AMPA receptor-mTORC1 signaling activation is required for neuroplastic effects of LY341495 in rat hippocampal neurons

The group II metabotropic glutamate 2/3 (mGlu2/3) receptor antagonist LY341495 produces antidepressant-like effects by acting on mammalian target of rapamycin complex 1 (mTORC1) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors in rodent. We investigated whether LY341495 affects neuroplasticity via these mechanisms in rat primary hippocampal cultures under conditions of dexamethasone (DEX)-induced neurotoxicity. Ketamine was used for comparison. Hippocampal cultures were treated with LY341495 under conditions of DEX-induced toxicity. Changes in mTORC1-mediated proteins were determined by Western blotting analyses. Changes in dendritic outgrowth and spine density were evaluated via immunostaining. LY341495 significantly prevented DEX-induced decreases in the levels of mTORC1, 4E-BP1, and p70S6K phosphorylation as well as the levels of the synaptic proteins. These effects were blocked by pretreatment with the AMPA receptor inhibitor 2,3-dihydroxy-6-nitro-7sulfamoyl-benzo(f)quinoxaline (NBQX) and the mTORC1 inhibitor rapamycin. LY341495 significantly attenuated DEX-induced decreases in dendritic outgrowth and spine density. Pretreatment with rapamycin and NBQX blocked these effects of LY341495. Further analyses indicted that induction of BDNF expression produced by LY341495 was blocked by pretreatment with NBQX and rapamycin. LY341495 has neuroplastic effects by acting on AMPA receptor-mTORC1 signaling under neurotoxic conditions. Therefore, activation of AMPA receptor and mTORC1 signaling, which enhance neuroplasticity, may be novel targets for new antidepressants.


Results
Effects of LY341495 on mTORC1 signaling. To investigate the effects of ketamine and LY341495 on mTORC1 signaling in DEX-treated hippocampal cells, the phosphorylation levels of mTORC1, 4E-BP1, and p70S6K, as well as the expression levels of the synaptic proteins PSD-95 and GluA1, were determined by Western blotting.
Effects of the AMPA receptor inhibitor NBQX on activation of mTORC1 signaling induced by LY341495. The antidepressant actions of ketamine and LY341495 require stimulation of the AMPA receptor 17,22 . In addition, the induction of mTORC1 signaling by ketamine is blocked by AMPA receptor inhibition 17,22 .
To determine whether the AMPA receptor is involved in activation of mTORC1 signaling induced by LY341495, DEX-treated hippocampal cells were pretreated with the AMPA receptor inhibitor NBQX and the mTORC1 inhibitor rapamycin for 30 min prior to adding ketamine (100 µM) or LY341495 (100 µM). After a 96-h incubation with ketamine or LY341495, the phospho-Ser 2448 -mTORC1, PSD-95, and GluA1 levels were measured via Western blotting.
Effects of LY341495 on hippocampal dendritic outgrowth and spine density. Ketamine increases mTORC1 signaling, and this effect induces increased synaptogenesis in the prefrontal cortex 8 . To examine whether LY341495 increases neuroplasticity, ketamine (100 μM) or LY341495 (1, 10, and 100 μM) was added to hippocampal cells cultured with DEX (500 μM) and incubated for 5 days. Total dendritic length and spine formation were measured using antibodies to the dendritic marker MAP-2 and the spine marker phalloidin, respectively.
