DOI: 10.5061/dryad.80gb5mkpx
title: Data from: Magnesium efflux from Drosophila Kenyon Cells is critical for normal and diet-enhanced long-term memory

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>Immunostaining </b>
 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-<i>GFP</i> (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.
 <b>FRET-based Mg<sup>2+</sup> concentration
 measurement in fixed fly brain</b> 1 to 2 day old
 flies with genotype c739; UAS-<i>MagFRET-1</i> were housed in
 vials with 1mM or 80mM [Mg<sup>2+</sup>] 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 Mg<sup>2+</sup> 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). <b>Confocal
 Mg<sup>2+</sup> imaging in explant fly
 brain</b> Explant brains expressing
 <i>c739</i>-GAL4 driven UAS-<i>MagIC</i> 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 NaHCO<sub>3</sub>, 1 mM
 NaH<sub>2</sub>PO<sub>4</sub>, 1.5 mM
 CaCl<sub>2</sub>, 4 mM MgCl<sub>2</sub>,
 osmolarity 275 mOsm [pH 7.3]) following dissection in calcium-free buffer
 (Barnstedt et al., 2016). To assay the
 Mg<sup>2+</sup> sensitivity of UAS-<i>MagIC</i> as
 well as the response of UAS-<i>MagIC</i> to other chemicals
 such as EDTA, EGTA and CaCl<sub>2</sub> (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 Mg<sup>2+</sup> 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/F<sub>0</sub>)” refers to the relative fluorescence
 intensity normalized to the mean intensity from the baseline period
 F<sub>0</sub>, calculated as
 (F-F<sub>0</sub>)/F<sub>0</sub>. The relative
 intensity ΔF/F<sub>0</sub> 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).