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Cognitive flexibility deficits in male mice exposed to neonatal hyperoxia followed by concentrated ambient ultrafine particles
Neurotoxicology and Teratology 70 (2018) 51–59
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Cognitive flexibility deficits in male mice exposed to neonatal hyperoxia followed by concentrated ambient ultrafine particles
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Keith Morris-Schaffera, , Marissa Sobolewskia, Kevin Welleb, Katherine Conrada, Min Yeec, Michael A. O'Reillyc, Deborah A. Cory-Slechtaa a
Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY 14642, United States of America Mass Spectrometry Resource Laboratory, University of Rochester Medical Center, Rochester, NY 14642, United States of America c Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, United States of America b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ultrafine particles Air pollution Neonatal hyperoxia Preterm birth Cognitive flexibility
Epidemiological evidence indicates an association between early-life exposure to air pollution and preterm birth. Thus, it is essential to address the subsequent vulnerability of preterm infants, who are exposed to unique factors at birth including hyperoxia, and subsequently to air pollution. Health effects of air pollution relate to particle size and the ultrafine particulate component (< 100 nm) is considered the most reactive. We previously reported neonatal mice exposed to hyperoxia (60% oxygen), mimicking preterm oxygen supplementation, for the first 4 days of life, followed by exposure to concentrated ambient ultrafine particles (CAPS) from postnatal day (PND) 4–7 and 10–13 exhibited deficits in acquisition of performance on a fixed interval (FI) schedule of reinforcement, a behavioral paradigm rewarding the first response at the end of a fixed interval of time. Specifically, mice exposed to hyperoxia followed by CAPS continued to respond earlier in the interval than controls, suggesting deficits in acquisition of timing of the interval. To further examine the extent of cognitive deficits produced by hyperoxia and CAPs exposures, performance under an intra- extradimensional shift discrimination paradigm was implemented, requiring the ability to respond to shifting rules for reward. Under these conditions, developmental exposure to hyperoxia and CAPS increased errors on both the reversal and extradimensional (ED) tasks in males but not females. Furthermore it altered the ratio of glutamate and GABA neurotransmitters in the frontal cortex, a region known to mediate cognitive flexibility, were observed immediately following neonatal hyperoxia and CAPS exposure on post-natal day 14 but not following behavioral experience. Collectively, the findings from this study suggests that combined developmental exposures to hyperoxia and CAPS leads to protracted and enhanced learning deficits consistent with cognitive inflexibility in males exclusively.
1. Introduction Individuals born preterm are exposed to environmental toxicants at critical windows of neurodevelopment typically protected by the maternal environment at low arterial oxygen saturation. However, very preterm infants (< 32 weeks) have underdeveloped pulmonary and cardiovascular systems and thus require oxygen supplementation to survive. Such supplementation is difficult to optimize, and consequently often leads to hyperoxia, particularly when considering concentrations to which the developing central nervous system would typically be exposed. Ambient particulate matter, generated from anthropogenic sources, is associated with both low birth weight and preterm birth (Sram et al., 2005; Glinianaia et al., 2004; Backes et al., 2013). A recent national study examining the association of preterm
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birth with PM2.5 (particulate matter < 2.5 μm in aerodynamic diameter) exposure found that nearly 16,000 premature births can potentially be attributed to air pollution with an estimated $4.33 billion in burden costs (Trasande et al., 2016). Given the link between air pollution and preterm birth, it is critical to assess the vulnerability of preterm infants exposed to hyperoxic environment followed by exposure to particulate matter in early development. With advances in medical care, the survival and major morbidities of very preterm infants has increased over the past two decades (Stoll et al., 2015), thus there is an urgent need to understand the unique toxicological challenges and susceptibility of individuals exposed to increased oxygen concentrations. Premature infants in a hyperoxic environment are at risk of hyperoxemia, an excess of oxygen in the blood; a direct threat to the CNS. The fetal environment is moderately
Corresponding author at: Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY, United States of America. E-mail address: keith_morrisschaff[email protected] (K. Morris-Schaffer).
https://doi.org/10.1016/j.ntt.2018.10.003 Received 19 June 2018; Received in revised form 7 September 2018; Accepted 10 October 2018 Available online 11 October 2018 0892-0362/ © 2018 Elsevier Inc. All rights reserved.
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extinction schedule, in which response rates were evaluated when reinforcement was withheld (Morris-Schaffer et al., 2018). Interestingly in the females, we observed only females exposed to both hyperoxia and CAPS exhibited learning deficits when the timing of the FI schedule was altered and when the shift was made from an FI to an extinction schedule, which was suggestive of inflexibility in rule learning (MorrisSchaffer et al., 2018). To directly assess the potential for an impaired cognitive flexibility phenotype, the current study examined the combined effects of neonatal hyperoxia and CAPS on an analog of the intraextradimensional shift set (Fray & Robbins, 1996), which required the mice to adapt to transitions in contingencies of reinforcement that ranged from a simple reversal of contingencies to a more complex extradimensional discrimination shift. Glutamatergic systems within the frontal cortex have been shown to influence extradimensional rule shifting (Marquardt et al., 2014; Brigman et al., 2009; Kos et al., 2011; Laurent & Podhorna, 2004). Consequently, to explore potential glutamatergic mechanisms in cognitive flexibility alterations produced by neonatal hyperoxia and CAPS, the ratio of glutamate, an excitatory neurotransmitter, to GABA, an inhibitory neurotransmitter, were measured in the frontal cortex following exposures and at the completion of behavioral testing.
hypoxemic, with fetal pulmonary arterial blood (PaO2) being ~18 mm Hg and oxygen saturation (SaO2) being ~50%, whereas a preterm infant with or without oxygen supplementation is maintained at > 80% SaO2 with a predicted PaO2 of 45–80 mm Hg, thus creating an inherent risk for hyperoxia in the premature CNS (Gao & Raj, 2010; Sola, 2015). Furthermore, preterm infants are shown to have higher cerebral oxygenation following birth than term infants, regardless of oxygen supplementation (Pichler et al., 2013; Almaazmi et al., 2013) and infants on long-term oxygen supplementation fluctuate above the desired level, with infants in some facilities spending up to 50% of their time in hyperoxic conditions (Sink et al., 2011; Deulofeut et al., 2006). Increased cerebral oxygenation and higher arterial oxygen saturation ranges following preterm birth have both been linked to poor cognitive outcomes measured using the Bayley Scales of Infant Development (Deulofeut et al., 2006; Verhagen et al., 2015). The direct effect of a hyperoxic environment on the developing CNS has the strong potential to enhance toxicity of environmental pollutants that share neurobehavioral outcomes. In humans, preterm birth and early-life exposure to air pollution share similar adverse neurodevelopmental outcomes. In a meta-analysis of 227 observational studies, one review found children born preterm had twice the risk for developing attention-deficit hyperactivity disorder (ADHD) (Bhutta et al., 2002). Correspondingly, increased exposure to elemental carbon attributed to traffic was associated with increase in hyperactivity scores on the Behavioral Assessment for Children (Newman et al., 2013). Preterm birth is also linked to cognitive deficits, with one study showing preterm birth decreased processing speed and reduced performance intelligence quotient on the Wechsler Intelligence Scales at adolescence (Soria-Pastor et al., 2008). Additionally, a study in Boston found ambient black carbon exposure in young children was associated with decreases in vocabulary level, and verbal and visual learning on a Wide Range Assessment Memory and Learning cognitive test (Suglia et al., 2008). Given the correspondence of exposures and outcome measures, it can be hypothesized common preterm risk factors, such as hyperoxia, might heighten vulnerability to developmental air pollution insults, such as particulate matter. Ambient ultrafine particles (UFPs: < 100 nm in aerodynamic diameter) are considered to be one of the most toxic components of air pollution and could be particularly toxic to the premature CNS. The toxicity of UFPs follows from their high surface area-to-mass ratio and high particle counts per unit of mass (Oberdörster et al., 2005; Oberdörster et al., 1994). These characteristics allow UFPs to adsorb large concentrations of toxicants, including heavy metals, endotoxins, and PAHs (Oberdörster et al., 2005). These toxicants can accumulate on the surface of UFPs and once taken up by an organism can have adverse biochemical reactions with the organisms' cells (Oberdörster et al., 2005). UFPs are capable of directly translocating to the CNS via uptake through nerve terminals in the olfactory mucosa to deposit within the parenchyma of the brain and induce neuroinflammation (Elder et al., 2006; Oberdorster et al., 2004). UFPs can also indirectly harm the CNS via long-term retention in the lung, which can trigger chronic inflammation (Sun et al., 2012; Park et al., 2015) and possibly lead to systemic inflammation. Our lab has shown in previous studies neonatal exposure to concentrated ambient ultrafine particles (CAPS) can induce glia activation (Allen et al., 2014c; Allen et al., 2014a), ventriculomegaly (Allen et al., 2014a), and differential responses on a variety of schedule-controlled responding paradigms (Allen et al., 2014c; Cory-Slechta et al., 2017; Allen et al., 2013). A previous study investigating the effects of neonatal hyperoxia and CAPS exposure showed cumulative and synergistic effects of combined exposures as measured on a fixed-interval (FI) schedule of reinforcement followed by an extinction paradigm, in a sex-specific manner. Males exposed to both neonatal hyperoxia and CAPS had temporal control deficits suggestive of additive interactions between both insults (Morris-Schaffer et al., 2018). However, in the males we did not observe any substantial combined treatment-related differences on a subsequent
2. Methods 2.1. Mice, reagents, & exposures Adult male and female C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME) were bred using a scheme designed to ensure timed births as previously described (Allen et al., 2014a). Newborn C57BL6 mice were birthed and maintained at 60% oxygen conditions until neonatal day 4, then maintained under normal animal room oxygen levels (21%). For this purpose, pure oxygen is humidified with sterile distilled water to 40–70%, filtered, and passed through into the chambers before venting out of the building. Mice birthed into unadulterated room air served as controls. Because adult mice are sensitive to hyperoxia, dams were rotated every 24 h between litters exposed to room air or hyperoxia. The neonatal hyperoxia exposure paradigm has been described and further detailed previously (Yee et al., 2009). Following hyperoxia exposure, mice were removed from dams and exposed to CAPS, as described previously (Allen et al., 2014a). Neonatal mice were exposed to filtered air or CAPS (< 100 nm in diameter) concentrated 10–20 fold using the Harvard Ultrafine Concentrated Ambient Particle System (HUCAPS). Exposures lasted for 4 h per day beginning at 9:00 a.m. on PND (postnatal day) 4–7 and 10–13. During these exposures pups were housed in small mesh chambers with four pups per chamber. This high-volume ambient sampling system utilizes condensational growth of the particulate phase in conjunction with virtual impaction to provide aerosols enriched for sizes smaller than 200 nm in diameter, with median sizes typically in the 70–90 nm range with concentrations of approximately 0.2–2 × 105/cm3. The gas-phase components of the ambient aerosol are not concentrated by the HUCAPS system. Particle counts were obtained using a condensation particle counter (model 3022A; TSI, Shoreview, MN), and mass concentration calculated using idealized particle density (1.5 g/cm3). The hyperoxia and CAPS exposures generated 4 treatment groups per sex: neonatal hyperoxia with and without CAPS (designated H Air and H CAPS, respectively) and neonatal room air with and without CAPS (designated A Air and A CAPS, respectively). A total of 50 litters were utilized and were culled to six mice per litter prior to exposures. Those litters were randomly assigned but, divided among the four exposure groups so each treatment group had minimum of 12 litters, with litter representing the primary unit of variability, n. Only two pups (one male and one female) were used per litter per behavioral and pathological endpoint. For the PND 14 pathology endpoint, n = 7 for all the treatment groups, except for the male H CAPS group which had an n = 6. For the behavioral groups n = 12, for all treatment groups across both 52
