Effects of ethanol are temporary.

<p>Behavioral recovery of learning ability after ethanol exposure was tested by dividing both the ethanol-exposed and water-exposed groups into two subgroups, one conditioned immediately after treatment and one conditioned three hours later. No significant difference was observed in the conditioning scores of alcohol and water treated groups at the end of three hours in contrast to the immediately-conditioned group (p = 0.8, n = 5).</p

Olfactory response and locomotion are unaffected by ethanol.

<p>(<b>A–C</b>) Response indices are shown for larvae when placed in the middle of an agar dish with ethyl acetate on one side and liquid paraffin on the other. The number of larvae in each odor zone was counted after 3 minutes. <b>A</b>. The Olfactory response shows a mild reduction with 30% ethanol treatment but not with 20% ethanol treatment. <b>B</b>. Larvae had been previously submerged for 20 minutes in either pure water or 20% EtOH. No significant difference was seen. <b>C</b>. Automated tracking. Left: Response index over a three-minute period when larvae are being tracked. Larvae had been previously submerged for 20 minutes in either pure water or 20% EtOH. <b>D</b>. Average speed in the absence of a stimulus is shown for larvae during the three minute tracking period. 20% EtOH did not cause a significant reduction in either locomotion speed or olfaction.</p

Ethanol treatment affects olfactory learning when the heat shock unconditioned stimulus is below the temperature optima.

<p><b>A</b>. Either heat alone or odor alone presentations resulted in the same response index (RI = #Larvae in odor zone/#Larvae total) as sham-treated larvae (p>0.05 for any comparison). <b>B</b>. Response indices for untrained (control) and trained larvae are shown for animals that either received water or 20% EtOH. All larvae were trained to associate the odor with a 41°C heat-shock. The response indices were similar for water-treated and ethanol-treated groups when comparisons were made for similar conditions such as the untrained group or the trained group. <b>C</b>. Learning indices (LI = (RI_control−RI_conditioned)/RI_control) calculated from the data in Panel B. <b>D</b>. Response indices for untrained (control) and trained larvae are shown for animals that either received water or 20% EtOH. All larvae were trained to associate the odor with a 35°C heat-shock. The conditioned response indices are significantly different in the ethanol treated groups (n = 32; p = 0.044). <b>E</b>. Learning indices calculated from the data in Panel D. Ethanol induced a significant reduction in learning.</p

Perdurance of internal ethanol.

<p><b>A</b>. Example standard curve for ethanol gas chromatography. All of the measurements noted in this document fall within the linear range of the gas chromatograph standard curve. <b>B</b>. Chromatographs of ethanol from larvae. Standard responses for known concentrations of ethanol (4.25 mM, 2.13 mM, 1.06 mM, 0.53 mM, and 0.26 mM) diluted in toluene as well as pure toluene are shown. Representative traces from larvae treated for 20 minutes with 20% ethanol (EtOH larvae 1 and 2) or water (control larvae) are also shown. <b>C</b>. The amount of ethanol absorbed by larvae depended on the amount of ethanol in the treatment solution. <b>D</b>. The brief heat shocks (41°C and 35°C) that were used in the conditioning experiments did not reduce internal ethanol below that measured in sham-treated larvae. Animals were treated for 20 minutes with 20% ethanol (Loading Dose) and then taken through the heat shock protocol at 35°C or 41°C as used in conditioning experiments. The loading dose is the same data shown in the panel C 20% bar graph and is repeated for comparison purposes. Sham-treated animals were taken through same protocol except that they did not receive the heat shocks but instead were moved to room temperature (24°C) plates.</p

Experimental design.

<p><b>A</b>. Schematic of the flow of the experiment. <b>B</b>. Schematic of the ethanol/water treatment protocol.</p