Mutation of the <i>rsh</i> gene reveals a major role in aversive olfactory memory (ARM) and is necessary for appetitive olfactory memory shortly after conditioning.

<p>Flies were either trained with odorants paired with electric shock or sugar reward. The training, cold-shock, retention intervals, and testing patterns (both pre and post) are diagrammed for each panel, the time axis is not to scale. (<i>A</i>) Olfactory memory tested three min after training is reduced in <i>rsh¹</i> flies compared to CS flies, although levels do not reach statistical significance (F(1,12)  = 3.5, P = 0.09). To reveal the <i>rsh</i> function in aversive olfactory memory, wild-type CS and <i>rsh¹</i> flies were trained with odorant / shock pairings, then after 2 hrs were given a cold-shock, memory was tested 1 hr later. Memory performance of <i>rsh¹</i> flies was significantly lower than wild-type CS flies with this procedure (F(1,10)  = 5.0, *  =  P = 0.04). (<i>B</i>) Appetitive olfactory short-term memory was tested at 3, 30, and 60 min after the odorant / sucrose training session. A <i>rsh¹</i> phenotype was evident at all tested time points after training (3 min: F(1,16)  = 29.2, ***  =  P<0.001; 30 min: F(1,14)  = 12.3, **  =  P<0.01; 60 min: F(1,14)  = 12.1, **  =  P<0.01). (<i>C</i>) The <i>rsh¹</i> appetitive short term olfactory memory phenotype is rescued with a transgenic copy of the wild-type version of the <i>rsh</i> gene (F(3,32)  = 13.0, P<0.0001; post-hoc tests: CS vs <i>rsh¹</i> ***  = P<0.001, <i>rsh¹</i> vs. <i>rsh¹</i>; hs-<i>rsh-1</i> *  = P<0.05, CS vs. <i>rsh¹</i>; hs-<i>rsh-1</i>, *  = P<0.05; <i>rsh¹</i> vs. CS; hs-<i>rsh-1</i> *  = P<0.05; CS vs. CS; hs-<i>rsh-1</i> *  = P<0.05). The values are means and error bars represent s.e.m.</p

Control behaviors of wild-type CS and <i>rsh¹</i> mutant flies.

<p>MCH avoidance: ANOVA F(3,32)  = 1.07, P = 0.4; Oct avoidance: F(3,20)  = 1.3, P = 0.3; Sugar attractiveness: ANOVA F(3,44) = 0.75, P = 0.53; Activity: F(1,561) = 3.3, P = 0.07.</p

Mutation of the <i>rsh</i> gene does not influence conditioning or place memory tested directly after training.

<p>Following a 30 s pre-test period (black bars), wild-type CS and <i>rsh¹</i> mutant flies were trained in two equal length periods for a total of either 6 or 20 min with 41°C (light gray bars). A 3 min memory was tested directly following in the post-test period (dark gray bars). The training, retention intervals, and testing patterns (both pre and post) are diagrammed for each panel, the time axis is not to scale. (<i>A</i>), Conditioning and memory tests were similar between the genotypes with 6 min of training (N = 331; pre-test: U = 12753.5, z = 1.07, P = 0.28; 1^st training period: U = 11877.0, z = 2.08, P = 0.04; 2^nd training period: U = 12888.5, z = 0.92, P = 0.36; post-test: U = 13237.0, z = 0.51, P = 0.61). (<i>B</i>) Conditioning and memory tests were also similar between the genotypes with 20 min of training (N = 232; pre-test: U = 6106.5, z = 1.22, P = 0.22; 1^st training period: U = 5740.5, z = 1.93, P = 0.06; 2^nd training period: U = 5802.0, z = −1.81,  = 0.07; post-test: U = 6463.0, z = −0.52, P = 0.60). (<i>C</i>) The <i>rsh</i> gene is necessary for normal short-term place memory. Flies were trained with intermittent training and then held for varying times (1 – 40 min) before being tested for memory with a short reminder training. The <i>rsh¹</i> flies had memory performance similar to wild-type CS levels with a 1 min delay between training and the memory test (N = 447, U = 24641.5, z = 0.24, P = 0.8). Significant differences were found at several time points following training (10 min: N = 295, U = 8637.0, z = .02, **  =  P<0.01; 20 min: N = 330, U = 10074.5, z = 3.95, ***  =  P<0.001; 30 min: N = 311, U = 10926.0, z = 1.45, P = 0.1; 40 min: N = 351, U = 12941.5, z = 2.48, **  =  P<0.01). The values are means and error bars represent s.e.m.</p

The <i>aru^8–128</i> allele reduces olfactory memory performance.

<p>Wild-type CS and Berlin (B) flies, as well as <i>aru^8–128</i> flies in either the CS or B genetic backgrounds were trained and tested for olfactory three minute memory. The memory performance was statistically different from wild-type only in the Berlin genetic background (CS vs. <i>aru^8–128</i> (CS), F(1,10) = 0.44, p>0.1, N = 12; Berlin vs. <i>aru^8–128</i> (B), F(1,12) = 27.5, *** = p<0.001, N = 14). The values are means and error bars represent SEMs.</p

Control behaviors in wild-type CS, Berlin, and different <i>aru</i> EPS8L3 mutant flies.