Changes similar to those observed in total dendritic length were observed for spine density (F [4,195] = 23.550, P < 0.001), which was significantly reduced in DEX-treated cells compared to control cells (0.5 vs. 1.4, respectively, P < 0.001) (Fig. 4). LY341495 significantly increased dendritic spine density in a concentration-dependent manner in DEX-treated cells (1 μM = 1.1, 10 μM = 1.2, 100 μM = 2.0, all P < 0.001). Ketamine at a dose of 100 μM also significantly increased spine density in DEX-treated cells (Supplemental Fig. S4A). The effects of rapamycin on increases in spine density induced by LY341495 or ketamine were examined, and two-way ANOVA revealed significant effects of both drug and drug × rapamycin interactions (Supplemental Table 2). Significant differences were also observed in spine density for ketamine, NBQX, and ketamine × NBQX interactions (Supplemental Table 2). Furthermore, statistical analyses of the effects of NBQX on the LY341495-induced increase in spine density revealed significant effects of LY341495 and LY341495 × NBQX interactions and a trend for the effects of NBQX (P = 0.074, Supplemental Table 2). Post hoc analyses (Fig. 4) showed that rapamycin and NBQX alone had no effect but inhibited the enhancement of spine density induced by LY341495 (LY341495 vs. rapamycin + LY341495, 2.8 vs. 2.0, respectively, P < 0.001; LY341495 vs. NBQX + LY341495, respectively, 3.5 vs. 2.5, Figure 3. Effects of LY341495 on total dendritic length in DEX-treated hippocampal cells: requirement for mTORC1 signaling and AMPA receptor activation. Cells were treated with LY341495 (1, 10, or 100 μM) or DMSO (non-drug treatment control, final concentration 0.5%) for 5 days with or without DEX (500 μM) (A). Cells were exposed to rapamycin (1 μM, B) or NBQX (50 μM, C) for 30 min prior to adding LY341495 (100 μM) or DMSO (control) for 5 days with DEX. In total, 400 cells from each group were analyzed. All data are expressed as the mean ± SEM. (A) * P < 0.01 vs. DMSO-treated, non-DEX-treated cells (control cells); † P < 0.01 vs. DMSO-treated, DEX-treated cells; (B,C). ** P < 0.01 vs. cells treated with DMSO alone; † † P < 0.01 vs. cells treated with LY31495 alone. P < 0.001) or ketamine (Supplemental Fig. S4B,C). These results indicate that the enhancement of dendritic outgrowth and spine density induced by LY341495 require activation of mTORC1 signaling and AMPA receptor.
Effects of LY341495 on BDNF expression: involvement with AMPA receptor-mTORC1 activation. Increases in spine formation induced by ketamine depend on BDNF release via activation of AMPA receptors and subsequent stimulation of mTORC1 signaling 23,24 . To determine whether LY341495 increases BDNF expression, hippocampal cells cultured with DEX (500 μM) were incubated with ketamine (100 μM) or LY341495 (1, 10, and 100 μM).

Discussion
The mGlu 2/3 antagonist LY341495 reversed the decreases in mTORC1, 4E-BP, and p70S6K phosphorylation and the decreases in synaptic proteins induced by DEX treatment in primary hippocampal neurons of rats. These effects were blocked by pretreatment of the cells with the mTORC1 inhibitor rapamycin and AMPA receptor inhibitor NBQX. In addition, LY341495 attenuated the decreases in dendritic outgrowth and spine density induced by DEX, and pretreatment with rapamycin and NBQX blocked these effects. LY341495 also increased BDNF expression, and this effect was blocked by rapamycin and NBQX.
Metabotropic glutamate (mGlu) receptors are G protein-coupled modulatory receptors in the glutamate receptor family 25 . There are eight subtypes of the mGlu receptor. The mGlu1 and mGlu2 receptors, which belong to Group I, are mainly excitatory, whereas the mGlu2 and mGlu3 receptors, which belong to Group II, and the mGlu4, mGlu6, mGlu7, and mGlu8 receptors, which belong to Group III, are inhibitory 26 . The following research drugs that bind to orthosteric mGlu 2/3 receptors have been shown to produce mGlu 2/3 receptor antagonistic effects: MGS0039, LY341495, and LY3020371 27 . Among these drugs, LY341495 is a highly potent and selective antagonist for mGlu2 and mGlu3 receptors, and several studies have shown that a group II metabotropic glutamate receptor antagonist has antidepressant effects similar to those of ketamine 28 . Bespalov et al. reported that intraperitoneal injection of LY341495 reduced the immobility time of mice in the FST 29 . Campo et al. reported that pre-administration of LY341495 dose-dependently reduced the immobility time of mice in the FST and that LY341495 also reduced their immobility time in the tail suspension test (TST) 30 . In addition to LY341495, MGS0039 has been shown to produce an increased antidepressant effect in the rat FST and mice TST as the dose increased 31 . Dwyer et al. also reported that administration of LY341495 to rats with anhedonia caused by chronic unpredictable stress improved anhedonia rapidly and for a relatively long time 32 . Another study confirmed that the antidepressant effect of LY341495 in the rat FST was blocked by the mTOR inhibitor rapamycin 16 . These findings suggest that mGlu2/3 antagonists may have antidepressant effects.
