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.

Immunostaining

Immunostaining was performed as described (Wu and Luo, 2006). Brains from 1-5 day old adult flies were dissected in PBS and fixed for 20 min in PBS with 4% paraformal­dehyde at room temperature. They were then washed twice briefly in 0.5% PBT (2.5ml Triton-X100 in 497.5ml PBS) and three 20 min washes. Brains were then blocked for 30 min at room temperature in PBT containing 5% normal goat serum and then incubated with primary and secondary antibodies with mild rotation (35 rpm) at 4 ˚C for 1 or 2 days. Primary antibodies were rabbit anti-GFP (1:250; Invitrogen A11122) and rabbit anti-HA (1:250, NEB 3724T). Alexa 488–conjugated goat anti-rabbit (1:250; Invitrogen, A11034) was the secondary antibody. Before and after the secondary antibody incubation, brains were subjected to two quick followed three 20-min washes in 0.5% PBT. Stained brains were mounted on glass slides in Vectashield (Vector Labs H1000) and imaged using a Leica TCS SP5 confocal microscope at 40X magnification (HCX PL APO 40X, 1.3 CS oil immersion objective, Leica). Image stacks were collected at 1024 × 1024 resolution with 1 μm step and processed using Fiji (Schindelin et al., 2012). For quantification in Figure 3G and 3H, rectangular ROIs of approximately 40 X 25 mm for the for γ lobe, or round ROIs with diameter of 15 μm for αβ, α'β' and EB were manually drawn on a single section of a z-stack scan of the fly brain. Corresponding ROIs were also drawn on the superior medial protocerebrum (SMP) as a background control region, and the mean fluorescence was assessed using ImageJ. ROI intensity of the MB lobes and the EB was normalized to that of the respective SMP intensity. An average between left and right brains was used for a single data point. For quantification in Figure 7C and 7D, ROIs are indicated in the figures and ROI intensity was calculated similarly to in Figure 3H.  In Figure 7C, a line was drawn through the fattest part of the tip of the α lobe. The intensity profile of this line was obtained through ImageJ. 30 data points in the middle of such a profile spanning about a 15μm line were extracted for each line profile. The profile was further normalized to the mean value of the first 5 data points (F0) and calculated as (F-F0)/F0. Mean values of these normalized profiles from different brains were plotted (Figure 7C, middle panel). Left and right profiles brains were calculated and are separately displayed. In Figure 7D, relative intensity from different ROIs representing different regions are added together to generate a total intensity measure for the MB.

FRET-based Mg²⁺ concentration measurement in fixed fly brain

1 to 2 day old flies with genotype c739; UAS-MagFRET-1 were housed in vials with 1mM or 80mM [Mg²⁺] food for 4 days before being collected. Fly brains were dissected in PBS and fixed for 20 min in PBS with 4% paraformal­dehyde at room temperature. They were then washed twice briefly in 0.5% PBT (2.5ml Triton-X100 in 497.5ml PBS) and three 10 min washes. Brains were then mounted on glass slides in Vectashield (Vector Labs H1000) and imaged using a wide-field Scientifica Slicescope with a 40x, 0.8 NA water-immersion objective and an Andor Zyla sCMOS camera with Andor Solis software (v4.27). In order to get the FRET ratio which indicates the Mg²⁺ concentration of the αβ neuron, time series were acquired alternatively between the cerulean channel and the citrine channel at 3 Hz with 512 x 512 pixels and 16 bit. The excitation wavelength for both channels is 436nm, while the emission filter for cerulean is 460-500nm and that for citrine is 520-550nm. Series acquisition starts from the cerulean channel and lasts for 5 seconds, then switches to the citrine channel and last for another 5 seconds, and this cycle is repeated for 2 more times. A total of 30 seconds (90 frames) image stack was therefore acquired for each brain. Image stacks were subsequently analyzed using ImageJ and custom-written Matlab scripts. In brief, rectangle ROIs (Figure 1E left panel) were manually drawn on the αβ lobes (one on α lobe and one on β lobe for each hemisphere, and outside the αβ lobes (one for each hemisphere) as background control. Fluorescence intensity from the cerulean channel was calculated by dividing each vertical or horizontal lobe ROI by the background ROI, and averaged between the two hemispheres for each lobe, and averaged over the 15 frames for each cycle. That from the citrine channel was obtained similarly. A FRET ratio was obtained from the above intensities, further averaged among the 3 cycles of acquisition, depicted as one data point in Figure 1E (right panel).

Confocal Mg²⁺ imaging in explant fly brain

Explant brains expressing c739-GAL4 driven UAS-MagIC were placed at the bottom of a 35 mm glass bottom microwell dish (Part No. P35G-1.5-14-C, MatTek corporation), beneath saline buffer solution (103 mM NaCl, 3 mM KCl, 5 mM N-Tris, 10 mM trehalose, 10 mM glucose, 7 mM sucrose, 26 mM NaHCO₃, 1 mM NaH₂PO₄, 1.5 mM CaCl₂, 4 mM MgCl₂, osmolarity 275 mOsm [pH 7.3]) following dissection in calcium-free buffer (Barnstedt et al., 2016). To assay the Mg²⁺ sensitivity of UAS-MagIC as well as the response of UAS-MagIC to other chemicals such as EDTA, EGTA and CaCl₂ (Figure 8B), brains were incubated in the saline buffer solution with 20 μg/mL digitonin for 6 min before subjected to imaging (Koldenkova et al., 2015). To assay the Mg²⁺ fluctuation in response to Forskolin (FSK) application (Figure 8C-I), brains were put in the saline buffer solution without digitonin or incubation. In both situations, saline refers to the buffer (either with or without digitonin) in which the brain is submerged.

Imaging was carried out in a LSM780 confocal microscope (Zeiss) with a 20x air objective using the ZEN 2011 software. The Venus part of MagIC was excited with a 488-nm laser and its emission was collected in the 520 to 560 nm range. mCherry was excited with a 561-nm laser and its emission was collected in the 600 to 640 nm range. Time series were acquired at 0.5 Hz with 512 x 512 pixels and 16 bit. Following 60s of baseline Venus/mCherry measurement, 2 to 20µl of saline or other relevant chemical solution was added via a micropipette to the dish with constant image capture. The effects of applied agents on Venus/mCherry emission were then recorded for 15 to 20 min.

Image stacks were subsequently analyzed using ImageJ and custom-written Python scripts. In brief, rectangle ROIs were manually drawn on the αβ neurons (one for each hemisphere, Figure 8A), and another ROI of same size was drawn in the middle but outside the MBs as background control. Fluorescence intensity from Venus (or mCherry) channel was calculated by subtracting the background ROI from the calyx ROIs, respectively, and averaged between the two hemispheres. This is referred as “Rel. Intensity (a.u.)” in Figure 8D-E. The ration between Venus and mCherry intensity was calculated as “MagIC Ratio” in Figure 8B-C and Figure 8F-G. Alternatively in Figure 8H, the intensity for the 2 channels was calculated separately. In this case, “Rel. Intensity (ΔF/F₀)” refers to the relative fluorescence intensity normalized to the mean intensity from the baseline period F₀, calculated as (F-F₀)/F₀. The relative intensity ΔF/F₀ of Venus was used to calculate the power spectral density (PSD, Figure 8I) through python function psd (under matplotlib.pyplot), which adopted a Welch’s average periodogram method (Bendat et al., 1989).