Learning and memory are fundamental brain functions affected by dietary and environmental factors. Here, we show that increasing brain magnesium using a newly developed magnesium compound (magnesium-L-threonate, MgT) leads to the enhancement of learning abilities, working memory, and short- and long-term memory in rats. The pattern completion ability was also improved in aged rats. MgT-treated rats had higher density of synaptophysin-/synaptobrevin-positive puncta in DG and CA1 subregions of hippocampus that were correlated with memory improvement. Functionally, magnesium increased the number of functional presynaptic release sites, while it reduced their release probability. The resultant synaptic reconfiguration enabled selective enhancement of synaptic transmission for burst inputs. Coupled with concurrent upregulation of NR2B-containing NMDA receptors and its downstream signaling, synaptic plasticity induced by correlated inputs was enhanced. Our findings suggest that an increase in brain magnesium enhances both short-term synaptic facilitation and long-term potentiation and improves learning and memory functions.

Here, we tested in vivo whether an increase in brain Mg 2+ by MgT could positively influence learning and memory functions in rats at different ages. We found that elevation of brain Mg 2+ led to significant enhancement of spatial and associative memory in both young and aged rats. To understand the molecular mechanisms underlying MgT-induced memory enhancement, we studied the changes in functional and structural properties of synapses in rats treated with MgT. The NR2B-containing NMDAR was increased, resulting in enhancement of NMDAR signaling and synaptic plasticity. At the cellular level, the number of presynaptic boutons was also increased. We propose that these positive changes in synaptic functions are the principal cellular mechanisms underlying the enhancement of learning and memory associated with the increase in brain Mg 2+ content. Thus, elevating brain Mg 2+ content via increasing magnesium intake might be a useful strategy to enhance cognitive abilities and prevent the age-dependent memory decline.

Mgconcentration is higher in the cerebrospinal fluid than in plasma. This concentration gradient is maintained by active transport process, which appears to regulate and limit the amount of Mgthat can be loaded into the brain. In fact, increasing plasma [Mg] by 3-fold via intravenous infusion of MgSOfor 5 days fails to elevate brain Mgcontent in rats (). In human, dramatic increase (100%–300%) in blood [Mg] via intravenous infusion of MgSOcorresponds to elevation in cerebrospinal fluid [Mg] only by 10%–19% (). Therefore, boosting brain Mgvia chronic oral magnesium supplement, the necessary condition for testing the influence of elevating brain Mgon memory function, is even more challenging. Therefore, we developed a new, highly bioavailable Mgcompound (magnesium-L-threonate, MgT; for chemical structure, see Figure S1 available online), that could significantly increase Mgin the brain via dietary supplementation.

Diet, in conjunction with environmental factors, has a crucial role in shaping brain cognitive capacity (for review, see). Therefore, searching for dietary components that can increase the number and plasticity of synapses might yield new strategies to enhance learning and memory functions. Magnesium (Mg), the fourth most abundant ion in body and a cofactor for more than 300 enzymes, is essential for the proper functioning of many tissues and organs, including the cardiovascular, neuromuscular, and nervous systems. In brain, one major action of Mgis modulating the voltage-dependent block of NMDA receptors (NMDAR), controlling their opening during coincidence detection that is critical for synaptic plasticity (). Our previous study suggests that Mgis a positive regulator of synaptic plasticity; increasing Mgconcentration in the extracellular fluid ([Mg) within the physiological range leads to permanent enhancement of synaptic plasticity in networks of cultured hippocampal neurons in vitro (). Therefore, it is tempting to investigate whether the increase in brain Mgcontent will enhance cognitive function in vivo.

The pattern and strength of synaptic connections are widely believed to code memory traces. Long-term potentiation of synaptic strength (LTP) is correlated with behaviorally relevant memory function: reductions in LTP cause memory impairments (), whereas increases in LTP are associated with enhancement of learning and memory (for reviews, see). However, the ability to store new information in neural networks depends on the degree of plasticity of synaptic connections, as well as the number of available connections. Therefore, number of synapses should be critical for learning and memory too. Indeed, loss of synapses is correlated with age-dependent memory decline in rats (for review, see), while hormones and neuropeptides, such as estrogen (), neurotophins (), insulin/IGF (), and ghrelin (), increase synaptic density and improve memory.

Altogether, these data suggest that increasing the density of synaptophysin-/synaptobrevin-containing presynaptic boutons might be a key structural change underlying the MgT-induced memory enhancement.

Next, we studied whether the density of Syn-(+) puncta is correlated to the memory score per individual rat. The short-term memory of aged rats (different from those used for T-maze experiments) was evaluated using the novel object recognition test with two objects. After behavioral assessment, rats were sacrificed and analyzed for Syn- / SNB1-(+) puncta density. The short-term memory among untreated aged rats varied significantly as revealed by their recognition index ( Figure 8 D), a phenomenon that had been reported previously (). MgT treatment enhanced memory and reduced the individual variation among treated aged rats ( Figure 8 D). Interestingly, the density of Syn-(+) puncta in DG correlated with the recognition index per individual rat of control group ( Figure 8 E, Pearson test, r= 0.50, p = 0.02). The density of Syn-(+) puncta in CA1 of control group also correlated with recognition index, though the correlation was slightly weaker than in DG ( Figure 7 F, r= 0.41, p = 0.04). The density of SNB1-(+) puncta in control group also correlated to short-term memory score (see Figures S6 B and S6C). No significant correlation between density of presynaptic puncta and memory score was found in MgT-treated group. This lack of correlation is expected since MgT treatment reduced interindividual variation of memory score within the group ( Figure 8 D).

The use of animal models to study the effects of aging on cognition.

