SEIX 17-20 octobre 2005 - Atelier Calcium

signalling to address the functions of calcium signalling components in Drosophila S2 cells.
This approach has proved successful in identifying STIM-1, a novel protein coupling store
depletion to capacitative calcium entry [7, 8].
CALCIUM MEASUREMENTS IN DROSOPHILA CHOLINERGIC NEURONS
We have used fura-2-based calcium imaging to examine nAChR-induced increases in [Ca 2+ ] i
in 3 rd instar larval primary cultured neurons derived from a transgenic Drosophila line in
which GFP is expressed in cholinergic (Cha-1) neurons of the central nervous system.
Cholinergic (Cha-1) neurons were isolated from flies homozygous for a 7.4 kb Cha-Gal4
driver and a UAS-GFP(S65T) responder transgene. Primary cultures from 3 rd instar larvae
were generated. Larvae were surface-sterilized in 70% ethanol, rinsed 3x in distilled water
and brains isolated in dissection medium (27% Leibovitz medium, 73% dissociation saline).
The composition of dissociation saline is as follows (in mM): CaCl 2 , 5.4; KCl, 21.4; MgCl 2 ,
12.3; NaCl, 36.0; NaHCO 3 , 4.8; KH 2 PO 4 , 1.5; glucose, 11.1; Tris, 24.8; penicillin G (50
units/ml), streptomycin sulphate (50 g/ml), pH 7.2, adjusted with NaOH and HCl. Brains
were incubated in 0.5 mg/ml collagenase in Ca 2+ - and Mg 2+ -free saline solution (in mM: NaCl
137; KCl, 2.7; NaH 2 PO 4·H 2 O, 0.36; NaHCO 3 , 12.0; glucose, 0.56) for 45 min in order to
disrupt the extracellular matrix. Optic and antennal lobes were removed prior to dissociation.
Larval brains were triturated using a sterile siliconized glass pipette. Cells were plated onto 22
mm diameter round glass coverslips and incubated in dissociation medium (dissection
medium plus 10% v/v heat-inactivated foetal bovine serum). Two day old neurons on
coverslips were rinsed three times in physiological saline (of composition in mM: NaCl, 140;
CaCl 2 , 2.0; MgCl 2 , 4.0; KCl, 3.0; HEPES 5; pH 7.2), and loaded with dye by immersion in
physiological saline containing the Ca 2+ -sensitive dye fura-2AM (1 M) and 0.002% pluronic
acid F-127 dispersing agent in saline for 1 h at room temperature.
GENE EXPRESSION PROFILING IN DROSOPHILA CHOLINERGIC NEURONS
USING MICROARRAYS
The only way to monitor and compare the full complement of genes being expressed in cells
under different conditions is by the use of microarrays. In a collaboration with The Salvaterra
lab we have used microarray-based gene expression to study the genes expressed in GFPtagged
cholinergic neurons of Drosophila. Based on studies with Affymetrix microarrays, we
have shown the presence of a number of ion channels and receptor subunits in these cells
including nAChRs. The expression of the D 2, D 6, D 7, D 1 and D 2 subunits has been
detected in embryonic and larval Cha-1 RNA by microarray analysis. The D 2 and D 2
subunits are the most abundantly expressed subunits at both developmental stages. The
mAChR receptor, DM1 is detected as well as voltage-gated sodium and calcium channels. IP3
receptors and ryanodine receptors are both present in these cells, in contrast to S2 cells where
only IP3 receptors are present.
SILENCING ENDOGENOUS RECEPTOR SUBUNITS AND MONITORING
FUNCTION USING FURA-2 BASED CALCIUM IMAGING
Using calcium imaging and patch-clamp electrophysiology, we have shown that Cha-1
(cholinergic) neurons respond to ACh, nicotine and choline. Responses to these agonists are
blocked by 10 M mecamylamine and 1 M -bungarotoxin ( -BTX). PCR amplification of
nAChR subunits from embryonic and larval Cha-1 cDNA shows that embryonic and larval
Cha-1 neurons express multiple nAChR subunits. We have detected developmental regulation
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