Intrahippocampal glutamine administration inhibits mTORC1 signaling and impairs long-term memory

Abstract

The mechanistic Target of Rapamycin Complex 1 (mTORC1), a key regulator of protein synthesis and cellular growth, is also required for long-term memory formation. Stimulation of mTORC1 signaling is known to be dependent on the availability of energy and growth factors, as well as the presence of amino acids. In vitro studies using serum- and amino acid-starved cells have reported that glutamine addition can either stimulate or repress mTORC1 activity, depending on the particular experimental system that was used. However, these experiments do not directly address the effect of glutamine on mTORC1 activity under physiological conditions in nondeprived cells in vivo. We present experimental results indicating that intrahippocampal administration of glutamine to rats reduces mTORC1 activity. Moreover, post-training administration of glutamine impairs long-term spatial memory formation, while coadministration of glutamine with leucine had no influence on memory. Intracellular recordings in hippocampal slices showed that glutamine did not alter either excitatory or inhibitory synaptic activity, suggesting that the observed memory impairments may not result from conversion of glutamine to either glutamate or GABA. Taken together, these findings indicate that glutamine can decrease mTORC1 activity in the brain and may have implications for treatments of neurological diseases associated with high mTORC1 signaling.

Figure 1.

Intrahippocampal administration of glutamine decreases mTORC1 activity. Intrahippocampal infusions of glutamine (37 μg/hippocampus, n = 6) or an equal volume of vehicle (saline) were administered to animals, and samples collected 30 min later. (A) Glutamine administration significantly decreased the phosphorylation, but not the total levels, of S6 (two-tailed, paired Student's t-test; P = 0.049). (B) The phosphorylation levels of S6K were also decreased, although it did not reach significance (two-tailed, unpaired Student's t-test; P = 0.16). (C) Glutamine administration did not significantly affect the phosphorylation levels of Tsc2, AMPK, ERK, or Akt assessed at 30 min post-infusion. (D) Intrahippocampal infusions of glutamine (52 μg/hippocampus, n = 3) or vehicle (saline) were administered and brain extracts collected 30 min later to be analyzed by HPLC. Glutamine administration significantly increased the levels of glutamine (two-tailed, unpaired Student's t-test; P = 0.008), but not glutamate or aspartate. Sample loading was corrected by normalization to the β-actin signal. Summary data are presented as the mean ± SEM.

Figure 2.

Glutamine bath application does not lead to significant changes in glutamatergic synaptic signaling. CA1 pyramidal neurons in horizontal hippocampal slice preparations were recorded at −65 mV in voltage-clamp mode using a cesium-based internal solution. (A) Representative experiment showing excitatory postsynaptic currents (EPSCs) evoked by paired pulses (10 Hz) applied to Schaffer collateral axons, prior to (black trace) and during bath application of L-glutamine (2.5 mM; red trace). Traces shown are the averages of 30 trials. (B) Representative trials showing spontaneous EPSCs recorded in the same CA1 pyramidal cell as in A, prior to (black trace) and during (red trace) application of L-glutamine (2.5 mM). (C) Summary data (mean ± SEM; n = 5 neurons) quantifing the evoked EPSC amplitude, paired-pulse ratio (EPSC2/EPSC1), and frequency of spontaneous EPSCs (sEPSCs) recorded in the presence of 2.5 mM glutamine, normalized to their respective values recorded in the control solution.

Figure 3.

Glutamine application does not lead to significant changes in GABAergic synaptic signaling. CA1 pyramidal neurons were held at 0 mV using a cesium-based internal recording solution. (A) A representative experiment showing disynaptic inhibitory postsynaptic currents (IPSCs) evoked by Schaffer collateral stimulation, prior to (black trace) and during application of L-glutamine (2.5 mM; red trace). Traces shown are the averages of 30 trials. (B) Representative trials showing spontaneous IPSCs recorded in the same CA1 pyramidal cell as A, prior to (black trace) and during (red trace) application of L-glutamine (2.5 mM). (C) Summary data (mean ± SEM; n = 5 neurons) quantifing the evoked IPSC amplitude and frequency of spontaneous IPSCs (sIPSCs) in the presence of 2.5 mM glutamine, normalized to their respective values recorded in the control solution.

