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Full Abstracts – NEUROPHYSIOLOGY AND BEHAVIOR
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The Drosophila vesicular monoamine transporter mutation provides a sensitized system to identify drugs that regulate aminergic
neurotransmission.
Hakeem O. Lawal, Traci Biedermann, Filmon Mehanzel, David E. Krantz. Psychiatry & Biobehavioral Sci, Univ California, Los
Angeles, Los Angeles, CA.
Most current treatments for neuropsychiatric disorders such as Parkinson’s disease, depression, and attention deficit disorder target proteins expressed at
aminergic synapses. However, methods to identify new molecular targets are limited. Using a mutation in the Drosophila vesicular monoamine transporter
(dVMAT) as a sensitized genetic background, we screened for novel drugs that might potentiate aminergic signaling. In flies, as well as mammals, VMATs
are required for packaging into synaptic vesicles all monoamine neurotransmitters, including dopamine, serotonin and in flies, octopamine. dVMAT mutants
show several behavioral deficits including reduced larval locomotion. We screened a panel of 1039 drugs and identified 40 compounds that increase larval
locomotion. In a secondary screen comparing the effects of dVMAT null and hypomorphic alleles, we showed that 7 drug are likely to act via increasing
octopamine release at presynaptic sites. A second set of drugs acts post-synaptically to activate octopamine receptors. A third set of drugs is likely to act via
cholinergic pathways, consistent with the proposed cholinergic input to glutamatergic motoneurons. Our screen represents an important new method to
identify new drugs and targets for the treatment of neuropsychiatric disorders. In addition, it provides a new set of tools for dissecting the molecular
mechanisms that control larval locomotion.
54
A non-binary expression approach to generating brain-dopamine deficient Drosophila.
Karol Cichewicz
1
, Magali Iché-Torres
2
, Serge Birman
2
, Jay
Hirsh
1
. 1) Biology, University of Virginia, Charlottesville, VA; 2) CNRS, ESPCI, Paris.
Drosophila tyrosine hydroxylase (DTH,
ple
), encoding the rate limiting enzyme in dopamine biosynthesis, expresses in the CNS and hypoderm, with
tissue-specific alternative splice forms. A recent study from our laboratories (Riemensperger et al, 2010) shows that a modified DTHg FS+/- gene that
selectively expresses in the hypoderm rescues the lethality of a DTH (
ple
) null mutation, generating healthy flies with normal lifespan. These flies have
undetectable brain dopamine, and show a number of behavioral defects that elucidate novel roles for neural dopamine (Hirsh et al., 2010; Riemensperger et
al, 2010). These flies were constructed with the GAL4:UAS binary expression system, such that further genetic manipulations with GAL4:UAS are difficult.
Here we present an approach, in which we switch off DTH expression in CNS without the use of binary expression tools, allowing for subsequent rescue
using GAL4-UAS in dopamine neuron subsets. The DTH FS mutations were recombineered into a large genomic BAC clone, which was integrated site-
specifically into a 3rd chromosome, and then recombined onto a ple mutant background. BAC plasmids containing the wild type 20kb DTH gene as well as
DTH FS successfully rescued
ple
lethality, showing that all DTH cis-regulatory elements are contained within this segment. Our preliminary data shows
dramatic reduction of activity and female infertility in flies rescued by DTH FS BAC the latter of which is rescued by L-DOPA feeding. The observed
female sterility indicates that the dopamine deficiency is likely more severe than with the initial GAL-UAS approach. With this rescue background,
dopamine neuron subset selective GAL4 drivers, in conjunction with UAS-DTH, will be used to elucidate the behavioral roles of dopamine in defined brain
regions.
55
Ryanodine receptor in neurons mediates volatile anesthetic sensitivity of Drosophila.
Shuying Gao, David Sandstrom, Qun Gu, Robert Scott, Howard
Nash. Laboratory of Molecular Biology, National Institute of Mental Health, NIH, Bethesda, MD.
Volatile anesthetics produce a profound and reversible alteration in the state of arousal in all motile metazoans that have been tested, yet few target
molecules have been identified and validated behaviorally. Previous work in the lab has indirectly implicated the Drosophila ryanodine receptor (dRyr), an
ion channel that governs Ca
2+
release from the endoplasmic reticulum, as a prominent component of a gene network that controls the response to the volatile
anesthetic halothane. Using an assay that evaluates the righting/climbing reflex, we found that heterozygous dRyr mutations conferred a strong and dominant
resistance to halothane and weaker resistance to three other volatile anesthetics: isoflurane, enflurane, and sevoflurane. Addition of a transgenic copy of dRyr
genomic DNA to a wild-type strain increased sensitivity to halothane. dRyr regulating halothane sensitivity in a gene dosage-dependent manner
demonstrates that dRyr is a limiting factor for halothane sensitivity. Altering key residues of dRyr can increase halothane sensitivity. The function of dRyr in
neurons, but not in muscle, is required for normal halothane sensitivity. RNAi knockdown dRyr in the nervous system induced halothane resistance.
Conversely, expressing dRyr under the control of a panneuronal driver was sufficient to rescue normal halothane sensitivity in a mutant background. To
determine whether anesthetics act directly on dRyr, anesthetic-induced Ca
2+
flux was measured using Ca
2+
-sensitive dyes and flow cytometry in Sf9 cells
stably transfected with dRyr. Although untransfected cells were completely insensitive to anesthetics, dRyr-expressing cells responded in a concentration-
dependent way. Halothane also induces
Ca2+
transients and hyperpolarization in larval RP2 motoneurons. This rise in Ca
2+
correlated with hyperpolarization
was blunted by expression of dRyr RNAi. Thus, Ryr function in neurons is critical for anesthetic responsiveness, and its expression in heterologous cells
confers anesthetic sensitivity, suggesting that Ryr is a bona fide target of volatile anesthetic action.