Solo testo.pdf - Fondazione Santa Lucia

A comparison of the pharmacological effects between carbamazepine and oxcarbazepine
Microfluorometry – Microfluorometric measurements of intracellular
[Na + ] and [Ca 2+ ] will be performed in cells loaded with the sodium-sensitive
indicator sodium-binding benzofuran isophthalate (SBFI) and fura-2
(K 5
salt), through the patch pipette. Fluorescence will be excited in fura-2
and SBFI-loaded neurons via a water immersion objective by epi-illumination
with light provided by a 75 W Xenon lamp bandpass, alternatively
filtered at 340 and 380 nm wavelength. Emission light will be monitored
using a 500 nm barrier filter and detected by a CCD camera. Each fluorescence
value will be obtained from pairs of 340 and 380 nm images and analyzed
off-line. Ratio images will be calculated from pairs of 340 and 380 nm
images corrected for background fluorescence, measured from image
regions free of dye fluorescence.
The levels of free intracellular calcium and sodium will be measured in
neocortical and hippocampal pyramidal neurons, in basal conditions, following
membrane depolarization and stimulation of specific glutamate receptors.
An increase of the intracellular ionic concentrations will be also obtained by
exposing the slices to brief episodes of ipoxia/ischemia. Specifically, we will follow
the amplitude and kinetics of calcium and sodium rise in response to local
application of glutamate agonists, in response to trains of afferent stimuli, and
during an episode of energy deprivation. Once we have standardized the
results obtained with these procedures, we will re-examine the sodium and calcium
dynamics in the presence of AMPA, mGluR and GABA receptor antagonists.
The results obtained will be then compared with those recorded in the
presence of Oxcarbazepine and Carbamazepine.
Extracellular recordings – For extracellular recordings test pulses will be
delivered through a bipolar nichrome electrode positioned in the stratum
radiatum of the CA1 region of the hippocampus. Evoked extracellular potentials
will be recorded with glass microelectrodes (2-4MΩ), filled with 3M NaCl.
Responses are amplified (Neurolog NL 104, Digitimer Ltd, Welwyn Garden
City, U.K.), digitized (sample rate, 33.33 kHz), and stored for later analysis
using pCLAMP 6 software facilities (Axon Instruments, Foster City, CA, USA).
In vitro OGD will be obtained by superfusing the slices with D-glucosedeficient
aCSF equilibrated with a 95% N 2
/5% CO 2
gas mixture. At the end
of the ischemic period, the slice will be again superfused with normal
(glucose-containing) oxygenated aCSF. The time course of fEPSPs amplitude,
before and during different time durations of OGD and after reperfusion
in normal oxygenated aCSF will be assessed. Our aim is to evaluate
whether Oxcarbazepine and Carbamazepine could partially or fully protect
synaptic transmission from an irreversible ischemic insult obtained with the
Oxygen-Glucose deprivation model given for a defined time course in control
experiments.
Drug application – Drugs will be applied by switching the standard aCSF
to one containing a known concentration of the drug(s). Full exchange of the
solution in the recording chamber occurs over about 1 minute. Drugs will be
applied beginning 30 min after the establishment of a stable baseline response.
A drug-naive hippocampal or neocortical slice will be used in each experiment.
2006 619