In addition to the research on antidepressant effects based on these animal models of depression, there have also been studies of the molecular effects of mGlu 2/3 antagonists. LY341495 increased the phosphorylation levels of mTOR, P70S6K, and 4E-BP1 and the expression of synaptic proteins PSD-95, GluA1, and Synapsin I in the rat prefrontal cortex 16 . LY341495 also increased phosphorylated ERK and phosphorylated S6K levels in primary cortical neurons as a function of dose; BDNF release also increased significantly as a function of time 13 . Chaki et al. observed that LY341495 stimulated 5-HT1A receptors in the medial prefrontal cortex (mPFC) of mice to activate PI3K/AKT signaling and induce antidepressant effects. The effect of LY341495 was reduced when rapamycin was administered by mPFC 33 . In addition, the decreased BDNF and PSD-95 expression in the prefrontal cortex, dentate gyrus, and hippocampus CA3 were reversed when MGS00039 was administered to socially-defeated stressed mice 34 . These results suggest that mGlu 2/3 antagonists can regulate neuroplasticity by activating mTORC1 signaling and increasing expression of synaptic proteins.
In this study, we conducted experiments in a DEX-induced toxicity environment. We confirmed that LY341495 increased mTORC1 signaling in primary hippocampal neurons of rats. DEX is a corticosteroid and has excitotoxic effects on nerve cells. Particularly, exposure to DEX in the prenatal period may be associated with the development of depression. Therefore, we applied the DEX toxicity model in this study [35][36][37] .
We observed that LY341495 treatment reversed the decreased phosphorylation levels of mTORC1, 4E-BP1 and p70S6K, and the expression of PSD95 and GluA1. Similar results were also observed with ketamine treatment. Li et al. observed that ketamine increased the number of synaptic proteins and activated mTORC1 signaling in the PFC of rats 8 . In addition, ketamine has also been shown to activate mTORC1 signaling and increase the expression of synaptic proteins in primary hippocampal neurons of rats 38 . In addition to the activation of www.nature.com/scientificreports www.nature.com/scientificreports/ mTORC1 signaling, recent studies have shown that ketamine rapidly increased glutamate release and activated AMPA receptors and mTORC1 signaling; the latter, in turn, stimulated BDNF release and Akt activation, facilitating synaptogenesis 39 . Increased BDNF release promotes the synthesis of synaptic proteins, resulting in the rapid antidepressant effect of ketamine. In the present study, we found that the decreases in the expression levels of mTORC1 signaling proteins in the DEX-induced toxic environment were reversed and the decreases in expression levels of BDNF, PSD-95, and GluA1 were also reversed by LY341495 treatment. These results suggest that LY341495, an mGlu 2/3 antagonist, has a mode of action similar to that of ketamine.
We also evaluated how LY341495 affected AMPA receptors. Several studies have shown that positive allosteric modulators of AMPA receptors have antidepressant effects 27,40 . In one study, rats' immobility time was significantly decreased by a single LY341495 injection before the FST 17 . In other studies, when NBQX was pre-administered before the FST, the antidepressant effects of LY341495 were dose-dependently decreased, and NBQX alone did not affect the immobility time 17,22 . In another study, NBQX reduced the antidepressant effects of ketamine and LY341495 co-administered in the FST 41 ; additionally, pre-administration of LY341495 decreased feeding latency in the novelty-suppressed feeding test, which was blocked by NBQX 42 , and pre-administration of NBQX also blocked increased dopamine release by LY341495 to the basal level in the nucleus accumbens of rats 43 . We observed that the increase in mTORC1 phosphorylation and increases in PSD-95 and GluA1 expression by LY341495 treatment in primary hippocampal neurons were blocked by NBQX. We also found that the increases in mTORC1 phosphorylation and in PSD-95 and GluA1 expression by ketamine were also blocked by NBQX. These results indicate that LY341495 has neuroplastic effects that increase synaptic protein expression by activating the AMPA receptor via a mode of action similar to ketamine 41,44,45 . To the best of our knowledge, this is the first study to examine how LY341495 affects the expression of synaptic proteins by acting on AMPA receptors in rat primary hippocampal neurons.