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deliveries for a total of 60 reinforcers. After lever press training was completed in all mice, the schedule was shifted to the intra-extra dimensional shift set task (Fig. 3). For the initial intradimensional discrimination task, pressing the back lever initiated a trial; a subsequent response on the left lever produced a house light flash and delivery of a reinforcer (Fig. 3b). Incorrect responses (back lever or right lever) were punished with a 10s time out period (TO) in which the house light turned off; the time out was reset by any additional lever presses within this period. Following a time out, a lever press on the back lever was required to re-initiate the trial. Incorrect responses included repeated back lever responses, a left or right lever response with no prior back lever response, or a right lever response after a back lever press (discrimination error). A 10 s inter-trialinterval (ITI) occurred after every reinforcer delivery during which the house light was turned off and any lever presses during the ITI reset the 10 s timer. A session ended after the delivery of 40 reinforcers. After 4 sessions, a reversal task was implemented in which a right lever response was required after trial initiation with a left lever response being scored as a reversal error (Fig. 3c). Following 4 sessions of the reversal task, a compounded reversal task was imposed in which trial initiation via a back lever response produced a yellow LED light displayed randomly across trials over the right or left lever but, did not signal the correct lever, as only left lever responses after trial initiation would be reinforced with right lever responses considered a learning error (Fig. 3d). After 4 sessions of the compounded reversal paradigm, an extradimensional discrimination paradigm was implemented. In this paradigm, a lever light would be displayed randomly over the right or left lever following initiation of a trial, and only a press on the lever with the illuminated lever light was reinforced (Fig. 3e). Incorrect responses (i.e., on the non-illuminated lever) initiated a TO and were followed by a correction procedure used to prevent perseverative responding on a single lever following trial initiation. The correction procedure required a back lever press followed by a response on the correct lever, which led to reinforcer delivery and the opportunity to start another new trial. During the correction procedure, the spatial location of the light and thus the correct lever was the same as the previous trial in which the learning error occurred; this would continue indefinitely until a correct response occurred. Errors produced the 10 s TO. Sessions ended following the delivery of 60 reinforcers and a total of 10 sessions were conducted. Cumulative learning errors in each session were the primary performance measurement, with an error defined only as the first error committed on an ED trial, ignoring the repeat errors made on the subsequent correction procedure trials. Incorrect responses on the ED paradigm correction procedure were not included in this measure, given it could not be determined whether correction occurred in response to the light or via simple reversal rules.
sexes. Note the PND 14 endpoint mice are randomly drawn from the same original litters used for the behavioral cohort. All mice used in this study were treated humanely with regard for alleviation of suffering and the study protocol was approved by the University of Rochester Institutional Animal Care and Use Committee. 2.2. Locomotor behavior To evaluate motor activity levels spontaneous locomotor activity was measured in photobeam chambers equipped with a transparent acrylic arena with a 48-channel infrared source, detector, and controller (Med Associates, St. Albans, VT). Locomotor behavior was assessed prior to the start of FI schedule-controlled behavior training (postnatal day 60). Locomotor activity was quantified in three 45-min sessions occurring once per day for 3 consecutive days, with the primary endpoint, ambulatory time, collected at 5 min epochs. Ambulatory time was defined as the cumulative time in which there were successive breaks of 2 × 2 photobeam virtual boxes within the chamber. A period of ambulation is stopped when the animal remained within a 2 × 2 virtual box for ≥300 ms. Habituation and average ambulatory activity in each session was explored. 2.3. Intra-extradimensional shift set task Following locomotor activity assessment, food restriction was imposed to provide motivation for food-rewarded behavior. Specifically, mice were placed on a food-restricted schedule for 3 days immediately prior to initiation of operant training to reach 85% of ad libitum weights. During the initial food restriction for 3 days, females were fed 1.0–1.5 g each and males were fed 1.5–2.0 g each per day with food restriction adjusted appropriately on an individual basis and their subsequent weight changes. Mice were maintained at 85% ad libitum body weight throughout the operant training schedule and feeding commenced immediately after each testing session. 2.4. Apparatus Behavioral testing was conducted in operant chambers (Med Associates, St. Albans, VT) housed in sound-attenuating cabinets equipped with white noise and fans for ventilation. Two fixed levers (right and left) were located on either side of a pellet dispenser for reinforcer delivery while on the opposite back wall, there was a fixed lever centered (back lever) across from the pellet dispenser. The primary incandescent house light was directly over the back lever and one yellow LED light placed over the left and right levers respectively. 2.5. Paradigm For this paradigm, elements of a previously reported model of reversal and extradimensional learning were adapted, in particular the spatial reversal component and the use of a light as a visual discrimination stimulus for the extradimensional shift (Boomhower & Newland, 2017). Mice were initially trained to press a lever for food reward using a fixed ratio 1 schedule (FR1), in which a reinforcer (20 mg food pellet) was delivered following a response on the designated correct lever, which would also trigger a house light flash and clicker sound cue. The correct (i.e., reinforced) lever was designated through a pseudo-randomized 3-trial block schedule in which reinforcement on the first trial was provided for a lever press on any of the available levers (right, left, or back). For the remaining two trials of the 3-trial block, reinforcement was only provided for presses to levers not previously reinforced. If the mouse pressed a previously reinforced lever, a 10s timeout was implemented in which the house light was extinguished, all reinforcement was withheld, and additional presses to any of the levers reset the timeout period. Twenty blocks were run in total in the training session, so each lever initiated twenty food
2.6. Glutamate (Glu) and γ-aminobutyric acid (GABA) neurochemistry analysis On PND 14, 24 h after the final CAPS exposure session, and on PND 240, two weeks after the final behavioral test session, mice were euthanized by cervical dislocation without sedation to preclude effects of anesthetic on neurochemistry. The frontal cortex was dissected from each animal, snap-frozen, and stored at −80 °C until analyses. Tissue was thawed and diluted in perchloric acid before being homogenized via ultra-sonication. The resulting homogenized solution was spun at 10,000g and the supernatant was used for mass spectrometry analysis. 10 uL of each sample was injected using a U3000 UHPLC (Dionex) onto a 2.1 × 150 mm PFP column (Thermo Scientific). Analytes were eluted isocratically with 0.1% formic acid in water and analyzed in positive mode on a TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Scientific). The column was washed with 0.1% formic acid in acetonitrile after analytes had eluted, then re53
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the extradimensional component. Females exposed to CAPS had significantly increased average errors (β = 1.25, SE = 0.56, p = 0.03, n = 12) in the intradimensional discrimination component, as compared to unexposed CAPS controls (Fig. 4A). Interestingly females exposed to hyperoxia had decreased average errors compared to their unexposed hyperoxia controls (β = −1.08, SE = 0.56, p = 0.06, n = 12) though it failed to reach statistical significance (Fig. 4A). CAPS-exposed males tended to have higher initial error levels but, showed a significantly steeper decrement in errors when compared to unexposed controls (β = −2.71, SE = 0.99, p < 0.01, n = 12), (Fig. 4E). A post-hoc ANOM on the first session showed A CAPS males had higher errors than the overall average in Session 1 (β = 8.84, 95% CI: −0.03, 17.71, n = 12), though it failed to reach significance. There were no significant ANOM post-hoc differences on Sessions 2 or Sessions 3 for males. No significant interactions between hyperoxia and CAPS were detected in either males or females for the discrimination task. In the intradimensional reversal task, no significant treatment-related learning or overall error differences were found in females on the reversal task (Fig. 4B). In males an interaction between hyperoxia, CAPS, and session was confirmed (β = 2.34, SE = 1.18, p = 0.05, n = 12) (Fig. 4F) which, the post-hoc ANOM analyses showed males exposed to both neonatal hyperoxia and CAPS had significantly higher errors than the overall mean of all four treatment groups during the second and third sessions (β = 2.03, 95% CI: 0.18, 3.87, n = 12). There were no significant treatment differences in either sex on the compounded reversal (Fig. 4C and F). No significant treatment-related differences in performance were found in females across the 10 sessions for the extradimensional shift component (Fig. 5A). In contrast, males exposed to CAPS had a significantly shallower slope across the 10 sessions (β = 0.26, SE = 0.12, p = 0.04, n = 12) consistent with a slower reduction in errors (Fig. 5B), as did males exposed to hyperoxia (β = 0.24, SE = 0.12, p = 0.04, n = 12). However, interactive or cumulative effects in the slope of error reduction were not observed in males exposed to both hyperoxia and CAPS.
equilibrated with 0.1% formic acid in water prior to the next injection. The flow rate was 400 μL/min, while the column oven was set at 30 °C. The precursor ion for GABA was 104.1 m/z, with fragment ions of 69.3 and 87.2 m/z with collision energies of 15, 10 respectively, at a tube lens voltage of 65 V. The precursor ion for glutamate was 148.1 m/z, with the fragment ions of 84.2 and 102.2 m/z, with collisions energies of 16, 10 respectively at a tube lens voltage of 71 V. Peak areas of each compound were compared to the internal standard to determine relative abundance between samples. 2.7. Statistical analysis Behavioral data were analyzed using a multi-level mixed-model while the glutamate:GABA ratios were assessed using a two-way ANOVA. All statistical analyses were done using JMP13 (Cary, NC) and stratified by sex. A random intercept and slope model was used to capture subject-level variability in behavioral performance. The intercept component was centered and used as a means of exploring the average errors across time, while the slope was used to define the linear function of learning across the sessions. CAPS and hyperoxia were designated as fixed factors and treatment effects were explored on both the intercept and slope components. Only the first 3 sessions of the discrimination and reversal tasks were analyzed to prevent inappropriate skewing of the analyses towards the final session, as it generally included a very prominent floor effect, when errors reached asymptotic levels as they approached zero. Interactions between the two factors were explored for each behavioral parameter, but if the interaction term had a p-value > 0.1, it was dropped from the model to avoid over-parameterization. Significant and main effects were further explored through a post-hoc analysis of means (ANOM), a multiple comparisons tests which, compares the means of each of the four groups with the overall mean across all groups. For behavioral findings, parameter estimates (β), standard errors (SE), along with the significance tests and for the ANOM are reported, as are the estimates and the confidence intervals. The reference groups for the parameter estimates were the unexposed controls with the estimates describing the magnitude of the change in the exposed mice. P ≤ 0.05 was considered statistically significant.
