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VOL. 71 (4), 821-833, 2005 CHOLINERGIC CURRENTS IN XENOPUS...

with a two-electrode system (Axoclamp-2A, Axon Instruments, USA)
20-48 h after the membrane transplant. Intracellular electrodes (1-3
MW resistance) were filled with 3 M KCl for voltage recording and
current injection. The volume of the oocyte recording chamber was
200 µl. Membrane currents were sampled by Lab PC+ (National
Instruments, USA) at twice the filter frequency, low-pass filtered
at 10 Hz and recorded on a PC using the Whole Cell Analysis v. 2.1.
program which was kindly provided by Prof. J. Dempster
(Strathclyde University, Scotland, UK). Currents were elicited by
challenges of 1-3 mM cholinergic agonist. The interval between
consecutive responses was systematically set to 10-12 min (flow
rate 6-8 ml. min–1), as we had previously established that this
was sufficient to ensure a complete recovery from receptor
desensitization. Oocytes from Xenopus donors were tested for
muscarinic acetylcholine receptors. To ensure the consistency of the
response amplitude, three consecutive agonist challenges were
applied before changing to other agonist or antagonist.

We tested the effects of the nicotinic agonists, nicotine,
acetylcholine, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), at
concentrations from 1 to 3 mM, on the Xenopus oocytes transplanted
with the human brain membranes. The membranes from frontal
cortex were obtained in two independent fractionations. The results
were recorded in oocytes from 12 Xenopus donors.

4. Measurement and calculations

The amplitude and time constant of responses was measured
through Whole Cell Analysis. Statistical analysis was done using
SigmaStat 2.0 (SPSS Inc, Chicago, IL), using the one way Anova
Test.

5. Drugs

(-)-Nicotine, acetylcholine chloride, 1,1-dimethyl-4-phenyl-
piperazinium iodide (DMPP), a-bungarotoxin and d-tubocurarine
chloride were obtained from Sigma, St Louis, MO, USA.

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