1. Using functional co-cultures of rat carotid body (CB) Oµ chemoreceptors and 'juxtaposed' petrosal neurones (JPNs), we tested whether ATP and ACh acted as co-transmitters. 2. Perforated-patch recordings from JPNs often revealed spontaneous and hypoxia-evoked (PO 2 •5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 ìÒ) or a nicotinic ACh receptor (nAChR) blocker (hexamethonium, 100 ìÒ; mecamylamine, 1 ìÒ) only partially inhibited these responses, but together, blocked almost all activity. 3. Under voltage clamp (−60 mV), fast perfusion of 100 ìÒ ATP over hypoxia-responsive JPNs induced suramin-sensitive (IC50 = 73 ìÒ), slowly-desensitizing, inward currents (IATP) with time constant of activation ôon = 30·6 ± 4·8 ms (n = 7). IATP reversed at 0·33 ± 3·7 mV (n = 4), and the dose-response curve was fitted by the Hill equation (EC50 = 2·7 ìÒ; Hill coefficient •0·9). These purinoceptors contained immunoreactive P2X2 subunits, but their activation by á,â-methylene ATP (á,â-meATP; EC50 = 2·1 ìÒ) suggests they are P2X2ÏP2X3 heteromultimers. 4. Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body-sinus nerve preparation in vitro. Further, P2X2 immunoreactivity was widespread in rat petrosal ganglia in situ, and co-localized in neurones expressing the CB chemo-afferent marker, tyrosine hydroxylase (TH). P2X2 labelling in the CB co-localized with nerve-terminal markers, and was intimately associated with TH-positive type 1 cells. 5. Thus ATP and ACh are co-transmitters during chemotransduction in the rat carotid body.

At clinical concentrations, the potent intravenous general anesthetic etomidate enhances ␥-aminobutyric acid, type A (GABA A ) receptor activity elicited with low ␥-aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA A receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channelactivating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(؉)-etomidate isomer and display parallel dependence on GABA A ␤ subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human ␣ 1 ␤ 2 ␥ 2L receptors. Etomidate at up to 100 M reduced GABA EC 50 values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC 50 and increased maximal efficacy, demonstrating that etomidate shifts the GABA A receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux coagonist mechanism with two equivalent etomidate sites that allosterically enhance GABA A receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA A receptors containing a point mutation that increases constitutive gating activity.

Rapid synaptic transmission requires close proximity of docked neurotransmitter-containing synaptic vesicles and voltage-gated Ca 2+ channels at presynaptic active zones. Here we show that the plasma membrane SNARE protein SNAP-25 specifically inhibited the activity of P/Q-type Ca 2+ channels, and formation of a mature SNARE complex containing syntaxin and synaptotagmin reactivated them. In a nerve terminal, this mechanism would ensure that Ca 2+ entry through P/Q-type Ca 2+ channels occurs primarily near active zones with docked synaptic vesicles and efficiently evokes neurotransmitter release.At many synapses in the central nervous system, Ca 2+ influx through P/Q-type Ca 2+ channels densely localized in presynaptic nerve terminals triggers rapid neurotransmitter release 1,2 . The synaptic vesicle proteins synaptotagmin and synaptobrevin/VAMP bind to the synaptic plasma membrane SNARE proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) to form a core complex implicated in synaptic vesicle docking and/or membrane fusion 3,4 . This core complex associates with N-type and P/Qtype Ca 2+ channels via the synaptic protein interaction ('synprint') site within the intracellular loop connecting homologous domains II and III of their α 1B and α 1A subunits 2 . Disrupting interactions between SNARE proteins and Ca 2+ channels inhibits neurotransmission, demonstrating that such interaction is required for efficient neurotransmitter release 2 , and co-expression of syntaxin with Fig. 1. Modulation of P/Q-type Ca 2+ -channel activity by SNARE proteins.Recombinant rat cDNAs for syntaxin 1A, SNAP-25, synaptotagmin I-CT (the cytoplasmic domain containing residues 80-421) and VAMP 2-NT (the cytoplasmic domain containing residues 1-92) were subcloned into the mammalian expression vector pEBV-his (Invitrogen, San Diego, California). cDNA encoding the Ca 2+ channel subunits α 1A (rbA isoform) and β 1b were cloned in pMT2, α 2 δ in pZEM228 and CD8 in EBO-pcD. Cells of the tsA-201 subclone of HEK 293 cells were transfected with the α 1A , β 1b and α2δ cDNAs in a 1:1:1 molar ratio, plus the indicated SNARE proteins and CD8, by the calcium phosphate method and incubated for 24 h. Transfection-positive cells were identified by labeling with a fluorophore-tagged anti-CD8 antibody and analyzed by whole-cell patch clamp as described 14 . (a) Expression of SNARE proteins. Cells transfected with P/Q-type Ca 2+ channel and a SNARE protein were harvested and lysed in hypotonic buffer. A P2 membrane fraction was solubilized in 1% CHAPS for syntaxin and SNAP-25 immunoblotting and for immunoprecipitation with anti-CNA2, an antibody against the carboxyl terminal sequence ([KY]RRAPGPREPLANDSPGR) of the rbA isoform of α 1A . The remaining S2 fraction was used for synaptotagmin and VAMP/synaptobrevin immunoblotting. Lane 1, SNAP-25; lane 2, syntaxin; lane 3, synaptotagmin; lane 4, VAMP/synaptobrevin. (b) Co-immunoprecipitation of Ca 2+ channels and SNARE proteins. Cells transfected with P/Q-type Ca 2+ channel and SNARE proteins w...