3.4. Glutamate/GABA As assessed on PND 14, i.e., one day following the final CAPS exposure, females exposed to CAPS had significantly elevated glutamate (Glu):GABA ratio in the frontal cortex (F(1, 27) = 13.644, p = 0.001, n = 7) than unexposed controls. Hyperoxia-exposed females had higher mean Glu:GABA ratio than unexposed controls, but the difference did not reach statistical significance (F(1,27) = 3.139, p = 0.089, n = 7) (Fig. 6A). The ANOM post-hoc showed H CAPS females had a higher Glu:GABA ratio than the overall average (β = 0.60, 95% CI: 0.10, 1.10, n = 7) and A Air females had lower Glu:GABA ratio than the overall average (β = −0.59, 95% CI: −1.09, −0.09, n = 7). CAPS-exposed males had significantly higher Glu:GABA ratio than unexposed controls (F(1, 26) = 8.054, p = 0.03, n = 6–7). The ANOM post-hoc showed H CAPS males had a higher Glu:GABA ratio than the overall average (β = 0.69, 95% CI: 0.20, 1.18, n = 6–7) and H Air males had lower Glu:GABA ratio than the overall average (β = −0.52, 95% CI: −1.01, −0.03, n = 6–7) (Fig. 6C). No significant treatment-differences were found in Glu:GABA ratio in the frontal cortex in adult mice of either sex that had undergone behavioral testing (Fig. 6B, D).
3. Results 3.1. CAPS exposure levels Fig. 1 shows particle concentrations (Fig. 1A) median particle diameter (Fig. 1B) and mean exposure mass concentration (Fig. 1C) cross the period of CAPS exposures. Particle sizes remained in the UFP range (< 100 nm), and mean exposure concentrations across treatment were approximately 41,184 particles/cm3 and the average mass was 22.6 μg/ m3. 3.2. Locomotor activity Time spent in ambulation during a 45-min locomotor session is shown in Fig. 2. Hyperoxia-exposed females exhibited a more rapid decline in ambulatory time across the session (β = 8.92, SE = 4.27, p = 0.04, n = 12) than corresponding unexposed controls. A hyperoxia × CAPS interaction on the overall average was found for males (β = 51.30, SE = 21.93, p = 0.02, n = 12) but, the ANOM post-hoc did not indicate average ambulatory time of any group differed from the overall mean.
4. Discussion In a previous study using this model of exposure, we found sexdependent differences in learning on an FI schedule followed by a subsequent extinction paradigm, which suggested the combined exposure altered the capacity of new rule learning (Morris-Schaffer et al., 2018). The objective of the current study was to directly ascertain the augmented risk of cognitive flexibility deficits that could result from
3.3. Intra-extradimensional shift set For all components of this paradigm, mice from all treatment groups obtained all available reinforcers, 40 per session for the intradimensional discrimination and reversal components, and 60 per session for 54
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Fig. 1. Characterization of CAPS exposures. Particle concentration is reported as number of particles × 105/cm3 ± SD (A). Daily diameter of CAPS is reported as nm ± SD (B). Particle mass concentrations are reported in μg/m3 ± SD (C).
(Morris-Schaffer et al., 2018). However the ID-ED shifts are testing rule learning under a different and specific context, all the tasks are learned through positive reinforcing stimuli, whereas extinction is the reduction of response rates in the absence of reinforcing stimuli, which may explain the discrepancy. Given the convergence of both neonatal hyperoxia and CAPS on a similar cognitive flexibility phenotype, and the potential for exposure to both insults to result in enhanced reversal and shift set learning deficits, these data suggest a unique vulnerability or preterm infants in regions with high air pollution. The alteration in learning patterns by hyperoxia and CAPS is particularly indicative of associative and potentially motivational deficits as opposed to general deficiencies in inhibitory functioning. Historically, reversal learning tasks in rodents have been separated into different learning phases, with early trial errors representative of
developmental exposure to neonatal hyperoxia followed by exposure to ambient ultrafine particulate matter using an analog of the intra-extradimensional shift set paradigm. Results showed males exposed to neonatal hyperoxia and CAPS had significantly increased errors on the later sessions in the intradimensional shift reversal component. Moreover, on the more difficult extradimensional shift, males exposed to neonatal hyperoxia, CAPS, or both had significant decreases in learning rate, as evidenced by a slower and incomplete reduction in errors. Interestingly, we did not see any treatment-related difficulties in the females with adjusting to the reversal or the extradimensional schedule, highlighting the robust sex-specific nature of the treatment effects. Furthermore this result is somewhat contrary to our previous study, which showed only combined exposure in females led to learning deficits when adjusting from a fixed interval to an extinction schedule
Fig. 2. Group mean ± S.E. ambulatory activity in female (A) and male mice (B) across 5 min blocks of a 45-min behavioral test session. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure, respectively. α ≤ 0.05. 55
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Fig. 3. Timeline of intradimensional-extradimensional shift set paradigm, 4 sessions of discrimination, 4 sessions of reversal, 4 sessions of compounded reversal, and 10 sessions of extra-dimensional shift. All sessions across all tasks were done consecutively. The initial intradimensional discrimination task reinforced a left lever response following trial initiation with a right lever response leading to a 10 s time out punishment (B). For the initial intradimensional reversal task, responses on the right lever were reinforced and the left lever produced timeout (C). For the compounded reversal, a yellow LED light over either lever was randomly displayed after trial initiation and responses on the left lever were always reinforced regardless of which lever light was illuminated, while responses on the right lever produced timeout (D). For the extradimensional shift set, responses were reinforced if they occurred on the lever beneath which, the light was illuminated, with the spatial location randomized across trials (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
to escape on the Barnes Maze was seen in Wistar rats exposed to 80% oxygen for 24 h on PND 6 indicative of memory deficits (Serdar et al., 2016). This was complemented by another study that examined the effects of 85% oxygen from PND 2 to PND 14, which found exposedC57BL/6 mice spent an increased amount of time to find the platform on the Morris Water Maze, a test of spatial learning and memory, and a decreased recognition index on the novel object paradigm, a test of recognition memory (Ramani et al., 2013). Although we did not find substantial hyperoxia-induced differences in ambulatory time in either sex beyond habituation alterations in females, another study found exposure to 80% O2 from PND 6 to PND 8 led to an increase in running wheel activity in C57BL/6 adolescent mice (Schmitz et al., 2012). One major limitation of relating the current study to previously published work, beyond the lack of a standardized exposure model, is none of these studies stratified analyses by sex or included it as an independent factor. Given these neonatal hyperoxia exposure windows involve critical periods of sexually dimorphic development within the CNS (Schulz & Sisk, 2016; Arnold & McCarthy, 2016), and our current study and previous work (Morris-Schaffer et al., 2018) consistently show a malespecific learning deficit, it is essential for further research to analyze and present the sexes separately. The extradimensional learning impairments observed in CAPStreated mice is supported by earlier published work using different behavioral paradigms but, the same time period of neonatal exposure to CAPS. In a two-lever repeated learning (RL) response chain schedule, in which mice were expected to learn a new two-lever sequence every
perseverative deficits and latent trials errors indicative of underlying associative or motivational behavioral domains (Hilson & Strupp, 1997; Widholm et al., 2003; Garavan et al., 2000). The only significant results in females were CAPS-exposed mice made more errors on the original discrimination task, which could potentially indicate early motivational deficiencies in engaging with the task rather than deficiencies in rule learning, which were not present on the more complex tasks for females. In males, although CAPS mice did show increased errors on the original discrimination task, the combined effect of hyperoxia and CAPS in the males was only seen in the last two sessions of the reversal task. The latency of the combined effect may indicate an initial deficiency or delay in the strength of association between the new spatial location and the reinforcing stimuli. This is further supported by the lack of combined treatment effect on the compounded reversal, which indicates general inhibitory control and flexibility is still maintained. Similarly on the ED shift in the males, prominent treatment differences were only present in the later sessions, again indicative of the treated mice's incapacity to generate and maintain a strong association between the new contingency and reinforcing stimulus. The observation that the deficit in the temporal learning pattern on the ED shift is virtually equivalent in the single exposure groups alone strongly suggests a common cognitive domain target between the two insults. To the best of our knowledge this is the first study directly examining cognitive flexibility deficits in mice neonatally exposed to hyperoxia. Previous behavioral studies have shown neonatal hyperoxia can lead to memory deficits and hyperactivity. Increased latency time 56
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Fig. 4. Group mean ± S.E. errors in female and male mice performing on the initial intradimensional discrimination (A, D), the reversal (B, E), and the compounded reversal (C, F) components. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. H × CAPS × Session indicates a statistical interaction of the treatments across the sessions. +/− denotes a statistical significant increase/decrease from the overall average as determined by analysis of means (ANOM). α ≤ 0.05.
reinforced; premature responses reset the timer. Sustained deficits were not seen across sessions at any given DRL value, but emerged only during the transitions to increasing DRL values, with CAPS-exposed males responding prematurely, again suggesting an initial difficulty in adjusting to new contingency of reinforcement (Cory-Slechta et al., 2017). As a potential underlying physiological mechanism that could
session, CAPS exposed males made significantly more errors during the earlier sessions of the schedule (Cory-Slechta et al., 2017). These errors were not persistent and were suggestive of initial difficulty in transitioning between different RL contingencies of reinforcement. A similar outcome was seen in the same study on a differential reinforcement of low rate (DRL) schedule of reward in which a specific length of time (DRL value) must elapse without a response before a response can be 57
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Fig. 5. Group mean ± S.E. errors in female (A) and male mice (B) in the extradimensional shift task. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. H × CAPS × Session indicates a statistically significant interaction of the treatments across the sessions. α ≤ 0.05.
transporter expression in astrocytes reducing their ability to uptake aspartic acid (Schmitz et al., 2011). The absence of treatment-related differences in Glu:GABA ratio in the adult behavior mice could be due to the transient nature of neurochemical changes in response to these exposures. Alternatively, an enriching learning environment alone is an extraneous factor known to heavily influence glutamate and GABA ratio within the CNS (He et al., 2010; Segovia et al., 2006). It is important to note the increase in Glu:GABA ratio in the frontal cortex at PND 14 in both males and females was insufficient to explain the prominent malespecific functional behavioral phenotype. Future studies should explore more static markers of frontal cortex activity in exposed mice, including protein expression of glutamate and GABA receptors, and dendritic branching complexity. Additionally, in mice that did not experience weight restriction and behavioral testing, extraneous factors that could modify toxicity, would be better suited for exploring the long-term
explain the behavioral deficits our lab explored the ratio of glutamate, and GABA, the classical excitatory and inhibitory neurotransmitters in the frontal cortex. Imbalances in inhibitory and excitatory signaling within the frontal cortex have been shown to modulate extradimensional shifts (Marquardt et al., 2014; Brigman et al., 2009; Kos et al., 2011; Laurent & Podhorna, 2004). Interestingly, PND 14 males and females exposed to both hyperoxia and CAPS had increased Glu:GABA ratio relative to the overall average. These results complement data from earlier neonatal CAPS-only exposures that revealed alterations in glutamate and GABA ratio in both sexes within the frontal cortex at PND 14, although the “dose” of particle concentration in those exposures was 3–4x as high as the current exposure (Allen et al., 2014c; Allen et al., 2014a; Allen et al., 2014b). The exact mechanism by which hyperoxia is enhancing the CAPS effect is unclear, although one study reported exposure to neonatal hyperoxia decreased glutamate-aspartate
Fig. 6. Group mean ± S.D. glutamate:GABA ratio in the frontal cortex of females (A) and males (C) at PND 14 and for females (B) and males (D) at PND 240 following behavioral testing. Raw data are reported for each treatment group as well as the mean and standard deviation. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. +/− denotes a statistically significant increase/decrease from the overall average as determined by analysis of means (ANOM). α ≤ 0.05.
