546 [5]). Anatomical studies have revealed that the AP sends dense efferent projections to the nucleus tractus solitarius, parabrachial nucleus, nucleus ambiguus, and the dorsal motor nucleus of the vagus, and receives afferent inputs from the hypothalamic para- ventricular and dorsomedial nucleii, and from the caudal nucleus tractus solitarius (Morest [6]; Kooy and Koda [7]; Shapiro and Miselis [8]). Thus, the AP is not only capable of responding to circulating hormones, but is also anatomically well situated to regulate a range of other central neurons, including those important in cardiovascular control (Sun and Spyer [9]). The low intrinsic ﬁring rates of AP neurons in vivo (Papas et al. [10]) suggests that under- standing and modulating excitatory inputs to AP could be particularly important in the functional out- put of AP neurons. A number of transmitters can evoke excitatory currents in the AP. Inward currents and increases in cytosolic Ca2+ ([Ca]in) via non- NMDA-glutamate receptors have been reported in rabbit and rat AP neurons, respectively (Jahn et al. [11]; Hay and Lindsley [12]). Our preliminary reports indicated that ATP also induces inward currents and [Ca]in increases via the activation of P2X receptor (P2XR) in isolated rat AP neurons (Sorimachi et al. [13, 14]). In addition, pre- and post-synaptic nicotinic ACh receptors (nAChR) have been demonstrated in the AP in rat brain slices (Funahashi et al. [15]), and we have recently reported the presence of nAChRs in dissociated rat AP neurons (Sorimachi and Wakamori [16]). During that study, we also noticed that many of these AP neurons also responded to ATP, which has prompted us to further investigate ATP responses in AP neurons, and potential interactions between nAChR and P2XR responses. In a variety of different peripheral neurons, including sympathetic neurons of bullfrog (Akasu and Koketsu [17]), rat (Nakazawa et al. [18]), and guinea-pig (Searl et al. [19]), cultured guinea-pig enteric and submucosal neurons (Zhou and Galligan [20] Barajas-Lopez et al. [21]), a negative interaction between P2XR- and nAChR responses has been reported. Such an interaction has also been observed for recombinant P2X2 and a3b4 nAChR channels in Xenopus oocytes and HEK cells (Khakh et al. [22, 23]; Boue-Grabot et al. [24]), where it has been recently suggested that this results from direct physical interactions between co-localized receptors (Khakh et al. [23]). In this study we more fully de- scribe P2XR responses in AP neurons, demon- strate cross-inhibition between P2XRs and nAChRs and characterize some of the features of this cross- inhibition. Purinergic Signalling (2006) 2:545–557 Experimental procedures Preparation of AP neurons The study was approved by the Committee on Animal Experimentation, Kagoshima University. Wistar rats (13–18 days-old) were anaesthetized with ether and decapitated. The brain was quickly removed from the skull and placed in ice-cold HEPES-buffered saline containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES and 5.5 mM glucose. The pH of the saline solution was adjusted to 7.4 by adding tris (hydroxymethyl) aminomethane. The brain was sliced at a thickness of 400 mm with a microslicer (DTK-1000, Dosaka, Kyoto, Japan), and the slices were kept in bicarbonate-buffered saline bubbled con- tinuously with 95% O2–5% CO2 at room temperature (21–26 -C). The bicarbonate-buffered saline contained 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM NaHCO3, 2 mM KH2PO4 and 5.5 mM glucose. Following 30–60 min incubation, the slices were treated with pronase (0.2 mg/ml) in bicarbonate-buffered saline for 20 min at 33 -C, followed by incubation for 30 min with bicarbonate-buffered saline contain- ing thermolysin (0.12–0.15 mg/ml). The bilateral AP regions were identiﬁed with a binocular microscope (Zeiss, Germany), and were cut out using the tip of an injection needle, and subsequently mechanically tritu- rated with ﬁre-polished glass pipettes of decreasing diameters. Dissociated neurons were placed on to the bottom of 35 mm culture dishes (Falcon, USA) for electrophysiological recordings, or on to glass coverslips (Matsunami, Japan) coated with poly-L-lysine for [Ca]in measurements. Electrophysiological recordings Electrical measurements were done using the whole- cell patch-clamp recording conﬁguration under volt- age-clamp conditions. Patch pipettes were fabricated from borosilicate glass tubes in ﬁve or six stages using a pipette puller (Model P-97, Sutter Instrument, San Rafael, CA, USA). The resistance between the re- cording electrode ﬁlled with the internal solution, and the reference electrode in the external solution, was 3– 6 MW. Ionic currents were measured, and voltages controlled, using a patch-clamp ampliﬁer (EPC-9, Heka, Darmstadt, Germany). All experiments were carried out at 24–26 -C. Culture dishes were placed on an inverted microscope (TE200, Nikon, Japan), and drugs were rapidly applied to single cells using a Y- tube perfusion device. The internal solution (patch