Wildfire-related smoke inhalation worsens cardiovascular risk in sleep disrupted rats.

Introduction: As a lifestyle factor, poor sleep status is associated with increased cardiovascular morbidity and mortality and may be influenced by environmental stressors, including air pollution. Methods: To determine whether exposure to air pollution modified cardiovascular effects of sleep disruption, we evaluated the effects of single or repeated (twice/wk for 4 wks) inhalation exposure to eucalyptus wood smoke (ES; 964 µg/m3 for 1 h), a key wildland fire air pollution source, on mild sleep loss in the form of gentle handling in rats. Blood pressure (BP) radiotelemetry and echocardiography were evaluated along with assessments of lung and systemic inflammation, cardiac and hypothalamic gene expression, and heart rate variability (HRV), a measure of cardiac autonomic tone. Results and Discussion: GH alone disrupted sleep, as evidenced by active period-like locomotor activity, and increases in BP, heart rate (HR), and hypothalamic expression of the circadian gene Per2. A single bout of sleep disruption and ES, but neither alone, increased HR and BP as rats transitioned into their active period, a period aligned with a critical early morning window for stroke risk in humans. These responses were immediately preceded by reduced HRV, indicating increased cardiac sympathetic tone. In addition, only sleep disrupted rats exposed to ES had increased HR and BP during the final sleep disruption period. These rats also had increased cardiac output and cardiac expression of genes related to adrenergic function, and regulation of vasoconstriction and systemic blood pressure one day after final ES exposure. There was little evidence of lung or systemic inflammation, except for increases in serum LDL cholesterol and alanine aminotransferase. These results suggest that inhaled air pollution increases sleep perturbation-related cardiovascular risk, potentially in part by increased sympathetic activity.

Impacts of a perinatal exposure to manganese coupled with maternal stress in rats: Learning, memory and attentional function in exposed offspring.

The developmental effects of chemicals that co-occur in vulnerable populations with elevated psychological stress are of increasing concern to the public. To investigate these concerns, we developed a rodent model of co-occurring perinatal manipulations and conducted a series of cognitive assessments in male and female offspring. Manganese (Mn), a neurodevelopmental toxicant when exceeding physiological requirements, was delivered in the drinking water (0, 2, or 4 mg Mn/mL) of rats from gestational day (GD) 7 to postnatal day (PND) 22. A variable perinatal stress paradigm was applied to half of the animals from GD13 to PND9. Novel object recognition (NOR), Morris water maze (MWM), differential reinforcement of low-rates procedure (DRL) and cued and uncued choice reaction time (CRT) tests were used to assess cognitive functions in offspring. Mn (4 mg/mL) and stress impaired NOR in adolescent males but facilitated NOR performance in females. However, when stress and Mn were combined these effects were attenuated in both sexes. During training for the DRL, Mn (2 mg/mL) facilitated, while stress impaired, lever press learning in both sexes. Few effects related to the treatments were found on DRL or MWM. During cued CRT, Mn (2 and 4 mg/mL) and stress reduced accuracy in males, while stress and Mn (2 mg/mL) increased anticipatory responding and slowed decision time in both sexes. Stress combined with Mn (2 mg/mL) improved cued accuracy and decision time, and Mn attenuated the effect of stress on anticipatory responding in both sexes. Stress slowed female movement time but when combined with Mn (4 mg/mL) the effect of stress was attenuated. During uncued CRT, except for decision time (which replicated effects observed with the cued task), no other effects of Mn or its combination with stress occurred. Females remained negatively affected by stress in most uncued CRT performance measures, while stressed improved male uncued accuracy. Taken together these data do not support increased cognitive impairment produced by Mn when combined with stress. However, the effects of perinatal stress alone, on these cognitive functions may hinder the detection of effects due to chemical exposures and underscores the need to consider the psychological health and wellbeing of the mother and her environment in risk assessment for developmental neurotoxicity of chemicals.

Prenatal exposure to vapors of gasoline-ethanol blends causes few cognitive deficits in adult rats.

