Nitric oxide synthase mediates the ability of darbepoetin alpha to improve the cognitive performance of STOP null mice

Abstract

STOP (stable tubule only polypeptide) null mice display neurochemical and behavioral abnormalities that resemble several well-recognized features of schizophrenia. Recent evidence suggests that the hematopoietic growth factor erythropoietin improves the cognitive performance of schizophrenics. The mechanism, however, by which erythropoietin is able to improve the cognition of schizophrenics is unclear. To address this question, we first determined whether acute administration of the erythropoietin analog known as darbepoetin alpha (D. alpha) improved performance deficits of STOP null mice in the novel objective recognition task (NORT). NORT performance of STOP null mice, but not wild-type littermates, was enhanced 3 h after a single injection of D. alpha (25 microg/kg, i.p.). Improved NORT performance was accompanied by elevated NADPH diaphorase staining in the ventral hippocampus as well as medial and cortical aspects of the amygdala, indicative of increased nitric oxide synthase (NOS) activity in these structures. NOS generates the intracellular messenger nitric oxide (NO) implicated in learning and memory. In keeping with this hypothesis, D. alpha significantly increased NO metabolite levels (nitrate and nitrite, NOx) in the hippocampus of both wild-type and STOP null mice. The NOS inhibitor, N (G)-nitro-L- arginine methyl ester (L-NAME; 25 mg/kg, i.p.), completely reversed the increase in hippocampal NOx levels produced by D. alpha. Moreover, L-NAME also inhibited the ability of D. alpha to improve the NORT performance of STOP null mice. Taken together, these observations suggest D. alpha enhances the NORT performance of STOP null mice by increasing production of NO.

Figure 1

Acute darbepoetin alfa (D. alfa; 25 μg/kg, i.p.) treatment reversed object recognition deficits in STOP null mice. (a) Object exploration time in the sample phase. The time spent exploring objects is shown in the bar graph as mean±SEM. Wild-type mice (WT) and STOP null mice (KO) injected with vehicle (10 ml/kg, i.p.; WT: N=12, KO: N=13) or D. alfa (25 μg/kg, i.p.; WT: N=12, KO: N=16) spent equal amounts of time investigating the objects during the sample phase. (b) Discrimination ratio (d2) in the choice phase is shown in the bar graph as mean±SEM. KO mice treated with an acute injection of D. alfa (25 μg/kg i.p.) displayed a significant improvement in NORT performance compared with vehicle controls. White bars, vehicle (10 ml/kg, i.p.; WT: N=12, KO: N=13); black bars, D.alfa (25 μg/kg, i.p.; WT: N=12, KO: N=16). Two-way ANOVA with Bonferroni post hoc t-test, ^*p<0.05.

Figure 2

Representative NADPH diaphorase staining in the forebrain of a WT mouse 3–4 h after D. alfa (25 μg/kg, i.p.) or vehicle (10 ml/kg, i.p.). (a and b) dorsal hippocampus; Bregma=−2.30 mm, (c and d) ventral hippocampus; Bregma=−3.16 mm, (e and f) amygdala (Am) and rhinal cortex (Rh); Bregma=−2.18 mm. Mice (WT or STOP null) treated with D. alfa (25 μg/kg i.p.) showed a marked increase in NADPH diaphorase staining in the ventral hippocampus as well as medial and cortical aspects of the amygdala relative to vehicle controls (indicated by arrowheads). Scale bars; 1 mm. (g) Quantification of signal intensity for NADPH-d staining, bars represent the means±SEM of groups composed of six mice each. Mann–Whitney U-test, ^*p<0.05.

Figure 3

Nitrate and nitrite (NOx) levels in brain tissue from wild-type (WT) and STOP null mice (KO) that received either D. alfa (25 μg/kg, i.p.) or vehicle (10 ml/kg, i.p.). (a) NOx levels in cortex. (b) NOx levels in hippocampus. NOx levels [μM/mg (protein)] are shown in the bar graph as mean±SEM. NOx levels in hippocampus were increased in both WT and KO mice 3–4 h after D. alfa treatment. White bars, vehicle (10 ml/kg; WT: N=8, KO: N=8); black bars, D. alfa (25 μg/kg i.p.; WT: N=10, KO: N=8). Two-way ANOVA with Bonferroni post hoc t-test, ^*p<0.05.