Stress affects neuroplasticity, resulting in neuronal changes that lead to chronic depression and altered synaptic density and synapse numbers, which lead to depression 46,47 . Decreased synaptic function and numbers in the dorsolateral prefrontal cortex of depressed patients have also been reported 48 . These results suggest that the depressed brain may show changes in neuroplasticity. In the present study, DEX treatment decreased the BDNF level, total dendritic outgrowth, and spine density of primary hippocampal cells of rats, and both LY341495 and ketamine treatment dose-dependently reversed these effects, confirming that LY341495 affects BDNF level, dendritic outgrowth, and spine density. Our results are similar to those of previous studies 38,49,50 . Zanos et al. reported that the ketamine metabolite (2R,6R)-hydroxynorketamine-(2R,6R)-HNK acts on mGlu2 receptor and that combined sub-effective doses of the mGlu 2/3 receptor antagonist LY341495 and (2R,6R)-HNK have synergistic effects on gamma oscillations and antidepressant-relevant behavioral actions 51 . They also reported that the ketamine metabolite (2R,6R)-hydroxynorketamine-(2R,6R)-HNK increases AMPA-GluA1 receptor expression and activity as well as the level of BDNF and phosphorylation of eEF2K 52 . Lepack et al. reported that LY341495 stimulates ERK signaling in primary cortical cultures to promote secretion of BDNF and that this mechanism of action is blocked by pretreatment with the TrkB inhibitor K252a 13 . We also observed that ketamine and LY341495 increased ERK phosphorylation levels (Supplemental Fig. S6). Therefore, although we did not observe antidepressant effects of LY341495 in animal models of depression, LY341495 may have an antidepressant effect with a synaptic mechanism similar to ketamine (Fig. 6).
This study had some limitations. First, LY341495 activated mTORC1 signaling only under toxic conditions. In contrast to our observations, a previous study showed that ketamine promotes mTORC1 signaling in PFC in non-depressed mice 53 . Further investigations, including additional animal model studies, are therefore necessary to clarify this issue. In addition, we used relatively high concentrations of LY341495 in this study. Although LY341495 has strong antagonistic activity against the mGlu 2/3 receptor, LY341495 does not act only on the mGlu 2/3 receptor, and LY341495 also has relatively potent antagonist activity and affinity for mGlu7 and mGlu8 receptors 54 . At the concentrations used in the present study, it is possible that LY341495 acted on mGlu7 and mGlu8 receptors as well. Furthermore, it is unclear whether the concentrations of LY341495 used in this study were sufficient for promoting mTORC1 signaling in vivo, especially in the human central nervous system. This discrepancy may be due to differences in the conditions of an in vivo human brain versus an in vitro cell culture. Finally, only 50 µM NBQX was used in this study. Therefore, it is necessary to investigate the effects of NBQX at various other concentrations. More advanced and well-designed studies are necessary to overcome these limitations.
This was the first study to investigate the effects of LY341495 on mTORC1 activation in the primary hippocampal neurons of rats. LY341495 activated the mTORC1 signaling pathway and neuroplastic changes, including increased BDNF expression, dendritic outgrowth, spine density, and synaptic proteins, under conditions of DEX-induced toxicity. These neuroplastic changes were blocked by the mTORC1 inhibitor rapamycin and the AMPA receptor antagonist NBQX. These findings suggest that LY341495 regulates neuroplasticity through AMPA receptors and mTORC1 signaling activation and that LY341495 has a mechanism of action similar to that of ketamine. Therefore, the mechanism of action of the mGlu 2/3 antagonists may be a suitable target for the development of new antidepressants. www.nature.com/scientificreports www.nature.com/scientificreports/ blotting analyses, cells were plated at 2 × 10 5 cells per six-well dish. For immunostaining, cells were plated on 18 × 18-mm coverslips in 12-well dishes at a density of 2 × 10 4 (dendritic outgrowth) and 5 × 10 3 cells (spine density). Cells were grown at 37 °C and 5% CO 2 for 10 days.

