View
499
Download
0
Tags:
Embed Size (px)
DESCRIPTION
Citation preview
2. Cellularmodelsused in Carbones lab
3. Cellularmodels
Chromaffincells: Derivedfrom the medullaof the adrenalglandof
Mouse, Rat and Bovine.
Representanimportantbranchof the sympatheticnervous system and from
a functionalpointofviewthey are Involved in
catecholaminesecretiondrivenbyvoltagegatedcalciumchannels.
Thesecells are physiologicallyrelevant in the stress response
4. Cellularmodels
DorsalRootGanglioncells: Derivedfrom the dorsalhornsof the
ratspinalcord . Theseneurons are important in the
transmissionofpainsignalsand are involved in
pathologiessuchasneuropathicpain. T typecalciumchannels are
thoughttocontributeconsiderably.
5. Cellularmodels
Hippocampalprimarycellcultures: Derivedfromnewbornmicepups .
Theseneurons create networks in culture.
Voltagegatedcalciumchannelsseemtobeimportant in
theirsynchronisationoffiringwhichisagedependent.Hippocampalcells
are involved in learning and memoryand in
pathologicalstatessuchasepilepsy
6. Itallcomes down to-VoltagegatedIonchannels( Ca2+ )-Ioncurrents
and actionpotentials-Secretion and endocytosis
7. contain the ion channel filter and has gating properties
modulate the V-dependent activation and steady-state inactivation
of the channel
membrane trafficking of 1-subunits
glycoprotein having four transmembrane segments
VoltagegatedCalciumchannels
8. VoltagegatedCalciumchannels
Families
Structure
9. Measuringioncurrents/actionpotentials
Patch Clamp: bymeansof a glassmicropipetteoneapproaches the cell,
appliessuction and obtains a gigaseal. Afterthat, bybreaking the
membrane, one can decide toclamp the voltagetomeasureioncurrents or
clamp the currenttomeasurevoltagefluctuationssuch
asactionpotentials
10. carbonfiber (5 mm diam)
stimulating pipette
50 ms
glass wall
chromaffin cell
Measuringsecretionbyamperometry
Amperometry: neurotransmitterssuchas dopamine, adrenaline,
noradrenaline, histamineetcundergooxidation/reductionreactions at
the carbonfibersurface and allow a sensitive measurementof the
secretionof single cells. Milisecondtimeresolution and a high
spatialresolutionofexocytoticevents are the
advantagesofthiselectrochemicaltechnique.
11. Non invasive measurementsofneuronalactivity
Multi ElectrodeArray (MEA): Cells are plated on top of a multi
electrodecontaining chip, spontaneousactivity can bemeasured (first
derivative of the intracellularsignal).An advantageisthatitconcerns
a non invasive techniquethatmaintains the physiologicalpropertiesof
the cell membrane lipidbilayer.
12. Projectsgoing on currently in Carbones Lab
13. L typecalciumchannel/ BK channelcoupling in
chromaffincellpacemaking
Andrea Marcantoni1, David H.F. Vandael1, Satyajit Mahapatra1,
Valentina Carabelli1, Martina J. Sinnegger-Brauns2, Joerg
Striessnig2, Emilio Carbone1
1 Department of Neuroscience, NIS Center, CNISM Research Unit,
10125 Torino, Italy
2 Institute of Pharmacy, Pharmacology and Toxicology, A-6020
Innsbruck, Austria
Abstract
We studied wild-type and Cav1.3-/- mouse chromaffin cells (WT-MCCs,
KO-MCCs) with the aim to determine the isoform of L-type
Ca2+channel (LTCC) and BK channels that underlie the pacemaker
current controlling spontaneous firing. Most WT-MCCs (80%) were
spontaneously active (1.5 Hz) and highly sensitive to nifedipine
and BayK-8644. Nifedipine blocked the firing while BayK-8644
increased 3-fold the firing rate. The two DHPs and the BK channel
blocker paxilline altered the shape of action potentials (AP)
suggesting close coupling of LTCCs to BK channels. WT-MCCs
expressed equal fractions of functionally active Cav1.2 and Cav1.3
channels. Cav1.3 channel deficiency decreased the number of
normally firing MCCs (30%; 2.0 Hz) suggesting a critical role of
these channels on firing, which derived from their slow
inactivation rate, sizeable activation at subthreshold potentials
and close coupling to fast-inactivating BK channels as determined
by using EGTA and BAPTA Ca2+buffering. By means of the action
potential-clamp, in TTX-treated WT-MCCs we found that the
interpulse pacemaker current was always net inward and dominated by
LTCCs. Fast inactivating and noninactivating BK currents sustained
mainly the afterhyperpolarization of the short APs (2-3 ms) and
only partially the pacemaker current during the long interspike
(300-500 ms). Deletion of Cav1.3 channels reduced drastically the
inward Ca2+ current and the corresponding Ca2+-activated BK current
during spikes. Our data highlight the role of Cav1.3, and to a
minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs
and open new interesting features about their role in the control
of firing and catecholamine secretion at rest and during sustained
stimulations matching acute stress.