Figure 4

Effect of -NAME on NOx levels and NORT performance in WT mice. -NAME (10, 25, or 50 mg/kg; i.p.) was administered 30 min before vehicle (10 ml/kg, i.p.) or D. alfa (25 μg/kg i.p.). NORT was performed 3 h after D. alfa treatment, and then hippocampi were subjected to measurement of NOx levels. (a) Dose-dependent inhibition of D. alfa (25 μg/kg, i.p.)-induced increases in nitrate and nitrite (NOx) levels in the hippocampus of wild-type mice by -NAME. NOx levels (μM/mg (protein)) are shown in the bar graph as mean±SEM. V; vehicle (10 ml/kg, i.p.); Darbepoetin alfa, D. alfa (25 μg/kg, i.p.), V/−: N=5, V/+: N=6, 10 mg/+: N=5, 25 mg/+: N=6, 50 mg/+: N=5. One-way ANOVA with Newman-Keuls test, ^*p<0.05. (b) Object exploration time in the sample phase. Each bar represents the mean±SEM. Animals that received vehicle or -NAME spent equivalent amounts of time exploring objects during sample phase. (c) Discrimination ratio (d2) during the choice phase for animals injected with vehicle (10 ml/kg, i.p.) or -NAME (10, 25, or 50 mg/kg; i.p.). Each bar graph represents the mean±SEM. There were no significant differences between groups that received vehicle (10 ml/kg, i.p.) or -NAME (10, 25, or 50 mg/kg; i.p.), one-way ANOVA. (d) Percent time spent exploring the novel object during the choice phase. Each bar graph represents the mean±SEM. V; vehicle (10 ml/kg); Darbepoetin alfa, D. alfa (25 μg/kg), V/−: N=5, V/+: N=6, 10 mg/+: N=5, 25 mg/+: N=6, 50 mg/+: N=5. There were no statistically significant differences among the groups, one-way ANOVA.

Figure 5

-NAME inhibits the improvement in NORT performance and the increase of NOx levels of STOP null mice that received D. alfa. Wild-type (WT) or STOP null mice (KO) were treated with -NAME (25 mg/kg, i.p.) and vehicle (10 mg/kg, i.p.; WT: N=8, KO: N=8) or -NAME (25 mg/kg, i.p.) and D. alfa (25 μg/kg, i.p.; WT: N=9, KO: N=8). The first injection occurred 30 min before the second. NORT was performed 3 h after D. alfa treatment, and then hippocampi were subjected to measurement of NOx levels. (a) Object exploration time in sample phase for WT and KO treated with -NAME (25 mg/kg, i.p.) and vehicle (10 mg/kg, i.p.) or -NAME (25 mg/kg, i.p.) and D. alfa (25 μg/kg, i.p.). Each bar represents mean±SEM. All four groups spent an equivalent amount of time investigating the objects during the sample phase. (b) Discrimination ratio (d2) in choice phase for WT or KO treated with -NAME (25 mg/kg, i.p.) and vehicle (10 mg/kg, i.p.) or -NAME (25 mg/kg, i.p.) and D. alfa (25 μg/kg, i.p.). Each bar represents mean±SEM. -NAME (25 mg/kg, i.p.) blocked the ability of D. alfa (25 μg/kg, i.p.) to improve NORT performance of KO. White bars, -NAME (25 mg/kg) and vehicle (10 ml/kg); black bars, -NAME (25 mg/kg) and D.alfa (25 μg/kg). Two-way ANOVA with Bonferroni post hoc t-test, ^*p<0.05. (c) -NAME inhibited the increase in nitrate and nitrite (NOx) levels induced by D. alfa in the hippocampus of wild-type and STOP null mice. The hippocampi from all animals were immediately removed and NOx measurements performed. NOx levels (μM/mg (protein)) are shown in the bar graph as mean±SEM. -NAME (25 mg/kg, i.p.) blocked the ability of D. alfa (25 μg/kg, i.p.) to increase NOx levels in both WT and KO mice. White bars, -NAME (25 mg/kg, i.p.) and vehicle (10 ml/kg, i.p.); black bars, -NAME (25 mg/kg, i.p.) and D.alfa (25 μg/kg, i.p.). There were no statistically significant differences among the groups, two-way ANOVA.