Evaluating the effects of chemical exposures on adaptive functioning

• 1.1. Although morphological and classical toxicological data have typically served as criteria for judging the effects of chemicals on the nervous system, there has been increasing interest in the use of behavioral end-points for examining the adverse effects of chemicals on nervous system function.
• 2.2. The present paper describes the current use of behavioral methods in neurotoxicity assessment and surveys some of the recent developments in the use of specialized methods for examining motor, sensory and cognitive changes following chemical exposures.

     

鱼类神经行为毒性研究进展

神经行为毒性是神经科学、神经药理学和神经毒理学的重要研究内容,对评价生态系统质量和研究有害因素或药物在生物神经系统作用机制具有重要理论和应用价值。鱼类中枢神经系统发达,对水环境中化合物极为敏感,其神经系统能够对各种刺激产生综合协调的应答反应,影响其运动功能、应激反应以及学习/记忆,改变游泳行为和社会行为,产生神经行为异常,诱发神经行为毒性效应。近年来,许多研究者开展了一系列以鱼类为受试对象的神经行为毒性研究,表明鱼类是开展神经行为毒性研究的重要物种,其成果在生态环境监测和评价、渔业生产、神经系统机制探究及药物开发等方面得到了广泛应用。鱼类作为神经行为毒性研究的物种,补充经典哺乳动物模型的不足,提供了高通量体外细胞分析和经典哺乳动物模型之间的关键模型。文章从鱼类生物学特征、实验鱼类品系和全基因组测序3个方面阐明鱼类是开展神经行为毒性研究的重要物种,综述了微塑料及其吸附污染物、有机污染物2类典型污染物和酒精、咖啡因、苯二氮卓类药物、选择性血清素再吸收抑制剂4类药物对鱼类的游泳行为和社会行为影响,探讨鱼类产生剂量或时间依赖性的神经行为毒性效应,并对未来研究方向进行了分析展望,以期为神经行为毒...     

Use of pharmacological challenges to disclose neurobehavioral deficits

Delineation of the neurotoxic effects of various environmental agents is often complicated by the considerable compensatory capacity of the central nervous system (CNS). Moreover, the degree to which such compensation occurs may be critical when one is examining dosages near the threshold for biological effect. A current approach has been to use pharmacological agents to unmask deficits that are reflected in performance on subsequent behavioral tasks. This noninvasive manipulation may also provide insight into the underlying neurochemical substrates of the CNS insult. This procedure has been successfully applied in experiments examining a wide range of compounds (e.g., lead, mercury, acrylamide, carbon disulfide) in animals exposed during development or adulthood. Even more striking is the ability of this technique to disclose latent effects, i.e., effects that are demonstrable long after exposure has ceased. This laboratory has attempted to further elaborate the use of pharmacological probes. Utilizing a drug discrimination paradigm, we have investigated the altered amphetamine sensitivity exhibited by offspring exposed to lead early in life. This paradigm has been frequently employed in psychopharmacology and has been well validated as a tool for assessing drug response thresholds. Furthermore, additional pharmacological manipulations (agonists, depletors, blockers) can be imposed on the original drug discrimination to refine hypotheses regarding neurochemical alterations underlying the shifts observed in drug discrimination thresholds.     

Silver impregnation of peripheral and central axons

A silver impregnation method suitable for peripheral and central nervous system axons is described. Essential features are the use of reagent grade chemicals only, a pretreatment solution to ensure optimal impregnation of different organs from different animals and species, and an unvarying procedure. The results are compared to those obtainable with a number of current impregnation methods and with modern immunocytochemical reactions.     

Silver Impregnation of Peripheral and Central Axons

A silver impregnation method suitable for peripheral and central nervous system axons is described. Essential features are the use of reagent grade chemicals only, a pretreatment solution to ensure optimal impregnation of different organs from different animals and species, and an unvarying procedure. The results are compared to those obtainable with a number of current impregnation methods and with modern immunocytochemical reactions.     

Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.     

Corticotropin-releasing factor antagonist blocks stress-induced fighting in rats

Corticotropin-releasing factor (CRF) has been shown to have potent central nervous system-activating effects when administered intracerebroventricularly (i.c.v.). In the present experiment, this activating effect was exaggerated by use of a stress-motivated behavioral paradigm. Low doses of CRF (0.01 and 0.1 micrograms/rat) administered i.c.v. facilitated stress-induced fighting. More importantly, alpha-helical CRF-(9-41), a CRF antagonist, blocked stress-induced fighting produced by higher levels of stress. These results suggest that CRF in the central nervous system may have a role in mediating behavioral responses to stress.     

Behavioral phenotyping of rodents

Established methods for analyzing behavioral traits in mutant lines of mice allow researchers to understand the outcomes of genetic manipulations in the nervous system. A rigorous six-tiered behavioral phenotyping strategy is described. Recommendations are offered for the design of mouse behavioral testing suites in animal housing facilities.     

Zebrafish model systems for developmental neurobehavioral toxicology

Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. Birth Defects Research (Part C) 99:14–23, 2013. © 2013 Wiley Periodicals, Inc.     

A neurophysiological study of sensitivity to a feeding deterrent in two sister species of Heliothis with different diet breadths

Heliothis subflexa and H. virescens are sister species that differ markedly in their hostplant specificity: the former is a specialist on one plant genus, the latter feeds on plants from many families. The behavioral threshold for rejection of deterrent chemicals is lower in larvae of H. subflexa than in those of H. virescens. In this paper, we examine the responses of the galeal styloconic sensilla of these larvae to stimulation by three chemicals, sucrose and inositol, which are phagostimulants, and sinigrin, a deterrent, in an attempt to determine the neural basis for the differences in feeding behavior between the species. The species difference could not be attributed to differences in firing rate of the deterrent-sensitive cells, differences in the ratio of responses to phagostimulants and deterrents, differences in the rates of adaptation of the sensory neurons, or differences in the extent of interactions between chemicals at the peripheral sensilla. We conclude that the differences between the species probably result from differences in processing sensory information within the central nervous system.     