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differences in how males and females differentially recover from neonatal hyperoxia and CAPS exposure. As with our previous study, we did not see any gross differences in activity levels in males or females, although interestingly in the current study, females exposed to hyperoxia had an altered habituation trajectory, which we did not observe previously (Morris-Schaffer et al., 2018). This new observation may be attributed to the greater sample size and power in the current study. Importantly, however, the lack of overall activity differences strengthens the assertion neonatal hyperoxia and CAPS exposures at the current levels are primarily influencing cognitive development in males. In summary, exposure to hyperoxic conditions followed by exposure to concentrated ambient ultrafine particles in mice produced male-preferential deficits in cognitive function. Further work is needed to elaborate the underlying physiological mechanisms through which hyperoxia and CAPS converge to produce an augmented deficit. However, the findings in the present studies suggest preterm infants born in regions with high air pollution may have a unique ongoing neurodevelopmental vulnerability to particulate matter
Gao, Y., Raj, J.U., 2010. Regulation of the pulmonary circulation in the fetus and newborn. Physiol. Rev. 90 (4), 1291–1335. Garavan, H., et al., 2000. Prenatal cocaine exposure impairs selective attention: evidence from serial reversal and extradimensional shift tasks. Behav. Neurosci. 114 (4), 725. Glinianaia, S.V., et al., 2004. Particulate air pollution and fetal health: a systematic review of the epidemiologic evidence. Epidemiology 15 (1), 36–45. He, S., et al., 2010. Early enriched environment promotes neonatal GABAergic neurotransmission and accelerates synapse maturation. J. Neurosci. 30 (23), 7910–7916. Hilson, J.A., Strupp, B.J., 1997. Analyses of response patterns clarify lead effects in olfactory reversal and extradimensional shift tasks: assessment of inhibitory control, associative ability, and memory. Behav. Neurosci. 111 (3), 532–542. Kos, T., et al., 2011. The effects of NMDA receptor antagonists on attentional set-shifting task performance in mice. Psychopharmacology 214 (4), 911–921. Laurent, V., Podhorna, J., 2004. Subchronic phencyclidine treatment impairs performance of C57BL/6 mice in the attentional set-shifting task. Behav. Pharmacol. 15 (2), 141–148. Marquardt, K., et al., 2014. Loss of GluN2A-containing NMDA receptors impairs extradimensional set-shifting. Genes Brain Behav. 13 (7), 611–617. Morris-Schaffer, K., et al., 2018. Effect of neonatal hyperoxia followed by concentrated ambient ultrafine particle exposure on cumulative learning in C57Bl/6J mice. Neurotoxicology 67, 234–244. Newman, N.C., et al., 2013. Traffic-related air pollution exposure in the first year of life and behavioral scores at 7 years of age. Environ. Health Perspect. 121 (736), 731–736. Oberdörster, G., Ferin, J., Lehnert, B.E., 1994. Correlation between particle size, in vivo particle persistence, and lung injury. Environ. Health Perspect. 102 (Suppl. 5), 173–179. Oberdorster, G., et al., 2004. Translocation of inhaled ultrafine particles to the brain. Inhal. Toxicol. 16 (6–7), 437–445. Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113 (7), 823–839. Park, E.J., et al., 2015. Chronic pulmonary accumulation of iron oxide nanoparticles induced Th1-type immune response stimulating the function of antigen-presenting cells. Environ. Res. 143 (Pt A), 138–147. Pichler, G., et al., 2013. Reference ranges for regional cerebral tissue oxygen saturation and fractional oxygen extraction in neonates during immediate transition after birth. J. Pediatr. 163 (6), 1558–1563. Ramani, M., et al., 2013. Neurodevelopmental impairment following neonatal hyperoxia in the mouse. Neurobiol. Dis. 50, 69–75. Schmitz, T., et al., 2011. Cellular changes underlying hyperoxia-induced delay of white matter development. J. Neurosci. 31 (11), 4327–4344. Schmitz, T., et al., 2012. Adolescent hyperactivity and impaired coordination after neonatal hyperoxia. Exp. Neurol. 235 (1), 374–379. Schulz, K.M., Sisk, C.L., 2016. The organizing actions of adolescent gonadal steroid hormones on brain and behavioral development. Neurosci. Biobehav. Rev. 70, 148–158. Segovia, G., et al., 2006. Environmental enrichment promotes neurogenesis and changes the extracellular concentrations of glutamate and GABA in the hippocampus of aged rats. Brain Res. Bull. 70 (1), 8–14. Serdar, M., et al., 2016. Fingolimod protects against neonatal white matter damage and long-term cognitive deficits caused by hyperoxia. Brain Behav. Immun. 52, 106–119. Sink, D.W., Hope, S.A., Hagadorn, J.I., 2011. Nurse:patient ratio and achievement of oxygen saturation goals in premature infants. Arch. Dis. Child. Fetal Neonatal Ed. 96 (2), F93–F98. Sola, A., 2015. Oxygen saturation in the newborn and the importance of avoiding hyperoxia-induced damage. NeoReviews 16 (7), e393–e405. Soria-Pastor, S., et al., 2008. Patterns of cerebral white matter damage and cognitive impairment in adolescents born very preterm. Int. J. Dev. Neurosci. 26 (7), 647–654. Sram, R.J., et al., 2005. Ambient air pollution and pregnancy outcomes: a review of the literature. Environ. Health Perspect. 113 (4), 375–382. Stoll, B.J., et al., 2015. Trends in care practices, morbidity, and mortality of extremely preterm neonates, 1993–2012. JAMA 314 (10), 1039–1051. Suglia, S.F., et al., 2008. Association of black carbon with cognition among children in a prospective birth cohort study. Am. J. Epidemiol. 167 (3), 280–286. Sun, Q., et al., 2012. Pulmotoxicological effects caused by long-term titanium dioxide nanoparticles exposure in mice. J. Hazard. Mater. 235–236, 47–53. Trasande, L., Malecha, P., Attina, T.M., 2016. Particulate matter exposure and preterm birth: estimates of us attributable burden and economic costs. Environ. Health Perspect. 124 (12), 1913–1918. Verhagen, E.A., et al., 2015. Cerebral oxygenation is associated with neurodevelopmental outcome of preterm children at age 2 to 3 years. Dev. Med. Child Neurol. 57 (5), 449–455. Widholm, J.J., et al., 2003. Effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin on spatial and visual reversal learning in rats. Neurotoxicol. Teratol. 25 (4), 459–471. Yee, M., et al., 2009. Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity. Am. J. Phys. Lung Cell. Mol. Phys. 297 (4), L641–L649.
Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was funded in part by National Institutes of Health Grants R01 ES025541 (D.A. Cory-Slechta), R01 HL091968 (M. A. O'Reilly). NIH Training Grant T32 ES007026 supported K. Morris-Schaffer. NIH Center Grant P30 ES001247 supported the animal inhalation facility and the animal behavior core. References Allen, J.L., et al., 2013. Developmental exposure to concentrated ambient particles and preference for immediate reward in mice. Environ. Health Perspect. 121 (1), 32–38. Allen, J.L., et al., 2014a. Early postnatal exposure to ultrafine particulate matter air pollution: persistent ventriculomegaly, neurochemical disruption, and glial activation preferentially in male mice. Environ. Health Perspect. 122 (9), 939–945. Allen, J.L., et al., 2014b. Consequences of developmental exposure to concentrated ambient ultrafine particle air pollution combined with the adult paraquat and maneb model of the Parkinson's disease phenotype in male mice. Neurotoxicology 41, 80–88. Allen, J.L., et al., 2014c. Developmental exposure to concentrated ambient ultrafine particulate matter air pollution in mice results in persistent and sex-dependent behavioral neurotoxicity and glial activation. Toxicol. Sci. 140 (1), 160–178. Almaazmi, M., et al., 2013. Cerebral near-infrared spectroscopy during transition of healthy term newborns. Neonatology 103 (4), 246–251. Arnold, A.P., McCarthy, M.M., 2016. Sexual differentiation of the brain and behavior: a primer. In: Neuroscience in the 21st Century, pp. 1–30. Backes, C.H., et al., 2013. Early life exposure to air pollution: how bad is it? Toxicol. Lett. 216 (1), 47–53. Bhutta, A.T., et al., 2002. Cognitive and behavioral outcomes of school-aged children who were born preterm: a meta-analysis. JAMA 288 (6), 728–737. Boomhower, S.R., Newland, M.C., 2017. Effects of adolescent exposure to methylmercury and d-amphetamine on reversal learning and an extradimensional shift in male mice. Exp. Clin. Psychopharmacol. 25 (2), 64. Brigman, J.L., et al., 2009. Effects of subchronic phencyclidine (PCP) treatment on social behaviors, and operant discrimination and reversal learning in C57BL/6J mice. Front. Behav. Neurosci. 3, 2. Cory-Slechta, D., et al., 2017. Developmental exposure to low level ambient ultrafine particle air pollution and cognitive dysfunction. Neurotoxicology. Deulofeut, R., et al., 2006. Avoiding hyperoxia in infants < or = 1250 g is associated with improved short- and long-term outcomes. J. Perinatol. 26 (11), 700–705. Elder, A., et al., 2006. Translocation of inhaled ultrafine manganese oxide particles to the central nervous system. Environ. Health Perspect. 114 (8), 1172–1178. Fray, P.J., Robbins, T.W., 1996. CANTAB battery: proposed utility in neurotoxicology. Neurotoxicol. Teratol. 18 (4), 499–504.
