Article Figures & Data
Figures
- Figure 3.
Courtship learning phenotypes of animals expressing T287D or T287A CaMKII in temporally and spatially restricted patterns. Animals expressing T287D or T287A CaMKII were trained for 1 hr with a mated female and then tested immediately with an anesthetized virgin to assess memory. A, 30Y- and MJ85b-driven expression of T287D CaMKII improved training with the mated female. Gray bar shows wild-type (wt) (Canton S) performance. Black bars show training indices (CIf/CIi) for the progeny of crosses between tetO-T287D; UAS-tTA and the GAL4 line indicated below the bar. 30Y- and MJ85b-driven expression of T287D significantly (ANOVA; F(6,127) = 6.7269; *p < 0.0001, with planned post hoc comparisons significant; α = 0.005) enhanced suppression compared with the UAS-tTA; tetO-T287D effector control. All animals were raised on tetracycline until eclosion. B, T287D CaMKII does not affect associative memory. Gray bar shows wild-type (Canton S) performance. Black bars show memory indices (CIt/mCIsh) for the progeny of crosses between tetO-T287D; UAS-tTA and the GAL4 line indicated below the bar. None of the flies carrying a GAL4 insert performed significantly differently from the UAS-tTA/+; tetO-T287D/+ control (ANOVA; F(6,124) = 6.3277; p < 0.0001, with planned post hoc comparisons not significant; α = 0.005). Wild-type flies performed significantly better than any animal carrying the effector transgenes (ANOVA; F(6,124) = 6.3277; *p < 0.0001, with planned post hoc comparisons significant; α = 0.005). All flies were grown on tetracycline until eclosion. C, Expression of UAS-T287A CaMKII under control of 30Y or MJ85b did not affect performance during training compared with the UAS alone control (ANOVA; F(2,44) = 1.5783; p > 0.2). All flies were grown on tetracycline-free medium. D, Expression of UAS-T287A CaMKII under control of 30Y or MJ85b did not affect memory compared with the UAS alone control (ANOVA; F(2,44) = 0.1734; p > 0.8). All flies were grown on tetracycline-free medium. For details of behavioral assays and statistics, see Materials and Methods.
- Figure 4.
Training enhancement can be rescued by suppressing T287D CaMKII expression during adulthood. All flies were fed tc during larval life and then either treated with a sucrose carrier solution or a tc-sucrose solution (100 μg/ml) until testing. Feeding tetO-T287D/+; UAS-tTA/30Y and MJ85b; tetO-T287D/+; UAS-tTA/+ flies tc during adult life abolishes the enhanced training phenotype. Asterisks indicate that the tc-fed flies performed significantly better than the sucrose-fed adults for both 30Y (ANOVA; F(1,38) = 3 8.1803; p < 0.0001) and MJ85b (ANOVA; F(1,45) = 16.9041; p < 0.0002).
- Figure 1.
The GAL4/UAS/tet-off system. A, Schematic of transgenes used. When a fly carrying a GAL4 driver is crossed to a fly with UAS-tTA and tetO gene constructs (GFP is shown here as an example), the resulting progeny are triply transgenic. When tetracycline is present, tTA is unable to activate the tetO promoter, and the tetO gene is kept silent. However, when tetracycline is absent, the triple-transgenic flies express the tetO gene in areas in which the GAL4 protein is expressed. B, When fed tetracycline in the larval medium at 10 μg/ml, tTA is unable to activate the tetO promoter, and the tetO gene is kept silent. Shown are third-instar larvae, early, and late pupae of the genotype MJ85b;UAS-tTA221/+;tetO-GFPB377/+, fed tetracycline throughout larval life (left) or not (right). Signal in tc-fed samples is attributable to nonspecific autofluorescence. C, Using a tetO-luciferase reporter, the time course of induction after removal of tetracycline food was determined in the adult fly. “% luciferase” on the y-axis indicates how much luciferase is expressed heads of MJ85b; UAS-tTA29/+; tetO-luc8B/+ flies fed tetracycline in the larval medium compared with the same genotype fed normal food, with back ground subtracted (see Materials and Methods). Flies were grown at 25°C. Each sample consists of protein from six heads; n = 3 for each time point. D, Feeding tc (100 μg/ml in a 4% sucrose solution) to MJ85b; UAS-tTA29/+; tetO-luc8B/+ adults for 5 d after eclosion suppresses tetO-luciferase expression.
- Figure 2.
Tetracycline feeding rescues T287D-induced morphological defects. Female (A) and male (B) flies of the genotype UAS-tTA29/+; 29BD/tetO-T287D3.4 are shown. Flies on left side of each panel were grown on 10 μg/ml tetracycline. Flies on the right side were grown on normal, tetracycline-free food.