Cav2.1 channels, which conduct P͞Q-type Ca 2؉ currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca 2؉ channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Cav2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with loss of presynaptic localization of the exogenously expressed channels. Cav1.2 channels, which conduct L-type Ca 2؉ currents, are ineffective in supporting synaptic transmission, but substitution of the synprint site from Cav2.1 channels in Cav1.2 was sufficient to establish synaptic transmission initiated by L-type Ca 2؉ currents through the exogenous Cav1.2 channels. Substitution of the synprint site from Cav2.2 channels, which conduct N-type Ca 2؉ currents, was even more effective than Cav2.1. Our results show that localization and function of exogenous Ca 2؉ channels in nerve terminals of superior cervical ganglion neurons require a functional synprint site and suggest that binding of soluble NSF attachment protein receptor (SNARE) proteins to the synprint site is a necessary permissive event for nerve terminal localization of presynaptic Ca 2؉ channels. E lectrophysiological and pharmacological studies have defined a diverse array of native Ca 2ϩ currents having different functions in neurons (1, 2). Voltage-gated Ca 2ϩ channels are complexes of a pore-forming ␣ 1 subunit with associated ␣ 2 ␦, ␤, and ␥ subunits (3-5). Ca v 2.1 channels that conduct P͞Q-type Ca 2ϩ currents and Ca v 2.2 channels that conduct N-type Ca 2ϩ currents are the primary initiators of fast synaptic transmission in vertebrate neurons (1, 6-12). These Ca 2ϩ channels bind directly to soluble NSF attachment protein (SNAP) receptor (SNARE) proteins involved in neurotransmitter release through a synaptic protein interaction (synprint) site in the large intracellular loop connecting domains II and III (L II-III ) of their ␣ 1 subunits (13-15). Disruption of this interaction by peptide inhibitors injected into presynaptic neurons reduces the efficiency of Ca 2ϩ entry in stimulating exocytosis (16,17). These results implicate the interaction of SNARE proteins with the synprint site in determining the efficiency of fast synaptic transmission, possibly by organizing docked synaptic vesicles close to the site of Ca 2ϩ entry.The molecular basis for the specific role of Ca v 2 channels in initiation of fast neurotransmission is not well understood. It may involve specific localization of Ca v 2 channels in nerve terminals, specific interactions with SNARE proteins or other proteins in the nerve terminal, or both. Results presented in the accompanying paper (18) show that exogenous Ca v 2.1 channels can be functionally expressed in superior cervical ganglion neurons (SCGNs) and can reconstitute synaptic transmission in neurons whose endogenous Ca v 2.2 channels have been blocked by -conotoxin GVIA. Here w...

To investigate synaptic mechanisms mediating chemosensory signalling in the carotid body, we developed co‐cultures of chemoreceptor type 1 cell clusters and dissociated petrosal neurones (PNs) from 7‐ to 14‐day‐old rat pups and tested for functional connectivity in CO2–HCO3− ‐or Hepes‐buffered medium at ∼35 °C. When cultured without type 1 cells, PNs were almost always quiescent (n= 104) and unresponsive to hypoxia (PO2= 5–25 mmHg) during perforated patch, whole‐cell recordings of membrane potential or voltage‐activated currents; in contrast, many PNs (77 out of 170) that were juxtaposed to type 1 cell clusters in co‐culture displayed spontaneous activity, comprising spikes and subthreshold potentials (SSPs) that resembled synaptic potentials. Additional tests suggested that de novo chemical synapses developed between PNs and type 1 cell clusters in vitro. For example: (i) the spontaneous activity was reversibly suppressed by substituting low calcium‐high magnesium in the bath; (ii) SSPs had variable amplitudes and persisted following action potential blockade with TTX (1 μm); (iii) the interval distribution between successive spontaneous events appeared random; and (iv) the frequency of spontaneous potentials was diminished (reversibly) by the nicotinic antagonist hexamethonium (100 /tM), suggesting contributions from the spontaneous release of ACh. Many complexes of ‘juxtaposed’ PNs and type 1 clusters were physiologically functional, since exposure to hypoxia caused a reversible depolarization and/or increased spike discharge in ∼30% of such neurones (n= 140). The hypoxia‐induced spike discharge persisted in the presence of the dopamine D2 receptor blocker spiperone (10–50 μm; n= 5); however, this discharge was reversibly inhibited by 100–200 μm hexamethonium, suggesting that it was mediated, at least in part, by ACh acting through nicotinic receptors. The hypoxia‐induced spike discharge and frequency of spontaneous potentials in co‐cultured PNs were reversibly suppressed when the buffer was switched from C02‐HC03∼ to Hepes (10 mm) at pH 7.4;′further, ‘functional’ PNs that displayed spontaneous activity and/or hypoxia‐induced responses in co‐culture were encountered more frequently in CO2–HCO3− (≥ 40 %) than in Hepes (≤26 %) buffer. We conclude that functional chemical synapses can develop de novo in cultures of carotid body type 1 cells and PNs and that ACh is probably an important excitatory neuro‐transmitter secreted from type 1 cells during hypoxic chemotransduction in the rat carotid body.

