Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14917–14921, December 1996 Neurobiology Single-channel properties of the nonselective cation conductance induced by neurotensin in dopaminergic neurons (cell cultureyG proteinyventral tegmental areayoutside–out patchyneurotensin antagonist) PEI-YU CHIEN*, RONALD H. FARKAS*, SHIGEHIRO NAKAJIMA†, AND YASUKO NAKAJIMA*‡ Departments of *Anatomy and Cell Biology and †Pharmacology, University of Illinois, College of Medicine, Chicago, IL 60612 Communicated by Susan E. Leeman, Boston University School of Medicine, Boston, MA, October 3, 1996 (received for review June 25, 1996) ABSTRACT Slow nonselective cation conductances play a described (14) except that 2% (instead of 5%) rat serum was central role in determining the excitability of many neurons, used, and neurons were dissociated with 12 (instead of 20) but heretofore this channel type has not been analyzed at the unitsyml of papain. We recorded from large neurons cultured single-channel level. Neurotensin (NT) excites cultured dopa- for '2 weeks (soma diameter ' 21 mm), '75% of which are minergic neurons from the ventral tegmental area primarily dopaminergic (9). by increasing such a cation conductance. Using the outside– Recordings were made using the outside–out or cell- out configuration of the patch clamp, we elicited single- attached configuration of the patch clamp (15). Single-channel channel activity of this NT-induced cation channel. Channel currents were recorded on videotape from a List EPC-7 activity was blocked by the nonpeptide NT antagonist amplifier (List Electronic, Darmstadt, Germany) and analyzed SR48692, indicating that the response was mediated by NT by using PCLAMP software (Axon Instruments, Foster City, receptors. The channel opened in both solitary form and in CA) as described (16). NT (10 nM) was applied by pressure bursts. The reversal potential was 24.2 6 1.7 mV, and the ejection (0.5 psi) from glass pipettes with a tip diameter of 3–4 1 elementary conductance was 31 pS at 267 mV with [Na ]o 5 mm, placed '45 mm from the patch. Experiments were con- 1 1 1 140 mM, [Cs ]o 5 5 mM, [Na ]i 5 88 mM, and [Cs ]i 5 74 duced at '238C. Values are given as mean 6 SEM. mM. Thus, the channel was permeable to both Na1 and Cs1. The standard pipette solution for whole-cell and outside– From these characteristics, it is likely that this channel is out recording contained 144 mM K-D-gluconate, 8.5 mM responsible for the whole-cell current we studied previously. In Na-D-gluconate, 1.5 mM NaCl, 5 mM HepeszKOH, 0.5 mM guanosine 5*-[g-thio]triphosphate-loaded cells, NT irrevers- EGTAzKOH, 0.25 mM CaCl2, 3 mM MgCl2,2mMNa2ATP, ibly activated about half of the channel activity, suggesting and 100 mMNa3GTP (pH 7.2). When guanosine 59-[g- that at least part of the response was mediated byaGprotein. thio]triphosphate (GTP[gS]; 100 mM) was used, GTP was Similar channel activity could be induced occasionally in the omitted. A Cs1-containing internal solution used for deter- cell-attached configuration by applying NT outside the patch mining the reversal potential contained 74 mM CsCl, 80 mM region. NaCl, 5 mM HepeszNaOH, 0.5 mM EGTAzNaOH, 0.25 mM CaCl2, 3 mM MgCl2,2mMNa2ATP, and 100 mMNa3GTP Neurotensin (NT), a peptide neurotransmitter originally iso- (pH 7.2). lated from bovine hypothalamus (1), excites many central In early experiments, Ca21 was omitted from the standard nervous system neurons (2–6) including dopaminergic neurons external solution because our previous whole-cell data sug- in the ventral tegmental area (VTA) (7–9). The mechanism by gested that the NT-induced conductance is blocked by external which NT excites VTA neurons has been shown to be an Ca21. This nominally Ca21-free external solution contained overlapping increase of nonselective cation conductance and 155 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 5 mM HepeszNaOH decrease of inward rectifier K1 conductance (8, 9). (pH 7.4) and had a Ca21 concentration of '1 mM, measured We previously used whole-cell recording to study the nonse- by Ca21 electrode. In later experiments, external Ca21 was lective conductance increased by NT in cultured VTA neurons buffered to 1 mM with EGTA. This Ca21-buffered external 1 1 (9). The conductance was equally permeable to Na ,K , and solution contained 140 mM NaCl, 5 mM KCl, 4.7 mM CaCl2, 1 2 Cs , but was impermeable to Cl , indicating that it was indeed a 5 mM EGTAzNaOH, 1.3 mM MgCl2, and 5 mM HepeszNaOH nonselective cation conductance. Activation of the NT-induced (pH 7.4). To determine the reversal potential, we used a nonselective current did not involve cAMP, cGMP, or internal Cs1-containing external solution, in which KCl was replaced Ca21, while the latency of activation was long, indicating the by CsCl. Tetrodotoxin (0.5 mM) was added for all recordings. involvement of second messenger(s). Therefore, this NT-induced The membrane potential was corrected for the liquid junction nonselective cation conductance might be a member of a unique potential as measured in reference to a saturated KCl micro- class of slow nonselective cation conductance, different from electrode. ligand-gated (10, 11), cyclic nucleotide-gated (12), and calcium- When analyzing burst time, we used a 3-kHz filter (23dbby activated nonselective (13) cation conductances. 