In the T-maze task, memory performance reached maximum 12 days after the onset of MgT treatment and dropped to baseline 12 days after the offset of MgT treatment in aged rats. If a change in the density of Syn-(+) puncta contributes to memory enhancement, both parameters are expected to change correspondingly. Indeed, when aged rats (22 month) were treated with MgT for 2 weeks, the density of Syn-(+) puncta increased in DG (∼44%, one-way ANOVA, p < 0.01, Figures 8 A and 8B) and in CA1 (∼30%, p < 0.0001, Figure 8 C) hippocampal areas. On the other hand, the density of Syn-(+) puncta in MgT-treated rats (2 weeks of treatment) returned to the control level 2 weeks after the end of MgT supplementation ( Figures 8 A, 8B, and 8C). Thus, the time course of change in the density of Syn-(+) puncta following on/off MgT treatment matched the time course of alterations in memory score.

(F) The density of Syn-(+) puncta in the CA1 also correlated with the recognition index of individual control rats (Pearson test). MgT-treated rats' data were not included in the correlation analysis. Data are presented as mean ± SEM. See also Figure S6

(E) The density of Syn-(+) puncta in the DG correlated with the recognition index of individual control rats. MgT-treated rats' data were not included in the correlation analysis, although data are displayed on the figure. No significant correlation was found in MgT-treated group.

(D) The short-term memory varied among individual aged rats as revealed by the recognition index of novel object recognition test. Rats treated with MgT spent more time exploring the novel object (n = 6) than controls (n = 10).

(B and C) Quantitative analysis of the density of Syn-(+) puncta in DG and CA1 of control (n = 10), MgT ON (n = 5) and MgT OFF (n = 5) rats (one-way ANOVA, DG, F 2,17 = 6.88, p = 0.0065; in CA1, F 2,17 = 11.45, p = 0.0007). ANOVA was followed by Bonferroni's post hoc test ( ∗∗ p < 0.01; ∗∗∗ p < 0.0001).

(A) Temporal relationship between “on/off” of MgT treatment and the density of Syn-(+) puncta. Syn-(+) puncta increased 2 weeks after MgT treatment (MgT ON) and returned to control level 2 weeks after stopping MgT treatment (MgT OFF). Scale bar, 10 μm.

The above data demonstrate that increase in brain Mg 2+ leads to increase in functional connectivity, synaptic plasticity, and enhancement of learning and memory. To further assess the impact of increase in density of presynaptic boutons to memory enhancement, we performed the following two sets of experiments.

Therefore, elevation of [Mgcan trigger increase in the number of functional presynaptic release sites with lower release probability. This synaptic reconfiguration maintains homeostasis of AMPA-mediated synaptic transmission for a single AP ( Figures 7 A and 7B) yet enhances transmission for bursting inputs ( Figure 7 E), similar to the observations in slices from MgT-treated rats ( Figure 5 B).

Notably, EPSCamplitude was the same under physiological [Ca(1.2 mM, Figure 7 B) in both groups of slices, meaning that Pr × n× q= Pr × n× q. Thus, the Pr of synaptic boutons in 1.2 Mg slices must be lower to counterbalance the increase in the number of release sites per connection. To test this possibility, the EPSCcoefficient of variation was used to estimate the Pr (Pr ∼ CV, []). CVwas 2.0-fold lower in 1.2 Mg slices (n = 10 cells, p < 0.05, Figure 7 D), suggesting that these synapses do have lower Pr under physiological [Ca. Application of the coefficient of variation to determine the quantal parameters involves several assumptions, such as nearly all variances are presynaptic, which cannot be verified directly (). We applied another independent method to estimate Pr of synapses by calculating the degree of short-term facilitation, which primarily depends on presynaptic factors. Low Pr synapses tend to have higher facilitation index (). Indeed, the synaptic facilitation of 1.2 Mg slices during bursts was 2.3-fold higher than that of 0.8 Mg slices (n = 10 cells, p < 0.001, Figures 7 and 7E). Therefore, Pr of synaptic boutons is likely to be lower in 1.2 Mg than in 0.8 Mg slices. To further verify the validity of both methods, we compared CVand the facilitation in 0.8 Mg and 1.2 Mg slices under high [Ca. Pr of synapses under high [Cais expected to be maximized. Under this condition, the differences in CVand the facilitation between 0.8 and 1.2 Mg slices should disappear. Consistent with this prediction, CVand facilitation were the same between both groups of slices under high [Ca(5 mM, Figures 7 D and 7E).

According to Katz's quantal hypothesis, the efficacy of a synaptic connection is determined by the product of the probability of release (Pr), the number of release sites (n), and the size of the postsynaptic response to a quantum of transmitter (q) (). Although the number of release sites can be estimated using the conventional quantal analysis, this approach requires a good voltage clamp of dendritic synapses, which is difficult to obtain for neurons in CNS (). Since presynaptic release is very sensitive to Ca, even without adequate voltage clamp, any difference in EPSC amplitudes under various [Cais most likely due to difference in Pr and/or n). Therefore, we inferred the number of functional release sites by studying synaptic transmission under various [Cashows recordings of the evoked EPSCfor a single AP under various [Cain 0.8 Mg versus 1.2 Mg slices. The amplitudes of EPSCwere almost identical under physiological concentrations of Ca(1.2 mM; Figure 7 gray, and Figure 7 B), suggesting that incubation of slices under higher [Mgdid not influence basal synaptic strength. When [Cawas elevated in test recording solution to 5 mM in order to maximize the Pr, EPSCamplitudes in both slices increased correspondingly; however, the degree of increase was 2.4-fold higher in 1.2 Mg than 0.8 Mg slices (n = 10 cells, t test, p < 0.0001, Figure 7 black and Figure 7 B). There was no difference in quantal size between 0.8 Mg and 1.2 Mg slices (n = 10 cells, Figure 7 C). Therefore, the increase in EPSCamplitudes at higher [Cain 1.2 Mg slices is likely due to higher number of functional release sites per axon.

(E) Synaptic facilitation, defined by the ratio of EPSC burst /EPSC single , was higher in 1.2-Mg slices at physiological [Ca 2+ ] o (n = 10, p < 0.001), but similar under high [Ca 2+ ] o . EPSC single is the amplitude of EPSC AMPA for single AP input, and EPSC burst is the mean amplitude of EPSC AMPA per single AP within the burst. Unpaired t test, ∗ p < 0.05, ∗∗∗ p < 0.001. Data are presented as mean ± SEM.

