Effect of olfactory manganese exposure on anxiety-related behavior in a mouse model of iron overload hemochromatosis

Abstract

Manganese in excess promotes unstable emotional behavior. Our previous study showed that olfactory manganese uptake into the brain is altered in Hfe(-/-) mice, a model of iron overload hemochromatosis, suggesting that Hfe deficiency could modify the neurotoxicity of airborne manganese. We determined anxiety-related behavior and monoaminergic protein expression after repeated intranasal instillation of MnCl2 to Hfe(-/-) mice. Compared with manganese-instilled wild-type mice, Hfe(-/-) mice showed decreased manganese accumulation in the cerebellum. Hfe(-/-) mice also exhibited increased anxiety with decreased exploratory activity and elevated dopamine D1 receptor and norepinephrine transporter in the striatum. Moreover, Hfe deficiency attenuated manganese-associated impulsivity and modified the effect of manganese on the expression of tyrosine hydroxylase, vesicular monoamine transporter and serotonin transporter. Together, our data indicate that loss of HFE function alters manganese-associated emotional behavior and further suggest that HFE could be a potential molecular target to alleviate affective disorders induced by manganese inhalation.

Keywords: Dopamine; Elevated plus maze; Impulsivity; Intranasal instillation; Norepinephrine; Serotonin.

Figure 1. Effect of intranasal manganese on the levels of manganese in different brain regions of Hfe-deficient mice

Microdissected brain tissues, including prefrontal cortex, striatum, hippocampus and cerebellum, were collected from mice that were intranasally instilled with MnCl₂ (5 mg/kg) or water. The steady-state concentrations of manganese were quantified by inductively coupled plasma mass spectrometry (ICP-MS; n = 7–8 per group). Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM and were analyzed using two-way ANOVA, followed by post-hoc comparisons. Hfe x Mn, an interaction effect between Hfe deficiency and olfactory Mn exposure.

Figure 2. Effect of intranasal manganese on the levels of iron in different brain regions of Hfe-deficient mice

The steady-state concentrations of iron in microdissected brain tissues were quantified by ICP-MS (n = 6–7 per group). Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM and were analyzed using two-way ANOVA, followed by post-hoc comparisons.

Figure 3. Effect of intranasal manganese on the levels of manganese and iron in the liver of Hfe-deficient mice

Metal levels in the liver collected from mice intranasally instilled with MnCl₂ or water were determined by ICP-MS (n = 6–8 per group). Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM and were analyzed using two-way ANOVA, followed by post-hoc comparisons.

Figure 4. Effect of intranasal manganese on emotional behavior in Hfe-deficient mice

Mice intranasally instilled with MnCl₂ (5 mg/kg, daily) or water for 3 weeks were tested on the elevated plus maze in order to determine anxiety- and impulsivity-related behavior, including time in the open arms (A), entries to the open arms (B), distance in the open arms (C), total distance in the whole maze (D) and latency of the first entry to an open arm (E). Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM (n = 10–13 per group) and were analyzed using two-way ANOVA, followed by post-hoc comparisons.

Figure 5. Effect of intranasal manganese on the expression of key proteins involved in dopaminergic pathway in the striatum from Hfe-deficient mice

Striatum collected from mice was analyzed by western blot to determine the expression levels of tyrosine hydroxylase (A), dopamine transporter (B) and dopamine D1 (C) and D2 (D) receptors. Relative intensities of protein bands normalized to actin were determined using Image Lab (version 4.1). Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM (n = 4 per group) and were analyzed using two-way ANOVA, followed by post-hoc comparisons.

Figure 6. Effect of intranasal manganese on the expression of norepinephrine and serotonin transporters in the striatum from Hfe-deficient mice

Striatum collected from mice was analyzed by western blot to determine the expression levels of norepinephrine transporter (A) and serotonin transporter (B). Relative intensities of protein bands were normalized to actin. Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM (n = 4 per group) and were analyzed using two-way ANOVA, followed by post-hoc comparisons.

Figure 7. Effect of intranasal manganese on the expression of proteins involved in monoaminergic turnover in the striatum from Hfe-deficient mice

Striatum collected from mice was analyzed by western blot to determine the expression levels of vesicular monoamine transporter (A) and catechol-O-methyltransferase (B). Relative intensities of protein bands were normalized to actin. Empty and closed bars represent wild-type (Hfe^+/+) and Hfe-deficient (Hfe^−/−) mice, respectively. Data were presented as mean ± SEM (n = 4 per group) and were analyzed using two-way ANOVA, followed by post-hoc comparisons.