strychnine and methyllycaconitine

Nicotinic receptors form a diverse group of ligand-gated ionotropic receptors with roles in both synaptic transmission and the control of excitability. In the bag cell neurons of Aplysia, acetylcholine activates an ionotropic receptor, which passes inward current to produce a long-lasting afterdischarge and hormone release, leading to reproduction. While testing the agonist profile of the cholinergic response, we observed a second current that appeared to be gated only by nicotine and not acetylcholine. The peak nicotine-evoked current was markedly smaller in magnitude than the acetylcholine-induced current, cooperative (Hill value of 2.7), had an EC50 near 500 μM, readily recovered from desensitization, showed Ca(2+) permeability, and was blocked by mecamylamine, dihydro-β-erythroidine, or strychnine, but not by α-conotoxin ImI, methyllycaconitine, or hexamethonium. Aplysia transcriptome analysis followed by PCR yielded 20 full-length potential nicotinic receptor subunits. Sixteen of these were predicted to be cation selective, and real-time PCR suggested that 15 of the 16 subunits were expressed to varying degrees in the bag cell neurons. The acetylcholine-induced current, but not the nicotine current, was reduced by double-strand RNA treatment targeted to both subunits ApAChR-C and -E. Conversely, the nicotine-evoked current, but not the acetylcholine current, was lessened by targeting both subunits ApAChR-H and -P. To the best of our knowledge, this is the first report suggesting that a nicotinic receptor is not gated by acetylcholine. Separate receptors may serve as a means to differentially trigger plasticity or safeguard propagation by assuring that only acetylcholine, the endogenous agonist, initiates large enough responses to trigger reproduction.

Topics: Acetylcholine; Aconitine; Action Potentials; Animals; Aplysia; Conotoxins; Dihydro-beta-Erythroidine; Hexamethonium; Mecamylamine; Neuroendocrine Cells; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Receptors, Nicotinic; Strychnine; Transcriptome

We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.

Topics: Aconitine; Animals; Atropine; DNA, Complementary; Membrane Potentials; Muscarinic Antagonists; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Oocytes; Patch-Clamp Techniques; Rats; Receptors, Nicotinic; Receptors, Serotonin, 5-HT3; Recombinant Fusion Proteins; Serotonin Antagonists; Strychnine; Xenopus laevis

Whole-cell patch clamp recordings were performed on neurones in the lamina X of rat spinal cord slices in order to characterize glycinergic synaptic currents and their modulation by nicotinic acetylcholine receptors. In the presence of TTX, bicuculline and kynurenic acid, glycine-induced currents and miniature glycinergic postsynaptic currents (mIPSCs) were recorded. These currents reversed near the chloride ion equilibrium potential and were blocked by strychnine (1 microM). A selective nicotinic acetylcholine receptor (nAChR) agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP), increased the frequency of glycinergic mIPSCs without altering significantly their amplitude distributions or their kinetic properties. The effects of DMPP were mimicked by different nAChRs agonists with the following apparent order of potency: ACh > DMPP > nicotine > cytisine. The effect of DMPP on mIPSCs was blocked by both d-tubocurarine and hexamethonium, and was reduced by dihydro-beta-erythroidine and methyllycaconitine (MLA), antagonists of non alpha7- and alpha7-containing nAChRs, respectively. In the absence of TTX, strychnine-sensitive glycinergic electrically evoked postsynaptic currents (eIPSCs) could be recorded. DMPP blocked the appearance of electrically evoked IPSCs while still inducing the appearance of spontaneous glycine IPSCs. These data demonstrate that neurones surrounding the central canal of the spinal cord present a glycinergic synaptic transmission which is modulated by terminal nAChRs.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aconitine; Alkaloids; Anesthetics, Local; Animals; Animals, Newborn; Azocines; Bicuculline; Dihydro-beta-Erythroidine; Dimethylphenylpiperazinium Iodide; Drug Interactions; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Hexamethonium; In Vitro Techniques; Kynurenic Acid; Membrane Potentials; Neurons; Nicotinic Agonists; Nicotinic Antagonists; Patch-Clamp Techniques; Quinolizines; Rats; Rats, Wistar; Receptors, Nicotinic; Spinal Cord; Strychnine; Tetrodotoxin; Time Factors; Tubocurarine

Ionotropic, nicotinic receptors have previously been shown to mediate both inhibitory (Cl-dependent) and excitatory (cationic) cholinergic responses in Aplysia neurons. We have used fast perfusion methods of agonist and antagonist application to reevaluate the effects on these receptors of a wide variety of cholinergic compounds, including a number of recently isolated and/or synthesized alpha toxins [alpha-conotoxin (alphaCTx)] from Conus snails. These toxins have been shown in previous studies to discriminate between the many types of nicotinic receptors now known to be expressed in vertebrate muscle, neuroendocrine, and neuronal cells. One of these toxins (alphaCTx ImI from the worm-eating snail Conus imperialis) revealed that two kinetically and pharmacologically distinct elements underlie the ACh-induced Cl-dependent response in Aplysia neurons: one element is a rapidly desensitizing current that is blocked by the toxin; the other is a slowly desensitizing current that is unaffected by the toxin. The two kinetically defined elements were also found to be differentially sensitive to different agonists. Finally, the proportion of the rapidly desensitizing element to the sustained element was found to be cell-specific. These observations led to the conclusion that two distinct nicotinic receptors mediate Cl currents in Aplysia neurons. The receptor mediating the rapidly desensitizing Cl-dependent response shows a strong pharmacological resemblance to the vertebrate alpha-bungarotoxin-sensitive, alpha7-containing receptor, which is permeable to calcium and mediates a rapidly desensitizing excitatory response.

Topics: Acetylcholine; Aconitine; Alkaloids; alpha7 Nicotinic Acetylcholine Receptor; Animals; Aplysia; Azocines; Bungarotoxins; Cations; Chlorides; Choline; Cholinergic Antagonists; Conotoxins; Dihydro-beta-Erythroidine; Electric Conductivity; Glycine Agents; Insecticides; Ion Channel Gating; Mollusk Venoms; Nicotinic Agonists; Nicotinic Antagonists; Oligopeptides; Patch-Clamp Techniques; Peptides; Quinolizines; Receptors, Nicotinic; Strychnine; Vertebrates