(D) CV −2 , calculated as variance 2 /mean 2 , was significantly higher (p < 0.05, n = 10) in 1.2 Mg slices at physiological [Ca 2+ ] o but similar at high [Ca 2+ ] o .

(B) Mean EPSC AMPA under low and high [Ca 2+ ] o (n = 10). The EPSC AMPA was similar under physiological [Ca 2+ ] o , but significantly higher (p < 0.0001) in 1.2-Mg slices under high [Ca 2+ ] o .

(A 1 and A 2 ) Mean EPSC AMPA recorded at −70 mV (average of 30 sweeps) and evoked by minimal stimulation at low frequency (upper traces, 0.1 Hz) and by bursting input (bottom traces; bursts of 5 APs, ISI = 20 ms, interburst interval 0.1 Hz) under low (1.2 mM, gray trace) and high (5 mM, black) [Ca 2+ ] o in 0.8 and 1.2 Mg slices.

In cultured hippocampal neurons, plasticity () and density (I.S., H. Zhou, and G.L., unpublished data) of functional presynaptic boutons increased 4 hr following elevation of [Mgfrom 0.8 mM to 1.2 mM. Therefore, freshly cut hippocampal slices (from 2-month-old rats) were incubated for 5 hr in ACSF containing different [Mg(0.8 and 1.2 mM, referred to as 0.8 and 1.2 Mg slices). Irrespective of incubation conditions, the baseline recording solution contained constant, 1.2 mM, [Mgbefore switching to test solutions. Whole-cell patch-clamp recordings were made on pyramidal CA1 neurons, and EPSCs were evoked by minimal stimulation of Shaffer collateral axons in the presence of an NMDAR blocker (50 μM AP-5).

Does Mg 2+ -induced increase in Syn- / SNB1-(+) puncta lead to increase in the number of functional release sites, and, subsequently, enhance synaptic transmission? To address this question, we studied the properties of synaptic transmission in hippocampal slices treated with different [Mg 2+ ] o .

It is worth noting that presynaptic boutons with very low concentration of synaptophysin/synaptobrevin will not be counted by our present approach, therefore, we might have underestimated the actual density of presynaptic Syn-/SNB1-(+) boutons. Since synaptobrevin is essential for synaptic vesicle fusion (), a presynaptic bouton with low synaptobrevin concentration may not be functional. Under these considerations, increase in the density of Syn- and/or SNB1-(+) puncta could be due to increase in either density of presynaptic boutons or amount of these proteins in the existing boutons.

To further characterize the potential cellular mechanisms that underlie MgT-induced memory enhancement, we investigated the effect of 1 month of MgT treatment on density of presynaptic boutons in aged rats (22 months). Rats were anesthetized and perfused, and the number of synaptophysin-positive (Syn-[+]) puncta in the stratum moleculare was measured. Figure 6 A shows Syn-(+) puncta in the DG of hippocampus. Indeed, the density of Syn-(+) puncta in MgT-treated rats was significantly higher than controls (∼67%, t test, p < 0.01, Figure 6 B). We also estimated the density of Syn-(+) puncta in CA1 and found that it was ∼25% higher in MgT-treated aged rats than aged controls (p < 0.05, Figure 6 C). A similar pattern of changes was observed with another presynaptic protein synaptobrevin (SNB1) ( Figure 6 D). MgT increased density of SNB1-(+) puncta by ∼43% in the DG and ∼34% in the CA1 subregions of hippocampus (p < 0.05, Figures 6 E and 6F). The density of Syn-(+) and SNB1-(+) puncta have been correlated per individual rat (Pearson test, r= 0.58, p = 0.0006, Figure S6 A). Thus, MgT treatment increased the density of presynaptic boutons containing vesicle proteins critical for transmitter release in DG and CA1 of aged rats.

(E and F) MgT treatment increased the density of SNB1-(+) puncta in the DG (E, p < 0.05) and CA1 (F, p < 0.05). The density was estimated as the number of immunostained puncta per 1000 μm 2 . Scale bar, 10 μm. Unpaired t test, ∗ p < 0.05, ∗∗ p < 0.01.

(B and C) The density of Syn-(+) puncta in the DG and CA1 of control (n = 10) and MgT-treated (n = 6) aged rats. MgT treatment increased the number of Syn-(+) puncta in the DG (B, p < 0.01) and CA1 (C, p < 0.05).

Several studies indicate that synaptic connections in hippocampus decline during aging, with the degree of loss of synapses correlating with the impairment of memory functions (). The reduction seems to be hippocampal subregional specific. For example, the stratum moleculare of the dentate gyrus (DG) is the most vulnerable brain region for age-related synaptic loss (). The loss of synaptic connections in the stratum radiatum of CA1 subregion is less than DG and remains controversial ().

Therefore, synapses in MgT-treated rats exhibited higher activation/expression of plasticity/memory-related proteins and enhancement of both short-term and long-term synaptic potentiation.

Long-term changes in synaptic strength are hypothesized to form the cellular basis of information storage and memory (). A synapse with increased amounts of NR2B-containing NMDAR () and strong synaptic facilitation () is expected to have higher magnitude of LTP. EPSCin CA1 neurons was recorded while stimulating Shaffer collaterals at low frequency (0.03 Hz). After stable recording was achieved (10 min), a spike-timing-dependent plasticity induction protocol () was applied. This protocol produced a persistent LTP in slices from both control and MgT-treated aged rats (control: n = 10 cells, p < 0.001 versus baseline responses before the pairing training; MgT: n = 12 cells, p < 0.01, Figure 5 C). However, the magnitude of LTP in slices from MgT-treated rats was higher than controls (∼52%, p < 0.0001, Figure 5 D). In slices that were not subjected to the pairing protocol, synaptic responses were not significantly altered over the entire recording period (last 5 min mean = 93.5 ± 8.9% of first 5 min baseline response, n = 5 cells).