Developmental exposure to inhaled ethanol-gasoline fuel blends is a potential public health concern. Here we assessed cognitive functions in adult offspring of pregnant rats that were exposed to vapors of gasoline blended with a range of ethanol concentrations, including gasoline alone (E0) and gasoline with 15% or 85% ethanol (E15 and E85, respectively). Rat dams were exposed for 6.5h daily to the vapors at concentrations of 0, 3000, 6000, or 9000 ppm in inhalation chambers from gestational day (GD) 9 through 20. Cage controls (offspring of non-exposed dams that remained in the animal facility during these exposures) were also assessed in the E0 experiment, but showed no consistent differences from the offspring of air-exposed controls. Offspring were tested as adults with trace fear conditioning, Morris water maze, or appetitive operant responding. With fear conditioning, no significant effects were observed on cue or context learning. In the water maze, there were no differences in place learning or escaping to a visible platform. However, during the reference memory probe (no platform) male rats exposed prenatally to E85 vapor (6000 and 9000 ppm) failed to show a bias for the target quadrant. Across studies, females (treated and some controls) were less consistent in this measure. Males showed no differences during match-to-place learning (platform moved each day) in any experiment and females showed only transient differences in latency and path length in the E0 experiment. Similarly, no differences were observed in delayed match-to-sample operant performance of E0 males or females; thus this test was not used to evaluate effects of E15 or E85 vapors. During choice reaction time assessments (only males were tested) decision and movement times were unimpaired by any prenatal exposure, while anticipatory responses were increased by vapors of E0 (9000 ppm) and E15 (6000 and 9000 ppm), and the latter group also showed reduced accuracy. E85 vapors did not disrupt any choice reaction time measure. Finally, no response inhibition deficit was observed in a differential reinforcement of low rate (DRL) response schedule in males or females in the E15 or E85 experiments. In summary, prenatal exposure to these fuel blends produced few deficits in adult offspring on these cognitive tests. Significant effects found during a water maze probe trial and choice reaction time tests were observed at vapor concentrations of 6000 ppm or higher, a concentration that is 4-6 orders of magnitude higher than those associated with normal automotive fueling operations and garages. Similar effects were not consistently observed in a previous study of inhaled ethanol, and thus these effects cannot be attributed to the concentration of ethanol in the mixture.

Selective cognitive deficits in adult rats after prenatal exposure to inhaled ethanol.

Increased use of ethanol blends in gasoline suggests a need to assess the potential public health risks of exposure to these fuels. Ethanol consumed during pregnancy is a teratogen. However, little is known about the potential developmental neurotoxicity of ethanol delivered by inhalation, the most likely route of exposure from gasoline-ethanol fuel blends. We evaluated the potential cognitive consequences of ethanol inhalation by exposing pregnant Long Evans rats to clean air or ethanol vapor from gestational days 9-20, a critical period of neuronal development. Concentrations of inhaled ethanol (5000, 10,000, or 21,000 ppm for 6.5h/day) produced modeled peak blood ethanol concentrations (BECs) in exposed dams of 2.3, 6.8, and 192 mg/dL, respectively. In offspring, no dose-related impairments were observed on spatial learning or working memory in the Morris water maze or in operant delayed match-to-position tests. Two measures showed significant effects in female offspring at all ethanol doses: 1) impaired cue learning after trace fear conditioning, and 2) an absence of bias for the correct quadrant after place training during a reference memory probe in the Morris water maze. In choice reaction time tests, male offspring (females were not tested) from the 5000 and 10,000 ppm groups showed a transient increase in decision times. Also, male offspring from the 21,000 ppm group made more anticipatory responses during a preparatory hold period, suggesting a deficit in response inhibition. The increase in anticipatory responding during the choice reaction time test shows that inhaled ethanol yielding a peak BEC of ~200mg/dL can produce lasting effects in the offspring. The lack of a dose-related decrement in the effects observed in females on cue learning and a reference memory probe may reflect confounding influences in the exposed offspring possibly related to maternal care or altered anxiety levels in females. The surprising lack of more pervasive cognitive deficits, as reported by others at BECs in the 200mg/dL range, may reflect route-dependent differences in the kinetics of ethanol. These data show that response inhibition was impaired in the offspring of pregnant rats that inhaled ethanol at concentrations at least 5 orders of magnitude higher than concentrations observed during normal automotive transport and fueling operations, which rarely exceed 100 ppb.

Extrapolating the acute behavioral effects of toluene from 1- to 24-h exposures in rats: roles of dose metric and metabolic and behavioral tolerance.

