In our laboratory we use an unbaited 6-arm radial tunnel maze (6-arm RTM) to assess working and reference memory in the course of neurotoxicity studies. The 6-arm RTM is believed to measure parameters comparable to those assessed in radial-arm mazes, but without the need of food deprivation and rewarding of animals. This is especially useful in the course of neurotoxicity studies as interferences of e.g. food deprivation with drug pharmacokinetics can be avoided. Since the 6-arm RTM is less evaluated than conventional mazes the aim of this study was to further confirm mean error score as measure of 'working memory', left-right discrimination within each radial arm (expressed as percent "blind-alley" visits) as measure of 'reference memory', and number of arm entries/min as a measure of motor activity. Therefore, hippocampal lesions were induced by injecting animals with the neurotoxicant trimethyltin (TMT). TMT at a dose of 5 mg/kg slightly lesioned hippocampal CA3 pyramidal cells in 3 of 8 animals, but did not affect behavioral measures in the 6-arm RTM. In all surviving animals treated with 7 or 9 mg/kg TMT moderate to marked loss of CA3 pyramidal cells was observed, while in 4 of these 7 rats CA4 pyramidal cells were also affected. Other brain lesions were not observed. TMT-induced brain lesions led to increased mean error score and number of arm visits during the retention phase and after changing maze configuration, whereas percent "blind-alley" visits were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Maze behavior of rodents provides insight into processes of learning and memory and also serves to assess cognitive functions in neurotoxicity tests. Neurotoxic agents may impair maze behavior by acting upon different parts of the nervous system. To assess the dependence of maze learning upon vestibular and/or auditory input, the two systems were lesioned. Daily treatment of rat pups with streptomycin (400 mg/kg sc) on postnatal day 11 to 22 caused irreversible impairment of vestibular and auditory functions, whereas, 20 injections of neomycin in adult rats (100 mg/kg sc, postnatal weeks 8 to 11) led to hearing loss only. Hearing loss was assessed by absence of Preyer's reflex and impaired vestibular function by loss or shortened duration of postrotatory nystagmus. Learning in the unbaited 6-arm radial maze was tested at the age of 2 to 3 mon using a maze configuration that allowed to assess order of arm entries ("working memory") and left-right discrimination within each arm ("reference memory"). Treatment with streptomycin but not with neomycin led to impaired order of arm entries. Since treatment with streptomycin failed to induce any signs of brain lesions, impaired maze learning is considered to result from destruction of vestibular hair cell receptors with subsequent vestibular impairment and not from hearing loss or cognitive impairment.

Motor activity is an important functional measure used in neurotoxicology. The effects of chemicals on motor activity, however, may depend on variables such as type of measurement apparatus, physical and environmental testing conditions, and many other experimental protocol and organismic variables. Due to the increasing use of motor activity in neurotoxicology, a major question concerns the potential for differences in experimental findings due to variations in sensitivity and reliability between different laboratories and devices used to measure motor activity. This study examined historical data from a number of laboratories that employed different devices and experimental protocols to measure motor activity. Four aspects of the motor activity data were compared: 1) within-laboratory control variability across time; 2) within-laboratory replicability of control data; 3) between-laboratory variability in the effects of chemicals; and 4) between-laboratory comparison of the control rates of habituation. The analyses indicated that there was a relatively restricted range of within-laboratory variability and reliability in control values, and that these ranges were comparable across laboratories. Similar profiles of habituation were also seen across the different laboratories. Moreover, in virtually every case, all laboratories were capable of detecting qualitatively similar changes in motor activity following acute exposure to a variety of chemicals. These data indicate a high degree of comparability in the data generated by the different devices and experimental protocols.

In the course of routinely performed subchronic toxicity studies with laboratory rodents, functional neurotoxicity, i.e., behavioral changes, usually are noted first during the daily cageside observations of all animals, Observation and neurologic examination of a few key animals provide a tentative diagnosis. Subsequent automated testing procedures for further characterization and quantifying behavioral changes might include motor activity, startle response, hurdle stepping, and maze behavior. Automated testing serves to assess the no observable effect under conditions of blind testing and provides further refinement of the diagnosis. Behavioral changes should be assessed as early as possible after onset of testing, i.e., during acute tests for mortality and the subsequent subchronic range-finding studies. Subsequent subchronic organ toxicity studies are then carried out by following a validated experimental protocol, including automated testing procedures and appropriate neuropathologic evaluation.

In the course of routinely performed subchronic toxicity studies with laboratory rodents, functional neurotoxicity, i.e. behavioral changes are usually noted first during the daily cage side observations of all animals. Observation and subsequent neurologic examination of a few key animals provide a primary tentative diagnosis. Additional automated testing might include: motor activity, startle response, hurdle stepping and maze behavior. Automated testing provides further refinement of the diagnosis and serves to determine the no observable effect level under conditions of blind testing. Behavioral changes should be assessed as early as possible after initiation of testing, i.e. during acute tests for mortality and the following subchronic range-finding studies. Subsequent subchronic organ toxicity studies are then carried out by following a valid experimental protocol for implementation of automated testing procedures and appropriate neuropathologic evaluation.