Next, we tested whether MgT treatment affects synaptic facilitation of hippocampal excitatory synapses. The input-output relationship in CA3-CA1 synaptic connections in hippocampal slices of control and MgT-treated aging rats was evaluated. Two types of input patterns—single action potentials (APs) and bursts (each burst contained 5 APs, ISI = 10 ms)—were applied through minimal stimulation of Shaffer collateral axons. Output was measured by whole-cell patch-clamp recordings in pyramidal CA1 neurons of AMPA-mediated excitatory postsynaptic current (EPSC). MgT treatment did not affect EPSCamplitude for single APs but significantly enhanced EPSCamplitude for bursts (control: n = 6 cells; MgT: n = 7 cells, p < 0.01, Figure 5 B).

To test the effects of enhancing NMDAR signaling on synaptic plasticity, we compared synaptic transmission and plasticity in control and MgT-treated rats. First, we determined if the increase in NR2B subunit expression was associated with changes in NMDAR-mediated synaptic transmission, and we recorded the EPSCbetween CA3-CA1 synaptic connections (Shaffer collaterals) in hippocampal slices using whole-cell patch-clamp recordings (V= −50 mV) in visually identified CA1 pyramidal neurons while stimulating Shaffer collaterals at low frequency (0.03 Hz). We measured the sensitivity of EPSCto ifenprodil, a selective antagonist of NR2B subunits (). Similar to our results in cultured hippocampal synapses (), the sensitivity of EPSCto ifenprodil (3 μM) significantly increased from 8.8 ± 3.1% to 24.1 ± 3.6% in slices from MgT-treated rats (n = 5 cells, p < 0.01, Figure 5 A). Therefore, the detected increase of NR2B expression is associated with an increase in the ratio of NR2B/NR2A in synapses.

(D) The magnitude of long-term potentiation (average over last 5 min) following “pairing training” was significantly higher in MgT-treated group (p < 0.001). Unpaired t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SEM.

(C) Long-term potentiation induced in pyramidal neurons in hippocampal slices of control rats (n = 10, left panel) by the pairing training protocol (indicated by an arrow). The insets show averages of six EPSC AMPAs 5 min before and 30 min after LTP induction. The dashed line indicates the mean basal synaptic responses. Right panel: LTP induced in hippocampal slices of MgT-treated rats (n = 12).

(B) Representative averaged (30 sweeps) traces of EPSC AMPA (−70 mV, 50 μM AP-5) evoked by two patterns of stimulation: single APs (black line, 0.1 Hz) and bursts (gray line, each burst contains 5 APs, ISI = 10 ms, interburst interval 10 s) in control and MgT-treated rats. Right panel: averaged EPSC AMPA amplitudes of control (n = 6) and MgT-treated (n = 7) rats. The averaged amplitude of EPSC AMPA per AP for bursts was significantly higher in MgT-treated rats (p < 0.01).

(A) Representative averaged (30 sweeps) traces of EPSC NMDA with/without blocking of the NR2B-containing NMDAR by ifenprodil (3 μM). In controls (n = 5), the EPSC NMDA amplitude slightly reduced by ifenprodil, while in MgT-treated rats (n = 5), the reduction of amplitude was significantly higher (p < 0.01, right panel).

We checked these predictions in rats treated with MgT. First, the expression levels of NMDAR subunits in control and MgT-treated rats were compared. Chronic MgT treatment selectively increased the expression of NR2B subunit (∼60% of control, t test, p < 0.001) in hippocampus homogenate, while the expression of other subunits of NMDAR (NR2A and NR1) was unchanged ( Figure 4 C). These data are consistent with our previous observations that increase in [Mgcan trigger upregulation of NR2B-containing NMDAR (). Next, we assessed whether the upregulation of NR2B-containing NMDAR leads to increase in activation of NMDAR-dependent signaling by examining the activation of α-CaMKII and CREB following NORT memory task (described above). After LTM test (24 hr retention interval), rats were decapitated and hippocampi were dissected. MgT treatment did not change the expression level of CaMKII and CREB but increased their activation. As a result, the ratio of phosphorylated-CaMKII/total CaMKII was increased (92%, p < 0.01), as was the ratio of phosphorylated-CREB/total CREB (57%, p < 0.01, Figure 4 D). Therefore, NMDAR-dependent signaling is enhanced in MgT-treated rats. To further confirm the beneficial effects of increase in NMDAR signaling, we quantified the expression level of the neurotrophic factor BDNF, a protein regulated by level of CREB activation. BDNF protein expression was significantly higher in MgT-treated rats (36%, p < 0.05, Figure 4 E).

To address these issues, we studied the NMDAR currents under different [Mg. Using iontophoretic application of glutamate to a putative postsynaptic site exactly as described before (), we isolated the postsynaptic Ifor biophysical studies in vitro. Figure 4 A shows the average Ifrom synapses grown under 0.8 mM [Mg(black line, n = 7 cells). When [Mgwas elevated to 1.2 mM acutely, amplitude of Inear resting membrane potential was reduced by ∼50%, suggesting that the amplitude of Inear resting membrane potential is very sensitive to small increase in [Mg. On the other hand, the size of Iunder positive membrane potentials remained the same (blue dashed line, n = 7 cells). Thus, at higher [Mg, strong depolarization is still capable of expelling Mgfrom mouth of NMDAR and removing Mgblock completely. Interestingly, when [Mgwere elevated chronically (cultures were grown and recorded under 1.2 mM [Mg, red line, n = 7 cells), the amplitudes of the Irecorded near resting membrane potential were almost identical with Ifrom synapses grown under 0.8 mM [Mg, while the amplitudes of Iat positive membrane potentials were significantly larger. This phenomenon can be visualized more directly in Figure 4 B, where the percentage of Ichanges in acute versus chronic elevation of [Mgis plotted as a function of membrane potential. These data suggest that reduction in Inear resting membrane potential triggers a compensatory upregulation of postsynaptic NMDAR (), which restores Ito its original level ( Figures 4 A and 4B, red line). However, the removal of the Mgblock during strong depolarization (correlated inputs) exposes these additional NMDAR, resulting in a selective increase in NMDAR activity during strong depolarization ( Figures 4 A and 4B, red line). Therefore, the ultimate effects of elevating [Mgwould be the upregulation of NMDAR and the enhancement of NMDAR-dependent signaling associated with correlated synaptic activity.

