Nicotinic Receptor Currents Research Articles

Xanomeline restores endogenous nicotinic acetylcholine receptor signaling in mouse prefrontal cortex

Cholinergic synapses in prefrontal cortex are vital for attention, but this modulatory system undergoes substantial pre- and post-synaptic alterations during adulthood. To examine the integrated impact of these changes, we optophysiologically probe cholinergic synapses ex vivo, revealing a clear decline in neurotransmission in middle adulthood. Pharmacological dissection of synaptic components reveals a selective reduction in postsynaptic nicotinic receptor currents. Other components of cholinergic synapses appear stable, by contrast, including acetylcholine autoinhibition, metabolism, and excitation of postsynaptic muscarinic receptors. Pursuing strategies to strengthen cholinergic neurotransmission, we find that positive allosteric modulation of nicotinic receptors with NS9283 is effective in young adults but wanes with age. To boost nicotinic receptor availability, we harness the second messenger pathways of the preserved excitatory muscarinic receptors with xanomeline. This muscarinic agonist and cognitive-enhancer restores nicotinic signaling in older mice significantly, in a muscarinic- and PKC-dependent manner. The rescued nicotinic component regains youthful sensitivity to allosteric enhancement: treatment with xanomeline and NS9283 restores cholinergic synapses in older mice to the strength, speed, and receptor mechanism of young adults. Our results reveal a new and efficient strategy to rescue age-related nicotinic signaling deficits, demonstrating a novel pathway for xanomeline to restore cognitively-essential endogenous cholinergic neurotransmission.

Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Reduced nicotinic receptor function in sympathetic ganglia is responsible for the hypothermia in the acetylcholinesterase knockout mouse

Cholinergic signalling in the sympathetic ganglia (SG) contributes to non-shivering thermogenesis by relaying the activation signal from the brain to SG neurons which activate many peripheral tissues to produce heat. Paradoxically, acetylcholinesterase (AChE) inhibitors, which should enhance cholinergic signalling, induce hypothermia. To understand the mechanism of how cholinergic signalling in the SG controls thermoregulation, we analysed infant AChE knockout mice, which are known to show hypothermia by postnatal day 15. Nicotinic receptor currents were reduced in acutely dissociated SG neurons of the AChE-deficient mice by over 40% compared with wild-type mice. When wild-type neurons were treated for 1 h with either oxotremorine-M, a muscarinic agonist, or nicotine, the amplitude of nicotinic receptor currents was also decreased by 40%. The hypothermia in AChE mutant mice was fully rescued by a peripheral injection of both ivermectin, which increases nicotinic receptor currents, and methyl-atropine, a muscarinic antagonist. Our results demonstrate that the hypothermia induced by the lack of AChE activity is primarily caused by a downregulation of nicotinic receptors via prolonged stimulation of muscarinic and nicotinic receptors in SG neurons. The stationary noise analysis of the nicotinic receptor current traces showed that the properties of single-channel activities were not different between the two genotypes, suggesting that the primary reason for downregulation of nicotinic receptors is due to a reduction of the receptors on the surface.

Blockade of nicotinic receptors of bovine adrenal chromaffin cells by nanomolar concentrations of atropine

Nanomolar concentrations of atropine have been considered up to now to be selective for blockade of muscarinic receptors for acetylcholine. A collateral finding indicated to us that these low concentrations of atropine could also target the neuronal nicotinic receptors. We report here a detailed study on this novel property of atropine. Catecholamine release, measured on-line with amperometry in chromaffin cells stimulated with acetylcholine pulses was blocked by atropine in a competitive manner. To corroborate a direct action of atropine on nicotinic receptors, we have employed N, N-dimethyl- N′-phenyl-piperazinium (DMPP), a pure nicotinic receptor agonist; atropine blocked its secretory responses with an IC 50 of 2.04 nM. Nicotinic currents, recorded with the whole cell configuration of the patch-clamp technique were blocked by atropine in a concentration-dependent manner (IC 50 of 11 nM), also showing a competitive nature. Nicotinic receptor currents in oocytes expressing bovine α7 and α3β4 nicotinic receptors were blocked by atropine with an IC 50 of 11.2 and 46.8 nM, respectively. Atropine (30 nM) also decreased the increment of the cytosolic calcium concentrations after stimulation with 30 μM DMPP in bovine chromaffin cells. However, action potentials evoked by DMPP were not modified by atropine. Our results demonstrate that nicotinic currents and their downstream consequences (i.e. cytosolic calcium elevations and catecholamine release) were blocked by nanomolar concentrations of atropine; although the blockade was partial, it must be considered when using atropine to study cholinergic neurotransmission, particularly at synapses where both nicotinic and muscarinic receptors are present i.e., the adrenal medulla and autonomic ganglia.

