Biochemical reagents were of analytical grade or better and were obtained from Sigma-Aldrich (St Louis, MO, USA), Fisher Scientific Company (Ottawa, ON, Canada) or Bioshop Canada Inc. (Burlington, ON, Canada) unless otherwise indicated. Oligonucleotides were obtained from Invitrogen (Burlington, ON, Canada). Rabbit polyclonal antibodies against the synthetic peptide MSGSGLDENPFGEPNLDN(C-amide) corresponding to the N-terminal 18 amino acids for Drosophila SCAMP were raised by YenZym Antibodies LLC (South San Francisco, CA, USA). Anti-myc-epitope mouse monoclonal 9E10 and rabbit polyclonal A14 antibodies that recognize EQKLISEEDL were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA), anti-β-tubulin and anti-nervana antibodies were obtained from Developmental Studies Hybridoma Bank (Iowa City, IA, USA). Horseradish Peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA, USA).

Strains used were: y1w67c23P{w[+mC]=GSV1}GS1041/Binsinscy (DGRC number 200072, Drosophila Genetic Resource Center; Kyoto, Japan), isogenized w¹¹¹⁸ (iso-w⁻), elav-GAL4^C155, w⁻;Sp/CyO;Δ2-3; Sb/TM6(±Δ2-3) and y[¹]w[*]P{UAS-mCD8::GFP.L}Ptp4E^LL4 Smox^MB388 P{neoFRT}19A/FM7c (Stock number 44384, Bloomington Drosophila Stock Center; Bloomington, IN, USA).

The P-element was mobilized from the y1w67c23P{w[+mC]=GSV1}GS1041 strain (Drosophila Genetic Resource Center) by crossing to Δ2-3 transposase. Approximately 300 mosaic-eyed progeny were crossed with the FM7c balancer y[¹]w[*]P{UAS-mCD8::GFP.L}Ptp4E^LL4 Smox^MB388 P{neoFRT}19A/FM7c (Bloomington Drosophila Stock Center) for two generations and the white eyed balanced progeny were PCR screened using the primers listed in Table 1. The molecular lesion generated by imprecise excision was determined by direct sequencing of genomic DNA from homozygous flies. For transgenic rescue experiments, Scamp^63A, elav-GAL4^c155 strain was generated.

A cDNA encoding Drosophila Scamp was isolated by RT-PCR using a Drosophila embryo mRNA (Clontech, Mountain View, CA, USA) as a template. Human SCAMP1 and SCAMP5 cDNAs were previously isolated in our laboratory as described (Lin et al., 2005). In brief, first strand cDNA synthesized from human brain total RNA by the use of random primers for reverse transcription was used as a template for PCR. N-terminal myc-tag was introduced by PCR using the primers summarized in Table 2 and ligated into the pUASTattB vector (Groth et al., 2004; Fish et al., 2007; Markstein et al., 2008). The sequence of the N-terminally myc-tagged clones was verified and transgenes were site specifically integrated into the attP2 genomic site by phiC31 recombinase by BestGene Inc. (Chino Hills, CA, USA).

Adult flies were quickly frozen in liquid nitrogen and the removed heads were lysed in RIPA buffer containing 140 mM NaCl, 20 mM Tris-HCl pH 8.0, 1% NP40, 0.1% SDS and 0.5% sodium deoxycholic acid freshly supplemented with proteinase inhibitor cocktails (Roche Diagnostics, Laval, PQ, Canada). The lysate was cleared by centrifugation at 16,000 r.c.f. for 15 min at 4°C twice, and the protein concentration of the supernatant was determined with the Bradford reagent (BioRad, Hercules, CA, USA). The denatured sample was resolved in a 12% SDS-PAGE gel and transferred to a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The blot was blocked in 5% skim milk in PBS-T (0.075% Tween 20 in PBS pH 7.4) and probed with anti-Drosophila SCAMP, anti-myc, or anti-nervana antibody at room temperature for 1 h. After extensive washing with PBS-T, the blot was incubated with HRP-conjugated secondary antibody. Following another washing in PBS-T, signal was detected with Luminata Forte Western chemilluminescent horseradish peroxidase substrate (Millipore, Billerica, MA, USA).