(E) Quantitative analysis of BDNF protein level in the hippocampus using ELISA kit. MgT significantly increased BDNF expression (p < 0.05, n = 10). Unpaired t test, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SEM.

(D) Same as (C), but of expression/phosphorylation of NMDAR downstream signaling molecules. MgT significantly increased phosphorylation of CamKII (p < 0.01, n = 7) and CREB (p < 0.01, n = 8) without altering the expression level of both proteins. Data are presented as percentage of change relative to control. β-actin was used as loading control.

(C) Quantitative analysis of expression of NMDAR subunits using western blots in the hippocampus of control and MgT-treated rats. MgT significantly increased NR2B subunit (p < 0.0001, n = 8), without altering NR2A and NR1 subunits.

(A) Voltage dependency of I NMDA evoked by glutamate iontophoresis at putative synapses in 0.8 Mg 2+ cultures (black line, n = 7), following acute (<1 hr, blue dashed line, n = 7) and chronic (red line, n = 7) elevation of [Mg 2+ ] o to 1.2 mM.

The above data indicate that elevating brain Mg has positive influence on learning and memory function. We have performed the following experiments to explore the possible molecular mechanisms underlying this memory enhancement. We focused on NMDAR-dependent signaling because its activation is critical for synaptic plasticity and memory (for review, see) and increase in NMDAR in synapses can enhance learning and memory (). Mg, an important regulator of NMDAR channel opening (), can have strong influence on NMDAR-dependent signaling. Thus, enhancement of learning and memory with elevating brain Mgmight be at least, in part, caused by alteration of NMDAR-dependent signaling. However, as NMDAR channel opening can be blocked by [Mg, increase in brain Mgwould increase NMDAR channel blockage, which might lead to downregulation of NMDAR-dependent signaling, resulting in impairment of learning and memory.

A crucial cognitive function requiring memory is pattern completion, i.e., the ability to retrieve memories based on incomplete information, a capability that declines profoundly during aging (). To test this capability, we compared the dependence of spatial memory recall on the integrity of extra-maze cues in young and aged control and MgT-treated (for 1 month) rats following the water maze training period. For control aged rats, the integrity of the extra-maze cues from surrounding curtains was essential for finding the hidden platform, as their performance significantly degraded under partial-cue conditions. In contrast, MgT-treated aged rats, as well as young rats, performed equally well under full- and partial-cue conditions (aged rats, Figure 3 H, unpaired t test, p < 0.05; young rats, Figure 3 D). Thus, MgT treatment enhanced memory recall under partial information conditions in aged but had no effect on young, rats.

The use of animal models to study the effects of aging on cognition.

To evaluate memory functions, we performed two test trials with the platform removed; the rats were allowed to search for 60 s. The first test trial commenced 1 hr after the end of training. All rats showed a remarkable preference for the target versus opposite quadrant (young, Figures 3 B and 3C, paired t test, p < 0.001; old, Figures 3 F and 3G, p < 0.001), suggesting that all, i.e., control and MgT-treated young and aged rats, could remember the platform location. To test long-term spatial memory, a second test trial was performed 24 hr later. Both young and aged control rats lost their preference for the target quadrant compared to other quadrants ( Figures 3 B and 3F). In contrast, MgT-treated young ( Figure 3 B) and aged ( Figure 3 G) rats retained their quadrant preference (p < 0.001; p < 0.01, respectively). Visual and locomotor functions were equal in both groups, as judged by latency of escape to a visible platform (data not shown) and swimming speed (see Table S1 ). Thus, MgT treatment significantly enhanced hippocampus-dependent spatial learning and memory in both young and aged rats.

We used the Morris water maze to perform further experiments to determine whether MgT leads to the improvement of spatial long-term memory (). Young rats underwent 8 trials of training within one day with a 1 hr intertrial interval. For aged rats, the training protocol was spread over two days: 5 trials on day 1, and 3 trials on day 2. This protocol was adopted because aged rats are not able to perform 8 trials within one day. During the training period, the performance of all rats gradually improved ( Figures 3 A and 3E). However, MgT-treated rats learned to find the hidden platform faster than controls (two-way ANOVA, MgT-treated versus control young rats, p < 0.01 and MgT-treated versus control aged rats, p < 0.001). In addition, the degree of learning ability enhancement by MgT was higher in aged ( Figure 3 E) than in young rats ( Figure 3 A).

(H) Partial removal of extra-maze cues impaired the ability of aged rats to find the hidden platform, while MgT-treated aged rats were still capable of locating the platform (unpaired t test, p < 0.05).p < 0.05,p < 0.01,p < 0.001. Data are presented as mean ± SEM. See also Table S1

(F and G) Performance of control (n = 16, F) and MgT-treated (n = 12, G) aged rats during the first (1 hr after training) and the second (24 hr after training) memory probe trials. During the first trial, both groups spent more time in the target quadrant (paired t test, p < 0.0001). Twenty-four hours later, only MgT-treated aged rats spent more time in the target quadrant (p < 0.01).

(D) Pattern completion test with partial extra maze cues of young rats. Partial cues did not impair the rats' ability to find the platform.

(B and C) Percentage of time spent in the target versus opposite quadrant during the first (1 hr after training) and the second (24 hr after training) probe trials in control (n = 14, B) and MgT-treated (n = 15, C) young rats. During the first probe trial, both groups spent significantly more time in the target quadrant (paired t test, p < 0.0001). On the other hand, only MgT-treated rats spent significantly more time in the target quadrant during the second test trial (24 hr after training, p < 0.0001).

(A) Escape time to find the hidden platform of young rats during the water maze training trials. MgT-treated (n = 15) rats learned faster than controls (n = 14, two-way ANOVA, F 1,216 = 7.85, p = 0.006). ANOVA was followed by Bonferroni's post hoc test.