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Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera
Cognitive flexibility deficits in male mice exposed to neonatal hyperoxia followed by concentrated ambient ultrafine particles
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Keith Morris-Schaffera, , Marissa Sobolewskia, Kevin Welleb, Katherine Conrada, Min Yeec, Michael A. O'Reillyc, Deborah A. Cory-Slechtaa a
Department of Environmental Medicine, University of Rochester Medical Center, Rochester, NY 14642, United States of America Mass Spectrometry Resource Laboratory, University of Rochester Medical Center, Rochester, NY 14642, United States of America c Department of Pediatrics, University of Rochester Medical Center, Rochester, NY 14642, United States of America b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ultrafine particles Air pollution Neonatal hyperoxia Preterm birth Cognitive flexibility
Epidemiological evidence indicates an association between early-life exposure to air pollution and preterm birth. Thus, it is essential to address the subsequent vulnerability of preterm infants, who are exposed to unique factors at birth including hyperoxia, and subsequently to air pollution. Health effects of air pollution relate to particle size and the ultrafine particulate component (< 100 nm) is considered the most reactive. We previously reported neonatal mice exposed to hyperoxia (60% oxygen), mimicking preterm oxygen supplementation, for the first 4 days of life, followed by exposure to concentrated ambient ultrafine particles (CAPS) from postnatal day (PND) 4–7 and 10–13 exhibited deficits in acquisition of performance on a fixed interval (FI) schedule of reinforcement, a behavioral paradigm rewarding the first response at the end of a fixed interval of time. Specifically, mice exposed to hyperoxia followed by CAPS continued to respond earlier in the interval than controls, suggesting deficits in acquisition of timing of the interval. To further examine the extent of cognitive deficits produced by hyperoxia and CAPs exposures, performance under an intra- extradimensional shift discrimination paradigm was implemented, requiring the ability to respond to shifting rules for reward. Under these conditions, developmental exposure to hyperoxia and CAPS increased errors on both the reversal and extradimensional (ED) tasks in males but not females. Furthermore it altered the ratio of glutamate and GABA neurotransmitters in the frontal cortex, a region known to mediate cognitive flexibility, were observed immediately following neonatal hyperoxia and CAPS exposure on post-natal day 14 but not following behavioral experience. Collectively, the findings from this study suggests that combined developmental exposures to hyperoxia and CAPS leads to protracted and enhanced learning deficits consistent with cognitive inflexibility in males exclusively.
1. Introduction Individuals born preterm are exposed to environmental toxicants at critical windows of neurodevelopment typically protected by the maternal environment at low arterial oxygen saturation. However, very preterm infants (< 32 weeks) have underdeveloped pulmonary and cardiovascular systems and thus require oxygen supplementation to survive. Such supplementation is difficult to optimize, and consequently often leads to hyperoxia, particularly when considering concentrations to which the developing central nervous system would typically be exposed. Ambient particulate matter, generated from anthropogenic sources, is associated with both low birth weight and preterm birth (Sram et al., 2005; Glinianaia et al., 2004; Backes et al., 2013). A recent national study examining the association of preterm
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birth with PM2.5 (particulate matter < 2.5 μm in aerodynamic diameter) exposure found that nearly 16,000 premature births can potentially be attributed to air pollution with an estimated $4.33 billion in burden costs (Trasande et al., 2016). Given the link between air pollution and preterm birth, it is critical to assess the vulnerability of preterm infants exposed to hyperoxic environment followed by exposure to particulate matter in early development. With advances in medical care, the survival and major morbidities of very preterm infants has increased over the past two decades (Stoll et al., 2015), thus there is an urgent need to understand the unique toxicological challenges and susceptibility of individuals exposed to increased oxygen concentrations. Premature infants in a hyperoxic environment are at risk of hyperoxemia, an excess of oxygen in the blood; a direct threat to the CNS. The fetal environment is moderately
Corresponding author at: Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY, United States of America. E-mail address: keith_morrisschaff[email protected] (K. Morris-Schaffer).
https://doi.org/10.1016/j.ntt.2018.10.003 Received 19 June 2018; Received in revised form 7 September 2018; Accepted 10 October 2018 Available online 11 October 2018 0892-0362/ © 2018 Elsevier Inc. All rights reserved.
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extinction schedule, in which response rates were evaluated when reinforcement was withheld (Morris-Schaffer et al., 2018). Interestingly in the females, we observed only females exposed to both hyperoxia and CAPS exhibited learning deficits when the timing of the FI schedule was altered and when the shift was made from an FI to an extinction schedule, which was suggestive of inflexibility in rule learning (MorrisSchaffer et al., 2018). To directly assess the potential for an impaired cognitive flexibility phenotype, the current study examined the combined effects of neonatal hyperoxia and CAPS on an analog of the intraextradimensional shift set (Fray & Robbins, 1996), which required the mice to adapt to transitions in contingencies of reinforcement that ranged from a simple reversal of contingencies to a more complex extradimensional discrimination shift. Glutamatergic systems within the frontal cortex have been shown to influence extradimensional rule shifting (Marquardt et al., 2014; Brigman et al., 2009; Kos et al., 2011; Laurent & Podhorna, 2004). Consequently, to explore potential glutamatergic mechanisms in cognitive flexibility alterations produced by neonatal hyperoxia and CAPS, the ratio of glutamate, an excitatory neurotransmitter, to GABA, an inhibitory neurotransmitter, were measured in the frontal cortex following exposures and at the completion of behavioral testing.
hypoxemic, with fetal pulmonary arterial blood (PaO2) being ~18 mm Hg and oxygen saturation (SaO2) being ~50%, whereas a preterm infant with or without oxygen supplementation is maintained at > 80% SaO2 with a predicted PaO2 of 45–80 mm Hg, thus creating an inherent risk for hyperoxia in the premature CNS (Gao & Raj, 2010; Sola, 2015). Furthermore, preterm infants are shown to have higher cerebral oxygenation following birth than term infants, regardless of oxygen supplementation (Pichler et al., 2013; Almaazmi et al., 2013) and infants on long-term oxygen supplementation fluctuate above the desired level, with infants in some facilities spending up to 50% of their time in hyperoxic conditions (Sink et al., 2011; Deulofeut et al., 2006). Increased cerebral oxygenation and higher arterial oxygen saturation ranges following preterm birth have both been linked to poor cognitive outcomes measured using the Bayley Scales of Infant Development (Deulofeut et al., 2006; Verhagen et al., 2015). The direct effect of a hyperoxic environment on the developing CNS has the strong potential to enhance toxicity of environmental pollutants that share neurobehavioral outcomes. In humans, preterm birth and early-life exposure to air pollution share similar adverse neurodevelopmental outcomes. In a meta-analysis of 227 observational studies, one review found children born preterm had twice the risk for developing attention-deficit hyperactivity disorder (ADHD) (Bhutta et al., 2002). Correspondingly, increased exposure to elemental carbon attributed to traffic was associated with increase in hyperactivity scores on the Behavioral Assessment for Children (Newman et al., 2013). Preterm birth is also linked to cognitive deficits, with one study showing preterm birth decreased processing speed and reduced performance intelligence quotient on the Wechsler Intelligence Scales at adolescence (Soria-Pastor et al., 2008). Additionally, a study in Boston found ambient black carbon exposure in young children was associated with decreases in vocabulary level, and verbal and visual learning on a Wide Range Assessment Memory and Learning cognitive test (Suglia et al., 2008). Given the correspondence of exposures and outcome measures, it can be hypothesized common preterm risk factors, such as hyperoxia, might heighten vulnerability to developmental air pollution insults, such as particulate matter. Ambient ultrafine particles (UFPs: < 100 nm in aerodynamic diameter) are considered to be one of the most toxic components of air pollution and could be particularly toxic to the premature CNS. The toxicity of UFPs follows from their high surface area-to-mass ratio and high particle counts per unit of mass (Oberdörster et al., 2005; Oberdörster et al., 1994). These characteristics allow UFPs to adsorb large concentrations of toxicants, including heavy metals, endotoxins, and PAHs (Oberdörster et al., 2005). These toxicants can accumulate on the surface of UFPs and once taken up by an organism can have adverse biochemical reactions with the organisms' cells (Oberdörster et al., 2005). UFPs are capable of directly translocating to the CNS via uptake through nerve terminals in the olfactory mucosa to deposit within the parenchyma of the brain and induce neuroinflammation (Elder et al., 2006; Oberdorster et al., 2004). UFPs can also indirectly harm the CNS via long-term retention in the lung, which can trigger chronic inflammation (Sun et al., 2012; Park et al., 2015) and possibly lead to systemic inflammation. Our lab has shown in previous studies neonatal exposure to concentrated ambient ultrafine particles (CAPS) can induce glia activation (Allen et al., 2014c; Allen et al., 2014a), ventriculomegaly (Allen et al., 2014a), and differential responses on a variety of schedule-controlled responding paradigms (Allen et al., 2014c; Cory-Slechta et al., 2017; Allen et al., 2013). A previous study investigating the effects of neonatal hyperoxia and CAPS exposure showed cumulative and synergistic effects of combined exposures as measured on a fixed-interval (FI) schedule of reinforcement followed by an extinction paradigm, in a sex-specific manner. Males exposed to both neonatal hyperoxia and CAPS had temporal control deficits suggestive of additive interactions between both insults (Morris-Schaffer et al., 2018). However, in the males we did not observe any substantial combined treatment-related differences on a subsequent
2. Methods 2.1. Mice, reagents, & exposures Adult male and female C57BL/6J mice from Jackson Laboratories (Bar Harbor, ME) were bred using a scheme designed to ensure timed births as previously described (Allen et al., 2014a). Newborn C57BL6 mice were birthed and maintained at 60% oxygen conditions until neonatal day 4, then maintained under normal animal room oxygen levels (21%). For this purpose, pure oxygen is humidified with sterile distilled water to 40–70%, filtered, and passed through into the chambers before venting out of the building. Mice birthed into unadulterated room air served as controls. Because adult mice are sensitive to hyperoxia, dams were rotated every 24 h between litters exposed to room air or hyperoxia. The neonatal hyperoxia exposure paradigm has been described and further detailed previously (Yee et al., 2009). Following hyperoxia exposure, mice were removed from dams and exposed to CAPS, as described previously (Allen et al., 2014a). Neonatal mice were exposed to filtered air or CAPS (< 100 nm in diameter) concentrated 10–20 fold using the Harvard Ultrafine Concentrated Ambient Particle System (HUCAPS). Exposures lasted for 4 h per day beginning at 9:00 a.m. on PND (postnatal day) 4–7 and 10–13. During these exposures pups were housed in small mesh chambers with four pups per chamber. This high-volume ambient sampling system utilizes condensational growth of the particulate phase in conjunction with virtual impaction to provide aerosols enriched for sizes smaller than 200 nm in diameter, with median sizes typically in the 70–90 nm range with concentrations of approximately 0.2–2 × 105/cm3. The gas-phase components of the ambient aerosol are not concentrated by the HUCAPS system. Particle counts were obtained using a condensation particle counter (model 3022A; TSI, Shoreview, MN), and mass concentration calculated using idealized particle density (1.5 g/cm3). The hyperoxia and CAPS exposures generated 4 treatment groups per sex: neonatal hyperoxia with and without CAPS (designated H Air and H CAPS, respectively) and neonatal room air with and without CAPS (designated A Air and A CAPS, respectively). A total of 50 litters were utilized and were culled to six mice per litter prior to exposures. Those litters were randomly assigned but, divided among the four exposure groups so each treatment group had minimum of 12 litters, with litter representing the primary unit of variability, n. Only two pups (one male and one female) were used per litter per behavioral and pathological endpoint. For the PND 14 pathology endpoint, n = 7 for all the treatment groups, except for the male H CAPS group which had an n = 6. For the behavioral groups n = 12, for all treatment groups across both 52
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deliveries for a total of 60 reinforcers. After lever press training was completed in all mice, the schedule was shifted to the intra-extra dimensional shift set task (Fig. 3). For the initial intradimensional discrimination task, pressing the back lever initiated a trial; a subsequent response on the left lever produced a house light flash and delivery of a reinforcer (Fig. 3b). Incorrect responses (back lever or right lever) were punished with a 10s time out period (TO) in which the house light turned off; the time out was reset by any additional lever presses within this period. Following a time out, a lever press on the back lever was required to re-initiate the trial. Incorrect responses included repeated back lever responses, a left or right lever response with no prior back lever response, or a right lever response after a back lever press (discrimination error). A 10 s inter-trialinterval (ITI) occurred after every reinforcer delivery during which the house light was turned off and any lever presses during the ITI reset the 10 s timer. A session ended after the delivery of 40 reinforcers. After 4 sessions, a reversal task was implemented in which a right lever response was required after trial initiation with a left lever response being scored as a reversal error (Fig. 3c). Following 4 sessions of the reversal task, a compounded reversal task was imposed in which trial initiation via a back lever response produced a yellow LED light displayed randomly across trials over the right or left lever but, did not signal the correct lever, as only left lever responses after trial initiation would be reinforced with right lever responses considered a learning error (Fig. 3d). After 4 sessions of the compounded reversal paradigm, an extradimensional discrimination paradigm was implemented. In this paradigm, a lever light would be displayed randomly over the right or left lever following initiation of a trial, and only a press on the lever with the illuminated lever light was reinforced (Fig. 3e). Incorrect responses (i.e., on the non-illuminated lever) initiated a TO and were followed by a correction procedure used to prevent perseverative responding on a single lever following trial initiation. The correction procedure required a back lever press followed by a response on the correct lever, which led to reinforcer delivery and the opportunity to start another new trial. During the correction procedure, the spatial location of the light and thus the correct lever was the same as the previous trial in which the learning error occurred; this would continue indefinitely until a correct response occurred. Errors produced the 10 s TO. Sessions ended following the delivery of 60 reinforcers and a total of 10 sessions were conducted. Cumulative learning errors in each session were the primary performance measurement, with an error defined only as the first error committed on an ED trial, ignoring the repeat errors made on the subsequent correction procedure trials. Incorrect responses on the ED paradigm correction procedure were not included in this measure, given it could not be determined whether correction occurred in response to the light or via simple reversal rules.
