Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain

Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new...

Identification of potential astrocytes in the teleost brain

Astrocytes are abundant star-shaped glial cells in the mammalian brain, with essential roles in metabolism, development, homeostasis, response to injury, behavior, and learning. Surprisingly, most regions of the teleost brain are thought to lack astrocytes, based primarily on the use of GFAP (glial fibrillary acidic protein) as a marker. Here, drawing on recent evidence that astrocytes are molecularly heterogeneous, we propose that astrocytes exist in the teleost brain, albeit of the olig2 subtype. Highly branched cells are present throughout the zebrafish brain, as shown here in Tg(sox10:EGFP) fish and previously in Tg(olig2:GFP) fish. Transcriptome data indicates the presence of brain cells that are olig2 and sox10 positive, which also express the astrocyte markers sox9b, sparcl1 and slc1a2b but lack gfap and the oligodendrocyte marker mbp. In situ hybridization confirms that stellate sox10:EGFP cells express olig2 and sox9b, while immunofluorescence indicates that they lack HuC/D and GFAP. We suggest that these cells be classified as astrocytes as this may more accurately reflect their functions.

Neuroprotective Role of Curcumin Against Benzo[a]pyrene-Induced Neurodegeneration in Zebrafish

Curcuma longa L. has been shown to exhibit neuroprotection in the brain. Curcuma longa L. has been used for its neuroprotective effect in humans of neurological disorders. The present study is aimed to evaluate the neuroprotective role of curcumin, a key component of Curcuma longa L., following exposure to waterborne B[a]P. Wild-type adult zebrafish (Danio rerio) were assigned as naïve, control (dimethyl sulfoxide), curcumin, B[a]P (Benzo[a]pyrene) and B[a]P+ curcumin group. B[a]P-induced altered antioxidant levels were enhanced by curcumin in the B[a]P+curcumin group. Findings showed that B[a]P induced anti-anxiety behavioral response and altered antioxidant activity in zebrafish is reduced by curcumin. The periventricular grey zone (PGZ) located on the optic tectum (TeO) in the zebrafish brain regulates anxiety-like behavior. Our histological study showed a significant increase in pyknotic neuronal counts in PGZ of TeO in adult zebrafish brain following B[a]P exposure and was improved by curcumin co-supplementation. Recent findings showed that curcumin improves glutathione production, an antioxidant necessary for maintaining redox homeostasis and shows a neuroprotective role in brain cells. The present study's findings address the potential role of curcumin co-supplementation as a herbal therapeutic against B[a]P-induced neurotoxicity in zebrafish.

A 3D adult zebrafish brain atlas (AZBA) for the digital age

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.