Andrew F.M. Johnstone

Microelectrode arrays: A physiologically based neurotoxicity testing platform for the 21st century §

Microelectrode arrays (MEAs) have been in use over the past decade and a half to study multiple aspects of electrically excitable cells. In particular, MEAs have been applied to explore the pharmacological and toxicological effects of numerous compounds on spontaneous activity of neuronal and cardiac cell networks. The MEA system enables simultaneous extracellular recordings from multiple sites in the network in real time, increasing spatial resolution and thereby providing a robust measure of network activity. The simultaneous gathering of action potential and field potential data over long periods of time allows the monitoring of network functions that arise from the interaction of all cellular mechanisms responsible for spatio-temporal pattern generation. In these functional, dynamic systems, physical, chemical, and pharmacological perturbations are holistically reflected by the tissue responses. Such features make MEA technology well suited for the screening of compounds of interest, and also allow scaling to high throughput systems that can record from multiple, separate cell networks simultaneously in multi-well chips or plates. This article is designed to be useful to newcomers to this technology as well as those who are currently using MEAs in their research. It explains how MEA systems operate, summarizes what systems are available, and provides a discussion of emerging mathematical schemes that can be used for a rapid classification of drug or chemical effects. Current efforts that will expand this technology to an influential, high throughput, electrophysiological approach for reliable determinations of compound toxicity are also described and a comprehensive review of toxicological publications using MEAs is provided as an appendix to this publication. Overall, this article highlights the benefits and promise of MEA technology as a high throughput, rapid screening method for toxicity testing.

Pyrethroid modulation of spontaneous neuronal excitability and neurotransmission in hippocampal neurons in culture

Pyrethroid insecticides have potent actions on voltage-gated sodium channels (VGSC), inhibiting inactivation and increasing channel open times. These are thought to underlie, at least in part, the clinical symptoms of pyrethroid intoxication. However, disruption of neuronal activity at higher levels of organization is less well understood. In order to characterize pyrethroid effects on neurotransmitter release and neuronal excitability in glutamatergic networks, we examined the effects of deltamethrin (DM) and permethrin (PM) on neuronal activity in hippocampal neuronal cultures using patch-clamp and microelectrode array (MEA) recordings. In the presence of inhibitors of GABA receptors, spontaneous excitatory post-synaptic currents (sEPSCs) and spontaneous spike rates were reduced in a concentration-dependent manner by both DM and PM. IC(50) values were 0.037 and 0.70microM for inhibition of sEPSCs and 0.60 and 21.8microM for inhibition of spontaneous spike rate by DM and PM, respectively. Both compounds altered burst activity by decreasing the number of spikes during spontaneous bursting, the number of sEPSCs within a bursting release event and the duration of sEPSC bursts while increasing both the interspike interval and the time between sEPSCs. Exposure of neurons to the VGSC-specific modulator veratridine had effects similar to both DM and PM, while inhibition of voltage-gated calcium channels had no effect on spontaneous spike rates. In the absence of GABA receptor antagonists, both DM and PM increased spontaneous spike rates. Altogether, these data demonstrate that DM and PM disrupt network activity in vitro, largely via a VGSC-dependent mechanism.

Episodic ozone exposure in adult and senescent Brown Norway rats: acute and delayed effect on heart rate, core temperature and motor activity

Abstract Setting exposure standards for environmental pollutants may consider the aged as a susceptible population but the few published studies assessing susceptibility of the aged to air pollutants are inconsistent. Episodic ozone (O3) is more reflective of potential exposures occurring in human populations and could be more harmful to the aged. This study used radiotelemetry to monitor heart rate (HR), core temperature (Tc) and motor activity (MA) in adult (9–12 months) and senescent (20–24 months) male, Brown Norway rats exposed to episodic O3 (6 h/day of 1 ppm O3 for 2 consecutive days/week for 13 weeks). Acute O3 initially led to marked drops in HR and Tc. As exposures progressed each week, there was diminution in the hypothermic and bradycardic effects of O3. Senescent rats were less affected than adults. Acute responses were exacerbated on the second day of O3 exposure with adults exhibiting greater sensitivity. During recovery following 2 d of O3, adult and senescent rats exhibited an elevated Tc and HR during the day but not at night, an effect that persisted for at least 48 h after O3 exposure. MA was elevated in adults but not senescent rats during recovery from O3. Overall, acute effects of O3, including reductions in HR and Tc, were attenuated in senescent rats. Autonomic responses during recovery, included an elevation in Tc with a pattern akin to that of a fever and rise in HR that were independent of age. An attenuated inflammatory response to O3 in senescent rats may explain the relatively heightened physiological response to O3 in younger rats.

