Adaptive Changes in Group 2 Metabotropic Glutamate Receptors Underlie the Deficit in Recognition Memory Induced by Methamphetamine in Mice

mGlu2/3 receptors genetic deletion or pharmacological blockade abrogates methamphetamine (Metha)-induced memory deficit in mice. Exploration time in seconds (sec) during the retention phase in the NOR task in wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. All mice were tested after 7 d of withdrawal (i.e., day 9 d 11). D–F, The performance on the NOR task for wild-type mice after 7 d of withdrawal following the same treatment with saline or Metha and injected 30 min before the training phase in the NOR paradigm with vehicle (DMSO, 40 µl i.p.), the selective negative modulator of the mGlu2 (VU6001966, 10 mg/kg, i.p.), or mGlu3 (VU0650786, 30 mg/kg, i.p.) receptor, respectively. Data are means ± SEM: Wilcoxon two-tailed matched-pair signed–rank test in A, B, D, and E (metha) and F (saline); two-way ANOVA for repeated measures with Bonferroni’s for multiple comparisons (novel vs familiar, F_(1,44)= 23.72; p = 1.5  *10⁻⁵; treatment, F_(1,44)= 0.0021; p = 0.96; interaction, F_(1,44)= 0.0059; p = 0.94) in C; paired two-tailed Student’s t test in F, metha (t = 2.375; df = 7). *p values are shown in the figure.

DI for wild-type, mGlu2^−/−, and mGlu3^−/− mice subjected to the NOR test following treatment with saline or methamphetamine (Metha). DI was calculated as a measure of relative novelty preference as follows: DI = (times spent exploring novel object) / (total time exploring both objects). A–C, Values of DI for wild-type, mGlu2^−/−, and mGlu3^−/− mice subjected to the NOR test after 7 d of withdrawal following treatment with saline or Metha. Values of DI for wild-type mice treated with saline or Metha (1 mg/kg, i.p. for 5 consecutive days) and subjected to the NOR test after 7 d of withdrawal in the presence or absence of NAMs of mGlu2 (VU6001966) or mGlu3 (VU0650786) receptors are shown in D–F, respectively. Vehicle (DMSO, 40 µl i.p.) or the respective compounds (VU6001966, 10 mg/kg, i.p.; VU0650786, 30 mg/kg, i.p.) were injected 30 min before the training phase in the NOR paradigm. Data are means ± SEM: unpaired t test in A (t = 2.485, df = 34); Mann–Whitney in D (U = 11). *p values are shown in the figure.

mGlu2/3 expression is upregulated in the PFC of methamphetamine (Metha)-treated wild–type mice. Western blot analysis for mGlu2/3 receptors expression in the PFC of wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. All mice were killed after 7 d of withdrawal (i.e., Day 11). Prestained protein standards were loaded on the first lane as molecular mass markers (M). Densitometric values of the dimer (250 kDa), the monomer (100 kDa), or the sum of dimer + monomer (total) were analyzed. Protein extracts from the PFC of wild-type mice or mGlu2^−/−, mGlu3^−/−, or mGlu2,3^−/− mice were used as control/negative controls. Data are means ± SEM: Mann–Whitney two-tailed test [U = 4 for A (total) and A (dimer); U = 5 for A (monomer); U = 13 for B (monomer)]. *p values are shown in the figure.

Methamphetamine (Metha) treatment does not affect the mGlu2/3 receptor expression in the NAc of wild-type, mGlu2^−/−, and mGlu3^−/− mice. Western blot analysis for mGlu2/3 receptors expression in the NAc of wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. Mice were subjected to the NOR test at 7 d of withdrawal following the treatment with saline or Metha. All mice were killed after the NOR test. Prestained protein standards were loaded on the first lane as molecular mass markers (M). Densitometric values of the dimer (250 kDa), the monomer (100 kDa), or the sum of dimer + monomer (total) were analyzed. Protein extracts from the NAc of wild-type mice or mGlu2^−/−, mGlu3^−/−, or mGlu2,3^−/− mice were used as control/negative controls. Data are means ± SEM.

Methamphetamine (Metha) treatment does not affect the mGlu2/3 receptor expression in the hippocampus of wild-type, mGlu2^−/−, and mGlu3^−/− mice. Western blot analysis for mGlu2/3 receptors expression in the hippocampus of wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. Mice were subjected to the NOR test at 7 d of withdrawal following the treatment with saline or Metha. All mice were killed after the NOR test. Prestained protein standards were loaded on the first lane as molecular mass markers (M). Densitometric values of the dimer (250 kDa), the monomer (100 kDa), or the sum of dimer + monomer (total) were analyzed. Protein extracts from the hippocampus of wild-type mice or mGlu2^−/−, mGlu3^−/−, or mGlu2,3^−/− mice were used as control/negative controls. Data are means ± SEM.

Exposure to NOR does not affect the mGlu2/3 receptor expression in the PFC and NAc of wild-type mice. Western blot analysis for mGlu2/3 receptors expression in the PFC (A) and NAc (B) of wild-type mice treated with saline or methamphetamine (Metha; 1 mg/kg, i.p.) for 5 consecutive days. All mice were killed after 7 d of withdrawal (i.e., Day 11). Mice were not subjected to the NOR test. Prestained protein standards were loaded on the first lane as molecular mass markers (M). Densitometric values of the dimer (250 kDa), the monomer (100 kDa), or the sum of dimer + monomer (total) were analyzed. Protein extracts from the PFC (A) or NAc (B) of mGlu2^−/−, mGlu3^−/−, or mGlu2,3^−/− mice were used as negative controls. Data are means ± SEM. Unpaired two-tailed Student’s t test t (t = 4.821; df = 14 for PFC dimer + monomer; t = 3.191; df = 14 for PFC monomer; t = 5.796; df = 14 for PFC dimer). *p values are shown in the figure.

