Depression-Associated Negr1 Gene-Deficiency Induces Alterations in the Monoaminergic Neurotransmission Enhancing Time-Dependent Sensitization to Amphetamine in Male Mice

Abstract

In GWAS studies, the neural adhesion molecule encoding the neuronal growth regulator 1 (NEGR1) gene has been consistently linked with both depression and obesity. Although the linkage between NEGR1 and depression is the strongest, evidence also suggests the involvement of NEGR1 in a wide spectrum of psychiatric conditions. Here we show the expression of NEGR1 both in tyrosine- and tryptophan hydroxylase-positive cells. Negr1^-/- mice show a time-dependent increase in behavioral sensitization to amphetamine associated with increased dopamine release in both the dorsal and ventral striatum. Upregulation of transcripts encoding dopamine and serotonin transporters and higher levels of several monoamines and their metabolites was evident in distinct brain areas of Negr1^-/- mice. Chronic (23 days) escitalopram-induced reduction of serotonin and dopamine turnover is enhanced in Negr1^-/- mice, and escitalopram rescued reduced weight of hippocampi in Negr1^-/- mice. The current study is the first to show alterations in the brain monoaminergic systems in Negr1-deficient mice, suggesting that monoaminergic neural circuits contribute to both depressive and obesity-related phenotypes linked to the human NEGR1 gene.

Keywords: Negr1; depression; dopamine; genetic models; serotonin.

Figure 1

Schematic overview of the cohorts of mice and tests/measurements performed in the current study. For the estimation of the treatment of acute and chronic amphetamine (cohort I), two subgroups of mice were used: cohort Ia for the estimation of the dose curve (data shown in Supplementary Figure S1) and cohort Ib for the chronic amphetamine administration. An open field test for baseline activity of cohort Ib mice was performed 7 days before the administration of amphetamine. Cohort II was used for the estimation of the treatment of chronic escitalopram and cohort III was used for baseline measurement of gene expression and IHC stainings.

Figure 2

Negr1^−/− mice are more sensitive to chronic amphetamine administration. Effect of chronic amphetamine on (A,D) distance traveled, (B,E) corner visits, and (C,F) rotations in the open field test. In G and H saline/amphetamine groups have not been separated yet, the body weight change is a reaction to non-pharmacological environmental manipulations. (G) Body weight dynamics measured during 1 week of the period before amphetamine injection (from day -10 until day -3) (H). Cumulative body weight change before saline/amphetamine injections (from day -10 until day -3). (I) Body weight change caused by chronic saline or amphetamine injections and behavioral testing (from day 1 until day 10). Data represents mean ± SEM, +—p < 0.01—the difference in treatment in WT mice, #—p < 0.05—the difference in treatment in Negr1^−/− mice (Tukey post hoc test, (A–C)); ++—p < 0.01—genotype effect, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Mann–Whitney, (D–F); Bonferroni post hoc test, (G–I)). AUC, area under curve.

Figure 3

The level of dopamine system-related genes in the VTA of mice. Relative mRNA expression levels of (A) dopamine transporter (Dat), (B) tyrosine hydroxylase (Th), (C) dopamine receptor D2 (Drd2), (D) catechol-O-methyltransferase (Comt) in Negr1^−/− mice and their WT littermates after 10 days of chronic saline or amphetamine i.p. injection (cohort I). The levels of (E) dopamine transporter (Dat), (F) tyrosine hydroxylase (Th), (G) dopamine receptor D2 (Drd2), (H) monoamine oxidase A (MaoA) and (I) monoamine oxidase B (MaoB) in home-cage Negr1^−/− mice and their WT littermates (cohort III). Data represents mean ± SEM, * p < 0.05, ** p < 0.01, ordinary two-way ANOVA (Bonferroni post hoc test) (A–D), Mann–Whitney U test (E–I).

Figure 4

Effect of chronic amphetamine in the dorsal striatum (DSTR) and ventral striatum (VSTR). The level of (A) dopamine (DA) and (B) dopamine turnover (3-MT/DA) in the DSTR. (C) Immunohistochemical DAT stainings of DSTR and VSTR. The level of (D) dopamine (DA), (E) dopamine turnover (3-MT/DA), (F) 3-MeOTyramine (3-MT), (G) 5-Hydroxyindoleacetic acid (5-HIAA), (H) dopamine transporter (Dat), and (I) catechol-O-methyltransferase (Comt) in the VSTR. Data represent mean ± SEM, ## p < 0.01—treatment effect, + p < 0.05, ++ p < 0.01—genotype effect, * p < 0.05—post hoc test, ordinary two-way ANOVA (Bonferroni post hoc test), Mann–Whitney U test (H,I), WT – wild-type.