Choice behavior of rats in a radially symmetrical 6-arm maze, without food reward, was validated for assessing changes in learning and memory following treatment with 4 psychoactive agents. The test is designed for future use in routine toxicity studies with laboratory rodents. Each radial main arm of the maze leads to a T-shaped choice-point with a blind alley on the left and a long angled alley on the right. Order of choice of the radial main arms served to score within-session working memory, by evaluating relative recency of arm reentries. The choice between blind alley and long-angled alley at the T-intersections provided a measure of between-session reference memory. Maze behavior as an indicator for impairment in learning and memory was validated by testing rats treated with d-amphetamine, chlorpromazine, scopolamine and physostigmine. Based on the above evaluations, working memory was found to be severely impaired by 0.3 and 1.0 mg/kg scopolamine, and reference memory to be improved by 0.02 mg/kg physostigmine and 1.0 mg/kg amphetamine. Locomotor activity, in terms of the total number of arm choices per test session, was altered by all substances as expected from previous reports in the literature. The test appears to be a valid and sensitive method for assessing learning and memory in the rat without the use of food reward, and thus well suited for implementation in routine toxicity studies with rodents.

To evaluate the validity and sensitivity of test procedures for routine assessment of chemically induced changes in motor activity in rats, studies were carried out with a commercially available photocell cage system. Testing of single, untreated rats showed that motor activity decreased within 10 min to a steady level. Overall activity was the same when testing was repeated at weekly intervals for 6 weeks. In groups of each 10 rats tested immediately after i.p. injection, chlorpromazine HCl (6 or 2 mg/kg) and physostigmine salicylate (0.5 mg/kg) decreased activity. d-Amphetamine sulfate (1.0 mg/kg) caused an increase of virtually all parameters, whereas scopolamine HCl (1.0 mg/kg) and physostigmine salicylate (0.02 mg/kg) led to an increase of particular aspects of motor activity only. Data generated with a mechanical calibrator varies 1–5% between the cages tested. It is concluded that the above procedures of recording of selected parameters of motor activity in groups of 10 rats for periods of up to 20 min would comply with the guidelines recommended by EPA TOSCA [1].

Basic mechanisms of the rat ERG were reinvestigated with contemporary methods of recording and photic stimulation via an optic fiber system connected with a contact lens. Flash stimulation and background illuminance were performed with photometrically defined light stimuli. ERGs recorded in darkness, from dark adapted rats, were similar with those observed in earlier work in which light flashes of much longer duration had been used. Flash stimulation carried out under stepwise increased background illuminance gave new information on the characteristics of oscillatory potentials of the ERG. In general the present observations agreed with the notion that the rods of the rat retina are as sensitive as in man, whereas the cones are functionally less efficient with respect to light sensitivity and temporal resolution. Differences in function of the cones from rat to man have to be kept in mind when using the rat ERG as a model for risk assessment in safety studies.

Neurotoxicity occurs during administration of high doses of compounds, in testing for acute toxicity (LD50) or on occasion of subchronic treatment for assessment of organ toxicity. Neurotoxicity induces behavioral disturbances, usually first noticed by the animal caretakers, who are observing daily the animals tested according to authorized guidelines. During the following neurological examination a few of the exposed animals, exhibiting distinct behavioral disturbances, are compared with controls. The neurological examination serves to characterize the observed changes, while the observation of the whole group of animals in the home cages gives indications about the highest non-toxic dose level. Further tests for additional clarification of neurotoxicity would be evaluation of neuromotor spontaneous activity in the photocell-cage, behavior in the maze or operant behavior, measurement of the maximum nerve conduction velocity or other especially developed tests.

Five beagle dogs were treated daily with 1 ml of trimethylphosphate (TMP) for 1 to 4 months. Neurotoxicity was evaluated by observation of behaviour, neurologic examination, neurophysiologic tests and by neuropathology. Neurotoxicity was first detected at the neurologic examination following four weeks of treatment: the most predictive indicators of neuropathy were the impairment of hopping, of tactile placing and the atactic gait. Electrophysiologic tests indicated a prolonged latency of neuromuscular impulse transmission after week nine, followed by a decrease of maximum conduction velocity of sensory fibers. At this time peripheral nerve fibers had abnormalities including paranodal and internodal swelling, paranodal demyelination and distal fragmentation. An additional dog treated with daily doses of 2 ml developed a severe distally accentuated degeneration of long spinal tracts and peripheral nerve fibers, manifested by impairment of sensory and central motor impulse conduction. Organophosphate neuropathy can thus be assessed in the dog by functional and morphological changes after prolonged exposure, in particular at doses causing severe morbidity.

Subchronic testing of laboratory animals in accordance with present regulatory guidelines involves maximum exposure with the chemical under investigation and serves for the evaluation of systemic toxicity as well as of lesions in organs and organ systems, including neurotoxicity. The primary assessment of neurotoxicity is essentially based on the overall observation of animal behaviour in the course of the customary toxicity studies and on the subsequent neuropathological evaluation with contemporary techniques. Under this maximum exposure the absence of symptoms and signs of neural abnormalities indicates that the material testd would be devoid of neurotoxicity.

Neurologic examination, electrophysiologic testing and microscopic post-mortem examination was used to study the neuropathy induced in the beagle dog by administration of excessive amounts of vitamin B6.

Sensory maximum nerve conduction velocity (MNCV) and motor MNCV were monitored in altogether 14 beagle dogs anaesthetized with pentobarbital. Sensory MNCV was determined by averaging cortically evoked potentials from somatosensory area I (SS I) in response to repeated electrical stimulation of digital paw pads, tibial nerve at calcaneus or sciatic nerve at trochanter.

In man, single fiber electromyography is used as a very sensitive indicator for the assessment of functional changes in motor nerves. The purpose of the present study was to adapt the above testing procedure to allow repeated investigations of dogs used in subchronic toxicity tests.

In the dog ERG flash-induced activation of the rod receptors can be selectively investigated due to relatively poor photic excitability of the cone component.