Homozygous Scamp deficiency mutant female flies were outcrossed with iso-w⁻ male, and the male Scamp hemizygous F1 progeny was tested for behavior, unless otherwise stated. Two independently-generated Scamp deficiencies were crossed and the heteroallelic Scamp deficient female F1 progeny was also tested for certain behaviors. Fly stocks were maintained at 22°C or 25°C and 70% relative humidity with 12 h/12 h light/dark cycles on standard cornmeal-yeast-based food. Flies to be tested in behavior assays were collected from freshly eclosed stocks, and transferred to fresh food vials prior to the experiment. All the experiments were done in the early to midafternoon in order to avoid variation due to circadian rhythm.

Null Scamp^63A and precise excision Scamp^211A homozygotes were collected within 16 h after eclosion and raised at 25°C in a moisture- and light-controlled environmental room. Surviving flies were counted every second day. Kaplan-Meier survival curves were generated by calculating the ratio of live flies and plotting the value as a function of incubation time.

Climbing assays were performed as described previously (Leal and Neckameyer, 2002; Martinez et al., 2007; Perkins et al., 2010) with some modifications. Age-matched adult flies of each genotype were transferred without anesthesia to an odorless, clean polystyrene climbing-test-tube and kept in the dark for approximately 10 min prior to the experiment. To assay climbing, the test chamber was tapped three times to bring all flies down to the bottom; we counted the number of flies that climbed past the 7 cm line, from the bottom, within 8 s. During climbing, flies were illuminated only from above to facilitate phototactic climbing. Mean numbers of flies crossing the 7 cm line was determined in 4 or 5× replicates per assay, and we performed 5 to 9 assays per genotype. All experiments were recorded with video for data collection.

House-made T-maze equipment and aversive odor associated learning assays were built and conducted as previously described (Tully and Quinn, 1985), with slight modifications. First, we entrained flies of all genotypes for odor-associated long-term memory. Approximately 130 post-eclosion flies (3–5 days old) of each genotype were transferred to a light-protected training chamber without anesthesia and exposed to a constant flow of odorless air (OLA) for 30 min. Thereafter, the following entrainment procedure was followed: (i) exposure to methylcyclohexanol (MCH) while being mildly electrically shocked with 0.5 s 60 V pulses (approximately 0.75 mA) given at 1.5 s intervals for 60 s, (ii) 45 s of OLA, (iii) 60 s of 3-octanol (OCT), and (iv) 45 s of OLA. After a 15 min spacing period, the entrainment cycle was repeated 10 times, each followed by a 15 min spacer. To test odor-associated long-term memory, trained flies were maintained at 22°C for 20 h before testing. Trained flies were transferred to the T maze apparatus without anesthesia, and exposed to MCH (shock-associated odor) and OCT (shock-unassociated odor) injected from opposite ends of the apparatus, but converging at the T maze choice point. Flies were forced to move from the choice point toward either MCH or OCT over a 2.5 min period by constant gentle agitation of the sliding center compartment at the choice point. At the end of the assay, the sliding center compartment was pulled up to separate flies on either end, and the number of flies on each side was counted. Experiments were carried out in the dark to minimize any phototaxis. Similar experiments using OCT as an associated odor and MCH as an non-associated odor were conducted and the learning index (LI) was calculated (Tully and Quinn, 1985). LI= (the fraction of flies avoiding the shock-associated odor) − (the fraction of flies avoiding the shock-unassociated odor). Reciprocal experiments were conducted using OCT as a shock-associated odor and MCH as a shock-unassociated odor, and the average value was defined as LI.

Heat-induced seizure assays were carried out as described previously (Hoeffer et al., 2003) with some modifications. Newly eclosed adult flies of each genotype were grouped in triplicate in regular fly food vials (10–12 flies per vials), and were grown for 4 days at 22°C prior to the experiments. When the UAS-GAL4 system was used, flies were incubated at 29°C for 3 days followed by 1-day incubation at 22°C. For seizure induction, flies were transferred to fresh polystyrene fly vials and immersed in a 42.3°C water bath for 3 min at which time all the flies were paralyzed. The fly vials were then transferred to 22°C and the number of standing flies that recovered from the heat-shock was counted every minute for up to 30 min. Experiments were repeated 7 times using different flies and all data were combined in the presented graphs of Kaplan-Meier failure function [1-S(t + 0)].