Figure 5

Expression of NEGR1 and TH in the striatum. Representative confocal images of the striatum show co-immunohistochemical stainings of anti-NEGR1 in green (A,A1), with anti-tyrosine hydroxylase in magenta (TH) (B,B1) and NEGR1 staining in the vicinity of dopaminergic projection area can be seen with white color merged images (C,C1). Boxed areas show the localization of the close-ups in the images (A1–C1). Scale bars: (A–C) 1 mm, (A1–C1) 300 µm. LV—lateral ventricles, Ctx—central cortex, Sept—septum, CPu—caudate putamen, NAc—nucleus accumbens, OT—olfactory tubercule, Pir—piriform cortex.

Figure 6

Hippocampi of Negr1^−/− mice weigh less compared to WT mice, and escitalopram restores the weight of the hippocampi of Negr1^−/− mice. (A) The weight of the hippocampi of the mice after receiving 10 days of saline or amphetamine. (B) Weight of hippocampi divided by the weight of mice (after receiving 10 days of saline or amphetamine). (C) Weight of hippocampi of the mice receiving 23 days of saline or escitalopram. (D) Weight of hippocampi divided by the weight of mice (after receiving 23 days of saline or escitalopram). Data represent mean ± SEM, ++ p < 0.01—genotype effect, * p < 0.05, ** p < 0.01—post hoc test, ordinary two-way ANOVA (Bonferroni post hoc test).

Figure 7

Effect of chronic administration of amphetamine or escitalopram on the level of monoamines and their metabolites in the hippocampus. The levels of (A) tyrosine, (B) dopamine (DA), (C) 3MeOTyramine (3-MT), (D) tyramine, (E) serotonin (5-HT), (F) 5-hydroxyindoleacetic acid (5-HIAA), (G) noradrenaline and (H) normetanephrine in the hippocampus of Negr1^−/− mice and their WT littermates after 10 days of chronic amphetamine i.p. injections. The levels of (I) tyrosine, (J) dopamine (DA), (K) 3MeOTyramine (3-MT), (L) tyramine, (M) serotonin (5-HT), (N) 5-Hydroxyindoleacetic acid (5-HIAA), (O) noradrenaline and (P) normetanephrine in the hippocampus of Negr1^−/− mice and their WT littermates after 23 days of chronic escitalopram i.p. injections. Data represent mean ± SEM, # p < 0.05, ## p < 0.01, ### p < 0.001- treatment effect, + p < 0.05, ++ p < 0.01—genotype effect, * p < 0.05, ** p < 0.01—post hoc test, ordinary two-way ANOVA (Bonferroni post hoc test).

Figure 8

Effect of chronic escitalopram on the level of monoamines and their metabolites in the raphe nuclei. Batch II mice (A–J) and batch III mice (K–N). Levels of (A) tryptophan (Trp), (B) serotonin (5-HT), (C) 5-Hydroxyindoleacetic acid (5-HIAA), (D) serotonin turnover (5-HIAA/5-HT), (E) tyrosine, (F) dopamine (DA), (G) 3,4-Dihydroxyphenylacetic acid (DOPAC), (H) dopamine turnover (DOPAC/DA), (I) tyramine and (J) 3MeOTyramine in raphe. The mRNA expression level of (K) serotonin transporter (Slc6a4), (L) tryptophan hydroxylase 2 (Tph2), (M) monoamine oxidase A (MaoA), and (N) monoamine oxidase B (MaoB). Data represent mean ± SEM, # p < 0.05, ### p < 0.001, #### p < 0.0001—treatment effect, + p < 0.05—genotype effect, * p < 0.05, ** p < 0.01, *** p < 0.001—post hoc test, ordinary two-way ANOVA (Bonferroni post hoc test) (A-J), Mann–Whitney U test (K-N). WT- wild-type.

Figure 9

Immunohistochemical staining of WT mouse brain coronal sections displaying an expression of tyrosine hydroxylase (TH), tryptophan hydroxylase 2 (TPH) with NEGR1. (A–E) epifluorescent images display localization of TH and TPH throughout dopaminergic and raphe nuclei. (F–K) laser scanning confocal microscope images displaying localization of NEGR1 in TH-positive cells in substantia nigra pars reticulata (SNr). (L–N) laser-scanning confocal microscope images show diffuse localization of NEGR1 in the dorsal raphe (DR), whereas (O–Q) the localization in medial raphe (MnR) is observable in cells expressing TH and TPH. (A–Q) Nuclei were stained using H33258 stain (blue). Scale bars: (A–E) 1 mm, (F–H,L–Q) 0.5 mm, (I–K) 100 µm. Aq—aqueduct, VTA—ventral tegmental area, IF—interfascicular nucleus, IPN—interpeduncular nucleus, SNc—substantia nigra pars compacta, SNr—substantia nigra pars reticulate, RL—rostral linear nucleus, DR—dorsal raphe nucleus, MnR—median raphe nucleus.