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.

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       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.

   
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         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]

   
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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]