Figure 4.

Post-training intrahippocampal administration of glutamine (37 μg/hippocampus) impairs long-term spatial memory. (A) Rats were trained in a 1-d abbreviated Morris water maze protocol and infused with either vehicle (saline; n = 8) or 37 μg/hippocampus of glutamine (n = 7) immediately after the last training trial. A probe trial was given 48 h later. (B) Glutamine (Gln)-infused mice had significantly fewer platform crossings during the probe trial (two-tailed, unpaired Student's t-test; P < 0.038). (C) Representative probe trial traces of a vehicle and a glutamine-infused animal showing their swim paths. (D) Dwell time in counter areas of decreasing diameters (4×, 3×, and 2× platform radius) during the probe trial was also significantly decreased in Gln-infused mice (two-way repeated-measures of ANOVA with treatment and ring number as between-subject factors: F_(3,36) = 5.015, P = 0.005). (E) There was no difference in swimming speed during the probe trial. (F) There was no difference in the latency to a visual platform performed after the probe trial. Data are presented as the mean ± SEM. (*) P < 0.05, (^‡) P < 0.01.

Figure 5.

Post-training intrahippocampal administration of glutamine (52 μg/hippocampus) impairs long-term spatial memory. Rats were infused with either vehicle solution (n = 9) or 52 μg/hippocampus of glutamine (n = 8) immediately after the last training trial. A probe trial was given 48 h later. Glutamine infused animals (A) required significantly longer search-time to find the hidden platform (two-tailed, unpaired Student's t-test; P = 0.04) and (B) crossed the platform fewer times than did vehicle-infused controls (two-tailed, unpaired Student's t-test; P = 0.03). (C) Representative probe trial traces of a vehicle and a glutamine-infused animal during the probe trial showing the paths taken. (D) Dwell time in counter areas of decreasing diameters (4×, 3×, and 2× platform radius) during the probe trial was also significantly decreased in Gln-infused mice (two-way repeated-measures of ANOVA with treatment and ring number as between-subject factors: group main effect F_(1,15) = 10.052, P = 0.006). (E) There was no difference in swimming speed during the probe trial. (F) There was no difference in latency to a visual platform performed after the probe trial. Data are presented as the mean ± SEM, (*) P < 0.05, (‡) P < 0.01.

Figure 6.

Intrahippocampal coadministration of leucine and glutamine resulted in no net change in mTORC1 activity. (A,B) Intrahippocampal infusions of leucine (27 μg/hippocampus, n = 5) or vehicle (saline) were administered and samples collected 30 min later. Leucine administration significantly increased the phosphorylation of mTORC1 targets (A) S6 (two-tailed, paired Student's t-test; P = 0.01) and (B) S6K (two-tailed, paired Student's t-test; P = 0.03). (C,D) Intrahippocampal coinfusion of leucine and glutamine (27 μg/hippocampus, 52 μg/hippocampus, respectively, n = 5) or vehicle (saline) were administered and samples collected 30 min later. There were no significant effects on the levels of C phosphorylated S6K or total S6K. (D) There were no significant effects of glutamine and leucine coadministration on the phosphorylation or total levels of S6. Sample loading was normalized to the β-actin signal. Summary data are presented as the mean ± SEM, (*) P < 0.05.

Figure 7.

Post-training intrahippocampal coadministration of leucine and glutamine resulted in no net effect on long-term spatial memory formation. (A) Rats were trained in a 1-d Morris water maze protocol and infused with either 27 μg/hippocampus of leucine + 52 μg/hippocampus of glutamine (n = 9) or vehicle solution (n = 9) immediately after the last training trial. A probe trial was given 48 h after training detected no difference in either the (B) number of crossings or the (C) dwell time in counter areas of decreasing diameters (4×, 3×, and 2× platform radius). (D) Representative image of a cresyl violet-stained section showing an infusion needle track (arrow) that terminates within the hippocampus. (E) Summary representation of cannula placements for the animals used in this experiment. Data are presented as the mean ± SEM.