Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Tests of untrained behaviors

Abstract

Manganese (Mn), an element that naturally occurs in the environment, has been shown to produce neurotoxic effects on the developing young when levels exceed physiological requirements. To evaluate the effects of this chemical in combination with non-chemical factors pregnant Long-Evans rats were treated with 0, 2, or 4 mg/mL Mn in their drinking water from gestational day (GD) 7 to postnatal day (PND) 22. Half of the dams received a variable stress protocol from GD13 to PND9, that included restraint, small cage with reduced bedding, exposure to predator odor, intermittent intervals of white noise, lights on for 24 h, intermittent intervals of lights on during dark cycle and cages with grid floors and reduced bedding. One male and one female offspring from each litter were tested to assess untrained behavior. Ultrasonic vocalizations (USV) were recorded from PND13 pups while they were isolated from the litter. Locomotor activity (MA) was measured in figure-eight mazes at PND 17, 29, and 79 (different set of rats at each time point). Social approach (SA) was tested at PND48. Acoustic startle response (ASR) and pre-pulse inhibition (PPI) were measured starting at PND58. At PND53 a sweetness preference for a chocolate flavored milk solution was assessed. There were sex related differences on several parameters for the USVs. There was also a Mn by stress by sex interaction with the females from the 4 mg/mL stressed dams having more frequency modulated (FM) call elements than the 4 mg/mL non-stressed group. There was an effect of Mn on motor activity but only at PND29 with the 2 mg/mL group having higher counts than the 0 mg/mL group. The social approach test showed sex differences for both the habituation and test phase. There was an effect of Mn, with the 4 mg/mL males having a greater preference for the stimulus rat than did the 0 mg/mL males. There was also a stress by sex interaction. The ASR and PPI had only a sex effect. Thus, with only the FM call elements having a Mn by stress effect, and the PND29 MA and SA preference index having a Mn effect but at different doses requires further investigation.

Keywords: Manganese (Mn); Neurobehavioral tasks; Perinatal stress.

Figure 1.

USV total call number for PND13 rat pups. No effect of sex, Mn, or stress was seen (A). Average number of elements in frequency modulated calls. NS = no stress group and PS = perinatally stressed group. There was a stress × Mn × sex effect with the females in the stress 4 mg/mL Mn group having more elements than the females in the non-stressed 4 mg/mL Mn group. Values are mean ± SE. * = p < .03 (B).

Figure 2.

Sex Differences in USV parameters - fundamental call frequency (A), call duration (B) and RMS call amplitude (loudness) (C). The females had a higher call frequency than the males at 40 kHz while the males had longer call durations at 40 kHz. The males had a louder response than the females at both 40 and 60 kHz (note than when graphing dBV, less negative is louder). No effect of Mn or stress. Values are LS mean ± SE. * = p < .02.

Figure 3.

Horizontal activity counts at PND17 (A), PND29 (B), and PND79 (C) for each Mn concentration. At PND29 the 2 mg/mL Mn group had increased activity compared to the 0 mg/mL Mn group. Values are mean ± SE. * = p < .02.

Figure 4.

Active velocity (only time spent moving) for day 1 of the social approach test. The 4 mg/mL Mn group moved faster than the 0 mg/mL Mn group for both sexes. The females moved faster than the males. No effect of stress was indicated in males or females. * = p < .05 (A). Preference Index for the stimulus rat as a function of time. The segmented line represents equal preference. All groups showed a preference for the stimulus rat. The 4 mg/mL Mn males had a greater preference than the 0 mg/mL males (B). The NS females had a greater preference than the NS males (C). Values are mean ± SE. * = p < .03.

Figure 5.

Habituation of the acoustic startle response (ASR) across the 8 blocks (5 trials/block) collapsed by Mn dose and stress condition. Females had a smaller amplitude across all blocks. Both sexes showed habituation. Values are LS mean ± SE. There were no effects of Mn or Stress on the ASR (A). Percent inhibition of the startle response by a pre-stimulus pulse as a function of sex and prepulse dB above a background level of 65 dB. The ASR was inhibited more in males than females when preceded by a prepulse noise (at all dB levels). No effects of stress or Mn were indicated. Values are LS mean ± SE (B).