Activity‐dependent changes in synaptic strength, such as long‐term potentiation (LTP) and long‐term depression (LTD) of synaptic transmission, are considered to be the cellular substrates of learning and memory (Neves et al , 2008 ; Collingridge et al , 2010 ). Competitive interactions between these forms of synaptic plasticity have been proposed to participate in memory storage (Diamond et al , 2005 ; Nicholls et al , 2008 ; Ge et al , 2010 ). Indeed, glycogen synthase kinase 3 beta (GSK3β) determines whether NMDA receptor activation induces or inhibits LTD (Peineau et al , 2007 ). Moreover, the activation of protein phosphatase 1 (PP1) during LTD dephosphorylates GSK3β at Ser9, resulting in its activation and receptor internalization. Since p‐Akt is a GSK3β inhibitor, PP1‐mediated Akt dephosphorylation further contributes to the enhancement of GSK3β activity. Significantly, GSK3β and Akt dephosphorylation are impaired by okadaic acid, a compound that also blocks LTD by inhibiting PP1 (Peineau et al , 2007 ). Conversely, activation of NMDA receptors leads to the stimulation of PI3K/Akt pathway during LTP, provoking the phosphorylation of GSK3β at Ser9 to prevent LTD. Thus, it appears reasonable to assume that the loss of cholesterol and the consequent enhancement of PI3K/Akt activity that occurs in the hippocampus during aging will have a detrimental effect on LTD and, consequently, on cognition. Since most studies coincide that aging is accompanied by a decreased propensity to develop LTD (Lee et al , 2005 ; Billard & Rouaud, 2007 ; Ahmed et al , 2011 ), we investigated here whether and how this is related to age‐associated constitutive cholesterol loss.

The accumulation of p‐Akt with age may be the result of different processes. We previously observed that the increase with age of the cholesterol hydroxylating enzyme cholesterol 24‐hydroxylase (CYP46A1), the major catabolic enzyme of cholesterol in the brain (Lund et al , 2003 ), triggers cholesterol loss‐dependent, ligand‐independent, activation of the TrkB receptor and, consequently, Akt phosphorylation (Martin et al , 2008 , 2011 ; Sodero et al , 2011a , b ). A second mechanism driving the increase in p‐Akt with age is also linked to cholesterol loss, and it involves the increase in plasma membrane sphingomyelin, similarly contributing to ligand‐independent TrkB phosphorylation and PI3K/Akt activation (Trovò et al , 2011 ). Increased PI3K/Akt activity in old neurons also seems to arise from the constitutive increase with age of interleukin 1B (IL‐1B) activity and from the age‐associated detachment from the plasma membrane of the PIP2 binding protein, myristoylated alanine‐rich C kinase substrate (MARCKS: Trovò et al , 2013 ). Altered binding of MARCKS to the membrane favors the accumulation of PIP3 in the synaptic fraction of old mice, with the subsequent increase in Akt phosphorylation (Trovò et al , 2013 ).

We previously showed that the phosphorylated form of the serine–threonine kinase Akt (p‐Akt) accumulates in old hippocampal neurons, both in vivo and in vitro (Martin et al , 2008 , 2011 ; Trovò et al , 2013 ), which probably reflects the strong need for survival signaling. Nevertheless, the counter effect of robust survival might be reduced performance. In fact, p‐Akt phosphorylation negatively regulates long‐term depression (LTD), a key process in learning and memory (see below and Peineau et al , 2007 ).

Aging is associated with cognitive decline, such that individuals older than 65 develop cognitive deficits or age‐associated memory impairments. The hippocampus, a brain structure that is central to the formation of declarative and other types of memory, is particularly sensitive to aging. However, these impairments are not paralleled by an increase in neuronal death (Burke & Barnes, 2006 ), indicative that more subtle mechanisms must be affected by aging to produce memory decline.