Recent research on the acute effects of volatile organic compounds suggests that extrapolation from short (∼1 h) to long durations (up to 4 h) may be improved by using estimates of brain toluene concentration (Br[Tol]) instead of cumulative inhaled dose (C × t) as a metric of dose. This study compared predictions of these two dose metrics on the acute behavioral effects of inhaled toluene in rats during exposures up to 24 h in duration. We first evaluated estimates of Br[Tol] with a physiologically based toxicokinetic (PBTK) model for rats intermittently performing an operant task while inhaling toluene for up to 24 h. Exposure longer than 6 h induced P450-mediated metabolism of toluene. Adjusting the corresponding parameters of the PBTK model improved agreement between estimated and observed values of Br[Tol] in the 24-h exposure scenario. Rats were trained to perform a visual signal detection task and were then tested while inhaling toluene (0, 1125, and 1450 ppm for 24 h and 1660 ppm for 21 h). Tests occurred at times yielding equivalent C × t products but different estimates of Br[Tol], and also at 1 and 6 h afterexposure. Effects of toluene were better predicted by Br[Tol] than by C × t. However, even using Br[Tol] as the dose metric (after accounting for metabolic induction), acute dose-effect functions during 24-h exposures were shifted to the right relative to 1-h exposures, indicating that a dynamic behavioral tolerance also developed during prolonged exposure to toluene.

Gender-dependent behavioral and sensory effects of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) in rats.

Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with behavioral and cognitive deficits in humans and animal models. Perinatal exposure to PCBs has also been associated with sensory deficits in animal models. These effects were hypothesized to be mediated in part by ortho-substituted PCBs, which do not or weakly bind to the aryl hydrocarbon (Ah) receptor. The present studies were designed to determine whether perinatal exposure to Aroclor 1254, a commercial mixture of > 99% ortho-substituted PCBs, would affect cognitive and sensory function in Long-Evans rats. Adult male and female offspring of female rats fed Aroclor 1254 (Lot #124-191; doses of 0, 1, or 6 mg/kg/day; gestational day 6 through postnatal day 21; n = eight/group) were trained to perform a signal detection task capable of assessing sensory thresholds. Training included autoshaping and operant conditioning. Thresholds for detecting a 1-s light stimulus were determined under background illuminations ranging from 2 lux to complete darkness. Female rats exposed to Aroclor 1254 autoshaped more rapidly than control females, at a rate akin to control males. Control females had lower thresholds than control males at all levels of background illumination. These differences were abolished by Aroclor 1254, which reduced thresholds in males and increased thresholds in females. These data extend previous findings of gender-specific effects of PCBs on neurobehavioral development to measures of acquisition and sensory function.

Characterizing tolerance to trichloroethylene (TCE): effects of repeated inhalation of TCE on performance of a signal detection task in rats.

Previous work showed that rats develop tolerance to the acute behavioral effects of trichloroethylene (TCE) on signal detection if they inhale TCE while performing the task and that this tolerance depends more upon learning than upon changes in metabolism of TCE. The present study sought to characterize this tolerance by assessing signal detection in rats during three phases of TCE exposures. Tolerance was induced in Phase 1 (daily 1-h test sessions concurrent with TCE exposure), extinguished in Phase 2 (daily tests in air with intermittent probe tests in TCE), and reinduced in Phase 3. Original induction in Phase 1 required 2 weeks, whereas reinduction in Phase 3 required less than 1 week. Tolerance persisted for 2 (accuracy) or 8 weeks [response time] in Phase 2 and was resistant to changes in test conditions in Phase 3. The slow induction, gradual extinction, savings during reinduction and lack of disruption from altered test conditions suggest mediation by instrumental learning processes. These data and most other evidence for behavioral tolerance to solvents can be explained by solvent-induced loss of reinforcement.

Behavioral components of tolerance to repeated inhalation of trichloroethylene (TCE) in rats.

