0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
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
. Recording
pipettes will be advanced towards individual striatal cells under visual
control in the slice and, on contact, tight GW seals were 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). 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 µM) will be added to the perfusing solution to study glutamate-mediated
eEPSCs, sEPSCs, and mEPSCs. Conversely, to detect GABAergic
eIPSCs, sIPSCs, and mIPSCs, MK-801 (30 µM) and CNQX (10 mM) will be
added to the external solution. 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).
To study the effects of microglia on striatal excitatory and inhibitory
transmission, microglia will be gently placed onto the surface of a striatal
slice, submerged in a continuously oxygenated recording chamber for at least
30 min before the experiment. The presence of microglial cells over the slices
will be monitored with a x40 water immersion objective combined with an
infra-red filter.
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. Statistical analysis will be performed using
a paired or unpaired Student’s t-test or Wilcoxon’s test. The significance level
will be established at p