Aplysia Neuron Recording Solutions

Methods for recording single K⁺ channels in neurons of the marine mollusk, Aplysia californica have been described in detail by Siegelbaum et al (1982), Shuster (1986), and Shuster & Siegelbaum (1987). See also Strong et al (1987) for methods to patch single voltage-gated Ca²⁺ channels in Aplysia neurons. Several special conditions apply for patch clamping Aplysia neurons. First, the osmolarity of the Aplysia recording and bath solutions is much higher than for mammalian solutions. I try to match the osmolarity and pH of my solutions to Aplysia hemolymph, which is ~980 mOsm and pH 7.8 for animals obtained from Marinus (Long Beach, CA). Note also that the high ionic strength of Aplysia solutions reduces the resistance of the recording pipette by about half from the expected value in mammalian solutions. The high ionic strength also makes it more difficult to obtain seals with resistances as high as those obtained in mammalian salines. The presence of stronger leak currents seems to be responsible. However, it is relatively easy to obtain seals with resistances in the 10 - 20 GOhm range. Even though you may only obtain a 10 GOhm seal, the high ionic strength of marine salines can also produce somewhat larger currents (given that the permeant ion concentration is greater than for mammalian solutions).

Besides problems associated with ionic strength, the osmolarity of marine salines is often adjusted by addition of sucrose, which can create other problems. When using sucrose-containing solutions be aware that the increased viscosity can appreciably slow down mixing of solutions. Another problem is that sucrose serves as a carbon source for the growth of microorganisms. Contaminated solutions will not initially appear discolored but instead will appear hazy and slightly luminescent. Solutions can go bad within days, therefore you may want to make up your solutions in small batches. Also, sterile filter the solutions immediately before use. If your filter gets clogged, this is a good indication that your solution is contaminated.

Detailed information on culture of Aplysia neurons can be found in Schacher & Proshansky (1983) and Goldberg (1991). While Aplysia neurons are quite beautiful (most are quite large and contain a bright orange pigment) I have had difficulty getting isolated neurons to attach to culture dishes. The relatively large size of many Aplysia neurons contributes to these and other difficulties in cell culture. An alternative is to record from neurons in intact ganglia which have been dissected from the animal and pinned in a recording chamber (e.g., see Shuster and Siegelbaum, 1987). The pinned ganglion preparation offers the advantage that the individual neurons can be identified (for review see Koester and Kandel, 1977).

ARTIFICIAL SEAWATER

500 mL, pH = 7.8, 1010 mOsm

Titrate with NaOH.

	MW	mM	g
NaCl	58.44	460	13.44
KCl	74.55	10	0.37
HEPES	238.31	10	1.19
CaCl2	110.99	11	0.61
MgCl2	95.23	54	2.57

APLYSIA NEURON DEPOLARIZING SOLUTION

250 mL, pH 7.8, 980 mOSm

May also be used as a recording pipette solution. Titrate the pH with methanesulfonic acid after addition of divalents (which can lower the pH). An alternative to methanesulfonic acid is to use aspartic acid. Note: for cultured neurons, adjust the osmolarity with sucrose to match the osmolarity of the culture medium.

	MW	mM	g
KOH	56.11	360	5.05
HEPES	238.31	10	0.60
EGTA	380.35	1.5	0.14
sucrose	342.30	250 (empirical - to adjust osmolarity)	15.57
CaCl2*	110.99	1	0.03
MgCl2	95.23	5	0.12
TEA-Cl**	165.7	10	0.41

* Free Ca²⁺ estimated to be ~80 nM (Brooks and Storey, 1992).

** External TEA blocks the Ca2+-activated K⁺ channel with an effective KD of ~300 µM. External TEA also acts as a fast open channel blocker of the S channel with an effective KD of ~90 mM. Internal TEA blocks the S channel with an effective KD of ~ 40 mM (Shuster and Siegelbaum, 1987).

APLYSIA L-15 SALINE FOR CELL CULTURE

250 mL, pH = 7.8, 1052 mOsm

This saline is used with Leibovitz's L-15 medium powder (GibcoBRL 41300-021) to make media for culture of Aplysia neurons. The final salt concentration after addition of L-15 approximates artificial seawater. The osmolarity after addition of L-15 is approximately 1215 mOsm.

	MW	mM	g
NaCl	58.44	307	4.49
KCl	74.55	5	0.09
HEPES	238.31	15	0.89
glucose	180.16	35	1.58
NaHCO3	84.00	2	0.04
CaCl2	110.99	10	0.28
McCl2	95.23	27	0.64
MgSO4	120.38	26	0.78

REFERENCES

Brooks SP, Storey KB. 1992. Bound and determined: a computer program for making buffers of defined ion concentrations. Anal Biochem, 201, 119-126.

Goldberg, DJ 1991. Culturing the Large Neurons of Aplysia. In G. Banker & K Goslin (eds), Culturing Nerve Cells. The MIT Press, Cambridge, Massachusetts. pp. 155-175.

Klein M. 1993. Differential cyclic AMP dependence of facilitation at Aplysia sensorimotor synapses as a function of prior stimulation: augmentation versus restoration of transmitter release. J. Neurosci., 13, 3793-3801.

Koester J, Kandel ER. 1977. Further identification of neurons in the abdominal ganglion of Aplysia using behavioral criteria. Brain Res, 121, 1-20.

Schacher S; Proshansky E. 1983. Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment. J. Neurosci., 3, 2403-13.

Shuster, M.J., J.S. Camardo, and S.A. Siegelbaum. 1991. Comparison of the serotonin-sensitive and Ca2+-activated K+ channels in Aplysia sensory neurons. J. Physiol. 440:601-621.

Shuster, M.J. and Siegelbaum, S.A. 1987. Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia neurons. J. Gen. Physiol., 90, 587-608.

Shuster, Michael Jeffrey. 1986. Modulation and characterization of the "S" K+ channel in cell-free membrane patches. Ph.D. Thesis. Columbia University. University Microfilms International. 300 Z. Zeeb Road, Ann Arbor, MI 48106.

Strong JA, Fox AP, Tsien RW, and Kaczmarek LK. 1987. Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons. Nature, 325, 714-717.