Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca2+ via T-type Ca2+ channels, as indicated by two-photon imaging. The enhanced Ca2+ levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing.
Neuronal signaling by G protein-coupled P2Y nucleotide receptors is not well characterized. We studied here the coupling of different molecularly defined P2Y receptors to neuronal G protein-gated inward rectifier K(+) (GIRK) channels. Individual P2Y receptors were coexpressed with GIRK1+GIRK2 (Kir3.1 + 3.2) channels by intranuclear plasmid injections into cultured rat sympathetic neurons. Currents were recorded using perforated-patch or whole-cell (disrupted patch) techniques, with similar results. P2Y(1) receptor stimulation with 2-methylthio ADP (2-MeSADP) induced activation of GIRK current (I(GIRK)) followed by inhibition. In contrast, stimulation of endogenous alpha(2)-adrenoceptors by norepinephrine produced stable activation without inhibition. P2Y(1)-mediated inhibition was also seen when 2-MeSADP was applied after I(GIRK) preactivation by norepinephrine or by expression of Gbeta(1)gamma(2) subunits. In contrast, stimulation of P2Y(4) receptors with UTP or P2Y(6) receptors with UDP produced very little I(GIRK) activation but significantly inhibited preactivated currents. Current activation was prevented by pertussis toxin (PTX) or after coexpression of the betagamma-scavenger transducin-Galpha.I(GIRK) inhibition by all three nucleotide receptors was insensitive to PTX and was significantly reduced after coexpression of RGS2 protein, known to inhibit G(q)alpha signaling. Inhibition was not affected 1) after coexpression of RGS11, which interferes with G(q)betagamma action; 2) after coexpression of phospholipase C (PLC) delta-Pleckstrin homology domain, which sequesters the membrane phospholipid phosphatidylinositol 4,5-bisphosphate; (3) after buffering intracellular Ca(2+) with 1,2-bis(2-aminiphenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM); and (4) after pretreatment with the protein kinase C inhibitor 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF 109203X). We conclude that activation of I(GIRK) by P2Y receptors is mediated by G(i/o)betagamma, whereas I(GIRK) inhibition is mediated by G(q)alpha. These effects may provide a mechanism for P2Y-modulation of neuronal excitability.
Muscarinic acetylcholine receptors (mAChRs) (nomenclature as agreed by the NC-IUPHAR Subcommittee on Muscarinic Acetylcholine Receptors [50]) are activated by the endogenous agonist acetylcholine. All five (M1-M5) mAChRs are ubiquitously expressed in the human body and are therefore attractive targets for many disorders. Functionally, M1, M3, and M5 mAChRs preferentially couple to Gq/11 proteins, whilst M2 and M4 mAChRs predominantly couple to Gi/o proteins. Both agonists and antagonists of mAChRs are clinically approved drugs, including pilocarpine for the treatment of elevated intra-ocular pressure and glaucoma, and atropine for the treatment of bradycardia and poisoning by muscarinic agents such as organophosphates. I
1. Membrane currents were recorded from voltage‐clamped, microelectrode‐impaled cells of the NG108‐15 mouse neuroblastoma x rat glioma clonal cell line, differentiated with prostaglandin E1. 2. A slow outward tail current reversing at post‐pulse potentials between ‐80 and ‐90 mV was evoked by depolarizing pre‐pulses to near 0 mV. The tail current was inhibited by Cd2+ ions (0.2‐1 mM) and hence attributed to activation of a Ca2+‐dependent K+ current by a priming voltage‐activated Ca2+ current. 3. Two components to this tail current could be distinguished pharmacologically: an early (less than or equal to 50 ms) component inhibited by 1‐5 mM‐tetraethylammonium (TEA), and a late component lasting several hundred milliseconds inhibited by apamin (0.1‐0.4 microM) or d‐tubocurarine (0.1‐0.5 mM). 4. Ionophoretic injection of Ca2+ ions evoked a transient outward current with an apparent reversal potential (from ramped current‐voltage curves) of ‐70 mV. This current was succeeded or sometimes replaced by an inward current with an apparent reversal potential between ‐20 and ‐10 mV. 5. The outward current induced by Ca2+ injections was unaffected or partly inhibited by TEA (1‐5 mM), but was strongly inhibited by apamin or d‐tubocurarine. 6. Hyperpolarizing voltage steps from between ‐30 and ‐40 mV induced inward current relaxations reversing at between ‐80 and ‐90 mV. These were considered to result from deactivation of the voltage‐dependent sustained K+ current, IM. 7. Application of methacholine, muscarine or Ba2+ ions produced an inward current, reduced input conductance and reduced IM deactivation relaxations. 8. It is concluded that differentiated NG108‐15 cells possess several of the K+ currents present in sympathetic neurones, including a delayed rectifier current, two species of Ca2+‐activated K+ current and the M‐current.
Abstract One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N‐type Ca 2+ currents: this reduces Ca 2+ ‐activated K + currents and facilitates high‐frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage‐dependent inhibition through activation of M 4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage‐independent inhibition via M 1 mAChRs ( Hille (1994) Trends in Neurosci ., 17, 531 –536]. We have analysed the mechanistic basis for this divergence at the level of the individual G‐proteins and their α and βγ subunits, using a combination of site‐directed antibody injection, plasmid‐driven antisense RNA expression, over‐expression of selected constitutively active subunits, and antagonism of endogenously liberated βγ subunits by over‐expression of βγ‐binding β‐adrenergic receptor kinase 1 (βARK1) peptide. The results indicate that: (i) M 4 mAChR‐induced inhibition is mediated by G oA; (ii) α and βγ subunits released from the activated G oA heterotrimer produce separate voltage‐insensitive and voltage‐sensitive components of inhibition, respectively; and (iii) voltage‐insensitive M 1 mAChR‐induced inhibition is likely to be mediated by the α subunit of G q . Hence, Ca 2+ current inhibition results from the concerted, but independent actions of three different G‐protein subunits.
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