Results

Impaired LTD‐induced Akt dephosphorylation in the aged hippocampus The magnitude of the LTD response seems to depend on the efficacy of AMPAR internalization, which is in turn dependent on Akt dephosphorylation, GSK3β activation, and receptor endocytosis. Accordingly, in an aged hippocampus in which the equilibrium is displaced toward pAkt (see 1), incomplete pAkt dephosphorylation would result in a poor LTD response. To test this prediction, p‐Akt levels were measured in hippocampal slices of young (4 month old; 4M) and old (20 month old; 20M) mice, in unstimulated control conditions, and after pharmacological induction of LTD. In control conditions, there was 1.89 ± 0.298 fold more p‐Akt in old than in young mice (P = 0.041, Fig 1A), and moreover, NMDA triggered a significant dephosphorylation of p‐Akt in hippocampal slices from young but not old mice (Fig 1A). In control experiments, the normalization of Akt to β‐actin showed that there was no change in total Akt following stimulation and that the amount of p‐Akt/β‐actin only decreased in young mice after NMDA stimulation (Fig 1A). Figure 1.p‐AKT dephosphorylation after LTD is impaired in old mice p‐Akt levels in acute hippocampal slices prepared from young (4M) and old (20M) mice in control conditions or 1 h after NMDA‐LTD induction were assessed in Western blots. The quantification shows that in control conditions, the levels of p‐Akt are 50% higher in 20M than in 4M mice. Stimulation with 20 μM NMDA induced p‐Akt dephosphorylation in 4M but not in 20M mice. The bar plots show the levels of p‐Akt in young and old mice, corrected for total Akt and for β‐actin, in controls and after stimulation. The quantification of total Akt/β‐actin shows that the total Akt levels do not change after stimulation. The levels of p‐Akt/Akt after stimulation were: 4M = 0.75 ± 0.081 (n = 5 animals, P = 0.014), and 20M = 1.01 ± 0.047, (n = 5 animals, P = 0.767). The levels of p‐Akt/β‐actin after stimulation were: 4M = 0.76 ± 0.065, (n = 5 animals, P = 0.004), and 20M = 1.05 ± 0.15, (n = 8 animals, P = 0.726). The levels of total Akt/β‐actin after stimulation were: 4M = 1.10 ± 0.101 (n = 5 animals, P = 0.340) and 20M = 1.08 ± 0.170, (n = 8 animals, P = 0.664). Western blot and quantification showing that addition of cholesterol to acute slices from 20M mice restores the basal levels of p‐Akt observed in young mice [20M + chol = 0.47 ± 10.3 (n = 5 animals, P = 0.0008)]. Control experiments show that the addition of cholesterol to acute hippocampal slices from 4M mice does not effect on the levels of p‐Akt and total Akt, or on p‐Akt dephosphorylation after 20 μM NMDA stimulation. p‐Akt/β‐actin: 4M + chol = 0.96 ± 0.139 (n = 5 animals, P = 0.805); Akt/β‐actin: 4M + chol = 1.16 ± 0.138 (n = 5 animals, P = 0.311). The levels of p‐Akt/Akt after stimulation were: 4M + chol = 0.81 ± 0.051 (n = 5 animals, P = 0.0143). Lower levels of the PIP2‐binding protein MARCKS were found in membrane preparations from 15 DIV cholesterol‐depleted neurons compared to controls. The plot shows the amount of MARCKS attached to the membrane relative to controls: control neurons = 1.00; MβCD‐treated neurons = 0.688 ± 0.11 (P = 0.045, n = 5 different cultures). The Western blots below show the membrane‐supernatant distribution of MARCKS in control neurons or after cholesterol depletion by MβCD. As can be seen in the blots, MβCD provokes a reduction in the MARCKS present in the membrane fraction (M) with a concomitant 26.33 ± 6.4% increase in the supernatant (SN, P = 0.02, n = 3 different cultures). The Western blot corresponds to control experiments showing that the addition of cholesterol to hippocampal slices from 4M mice does not affect the amount of MARCKS found in membrane fractions: 4M + chol = 0.80 ± 0.124 (P = 0.187, n = 3 animals). Data information: In (A‐E), the presented values are relative to controls, considered as 1. The P‐values correspond to 2‐sided t‐test. Data information: In (A‐E), the presented values are relative to controls, considered as 1. The‐values correspond to 2‐sided‐test. Source data are available for this figure. Source Data for Figure 1 [emmm201303711-SourceData-Fig1.pdf] To test the possibility that the reduced capacity to dephosphorylate Akt upon stimulation of the aged hippocampus is related to the loss of cholesterol in the synaptic fraction (Martin et al, 2008; Sodero et al, 2011a,b), we replenished cholesterol in the membranes of hippocampal slices from 20M mice. Exposing slices to 30 μM cholesterol‐methyl‐β‐cyclodextrin (cholesterol‐MβCD) and 5 μM cholesterol for 60 min reduced the levels of p‐Akt to 47% ± 10.3 of the 20M controls, levels similar to those found in slices from young mice (Fig 1B). To ascertain whether this replenishment strategy did in fact restore cholesterol in the plasma membrane, the levels of this sterol were measured in hippocampal membrane fractions from young and old mice, before