To monitor the time-course of MgT treatment on working spatial memory, task performance was evaluated every sixth day ( Figures 2 B and 2D). Since the largest difference in choice accuracy between the treated versus control rats was obtained at 5 min delay interval, we monitored choice accuracy only at this delay interval for the remaining experiments. A significant increase in choice accuracy of MgT-treated young rats was apparent 6 days after the onset of treatment (one-way ANOVA, p < 0.05), peaked on day 12 (p < 0.001), and did not decline over 1 month after MgT treatment was stopped (days 30 to 60, Figure 2 B). For MgT-treated aged rats, a significant increase in choice accuracy occurred 12 days after the onset of treatment (p < 0.001) and remained stable until MgT was stopped (day 30). In contrast to young rats, the working memory performance of aged rats declined to the baseline value within 12 days following interruption of MgT treatment ( Figure 2 D). Therefore, the on/off kinetics of MgT-induced spatial memory enhancement seems symmetric in aged rats. To test if MgT could re-enhance spatial memory functions of MgT-treated aged rats, following 30 days of drinking plain water (days 30–60), they drank water supplemented with MgT again. Strikingly, aged rat performance was re-enhanced within 12 days of treatment ( Figure 2 D). Thus, MgT consumption enhanced spatial working memory of young and aged rats (For the detailed time course curves for each group, see Figures S5 C and S5D).

At the end of the training, rats were assigned to control and MgT-treated groups to assure each group had a comparable average working memory capability. Spatial working memory was tested by a gradual increase of the delay between sample and choice trials, before (day 0) and after (day 24) MgT treatment ( Figures 2 A and 2C). The choice accuracy of control young rats did not change significantly (see Figure S5 A). On the other hand, MgT-treated young rats had significantly better performance than untreated rats at the longest delay interval (5 min, p < 0.05, Figures 2 A). For control aged rats, their choice accuracy declined slightly during the experimental period (see Figure S5 B). However, MgT-treated aged rats displayed significantly better performance than untreated aged rats in 5 min delay interval (p < 0.05, Figure 2 C). Thus, MgT treatment can enhance spatial working memory in young and aged rats. Spatial working memory evaluated by T-maze did not significantly decline with aging under our experimental conditions. However, aged rats learned the alternating T-maze task slower than young rats and MgT treatment in aged rats prevented such deficit (data not shown).

(A) Spatial working memory of young rats (2 months) in the T-maze before (day 0) and after (day 24) MgT treatment. MgT-treated rats showed significant improvement in performance at 5 min delay as a retention interval compared to control rats (unpaired t test,p < 0.05, n = 12). Fifty percent of correct responses represent chance levels of performance. (C) Spatial working-memory of aged rats (22 months) in the T-maze before (day 0) and after (day 24) MgT treatment. MgT-treated rats showed significant improvement in performance after 5 min delay as a retention interval compared to control rats (t test, p < 0.05, n = 12). (B and D) Time course of MgT effect on spatial working memory of young and aged rats. Data are calculated as the difference in correct choices between control (n = 12) and MgT-treated (n = 12) rats. One-way ANOVA analysis revealed significant differences (compared with day 0) as follows: young rats F= 7.08, p < 0.0001; aged rats F= 16.38, p < 0.0001). Bonferroni's post hoc test,p < 0.05,p < 0.001. Data are presented as mean ± SEM. See also Figure S5

We tested rats for several hippocampus-dependent forms of memory. Spatial working memory was assessed using a T-maze non-matching-to-place task (). Naive untreated rats were trained for 10 days on a reward forced-choice alternation task (see Supplemental Experimental Procedures ). The percentage of correct choices (alternations) was recorded for each daily session. Following 8 days of training, all rats attained an asymptotic choice accuracy level of ∼94%, indicating that they learned the task. In these experiments, the rats likely used a spatial strategy because when the maze was rotated by 180°, the rats went to the arm predicted by allocentric, rather than egocentric, coordinates (data not shown).

Next, rats were treated with various magnesium compounds for 1 month via drinking water at a dose of 50 mg/kg/day elemental Mg. We used aging rats (18 month) because they already have memory decline (compared to younger rats) in order to increase the possibility of observing memory improvement. Rats treated with MgT showed significant enhancement of short-term memory (10 min retention interval, one-way ANOVA analysis, p < 0.05) using a modified NORT (see Figure S4 and Supplemental Experimental Procedures ). Rats treated with magnesium-chloride or -citrate displayed enhanced short-term memory as well, but this enhancement was not statistically significant ( Figure 1 B). Surprisingly, although magnesium-gluconate in milk has a comparable bioavailability to MgT (X.Z., F. Mao, Y. Shang, N.A., and G.L., unpublished data), it failed to enhance memory ( Figure 1 B). For the long-term memory test (12 hr retention interval), only MgT-treated rats exhibited enhanced performance (p < 0.05, Figure 1 C). To test whether threonate per se has any positive effect on memory, we assessed the effect of sodium-L-threonate on memory; no effect was observed ( Figures 1 D and 1E). We also examined the effectiveness of the combination of magnesium-chloride with sodium-L-threonate, as the mixture of both forms the MgT complex in aqueous solution. To our surprise, this combination was ineffective ( Figures 1 D and 1E). Hence, we chose MgT as the optimal testing compound to study the effects of elevating brain Mgon memory and its underlying molecular and cellular mechanisms.

Control rats were fed ordinary rat chow containing 0.15% Mg(see Experimental Procedures ), which is considered standard. Different MgT doses were given to rats and their performances on learning behavior in the water maze task were compared. We found that 50 mg/kg/day (elemental Mg) is the minimum effective dose (see Figure S2 A). In a separate memory test, the novel object recognition test (NORT), when the actual dose consumed by individual rats varied due to the difference in daily fluid intake, the effective dose was still around 50 mg/kg/day (see Figure S2 B). Therefore, 50 mg/kg/day was used in this study. Chronic (1 month) MgT treatment at this dose did not influence water and food intake, body weight, and overall mobility (see Figure S3 ).

