Methods for Assessing Neurotoxicity in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 113

10.2 LIMITATIONS OF CURRENT NEUROTOXICITY
TESTING
Approaches for assessing neurotoxicity are categorized into three general groups:
behavioral, morphological (neurohistopathology), and biochemical (measurements
of altered cellular metabolism and function). Current preclinical neurotoxicity testing
generally relies on detection of behavioral abnormalities and/or the appearance of
overt histopathological lesions in nerve tissue. Animal behavioral studies are most
effective in detecting neurotoxicity that adversely affects well-defined and easily
detectable parameters such as survival, motor function, aggression, feeding, groom-
ing, and reproductive and maternal behavior. However, it is likely that a majority of
the effects on the central nervous system are silent and produce changes in function
that result in subtle alterations in parameters such as emotion, cognition, tempera-
ment, or mood that cannot be adequately evaluated in animals and may not be easily
identifiable in human studies. Histological methods are effective in detecting
neurotoxicity only when lesions in the nervous system are extensive and can be
detected by immunohistochemical staining methods. The number of sections that can
be analyzed and the availability of skilled neuropathologists are limiting factors. The
application of biochemical markers for neurotoxicity is an ongoing area of research
within both government agencies and the academic community. Several specific
biochemical markers (e.g., changes in enzyme activity, protein phosphorylation)
have been examined but have proven useful for detecting only specific types of
neurotoxicity. Other biochemical assessments that correlate brain functions with
metabolism have not been rigorously tested. More recently, a variety of “-omic”
technologies are increasingly applied for preclinical safety assessment; however,
these approaches have not yet been implemented in neurotoxicity safety evaluations
(O'Callaghan and Sriram, 2005). Development of rapid assay methods and new
animal models to predict neurotoxicity are urgently needed.
Zebrafish is exceptionally well suited for neurotoxicity studies that combine
cellular, molecular, and genetic approaches. Because the embryo is transparent
and develops rapidly, development of specific neurons and axon tracts can be
visualized in live embryos using differential interference contrast (DIC) microscopy
or by injecting live dyes (Kuwada and Bernhardt, 1990). Specific types of neurons can
be visualized in whole, fixed embryos by immunohistochemistry or in situ hybrid-
ization (Chandrasekhar et al., 1997; Moens and Fritz, 1999). Motor neuron activity
can be monitored in vivo by calcium imaging and patch clamp recording (Drapeau
et al., 2002). Function of individual neurons can be elucidated by specific neural lesion
using toxic dye injection (Gahtan and O'Malley, 2001) or laser ablation (Fetcho and
Liu, 1998). In addition, mutants exhibiting observable morphological phenotypes or
behaviors have been isolated from large-scale screens and have been useful in
understanding early CNS patterning (Jiang et al., 1996; Schier et al., 1996).
10.3 ASSESSING NEUROTOXICITY IN ZEBRAFISH
Recently, zebrafish has been shown to be a useful animal model for assessing
compound-induced neurotoxicity. During the first 2 weeks of development, zebrafish