and after exposure to the cholesterol‐MβCD/cholesterol mix. The cholesterol:total protein ratio was 0.420 ± 0.053 in hippocampal membranes from 4M mice and 0.326 ± 0.044 in hippocampal membranes from 20M mice, confirming a loss of more than 20% of the cholesterol with age (see Martin et al, 2008 and Sodero et al, 2011a,b). After replenishment, the cholesterol levels in membranes from 20M mice increased the cholesterol:protein ratio to 0.398 (±0.070 μg), representing a recovery of 17.12% and a replenishment to 95% the levels in hippocampal membranes from young mice. In a control experiment, we tested the effect of adding cholesterol to slices prepared from young mice, and in 4M acute hippocampal slices, this did not alter the amount of p‐Akt or Akt in basal conditions (Fig 1C). Moreover, NMDA triggered p‐Akt dephosphorylation in both control and cholesterol‐replenished slices, maintaining similar levels of p‐Akt after stimulation in both cases (4M control + NMDA: 75 ± 8%; 4M cholesterol + NMDA: 81 ± 5%; P = 0.694). We recently demonstrated that in old mice, hippocampal synapses contain less MARCKS in membranes, resulting in reduced PI(4,5)P2 clustering and increased levels of PI(3,4,5)P3 and p‐Akt (Trovò et al, 2013). To gain further insight into how cholesterol loss during aging may increase pAkt, we investigated the relationship between cholesterol loss and the association of MARCKS with the plasma membrane. When hippocampal neurons were treated with a low dose of the cholesterol‐extracting drug MβCD, there was a 23.63 ± 5.60% loss of cholesterol and a significant reduction in membrane‐bound MARCKS (Fig 1D). This reduction of MARCKS in low‐cholesterol membranes was not due to protein degradation, as analyzing the membrane and supernatant fractions from control and cholesterol‐depleted neurons in Western blots demonstrated that MβCD treatment provoked MARCKS detachment from the membranes with its concomitant increase in the soluble fraction (Fig 1D). Finally, we assessed whether the addition of cholesterol to hippocampal slices prepared from young mice affected the distribution of MARCKS. Accordingly, we found that the addition of cholesterol to acute hippocampal slices from 4M mice did not affect the levels of MARCKS in the membrane fractions of those slices (Fig 1E). To demonstrate a functional connection between the reduction in MARCKS at the membrane and Akt phosphorylation, the amount of p‐Akt was measured in 15 DIV neurons infected with lentiviral particles that express a shRNA designed to knockdown MARCKS expression (shMARCKS). MARCKS knockdown was associated with increased p‐Akt levels (Supplementary Fig S1), and the same effect observed following the overexpression of a mutant form of MARCKS that cannot bind PI(4,5)P2 (Trovò et al, 2013). When cholesterol was extracted from shMARCKS‐treated neurons, a further increase of Akt phosphorylation was observed, supporting our hypothesis that cholesterol loss and membrane MARCKS detachment both contribute to Akt phosphorylation (Supplementary Fig S1). The phosphatase and tensin homolog deleted on chromosome ten (PTEN) catalyzes the conversion of PI(3,4,5)P3 to PI(4,5)P2. It was shown previously that during NMDA‐induced LTD, PTEN is recruited to the postsynaptic density (PSD) and that this is a strict requirement for this type of LTD (Jurado et al, 2010). Hence, we tested whether impaired p‐Akt dephosphorylation after LTD in old mice may be also due to deficits in PTEN activity. It might be expected that deficient PI(3,4,5)P3 degradation by PTEN would lead to the subsequent accumulation of PI(3,4,5)P3 and p‐Akt hyperactivation. Hence, PTEN levels were analyzed in the PSD fraction of hippocampal slices from 4M and 20M mice, 30 min after NMDA‐induced LTD. Recruitment of PTEN to the PSD fraction after NMDA‐LTD was similar in the membranes from young and old hippocampal slices (Fig 2). Figure 2.Impaired Akt dephosphorylation in old mice is not due to deficits in PTEN No differences were observed in PTEN recruitment to the PSD after LTD induction in young and old mice. The Western blot shows the levels of PTEN found in the PSD purified from acute hippocampal slices at 30 min after LTD. No difference in the levels of recruited PTEN was observed between 4‐ and 20‐month‐old mice (mean ± standard error, n = 5 animals from each age). Total levels of PSD95 and PTEN do not change comparing young and old mice (mean ± standard error, n = 5 animals from each age). Source data are available for this figure. Source Data for Figure 2 [emmm201303711-SourceData-Fig2.pdf] Together, these studies indicated that the loss of cholesterol in the hippocampus of aged mice produces a strong increase in PI3K/pAkt activity. Moreover, MARCKS detachment driven by cholesterol loss, a protein previously shown to be associated with high pAkt levels in the aged (Trovò et al, 2013), seems also to be another possible upstream determinant.