The possibility that the acute neurotoxic effects of organic solvents change with repeated exposure will affect risk assessment of these pollutants. We observed previously that rats inhaling trichloroethylene (TCE) showed a progressive attenuation of impairment of signal detection behavior across several weeks of intermittent exposure, suggesting the development of tolerance. Here, we explored the development of tolerance to TCE during two weeks of daily exposures, and the degree to which learned behavioral modifications ("behavioral tolerance") could account for the effect. Adult Long-Evans rats were trained to perform a visual signal detection task (SDT) in which a press on one lever yielded food if a visual stimulus (a "signal") had occurred on that trial, and a press on a second lever produced food if no signal had been presented. In two experiments, with 2000 and 2400 ppm of TCE respectively, trained rats were divided into two groups (n = 8/group) with equivalent accuracy and then exposed to TCE in two-phase studies. In Phase 1, one group of rats received daily SDT tests paired with 70-min TCE exposures, followed by 70-min exposures to clean air after testing. The other group received daily SDT tests in clean air, followed by 70-min exposures to TCE (unpaired exposure and testing). All rats thus received the same number and daily sequence of exposures to TCE that differed only in the pairing with SDT testing. Both concentrations of TCE disrupted performance of the paired groups and this disruption abated over the 9 days of exposure. In Phase 2, the pairing of exposure and test conditions were reversed for the two groups. The groups that were shifted from unpaired to paired exposures (Unpaired-Paired groups) showed qualitatively similar patterns of deficit and recovery as did the rats whose tests were initially paired with TCE (Paired-Unpaired groups), indicating that task-specific learning was involved in the development of tolerance. Quantitative differences in the magnitude and duration of the effects of TCE in the two groups indicated that other factors, not specific to the SDT, also contributed to the development tolerance to TCE. Published by Elsevier Science Inc.

A comparison of the effects of bilateral and unilateral infusions of muscimol into the basal forebrain on cued detection of visual targets in rats.

This study investigated the role of the basal forebrain cholinergic system (BFCS) in rats' performance of a visuospatial attention task. Muscimol was infused bilaterally and unilaterally into the BFCS to inhibit cholinergic projections to the cortex. Muscimol slowed responding without significantly affecting side-bias. Bilateral infusions increased accuracy for all targets, whereas unilateral infusions reduced accuracy for targets contralateral to the infusion and increased accuracy for targets ipsilateral to the infusion. After a low unilateral dose of muscimol, invalid cues impaired detection of contralateral targets and spared detection of ipsilateral targets. A high unilateral dose of muscimol impaired detection of contralateral targets independently of cueing. These results suggest that interhemispheric imbalance in cortical activity by pharmacological manipulation of the BFCS can impair the detection of lateralized visual stimuli.

Selective removal of cholinergic neurons in the basal forebrain alters cued target detection.

A spatial orienting task was used to assess attention in rats with selective cholinergic lesions of the basal forebrain. The task required each rat to press a lever in response to a visual target that could occur in one of two locations. A target could be preceded by a cue that either accurately predicted the location of the target (valid) or appeared in the location opposite the target (invalid). Target detection was facilitated by valid cues and degraded by invalid cues in control rats. Performance of rats with lesions was equivalent to that of control rats for valid cues, but reflected an increased cost of invalid cueing. These data support a modulatory role for the basal forebrain cholinergic system in visuospatial attention.

Effects of unilateral removal of basal forebrain cholinergic neurons on cued target detection in rats.

Corticopetal cholinergic neurons in the basal forebrain (BF) were removed unilaterally from rats by infusing the cholinergic immunotoxin 192 IgG-saporin into the substantia innominata. After 2 weeks, the rats with right-hemisphere infusions showed signs of visuospatial neglect for targets in the left visual field in a cued visual target detection task based upon human covert orienting procedures. No behavioral effects were evident 4-6 weeks post-infusion. Ten to 22 weeks post-infusion all rats responded more quickly and less accurately to targets in the visual field contralateral to the infusion than to targets ipsilateral to the infusion: further, accuracy for contralateral targets decreased with increasing time between trial initiation and target presentation (target delay), whereas accuracy for ipsilateral targets increased with target delay. Cues did not affect responding to targets in the contralateral visual field more than to targets in the ipsilateral field. The changes in performance could not be attributed to sensory or mnemonic impairment or to response bias. The temporal characteristics of response accuracy and latency suggest the competitive interaction of two time-dependent processes: an attentional process which relies upon cholinergic input from the BF, and a response preparation process which is normally inhibited by the attentional process. These results suggest a role for corticopetal cholinergic pathways in maintaining attention to salient stimuli by inhibiting subcortical motor circuits.

Detection of visual signals by rats: effects of chlordiazepoxide and cholinergic and adrenergic drugs on sustained attention.