Statistical analyses were conducted using Stata/IC 10.1 (StataCorp LP, College Station, TX, USA). Log-rank tests were used for Drosophila survival analysis and for the analysis of recovery time of heat-induced seizure assays. Means of two groups were compared with Student's t-tests or Wilcoxon rank-sum tests. All hypothesis tests are two-sided. Repeated measures ANOVA was used to analyze the longitudinal climbing assay data, with age as a within-subject factor, taking the proportion of climbed flies in each vial as the unit of analysis.

By mobilizing the P{GSV1}GS1041, several imprecise excision mutants were isolated (Figure 1A). In the current study, Scamp^63A was used for most of the behavioral analysis whereas Scamp^50B was used to generate heterozygous Scamp^63A/Scamp^50B null alleles to characterize female behaviors. As controls, precise excision revertant lines, Scamp^211A and Scamp^209A were used. To test protein expression in these mutants, a polyclonal antibody against the N-terminal 18 amino acids of Drosophila SCAMP was raised in rabbit. A single band approximately 35 kD in size was detected by western blot in lysates isolated from Scamp^211A and iso-w (Figure 1B). Pre-incubation of the primary antibody with the epitope peptide eliminated this band. No appreciable signal was detected in Scamp^63A and Scamp^50B, demonstrating the antibody's specificity. Homozygous deletion mutant flies are viable, fertile and have no apparent morphological abnormalities.

Decreased lifespan is frequently associated with neurodegenerative disorders in Drosophila and is often used as a “straightforward first look” at the phenotype (Lessing and Bonini, 2009). We therefore tested the survival of adult flies. The median lifespan for revertant Scamp^211A males was 52 days whereas that of Scamp^63A males was substantially reduced to 26 days (−40.9%) (p < 0.001, log-rank test) (Figure 2). A similar reduction of life span was observed in Scamp^63A homozygous females (34.1% reduction compared to controls, log-rank test p < 0.001).

Impaired climbing behavior is frequently associated with neurological disorders, and climbing is one of the most commonly assayed behaviors because of the sensitive and robust nature of the assay that allows for testing a large number of flies (Lessing and Bonini, 2009). Homozygous Scamp deficient females (Scamp^63A and Scamp^50B) and homozygous revertant Scamp^211A females were crossed with isogenized (isow⁻) males, and the F1 male nulls (Scamp^63A/Y and Scamp^50B/Y) and Scamp^211A/Y controls were subjected to climbing assays 5–8 days after eclosion. Climbing of Scamp^63A/Y and Scamp^50B/Y was significantly slower than the Scamp^211A/Y (p < 0.001 by Student's t-test, Figure 3A) where the two null mutants exhibited almost identical degrees of impairment. We next asked whether transgenic expression of Drosophila Scamp, human SCAMP1 and SCAMP5 can rescue the climbing phenotype of Drosophila Scamp nulls. When pan-neuronally expressed by elav-GAL4^c155, all the heterologous human and Drosophila proteins were expressed similarly (Supplementary Figure 1). Transgenic expression of Drosophola Scamp under pan-neuronal promoter almost fully rescued the Scamp null climbing phenotype (Figure 3B). Either human SCAMP1 or SCAMP5 in Scamp null mutants substantially ameliorated the climbing impairment (p < 0.05 by Student's t-test), suggesting the conserved role of SCAMPs in climbing behavior. The neuronal expression of any SCAMP gene in heterozygous Scamp^63A/+ females did neither deteriorate nor enhance the climbing behavior, further supporting the specificity of rescue experiments (data not shown). We next examined whether female Scamp^63A/Scamp^50B heteroallelic nulls at different ages also elicit retarded climbing. Scamp nulls showed significantly slower climbing than Scamp^63A/Scamp^50B heteroallelic mutants at all ages except for 1–3 days post-eclosion (p < 0.001 or p < 0.01 by Student t-tests, Figure 3C). Climbing ability of heterozygous Scamp^63A/Scamp^211A flies started to show only a slight decline (−11% on average) at 22–24 days after eclosion whereas Scamp nulls (Scamp^63A/Scamp^50B) exhibited a significant decline (−24% compared to the initial proportion) as early as 8–10 days post-eclosion. Results from the repeated measures ANOVA supported that Scamp nulls perform worse and the degree of age-dependent decline significantly differs between the two strains [the interaction term of age and strain was statistically significant with F_{(4, 354)} = 15.85, p < 0.001], indicating that Scamp deficiency accelerates age-dependent climbing impairment.