Cholesterol loss results in surface accumulation and impaired endocytosis of AMPARs To determine the extent to which the enhanced PI3K/pAkt driven by cholesterol loss in the old affects the basic molecular machinery of cognition, we analyzed AMPAR dynamics in hippocampal neurons in vitro. In general, cells in vitro are particularly useful to study and quantify receptor dynamics, and hippocampal neurons in vitro are particularly suited as, like hippocampal cells in situ, they also undergo a significant reduction in plasma membrane cholesterol over time in vitro, which in turn is due to the increased expression of the enzyme CYP46A1 (Martin et al, 2008). Furthermore, hippocampal neurons also experience a time‐dependent increase in p‐Akt levels driven by cholesterol loss (Martin et al, 2011). To study AMPAR dynamics, we measured the surface levels and internalization rate of GluA2 containing receptors, since these are the most abundant in the mature hippocampal neurons (Wenthold et al, 1996; Malenka, 2003). Significantly more AMPARs were present on the cell surface of 30 DIV neurons compared to 15 DIV neurons (Fig 3A and B), and measurements of the area covered indicated that GluA2‐AMPARs were present in larger clusters on 30 DIV neurons (Fig 3C). By contrast, 15 and 30 DIV neurons contained similar amounts of total GluA2 (Supplementary Fig S2A), excluding any possibly differences in expression. Figure 3.Surface GluA2‐AMPARs accumulate in low‐cholesterol hippocampal neurons A, B. The surface staining of GluA2‐containing AMPARs (A) is significantly higher in 30 DIV than in 15 DIV neurons (15 DIV = 1.00 ± 0.28; 30 DIV = 2.73 ± 0.27; P < 0.0001, n = 3). Stimulation with glutamate (100 μM) resulted in decreased GluA2 staining of 15 DIV (15 DIV glut = 0.62 ± 0.095; P control, glut = 0.0310; n = 3) but not 30 DIV neurons (30 DIV glut = 2.86 ± 0.219; P control, glut = 0.053; n = 3), indicating that low‐cholesterol neurons have a reduced capability to endocytose AMPARs in response to ligand. Fluorescence intensity (FI/area) quantified in the processes of neurons is shown in (B).

C. Quantification of the area of GluA2‐AMPARs clusters in 15 and 30 DIV neurons indicates that larger receptor clusters form in low‐cholesterol neurons (average area 15DIV: 0.055 ± 0.0042 μm 2 , 30 DIV: 0.086 ± 0.0053 μm 2 , P = 0.0009, n = 3 different cultures).

