Involvement of transient receptor potential-like channels in responses to mGluR-I activation in midbrain dopamine neurons Alessandro Tozzi, 1,y C. Peter Bengtson, 1,y Patrizia Longone, 1 Corrado Carignani, 1, Francesca R. Fusco, 1 Giorgio Bernardi 1,2 and Nicola B. Mercuri 1,2 1 Experimental Neurology Laboratory, I.R.C.C.S. Fondazione Santa Lucia, Via Ardeatina 306, Rome, Italy 2 Clinica Neurologica, University of Rome Tor Vergata, Rome, Italy Keywords: brain slice, patch clamp, rat, substantia nigra, whole cell Abstract Weinvestigatedtheinvolvementofstore-operatedchannels(SOCs)andtransientreceptorpotential(TRP)channelsintheresponseto activationofthegroupImetabotropicglutamatereceptorsubtype1(mGluR1)withtheagonist(S)-3,5-dihydroxyphenylglycine(DHPG, puffapplication)indopamineneuronsinratbrainslices.ThemGluR1-inducedconductancereversedpolaritycloseto0mVandatmore positive potentials when extracellular potassium concentrations were increased, indicating the involvement of a cationic channel. DHPGcurrentsbutnotintracellularcalciumresponseswerereducedbylowextracellularsodiumconcentrationsbutwerenotaffected bysodiumchannelblockers,tetrodotoxinandsaxitoxinorbyinhibitionoftheh-currentwithcesium.Abolitionofcalciumresponseswith intracellular BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid; 10mM) did not affect current responses, indicating they were not calcium activated. Extracellular application of non-selective SOCs and TRP channel blockers 2-aminoethoxydiphe- nylborane(2-APB),SKF96365,rutheniumredand¯ufenamicacid(butnotgadolinium)reducedDHPGcurrentandcalciumresponses. Intracellularapplicationofrutheniumredand2-APBdidnotaffectDHPGcurrents,indicatingthatIP3andryanodinereceptorsdidnot mediatetheiractions.Single-cellPCRrevealedthepresenceofTRPC1and5mRNAinmostdopamineneuronsandsubtypes3,4and 6insome.StoredepletionevokedcalciumentryindicativeofSOCs,providingthe®rstfunctionalobservationofsuchchannelsinnative centralneurons.StoredepletionwitheithercyclopiazonicacidorryanodineabolishedcalciumbutnotcurrentresponsestoDHPG.The electrophysiologicalandpharmacologicalpropertiesofthemGluR1-inducedinwardcurrentareconsistentwiththeinvolvementofTRP channels whereas calcium responses are dependent on the function of SOCs in voltage clamp recordings. Introduction Complex interactions between group I metabotropic glutamate recep- tors (mGluR-I), intracellular Ca 2 stores and Ca 2 -permeable cation channels in the plasma membrane of neurons have been identi®ed in the cortex, hippocampus and cerebellum (Linden et al., 1994; Vignes et al., 1995; Abdul-Ghani et al., 1996; Fagni et al., 2000; Nakamura et al., 2000; Gee et al., 2003). In voltage clamped dopamine neurons of the substantia nigra pars compacta (SNc) the selective mGluR-I agonist (S)-3,5-dihydroxyphe- nylglycine (DHPG) can produce an excitation that is caused by the activation of a G-protein-dependent inward current associated with an increase of intracellular [Ca 2 ]. These DHPG-mediated responses are antagonized by the subtype 1 metabotropic glutamate receptor (mGluR1)antagonistCPCCOEt,butnotthesubtype5mGluRantago- nist MPEP, indicating the selective involvement of mGluR1 (Guatteo et al., 1999; Tozzi et al., 2001). Ca 2 mobilization accompanying mGluR-I-activated inward cur- rents diminishes following depletion of intracellular Ca 2 stores with thapsigargin or following the removal of extracellular Ca 2 with an EGTA-containingnominallyCa 2 -freesolution(Guatteo etal.,1999). However, intracellular Ca 2 transients accompanying outward mGluR-I-activated currents appear not to be affected by the removal ofextracellularCa 2 (Morikawa etal.,2003).ThusextracellularCa 2 maycontributetoCa 2 responsesassociatedwithinwardcurrentsand/ or may be necessary to replenish intracellular Ca 2 stores between strong activation of mGluR-I. In voltage clamped neurons in which voltage-activated Ca 2 channels are largely inactive, a potential mechanism to explain such Ca 2 in¯ux is capacitive Ca 2 entry mediated by store-operated calcium channels (SOCs). Physical and chemical interactions between store-located receptors oftheendoplasmicreticulumandchannelsontheplasmaticmembrane havebeenextensivelystudiedininvertebrateandculturedmammalian cellstoaccountfortheactivationofCa 2 -permeablecationicchannels (Patterson et al., 1999; Putney, 1999; Ma et al., 2000; McFadzean & Gibson, 2002; Venkatachalam et al., 2002); however, little is known about the existence, function and mechanisms of activation of such channels in neurons. The molecular, pharmacological and functional characteristics of SOCs have identi®ed them as members of the transient receptor potential (TRP) channel family (McFadzean & Gibson, 2002). In European Journal of Neuroscience, Vol. 18, pp. 2133±2145, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02936.x Correspondence: Dr Nicola B. Mercuri, 1 Experimental Neurology Laboratory, as above. E-mail: Mercurin@med.uniroma2.it  Present address: GlaxoSmithKline Medicines Research Centre, Verona, Italy. y A.T. and C.P.B. contributed equally to this work. Received 3 July 2003, revised 30 July 2003, accepted 4 August 2003