Conformational changes in proteins in vitro as a means of predicting the acute toxicities of chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Bj?rn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.     

Linoleic acid increases chorda tympani nerve responses to and behavioral preferences for monosodium glutamate by male and female rats

Previous studies suggest that the chorda tympani nerve (CT) is important in transmitting fat taste information to the central nervous system. However, the contribution of the CT in this process may depend upon the presence of other taste stimuli and/or differ in males and females. Accordingly, the present study investigated the role of the CT in free fatty acid taste processing by examining electrophysiological activity of the CT in response to the free fatty acid linoleic acid (LA), as well as by measuring behavioral responses to LA-taste mixtures. We recorded whole nerve responses from the CT in response to lingual application of LA with or without monosodium glutamate (MSG) in anesthetized male and female rats. In addition, we examined preferences for MSG + LA taste mixtures in behavioral tests. Although lingual application of LA alone did not produce CT whole nerve responses, coapplication of LA and MSG elicited greater CT responses than did MSG alone. These findings were paralleled by greater preferences for MSG + LA taste mixtures than for MSG alone. In both cases, the effect was particularly pronounced in male rats. Thus LA enhances CT activity and behavioral responses to LA + MSG taste mixtures, although there are sex differences in the effects. These results suggest that CT input is important in mediating behavioral responses to fat taste, but the effects depend upon other taste stimuli and differ in males and females.     

Structure-activity relationships of 18 endogenous neuropeptides on the motor nervous system of the nematode Ascaris suum

Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motor nervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motor neurons. These motor neurons are part of the final common output pathway from the motor nervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motor neurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motor neuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motor neurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motor neurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motor neurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motor neurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motor neuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motor neuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motor neurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.     

Designing and Implementing Nervous System Simulations on LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal''s taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.¹ The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.     

Viral vectors as part of an integrated functional genomics program.

Over the past decade, viral vectors have slowly gained mainstream acceptance in the neuroscience and genetics communities for the in vivo study of gene function [1]. Using stereotactic techniques, it is possible to characterize neuroanatomical relationships through the delivery of neurotropic viral vectors to specific brain regions. More sophisticated studies combine viral vectors with other methods of genetic manipulation such as germline transgenic mice. As more is learned about the properties of different viral vectors, it has become possible to use viral vectors to test hypotheses about the function of genes, through targeted in vivo delivery to the central nervous system (CNS). The effects of gene expression in the brain can be measured on the molecular, biochemical, electrophysiological, morphological, and behavioral levels. We propose that viral vectors should be considered as part of an integrated functional genomics platform in the CNS.     

Croton zehntneri essential oil: effects on behavioral models related to depression and anxiety.

Croton zehntneri (Cz), a popular plant used to treat "nervous disturbance", contains a complex mixture of compounds, including substances exhibiting central nervous system activity. The effects of Cz essential oil administration (p.o.) on the rat's central nervous system were studied in behavioral models used to evaluate anxiety and antidepressive drugs. The results showed that administration of Cz essential oil: 1) increased the immobility duration measured in the forced swimming test as compared to control group (control = 89.8 +/- 45.8; 1 microl = 153.0 +/- 48.7; 3 microl = 157.4 +/- 45.3; 10 microl = 145.3 +/- 51.0); 2) reduced the locomotion frequency observed in the open field (control = 62.5 +/- 22.7; 3 microl = 38.0 +/- 13.5; 10 microl = 39.2 +/- 22.2); 3) had no effect on the experimental group (1 microl) observed in open field; 4) had no effect on animals tested in social interactions, plus-maze and holeboard tests. These data suggest that Cz oil produced central depressor effects in rats without any anxiety alterations. These results may explain the popular use of this plant in Brazilian folk medicine for treating "nervous disturbances".     

The use of pharmacological screening for characterizing the toxicity of pertussis vaccines

The influence of pertussis preparations, introduced by oral and parenteral routes, on the detoxifying function of the liver and the state of the nervous system of the animals was studied by methods used in pharmacology and toxicology. The use of these methods made it possible to find out side effects produced by corpuscular pertussis vaccine, introduced parenterally, on the detoxifying function of the liver and the state of the nervous system of the animals. The negative influence on the nervous system was more pronounced after the injection of the commercial adsorbed diphtheria-pertussis-tetanus vaccine used in this investigation than after the injection of pertussis monovaccine. The oral administration of corpuscular pertussis vaccine exerted no negative influence on the above-mentioned body functions of the animals.     

A tier system for developmental toxicity evaluations based on considerations of exposure and effect relationships

The backlog of untested chemicals and the rate at which new substances enter the marketplace exceed our capacity for developmental effects testing by standard in vivo methods. However, conservative use of two observations in a manner consistent with present day understanding of abnormal developmental biology can more accurately focus attention and resources on those agents in greatest need of complex testing for effects on in utero development. These two observations are 1) most chemicals are no more toxic to embryonic development than they are to adult homeostasis and 2) most human exposure to chemicals is de minimus, i.e., so small that it is inconsequential. Recently devised in vitro assays to quantitatively rank chemicals according to their developmental hazard index, when used in conjunction with more conventional in vivo methods and appropriate considerations of exposure, permit evaluation of a significantly larger number of chemicals than is currently achieved. The methods described apply a tier approach to establish testing priorities that markedly reduce the time, cost, and number of laboratory animals needed for evaluation of developmental toxicity.