Central cholinergic and adrenergic pathways support the attentional processes necessary for detecting and reporting temporally unpredictable stimuli. To assess the functional effects of pharmacological manipulations of these pathways, male Long-Evans rats performed a two-choice, discrete-trial signal-detection task in which food was provided for pressing one lever after presentation of a signal (a 300-ms light flash), and for pressing a second lever at the end of a trial lacking a signal. Seven signal intensities were presented during each 1-h session in a pseudo-random order across three 100-trial blocks. After acquisition of a stable performance baseline, the acute effects of chlordiazepoxide (0, 3, 5, 8 mg/kg i.p.), pilocarpine (0, 1.0, 1.8, 3.0 mg/kg s.c.), scopolamine 0, 0.030, 0.056, 0.100 mg/kg s.c.), nicotine (0, 0.08, 0.25, 0.75 mg/kg s.c.), mecamylamine (0, 1.8, 3.0, 5.6 mg/kg i.p.), clonidine (0, 0.003, 0.010, 0.030 mg/kg s.c.), and idazoxan (0, 1, 3, 10 mg/kg s.c.) were assessed. Five measures of performance were analyzed: response failures; the proportion of "hits" [P(hit): the proportion of correct responses on signal trials]; the proportion of "false alarms" [P(fa): the proportion of incorrect responses on non-signal trials]; and response times (RT) for hits and for correct rejections. All drugs which slowed responding affected RT for hits and correct rejections equivalently, suggesting little or no influence of motor slowing on choice accuracy. Chlordiazepoxide reduced P(hit) at low signal intensities only, without affecting P(fa) or RT, consistent with sensory impairment (reduced visual sensitivity). All other drugs except nicotine reduced P(hit) at high signal intensities preferentially, suggesting a non-visual source of the impairment. Scopolamine, mecamylamine and clonidine affected both P(hit) and P(fa); pilocarpine and idazoxan reduced P(hit) without affecting P(fa). Nicotine at 0.75 mg/kg decreased P(hit) in the first block of trials; at 0.08 mg/kg it increased P(hit) in the second block; no dose affected P(fa). RTs were increased by pilocarpine, scopolamine, mecamylamine and clonidine, but not by nicotine or idazoxan. The data suggest that drugs which reduce cholinergic or adrenergic tone (scopolamine, mecamylamine and clonidine) impair sustained attention by decreasing the detection of signals and by increasing the false alarm rate, whereas drugs which elevate cholinergic or adrenergic tone (pilocarpine, nicotine and idazoxan) decrease attention by impairing detection of signals without affecting the false alarm rate. In contrast, the GABA-facilitating drug chlordiazepoxide appeared to affect visual thresholds rather than attention.

2,4-Dithiobiuret in rats: cognitive facilitation after acute injection precedes motor impairment after repeated daily injections.

2,4-Dithiobiuret (DTB) is a sulfonated derivative of urea that is used as a reducing agent in chemical manufacture. Its low acute toxicity to rodents belies a peripherally mediated, delayed-onset muscle weakness which develops during repeated daily exposure. In experiment 1, a standard dose regimen of DTB (0.5 mg/kg per day IP for 5 days) was used to induce motor dysfunction as a way to dissociate peripheral and central influences on a test of cognitive and motor function in rats. Sixteen male rats were trained to perform a Delayed Matching-to-Position/Visual Discrimination (DMTP/VD) task which permits quantification of working memory (matching accuracy), reference memory (discrimination accuracy), and motor function (choice response latency and nosepoke inter-response time, IRT). The first dose of DTB significantly increased matching accuracy; during the following week, DTB reduced matching accuracy, increased choice response latency and nosepoke IRT, and reduced trial completion. Discrimination accuracy remained unaffected. Experiment 2 explored the effects of single administrations of DTB on DMTP/VD. Sixteen other trained rats were divided into two groups with equal matching accuracy. One group received DTB (0.5,1.0, and 2.0 mg/kg, IP) in separate injections at least 1 week apart; the other group received vehicle at the same times. Matching accuracy increased significantly in the treated rats and not in the controls following each dose of DTB. The magnitude of this increase was dose dependent, and lasted from 1 to 8 weeks after each injection. Discrimination accuracy, response latency, nosepoke IRT and trial completion remained unaffected throughout the study. After DTB, matching accuracy was less easily disrupted by scopolamine (0.1-0.3 mg/kg, IP). However, DTB did not alter the rats' response to reducing the distance between the response levers, to reversal of the matching rule to a nonmatching rule, or to challenge with MK-801 (0.05-0.10 mg/kg, IP). These data indicate that acute DTB causes a long-lasting facilitation of working memory in rats in the absence of any of the indications of motor impairment which follow repeated, daily injections of the chemical.