D, E. Fluorescence images show the internalized AMPARs in 15DIV and 30DIV neurons, before and after glutamate stimulation. The fluorescence images correspond to higher magnifications of the regions indicated in the insets. Glutamate exposure resulted in increased AMPAR internalization in 15 DIV neurons (D) and 5 min after glutamate addition the fluorescence intensity (FI)/area measured in the processes increased from 1 ± 0.06 to 1.33 ± 0.12 (t‐test, P = 0.0082, n = 4). Glutamate addition did not increase AMPAR internalization in 30 DIV neurons (E). The values of FI/area in the processes were 1 ± 0.082 and 0.88 ± 0.048 (t‐test, P = 0.252, n = 3). Data information: The values represent the mean ± s.e.m. relative to controls. n: number of different experiments. The data were compared using Mann‐Whitney non‐parametric t‐test. Data information: The values represent the mean ± s.e.m. relative to controls.: number of different experiments. The data were compared using Mann‐Whitney non‐parametric‐test. We analyzed the effect of neuronal stimulation on AMPAR endocytosis in 15 and 30 DIV neurons. In the presence of 100 μM glutamate, which induces AMPAR endocytosis in hippocampal cultures through a mechanism also employed during LTD (Beattie et al, 2000; Man et al, 2000), weaker surface GluA2 staining was evident in 15 DIV neurons. By contrast, there was no such effect on the number of surface AMPARs in 30 DIV neurons (Fig 3A and B), suggesting that internalization is impaired in the older neurons. Indeed, when the levels of internalized GluA2‐AMPARs were measured using an antibody‐feeding strategy followed by acid wash (see 4), receptor internalization in 15 DIV neurons increased by 33% in the presence of 100 μM but not in 30 DIV neurons (Fig 3D–G). To test whether the reduction in the levels of cholesterol in these older cells might be responsible for the weaker receptor internalization of 30 DIV neurons, these cells were incubated with the cholesterol‐MβCD (30 μM) and cholesterol (5 μM) replenishment mix. Under these conditions, there was a significant loss of surface AMPARs on 30 DIV neurons stimulated with glutamate (Fig 4A and B). Indeed, the levels of cholesterol in the neuronal membranes were measured after replenishment, and in 30 DIV hippocampal neurons, the cholesterol‐MβCD/cholesterol mix produced a 19% increase in membrane cholesterol: the cholesterol levels in membrane preparations rose from 0.144 ± 0.002 to 0.171 ± 0.011 μg cholesterol/μg of protein. By contrast, the total levels of GluA2 were not affected by cholesterol replenishment in vitro or in vivo (Supplementary Figs S2A and S3). Figure 4.Glutamate addition decreases the level of surface AMPARs in cholesterol‐replenished 30 DIV neurons The surface AMPAR staining decreased in cholesterol‐replenished 30 DIV neurons after a 10‐min incubation with glutamate. The boxes on the right correspond to high‐magnification images of the regions indicated in the insets. The bar plot shows the quantification of the fluorescence intensity (FI)/area (mean ± s.e.m., relative to the cholesterol + glutamate condition) in the processes of control and cholesterol‐replenished neurons. No differences in the FI/area values were found in control, glutamate‐stimulated (control = 1.67 ± 0.12, glutamate = 1.35 ± 0.07; P = 0.053; n = 3) or cholesterol‐replenished unstimulated neurons (control = 1.67 ± 0.12; cholesterol = 1.63 ± 0.19; P = 0.87; n = 3). Stimulation of cholesterol‐replenished neurons (chol + glut) provoked a significant decrease in the surface GluA2 staining (control = 1.67 ± 0.12; cholesterol + glutamate = 1.00 ± 0.12; P = 0.0018; n = 3). The data were compared using Mann‐Whitney non‐parametric t‐test. This second series of experiments indicates that the loss of cholesterol with aging may perturb certain aspects of cognition by virtue of defective AMPAR internalization, justifying the reduced LTD typical at this stage of life.

Cholesterol loss affects the lateral mobility of AMPA receptors In order to study how changes in cholesterol levels may affect AMPAR behavior in more detail, we next measured AMPAR lateral diffusion. LTD requires the rapid redistribution of receptors away from the synapse by lateral diffusion (Tardin et al, 2003), and the subsequent internalization of the displaced receptors by endocytosis is essential to sustain LTD (Carroll et al, 1999; Beattie et al, 2000). Mechanistically, it has been proposed that the mobility of synaptic but not extra‐synaptic receptors during LTD requires PI(3,4,5)P3 depletion (Arendt et al, 2010). Hence, the loss of cholesterol in the older cells would probably affect AMPAR displacement by lateral diffusion, similar to, and possibly as a consequence of reduced internalization (see Figs 3 and 4). To test this prediction, we used quantum‐dot‐based single molecule tracking (Fig 5 and Supplementary Methods). Figure 5.Lateral diffusion of GluA2‐AMPARs is altered in low‐cholesterol hippocampal neurons after LTD induction A. Phase contrast and fluorescence images showing the processes of hippocampal neurons where GluA2 subunits were labeled with quantum dots.

