Effects of intensity and type of prepulse stimulus on prepulse inhibition in scopolamine treated rats.

Prepulse inhibition (PPI) of the auditory startle response (ASR) is a behavioral test that has been used to measure auditory thresholds, to assess sensory-motor integration functions, and its use has been recommended in the United States Environmental Protection Agency Developmental Neurotoxicity Guideline (OPPTS 870.6300). The purpose of the present study was to determine to what extent the intensity and/or type of prepulse stimuli modulate PPI in scopolamine-treated rats. The PPI of the ASR peak amplitude was measured when the intensity of a 10-kHz prepulse tone was varied (69-, 80-, and 90 dB[A]; Experiment 1) and when both the intensity and type of auditory prepulse (a 10-kHz tone vs. a white noise burst) were varied (Experiment 2). Scopolamine treatment attenuated PPI in both experiments and interacted significantly with the prepulse stimulus intensity in Experiment 1. In Experiment 2, the percent of PPI was linearly related to prepulse stimulus intensity for trials using a tone, but was biphasic on trials using a white-noise prepulse stimulus. Prepulse stimuli of certain intensities elicited a response, and this response was greater when the prepulse stimulus was a white noise burst versus a tone of the same intensity. Further, the response to the prepulse altered the amount of inhibition and, therefore, confounded the overall measure of PPI at the higher prepulse stimulus intensity levels. Overall, these results indicate that careful consideration of the intensity and type of prepulse stimuli be taken in the context of their potential to induce a prepulse-elicited response, as well as providing the appropriate measures of such a response, when designing and interpreting PPI experiments.

Differential sensitivity of blood, peripheral, and central cholinesterases in beagle dogs following dietary exposure to chlorpyrifos.

Two studies were performed to find out whether exposure limits that protect brain acetylcholinesterase (AChE) will protect peripheral tissue AChE after exposure to chlorpyrifos (CPF), an organophosphate insecticide. In a methods-development study, male dogs (3/dose) were exposed to 0.0, 0.3, 0.6, or 1.2mg/kg/day CPF in their diets for 4 weeks. Mixed cholinesterase (mChE), AChE, and butyrylcholinesterase (BuChE) activities were measured in plasma, RBC, brain, left atrium and ventricle, diaphragm, quadriceps, and nodose ganglia. Plasma, brain and peripheral tissue BuChE was inhibited at all dose levels. While RBC AChE was inhibited at all doses, brain and peripheral AChE activities were unaffected. In the main study, dogs (4/sex/dose) were exposed to 0.0, 0.5, 1.0, or 2.0mg/kg/day CPF in their diets for six weeks and RBC AChE was significantly inhibited at all doses in both sexes. Diaphragm, quadriceps, and nodose ganglia AChE was unaffected by treatment. Brain AChE was decreased by approximately 6% compared to controls in high-dose groups, and this was considered a threshold effect. Left atrium AChE in high-dose dogs was 25.5% less (males) and 32.1% greater (females) than controls; these differences were attributed to chance. While peripheral tissue and brain AChE were not affected following exposure to 1.0mg/kg/day, RBC AChE was inhibited at all doses. These results show that RBC AChE is more sensitive than brain or peripheral tissue AChE to inhibition by CPF, and that protection of brain AChE would protect peripheral tissue AChE.

Lack of trigeminal nerve toxicity in rats exposed to trichloroethylene vapor for 13 weeks.

Male and female Fischer-344 rats were exposed to 1,1,2-trichloroethylene (TCE) at 250, 800, or 2500 ppm for 6 h/day, 5 days/week, for 13 weeks. Weekly body weights and daily clinical observations were recorded and a functional observational battery (FOB) was performed monthly. Postexposure neurotoxicological evaluations included an electrodiagnostic evaluation of auditory function, the trigeminal nerve, and a comprehensive neuropathological examination. After 8 weeks of exposure, female, but not male, rats exposed to 2500 ppm were slightly more reactive to handling than the controls but not after 13 weeks of exposure. After 13 weeks, female rats exposed to 2500 ppm TCE were slightly more active during the 1-min observation period than the controls. There were no treatment-related differences in grip performance, landing foot splay, or on the trigeminal nerve-evoked potential at any dose. At 2500 ppm TCE, mild frequency-specific hearing deficits were observed, including elevated tone-pip auditory brainstem response thresholds. Focal loss of hair cells in the upper basal turn of the cochlea was observed in 2500 ppm-exposed rats. Except for the cochleas of 2500 ppm-exposed rats, no treatment-related lesions were noted during the neuro-histopathologic examination. The no-observable-adverse-effect level for this study was 800 ppm based on ototoxicity at 2500 ppm.