Methods
Drug treatment. After 10 days of incubation, the cells were cultured with LY341495 (1, 10, 100 µM; Tocris Bioscience) or ketamine (100 µM; Huons) in the presence of DEX (500 µM; Sigma) for 4 days (Western blotting analyses) and 5 days (immunostaining analyses). To study the blocking effects, cells were treated with 50 µM NBQX (Calbiochem) or 1 µM rapamycin (Calbiochem) 30 min prior to LY341495 or ketamine. The culture medium and these drugs were changed every 2 days. A concentration of 500 µM DEX was selected because cell viability was 75-80% at this dose 56 . The concentrations of LY341495 used in this study were based on the observation that these concentrations (1, 10, and 100 µM) lead to concentration-dependent increases in the levels of mTORC1 phosphorylation under DEX-induced toxic conditions; lower concentrations (<1 µM) have no effect on the level of mTORC1 phosphorylation (data not shown). The concentrations of ketamine were based on a previous study that found that this concentration enhances mTORC1 phosphorylation as well as dendritic outgrowth in hippocampal cells 38 .

Western blotting analyses.
Western blotting experiments were performed as described previously 57 . Immunostaining analyses for dendritic outgrowth and spine density. Cells were fixed in 4% paraformaldehyde. For dendritic outgrowth, cells were incubated with anti-microtubule-associated protein (MAP)-2 antibody (1:1000, MAB3418; Millipore). Alexa Fluor ® 568 goat anti-mouse IgG (Invitrogen) was used as a secondary antibody, and Hoechst 33258 (Invitrogen) was used for nuclear staining. Stained cells were mounted on cover glasses and observed through a fluorescence microscope (Olympus). To analyze the total dendritic length, www.nature.com/scientificreports www.nature.com/scientificreports/ five fields were randomly selected from each group, and two independent cultures were performed. All neurons in a given field were counted, including both basal and apical dendrites. Dendritic length was determined as the distance between the edge of the cell body and the tip of the growth cone. Total dendritic length was obtained by summing the lengths of all dendrites from a single neuron and then averaging this measure in each group using the automated image-analysis program MetaMorph (Molecular Devices) 58 . At least 400 cells were analyzed in 10 fields by a researcher blinded to the groups. To determine spine density, cells were incubated with Alexa Fluor ® 488 Phalloidin (1:40; Molecular Probes) and mounted for image acquisition. Images were captured at a resolution of 1024 × 1024 pixels, using an LSM 510 META laser-scanning confocal microscope (Carl Zeiss) with a 63 × oil immersion objective lens. To analyze spine density, we differentiated between spines and filopodia by shape and length. Spines were defined as less than 3 µm in length with a round or mushroom shape, and filopodia were defined as between 3 and 10 µm in length with a narrow shape. Ten neurons were randomly selected from each group, and three independent cultures were performed. In each 30 neuron group, two 50 µm dendritic segments per neuron were analyzed (50-60 dendritic segments per group) by a researcher blinded to the groups. To represent the average spine density in a 10 µm dendrite, the spine density of 50 µm dendritic segments was divided by 5.
Statistical analyses. All statistical analyses were performed using GraphPad Prism software (ver. 7.03; GraphPad Software). Dose studies for ketamine and LY341495 were analyzed by one-way analysis of variance (ANOVA). For blockade experiments, two-way ANOVA was used to assess the main effect of drug (ketamine or LY341495) or inhibitor (rapamycin or NBQX) and the interaction between drug and inhibitor (ketamine × rapamycin and ketamine × NBQX; LY341495 × rapamycin and LY341495 × NBQX). For post hoc comparison, Tukey's multiple-comparison test was carried out. In all analyses, P < 0.05 was taken to indicate statistical significance.

Data availability
The data are available from the corresponding author upon request.