Methylphenidate and Guanfacine Ameliorate ADHD-Like Phenotypes in Fez1-Deficient Mice

Abstract

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that, while prevalent, has a stagnant track record for advances in treatment. The limited availability of animal models with appropriate face and predictive validities has hampered progress in developing novel neurobiological hypotheses and testing new therapeutic options for this condition. Here, we report that mice deficient in Fez1, a gene specifically expressed in the nervous system with documented functions in neurodevelopment, show hyperactivity and impulsivity phenotypes, which are ameliorated by administering methylphenidate (MPH) or guanfacine (GFC), two pharmacological agents used for ADHD treatment. Fez1-knockout (KO) mice show reduced expression of tyrosine hydroxylase in the midbrain and the brain stem and have reduced levels of dopamine, norepinephrine, or their metabolites in both the nucleus accumbens and the prefrontal cortex. These neurochemical changes in Fez1-KO mice were normalized by MPH or GFC. We propose that Fez1-KO mice can be used as a model to evaluate the role of altered neurodevelopment in the manifestation of ADHD-like behavioral phenotypes, as well as to investigate the neurobiological mechanisms of existing and new pharmacotherapeutic agents for ADHD.

Keywords: Attention-deficit/hyperactivity disorder; Dopamine; FEZ1; Guanfacine; Hyperactivity; Impulsivity; Methylphenidate; Neurodevelopment; Norepinephrine; Tyrosine hydroxylase.

Fig. 1

Hyperlocomotion is ameliorated by methylphenidate (MPH) or guanfacine (GFC) in Fez1-knockout (KO) mice. Locomotor activities in the open field box were evaluated for wild-type (WT) control mice treated acutely with saline (n = 10), MPH (0.5 mg/kg body weight) (n = 5), and GFC (0.1 mg/kg body weight) (n = 5), and for Fez1-KO mice treated with saline (n = 8), MPH (n = 8), and GFC (n = 8) (male, 6–7 weeks old). a Locomotor activities shown in 5-min bins for WT mice treated with saline or MPH, and Fez1-KO mice treated with saline or MPH. Statistical significance: F(55, 418) = 2.531, p < 0.0001 (two-way ANOVA with repeated measures); * p < 0.05 (Bonferroni post hoc test). b Total distance traveled (cm) over the 60-min test period for the mice used in a. Statistical significance: F(5, 38) = 4.511, p = 0.0025 (one-way ANOVA); * p < 0.05 (Bonferroni post hoc test). c Locomotor activities shown in 5-min bins for WT mice treated with saline or GFC, and Fez1-KO mice treated with saline or GFC. Statistical significance: F(55, 418) = 2.531, p < 0.0001 (two-way ANOVA with repeated measures); * p < 0.05, ** p < 0.01. d Total distance traveled (cm) over the 60-min test period for the mice used in c. Statistical significance: F(5, 38) = 4.511, p = 0.0025 (one-way ANOVA); * p < 0.05, ** p < 0.01.

Fig. 2

Impulsivity is ameliorated by guanfacine (GFC) in Fez1-knockout (KO) mice. a, b The elevated plus maze was used to test wild-type (WT) control mice treated acutely with saline (n = 11), methylphenidate (MPH) (0.5 mg/kg body weight) (n = 7), and GFC (0.1 mg/kg body weight) (n = 6), and Fez1-KO mice treated with saline (n = 8), MPH (n = 10), and GFC (n = 8). Statistical significance: total distance traveled (cm) (a), F(5, 44) = 2.818, p = 0.0272; time spent in open arms (%) (b), F(5, 44) = 5.404, p = 0.0006 (one-way ANOVA); * p < 0.05, ** p < 0.01 (Bonferroni post hoc test). c Cliff avoidance behaviors were scored for 30 min for WT mice treated acutely with saline (n = 16), Fez1-KO mice treated with saline (n = 16), and Fez1-KO mice treated with GFC (n = 13). The percentage of mice that stayed on the platform during the test period was plotted. Statistical significance: F(58, 1,218) = 2.302, p < 0.0001 (two-way ANOVA with repeated measures); ** p < 0.01. d Percentage of cliff avoidance reaction (CAR) at the end of the test. Statistical significance: F(2, 42) = 4.489, p = 0.0171 (one-way ANOVA); * p < 0.05.

Fig. 3

Profiles of catecholamines and their metabolites in the nucleus accumbens (NAc) of Fez1-knockout (KO) mice. Levels of catecholamines and their metabolites in the NAc were measured by high-performance liquid chromatography using wild-type (WT) mice injected acutely with saline (n = 5), Fez1-KO mice treated with saline (n = 5), Fez1-KO mice treated with methylphenidate (MPH) (0.5 mg/kg body weight) (n = 5), and Fez1-KO mice treated with guanfacine (GFC) (0.1 mg/kg body weight) (n = 5) (male, 7 weeks old). a–c Levels of dopamine (DA) (a), DOPAC (b), and homovanillic acid (HVA) (c) measured in the NAc. d–f Levels of norepinephrine (NE) (d), normetanephrine (NM) (e), and HMPG (f) measured in the NAc. Statistical significance: * p < 0.05, ** p < 0.01 (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Fig. 4

Reduced expression of tyrosine hydroxylase (TH) in the midbrain is augmented by methylphenidate (MPH) or guanfacine (GFC) in Fez1-knockout (KO) mice. a Expression of TH in the ventral tegmental area and substantia nigra pars compacta in wild-type (WT) and Fez1-KO mice treated with saline, Fez1-KO mice treated with MPH (0.5 mg/kg body weight), and Fez1-KO mice treated with GFC (0.1 mg/kg body weight) (daily i.p. for 2 weeks, 7–8 weeks old when perfused). Scale bar, 200 μm. b Fluorescence intensities of anti-TH immunostaining per soma (80–100 somas scored per section, 3–4 serial sections analyzed per animal; n = 4 animals used per group) above the background level were quantified by ImageJ, and normalized by the average fluorescence intensity in WT mice within each cohort (#1, #2, #3, and #4). Relative fluorescence intensities per soma from a representative section are plotted in the graph, and the overall average fluorescence intensities (±SEM) across multiple sections per group are shown in red in the graph. Statistical significance: ** p < 0.01 (Kruskal-Wallis test followed by Dunn's multiple comparison test).