We further verified that the compound with high bioavailability and loading ability into brain is the best Mg 2+ compound for studying the effects of elevating brain Mg 2+ on memory. For this propose, we determined the effective dose for memory enhancement first.

Here, we explored the ability of our newly developed compounds to increase the cerebrospinal fluid (CSF) Mgconcentration ([Mg). CSF was collected before treatment to determine the baseline [Mgof each individual rat. The CSF was then collected 12 and 24 days after magnesium treatment from the same rats. Total [Mgincreased gradually in MgT-treated rats. At day 24, [Mgwas 7% higher than baseline (one-way ANOVA, p < 0.05, Figure 1 A). To monitor the effect of repeated CSF sampling on [Mg, we measured [Mgin control rats without any magnesium supplementation. [Mgdropped by ∼9% at the third sampling point (24 days, Figure 1 A). This drop might be due to the loss of CSF Mgassociated with CSF sampling; as the CSF volume in rat brain is about 300–400 μl, each sampling (50-100 μl) can lead to 15%–30% loss of CSF Mg. Considering this reduction of [Mgassociated with the repeated sampling, the actual increase in [Mgshould be ∼15% (two-way ANOVA, p < 0.001, Figure 1 A). Other magnesium compounds did not elevate [Mgsignificantly when compared to control ( Figure 1 A).

A modified version of novel object recognition test was used to increase the difficulty of the task (see Supplemental Experimental Procedures and Figure S4 ). Dashed line indicates no memory. MgG, magnesium gluconate. Bonferroni's post hoc test,p < 0.05,p < 0.001. Data are presented as mean ± SEM.

(D and E) Effects of sodium-L-threonate (NaT) alone and when combined with magnesium chloride (MgCl) on short-term memory (D) and long-term memory (E). ANOVA analysis revealed a significant effect of treatment on short-term memory (F= 2.90, p = 0.049, n = 8–10) and long-term memory (F= 3.23, p = 0.034, n = 9–10). Post hoc test revealed significant effect of magnesium-L-threonate (MgT). See also Figures S2 and S3

(C) Long-term memory test (12 hr) using novel object recognition test. One-way ANOVA analysis revealed significant effect of treatment on short-term memory (F 4,34 = 2.89, p = 0.037, n = 7–9) and long-term memory (F 4,31 = 4.50, p = 0.005, n = 5–10). Post hoc test revealed significant effect of magnesium-L-threonate (MgT) on short term and long-term memory.

(A) Elevation of magnesium concentration in the cerebrospinal fluid ([Mg 2+ ] CSF ) following treatment with different magnesium compounds. Total Mg 2+ in CSF was measured before magnesium treatment (day 0), 12, and 24 days after magnesium treatment. Two-way ANOVA analysis revealed significant effect of treatment (F 3,69 = 4.76, p = 0.0045, n = 6–8). Data were calculated and presented as a percentage of baseline level.

To study the effect of elevating brain Mg 2+ on learning and memory, we needed to identify a suitable Mg 2+ compound that enhances loading of Mg 2+ into the brain. To achieve this goal, the Mg 2+ compound needs high efficacy to transport Mg 2+ from the digestive tract into the blood and, ultimately, into the central nervous system. In a separate study, the bioavailability (evaluated by absorption, excretion, and retention rate of magnesium) of four commercially available Mg 2+ compounds (magnesium-chloride, -citrate, -glycinate, and -gluconate) and two Mg 2+ preparations we developed (magnesium-L-threonate, MgT, and magnesium-gluconate in milk) was compared in rats. We found that both MgT and magnesium-gluconate in milk have higher bioavailability (X.Z., F. Mao, Y. Shang, N.A., and G.L., unpublished data).

Discussion

2+ in both young and aged rats can enhance different forms of learning and memory (Figure 2, 2+ induced reconfiguration of synaptic networks from a small number of synapses with high release probability to a larger number of synapses with low release probability ( We found that increasing brain Mgin both young and aged rats can enhance different forms of learning and memory ( Figure 1 Figure 3 ). Chronic MgT treatment upregulated NR2B-containing NMDAR and increased activation/expression of downstream signaling molecules in the hippocampus ( Figure 4 ). This was associated with a dramatic increase in short-term synaptic facilitation and long-term potentiation that are critical for learning and memory ( Figure 5 ). At the cellular level, on the other hand, MgT treatment also increased number of synaptophysin-/synaptobrevin-containing presynaptic boutons ( Figure 6 ). Therefore, elevated Mginduced reconfiguration of synaptic networks from a small number of synapses with high release probability to a larger number of synapses with low release probability ( Figure 7 ). Finally, increase in the density of synaptophysin-/ synaptobrevin-containing presynaptic boutons correlated with improvement of memory functions ( Figure 8 ).

Mamiya et al., 2003 Mamiya T.

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Takeshima H. Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors. 2+ influx through synaptic NMDAR triggers activation of CREB transcription factor, leading to the expression of genes that promote cell survival and synaptic plasticity such as the neurotrophic factor BDNF ( Vanhoutte and Bading, 2003 Vanhoutte P.

Bading H. Opposing roles of synaptic and extrasynaptic NMDA receptors in neuronal calcium signalling and BDNF gene regulation. Fukushima et al., 2008 Fukushima H.

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et al. Upregulation of calcium/calmodulin-dependent protein kinase IV improves memory formation and rescues memory loss with aging. Pang and Lu, 2004 Pang P.T.

Lu B. Regulation of late-phase LTP and long-term memory in normal and aging hippocampus: role of secreted proteins tPA and BDNF. Silva et al., 1998 Silva A.J.

Kogan J.H.