sexes. Note the PND 14 endpoint mice are randomly drawn from the same original litters used for the behavioral cohort. All mice used in this study were treated humanely with regard for alleviation of suffering and the study protocol was approved by the University of Rochester Institutional Animal Care and Use Committee. 2.2. Locomotor behavior To evaluate motor activity levels spontaneous locomotor activity was measured in photobeam chambers equipped with a transparent acrylic arena with a 48-channel infrared source, detector, and controller (Med Associates, St. Albans, VT). Locomotor behavior was assessed prior to the start of FI schedule-controlled behavior training (postnatal day 60). Locomotor activity was quantified in three 45-min sessions occurring once per day for 3 consecutive days, with the primary endpoint, ambulatory time, collected at 5 min epochs. Ambulatory time was defined as the cumulative time in which there were successive breaks of 2 × 2 photobeam virtual boxes within the chamber. A period of ambulation is stopped when the animal remained within a 2 × 2 virtual box for ≥300 ms. Habituation and average ambulatory activity in each session was explored. 2.3. Intra-extradimensional shift set task Following locomotor activity assessment, food restriction was imposed to provide motivation for food-rewarded behavior. Specifically, mice were placed on a food-restricted schedule for 3 days immediately prior to initiation of operant training to reach 85% of ad libitum weights. During the initial food restriction for 3 days, females were fed 1.0–1.5 g each and males were fed 1.5–2.0 g each per day with food restriction adjusted appropriately on an individual basis and their subsequent weight changes. Mice were maintained at 85% ad libitum body weight throughout the operant training schedule and feeding commenced immediately after each testing session. 2.4. Apparatus Behavioral testing was conducted in operant chambers (Med Associates, St. Albans, VT) housed in sound-attenuating cabinets equipped with white noise and fans for ventilation. Two fixed levers (right and left) were located on either side of a pellet dispenser for reinforcer delivery while on the opposite back wall, there was a fixed lever centered (back lever) across from the pellet dispenser. The primary incandescent house light was directly over the back lever and one yellow LED light placed over the left and right levers respectively. 2.5. Paradigm For this paradigm, elements of a previously reported model of reversal and extradimensional learning were adapted, in particular the spatial reversal component and the use of a light as a visual discrimination stimulus for the extradimensional shift (Boomhower & Newland, 2017). Mice were initially trained to press a lever for food reward using a fixed ratio 1 schedule (FR1), in which a reinforcer (20 mg food pellet) was delivered following a response on the designated correct lever, which would also trigger a house light flash and clicker sound cue. The correct (i.e., reinforced) lever was designated through a pseudo-randomized 3-trial block schedule in which reinforcement on the first trial was provided for a lever press on any of the available levers (right, left, or back). For the remaining two trials of the 3-trial block, reinforcement was only provided for presses to levers not previously reinforced. If the mouse pressed a previously reinforced lever, a 10s timeout was implemented in which the house light was extinguished, all reinforcement was withheld, and additional presses to any of the levers reset the timeout period. Twenty blocks were run in total in the training session, so each lever initiated twenty food
2.6. Glutamate (Glu) and γ-aminobutyric acid (GABA) neurochemistry analysis On PND 14, 24 h after the final CAPS exposure session, and on PND 240, two weeks after the final behavioral test session, mice were euthanized by cervical dislocation without sedation to preclude effects of anesthetic on neurochemistry. The frontal cortex was dissected from each animal, snap-frozen, and stored at −80 °C until analyses. Tissue was thawed and diluted in perchloric acid before being homogenized via ultra-sonication. The resulting homogenized solution was spun at 10,000g and the supernatant was used for mass spectrometry analysis. 10 uL of each sample was injected using a U3000 UHPLC (Dionex) onto a 2.1 × 150 mm PFP column (Thermo Scientific). Analytes were eluted isocratically with 0.1% formic acid in water and analyzed in positive mode on a TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Scientific). The column was washed with 0.1% formic acid in acetonitrile after analytes had eluted, then re53
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the extradimensional component. Females exposed to CAPS had significantly increased average errors (β = 1.25, SE = 0.56, p = 0.03, n = 12) in the intradimensional discrimination component, as compared to unexposed CAPS controls (Fig. 4A). Interestingly females exposed to hyperoxia had decreased average errors compared to their unexposed hyperoxia controls (β = −1.08, SE = 0.56, p = 0.06, n = 12) though it failed to reach statistical significance (Fig. 4A). CAPS-exposed males tended to have higher initial error levels but, showed a significantly steeper decrement in errors when compared to unexposed controls (β = −2.71, SE = 0.99, p < 0.01, n = 12), (Fig. 4E). A post-hoc ANOM on the first session showed A CAPS males had higher errors than the overall average in Session 1 (β = 8.84, 95% CI: −0.03, 17.71, n = 12), though it failed to reach significance. There were no significant ANOM post-hoc differences on Sessions 2 or Sessions 3 for males. No significant interactions between hyperoxia and CAPS were detected in either males or females for the discrimination task. In the intradimensional reversal task, no significant treatment-related learning or overall error differences were found in females on the reversal task (Fig. 4B). In males an interaction between hyperoxia, CAPS, and session was confirmed (β = 2.34, SE = 1.18, p = 0.05, n = 12) (Fig. 4F) which, the post-hoc ANOM analyses showed males exposed to both neonatal hyperoxia and CAPS had significantly higher errors than the overall mean of all four treatment groups during the second and third sessions (β = 2.03, 95% CI: 0.18, 3.87, n = 12). There were no significant treatment differences in either sex on the compounded reversal (Fig. 4C and F). No significant treatment-related differences in performance were found in females across the 10 sessions for the extradimensional shift component (Fig. 5A). In contrast, males exposed to CAPS had a significantly shallower slope across the 10 sessions (β = 0.26, SE = 0.12, p = 0.04, n = 12) consistent with a slower reduction in errors (Fig. 5B), as did males exposed to hyperoxia (β = 0.24, SE = 0.12, p = 0.04, n = 12). However, interactive or cumulative effects in the slope of error reduction were not observed in males exposed to both hyperoxia and CAPS.
equilibrated with 0.1% formic acid in water prior to the next injection. The flow rate was 400 μL/min, while the column oven was set at 30 °C. The precursor ion for GABA was 104.1 m/z, with fragment ions of 69.3 and 87.2 m/z with collision energies of 15, 10 respectively, at a tube lens voltage of 65 V. The precursor ion for glutamate was 148.1 m/z, with the fragment ions of 84.2 and 102.2 m/z, with collisions energies of 16, 10 respectively at a tube lens voltage of 71 V. Peak areas of each compound were compared to the internal standard to determine relative abundance between samples. 2.7. Statistical analysis Behavioral data were analyzed using a multi-level mixed-model while the glutamate:GABA ratios were assessed using a two-way ANOVA. All statistical analyses were done using JMP13 (Cary, NC) and stratified by sex. A random intercept and slope model was used to capture subject-level variability in behavioral performance. The intercept component was centered and used as a means of exploring the average errors across time, while the slope was used to define the linear function of learning across the sessions. CAPS and hyperoxia were designated as fixed factors and treatment effects were explored on both the intercept and slope components. Only the first 3 sessions of the discrimination and reversal tasks were analyzed to prevent inappropriate skewing of the analyses towards the final session, as it generally included a very prominent floor effect, when errors reached asymptotic levels as they approached zero. Interactions between the two factors were explored for each behavioral parameter, but if the interaction term had a p-value > 0.1, it was dropped from the model to avoid over-parameterization. Significant and main effects were further explored through a post-hoc analysis of means (ANOM), a multiple comparisons tests which, compares the means of each of the four groups with the overall mean across all groups. For behavioral findings, parameter estimates (β), standard errors (SE), along with the significance tests and for the ANOM are reported, as are the estimates and the confidence intervals. The reference groups for the parameter estimates were the unexposed controls with the estimates describing the magnitude of the change in the exposed mice. P ≤ 0.05 was considered statistically significant.
