1. Picrotoxin (100 |M) and DL-2-Amino-5-phosphonopentanoic acid (APV) (40 |M) are included in the buffer when isolating (RS)-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated excitatory postsynaptic current (EPSCs). APV and 6,7-Dinitroquinoxaline-2,3-dione (DNQX) (10 |M) are included when isolating GABAA-mediated IPSCs.
2. Typical internal solution for isolation of inhibitory postsynaptic currents (IPSCs): 125 mM CsCl, 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonate (HEPES), 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM CaCl2, 2.0 mM Mg2+-ATP, 0.2 mM Na+-Guanosine 5-triphosphate (GTP). For isolation of AMPA-
mediated EPSCs: 100 mM CsCH3SO3, 60 mM CsCl, 0.2 mM EGTA, 10 mM HEPES, 1.0 mM MgCl2, 1.0 mM Mg2+-ATP, 0.3 mM Na+-GTP.
3. Recording chambers and all tubing should be flushed well with distilled, deionized H2O at the end of each day, and with 70% EtOH solution (followed by H2O) on a weekly basis.
4. Although many of the cannabinoid ligands have affinities in the low nM range for the CB1 receptor, it is not uncommon in slice studies to utilize bath concentrations in excess of 1 |M. This probably reflects the ability of the drug to partition from bath to tissue, and specifically to receptor sites within the tissue.
5. Concentration-response curves are readily constructed using any curve-fitting software, such as Prism (GraphPad Scientific, San Diego CA). Typically 4-10 slices per concentration will be used, and slices should be obtained from several animals, rather than from a single subject. We have not routinely observed desensitization of responses using a cumulative concentration protocol, and therefore find this approach to be useful, especially in extracellular recordings that can be maintained for several hours.
1. Dunwiddie, T. V. (1986) The use of in vitro brain slices in neuropharmacology, in Electrophysiological Techniques in Pharmacology (Geller, H. M., ed), Alan R. Liss, Inc., New York, pp. 65-90.
3. Brundege, J. M. and Williams, J. T. (2002) Differential modulation of nucleus accumbens synapses. J. Neurophysiol. 88, 142-151.
4 Johnson, S. W. and North, R. A. (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J. Neurosci. 12, 483-488.
5. Weiner, J. L., Gu, C., and Dunwiddie, T. V. (1997) Differential ethanol sensitivity of subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal neurons. J. Neurophysiol. 77, 1306-1312.
6. Nicola, S. M., Kombian, S. B., and Malenka, R. C. (1996) Psychostimulants depress excitatory synaptic transmission in the nucleus accumbens via presynaptic D1-like dopamine receptors. J. Neurosci. 16, 1591-1604.
7. Zhang, X. F., Hu, X. T., and White, F. J. (1998) Whole-cell plasticity in cocaine withdrawal: Reduced sodium currents in nucleus accumbens neurons. J. Neurosci. 18, 488-498.
8. Hoffman, A. F. and Lupica, C. R. (2000) Mechanisms of cannabinoid inhibition of GABA(A) synaptic transmission in the hippocampus. J. Neurosci. 20, 2470-2479.
9. Hoffman, A. F., Riegel, A. C., and Lupica, C. R. (2003) Functional localization of cannabinoid receptors and endogenous cannabinoid production in distinct neuron populations of the hippocampus. Eur. J. Neurosci. 18, 524-534.
10. Edwards, F. A. and Konnerth, A. (1992) Patch-clamping cells in sliced tissue preparations. Methods Enzymol. 207, 208-222.
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