Methamphetamine (Metha) treatment does not affect the mGlu2/3 receptor expression in the perihinal cortex of wild-type, mGlu2^−/−, and mGlu3^−/− mice. Immunohistochemical analysis for mGlu2/3 receptors expression in the perirhinal cortex of wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. Mice were subjected to the NOR test at 7 d of withdrawal following the treatment with saline or Metha. All mice were killed after the NOR test. Densitometric values of mGlu2/3 immunoreactivity (ir) in the perirhinal cortex of wild-type, mGlu2^−/−, and mGlu3^−/− mice are shown in A′–C′, respectively.

Effect of methamphetamine treatment on signal transduction mechanisms of mGlu2 and mGlu3 receptors in the PFC. cAMP levels in PFC slices from wild-type, mGlu2^−/−, and mGlu3^−/− mice treated with saline or methamphetamine (1 mg/kg, i.p.), for 5 consecutive days and killed after 7 d of withdrawal are shown in A–C, respectively. Slices were pretreated with LY379268 (3 µM, 10 min before FSK) and then stimulated for 20 min with FSK (10 µM). Graphs in A′–C′ show the percentage of reduction of FSK-induced cAMP formation by LY379268 for each genotype. Values are means ± SEM: (A) One-way ANOVA with Bonferroni's multiple-comparison test (F_(2,30)= 46.11; p = 7.1  *10⁻¹⁰ for saline; F_(2,36)= 49.54; p = 4.6  *10⁻¹¹ for methamphetamine); (B,C) Kruskal–Wallis test with Dunn's multiple-comparison test (Kruskal–Wallis statistic, 13.55 in B, saline; 14.9 in B, methamphetamine; 12.29 in C, saline; 14.01 in C, methamphetamine). *p values are shown in the figure. D, Stimulation of PI hydrolysis by DHPG combined or not with LY379268 in slices of PFC prepared from wild-type mice treated with saline or methamphetamine (1 mg/kg, i.p.), for 5 consecutive days and killed after 7 d of withdrawal. Slices were treated with DHPG (200 µM, 60 min) in the absence or presence of LY379268 (3 µM). LY379268 was not tested in the absence of DHPG because it is known to be ineffective on PI hydrolysis on its own in the adult mouse brain (Di Menna et al., 2018). Values are means ± SEM: saline, One-way ANOVA with Bonferroni's multiple-comparison test (F_(2,21)= 46.22; p = 2.03  *10⁻⁸); methamphetamine, Kruskal–Wallis test with Dunn's multiple-comparison test (Kruskal–Wallis statistic, 12.5). *p values are shown in the figure.

Methamphetamine treatment alters the modulation of [³H]-D-aspartate release by mGlu2/3 receptors in PFC synaptosomes. Depolarization-induced [³H]-D-aspartate (Asp) release in superfused synaptosomes prepared from the PFC of wild-type (A), mGlu2^−/− (B), and mGlu3^−/− (C) mice treated with saline or methamphetamine (1 mg/kg, i.p.), for 5 consecutive days and killed after 7 d of withdrawal. Synaptosomes were transiently (90 s) exposed to 15 mM KCl containing medium. When indicated, LY379268 (1–10 nM) was added concomitantly to the depolarizing stimulus. Fractions were collected as follows: two 3 min fractions (basal release), one before (t = 36–39 min) and one after (t = 45–48 min) a 6 min fraction (t = 39–45 min) containing the evoked release. The KCl-evoked [³H]-D-aspartate overflow was estimated by subtracting the neurotransmitter content into the first and the third fractions collected (basal release) from that in the 6 min fraction collected during and after the depolarization pulse (evoked release), and it is expressed as induced overflow over basal release (%). Values are means + SEM: one-way ANOVA with Bonferroni's multiple-comparison test for A, saline (F_(2,21)= 13.41; p = 1.8  *10⁻⁴), and B, saline (F_(2,21)= 7.31; p = 3.9 1.8  *10⁻³); Kruskal–Wallis test with Dunn's multiple-comparison test for A, methamphetamine (Kruskal–Wallis statistic, 10.15). *p values are shown in the figure.

vGlut1 (vesicular glutamate transporter 1), Rab3A (Ras-related protein 3A), and AGS3 (activator of G-protein signaling 3) expression is upregulated in the PFC of methamphetamine (Metha)-treated wild-type mice. Western blot analysis for vGlut1 (A), Rab3A (B), and AGS3 (C) expression in the PFC of wild-type, mGlu2^−/−, and mGlu3^−/− mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. All mice were killed after 7 d of withdrawal (i.e., Day 11). Data are means ± SEM: Mann–Whitney two-tailed test (U = 0 for A wild-type mice; U = 1 for A mGlu3^−/− mice, and C wild-type mice; U = 8 for B wild-type mice). *p values are shown in the figure.

Methamphetamine (Metha) treatment does not affect the expression level of STX 1A (syntaxin 1A), Syn (synaptophysin), Munc-18 (mammalian homolog of the unc-18 gene), and xCT (cystine-glutamate antiporter) in the PFC of Metha-treated of wild-type, mGlu2^−/−, and mGlu3^−/− mice. Western blot analysis for STX 1A (A), Syn (B), Munc-18 (C), and xCT (D) expression in the PFC of wild-type, mGlu2^−/−, and mGlu3^−/− mice treated with saline or Metha (1 mg/kg, i.p.) for 5 consecutive days. All mice were killed after 7 d of withdrawal (i.e., Day 11). Prestained protein standards were loaded on the first lane as molecular mass markers (M). Data are means ± SEM.