Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain

Abstract

Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently gamma-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased (3)H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35mg Fe/kg diet), iron-deficient (ID; 6mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1g Mn/l) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABA(A), and GABA(B) differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor proteins, which may be the basis of the neurological characteristics of manganism.

Figure 1

Microdialysate analysis. Mean concentrations ± SEM are shown for (A) extracellular GABA, (B) manganese, and (C) iron in microdialysate samples from the striatum after six weeks of dietary treatment. Inset: Correlational analysis of extracellular Fe and GABA concentrations (R= −0.32; p=0.08). *p<0.001; †p<0.10

Figure 2

Plasma metal concentrations at six weeks. (A) Plasma manganese concentrations were significantly increased in those animals receiving manganese supplementation versus those animals receiving deionized water alone (p=0.02). (B) A significant decrease in plasma iron concentration was observed in animals receiving the ID diet versus the CN diet (p=0.007).

Figure 3

Brain metal concentrations at six weeks. Mean concentrations ± SEM are shown for manganese (A) and iron (B) for caudate putamen (Cp), globus pallidus (GP), hippocampus (HC), substantia nigra (SN), and cerebellum (Cb). CN is represented in black, CNMn in gray, ID in white, and IDMn in dotted area. *p<0.05 according to Dunnet’s post-hoc analysis

Figure 4

Effect of dietary treatment on GAT-1 protein and mRNA expression. Mean expression as percentage of control ± SEM for GAT-1 (A) protein and (B) mRNA relative to β-actin are shown for caudate putamen (Cp), globus pallidus (GP), hippocampus (HC), substantia nigra (SN), and cerebellum (Cb). CN is represented in black, CNMn in gray, ID in white, and IDMn in dotted area. (C) Representative blots for GAT-1 and β-actin for each region are shown, with each band representing an individual animal. *p<0.05 according to Dunnet’s post-hoc analysis

Figure 5

Effect of dietary treatment on GABA_A protein and mRNA expression. Mean expression as percentage of control ± SEM for GABA_A (A) protein and (B) mRNA relative to β-actin are shown for caudate putamen (Cp), globus pallidus (GP), hippocampus (HC), substantia nigra (SN), and cerebellum (Cb). CN is represented in black, CNMn in gray, ID in white, and IDMn in dotted area. (C) Representative blots for GABA_A and β-actin for each region are shown, with each band representing an individual animal. *p<0.05 according to Dunnet’s post-hoc analysis

Figure 6

Effect of dietary treatment on GABA_B protein and mRNA expression. Mean expression as percentage of control ± SEM for GABA_B (A) protein and (B) mRNA relative to β-actin are shown for caudate putamen (Cp), globus pallidus (GP), hippocampus (HC), substantia nigra (SN), and cerebellum (Cb). CN is represented in black, CNMn in gray, ID in white, and IDMn in dotted area. (C) Representative blots for GABA_B and β-actin for each region are shown, with each band representing an individual animal. *p<0.05 according to Dunnet’s post-hoc analysis

Figure 7

GABA biology during Mn overload. This simple schematic of the basal ganglia represents the potential consequences of the increased extracellular GABA concentrations in the striatum due to alterations of Mn and Fe status observed in the current study. (1) Increased extracellular GABA concentrations in the striatum would reduce the activity of the GABA striatopallidal projection neurons (2) (dotted line). This reduction in activity would (3) increase the GABAergic inhibitory firing from the globus pallidus (GP) to the subthalamic nucleus (STN) (heavy black line), in turn (4) decreasing the excitatory glutamatergic firing from this region to the substantia nigra (SN) (dotted line). (5) Decreased glutamatergic excitation in the substantia nigra, along with decreased GABAergic inhibition from the striatonigral projection neurons (dotted line) and decreased protein expression of GAT-1 and GABA_B, would lead to a dysregulation of dopaminergic firing to the striatum (alternating line). This decrease in GABAergic firing to the substantia nigra may also contribute to the dopaminergic alterations observed during ID (Nelson et al., 1997; Erikson et al., 2000; 2001).