The peripheral control of breathing is mediated by O2-sensitive carotid body (CB) type 1 cells, which express multiple neurotransmitters including the monoamines, dopamine and serotonin (5-HT). Whereas dopamine has been extensively studied, 5-HT has received little attention. Here, to elucidate the role of 5-HT in CB chemotransmission, we used perforated-patch recording from rat type 1 cell clusters and co-cultured petrosal (afferent) neurones. 5-HT induced action potentials and/or membrane depolarization associated with a conductance decrease in approximately 40% of recordings from type 1 cells (n = 78/192). These responses were markedly inhibited by the 5-HT2 receptor antagonist ketanserin (10-50 microM) and by the protein kinase C (PKC) inhibitor chelerythrine (50 microM). The PKC activator 1-oleoyl-2-acetylglycerol (OAG; 50 microM) mimicked the 5-HT-induced depolarization, and the combined effects of 5-HT and OAG were non-additive. The 5-HT-induced responses reversed near the potassium (K+) equilibrium potential (at approximately -82 mV; EK = -83 mV), suggesting inhibition of a resting K+ conductance. In type 1 cells (n = 7), voltage-activated outward K+ current was also inhibited by 1-50 microM 5-HT, an effect that was prevented by PKC inhibitors (chelerythrine and NPC 15437) and mimicked by OAG; the outward K+ current inhibited by 5-HT appeared to be predominantly a Ca(2+)-dependent K+ current. The 5-HT2 receptor blockers ketanserin and ritanserin reversibly inhibited spontaneous action potentials and the hypoxia-induced receptor potential recorded from clustered type 1 cells. Moreover, these blockers reversibly inhibited the hypoxic chemosensory response recorded postsynaptically in petrosal neurones that functionally innervated type 1 clusters in co-culture. RT-PCR and confocal immunofluorescence techniques revealed 5-HT2a receptor expression in rat CB type 1 cells. These results suggest that release of endogenous 5-HT regulates CB chemoreceptor function presynaptically, by a positive feedback mechanism involving autocrine-paracrine stimulation of 5-HT2a receptors and PKC modulation of resting and Ca(2+)-dependent K+ conductances.

The incidence of delirium was 14.8% in the first week after admission with acute ischemic stroke. Age, having a previous stroke, stroke severity, and left-cortical infarction were independently predictors of PSD. PSD may result in a significantly worse functional outcome.

N-type and P͞Q-type Ca 2؉ channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membranedelimited pathway involving G␤␥ subunits. Inhibition is caused by a shift from an easily activated ''willing'' (W) state to a moredifficult-to-activate ''reluctant'' (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca 2؉ channels containing Cav2.2a ␣1 subunits and P͞Q-type Ca 2؉ channels containing Cav2.1 ␣1 subunits revealed substantial differences. In the absence of G protein modulation, Ca v 2.1 channels containing Ca v ␤ subunits were tonically in the W state, whereas Cav2.1 channels without ␤ subunits and Cav2.2a channels with ␤ subunits were tonically in the R state. Both Cav2.1 and Ca v2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca v␤ subunits are intrinsic properties of the Ca 2؉ channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca 2؉ -channel activity incorporating an intrinsic equilibrium between the W and R states of the ␣1 subunits and modulation of that equilibrium by G proteins, Ca v␤ subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P͞Q-type Ca 2؉ channels to initiate neurotransmitter release.neuromodulation ͉ calcium channels ͉ G␤␥ subunits ͉ protein phosphorylation N euronal voltage-gated Ca 2ϩ channels are involved in multiple cellular functions including neurotranstransmitter release, Ca 2ϩ -mediated regulatory processes, and generation of dendritic action potentials. Electrophysiological and pharmacological studies distinguish at least six classes of Ca 2ϩ currents designated L-, N-, P-, Q-, R-, and T-type (1, 2). Ca 2ϩ channels consist of complexes of a pore-forming ␣ 1 subunit with ␣ 2