- Figure 5.
T287D CaMKII abolishes the lag to courtship suppression during training. CIs are shown for the initial (CI0-10, 0-10 min), middle (CI20-30, 20-30 min), and final (CI50-60, 50-60 min) portions of the training phase. Halfway through training, wild-type males have not begun to suppress courtship. tetO-T287D/+; UAS-tTA/30Y and MJ85b; tetO-T287D/+; UAS-tTA/+ males start at a lower courtship level and are able to immediately begin to further suppress courtship.
- Figure 6.
GAL4 expression patterns. GAL4-driven expression of UAS-mCD8GFP in adult male brains (first column), antennae (second column), proboscises (third column), and foretarsi (fourth right). A, 30Y expresses in mushroom bodies, antennal lobes, lateral protocerebrum, subesophageal ganglion, and optic lobes but not in the chemosensory organs. B, MJ85b expresses throughout the brain, as well as the antenna, proboscis, and foreleg. C, 201Y expresses primarily in the mushroom body, with expression in a few cells of the subesophageal ganglion, antennal lobe, and lateral protocerebrum, and no expression in chemosensory organs. D, 29BD expresses throughout the brain and chemosensory organs. E, MJ94 expresses in the subesophageal ganglion, with innervations of the antennal lobe, and in the antenna, proboscis, and foreleg. Images were collected as z-stacks on a Leica TCS SP2 confocal scanning microscope. Gain settings were not matched between genotypes. Scale bars: first and second columns, 40 μm; third and fourth columns, 80 μm.
Tables
- Table 1.
Tetracycline feeding rescues toxic effects of overexpression of activated CaMKII
GAL4 UAS-tTA tet0-T287D −tc phenotype +tc rescue C155 220 7.1 Lethal Yes C155 220 9.3 Lethal Yes 29BD 220 7.1 Lethal Yes 29BD 220 9.3 Lethal Yes 29BD 221 3.4 Infantile Yes 29BD 29 3.4 Infantile Yes MJ85b 220 7.1 Lethal Yes MJ85b 221 3.4 Small Yes MJ85b 221 3.3 Small Yes MJ85b 29 3.4 Small Yes -
Lethality of T287D CaMKII is rescued by tetracycline suppression of expression during development. Females of each GAL4 line were crossed to flies carrying UAS-tTA and tet0-T287D transgenes and grown with or without tetracycline in the larval medium. Adult progeny were counted, and the percentage viability and/or display of infantile characteristics of triple-transgenic progeny was recorded. All tetracycline-fed crosses completely rescued lethality or infantile characteristics, producing flies with wild type-like appearance. Data are shown for three different GAL4 lines, three different UAS-tTA insertions, and four different tet0-T287D insertions. −tc phenotype, Flies crossed without tc medium; +tc rescue, flies crossed with tc medium.
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Supplemental data
Files in this Data Supplement:
- supplemental material - Table 1 (Supplementary). Locomotor activity of T287D CaMKII males. Spontaneous activity was measured in the line-crossing assay.� Individual male flies were placed in courtship chambers, and the number of times the fly crossed a line drawn through the center of the chamber was counted during a 3 minute period, in red light.� Raw data was subjected to a one-way ANOVA with genotype as the main effect (F[9, 228] = 4.43, P < 0.0001).� The one-way ANOVA showed significant differences, and was followed by means comparisons with an adjusted alpha level of 0.003 for 17 planned comparisons (Sokal and Rohlf, 1995).� MJ85b, 30Y/+, and 201Y/+ were significantly less active than wild type (P < 0.003).
- supplemental material - Figure 1 (Supplementary). Behavioral effects of GAL4 elements. To determine if the GAL4 elements used in this study had effects on behavior, we trained males that were hemi- or heterozygous for each of the GAL4 elements for 1 h with a mated female. Memory was assessed immediately following training with an anesthetized virgin. Flies were raised on tetracycline-free food. (A) GAL4 insertions training effects. Gray bar shows wild type (Canton S) performance. Black bars show training indices (CIf/CIi) for the progeny of crosses between wild type and the GAL4 line indicated below the bar. Compared to wild type, 201Y and 29BD males were significantly less able to suppress courtship during training (ANOVA F[5, 107] = 5.9296, P < 0.0001, with planned post-hoc comparisons significant, a = 0.01). (B) GAL4 insertions do not effect memory. Gray bar shows wild type (Canton S) performance. Black bars show memory indices (CIt/mCIsh) for the progeny of crosses between wild type and the GAL4 line indicated below the bar. There were no significant differences between GAL4 and wild type animals (ANOVA F[5, 105] = 3.2097, P = 0.0098, with planned post-hoc comparisons not significant, a = 0.01).
