Chronic unpredictable stress influenced the behavioral but not the neurodegenerative impact of paraquat

Abstract

The impact of psychological stressors on the progression of motor and non-motor disturbances observed in Parkinson's disease (PD) has received little attention. Given that PD likely results from many different environmental "hits", we were interested in whether a chronic unpredictable stressor regimen would act additively or possibly even synergistically to augment the impact of the toxicant, paraquat, which has previously been linked to PD. Our findings support the contention that paraquat itself acted as a systemic stressor, with the pesticide increasing plasma corticosterone, as well as altering glucocorticoid receptor (GR) expression in the hippocampus. Furthermore, stressed mice that also received paraquat displayed synergistic motor coordination impairment on a rotarod test and augmented signs of anhedonia (sucrose preference test). The individual stressor and paraquat treatments also caused a range of non-motor (e.g. open field, Y and plus mazes) deficits, but there were no signs of an interaction (neither additive nor synergistic) between the insults. Similarly, paraquat caused the expected loss of substantia nigra dopamine neurons and microglial activation, but this effect was not further influenced by the chronic stressor. Taken together, these results indicate that paraquat has many effects comparable to that of a more traditional stressor and that at least some behavioral measures (i.e. sucrose preference and rotarod) are augmented by the combined pesticide and stress treatments. Thus, although psychological stressors might not necessarily increase the neurodegenerative effects of the toxicant exposure, they may promote co-morbid behaviors pathology.

Keywords: AAR, alternate arm return; ANOVA, analysis of variance; BCA, bicinchoninic acid; BDNF, brain derived neurotrophic factor; CUS, chronic unpredictable stress; Cytokine; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EPM, elevated plus maze; FST, forced swim test; GR, glucocorticoid receptor; HPA, hypothalamus-pituitary adrenal; IBA1, ionized calcium-binding adapter molecule 1; Inflammatory; MMx, Micromax; Microglia; PB, phosphate buffer; PBS, phosphate buffered saline; PD, Parkinson's disease; PFA, paraformaldehyde; PVDF, polyvinylidene difluoride; Parkinson's; RIPA, Radio Immuno Precipitation Assay; RR, rotarod; SAB, spontaneous alternation behavior; SAR, same arm return; SDS, sodium dodecyl sulphate; SNc, substantia nigra pars compacta; SPT, sucrose preference test; Stress; TH, tyrosine hydroxylase; Toxicity; VTA, ventral tegmental area; pGR, phosphate glucocorticoid receptor.

Fig. 1

Schematic timeline of study. Schematic timeline (CUS = chronic unpredictable stress; EPM = elevated plus maze; FST =forced swim test; MMx = Micromax; PQ = paraquat; RR = rotarod; SAB = spontaneous alternation behavior Y maze; SPT = sucrose preference test).

Fig. 2

Three weeks of chronic unpredictable stress induces anxiety-like behavior and working memory deficits. Mice that received three weeks of chronic unpredictable stress spent significantly less time in the open zones of the elevated plus maze (panel A) indicating an anxious-like state. Stressed mice also displayed working memory deficits as made evident by reduced overall spontaneous alternations (panel B). *p < 0.05, relative to non-stressed mice. n = 24 stressed mice compared to 8 controls. All data is expressed as mean ± SEM.

Fig. 3

Chronic unpredictable stress enhanced motor impairment in paraquat exposed mice. Mice were given three weeks of chronic unpredictable stress followed by paraquat (10 mg/kg; ip) or saline injections twice/week over three weeks upon which the stressor regimen continued. As shown in panel A, stressed mice had a significant reduction in home-cage activity beginning at Week 2 which continued until the end of the study which is depicted by the top horizontal line (n = 11 non-stressed saline mice, n = 11 non-stressed paraquat mice, n = 11 stressed saline, n = 11 stressed paraquat mice). Additionally, paraquat alone provoked a significant reduction in motor activity by Week 5 relative to non-stressed counterparts, however the stress did not enhance motor deficits provoked by the toxin. Panel B shows that both paraquat and stress exposure alone modestly reduced rotarod retention time. However, the combination of the chronic stress and paraquat exposure produced the most robust coordination deficit, that differed from all other groups (n = 12/group). + p < 0.05 relative to non-stressed mice irrespective of saline or paraquat exposure; #p < 0.05 relative to all other groups; †p < 0.05 relative to non-stressed saline treated mice irrespective of stress exposure. All data is expressed as mean ± SEM, post-hoc tests were analyzed using Fisher's LSD.

Fig. 4

Chronic unpredictable stress did not alter paraquat provoked SNc neurodegeneration accompanied by regional microglia activation. As shown in panel A, paraquat induced a significant reduction in TH + SNc dopamine neurons, but this affect was not further influenced by stressor exposure. Similarly, paraquat increased microglial morphological ratings indicative of and activated state, but the stressor had absolutely no effect (panel B). Representative images are shown in the photomicrograph. *p < 0.05 relative to saline treated mice irrespective of stress exposure. n = 5/group. All data is expressed as mean ± SEM.

Fig. 5

Paraquat augmented the impact of a stressor on anhedonia (sucrose preference) and provoked behavioral despair, anxiety, and cognitive-like deficits similar to a chronic unpredictable stressor regimen. As shown in panel A, beginning on Week 5, the individual paraquat and stress treatments significantly reduced sucrose preference. Moreover, the combination of paraquat in mice that had also been receiving the chronic stress exposure produced a further robust reduction in sucrose preference that was significantly greater than all other groups. Panel B reveals that the individual paraquat and stressor treatments each significantly increased the time spent immobile in a forced swim test (n = 12 non-stressed saline mice, n = 11 non-stressed paraquat mice, n = 12 stressed saline, n = 12 stressed paraquat mice). However, there appeared to be no interaction between these two treatments on this behavioral outcome. Similarly, panels C and D, show that paraquat and chronic stressor treatments each reduced time in open zone of an elevated plus maze (n = 12/group) and number of spontaneous alternations in a Y-maze, respectively. But there was no evidence of an interaction between the two treatments, + p < 0.05 relative to non-stressed saline treated animals, †p < 0.05, relative to all other groups. All data is expressed as mean ± SEM, post-hoc tests were analyzed using Fisher's LSD.

Fig. 6

Paraquat and stressor treatment increased corticosterone and provoked hippocampal alterations in BDNF and GR levels. As shown in panel A, paraquat and chronic unpredictable stress individually significantly increased plasma corticosterone levels, but there was no interaction evident between the two (panel A; n = 6/group). With regards to BDNF, paraquat and the stressor regimen each reduced hippocampal BDNF levels, but again there was no indication of an interaction between the insults (panel C; n = 5/group). There was however, a significant interaction for hippocampal glucocorticoid (GR) levels, such that paraquat and the stressor individually reduced levels; whereas the combination of paraquat on top of the stressor regimen actually produced GR levels comparable to control animals (panel B, n = 4/group). However, the phosphorylated form of GR (pGR) was not significantly altered by the treatments (panel D; n = 4/group). *p < 0.05 relative to saline treated mice irrespective of stress exposure; + p < 0.05 relative to non-stressed mice irrespective of paraquat exposure; †p < 0.05 relative to all other groups; # and $ p < 0.05 relative to non-stressed paraquat treated mice and saline treated mice exposed to chronic unpredictable stress. All data is expressed as mean ± SEM, post-hoc tests were analyzed using Fisher's LSD.