0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
tory postsynaptic currents (mEPSCs). Mice will be killed under halothane
anesthesia, and corticostriatal coronal slices (200 mm) will be prepared from
fresh tissue blocks of the brain with the use of a vibratome. A single slice will
then be transferred to a recording chamber and submerged in a continuously
flowing artificial cerebrospinal fluid (ACSF) (34°C, 2-3 ml/min) gassed with
95% O 2
- 5% CO 2
. The composition of the control solution will be (in mM): 126
NaCl, 2.5 KCl, 1.2 MgCl 2
, 1.2 NaH 2
PO 4
, 2.4 CaCl 2
, 11 Glucose, 25 NaHCO 3
.
Whole-cell patch clamp recordings will be made with borosilicate glass
pipettes (1.8 mm o.d.; 2-4 MW), in voltage-clamp mode, at the holding potential
(HP) of –80 mV. Bicuculline (10 mM) will be added to the perfusing solution
to study glutamate-mediated eEPSCs, sEPSCs, and mEPSCs. Intraelectrode
solution will have the following composition (mM): CsCl (110), K + -gluconate
(30), ethylene glycol-bis (b-aminoethyl ether)-N,N,N’,N’-tetra-acetic
acid (EGTA; 1.1), HEPES (10), CaCl 2
(0.1), Mg-ATP (4), Na-GTP (0.3). Synaptic
events will be stored by using P-CLAMP 9 (Axon Instruments) and analyzed
off line on a personal computer with Mini Analysis 5.1 [Synaptosoft,
Leonia, NJ, USA] software.
In both control and EAE brains, the striatum will be readily identified
under low power magnification, whereas individual neurons will be visualized
in situ using a differential interference contrast (Nomarski) optical system.
This will employ an Olympus BX50WI (Japan) non-inverted microscope with
x40 water immersion objective combined with an infra-red filter, a monochrome
CCD camera (COHU 4912), and a PC compatible system for analysis
of images and contrast enhancement [WinVision 2000, Delta Sistemi, Italy].
Recording pipettes will be advanced towards individual striatal cells in the
slice under positive pressure and, on contact, tight GW seals will be made by
applying negative pressure. The membrane patch will then be ruptured by
suction and membrane current and potential monitored using an Axopatch
1D patch clamp amplifier (Axon Instruments, Foster City, CA, USA). Wholecell
access resistances measured in voltage clamp will be in the range of 5-20
MW.
Synaptic events will be stored by using P-CLAMP 9 (Axon Instruments)
and analyzed off line on a personal computer with Mini Analysis 5.1 [Synaptosoft,
Leonia, NJ, USA] software. The detection threshold of spontaneous
and miniature events will be set at twice the baseline noise. The fact that no
false events would be identified will be confirmed by visual inspection for
each experiment. Offline analysis will be performed on spontaneous and
miniature synaptic events recorded during fixed time epochs (3-5 min, 3-6
samplings), sampled every 5 or 10 minutes. Only cells that will exhibit stable
frequencies (less than 20% changes during the control samplings) will be
taken into account. For kinetic analysis, events with peak amplitude between
10 and 50 pA will be grouped, aligned by half-rise time, and normalized by
peak amplitude. Events with complex peaks will be eliminated. In each cell,
all events between 10 and 50 pA will be averaged to obtain rise times, decay
times, and half widths [Centonze et al. 2009].
One to six cells per animal will be recorded. For each type of experiment
and time point, at least four distinct animals will be employed for each experi-
652 2009