Activation and block of mouse muscle-type nicotinic receptors by tetraethylammonium

We have studied the activation and inhibition of the mouse muscle adult-type nicotinic acetylcholine receptor by tetraethylammonium (TEA) and related quaternary ammonium derivatives. The data show that TEA is a weak agonist of the nicotinic receptor. No single-channel clusters were observed at concentrations as high as 5 mM TEA or in the presence of a mutation which selectively increases the efficacy of the receptor. When coapplied with 1 mM carbamylcholine (CCh), TEA decreased the effective opening rate demonstrating that it acts as a competitive antagonist of CCh-mediated activation. Kinetic analysis of currents elicited by CCh and TEA allowed an estimate of receptor affinity for TEA of about 1 mM, while an upper limit of 10 s−1 could be set for the wild-type channel-opening rate constant for receptors activated by TEA alone. At millimolar concentrations, TEA inhibited nicotinic receptor currents by depressing the single-channel amplitude. The effect had an IC50 of 2–3 mM, depending on the conditions of the experiment, and resembled a standard open-channel block. However, the decrease in channel amplitudes was not accompanied by an increase in the mean burst duration, indicating that a linear open-channel blocking mechanism is not applicable. Upon studying block by other nicotinic receptor ligands it was found that block by CCh, tetramethylammonium and phenyltrimethylammonium can be accounted for by the sequential blocking mechanism while block in the presence of methyltriethylammonium, ethyltrimethylammonium or choline was inconsistent with such a mechanism. A mechanism in which receptors blocked by TEA can close would account for the experimental findings.

Alpha 2-Adrenoreceptor-mediated inhibition of acetylcholine-induced noradrenaline release from rat sympathetic neurons: an action at voltage-gated Ca2+ channels.

[3H]Noradrenaline release was studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Acetylcholine elicited a concentration- and time-dependent increase in 3H outflow which was half-maximal at about 300 microM and within 5 s. The overflow induced by 10 s exposure to 300 micro A acetylcholine was reduced by the nicotinic antagonist hexamethonium, but increased by the muscarinic antagonist atropine. Cd2+ (300 microM) prevented the overflow evoked by electrical field stimulation, but reduced acetylcholine-induced overflow by less than 50%. Removal of extracellular Ca2+ abolished stimulation-evoked tritium overflow irrespective of the stimulus. The selective alpha2-adrenoceptor agonist UK 14,304 inhibited acetylcholine-evoked overflow to a significantly smaller extent (approximately 25% maximal inhibition) than electrically induced overflow ( > or = 45% maximal inhibition). These inhibitory effects were antagonized by the alpha2-adrenoceptor antagonist yohimbine. Noradrenaline (0.1 microM) reduced acetylcholine-evoked overflow to the same extent as did UK 14,304 (0.1 microM). UK 14,304 had no effect when 3H overflow was evoked by acetylcholine in the presence of 300 microM Cd2+. Currents through nicotinic acetylcholine receptors and voltage-activated Ca2+ currents were studied with the whole-cell variant of teh patch-clamp technique. UK 14,304 reduced nicotinic acetylcholine receptor currents and voltage-activated Ca2+ currents with similar potency and efficacy. Yohimbine, however, antagonized only the inhibition of voltage-activated Ca2+ currents, but not the effects of UK 14,304 on nicotinic receptor currents. Furthermore, yohimbine per se reduced currents through nicotinic receptors. Noradrenaline (10 microM) inhibited voltage-dependent Ca2+ currents just as did UK 14,304 (10 microM), but failed to reduce currents through nicotinic acetylcholine receptor channels. Cd2+ (300 microM) abolished voltage-activated Ca2+ currents and reduced nicotinic acetylcholine receptor currents by 65%. These results indicate that acetylcholine evokes noradrenaline release from rat sympathetic neurons by activation of nicotinic receptors and restricts this release via muscarinic receptors. The acetylcholine-induced transmitter release is based on two mechanisms, one involving and the other one bypassing voltage-dependent Ca2+ channels. alpha2-Adrenoceptor activation reduces voltage-activated Ca2+ currents and effects exclusively the component of acetylcholine-induced release which involves voltage-dependent Ca2+ channels. These results support the hypothesis that voltage-activated Ca2+ channels are the sole site of autoinhibitory alpha2-adrenergic effects on transmitter release from rat sympathetic neurons. The inhibitory effects of alpha2-adrenoceptor agonists and antagonists on currents through nicotinic acetylcholine receptors are not mediated by an alpha2-adrenoceptor.

Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed.

1. A general approach to the analysis of ensemble currents of ligand-gated channels is presented, with a variety of examples that include single and multiple agonist binding steps, desensitization and several blocking pathways. 2. The use of matrix methods to describe model reaction schemes leads to a simplification if the reaction scheme is irreversible: the product of the exponential relaxation rate constants is exactly equal to the product of the forward reaction steps. 3. This method of analysis applied to the bovine adrenal nicotinic receptor suggests that in the range of acetylcholine concentrations from 1 microM to 2 mM, a model with a single kinetically relevant agonist binding step is appropriate. 4. Complex models, to explain the presence of two desensitizing components in currents recorded from whole cells, may be discounted in favour of two distinct receptor types. 5. A simple model of open channel block is discounted, and desensitization of the blocked state proposed.