid influx of Na+) while maintaining the ionic gradient. The chloride comes using HCl to titrate the pH. NMDG ACSF can be used as a perfusion, cutting and short duration (~10 min) recovery solution. When used as a cutting solution it is generally chilled and when used as a recovery solution it is generally heated to ~32 C. NMDG ACSF also contains thiourea, Na-pyruvate and Na-ascorbate. NMDG ACSF can be chilled or used at room temperature for the slicing step. Freezing the NMDG to the point of being slushy is not recommended. There is one study comparing choline and NMDG ACSF and found no difference in the survival of interneurons between the two solutions and both were better than ACSFTanaka et al., 2008. Some studies claim to find that NMDG is superior to sucrose ACSF and others find no differenceTanaka et al., 2008Pan et al., 2015Martina & Taverna, 2014. Do not leave your slices in the NMDG recovery solution for more than 12 min Martina & Taverna, 2014. NMDG recovery seems to promote good neuronal morphology at the expense of good neuronal function (i.e. your good neurons are easier to see but so are the bad neurons even if they don’t look bad). Lastly the recipe here does is not the only NMDG ACSF. You can lower the glucose by 1/2 and substitute MgSO4 with MgCl2. MgCl2 will bring your chloride levels close to normal ACSF (the rest of the Cl- comes from the HCl you use to pH the solution). I have never found out why the modern NMDG recipe uses MgSO4 instead of MgCl2. One of the earliest NMDG recipes I find uses MgCl2. The only reason I can guess is to lower the osmolarity. There are problems using MgSO4. One problem is SO4 salts tend to be less soluble, which is probably why MgSO4 is used at 10 mM instead of 7 mM like in the choline ACSF. Since SO4 salts tend to be less soluble SO4 can chelate ions in solution. CaSO4 is fairly insoluble so it is possible that Ca++ is being chelated in the NMDG solution which is not good. No Ca++ can destabilize cells. Someone I need was trying to study how ER calcium levels change synaptic release probability and to do so they had to switch to Ca++ free ACSF. The cells they were recording would get extremely unstable. Lastly, when using the NMDG there is modification that spikes in a high NaCl NMDG ACSF during recovery.

Recipe notes

• You can make a stock solution of KCl, MgCl2, CaCl2, HEPES, NMDG and sodium phosphate just make sure to pH to 7.4 before adding the ions.

• The solution needs to have the pH adjusted with HCl after adding NMDG.

• Once you add sodium bicarbonate you need you need to bubble the ACSF with carbogen before adding MgCl2 or CaCl2 as these can precipitate out when the pH above ~7.6.

HEPES Holding ACSF¶

HEPES holding ACSF is like standard ACSF except that it has lower Na+ and has HEPES added to prevent edema. Edema primarily occurs in adult brain slices, the older the mouse/rat the tissue came from the worse the edema. There are some labs that use the HEPES holding ACSF no matter what. I am not sure it makes a difference for most recordings but that could depend on the cell type, brain region and type of tissue. HEPES ACSF also contains thiourea, Na-pyruvate and Na-ascorbate similar to what you would find in the choline and NMDG ACSF.

Recipe notes:

• You can make a stock solution of KCl, MgCl2, CaCl2, HEPES, NaCl and sodium phosphate.

Sucrose ACSF¶

Sucrose ACSF has all or some of the NaCl is replaced with sucrose. The recipe here replaces all the NaCl with sucrose but I have seen as little as 32 mM of NaCl replaced with sucrose (64 mM). The sucrose is primarily used to keep the osmolarity the same when the NaCl is removed. Remember to keep the osmolarity the same you need to add about 2 mM of sucrose for every 1 mM of NaCl you leave out. Sucrose ACSF seems to be used primarily for cutting, but not for holding or recording ACSF. Sucrose was the traditional choice for cutting tissue in. Many labs have switched over to NMDG or choline based ACSF.

Glycerol ACSF¶

Glycerol replaces NaCl in this recipe. I have not seen this recipe used much. One paper claims improvements over regular ACSF and sucrose ACSF Ye et al., 2006. Replacing NaCl with glycerol follows the same idea as sucrose ACSF. Why use glycerol instead of sucrose? The idea is that if sucrose gets into a cell it will pull water with it and cause the cells to swell (sucrose is very good a pulling water towards it due to the large number of OH groups) whereas glycerol apparently does not readily pull water towards (has about 1/2 the number of OH groups compared to sucrose). This apparent benefit is on top of reducing excitotoxicity due to Na+ flooding cells after cutting. Glycerol is also a naturally molecule in the body so it is biologically acceptable.

