Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Abstract

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

The effects of aging on Amyloid-β42-induced neurodegeneration and regeneration in adult zebrafish brain

“…Microinjections: Cerebroventricular microinjections (CVMI) were performed as described. 48,49 Tissue preparation and immunohistochemistry: Tissue preparations and stainings were performed as described previously using the following antibodies: anti-ß-Amyloid (Cell Signaling, 8243, 1:500), anti-HuC/D (Life Technologies, A21271, 1:500), anti-PCNA (Dako, M0879, 1:500), anti-S100b (Dako, Z0311, 1:500), anti-L-Plastin (gift from Michael Redd, 1:5000), anti-acetylated-tubulin (Sigma, T6793, 1:500), anti-BrdU (Abd Serotec, MCA2060, 1:500), Synaptophysin (Abcam, Ab32594, 1:500). All secondary antibodies were from Molecular probes and were used at 1:500 dilution.…”

See-Thru-Gonad zebrafish line: developmental and functional validation

“…In vivo methods for functional analysis of miRNA in zebrafish gonad have yet to be developed. Recently, targeted gene knockdown by injection of morpholino oligonucleotides antisense for an miRNA precursor was developed for the zebrafish brain (Kizil et al 2013). There is potential that a zebrafish line optimized for reproductive biology could allow this technique to be adapted for gonadal studies as well.…”

A Novel, Low-Cost Anesthesia and Injection System for Zebrafish Researchers

“…This type of system with gradual cooling and warming could likely be used for performing procedures, such as cerebroventricular microinjections into the adult zebrafish brain that are currently and can only be done using chemical anesthetics. [18][19][20][21]…”