B. GluA2‐AMPARs were labeled using an anti GluA2 antibody and a biotinylated anti‐mouse Fab fragment conjugated to streptavidin‐coated quantum dots.

C. Image showing the reconstructed trajectories of individual GluA2‐AMPARs.

D, E. Glutamate stimulation increases the diffusion of synaptic and extra‐synaptic AMPARs in 15 DIV but not 30 DIV neurons. 15DIV: D i control = 0.023 ± 0.0028 μm 2 /s, D i glut = 0.0298 ± 0.0045 μm 2 /s ( P = 0.0201), D out control = 0.0613 ± 0.0099 μm 2 /s, D out glut = 0.0917 ± 0.0149 μm 2 /s ( P = 0.0054). 30 DIV: D in control = 0.0269 ± 0.0085 μm 2 /s, D in glut = 0.0231 ± 0.0044 μm 2 /s ( P = 0.4048) and D out control = 0.086 ± 0.0213 μm 2 /s, D out glut = 0.0771 ± 0.0207 μm 2 /s ( P = 0.5228).

F. The addition of cholesterol to 30 DIV neurons restores the response of synaptic AMPARs to glutamate. D in chol = 0.0250 ± 0.0035 μm2/s, D in chol + glut = 0.0337 ± 0.0047 μm2/s (P = 0.0254). Data information: In individual AMPAR tracking experiments the diffusion coefficients calculated from all the trajectories analyzed (ranging from 250 to 500 obtained from at least five different cultures) have a one‐tail distribution. The effect of glutamate in single experiments has been studied using non‐parametric Mann‐Whitney tests. The mean values of the medians follow a normal distribution and thus groups of experiments were compared using unpaired t‐test. Data information: In individual AMPAR tracking experiments the diffusion coefficients calculated from all the trajectories analyzed (ranging from 250 to 500 obtained from at least five different cultures) have a one‐tail distribution. The effect of glutamate in single experiments has been studied using non‐parametric Mann‐Whitney tests. The mean values of the medians follow a normal distribution and thus groups of experiments were compared using unpaired‐test. The instantaneous diffusion coefficients (D) for confined (within synapses, D in ) and non‐confined (beyond synapses, D out ) AMPARs were 0.023 ± 0.0028 μm2/s and 0.0613 ± 0.0099 μm2/s, respectively, in 15 DIV neurons (Fig 5D), similar to those in neurons cultured for 30 DIV: D in = 0.0269 ± 0.0085 μm2/s and D out = 0.086 ± 0.0213 μm2/s (Fig 5E). Control experiments where synapses were stained using the synaptic marker mitotracker (Groc et al, 2004) showed that the D in obtained for sites of confinement corresponded to D in particles within synapses (Supporting Information). As opposed to the basal levels, in the presence of 100 μM glutamate, there was an increase of the mean D in and D out in 15 DIV neurons (Fig 5D), while glutamate was unable to induce any change in AMPAR mobility in 30 DIV neurons, neither within nor beyond synapses (Fig 5E). To determine whether this reduced mobility in 30 DIV neurons was the consequence of these cells' lower cholesterol content, we repeated these measurements in the presence of the cholesterol‐replenishing solution (see above). Cholesterol replenishment did not affect AMPAR diffusion in control conditions, although glutamate stimulation increased the mobility of synaptic AMPARs, increasing the D in value in the cholesterol reinforced cells from 0.0250 ± 0.0035 μm2/s to 0.0337 ± 0.0047 μm2/s (P = 0.0254, Fig 5F). Since glutamate was used instead of NMDA to stimulate hippocampal neurons in culture, control experiments were performed to compare the effect of NMDA and glutamate on AMPAR lateral diffusion. We observed that the addition of 20 μM NMDA to 15 DIV neurons also increased the values of D in and D out , and no differences were observed with respect to the D values obtained by glutamate stimulation (Supplementary S4A and B). However, NMDA stimulation of cholesterol‐replenished 15 DIV neurons did not produce any differences compared to controls (Supplementary Fig S4B).

