Solutions

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 1st stop measures the volume you need, but if you press harder there will be the 2nd stop that empties the pipette entirely, and if you press even harder - there will be yet the 3d stop that ejects the tip. Beware that the scales take long time to switch on for the 1st 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)

Procedure

  1. We first try to make 100 ml or internal solution without ATP and GTP. Start with ~70 ml of water, and add all the required drugs.
  2. Drive the solution pH to the target (7.2), using KOH as a base (HCl in case of overshoot).
  3. Measure osmolarity, and add water to make the stock 250 ± 5 Osm. We should usually end up with 90+ ml of solution.
  4. Aliquot in 10 tubes, 10 ml each, using a syringe with a disk-shaped filter tip (draw without a filter, expel through the filter). All tubes but one are to be frozen at −20°C.
  5. Measure ATP and GTP in amounts due for 10 ml of solution, and add them to the single tube that would go into further processing. Use small hexagonal weighload, and when the desired weight is achieved, take 200-300 μl of solution from a tube with a micropipette, and move it to the weighload; dissolve the grains, and move the solution back. To dissolve the grains quicker, you may try to suck and expel the liquid through the tip several times. If you have problems with measuring the required weight precisely, you may also weigh slightly more, then move some volume as described, and then move back only part of the volume, proportional to the ratio of desired weight to the actual weight, thus ensuring correct concentration in the solution.
  6. Aliquot this full solution into 20 2.5 ml conical tubes, .5 ml in each. 19 of them go into deep freeze at −80°C, while one goes into experiment. The fridge is located in the lab nearby.
  7. To unfreeze a full stock .5 ml tube, just take it from the −80°C fridge, and let it thaw. To unfreeze the 10 ml of ATP/GTP-less stock, keep it in hot water for ~20 min, applying vortex shake from time to time.

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.