Zebrafish are exceptionally well suited for neurobiology studies that combine cellular, molecular, and genetic approaches. The ability to visually assess the structure of the nervous system in vivo is a unique advantage for using the zebrafish model. We provide a family of neural assays for assessing neurotoxicity and drug effects in preventing neuronal damages.

Neuron-specific Antibody Staining

Toxic effects on the central nervous system (CNS) and peripheral nervous system (PNS) are assessed by staining with neuronal specific antibodies. Specific populations of neurons, such as motor neurons and dopaminergic neurons, can be analyzed by specific antibodies.

An example of assessment of nervous system defects using a neuron specific antibody. (A), Control. (B), Drug treated. Note the reduction of PNS neurons in the drug treated zebrafish.

Neuronal Proliferation Assessment

Toxic effects on the proliferation of the CNS neurons are assessed by staining with an anti-PCNA antibody.

Proliferation zone in the brain. Zebrafish at 2 hpf were treated with 1% ethanol (B) for 5 days, followed by fixation and staining with an anti-PCNA antibody to label the proliferation zone in the brain (bright green fluorescence). Abnormal brain shape and proliferation zone were observed in ethanol treated zebrafish. (A), control. Anterior is up; the positions of the eyes are labeled with letter E.

Apoptosis Assessment

Apoptosis can be detected in live zebrafish embryos using acridine orange staining.

Detection of drug-modulated apoptosis in zebrafish by acridine orange staining. Increased apoptosis in the brain was observed after Brefeldin A treatment (B). The arrow in (B) points to the apoptotic cells in the brain. (A), Control. Anterior to the left. The positions of the eyes are indicated.

Quantitative Analysis of Apoptosis

The acridine orange in the apoptotic cells is extracted by solvent, and the amount of acridine orange is quantitated with a fluorescence plate reader. The relative fluorescence unit (RFU) reflects the level of apoptosis. Three concentrations are tested for each compound, with 10 zebrafish per concentration.

Quantitative apoptosis assay. Fluorescence from apoptotic cells in zebrafish embryos was quantitated in a fluorescence microplate reader. Neomycin treatment increased RFU (relative fluorescence unit) in a concentration-dependent manner.

Ototoxicity

Although the zebrafish ear does not contain a specialized hearing organ, many features are conserved with other vertebrate species. Zebrafish also possess a series of mechanosensory structures called neuromasts, which are located on the surface of the body and contain hair cells that are equivalent to mammalian inner ear hair cells. The hair cells are examined by staining with 2, 4-dimethyl-aminostyryl-N-ethyl pyridinium iodide (DASEPI).

Inner Ear Hair Cell Assessment: The hair cells are stained with DASEPI (black dots in A). Gentamicin, which is known to induce hair cell injury in humans, also caused hair cell loss in zebrafish (B).

Other neural assays are available, and studies can be custom designed. Some examples of our neural assays are listed below. Please inquire.

Organization of the nervous system can be examined by antibody staining. (A) is a schematic drawing of the optic nerve, optic track and commissure in zebrafish. Compared to the untreated control (B), reduced staining of the optic nerve (ON) was observed after exposing 48 hour post fertilization (hpf) embryos to 1% ethanol (C).

Legend:
ON=optic nerve                        OT=optic tract
AC=anterior commissure          POC= postoptic commissure
RGCs=retinal ganglion cells      P=posterior
A=anterior                               TPOC=tract postoptic commissure

Motor neuron morphology can be assessed using antibody staining. After ethanol exposure, axon loss on peripheral motor neurons was observed.