<p>Control behaviors of wild-type and <i>aru</i> EPS8L3 mutant flies were largely similar. A) The avoidance of 41°C high temperature was similar between wild-type flies and all other flies with the three different <i>aru</i> EPS8L3 alleles (p's>0.1, N's between 100 and 240 for each genotype). B) Shock avoidance for flies with different <i>aru</i> EPS8L3 alleles were not statistically significantly different (CS compared to the three other <i>aru</i> EPS8L3 alleles: F(3,20) = 0.32, p>0.1; Berlin compared to the three other <i>aru</i> EPS8L3 alleles: F(3,24) = 1.29, p>0.1). C) Avoidance of MCH compared to ambient air was not statistically different between wild-type flies and flies with the three other <i>aru</i> EPS8L3 alleles (CS compared to the three other alleles: F(3,20) = 0.54, p>0.1; Berlin compared to the three other alleles: F(3,22) = 1.28, p>0.1). D) The only statistically significant difference in the different genotypes in the avoidance of octanol (OCT) was between flies from the CS and <i>aru^S13</i> genotypes (CS background: F(3,20) = 3.5, p = 0.04, * = p<0.05 with Newman-Keuls <i>post-hoc</i> test with <i>aru^S13</i> (CS) and CS; Berlin genetic background: F(3,20) = 0.57, p>0.1).</p

Place memory phenotypes of <i>aru^8–128</i>, <i>aru^S8</i>, and <i>aru^S13</i> flies.

<p>Wild-type CS and Berlin, as well as flies with a precise excision (<i>aru^S8</i>) and imprecise excision (<i>aru^S13</i>) in both genetic backgrounds, were trained in the heat-box and tested for place memory. A) The memory score of flies from wild-type, <i>aru^S8</i> and <i>aru^S13</i>genotypes are presented, where there were no statistically significant differences detected in any of the genotypes (CS with <i>aru^S8</i> (CS), and <i>aru^S13</i> (CS) p's>0.1, N = 371). B) Flies with the <i>aru^S8</i> and <i>aru^S13</i>alleles in the wild-type Berlin background were also not significantly different (p's>0.1, N = 341). The values are means and error bars represent SEMs.</p

Molecular characterization of new <i>aru</i> alleles.

<p>A) The <i>aru^8–128</i> allele is an insertion of a PGawB element in the genome corresponding to either the first intron of the RD and RC transcripts or 5′ of the RA transcripts. PCR primer pairs corresponding to the P-element inverted repeats (primer 1) and adjacent genomic sequence (primers 2 and 3) were used to characterize two new <i>aru</i> alleles, <i>aru^S8</i>and <i>aru^S13</i> (which were generated by re-mobilizing PGawB element in <i>aru^8–128</i>flies). B) Amplification across the PGawB insertion site using primers 2 and 3 was possible from wild-type CS and <i>aru^S8</i>, but not <i>aru^S13</i>, genomic DNA. Amplification with the 1–2 and 1–3 primer pairs amplified the expected size products from genomic DNA from <i>aru^S13</i> flies.</p

The olfactory short-memory defect of <i>aru^8–128</i> flies is reverted to normal in flies with a precise excision allele (<i>aru^S8</i>) but reduced in flies with an imprecise excision allele (<i>aru^S13</i>).

<p>Wild-type CS and Berlin flies, as well as flies with a precise excision (<i>aru^S8</i>) and imprecise excision (<i>aru^S13</i>), were trained and tested for olfactory three minute memory. The short-term memory score of flies from wild-type, <i>aru^S8</i> and <i>aru^S13</i>genotypes are presented. A) The memory performance was statistically different in flies with the imprecise excision allele and their corresponding wild-type strain (CS with <i>aru^S8</i> (CS) and <i>aru^S13</i> (CS) F(2,15) = 5.8, p<0.01, * = p<0.05 with a Newman-Keuls <i>post-hoc</i> test of CS and <i>aru^S8</i> (CS) with <i>aru^S13</i> (CS), N = 18). B) Differences were also identified in flies from the wild-type Berlin backgrounds (Berlin with <i>aru^S8</i> (B) and <i>aru^S13</i> (B) F(2,29) = 7.3, p<0.002, * = p<0.05 with a Newman-Keuls <i>post-hoc</i> test of Berlin and <i>aru^S8</i> (B) with <i>aru^S13</i> (B), N = 32). The values are means and error bars represent SEMs.</p

The <i>aru^8–128</i> allele reduces place memory performance.

<p>Wild-type CS and Berlin (B) flies, as well as <i>aru^8–128</i> flies in either the CS or B genetic backgrounds were trained for 20 min and then examined for place memory directly afterward. The mutant memory performance was statistically different from wild-type only in the CS genetic background (CS vs. <i>aru^8–128</i> (CS), ** = p<0.01, N = 215; Berlin vs. <i>aru^8–128</i> (B), p>0.1, N = 643). The values are means and error bars represent SEMs.</p