3.4. Glutamate/GABA As assessed on PND 14, i.e., one day following the final CAPS exposure, females exposed to CAPS had significantly elevated glutamate (Glu):GABA ratio in the frontal cortex (F(1, 27) = 13.644, p = 0.001, n = 7) than unexposed controls. Hyperoxia-exposed females had higher mean Glu:GABA ratio than unexposed controls, but the difference did not reach statistical significance (F(1,27) = 3.139, p = 0.089, n = 7) (Fig. 6A). The ANOM post-hoc showed H CAPS females had a higher Glu:GABA ratio than the overall average (β = 0.60, 95% CI: 0.10, 1.10, n = 7) and A Air females had lower Glu:GABA ratio than the overall average (β = −0.59, 95% CI: −1.09, −0.09, n = 7). CAPS-exposed males had significantly higher Glu:GABA ratio than unexposed controls (F(1, 26) = 8.054, p = 0.03, n = 6–7). The ANOM post-hoc showed H CAPS males had a higher Glu:GABA ratio than the overall average (β = 0.69, 95% CI: 0.20, 1.18, n = 6–7) and H Air males had lower Glu:GABA ratio than the overall average (β = −0.52, 95% CI: −1.01, −0.03, n = 6–7) (Fig. 6C). No significant treatment-differences were found in Glu:GABA ratio in the frontal cortex in adult mice of either sex that had undergone behavioral testing (Fig. 6B, D).
3. Results 3.1. CAPS exposure levels Fig. 1 shows particle concentrations (Fig. 1A) median particle diameter (Fig. 1B) and mean exposure mass concentration (Fig. 1C) cross the period of CAPS exposures. Particle sizes remained in the UFP range (< 100 nm), and mean exposure concentrations across treatment were approximately 41,184 particles/cm3 and the average mass was 22.6 μg/ m3. 3.2. Locomotor activity Time spent in ambulation during a 45-min locomotor session is shown in Fig. 2. Hyperoxia-exposed females exhibited a more rapid decline in ambulatory time across the session (β = 8.92, SE = 4.27, p = 0.04, n = 12) than corresponding unexposed controls. A hyperoxia × CAPS interaction on the overall average was found for males (β = 51.30, SE = 21.93, p = 0.02, n = 12) but, the ANOM post-hoc did not indicate average ambulatory time of any group differed from the overall mean.
4. Discussion In a previous study using this model of exposure, we found sexdependent differences in learning on an FI schedule followed by a subsequent extinction paradigm, which suggested the combined exposure altered the capacity of new rule learning (Morris-Schaffer et al., 2018). The objective of the current study was to directly ascertain the augmented risk of cognitive flexibility deficits that could result from
3.3. Intra-extradimensional shift set For all components of this paradigm, mice from all treatment groups obtained all available reinforcers, 40 per session for the intradimensional discrimination and reversal components, and 60 per session for 54
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Fig. 1. Characterization of CAPS exposures. Particle concentration is reported as number of particles × 105/cm3 ± SD (A). Daily diameter of CAPS is reported as nm ± SD (B). Particle mass concentrations are reported in μg/m3 ± SD (C).
(Morris-Schaffer et al., 2018). However the ID-ED shifts are testing rule learning under a different and specific context, all the tasks are learned through positive reinforcing stimuli, whereas extinction is the reduction of response rates in the absence of reinforcing stimuli, which may explain the discrepancy. Given the convergence of both neonatal hyperoxia and CAPS on a similar cognitive flexibility phenotype, and the potential for exposure to both insults to result in enhanced reversal and shift set learning deficits, these data suggest a unique vulnerability or preterm infants in regions with high air pollution. The alteration in learning patterns by hyperoxia and CAPS is particularly indicative of associative and potentially motivational deficits as opposed to general deficiencies in inhibitory functioning. Historically, reversal learning tasks in rodents have been separated into different learning phases, with early trial errors representative of
developmental exposure to neonatal hyperoxia followed by exposure to ambient ultrafine particulate matter using an analog of the intra-extradimensional shift set paradigm. Results showed males exposed to neonatal hyperoxia and CAPS had significantly increased errors on the later sessions in the intradimensional shift reversal component. Moreover, on the more difficult extradimensional shift, males exposed to neonatal hyperoxia, CAPS, or both had significant decreases in learning rate, as evidenced by a slower and incomplete reduction in errors. Interestingly, we did not see any treatment-related difficulties in the females with adjusting to the reversal or the extradimensional schedule, highlighting the robust sex-specific nature of the treatment effects. Furthermore this result is somewhat contrary to our previous study, which showed only combined exposure in females led to learning deficits when adjusting from a fixed interval to an extinction schedule
Fig. 2. Group mean ± S.E. ambulatory activity in female (A) and male mice (B) across 5 min blocks of a 45-min behavioral test session. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure, respectively. α ≤ 0.05. 55
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Fig. 3. Timeline of intradimensional-extradimensional shift set paradigm, 4 sessions of discrimination, 4 sessions of reversal, 4 sessions of compounded reversal, and 10 sessions of extra-dimensional shift. All sessions across all tasks were done consecutively. The initial intradimensional discrimination task reinforced a left lever response following trial initiation with a right lever response leading to a 10 s time out punishment (B). For the initial intradimensional reversal task, responses on the right lever were reinforced and the left lever produced timeout (C). For the compounded reversal, a yellow LED light over either lever was randomly displayed after trial initiation and responses on the left lever were always reinforced regardless of which lever light was illuminated, while responses on the right lever produced timeout (D). For the extradimensional shift set, responses were reinforced if they occurred on the lever beneath which, the light was illuminated, with the spatial location randomized across trials (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
to escape on the Barnes Maze was seen in Wistar rats exposed to 80% oxygen for 24 h on PND 6 indicative of memory deficits (Serdar et al., 2016). This was complemented by another study that examined the effects of 85% oxygen from PND 2 to PND 14, which found exposedC57BL/6 mice spent an increased amount of time to find the platform on the Morris Water Maze, a test of spatial learning and memory, and a decreased recognition index on the novel object paradigm, a test of recognition memory (Ramani et al., 2013). Although we did not find substantial hyperoxia-induced differences in ambulatory time in either sex beyond habituation alterations in females, another study found exposure to 80% O2 from PND 6 to PND 8 led to an increase in running wheel activity in C57BL/6 adolescent mice (Schmitz et al., 2012). One major limitation of relating the current study to previously published work, beyond the lack of a standardized exposure model, is none of these studies stratified analyses by sex or included it as an independent factor. Given these neonatal hyperoxia exposure windows involve critical periods of sexually dimorphic development within the CNS (Schulz & Sisk, 2016; Arnold & McCarthy, 2016), and our current study and previous work (Morris-Schaffer et al., 2018) consistently show a malespecific learning deficit, it is essential for further research to analyze and present the sexes separately. The extradimensional learning impairments observed in CAPStreated mice is supported by earlier published work using different behavioral paradigms but, the same time period of neonatal exposure to CAPS. In a two-lever repeated learning (RL) response chain schedule, in which mice were expected to learn a new two-lever sequence every
perseverative deficits and latent trials errors indicative of underlying associative or motivational behavioral domains (Hilson & Strupp, 1997; Widholm et al., 2003; Garavan et al., 2000). The only significant results in females were CAPS-exposed mice made more errors on the original discrimination task, which could potentially indicate early motivational deficiencies in engaging with the task rather than deficiencies in rule learning, which were not present on the more complex tasks for females. In males, although CAPS mice did show increased errors on the original discrimination task, the combined effect of hyperoxia and CAPS in the males was only seen in the last two sessions of the reversal task. The latency of the combined effect may indicate an initial deficiency or delay in the strength of association between the new spatial location and the reinforcing stimuli. This is further supported by the lack of combined treatment effect on the compounded reversal, which indicates general inhibitory control and flexibility is still maintained. Similarly on the ED shift in the males, prominent treatment differences were only present in the later sessions, again indicative of the treated mice's incapacity to generate and maintain a strong association between the new contingency and reinforcing stimulus. The observation that the deficit in the temporal learning pattern on the ED shift is virtually equivalent in the single exposure groups alone strongly suggests a common cognitive domain target between the two insults. To the best of our knowledge this is the first study directly examining cognitive flexibility deficits in mice neonatally exposed to hyperoxia. Previous behavioral studies have shown neonatal hyperoxia can lead to memory deficits and hyperactivity. Increased latency time 56
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Fig. 4. Group mean ± S.E. errors in female and male mice performing on the initial intradimensional discrimination (A, D), the reversal (B, E), and the compounded reversal (C, F) components. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. H × CAPS × Session indicates a statistical interaction of the treatments across the sessions. +/− denotes a statistical significant increase/decrease from the overall average as determined by analysis of means (ANOM). α ≤ 0.05.
reinforced; premature responses reset the timer. Sustained deficits were not seen across sessions at any given DRL value, but emerged only during the transitions to increasing DRL values, with CAPS-exposed males responding prematurely, again suggesting an initial difficulty in adjusting to new contingency of reinforcement (Cory-Slechta et al., 2017). As a potential underlying physiological mechanism that could
session, CAPS exposed males made significantly more errors during the earlier sessions of the schedule (Cory-Slechta et al., 2017). These errors were not persistent and were suggestive of initial difficulty in transitioning between different RL contingencies of reinforcement. A similar outcome was seen in the same study on a differential reinforcement of low rate (DRL) schedule of reward in which a specific length of time (DRL value) must elapse without a response before a response can be 57
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Fig. 5. Group mean ± S.E. errors in female (A) and male mice (B) in the extradimensional shift task. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. H × CAPS × Session indicates a statistically significant interaction of the treatments across the sessions. α ≤ 0.05.
transporter expression in astrocytes reducing their ability to uptake aspartic acid (Schmitz et al., 2011). The absence of treatment-related differences in Glu:GABA ratio in the adult behavior mice could be due to the transient nature of neurochemical changes in response to these exposures. Alternatively, an enriching learning environment alone is an extraneous factor known to heavily influence glutamate and GABA ratio within the CNS (He et al., 2010; Segovia et al., 2006). It is important to note the increase in Glu:GABA ratio in the frontal cortex at PND 14 in both males and females was insufficient to explain the prominent malespecific functional behavioral phenotype. Future studies should explore more static markers of frontal cortex activity in exposed mice, including protein expression of glutamate and GABA receptors, and dendritic branching complexity. Additionally, in mice that did not experience weight restriction and behavioral testing, extraneous factors that could modify toxicity, would be better suited for exploring the long-term
explain the behavioral deficits our lab explored the ratio of glutamate, and GABA, the classical excitatory and inhibitory neurotransmitters in the frontal cortex. Imbalances in inhibitory and excitatory signaling within the frontal cortex have been shown to modulate extradimensional shifts (Marquardt et al., 2014; Brigman et al., 2009; Kos et al., 2011; Laurent & Podhorna, 2004). Interestingly, PND 14 males and females exposed to both hyperoxia and CAPS had increased Glu:GABA ratio relative to the overall average. These results complement data from earlier neonatal CAPS-only exposures that revealed alterations in glutamate and GABA ratio in both sexes within the frontal cortex at PND 14, although the “dose” of particle concentration in those exposures was 3–4x as high as the current exposure (Allen et al., 2014c; Allen et al., 2014a; Allen et al., 2014b). The exact mechanism by which hyperoxia is enhancing the CAPS effect is unclear, although one study reported exposure to neonatal hyperoxia decreased glutamate-aspartate
Fig. 6. Group mean ± S.D. glutamate:GABA ratio in the frontal cortex of females (A) and males (C) at PND 14 and for females (B) and males (D) at PND 240 following behavioral testing. Raw data are reported for each treatment group as well as the mean and standard deviation. H or CAPS indicates statistical main effect of postnatal hyperoxia and CAPS exposure respectively. +/− denotes a statistically significant increase/decrease from the overall average as determined by analysis of means (ANOM). α ≤ 0.05.
