DOI: 10.5061/dryad.q2bvq83hs
title: Behavior data from T-maze assay

description: Dietary magnesium (Mg2+) supplementation can enhance memory in young and
 aged rats. Memory-enhancing capacity was largely ascribed to increases in
 hippocampal synaptic density and elevated expression of the NR2B subunit
 of the NMDA-type glutamate receptor. Here we show that Mg2+ feeding also
 enhances long-term memory in Drosophila. Normal and Mg2+ enhanced fly
 memory appears independent of NMDA receptors in the mushroom body and
 instead requires expression of a conserved CNNM-type Mg2+-efflux
 transporter encoded by the unextended (uex) gene. UEX contains a putative
 cyclic nucleotide-binding homology domain and its mutation separates a
 vital role for uex from a function in memory. Moreover, UEX localization
 in mushroom body Kenyon Cells is altered in memory defective flies
 harboring mutations in cAMP-related genes. Functional imaging suggests
 that UEX-dependent efflux is required for slow rhythmic maintenance of
 Kenyon Cell Mg2+. We propose that regulated neuronal Mg2+ efflux is
 critical for normal and Mg2+ enhanced memory.
description: <b>METHOD DETAILS</b>
 <b>Behavioral experiments</b>
 1-7 day old mixed
 sex flies were used for behavioral T-maze experiments. Odors were
 4-methylcyclohexanol (MCH) and 3-octanol (OCT), diluted
 ~1:10<sup>3</sup> (specifically, 9μl MCH, or 7μl OCT in 8ml
 mineral oil). All experiments were performed at 23˚C and 55%-65% relative
 humidity.    Appetitive immediate and later
 memory experiments were performed essentially as described (Krashes and Waddell,
 2008; Perisse et al.,
 2013). Batches of 100-120 flies were starved for
 21-23 h before training in 35 ml starvation vials containing ~2ml 1% agar
 (as a water source) and a 2 cm x 4 cm filter paper. Sugar papers (5 cm x
 7.5 cm) for training were prepared by soaking with 4 ml of 2 M sucrose and
 drying overnight. Water papers of same size were soaked with water and
 left overnight. For appetitive training, flies were transferred from a
 starvation tube to a training tube with a dry ‘water’ paper, and
 immediately attached to the training arm of the T-maze and exposed to the
 CS− odor for 2 min, followed by 30 s of clean air. Flies were then
 transferred to another training tube with dry sugar paper, attached to the
 T-maze and exposed to the CS+ odor for 2 min. Immediate memory was tested
 by transporting flies to the T-choice point and allowing them 2 min to
 choose between the two odor streams. To assay 24 h memory, flies were
 removed from the training tube and transferred to standard cornmeal food
 vials for 1 h, then transferred back into starvation vials for 23 h until
 testing. Performance Index was calculated as the number of flies in the
 CS+ arm minus the number in the CS- arm, divided by the total number of
 flies. MCH and OCT, were alternately used as CS+ or CS- and a single
 sample, or n, represents the average performance score from two
 reciprocally trained groups.    For behavior tests after
 Mg<sup>2+</sup> feeding,1 to 2 day old flies were housed in
 vials with Mg<sup>2+</sup> supplemented food for 1-5 days
 before being starved for appetitive training and testing, as described
 above. To make 80 mM [Mg<sup>2+</sup>] food, 40 ml of 1 M
 MgCl<sub>2 </sub>solution was added to 460 ml of normal liquid
 fly food.1 mM [Mg<sup>2+</sup>] food was made by diluting 0.5
 ml 1 M MgCl<sub>2</sub> in 39.5 ml MilliQ water and adding it
 to 460 ml liquid food. Food was aliquoted and cooled to solidify.
 MgSO<sub>4</sub> and CaCl<sub>2</sub> supplemented
 food was prepared the same way.
    Aversive immediate and 24 h memory experiments
 were conducted as previously described (Hirano et al.,
 2013; Perisse et al.,
 2016; Tully and Quinn,
 1985). Groups of 100-120 flies were trained with
 either one cycle of aversive training, or 5 cycles spaced by 15 min
 inter-trial intervals (spaced training). For aversive immediate memory,
 flies were tested after one-cycle training. Aversive 24 h memory was
 tested using two different protocols. In the fasting-facilitated protocol,
 flies were starved for 16 h before one-cycle training (Hirano et al., 2013). For
 spaced training flies were not starved before training. Flies were fed on
 normal fly food for 24 h after fasting-facilitated and spaced training,
 before being tested for memory performance. During each aversive training
 cycle, flies were exposed to a first odor (CS+) for 1 min paired with
 twelve 90 V electric shocks at 5 s intervals. Following 45 s of clean air,
 a second odor (CS−) was presented for 1 min without shock. Performance
 Index was calculated as the number of flies in the CS- arm minus the
 number in the CS+ arm, divided by the total number of flies. MCH and OCT,
 were alternately used as CS+ or CS- and a single sample, or n, represents
 the average performance score from two reciprocally trained
 groups.    Sensory acuity tests (Table S1)
 were performed as described (Keene et al.,
 2006; Keene et al.,
 2004; Schwaerzel et al.,
 2003) with modifications. To test olfactory acuity,
 untrained flies were given 2 min to choose between a diluted odor as used
 in conditioning and air bubbled through mineral oil in the T maze. An
 Avoidance Index was calculated as the number of flies in the air arm minus
 the number in the odor arm, divided by the total number of flies. Electric
 shock avoidance was performed and calculated similarly. Untrained flies
 chose for 1 min between two tubes containing electric grids, but only one
 was connected to the power source. An avoidance index was calculated as
 the number of flies in the non-electrified arm minus the number in the
 electrified arm, divided by the total number of flies. To assess sugar
 acuity, starved flies were given 2 min to choose between an arm of the
 T-maze containing a dried sugar paper and the other containing a dried
 ‘water’ filter paper. Both papers were prepared as in the appetitive
 memory assays. A Preference Index was calculated as the number of flies in
 the sugar arm minus that in the other arm, divided by the total number of
 flies. We found that keeping the light on in the behavioral room and
 having air flow running through the testing tubes greatly enhanced the
 Preference Index in wild-type flies and therefore applied those conditions
 for all sugar preference testing.
 <b>QUANTIFICATION AND STATISTICAL
 ANALYSES</b> Behavior data were
 analyzed using Excel and Prism 6. Unpaired two-tailed t-tests were used
 for comparing two groups, and one-way ANOVA followed by a Tukey’s post-hoc
 test was used for comparing multiple groups. Definition of statistical
 significance is set at p&lt;0.05.