External Solution

All components of the External Solution (also known as Artificial Cerebro-Spinal Fluid, or ACSF) fall into 2 groups. The majority of components are stable, and so you can add them to deionized water, and store the solution in the fridge (at 2°C). Some of the components would however precipitate if you try to store them for long, so you have to add them to the solution immediately before the experiment. First, here's the stable stuff:

Substance  Concentration, mM      g/l

NaCl       115                    6.7206

KCl        4                      0.298

HEPES      5                      1.191

Glycine    10 μM                  1ml of 10 mM stock

Glucose    10                     1.802

And second, immediately before the experiment you also add proportional amount of the following unstable components:

CaCl2      3 3ml of 1M stock

MgCl2      3 3ml of 1M stock

pH=7.2; Osmolarity=250

Alternative ionic concentrations:

• To hyperpolarize all cells slightly (may decrease total N spikes, but improves spike-timing): 6 Mg, 3 Ca.
• To depolarize the cells (hopefully to somewhat deactivate Na channels): 6 K, 1.5 Mg.

Preparation

Here's some general tactics for the solution preparation:

Take deionized water, about ~80-85% of the final solution volume, and mix the stable reagents in;

Bring pH to the target by carefully adding suitable bases (or acids). For the external a suitable pare is obviously NaOH/HCl, as external is Na-based, while for the typical internal you would use KOH/KCl instead.

Bring osmolarity to the target by adding di water. You'd better do it in 2-3 iterations, in order not to overshoot (as all measurements are noisy, including that from the osmometer). To be on a safe side, when the calculation tells you that X ml of distilled water is to be added to bring osmolarity to the target, add only 80% of this volume. This way you'll approach the target slowly, and the ultimate result will be more precise.

Detailed instructions for the pHmeter and osmometer are provided below. With amounts shown in the tables above the final solution volume may be slightly (2-5%) less than the target, but the osmolarity should be correct.

Use a 1 liter beaker with a magnetic stirrer working at ~300 rpm ongoing. Use big hexagonal weighboats for NaCl, and square pieces of waxed paper for everything else. To transfer the stock solutions, choose a proper pipetter; the color of the pipetter button corresponds to the color of conical tips to use. When pressing the button, the 1^st stop measures the volume you need, but if you press harder there will be the 2^nd stop that empties the pipette entirely, and if you press even harder - there will be yet the 3^d stop that ejects the tip. Beware that the scales take long time to switch on for the 1^st time, and they can't zero the TARE until the weight is really stable (e.g. when the window is open they cannot do that, as the wind from the ventilation system seems to be strong enough to change the "weight" at the scales; they also seem to be sensitive to your steps if you are jumping around). So be patient. Note also that while all the beakers have volume marks on them, these marks are actually ridiculously incorrect. All volumes are to be measured with the measuring cylinders, and only with them.

After the osmolarity is measured, and the stock is brought to its final volume, it should be filtered with a vacuum-driven huge orange disposable filter right into a bottle where it will be stored. Label the bottle, indicating the owner, the date, and the solution type.

As mentioned above, immediately before the experiment, Ca and Mg are to be added to the ACSF from stock solutions. Make a point of always adding Ca and Mg in the same order, so that even if you are sleepy or get distracted, and forget what you were doing midway in the process, you would always know which of the salts you have added already, and which is still missing. Some people in the lab traditionally add MgCl₂first, and CaCl₂ second; some other people always do Ca first and Mg second (which is also incidentally the alphabetical order), but the point is in always doing it the same way. No need for guessing or hectic recollection. If you are sure that you have added some ion already, you'll always know which one it was.

Internal Solution

For the internal, these substances are to be added to the stock from the beginning:

What            Molarity, mM              g / 100 ml of solution

K-gluconate     100                       2.343

KCl             8                         0.0596

NaCl            5                         0.0292

MgCl2 · 6H2O    1.5                       0.0305

HEPES           20                        0.4766

EGTA            10                        0.3804

And these 2 substances, being unstable, are prepared and added separately, as it is described below:

ATP             2                         11 mg / 10 ml

GTP             0.3                       1.57 mg / 10 ml

Target pH=7.2; Osm=250 (would be ~255 after ATP+GTP are added)

Predicted membrane potentials for these ACSF and Internal

• EK: −102 mV
• ENa: 72 mV
• ECl: −53 mV
• Junction Potential: either +12 or +14 mV (depending on calculation). It means that when you zero your offset, and then think that you are clamping the cell at 0 mV, actually it is clamped at about −12 mV (see this link for details)

Cs-based Internal

Per 100 ml:

CsMethaneSulfonate           80 mM *            1825 mg

MgCl2                        5 mM               101.7

TEA                          20 mM              331.4

EGTA                         10 mM              380.4

HEPES                        20 mM              476.6

ATP                          2 mM               11 mg / 10 ml

GTP                          0.3 mM             1.57 mg / 10 ml

To be brought to pH 7.2 by CsOH.

* Supposedly after you add CsOH, the concentration of Cs reaches about 90 mM.

Anesthetic (MS-222) solution

Anesthetic cannot be dissolved in deionized water (it will hurt tadpole skin and membranes), and it cannot be dissolved in blue rearing media (it reacts with methylene blue and forms some orange compound that seem to be toxic).

Instead, we dissolve it in "reconstructed water" that has higher osmolarity than diH2O. Recipe: 1L dH2O + 50 mM NaCl (2.92 g) + 0.02% by weight of tricaine methane sulfonate (MS-222, 200 mg). Because every tadpole spends only 1-5 min in the anesthetic solution, we can afford not to add other salts, and we don't buffer it.

References

Pratt, K. G., Dong, W., & Aizenman, C. D. (2008). Development and spike timing–dependent plasticity of recurrent excitation in the Xenopus optic tectum.Nature neuroscience, 11(4), 467-475.

Khakhalin, A. S., & Aizenman, C. D. (2012). GABAergic transmission and chloride equilibrium potential are not modulated by pyruvate in the developing optic tectum of Xenopus laevis tadpoles. PLoS One, 7(4), e34446-e34446.