14. Multi-walled carbon nanotubesand chromaffin cell excitability
by enhancing functional noninactivating BK channels
Daniela Gavello1,2, Roberta Cesa1,4, Federica Premoselli1, Federico
Cesano2,5, Domenica Scarano2,5, Bice Fubini2,3,5, Emilio
Carbone1,2,4, Ivana Fenoglio2,3,5*, Valentina Carabelli1,2,4*
1 Department of Neuroscience, University of Torino, 10125 Torino,
Italy
2Interdepartimental Center for Nanostructured Interfaces and
Surfaces, Torino, Italy
3InterdepartimentalCenter G. Scansetti for Studies on Asbestos and
other Toxic Particulates, Torino, Italy
4 National Institute of Neuroscience, Torino, Italy
5Department of Chemistry IFM, University of Torino, 10125, Torino,
Italy
Abstract
We describe the effects of multi-walled carbon nanotubes (MWCNTs)
on the excitability of cultured neuroendocrinechromaffin cells.
After 24 h exposure, MWCNTs enter chromaffin cells and affect their
excitability by decreasing their spontaneous firing frequency.
These effects are not associated to changes of Na+and Ca2+channels
conductance but rather to an increased contribution of
Ca2+-activated Big-K+ channels with noninactivating kinetics.
15. Cav1.2 and Cav1.3 channels in catecholamine secretion in
isolated mouse chromaffin cells (MCC)
Victor Navarro1, Valentina Carabelli1, Joerg Striessnig2, Emilio
Carbone1
1 Department of Neuroscience, NIS Center, CNISM Research Unit,
10125 Torino, Italy
2 Institute of Pharmacy, Pharmacology and Toxicology, A-6020
Innsbruck, Austria
INTRODUCTION
Chromaffin cells release catecholamines (CA) by exocytosis, a
Ca2+-dependent process that entails the fusion of secretory
vesicles and the cell membrane. L-type Ca2+ channels sustain the CA
secretion and play a major role in the control of chromaffin cell
excitability (Garca et al., 1984;2006). L-type calcium channels
represent the major Ca2+ current component in mouse chromaffin
cells (Hernndez-Guijo et al; 1998), but the individual role of
Cav1.2 and Cav1.3 channels in the exocytosis has not yet been
described.Here we measured single vesicles exocytosis, capacitance
changes and Ca2+ currents by combining amperometric techniques
(with carbon fiber microelectrodes placed on the cell surface) and
simultaneous patch-clamp recordings (Wightman et al, 1995; Rosa et
al., 1995; Marcantoni et al., 2009).
OBJECTIVE
Characterize the role of Cav1.2 and Cav1.3 channels on
catecholamines secretion in isolated mouse chromaffin cells (MCC),
comparing the different exocytotic and endocytotic activity which
derives from the Ca2+entry during voltage-clamp pulses of fixed
duration and variable amplitude.
METHODS
We designed a series of measurements based on the simultaneous
recordings of Ca2+ currents and secretory events, using a double
patch-clamp amplifier as voltage-clamp controller and amperometric
detector. In this system, carbon fiber acts as an amperometric
sensitive electrode, detecting the exocytosis of CA from a
chromaffin granule. The oxidation of a CA molecule produces two
electrons which are taken up by the positively charged working
electrode (+800 mV), thereby creating a current directly
proportional to the CA concentration surrounding the electrode tip.
The system maintains a fixed potential and amplifies the current
producing an output voltage signal, which can be acquired on-line.
At the same time a sinusoidal wave was added to analyze the changes
capacitance membrane simultaneously with calcium influx and/or
exocytosis.To reach this aim, we used wild type (WT) and Cav1.3-/-
mouse chromaffin cells (MCCs) and analyzed the exocytotic activity
induced by Ca2+influx during a series of voltage-clamp pulses. We
recorded total Ca2+currents and the associatedexocytotic events
simultaneously as well as endocytosis evoked by calcium.
16. STUDY OF L-TYPE CALCIUM CHANNELS IN MOUSE CHROMAFFIN AND
HYPPOCAMPAL CELLS BY MULTIELECTRODE ARRAY (MEA) RECORDING
TECHNIQUE
Jonathan Rojo Ruiz, Emilio Carbone
Abstract:
Adrenal chromaffin cells represent an ideal system for studying the
biophysics of voltage-gated Ca2+ channels and their role on
neurotransmitter release. Chromaffin cells express mainly
high-threshold Ca2+ channels (L, N, P/Q and R-types) and under
chronic hypoxic conditions (3% O2) or following long-term
-adrenergic stimulation they also express low-threshold T-type
channels [1]. Moreover, rat chromaffin cells (RCCs) have high
access resistance at rest and possess sufficient high densities of
Na+ and K+ channels to generate all-or-none action potential spikes
when injecting small amounts of current (3-5 pA) through the cell
membrane. In normal culture conditions, RCCs preserve a round
spherical shape and do not develop neuronal processes for days in
culture. Moreover, they express a homogeneous distribution of
voltage-gated ion channels which are involved in the generation of
the action potential. This is at variance to that occurring on
neurons where ion channels distribution varies greatly from the
soma to the dendrities or axons, increasing the number of possible
extracellular waveforms that can be detected with MEAs. These
properties make the chromaffin cells an interesting model for the
study of the single extracellular action potential.Considering the
different role of Na+, Ca2+ and K+ channels in the generation of
action potentials we initiated a detailed study of the spontaneous
(autorhythmic) electrical activity of RCCs using a 60 MEA system
made of 30 m electrode diameter separated by 200 m distance (MCS
GmbH, Reutlingen, Germany). The purpose of the study was dual: 1)
to monitor the spontaneous electrical activity by means of
extracellular recordings, thus leaving unaltered, and then more
similar to the physiological conditions, the intracellular medium;
2)To Identify the role of L-type calcium channels controlling
action potencial firings by analyzing the changes of extracellular
recordings induced by selective ion channel blockers.
17. The Lab members