Methods for Assessing Neurotoxicity in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 114

are transparent, providing excellent accessibility for visualization of vital dyes,
fluorescent tracers, antibodies, and riboprobes in live and whole mount fixed speci-
mens. Zebrafish have an early population of neurons, called primary neurons, which
are part of a relatively simple nervous system that differentiates in order to coordinate
larval movement (Westerfield et al., 1986; Kimmel et al., 1988). The distribution
and projection patterns of specific primary neurons have been described in detail
(Eisen, 1991). By 24 h post fertilization (hpf), these primary zebrafish neurons
differentiate and establish many of their projections. During this period, the relatively
large neuronal cell bodies can be identified in vivo using Normarski optics. By 48hpf,
zebrafish brain ventricles have formed (Jiang et al., 1996; Schier et al., 1996).
Zebrafish body length increases from 1mm on 1dpf (day post fertilization) to 5mm
by 6dpf and the animals do not have skulls.Whole animal staining can be performed to
examine the entire nervous system in extensive detail. Moreover, zebrafish motor
behavior develops in a predictable sequence (Drapeau et al., 2002), supporting
assessment of drug effects on locomotion. Classical neurotoxins tested in zebrafish
include (1) dopaminergic neurotoxins such as MPTP (1-methyl-4-phenyl-1,2,3,
6-tetrahydropyridine), 6-hydroxydopamine (6-OHDA), rotenone, and paraquat;
(2) non-NMDA-type glutamate receptor (AMPA) agonists or antagonists such as
domoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, alpha-latrotoxin, and picrotoxin;
(3) nicotinic acetylcholine receptor (nAChR) antagonists such as bungarotoxins and
cobratoxins or acetylcholinesterase (AChE) inhibitors; and (4) NMDA receptor
antagonist, DL -2-amino-5-phosphonovalerate (AP-5) (Legendre, 1997; Ali et al.,
2000; Hatta et al., 2001; Rigo et al., 2003).
10.3.1 Compound Effects on Motor Neurons
and Neuronal Proliferation
In vertebrates, motor neurons are defined as neurons located in the central nervous
system that project their axons outside the CNS and directly or indirectly control
muscles. Drug-induced neurotoxicity on motor neurons and neuronal proliferation
has been extensively reported in mammalian models; for example, ethanol has been
shown to induce motor neuron death and inhibit neuronal proliferation. Ethanol has
also been found to affect the brain and motor functions in humans. To assess
compound effects on morphology of motor neurons and pattern of neuronal prolif-
eration, 5hpf zebrafish were exposed to 2.5% ethanol for 1 h and zebrafish were fixed
at 2dpf for whole mount immunostaining using Znp-1, a mouse monoclonal antibody
specific to zebrafish primary motor neurons or a mouse monoclonal antibody against
PCNA (proliferating cell nuclear antigen) (Parng et al., 2007). Primary motor neurons
in untreated, wild-type zebrafish exhibited an organized, stereotypical vertical
pattern. After ethanol treatment, primary motor neuron staining in the somite region
was absent (Fig. 10.1a and b) and the pattern of neuronal proliferation was abnormal
(Fig. 10.1c and d). These results suggest that zebrafish is a suitable animal model for
visually assessing compound effects on motor neurons and neuron proliferation
(Parng et al., 2007).