Low magnesium ACSF¶

Magnesium-free ACSF is ACSF with very little or no magnesium added. Some recipes increase Ca++ to make up for the osmolarity and ionic gradient difference. The only time I have seen this used is if you want to record NMDAR currents at -70 mV or if you want to induce epileptic-like activity in the cortex. As mentioned above there is a Ringer’s solution that still contains Mg++ that might be better for moderate amounts of spontaneous activity if you want to avoid epileptic-like activity. You will need to add TTX to the ACSF to prevent seizure-like activity if you are recording without any Mg++.

Strontium ACSF¶

Strontium ACSF has the calcium replaced with Sr2+. Generally, strontium ACSF recording projection/cell-type specific quantal events. Sr2+ is less effectively bound by proteins that are involved in synaptic vesicle release. As such when doing electrical or optogenetic stimulation you can get a large peak followed by small quantal events that occur during the decay of the event. It is not recommended to use this ACSF for holding tissue for long periods of time. See the optogenetic stimulation section for more about this experiment.

Tissue generally needs to sit in Sr2+ ACSF for 15-30 min before enough Sr2+ had diffused intracellularly. You may be able to shorten the incubation period by increasing the Sr2+ concentration above what you normally use for Ca++.

Recipe notes:

• It is recommended to add ~20 uM EGTA in the ACSF to bind any extra free Ca++ that may be introduced by transferring the tissue to the bath or by Ca++ that is released by the cells internal stores.

Comparing recovery solutions¶

I think it is useful to compare the osmolarity and ionic charge of standard ACSF, choline ACSF and NMDG ACSF to understand why and how these solutions were created. Below is a table that shows the different osmolarities of each reagent.

One thing to note is that sucrose has very little Cl- and Na+ or other postively charged ions. NMDG also does not have as much Cl- as ACSF but the charge is somewhat balanced since SO4-- has two free electrons. As mentioned above you can substitute MgSO4 with MgCl2 to bring chloride levels closer to standard ACSF. Both choline and NMDG ACSF solutions have lower amount of positively charged ions compared to standard ACSF.

What solution should you choose?¶

There are a lot papers using sucrose, NMDG and choline ACSF recipes all of which seem to have acceptable data. In my hands, the modern NMDG version does not seem to be any better than choline ACSF. I have noticed the tissue is grainer and the cells less healthy looking (shrunken and less plump). But this is not a quantitative assessment between the two solutions. The real question is what should the tissue look like if it were not sectioned? Additionally, the choice of some reagents in the NMDG solution like substituing MgSO4 for MgCl2 I feel like were never fully explained. So if you have time, I encourage you to try out different solutions keeping in mind the potential draw backs of each.

• Sucrose: Loss of total charge (no Na+ and Cl-)

• NMDG: Change in species of charged molecules (SO4-- replaces Cl-), potential chelation of Ca++ (due to SO4-- forming insoluble precipitate with Ca++), fairly different Cl- levels compared to ACSF.

• Choline: Potential activation and desensitization of $\alpha7$ containing receptors and the associated downstream effects of activating those receptors.

Drugs included in the external solutions¶

There are a variety drugs and their concentrations that you can include in your external solutions. These drugs are primarily used to target different receptors. Below is a list of drugs and why they are used. Note that this list is not comprehensive.

• (RS)-CPP/(R)-CPP (10 uM): Blocks NMDA receptors

• AP5 (50 uM): Blocks NMDA receptors

• NBQX (1 uM): Blocks AMPA and Kainate receptors

• CNQX (10 uM): Blocks AMPA and Kainate receptors

• DNQX (10 uM): Block AMPA and Kainate receptors

• TTX (0.3-1 uM): Blocks voltage-gated Na+ channel

• Picrotoxin (50 uM): Blocks GABAA receptors

• Gabazine/SR-95531 (2-20 uM): GABAA receptors

• 4-AP (50-1000 uM): Blocks voltage-gated K+ channels

• Strychnine: Blocks glycine receptors

• ZD-7288 (20 uM): Blocks HCN channels

• TEA (5 uM): Blocks K+ channels

• Cadmium chloride (0.1 uM): Blocks Ca++ channels

• Barium chloride (1 mM): Blocks K+ channels

DMSO¶

DMSO is usually needed for nonpolar drugs that you want to bath apply to your slices. I have seen it used at a concentrate of 1 uM in the ACSF. I would not recommend including this in your holding solutions and only using it in your recording solution if you need it. I have heard that it can make is harder to patch cells so some labs will first patch a cell in regular ACSF, then add DMSO and do their baseline recordings, and finally wash in their drug. Since you will need to flush the rig between bath applications of your drug it is pretty easy to just add some DMSO to your recording ACSF when needed. Also, I have seen that DMSO may built up in the tubing and in the incubation well. The DMSO/drug residue can be cleaned by just flushing the rig with 70% EtOH.