Developmental manganese neurotoxicity in rats: Cognitive deficits in allocentric and egocentric learning and memory

Abstract

Manganese (Mn) is an essential element but neurotoxic at higher exposure levels. The effects of Mn overexposure (MnOE) on hippocampal and striatal-dependent learning and memory in rats were tested in combination with iron deficiency (FeD) and developmental stress that often co-occur with MnOE. Moderate FeD affects up to 15% of U.S. children and developmental stress is common in lower socio-economic areas where MnOE occurs. Pregnant Sprague-Dawley rats and their litters were housed in cages with or without (barren cage (BAR)) standard bedding from embryonic day (E)7 to postnatal day (P)28. Dams were fed a 90% FeD or iron sufficient (FeS) diet from E15-P28. Within each litter, separate offspring were treated with 100mg/kg Mn (MnOE) or vehicle (VEH) by gavage on alternate days from P4-28. Offspring were tested as adults in the Morris and Cincinnati water mazes. FeD and developmental stress interactively impaired spatial learning in the Morris water maze. Developmental stress and MnOE impaired learning and memory in both mazes. MnOE resulted in reduced CA1 hippocampal long-term potentiation (LTP) and increased levels of α-synuclein. Preweaning MnOE resulted in cognitive deficits on multiple domains of learning and memory accompanied by impaired LTP and α-synuclein changes, effects worsened by developmental stress.

Keywords: Alpha-synuclein; Egocentric learning; Long-term potentiation; Manganese; Neurotoxicity; Spatial learning.

Fig. 1

Study Design. A, Experimental paradigm showing cage, diet, and dosing exposures. All treatments ended on P28 when pups were separated from their dams. B, Behavioral testing layout. Litters were split into two arms; each rat was given each test. Group sizes are shown in parentheses. This report only pertains to the straight channel, CWM, and MWM data. Data on the other behaviors are reported elsewhere (Amos-Kroohs et al., 2016).

Fig. 2

Cincinnati Water Maze: Latency to escape and errors as a function of MnOE (A,B), cage rearing (C,D), and dietary iron (E,F). Rats were tested starting on P66. VEH = vehicle-treated controls; MnOE (100 mg/kg) was by gavage every other day from P4-28; STD = standard cage; BAR = barren cage; FeS = Fe sufficient diet; FeD = Fe deficient diet (90% reduction relative to FeS diet). Note that although the general pattern of effect on errors is similar to that found on latency, there are differences, including that BAR cage effects were similar in magnitude to those seen on latency for MnOE but the BAR effect was less pronounced for errors than on latency. *p <0.05; **p<0.01; ***p<0.001; †p<0.10 compared with VEH, STD, or FeS.

Fig. 3

MWM path length (m) for three phases of testing following developmental MnOE: MWM-acquisition (initial learning); MWM-reversal (platform moved to the opposite quadrant); MWM-Shift (platform moved to an adjacent quadrant relative to the one used during reversal). Platform sizes were 10, 7, and 5 cm in diameter during acquisition, reversal, and shift, respectively. **p<0.01; ***p<0.001; compared with VEH.

Fig. 4

Morris water maze probe: A,B,C: Effect of MnOE on average distance to the former platform site during acquisition (A), reversal (B), and shift (C) probe trials of each phase. For each phase the 45 s probe trial was given on day-7 with the platform removed. D,E,F: Effect of rearing condition on probe performance during acquisition (D), reversal (E), and shift (F), respectively. *p<0.05; **p<0.01 compared with VEH or STD cage controls.

Fig. 5

Morris water maze interactions. A,B,C: path length (m) showing the interaction between cage condition and diet in the MWM during acquisition (A), reversal (B), and shift (C) phases of testing. D, interaction of MnOE and diet on reversal probe performance for average distance to the platform site (m). E, shows the result for the MWM Shift phase in females for the MnOE X sex interaction (female results shown) as a function of BAR vs. STD cage rearing. F, shows the result for MWM cued (proximal cue) learning as a function of MnOE and sex conducted after hidden platform testing to determine if rats could find a visible platform moved randomly from trial-to-trial and with curtains closed around the pool to obscure distal cues. *p <0.05; **p<0.01; ***p<0.001 compared with the relevant control condition in each panel.

Fig. 6

Western blots of α-synuclein in hippocampus and neostriatum of VEH and MnOE rats at two ages: P29 and P60. Semiquantitative analysis of band density using ImageJ software and expressed against actin are presented in Fig. 7. Top panel: P29; bottom panel: P60. Left panels: hippocampus; right panels: neostriatum. N = 4/age (males).

Fig. 7

Semiquantitative concentrations estimates of α-synuclein in two brain regions and at two ages in a group of naïve animals from MnOE or VEH groups. A, α-synuclein relative band density by Western analysis in hippocampus in MnOE and VEH treated rats gavaged every other day from P4-28 and assessed on P29 or P60. B, α-synuclein relative density in neostriatum of the same animals as in A. *p <0.05; **p< 0.01; †p<0.10 trend in MnOE rats compared with VEH controls.

Fig. 8

Long-term potentiation (LTP). LTP in the CA1 region of the hippocampus in rats exposed to Mn by gavage every other day from P4-22 and brain slices analyzed on P23 following a tetanizing stimulus. There was a significant interaction of MnOE x Time (min). As can be seen, most of the effect of Mn was to blunt the induction phase of LTP during the first 35 min. After 35 min, LTP continued but the two groups’ responses converged at approximately 150% above pre-tetanus baseline.