8-pole Bessel filter) with 25-kHz digitization, and the level The purpose of this paper was to characterize the single- changes registered if longer than 40 msec. As will be described, channel properties of the nonselective cation conductance the close time histogram was fit by two exponentials with time induced by NT. We also examined the involvement of G constants tg,f (short gaps) and tg,s (long closings), indicating proteins in the NT response. that the channel opened in bursts, with the long openings interrupted by brief closures. After measuring close times, MATERIALS AND METHODS burst times were determining by analyzing the same data file, but this time a level change from the open state was registered VTA neurons were cultured from 2- to 4-day-old postnatal only when the transition from the open state lasted longer than Long-Evans rats (Charles River Breeding Laboratories) as a critical time (tc) (17). For the rest of the experiments, the The publication costs of this article were defrayed in part by page charge Abbreviations: NT, neurotensin; VTA, ventral tegmental area; payment. This article must therefore be hereby marked ‘‘advertisement’’ in GTP[gS], guanosine 59-[g -thio]triphosphate. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. 14917 Downloaded by guest on September 24, 2021 14918 Neurobiology: Chien et al. Proc. Natl. Acad. Sci. USA 93 (1996) frequency response was 2 kHz with 20-kHz digitization, and Block by NT Antagonists. The nonpeptide NT antagonist level changes registered if longer than 100 msec. SR48692 was used to characterize the receptor mediating the Npo was calculated by (18): NT response. This antagonist competitively inhibits NT bind- ing to the high-affinity binding site present in the rat mesen- N cephalon (19). NT was first applied to the patch to establish a baseline response. Two hundred seconds later, SR48692 (10 Npo 5 O $nP~n!% n51 nM) was applied for 5 sec, followed in 15–23 sec by a second application of NT. After another 200 sec, NT was applied for in which N is the number of the channels in the patch, po is the a third time (Fig. 2). Using different patches as controls, the open probability of an individual channel, and P(n)isthe solvent alone (0.001% dimethyl sulfoxide) instead of SR48692 probability of the record staying at a level at which n channels was applied before the second NT application. The results open simultaneously. were summarized in Fig. 2. For control patches (open bars), The chemicals used were NT (Peninsula Laboratories) and the average peak Npo for the first NT response was 0.11 6 0.03 GTP[gS] (Sigma). The nonpeptide NT antagonist SR48692 (n 5 5). The second NT response decreased to 0.06 6 0.02 was obtained from Sanofi Recherche (Tolouse, France). because of desensitization. The third response decreased fur- ther to 0.03 6 0.01. For the NT antagonist experiment (solid bars), the average peak Npo of the first NT response was 0.14 6 RESULTS 0.04 (n 5 7), fairly close to the response in control patches. NT-Induced Channel Activity. As previously described (9), After SR48692 was applied, the second NT response was NT induced an inward current in whole-cell recording (Fig. dramatically decreased to 0.007 6 0.002. Compared with the 1A). The average whole-cell response induced by 10 nM NT control, the difference was significant (P 5 0.0045). After washout of the NT antagonist, the third response partially was 240 6 154 pA (n 5 5). NT applied to outside–out recovered, to 0.05 6 0.02. The single-channel current ampli- membrane patches induced long-lasting channel activity (Fig. tude was not affected by the NT antagonist (data not shown). 1B1). On an expanded time base, Fig. 1B2 shows that there was These results showed that the channel activity was blocked by no channel activity before NT application, while channel the NT antagonist, indicating that NT was acting through NT activity occurred after NT was applied (Fig. 1B3). Fig. 1B4 receptors. shows the change of Npo induced by NT. Npo reached a peak Kinetic Analysis of the Burst Behavior. Kinetic properties of ('0.1) 25 sec after NT application, then decreased to zero the NT-induced channel were studied in standard external and after '60 sec.