A previous study showed that trans-heterozygosity for the long-term memory defective mutant CG32594 (ben) and Scamp (a P-element insertional mutant) led to a defect in long-term memory (Zhao et al., 2009). However, the effects of a Scamp null on long-term memory formation are unknown. To address this, we used an established aversive olfactory associated learning assay that uses electric shock for reinforcement. When exposed to 4-methylcyclohexanol (MCH) or 3-octanol (OCT) in the T-maze test tube under the optimized experimental condition, all genotypes evaded the odor source and moved into the opposite chamber within 2.5 min, suggesting that Scamp mutants sense odors and their odor-avoidance movement is intact. Scamp null adults and age-matched revertant controls were subjected to 10 cycles of training sessions consisting of an exposure with a shock-associated- and unassociated-odors. Both genotypes reacted similarly to electric shock by freezing, falling and jumping. This memory persisted in male revertant flies after 24 h, as they showed a strong tendency to avoid the shock-associated odor 24 h after training [Learning Index (LI) = 0.72 ± 0.22 (mean ± SD) Figure 4A]. In contrast, Scamp nulls displayed an almost even distribution between the shock-associated and unassociated odors, showing a significant difference in LI (LI = 0.046 ± 0.092, p < 0.01 by Wilcoxon rank-sum test). Similarly, heteroallelic null females exhibited significant impairment in long-term memory, as opposed to female homozygous revertants (LI = 0.015 ± 0.11 vs. 0.84 ± 0.049, p < 0.01 by Wilcoxon rank-sum test, Figure 4B). These results indicate that Scamp functions in learning and long-term memory in both males and females. We next tested the effect of neuronal restoration of Drosophila Scamp, human SCAMP1 or SCAMP5 on the long-term memory deficit of Scamp null males. Inclusion of the Drosophila UAS-Scamp (dmSc) significantly improved the Learning Index (LI), compared to the Scamp nulls that had elav-GAL^C155 (LI = 0.35 ± 0.16 vs. −0.016 ± 0.14, p < 0.01 by Wilcoxon rank-sum test, Figure 4C). Interestingly, transgenic expression of human SCAMP1 (hSc1) but not SCAMP5 (hSc5) also restored the LI of Scamp nulls albeit to a lesser extent, suggesting some conserved roles between humans SCAMP1 and Drosophila SCAMP in long-term memory formation.

Elevated temperatures are a major environmental stress that influences cellular processes including enzymatic activity and the ion flux (Garrity et al., 2010), which affects membrane excitability (Peng et al., 2007) and triggers seizures (Wu et al., 1978; Hoeffer et al., 2003; Wang et al., 2004). Raising the temperature triggers seizures to flies, and the experimentally induced seizures was suggested to mimic some aspects of human epilepsy (Song and Tanouye, 2008). The outcrossed male progeny (Scamp^63A/Y and Scamp^211A/Y) was subjected to heat-induced seizure susceptibility assay 5–7 days after eclosion. The median recovery time for Scamp^211A/Y revertant flies was 2.5 min and 100% of these flies recovered within 23.5 min (N = 52). In contrast, the median recovery time of Scamp^63A/Y (N = 35) was 17.5 min, which is considerably longer than that of revertants (p < 0.001 by log-rank test) (Figure 5A). Moreover, 28.6% of Scamp^63A/Y flies did not recover even after a 30-min incubation at 22°C (p < 0.001 by Pearson's chi²-test). Neuronal UAS-Scamp restoration experiments showed partial rescue of heat shock recovery. Median recovery time for elav-GAL4^C155, Scamp^63A/Y flies was 18.5 min (N = 81). Inclusion of the UAS-Scamp improved the median recovery time to 14 min (N = 82), a small but statistically-significant difference (p < 0.001 by log-rank test) (Figure 5B). Only 75 out of 81 (92.6%) elav-GAL4^C155, Scamp^63A/Y flies recovered from heat-shock within 30 min whereas 81 of 82 (98.8%) of the UAS-Scamp restored flies did (p < 0.05 by Pearson's chi²-test).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.