The arouser EPS8L3 Gene Is Critical for Normal Memory in Drosophila

The genetic mechanisms that influence memory formation and sensitivity to the effects of ethanol on behavior in Drosophila have some common elements. So far, these have centered on the cAMP/PKA signaling pathway, synapsin and fas2-dependent processes, pumilio-dependent regulators of translation, and a few other genes. However, there are several genes that are important for one or the other behaviors, suggesting that there is an incomplete overlap in the mechanisms that support memory and ethanol sensitive behaviors. The basis for this overlap is far from understood. We therefore examined memory in arouser (aru) mutant flies, which have recently been identified as having ethanol sensitivity deficits. The aru mutant flies showed memory deficits in both short-term place memory and olfactory memory tests. Flies with a revertant aru allele had wild-type levels of memory performance, arguing that the aru gene, encoding an EPS8L3 product, has a role in Drosophila memory formation. Furthermore, and interestingly, flies with the aru8–128 insertion allele had deficits in only one of two genetic backgrounds in place and olfactory memory tests. Flies with an aru imprecise excision allele had deficits in tests of olfactory memory. Quantitative measurements of aru EPS8L3 mRNA expression levels correlate decreased expression with deficits in olfactory memory while over expression is correlated with place memory deficits. Thus, mutations of the aru EPS8L3 gene interact with the alleles of a particular genetic background to regulate arouser expression and reveals a role of this gene in memory

The Radish Gene Reveals a Memory Component with Variable Temporal Properties

Memory phases, dependent on different neural and molecular mechanisms, strongly influence memory performance. Our understanding, however, of how memory phases interact is far from complete. In Drosophila, aversive olfactory learning is thought to progress from short-term through long-term memory phases. Another memory phase termed anesthesia resistant memory, dependent on the radish gene, influences memory hours after aversive olfactory learning. How does the radish-dependent phase influence memory performance in different tasks? It is found that the radish memory component does not scale with the stability of several memory traces, indicating a specific recruitment of this component to influence different memories, even within minutes of learning

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

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

Genetic Dissociation of Ethanol Sensitivity and Memory Formation in Drosophila melanogaster

The ad hoc genetic correlation between ethanol sensitivity and learning mechanisms in Drosophila could overemphasize a common process supporting both behaviors. To challenge directly the hypothesis that these mechanisms are singular, we examined the learning phenotypes of 10 new strains. Five of these have increased ethanol sensitivity, and the other 5 do not. We tested place and olfactory memory in each of these lines and found two new learning mutations. In one case, altering the tribbles gene, flies have a significantly reduced place memory, elevated olfactory memory, and normal ethanol response. In the second case, mutation of a gene we name ethanol sensitive with low memory (elm), place memory was not altered, olfactory memory was sharply reduced, and sensitivity to ethanol was increased. In sum, however, we found no overall correlation between ethanol sensitivity and place memory in the 10 lines tested. Furthermore, there was a weak but nonsignificant correlation between ethanol sensitivity and olfactory learning. Thus, mutations that alter learning and sensitivity to ethanol can occur independently of each other and this implies that the set of genes important for both ethanol sensitivity and learning is likely a subset of the genes important for either process

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