Ketamine Info
Newsgroups: alt.drugs Subject: Ketamine for Worms! Date: 2 Oct 1994 02:10:59 GMT I came across these articles on the net -- reminded me of the "Ketamine for Children" thread. ps. the site was "http://eatworms.swmed.edu/", and the worm in question is C. elegans. ============================================ MUTATIONS THAT CAUSE CONVULSIONS AGAINST AN ANESTHETIC, KETAMINE Hideki Ando and Hiroaki Kagawa As a part of study on neuro-muscular function, we tried to isolate mutants having abnormal response against some anesthetics. Sixteen strains of three independent mutations were isolated with EMS mutagenesis. Those had specific response and convulsive movement in 3OmM Ketamine-Hydrochloride(C13H16ClNO.HCl). All mutants had similar response to another drugs; serotonine, octopamine, as N2. In Ketamine, N2 worm had two phased kinetics between time and paralytic states, but responded no obvious convulsion to ketamine. Mutants basically show N2 like two phased kinetics, but were accompanied by clear convulsion during almost all stages. Modes of these convulsion was classified into two classes; quick and vibration-like convulsions in 15 strains. But nine of those show twitcher in the absence of ketamine and other strains had similar as N2. Another one shows wave- like convulsive movement in 30mM ketamine and had cold-sensitive uncoordinated movement in the absence of ketamine. This strain had almost paralytic phenotype at 16 C and recovered perfect motility after 40 min at 30 C. Genetic analysis shows that 16 strains divided into three independent mutations. Three of nine strains having twitcher in the absence of ketamine were mapped on LG-IV and could not complement to unc-22(e66). Strain J030 having wave-like convulsion was mapped on LGV. Double mutant from trans configuration to dpy-11(e224) was not obtained. This means that mutation site closed to dpy-11(e224), or might be the same cluster. Other strains showing vibration-like convulsions in 30mM ketamine but normal behavior without ketamine was in progress. One of them might be on LGII. These results indicate that ketamine had multiple function to neuro-muscular mechanisms in the worm. Further investigation of defectivity of these mutants and molecular characterization of defective genes allows us to know new aspects about mechanisms of receptor-effecter circuit and cold- sensitive uncoordinated movement of C. elegans. _________________________________________________________________ PHARMACOLOGICAL CHARACTERIZATION AND CLONING OF KRA-1 MUTANT Hideki Ando and Hiroaki Kagawa We have previously reported on isolation of ketamine response abnormal (KRA) mutants by temporary convulsive phenotypes (WBG vol10, No.3, 1988 and CSH 1989). Ketamine, a general anesthetic, is one of the non-competitive antagonist of N-methyl-D-aspartate (NMDA). Genetic and molecular study on genes that have influence on normal pharmacological response to such drugs must be an adequate approach to understanding the NMDA class of glutamate receptor. As reported previously, we identified a gene, kra-1 on LGV by genetic study on a strain kh-30. Genetic locus of kh-30 mutation site was determined between right side breakpoints of nDf32 and sDf20 (0.08mu in span). This mutation expresses a semidominant convulsive phenotype in 30mM ketamine solution or other NMDA antagonists, a strong inhibition of postembryonic development of 10mM ketamine containing NGA, a recessive cold sensitive Unc phenotype, and variable motility in usual condition. As suggested by John White, we also tested previously isolated strains including 26 unc loci derived from over 30 genes and some levamisole resistant strains appeared to be blocked their postembryonic development by ketamine. In addition, some mutant that have abnormal neuron networks showed ketamine resistance in postembryonic development. On the other hand, in immediate response to ketamine, hypersensitive paralyzing strains (whole body and head region restricted) could be found. We are observing pharmacological responses of KRA and other strains to acetylcholine antagonists or other reagents. Pharmacological and anatomical data will give us any suggestions about the ketamine functional site on neuron networks in the worm. Tc1 tagging of mec-1 gene is in progress with generous supply of Tc1 clones by Marty Chalfie. We do hope mec-1 linked fragment will be cloned for matching physical map and genetic map very soon. [See Figure 1] _________________________________________________________________ A KRA-1 GENE MIGHT ENCODE A NICOTINIC RECEPTOR-ASSOCIATED NMDA TYPE ION CHANNEL IN C. ELEGANS Hideki Ando and Hiroaki Kagawa Recent studies in invertebrate glutamate receptors indicate the presence of at least three types of glutamate receptors, none of which are of the NMDA type. Pharmacological data have shown invertebrate glutamate receptor ion channels have less selectivity for noncompetitive antagonists than that of NMDA receptor ion channels and are not shared a voltage-sensitive block by Mg2+ ions, a property of NMDA receptor ion channel. We have tested pharmacological effect of noncompetitive antagonist of NMDA receptors to the worm. In competition assay using cut worm bathing in drug solution, nicotinic transmission appeared to be specifically antagonized by ketamine, a non-competitive antagonist of NMDA receptor. We have isolated sixteen EMS induced mutants showing convulsions against 30mM ketamine and other NMDA noncompetitive antagonists; 1mM PCP and MK-801. In the case of cut worm assay, convulsion could be induced at less than one tenth concentrations. Genetic study to one strain kh30 identified a gene kra-1 which was mapped on the locus closed to mec-1 and unc-68 loci on chromosome V. kra-1(kh30) animal was weak resistant to nicotine (0.1mM), suggesting this convulsion was derived from defective function of nicotine receptor system. Defective site of kra- 1(kh30) might be on nicotinic receptor-associated ion channel because kra-1(kh30) was also weak resistant to channel activator ouabine (0. 1mM). Competitive nicotinic antagonist d-tubocuraine suppressed ketamine function to kra-1(kh30) animal. This suggests that binding affinity of ketamine to ion channel decreased in inactivated state. Finally, convulsion expression of kra-1(kh30) animal with ketamine was also suppressed by Mg2+ ion. These data permit us to conclude that this ion channel could be associated by nicotinic receptor with pharmacological homology to vertebrate NMDA ion channel. Cloning and molecular study of the gene will confirm a pharmacological results. [See Figure 1]