Neurotoxicity test validation, positive controls and proficiency: are chemicals necessary?

The USEPA neurotoxicity guidelines require the use of positive control data in support of toxicology studies submitted to the Agency and emphasize the use of chemicals to accomplish this requirement. These guidelines, though, propose a number of different rationales for the use of chemicals as positive control agents. We re-evaluated the potential roles of positive control data in addressing three questions: 1) what does the test measure? 2) is the performing laboratory proficient in the use of the test? 3) do the complementary data submitted in support of neurotoxicity studies conducted with the test material provide enough context for the interpretation of the biological significance of an effect? While, for most types of guideline neurotoxicity tests, the use of test chemicals has been emphasized for positive control testing, the use of non-chemical procedures (i.e., systematic manipulation of the experimental parameters of a test, which poses less risk of adverse effects to the test animals) should be strongly considered as a potential alternative.

Validation of an auditory startle response system using chemicals or parametric modulation as positive controls.

Neurotoxicity regulatory guidelines mandate that automated test systems be validated using chemicals. However, in some cases, chemicals may not necessarily be needed to prove test system validity. To examine this issue, two independent experiments were conducted to validate an automated auditory startle response (ASR) system. In Experiment 1, we used adult (PND 63) and weanling (PND 22) Sprague-Dawley rats (10/sex/dose) to determine the effect of either d-amphetamine (4.0 or 8.0 mg/kg) or clonidine (0.4 or 0.8 mg/kg) on the ASR peak amplitude (ASR PA). The startle response of each rat to a short burst of white noise (120 dB SPL) was recorded over 50 consecutive trials. The ASR PA was significantly decreased (by clonidine) and increased (by d-amphetamine) compared to controls in PND 63 rats. In PND 22 rats, the response to clonidine was similar to adults, but d-amphetamine effects were not significant. Neither drug affected the rate of the decrease in ASR PA over time (habituation). In Experiment 2, PND 31 Sprague-Dawley rats (8/sex) were presented with 150 trials consisting of either white noise bursts of variable intensity (70-120 dB SPL in 10 dB increments, presented in random order) or null (0 dB SPL) trials. Statistically significant sex- and intensity-dependent differences were detected in the ASR PA. These results suggest that in some cases, parametric modulation may be an alternative to using chemicals for test system validation.

Factors affecting grip strength testing.

The rodent grip strength test was developed decades ago and is a putative measure of muscular strength. This test has been included in the functional observational battery (FOB) to screen for neurobehavioral toxicity, and changes in grip strength have been interpreted as evidence of motor neurotoxicity. Despite its widespread use, questions remain about what the grip strength test actually measures. In this study, potential confounders of the grip strength test were identified and tested, including operational parameters, disruption of peripheral sensory function and changes in body weight. Operational parameters (sampling rate, system type and trial angle but not trial speed) had dramatic effects on grip strength data. Doxorubicin (DX, 10 mg/kg iv) was used to cause sensory impairment. It decreased forelimb and hindlimb grip strength (by 27% and 32%, respectively, compared with controls), an effect that was correlated with degeneration of peripheral and central sensory components (distal tibial and sural nerves, dorsal funiculus of the spinal cord and dorsal, but not ventral, spinal roots). Feed restriction-induced loss of body weight (26% compared with controls) and muscle mass (20% compared with controls) reversibly decreased both forelimb and hindlimb grip strength (18% and 17%, respectively, compared with controls). Ignoring these confounding factors could potentially lead to increased data variability and inconsistency within single studies, across studies and in historical control data sets. To assist in data interpretation and evaluation of grip strength results, it is suggested that exact conditions of application of the test be reported in greater detail. Furthermore, given that the grip strength test can be influenced by factors other than true muscular strength, use of the term grip performance is proposed to better reflect the apical nature of this test.