1. Carbone’s Lab: Topicsof interest Technicalapproach

2. Cellularmodelsused in Carbone’s 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 α1-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, histamineetc…undergooxidation/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 Carbone’s 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 (García et al., 1984; 2006). L-type calcium channels represent the major Ca2+ current component in mouse chromaffin cells (Hernández-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 µm electrode diameter separated by 200 µm 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