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Kida S. CREB and memory. 2+ enhances CREB activation by acting on other signaling pathways. One important effector downstream of NMDAR signaling is CaMKII. Increased activation of CaMKII was shown to underlie the enhancement of LTP and learning and memory observed in mice lacking the nociceptin opioid receptor (). In MgT-treated rats, upregulation of NR2B is associated with enhancement of CaMKII activation following memory task, suggesting that NMDAR signaling is enhanced. CREB is another important downstream molecule critical for learning and memory. Cainflux through synaptic NMDAR triggers activation of CREB transcription factor, leading to the expression of genes that promote cell survival and synaptic plasticity such as the neurotrophic factor BDNF (). Increased activation of CREB and/or expression of BDNF enhance LTP in hippocampus and learning and memory (). Here, we found increased activation of CREB and higher level of BDNF in MgT-treated rats too. Because CREB can also be activated by other molecular pathways such as cAMP pathway (), we cannot exclude the possibility that Mgenhances CREB activation by acting on other signaling pathways.

In addition to voltage-dependent inhibition of NMDAR, Mg2+ may act at other targets that, synergistically or independently, might have led to the observed effects. For instance, in the intracellular compartment, an increase in Mg2+ could compete with Ca2+, altering Ca2+ signaling. Furthermore, increased intracellular Mg2+ might influence Mg2+-dependent enzymatic reactions, which might affect other cellular processes such as cell excitability and/or cell metabolism that might contribute to the observed enhancement of memory.

2+, increase in density of synaptic boutons, and enhancement of memory functions (12 days time-course, Figure 2, 2+] in vitro can alter synaptic configuration in hippocampal slices within 5 hr (2+ loading into the brain (2+ excretion rate in young animals ( Corman and Michel, 1987 Corman B.

Michel J.B. Glomerular filtration, renal blood flow, and solute excretion in conscious aging rats. At the cellular level, we hypothesize that increase in bouton density might be a key change underlying memory enhancement by MgT. In support of this hypothesis, on one hand, is the temporal correlation among the onset of MgT treatment, elevation of brain Mg, increase in density of synaptic boutons, and enhancement of memory functions (12 days time-course, Figure 1 Figure 8 ). On the other hand, ending of MgT supplementation leads to a reduction in bouton density and memory performance back to the baseline in aged rats ( Figure 2 Figure 8 ). Although changing extracellular [Mg] in vitro can alter synaptic configuration in hippocampal slices within 5 hr ( Figure 7 ), slow time-course of Mgloading into the brain ( Figure 1 A) might be the factor that delays the onset of MgT effect in vivo. In young rats the enhanced memory functions persisted for 60 days after the end of MgT treatment. This is possibly due to lower Mgexcretion rate in young animals (). The exact mechanisms underlying the prolonged effect of MgT on memory functions in young rats remain to be investigated.

Gómez-Pinilla, 2008 Gómez-Pinilla F. Brain foods: the effects of nutrients on brain function. 2+. It is worth noting that the control rats in the present study had a normal diet, which is widely accepted as containing a sufficient amount of Mg2+. The effects we observed were due to elevation of body Mg2+ content to higher levels than a normal diet. Improvement of memory functions in aged rats by a high dosage of Mg2+ diet (2% elemental Mg2+) has been reported before ( Landfield and Morgan, 1984 Landfield P.W.

Morgan G.A. Chronically elevating plasma Mg2+ improves hippocampal frequency potentiation and reversal learning in aged and young rats. 2+-induced diarrhea, hindering further mechanistic studies. Having studied the biophysical effects of Mg2+ on synaptic plasticity in cultured hippocampal neurons in vitro ( Slutsky et al., 2004 Slutsky I.

Sadeghpour S.

Li B.

Liu G. Enhancement of synaptic plasticity through chronically reduced Ca2+ flux during uncorrelated activity. 2+ in intact rats, we concluded that development of a new compound that efficiently loads Mg2+ into the brain was essential. With this Mg2+ compound (MgT), we are able to study the influences of long-term elevation of brain magnesium on cognitive functions without disrupting other physiological functions. In the current study, we did not test the effects of Mg2+ deficiency on synaptic plasticity and memory function. A previous study already showed that chronic reduction of dietary magnesium impairs memory ( Bardgett et al., 2005 Bardgett M.E.

Schultheis P.J.

McGill D.L.

Richmond R.E.

Wagge J.R. Magnesium deficiency impairs fear conditioning in mice. 2+ is an essential ion for normal cellular functions and body health, many physiological functions are impaired with the reduction of body Mg2+. Therefore, it is difficult to establish a casual relationship between brain Mg2+ and memory functions by induction of Mg2+ deficiency. Nonetheless, our “on/off” experiments in the T-maze provide evidence for the possible causal relationship between high Mg2+ intake and memory enhancement in aged rats ( Diet, exercise, and environmental enrichment can affect brain health and cognitive function (for review, see). Here, we introduce a new strategy to enhance learning and memory and prevent age-related memory decline by increasing brain Mg. It is worth noting that the control rats in the present study had a normal diet, which is widely accepted as containing a sufficient amount of Mg. The effects we observed were due to elevation of body Mgcontent to higher levels than a normal diet. Improvement of memory functions in aged rats by a high dosage of Mgdiet (2% elemental Mg) has been reported before (). However, it triggered weight loss due to Mg-induced diarrhea, hindering further mechanistic studies. Having studied the biophysical effects of Mgon synaptic plasticity in cultured hippocampal neurons in vitro (), and after studying the homeostatic regulation of Mgin intact rats, we concluded that development of a new compound that efficiently loads Mginto the brain was essential. With this Mgcompound (MgT), we are able to study the influences of long-term elevation of brain magnesium on cognitive functions without disrupting other physiological functions. In the current study, we did not test the effects of Mgdeficiency on synaptic plasticity and memory function. A previous study already showed that chronic reduction of dietary magnesium impairs memory (). However, because Mgis an essential ion for normal cellular functions and body health, many physiological functions are impaired with the reduction of body Mg. Therefore, it is difficult to establish a casual relationship between brain Mgand memory functions by induction of Mgdeficiency. Nonetheless, our “on/off” experiments in the T-maze provide evidence for the possible causal relationship between high Mgintake and memory enhancement in aged rats ( Figure 2 D).