NMDA‐LTD is impaired in aged animals in a manner dependent on cholesterol loss In light of the fact that the loss of membrane cholesterol in old neurons perturbs AMPAR membrane internalization and lateral diffusion, two key events needed for LTD, this event was further studied by electrophysiology. Application of NMDA (30 μM) for 4 min induced strong depression in hippocampal slices obtained from 2‐month‐old (2M) mice, whereas no LTD was observed in slices from 20M mice (Fig 6A). Furthermore, LTD was efficiently induced in slices from middle‐aged, 10‐month‐old (10M) mice, confirming that reduced LTD in the 20M mice is part of the aging phenotype and manifested in early adulthood (Fig 6B). Figure 6.NMDA‐LTD is lost with age and restored by cholesterol replenishment NMDA‐LTD was observed in slices of 2‐month‐old mice (2M, ○), but it was absent in 20‐month‐old mice (20M, ♦), yet cholesterol replenishment restored LTD in old slices (20M + chol, ▵ gray filled). Cholesterol perfusion did not have any effect on 2M slices (2M + chol, □ gray filled). Empty boxes indicate the period of cholesterol perfusion. Gray squares represent the time of NMDA application. The plot shows the average ± s.e.m. of the responses collected from the last 20 min of the recordings. Values, 2M: 49.05 ± 2.94%; 2M + chol: 30.82 ± 7.68% (P2M, 2M+chol = 0.137); 20M: 82.69 ± 8.02%; 20M + chol: 48.61 ± 5.50% (P2M, 20M = 0.014; P20M, 20M+chol = 0.0093). Cholesterol depletion impaired NMDA‐LTD in middle‐aged hippocampal slices. Treatment with MβCD 2 h prior to NMDA addition abolished NMDA‐LTD in 10‐month‐old mice. Control (○), cholesterol depleted (■). The average ± s.e.m. of the responses collected from the last 20 min of the recordings for the control (38.43 ± 3.68%) and MβCD‐treated groups (90.82 ± 8.45%; P = 0.0003) are plotted on the right. Intraventricular infusion of cholesterol in old mice restored NMDA‐LTD. Field excitatory postsynaptic potentials (fEPSPs) were recorded from hippocampal slices of 24‐month‐old mice infused with cholesterol (♢) or vehicle (♦) in the lateral ventricle for 14 days. A significant NMDA‐LTD was obtained in slices prepared from cholesterol‐infused mice, but no LTD was observed in the vehicle‐treated group. The plot shows the average ± s.e.m. of the responses collected from the last 20 min of the recordings for control (IV vehicle: 104.02 ± 3.64%) and cholesterol‐treated group (IV chol: 68.05 ± 17.34%; P = 0.0286). Data information: The sample traces recorded at the times indicated as 1, 2, and 3 for each condition are shown in (A), (B), and (C). In all the cases, the data were compared using Mann‐Whitney non‐parametric t‐tests and the number of animals (n) is shown in the figure. Data information: The sample traces recorded at the times indicated as 1, 2, and 3 for each condition are shown in (A), (B), and (C). In all the cases, the data were compared using Mann‐Whitney non‐parametric‐tests and the number of animals () is shown in the figure. To determine whether the impaired LTD in the old was due to the loss of cholesterol that occurs naturally in the hippocampus in later life (see 1), hippocampal slices from old mice were incubated with the cholesterol replenishment mix (see above) and cholesterol replenishment was seen to rescue NMDA‐induced LTD in old hippocampal slices (Fig 6A). Strikingly, the LTD in slices from old animals treated with the cholesterol was very similar to that in untreated slices from young mice. To rule‐out any unspecific effects, slices from young mice (i.e., with normal cholesterol levels) were exposed to the cholesterol replenishment mix and their electrophysiological response was indistinguishable from that of the controls (Fig 6A). To determine whether cholesterol loss is sufficient to impair LTD, cholesterol was removed from hippocampal slices obtained from 10M mice with the drug MβCD, resulting in a net loss of 28.65 ± 16.77% cholesterol and a marked reduction in LTD (Fig 6B). Next, we infused cholesterol for 14 days into the lateral ventricle of 20M mice, after which the animals were sacrificed and hippocampal slices were prepared for electrophysiological recordings. While NMDA induced a brief and transient LTD in the vehicle‐treated group, a significant and long‐lasting depression was registered in slices from cholesterol‐infused aged mice (Fig 6C). No significant differences in the input‐output curves were observed in the conditions tested, indicating that basal synaptic transmission is not affected by changes in cholesterol (Supplementary Fig S5).

