1. OCTOPAMINE-DEFICIENT MUTATION SUPPRESSES SHAKER PHENOTYPES IN DROSOPHILA P.K.Rivlin, J.L. Krans, K.D. Gawera, S.W. Wong and R.R.Hoy. Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853 We are using Drosophila mutants with altered levels of bioamines (in particular, octopamine, dopamine, serotonin) to investigate the role that bioamines play in controlling behavior, synaptic development/function and lifespan. It is well established that ion channel mutants alter neural activity and display a number of behavioral, developmental and physiological phenotypes. Using double mutant combinations, we are investigating how bioamines contribute to the phenotypes observed in ion channel mutants, and whether bioamines play a role in the homeostatic regulation of activity at the nerve terminal. Shaker (Sh) potassium channel mutants display a number of behavioral and physiological phenotypes that include leg-shaking under ether anesthesia (Kaplan and Trout 1969), altered larval locomotion (Wang et al. 2002), repetitive firing of action potentials in larval nerves, prolonged neurotransmitter release at the larval neuromuscular junction (Jan et al. 1977; Ganetzky and Wu 1982), and reduced lifespan (Trout and Kaplan 1970). These phenotypes are enhanced when Sh is combined with another potassium channel mutant, ether-a-go go (eag). In addition, expansion of octopamine-containing nerve terminals (type II) is observed at the neuromuscular junction in eag Sh mutants (Budnik et al. 1990). This raises the possibility that octopamine may contribute, in part, to the enhanced phenotypes observed in eag Sh. To address this question, we have begun an analysis of Sh mutants with genetically altered octopamine levels. Here we present an analysis of a Tßh M18 Sh 5 double mutant. Tßh M18 is a null mutation in the tyramine beta hydroxylase gene and is unable to synthesize octopamine (Monostirioti et al. 1990). 1. Tßh M18 partially rescues lifespan in Sh 5 mutant. 2. Oxidative stress resistance is increased in Tßh M18 Sh 5 double mutant. Figure 1. Survival curves at 25 o C. Sh 5 (red, n = 571); Tßh M18 Sh 5 (green, n = 322); Tßh M18 (blue, n = 376). Sh 5 displays a 43% reduction in mean lifespan as compared to Tßh M18 (32.2 vs. 56.8 days). Tßh M18 Sh 5 displays a 26% extension in lifespan as compared to Sh 5 (40.5 vs. 32.2 days). Tßh M18 lives a normal lifespan (unpublished observations). Genotype CS Tßh M18 Tßh M18 Sh 5 Sh 5 eag Sh 133 % Survival 90.0 95.8 87.5 51.3 30.0 n 70 230 120 230 187 Figure 2. Suspectibility to 10 mM paraquat, a compound that generates free-radicals. Order of severity correlates with lifespan: eag Sh 133 < Sh 5 < Tßh M18 Sh 5 < Tßh M18 ≈ CS. The hyperexcitable mutant, eag Sh 133 serves as a control with severely reduced lifespan. 3. Genetic screen for suppressors of Shaker Figure 3. EP misexpression screen to identify genes that rescue the shortened lifespan of Shaker. Briefly, an EP element is a transposable P element containing a basal promoter and 14 copies of the yeast UAS sequence, which responds to the transcription factor GAL4. Each EP line is crossed to C155, a transgenic fly that expresses GAL4 in all post-mitotic neurons. F1 flies are generated in which GAL4 drives high expression of the genes adjacent to the EP element. Each EP line has been sequenced and the associated gene identified. F1 males are tested for resistance to paraquat. Thus far, we have identified one putative suppressor, glutathione S1 transferase (GSTS1). GSTS1 is an enzyme involved in free-radical homeostasis. OVERVIEW Figure 5. Evoked potentials (ejps) from wildtype and Shaker genotypes. Right: Sh 5 mutant Left: CantonS strain. Each trace is the average of 20 repetitions of electrical stimulation to the segmental nerve in third instar larval preparations. Voltage recordings are acquired in modified Standard Saline (Jan and Jan 1976) using a single sharp electrode. Mean amplitude of Sh 5 ejps was always greater than that of CS, and this relationship was heavily [Ca++] dependent. Note: Since Jan, Jan and Dennis (1977) reported these differences at the NMJ, several new salines have been developed for the larval preparation. It is our experience that these at least partially mask the Sh 5 NMJ phenotype, even at lowered [Ca++], where the magnitude difference is typically greater than 10-fold. 5. Synaptic physiology of wildtype and Sh 5 6. Tßh M18 suppresses the Sh 5 NMJ phenotype. Figure 6. Tßh M18 mutation suppresses Shaker phenotype. Recordings were made from both muscle 6 and muscle 12 in larval preparations (number of muscle recordings per strain, left to right: 12, 7, 11, 6). In all cases, muscle 12 showed smaller mean ejps than muscle 6 (not significant; p>0.05, all). Tßh M18 ( M18 ) showed a non-significant increase in ejp amplitude over CS (m6: p>0.3, m12: p>0.05), but when combined with the Sh 5 mutation ( M18Sh5 ), the double mutant abolished the increased ejp size that is characteristic of Shaker mutants ( Sh5 ) (*: p<<0.01). Insets: examples of voltage recordings from mutants not shown above, Tßh M18 and Tßh M18 Sh 5, and CS. Scalebar: 25ms, 5mV. Figure 4. Schematic of larval neuromuscular preparation and recording paradigm. Larval muscles are shown in gray. The segmental nerve was stimulated to evoke synaptic potentials. Minimum intensity stimuli to evoke full response were determined experimentally. Excitatory junctional potentials (EJPs) were collected at muscles 6 and 12. Note that octopamine is present at the NMJ of muscle 12, but not at muscle 6. 4. Larval Neuromuscular Junction (NMJ) Does octopamine contribute to Shaker phenotypes? [C155-GAL4] eag Sh 133 EP Line (UAS-gene X) enhancerno effectsuppressor “increased stress resistance” (1)Our analysis of Tßh M18 Sh 5 suggests a link between synaptic transmission, oxidative stress and aging. In addition, our finding that overexpression of GSTS1 in the CNS rescues stress resistance in Shaker suggests that nervous system function is a critical determinant of aging. (2)It is well established that cAMP plays a role in regulating synaptic function and development. The absence of OA and/or the elevated levels of its precursor, tyramine may result in altered cAMP levels which act to suppress neurotransmitter release as well as increase stress resistance in Sh 5 mutants. (2)To address the role of cAMP in aging, we are examining whether the cAMP mutants dunce (dnc) and rutabaga (rut) affect lifespan and stress resistance. (3)To address the role of synaptic transmission in aging, we are extending our studies to synaptic machinery mutants. (4)Lastly, we are investigating whether there is a critical period for lifespan rescue in Tßh M18 Sh 5. ACKNOWLEDGEMENTS We thank Drs. M. Monostirioti and C-F Wu, and the Bloomington Stock Center for providing fly stocks. This work was supported by a HHMI Professor Award to RRH. CONCLUSIONS & FUTURE DIRECTIONS