Acute toluene exposure alters expression of genes in the central nervous system associated with synaptic structure and function

Toluene is a volatile organic compound (VOC) and a ubiquitous air pollutant of interest to EPA regulatory programs. Whereas its acute functional effects are well described, several modes of action in the CNS have been proposed. Therefore, we sought to identify potential pathways mediating direct or indirect effects of VOCs by investigating the genomic response of the rat CNS to acutely-inhaled toluene. Adult male Long-Evans rats inhaled clean air or 1000 ppm toluene vapor for 6 h. Specific brain regions were collected from the rats either immediately after 6 h of treatment or 18 h after removal from the exposure chambers (n=6/group/time). Total mRNA was extracted from the striatum and hybridized to Rat 230A Affymetrix arrays. Statistical analyses showed 226 and 3352 transcripts altered in the toluene-exposed groups relative to controls at the 6 h time point and after the 18 h recovery period, respectively. Relative to controls, toluene exposure was associated with induction or repression of genes in pathways associated with synaptic plasticity, including long-term depression, GABA receptor signaling and mitochondrial function. In each of these pathways, responses were characterized by changes in a small number of transcripts following the 6 h toluene inhalation and with substantial increases in numbers of changed transcripts at 18 h recovery following termination of exposure. This report provides the first global genomic evidence that CNS pathways affected by toluene are strongly associated with neurological processes participating in synaptic transmission and plasticity.

A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing ☆

HIGHLIGHTSFour laboratories tested 6 compounds for neuroactivity using microelectrode arrays.All four laboratories were able to correctly identify the three neurotoxic compounds.Three non‐neuroactive compounds were correctly identified 10/12 times.Despite methodological differences, results were consistent across laboratories.These results support use of microelectrode arrays for neurotoxicity screening. ABSTRACT There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non‐neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non‐neurotoxic (“negative controls”), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration‐dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration‐response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function.

Thermal Stress and Toxicity

Elevating ambient temperature above thermoneutrality exacerbates toxicity of most air pollutants, insecticides, and other toxic chemicals. On the other hand, safety and toxicity testing of toxicants and drugs is usually performed in mice and rats maintained at sub-thermoneutral temperatures of ~22∘C. When exposed to chemical toxicants under these relatively cool conditions, rodents typically undergo a regulated hypothermic response, characterized by preference for cooler ambient temperatures and controlled reduction in core temperature. Reducing core temperature delays the clearance of most toxicants from the body; however, a mild hypothermia also improves recovery and survival from the toxicant. Raising ambient temperature to thermoneutrality and above increases the rate of clearance of the toxicant but also exacerbates toxicity. Furthermore, heat stress combined with work or exercise is likely to worsen toxicity. Body temperature of large mammals, including humans, does not decrease as much in response to exposure to a toxicant. However, heat stress can nonetheless worsen toxic outcome in humans through a variety of mechanisms. For example, heat-induced sweating and elevation in skin blood flow accelerates uptake of some insecticides. Epidemiological studies suggest that thermal stress may exacerbate the toxicity of airborne pollutants such as ozone and particulate matter. Overall, translating results of studies in rodents to that of humans is a formidable task attributed in part to the interspecies differences in thermoregulatory response to the toxicants and to thermal stress.

Impact of genetic strain on body fat loss, food consumption, metabolism, ventilation, and motor activity in free running female rats.

Chronic exercise is considered as one of the most effective means of countering symptoms of the metabolic syndrome (MS) such as obesity and hyperglycemia. Rodent models of forced or voluntary exercise are often used to study the mechanisms of MS and type 2 diabetes. However, there is little known on the impact of genetic strain on the metabolic response to exercise. We studied the effects of housing rats with running wheels (RW) for 65 days compared to sedentary (SED) housing in five female rat strains: Sprague-Dawley (SD), Long-Evans (LE), Wistar (WIS), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY). Key parameters measured were total distance run, body composition, food consumption, motor activity, ventilatory responses by plethysmography, and resting metabolic rate (MR). WKY and SHR ran significantly more than the WIS, LE, and SD strains. Running-induced reduction in body fat was affected by strain but not by distance run. LEs lost 6% fat after 21 d of running whereas WKYs lost 2% fat but ran 40% more than LEs. LE and WIS lost body weight while the SHR and WKY strains gained weight during running. Food intake with RW was markedly increased in SHR, WIS, and WKY while LE and SD showed modest increases. Exploratory motor activity was reduced sharply by RW in all but the SD strain. Ventilatory parameters were primarily altered by RW in the SHR, WKY, and WIS strains. MR was unaffected by RW. In an overall ranking of physiological and behavioral responses to RW, the SD strain was considered the least responsive whereas the WIS was scored as most responsive. In terms of RW-induced fat loss, the LE strain appears to be the most ideal. These results should be useful in the future selection of rat models to study benefits of volitional exercise.