Constitutive cholesterol loss impairs LTD in the aged: in vivo studies To demonstrate the relevance of the age‐associated cholesterol loss in vivo, we measured LTD in the hippocampus of anesthetized 2M and 20M Wistar rats. This animal model was used as old rats are more resistant than mice to the anesthetic and recording procedures, and as rat aging is also accompanied by a loss of cholesterol in the hippocampus (Martin et al, 2008). To induce hippocampal LTD in rats, concentric bipolar stimulating electrodes were placed in the ipsilateral CA3 field for orthodromic activation of the CA1 field, where evoked potentials were recorded (Fig 7A). In this way, it could be seen that the LTD induced by local application of NMDA was robust in young adult rats and impaired in old animals (Fig 7B). Figure 7.Cholesterol rescues LTD and cognition in old rats Sagittal section of the hippocampus of anesthetized rats injected with fluorescent Bodipy‐cholesterol. The upper panel shows the merged phase contrast and fluorescence images. The sites where Bodipy‐cholesterol was injected (1), the Schaffer stimulation site (2), and the recording site (3) are indicated. Lower panel: magnification of the fluorescence image showing that Bodipy‐cholesterol diffuses from the injection sites (1) to the recording zone (2). The image corresponds to the magnification of one section of hippocampus, 60 min after cholesterol injection into the st. radiatum (40× 1.25 IMM OIL Leica laser‐scanning confocal microscopy). LTD was registered in 2‐month‐old anesthetized animals (2M, ○), but it was impaired in 20‐month‐old animals (20M, ♦). Cholesterol injection into the stratum radiatum of the old animals rescued LTD (20M + cholesterol, ▵ gray filled), but it had no effect in young animals (2M + chol, ☐ gray filled). The plot shows the evolution of the fEPSPs following the NMDA‐inducing LTD protocol (n = 5). Average fEPSPs were built over 10‐min periods (1 trial per min), and the same individuals were employed for control and cholesterol treatment using the left or right hippocampus. Representative traces are shown of fEPSPs recorded in the stratum radiatum in vivo before (solid traces) and 40 min after NMDA‐LTD (dotted traces) in 2M, 20M, and 20M rats injected with cholesterol (20M + cholesterol). The plot shows the average ± s.e.m. values of the responses collected from the last 40 min of the recordings from 2M (51.11 ± 4.73), 2M + cholesterol (2M + cholesterol: 40.83 ± 3.74; P2M, 2M + chol = 0.151), 20M (107.03 ± 2.78; P2M, 20M = 0.007), and 20M rats injected with cholesterol (20M + chol: 64.12 ± 6.30; P20M, 20M + chol = 0.008). The data were compared using Mann‐Whitney non‐parametric t‐tests. We tested whether the impaired LTD in old rats could be rescued by enhancing the availability of cholesterol, as observed in mouse hippocampal slices (Fig 6). Before proceeding with the study, we established optimal conditions to guarantee that cholesterol injected into the hippocampus would be able to infuse into a wide enough region to include the area recorded by the electrode. Thus, two injections of a cholesterol solution containing the fluorescent cholesterol derivative Bodipy‐cholesterol (3.5 μM) were applied to the apical dendritic layer of the CA1 region of an adult rat, 1 mm apart (Fig 7A). To verify adequate diffusion of Bodipy‐cholesterol to the recording site, hippocampal sections were examined by fluorescence microscopy 60 min later, when the injected Bodipy‐cholesterol could be seen to have infused into all the tissue located between the injection sites (Fig 7A). In addition, to avoid tissue damage in the recording zone, the evoked potentials were recorded from an intermediate location between the injection sites (Fig 7A). Having established these parameters, when we examined the electrophysiological recordings, a very strong LTD was evident in old animals that received a hippocampal injection of cholesterol 60 min before LTD induction (Fig 7B). As a control, young rats were also injected with cholesterol 60 min before the LTD induction, although no differences were observed between the treated and untreated animals (Fig 7B). To rule‐out variability between animals, the same rat was used as both the experimental and control case, that is, the cholesterol solution was injected into the hippocampus of one hemisphere and control solution into the contralateral hippocampus. The sterol specificity was tested by measuring LTD in aged rats injected in the hippocampus with oleic acid‐MβCD or with stigmasterol‐MβCD, neither of which restored LTD (Supplementary Fig S6), thereby confirming the specificity of cholesterol in the phenotype observed.