The contributions of neonatal inhalation of copper to air pollution-induced neurodevelopmental outcomes in mice

Abstract

Exposures to ambient ultrafine particle (UFP) air pollution (AP) during the early postnatal period in mice (equivalent to human third trimester brain development) produce male-biased changes in brain structure, including ventriculomegaly, reduced brain myelination, alterations in neurotransmitters and glial activation, as well as impulsive-like behavioral characteristics, all of which are also features characteristic of male-biased neurodevelopmental disorders (NDDs). The purpose of this study was to ascertain the extent to which inhaled Cu, a common contaminant of AP that is also dysregulated across multiple NDDs, might contribute to these phenotypes. For this purpose, C57BL/6J mice were exposed from postnatal days 4-7 and 10-13 for 4 hr/day to inhaled copper oxide (Cu_xO_y) nanoparticles at an environmentally relevant concentration averaging 171.9 ng/m³. Changes in brain metal homeostasis and neurotransmitter levels were determined following termination of exposure (postnatal day 14), while behavioral changes were assessed in adulthood. Cu_xO_y inhalation modified cortical metal homeostasis and produced male-biased disruption of striatal neurotransmitters, with marked increases in dopaminergic function, as well as excitatory/inhibitory imbalance and reductions in serotonergic function. Impulsive-like behaviors in a fixed ratio (FR) waiting-for-reward schedule and a fixed interval (FI) schedule of food reward occurred in both sexes, but more prominently in males, effects which could not be attributed to altered locomotor activity or short-term memory. Inhaled Cu as from AP exposures, at environmentally relevant levels experienced during development, may contribute to impaired brain function, as shown by its ability to disrupt brain metal homeostasis and striatal neurotransmission. In addition, its ability to evoke impulsive-like behavior, particularly in male offspring, may be related to striatal dopaminergic dysfunction that is known to mediate such behaviors. As such, regulation of air Cu levels may be protective of public health.

Keywords: Air pollution; Copper; Dopamine; Metal dyshomeostasis; Neurodevelopmental disorders; Ultrafine particulate matter.

Figure 1.. Experimental design for exposures, micro-dissections, and adulthood behavior.

A. Schematic depicting the experimental design of postnatal Cu_xO_y exposures and micro-dissected brain tissue from a subset of offspring. B. Schedule of behavioral testing for remaining offspring in litters used during PND14 micro-dissections. Created with BioRender.com.

Figure 2.. Exposure characteristics at ages PND4-7 and 10-13.

A. Mean ± standard deviation values for mass concentration of Cu_xO_y exposures (ng/m³; open triangles) and of particle diameter (nm; gray circles) across the 8 days of postnatal exposure. B. Mean ± standard deviation values for particle number concentrations (particles /cm³) across the 8 days of postnatal exposure.

Figure 3.. The effects of inhalational Cu exposure on correlations between metals within the olfactory bulb and cortex of PND14 male and female offspring.

Color maps of the Pearson Product-Moment correlation coefficient (r) values for olfactory bulb (left two columns) and cortex (right two columns) metals of male offspring (Panel A) and female offspring (Panel B). The strength and directionality of correlations are indicated by the intensity and color hue (red = positive, blue = negative) of the square, respectively. N = 5-7/sex/region/treatment/metal (see Supplementary Table 2). * = p-value < 0.05, # = p-value < 0.10.

Figure 4.. Changes in striatal, frontal cortical, midbrain, and cerebellar neurotransmitter levels in Cu_xO_y- exposed PND14 male offspring.

Group mean ± standard error percent of filtered air control mean values for glutamatergic (Gln=glutamine; Glu=glutamate; GABA=γ aminobutyric acid; Gln/Glu=glutamine/glutamate; Glu/GABA=glutamate/γ aminobutyric acid), serotonergic (Trp=tryptophan; Kyn=kynurenine; 5HTP=5 hydroxytryptophan; 5HT=serotonin, 5HIAA=5 hydroxyindoleacetic acid; 5HIAA/5HT=5 hydroxyindoleacetic acid/ serotonin) and dopaminergic (tyr=tyrosine; HVA=homovanillic acid; DOPAC=3,4-dihydroxyphenylacetic acid; DA=dopamine; NE=norepinephrine; HVA/DA= homovanillic acid/dopamine; DOPAC/DA=3,4-dihydroxyphenylacetic acid/dopamine; DA/Tyr=dopamine/tyrosine) neurotransmitters from male PND14 striatum (Panel A), frontal cortex (Panel B), midbrain (Panel C) and cerebellum (Panel D) following exposure to Cu_xO_y. N = 4-7/sex/region/treatment/neurotransmitter (see Supplementary Tables 3-5). * = p-value < 0.05, # = p-value < 0.10.

Figure 5.. Changes in striatal, frontal cortical, midbrain, and cerebellar neurotransmitter levels in Cu_xO_y- exposed PND14 female offspring.