Behaviorally mediated, warm adaptation: A physiological strategy when mice behaviorally thermoregulate☆

Laboratory mice housed under standard vivarium conditions with an ambient temperature (Ta) of ~22°C are likely to be cold stressed because this Ta is below their thermoneutral zone (TNZ). Mice raised at Tas within the TNZ adapt to the warmer temperatures, developing smaller internal organs and longer tails compared to mice raised at 22°C. Since mice prefer Tas equal to their TNZ when housed in a thermocline, we hypothesized that mice reared for long periods (e.g., months) in a thermocline would undergo significant changes in organ development and tail length as a result of their thermoregulatory behavior. Groups of three female BALB/c mice at an age of 37 days were housed together in a thermocline consisting of a 90cm long aluminum runway with a floor temperature ranging from 23 to 39°C. Two side-by-side thermoclines allowed for a total of 6 mice to be tested simultaneously. Control mice were tested in isothermal runways maintained at a Ta of 22°C. All groups were given cotton pads for bedding/nest building. Mass of heart, lung, liver, kidney, brain, and tail length were assessed after 73 days of treatment. Mice in the thermocline and control (isothermal) runways were compared to cage control mice housed 3/cage with bedding under standard vivarium conditions. Mice in the thermocline generally remained in the warm end throughout the daytime with little evidence of nest building, suggesting a state of thermal comfort. Mice in the isothermal runway built elaborate nests and huddled together in the daytime. Mice housed in the thermocline had significantly smaller livers and kidneys and an increase in tail length compared to mice in the isothermal runway as well as when compared to the cage controls. These patterns of organ growth and tail length of mice in the thermocline are akin to warm adaptation. Thus, thermoregulatory behavior altered organ development, a process we term behaviorally mediated, warm adaptation. Moreover, the data suggest that the standard vivarium conditions are likely a cold stress that alters normal organ development relative to mice allowed to select their thermal preferendum.

Thermoregulatory deficits in adult Long Evans rat exposed perinatally to the antithyroidal drug, propylthiouracil.

Developmental exposure to endocrine disrupting drugs and environmental toxicants has been shown to alter a variety of physiological processes in mature offspring. Body (core) temperature (T(c)) is a tightly regulated homeostatic system but is susceptible to disruptors of the hypothalamic pituitary thyroid (HPT) axis. We hypothesized that thermoregulation would be disrupted in adult offspring exposed perinatally to an HPT disruptor. Propylythiouracil (PTU) was used as a prototypical compound because of its well known antithyroidal properties. PTU was added to the drinking water of pregnant rats in concentrations of 0, 1, 2, 3, and 10 ppm from gestational day (GD) 6 through postnatal day (PND) 21. Adult male offspring were implanted with radiotransmitters to monitor Tc and motor activity (MA) and were observed undisturbed at an ambient temperature of 22 °C for 12 consecutive days. Data were averaged into a single 24 hour period to minimize impact of ultradian changes in T(c) and MA. All treatment groups showed a distinct circadian temperature rhythm. Rats exposed to 10 ppm PTU exhibited a marked deviation in their regulated T(c) with a reduction of approximately 0.4 °C below that of controls throughout the daytime period and a smaller reduction at night. Rats exposed to 1 or 2 ppm also had smaller but significant reductions in T(c). MA was unaffected by PTU. Overall, developmental exposure to moderate doses of an antithyroidal drug led to an apparent permanent reduction in T(c) of adult offspring that was independent of changes in MA.

Age-related differences in pulmonary effects of acute and subchronic episodic ozone exposures in Brown Norway rats

Abstract Ozone (O3) is known to induce adverse pulmonary and systemic health effects. Importantly, children and older persons are considered at-risk populations for O3-induced dysfunction, yet the mechanisms accounting for the age-related pulmonary responses to O3 are uncertain. In this study, we examined age-related susceptibility to O3 using 1 mo (adolescent), 4 mo (young adult), 12 mo (adult) and 24 mo (senescent) male Brown Norway rats exposed to filtered air or O3 (0.25 and 1.00 ppm), 6 h/day, two days/week for 1 week (acute) or 13 weeks (subchronic). Ventilatory function, assessed by whole-body plethysmography, and bronchoalveolar lavage fluid (BALF) biomarkers of injury and inflammation were used to examine O3-induced pulmonary effects. Relaxation time declined in all ages following the weekly exposures; however, this effect persisted only in the 24 mo rats following a five days recovery, demonstrating an inability to induce adaptation commonly seen with repeated O3 exposures. PenH was increased in all groups with an augmented response in the 4 mo rats following the subchronic O3 exposures. O3 led to increased breathing frequency and minute volume in the 1 and 4 mo animals. Markers of pulmonary permeability were increased in all age groups. Elevations in BALF γ-glutamyl transferase activity and lung inflammation following an acute O3 exposure were noted in only the 1 and 4 mo rats, which likely received an increased effective O3 dose. These data demonstrate that adolescent and young adult animals are more susceptible to changes in ventilation and pulmonary injury/inflammation caused by acute and episodic O3 exposure.