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differences in how males and females differentially recover from neonatal hyperoxia and CAPS exposure. As with our previous study, we did not see any gross differences in activity levels in males or females, although interestingly in the current study, females exposed to hyperoxia had an altered habituation trajectory, which we did not observe previously (Morris-Schaffer et al., 2018). This new observation may be attributed to the greater sample size and power in the current study. Importantly, however, the lack of overall activity differences strengthens the assertion neonatal hyperoxia and CAPS exposures at the current levels are primarily influencing cognitive development in males. In summary, exposure to hyperoxic conditions followed by exposure to concentrated ambient ultrafine particles in mice produced male-preferential deficits in cognitive function. Further work is needed to elaborate the underlying physiological mechanisms through which hyperoxia and CAPS converge to produce an augmented deficit. However, the findings in the present studies suggest preterm infants born in regions with high air pollution may have a unique ongoing neurodevelopmental vulnerability to particulate matter
Gao, Y., Raj, J.U., 2010. Regulation of the pulmonary circulation in the fetus and newborn. Physiol. Rev. 90 (4), 1291–1335. Garavan, H., et al., 2000. Prenatal cocaine exposure impairs selective attention: evidence from serial reversal and extradimensional shift tasks. Behav. Neurosci. 114 (4), 725. Glinianaia, S.V., et al., 2004. Particulate air pollution and fetal health: a systematic review of the epidemiologic evidence. Epidemiology 15 (1), 36–45. He, S., et al., 2010. Early enriched environment promotes neonatal GABAergic neurotransmission and accelerates synapse maturation. J. Neurosci. 30 (23), 7910–7916. Hilson, J.A., Strupp, B.J., 1997. Analyses of response patterns clarify lead effects in olfactory reversal and extradimensional shift tasks: assessment of inhibitory control, associative ability, and memory. Behav. Neurosci. 111 (3), 532–542. Kos, T., et al., 2011. The effects of NMDA receptor antagonists on attentional set-shifting task performance in mice. Psychopharmacology 214 (4), 911–921. Laurent, V., Podhorna, J., 2004. Subchronic phencyclidine treatment impairs performance of C57BL/6 mice in the attentional set-shifting task. Behav. Pharmacol. 15 (2), 141–148. Marquardt, K., et al., 2014. Loss of GluN2A-containing NMDA receptors impairs extradimensional set-shifting. Genes Brain Behav. 13 (7), 611–617. Morris-Schaffer, K., et al., 2018. Effect of neonatal hyperoxia followed by concentrated ambient ultrafine particle exposure on cumulative learning in C57Bl/6J mice. Neurotoxicology 67, 234–244. Newman, N.C., et al., 2013. Traffic-related air pollution exposure in the first year of life and behavioral scores at 7 years of age. Environ. Health Perspect. 121 (736), 731–736. Oberdörster, G., Ferin, J., Lehnert, B.E., 1994. Correlation between particle size, in vivo particle persistence, and lung injury. Environ. Health Perspect. 102 (Suppl. 5), 173–179. Oberdorster, G., et al., 2004. Translocation of inhaled ultrafine particles to the brain. Inhal. Toxicol. 16 (6–7), 437–445. Oberdörster, G., Oberdörster, E., Oberdörster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113 (7), 823–839. Park, E.J., et al., 2015. Chronic pulmonary accumulation of iron oxide nanoparticles induced Th1-type immune response stimulating the function of antigen-presenting cells. Environ. Res. 143 (Pt A), 138–147. Pichler, G., et al., 2013. Reference ranges for regional cerebral tissue oxygen saturation and fractional oxygen extraction in neonates during immediate transition after birth. J. Pediatr. 163 (6), 1558–1563. Ramani, M., et al., 2013. Neurodevelopmental impairment following neonatal hyperoxia in the mouse. Neurobiol. Dis. 50, 69–75. Schmitz, T., et al., 2011. Cellular changes underlying hyperoxia-induced delay of white matter development. J. Neurosci. 31 (11), 4327–4344. Schmitz, T., et al., 2012. Adolescent hyperactivity and impaired coordination after neonatal hyperoxia. Exp. Neurol. 235 (1), 374–379. Schulz, K.M., Sisk, C.L., 2016. The organizing actions of adolescent gonadal steroid hormones on brain and behavioral development. Neurosci. Biobehav. Rev. 70, 148–158. Segovia, G., et al., 2006. Environmental enrichment promotes neurogenesis and changes the extracellular concentrations of glutamate and GABA in the hippocampus of aged rats. Brain Res. Bull. 70 (1), 8–14. Serdar, M., et al., 2016. Fingolimod protects against neonatal white matter damage and long-term cognitive deficits caused by hyperoxia. Brain Behav. Immun. 52, 106–119. Sink, D.W., Hope, S.A., Hagadorn, J.I., 2011. Nurse:patient ratio and achievement of oxygen saturation goals in premature infants. Arch. Dis. Child. Fetal Neonatal Ed. 96 (2), F93–F98. Sola, A., 2015. Oxygen saturation in the newborn and the importance of avoiding hyperoxia-induced damage. NeoReviews 16 (7), e393–e405. Soria-Pastor, S., et al., 2008. Patterns of cerebral white matter damage and cognitive impairment in adolescents born very preterm. Int. J. Dev. Neurosci. 26 (7), 647–654. Sram, R.J., et al., 2005. Ambient air pollution and pregnancy outcomes: a review of the literature. Environ. Health Perspect. 113 (4), 375–382. Stoll, B.J., et al., 2015. Trends in care practices, morbidity, and mortality of extremely preterm neonates, 1993–2012. JAMA 314 (10), 1039–1051. Suglia, S.F., et al., 2008. Association of black carbon with cognition among children in a prospective birth cohort study. Am. J. Epidemiol. 167 (3), 280–286. Sun, Q., et al., 2012. Pulmotoxicological effects caused by long-term titanium dioxide nanoparticles exposure in mice. J. Hazard. Mater. 235–236, 47–53. Trasande, L., Malecha, P., Attina, T.M., 2016. Particulate matter exposure and preterm birth: estimates of us attributable burden and economic costs. Environ. Health Perspect. 124 (12), 1913–1918. Verhagen, E.A., et al., 2015. Cerebral oxygenation is associated with neurodevelopmental outcome of preterm children at age 2 to 3 years. Dev. Med. Child Neurol. 57 (5), 449–455. Widholm, J.J., et al., 2003. Effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-pdioxin on spatial and visual reversal learning in rats. Neurotoxicol. Teratol. 25 (4), 459–471. Yee, M., et al., 2009. Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity. Am. J. Phys. Lung Cell. Mol. Phys. 297 (4), L641–L649.
Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was funded in part by National Institutes of Health Grants R01 ES025541 (D.A. Cory-Slechta), R01 HL091968 (M. A. O'Reilly). NIH Training Grant T32 ES007026 supported K. Morris-Schaffer. NIH Center Grant P30 ES001247 supported the animal inhalation facility and the animal behavior core. References Allen, J.L., et al., 2013. Developmental exposure to concentrated ambient particles and preference for immediate reward in mice. Environ. Health Perspect. 121 (1), 32–38. Allen, J.L., et al., 2014a. Early postnatal exposure to ultrafine particulate matter air pollution: persistent ventriculomegaly, neurochemical disruption, and glial activation preferentially in male mice. Environ. Health Perspect. 122 (9), 939–945. Allen, J.L., et al., 2014b. Consequences of developmental exposure to concentrated ambient ultrafine particle air pollution combined with the adult paraquat and maneb model of the Parkinson's disease phenotype in male mice. Neurotoxicology 41, 80–88. Allen, J.L., et al., 2014c. Developmental exposure to concentrated ambient ultrafine particulate matter air pollution in mice results in persistent and sex-dependent behavioral neurotoxicity and glial activation. Toxicol. Sci. 140 (1), 160–178. Almaazmi, M., et al., 2013. Cerebral near-infrared spectroscopy during transition of healthy term newborns. Neonatology 103 (4), 246–251. Arnold, A.P., McCarthy, M.M., 2016. Sexual differentiation of the brain and behavior: a primer. In: Neuroscience in the 21st Century, pp. 1–30. Backes, C.H., et al., 2013. Early life exposure to air pollution: how bad is it? Toxicol. Lett. 216 (1), 47–53. Bhutta, A.T., et al., 2002. Cognitive and behavioral outcomes of school-aged children who were born preterm: a meta-analysis. JAMA 288 (6), 728–737. Boomhower, S.R., Newland, M.C., 2017. Effects of adolescent exposure to methylmercury and d-amphetamine on reversal learning and an extradimensional shift in male mice. Exp. Clin. Psychopharmacol. 25 (2), 64. Brigman, J.L., et al., 2009. Effects of subchronic phencyclidine (PCP) treatment on social behaviors, and operant discrimination and reversal learning in C57BL/6J mice. Front. Behav. Neurosci. 3, 2. Cory-Slechta, D., et al., 2017. Developmental exposure to low level ambient ultrafine particle air pollution and cognitive dysfunction. Neurotoxicology. Deulofeut, R., et al., 2006. Avoiding hyperoxia in infants < or = 1250 g is associated with improved short- and long-term outcomes. J. Perinatol. 26 (11), 700–705. Elder, A., et al., 2006. Translocation of inhaled ultrafine manganese oxide particles to the central nervous system. Environ. Health Perspect. 114 (8), 1172–1178. Fray, P.J., Robbins, T.W., 1996. CANTAB battery: proposed utility in neurotoxicology. Neurotoxicol. Teratol. 18 (4), 499–504.
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