Group mean ± standard error percent of filtered air control mean values for glutamatergic (Gln=glutamine; Glu=glutamate; GABA=γ aminobutyric acid; Gln/Glu=glutamine/glutamate; Glu/GABA=glutamate/γ aminobutyric acid), serotonergic (Trp=tryptophan; Kyn=kynurenine; 5HTP=5 hydroxytryptophan; 5HT=serotonin, 5HIAA=5 hydroxyindoleacetic acid; 5HIAA/5HT=5 hydroxyindoleacetic acid/ serotonin) and dopaminergic (tyr=tyrosine; HVA=homovanillic acid; DOPAC=3,4-dihydroxyphenylacetic acid; DA=dopamine; NE=norepinephrine; HVA/DA= homovanillic acid/dopamine; DOPAC/DA=3,4- dihydroxyphenylacetic acid/dopamine; DA/Tyr=dopamine/tyrosine) neurotransmitters from female PND14 striatum (Panel A), frontal cortex (Panel B), midbrain (Panel C) and cerebellum (Panel D) following exposure to Cu_xO_y. N = 5-7/sex/region/treatment/neurotransmitter (see Supplementary Tables 3-5). * = p-value < 0.05.

Figure 6.. The effects of inhalational Cu exposure on spontaneous locomotor activity assessments in PND60 male and female offspring.

Group mean ± standard error levels of ambulatory distance traveled (centimeters), stereotypic counts (number), jump time (seconds) and vertical time (seconds) in locomotor activity assessments plotted in 5-minute bins over the course of a 60 min session in males (Panel A) and females (Panel B) of air-exposed (filled-in symbols) and Cu_xO_y -exposed (open symbols) offspring. N = 8/sex/treatment. * = p-value < 0.05, # = p-value < 0.10.

Figure 7.. The effects of inhalational Cu exposure on the performance of male and female offspring in a Fixed Ratio wait-for-reward schedule of reinforcement (initial wait = 2.5 seconds, incremental increase = 2.5 seconds).

Group mean ± standard error levels of FR response rate (responses/minute; first column), total FR resets (number; second column), mean long wait time (seconds; third column), and mean responses per reinforcer (number; fourth column) of male (Panel A) and female (Panel B) air-exposed (filled-in symbols) and Cu_xO_y - exposed (open symbols) offspring across 4 sessions of the FR wait schedule with an initial wait of 2.5 seconds and subsequent increments of 2.5 seconds. Time x treatment=interaction effect in repeated measures analyses of variance; N = 8/sex/treatment. * = p-value < 0.05, # = p-value < 0.10.

Figure 8.. The effects of inhalational Cu exposure on the performance of male and female mice in a Fixed Ratio wait-for-reward schedule of reinforcement (initial wait = 5.0 seconds, incremental increase = 5.0 seconds).

Group mean ± standard error levels of FR response rate (responses/minute; first column), total FR resets (number; second column), mean long wait time (seconds; third column), and mean responses per reinforcer (number; fourth column) of male (Panel A) and female (Panel B) air-exposed (filled-in symbols) and Cu_xO_y -exposed (open symbols) offspring across 4 sessions of the FR wait schedule with an initial wait of 5.0 seconds and subsequent increments of 5.0 seconds. Treatment = main effect of treatment in repeated measures analyses of variance; time x treatment = interaction effect in repeated measures analyses of variance; N = 8/sex/treatment. * = p-value < 0.05, # = p-value < 0.10.

Figure 9.. The effects of inhalational Cu exposure on the performance of male and female mice in a Fixed Interval 60-second schedule.

Group mean ± standard error levels of overall response rate (responses per minute; first column), run rate (responses per minute; second column), mean post-reinforcement pause time (seconds; third column) and mean inter-response time (seconds; fourth column) across the course of 16 sessions on a Fixed Interval (FI) 60-second schedule of food reward in male (Panel A) and female (Panel B) air-exposed (filled-in symbols) and Cu_xO_y -exposed (open symbols) offspring. Treatment = main effect of treatment in repeated measures analyses of variance; time x treatment = interaction effect in repeated measures analyses of variance; N = 8/sex/treatment. * = p-value < 0.05, # = p-value < 0.10.

Figure 10.. The effects of inhalational Cu exposure on the performance of male and female mice in a Fixed Interval 60-second schedule of extinction.

Group mean ± standard error levels of overall response rate (responses per minute; first column), run rate (responses per minute; second column), mean post-reinforcement pause time (seconds; third column) and mean inter-response time (seconds; fourth column) across the course of 5 sessions on a Fixed Interval (FI) 60 second schedule of extinction in male (Panel A) and female (Panel B) air-exposed (filled-in symbols) and Cu_xO_y -exposed (open symbols) offspring. Treatment = main effect of treatment in repeated measures analyses of variance; time x treatment = interaction effect in repeated measures analyses of variance; N = 7-8/sex/treatment. * = p-value < 0.05, # = p-value < 0.10.