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.

METHOD DETAILS

Behavioral experiments

1-7 day old mixed sex flies were used for behavioral T-maze experiments. Odors were 4-methylcyclohexanol (MCH) and 3-octanol (OCT), diluted ~1:10³ (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 Mg²⁺ feeding,1 to 2 day old flies were housed in vials with Mg²⁺ supplemented food for 1-5 days before being starved for appetitive training and testing, as described above. To make 80 mM [Mg²⁺] food, 40 ml of 1 M MgCl₂ solution was added to 460 ml of normal liquid fly food.1 mM [Mg²⁺] food was made by diluting 0.5 ml 1 M MgCl₂ in 39.5 ml MilliQ water and adding it to 460 ml liquid food. Food was aliquoted and cooled to solidify. MgSO₄ and CaCl₂ 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.

QUANTIFICATION AND STATISTICAL ANALYSES

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<0.05.