Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development

BACKGROUND: Electroporation is a technique for the introduction of nucleic acids and other macromolecules into cells. In chick embryos it has been a particularly powerful technique for the spatial and temporal control of gene expression in developmental studies. Electroporation methods have also been reported for Xenopus, zebrafish, and mouse. RESULTS: We present a new protocol for zebrafish brain electroporation. Using a simple set-up with fixed spaced electrodes and microinjection equipment, it is possible to electroporate 50 to 100 embryos in 1 hour with no lethality and consistently high levels of transgene expression in numerous cells. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Explants containing transfected neurons can be cultured for in vitro analysis. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry. CONCLUSION: Building on previously described methods, we have optimized several parameters to allow for highly efficient unilateral or bilateral transgenesis of a large number of cells in the zebrafish brain. This method is simple and provides consistently high levels of transgenesis for large numbers of embryos

Deletion of the WD40 Domain of LRRK2 in Zebrafish Causes Parkinsonism-Like Loss of Neurons and Locomotive Defect

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD

The thalamo-habenula projection revisited

The thalamus is one of the most highly connected hubs of the vertebrate brain, with roles in perception, arousal, navigation, memory and consciousness. One connection that is missing from contemporary maps is a link to the habenula. This link was reported in the early part of the last century, but appears to have slipped into obscurity. Here, I review the evidence for the existence of this innervation and consider the potential roles it could play. In particular, the possibility that this pathway is involved in non-visual responses to ambient illumination, including emotional responses, is examined.Accepted versio

The vertebrate habenula

A renowned neuroscientist, now retired from Cambridge University, once told me that he had been a graduate student in North America when he first came across the term “habenula”. For him and his course mates, the habenula provided a source of humor. Nobody knew what it did, but the word sounded unusual and provoked a lot of laughter. The anatomy of habenula neurons, with their unusual terminations of loops and spirals at the midline, was a source of wonderment. There was nothing else like it in the vertebrate brain.Accepted versio

Masking of a circadian behavior in larval zebrafish involves the thalamo-habenula pathway

Changes in illumination can rapidly influence behavior that is normally controlled by the circadian clock. This effect is termed masking. In mice, masking requires melanopsin-expressing retinal ganglion cells that detect blue light and project to the thalamus. It is not known whether masking is wavelength-dependent in other vertebrates, nor is it known whether the thalamus is also involved or how it influences masking. Here, we address these questions in zebrafish. We find that diel vertical migration, a circadian behavior in larval zebrafish, is effectively triggered by blue, but not by red light. Two-photon calcium imaging reveals that a thalamic nucleus and a downstream structure, the habenula, have a sustained response to blue but not to red light. Lesioning the habenula reduces light-evoked climbing. These data suggest that the thalamo-habenula pathway is involved in the ability of blue light to influence a circadian behavior.MOE (Min. of Education, S’pore)Published versio

Identification of GABAergic neurons innervating the zebrafish lateral habenula

Habenula neurons are constantly active. The level of activity affects mood and behaviour, with increased activity in the lateral habenula reflecting exposure to punishment and a switch to passive coping and depression. Here, we identify GABAergic neurons that could reduce activity in the lateral habenula of larval zebrafish. GAD65/67 immunohistochemistry and imaging of gad1b:DsRed transgenic fish suggest the presence of GABAergic terminals in the neuropil and between cell bodies in the lateral habenula. Retrograde tracing with the lipophilic dye DiD suggests that the former derives from the thalamus, while the latter originates from a group of cells in the posterior hypothalamus that are located between the posterior tuberal nucleus and hypothalamic lobes. Two-photon calcium imaging indicates that blue light causes excitation of thalamic GABAergic neurons and terminals in the neuropil, while a subpopulation of lateral habenula neurons show reduced intracellular calcium levels. Whole-cell electrophysiological recording indicates that blue light reduces membrane potential of lateral habenula neurons. These observations suggest that GABAergic input from the thalamus may mediate inhibition in the zebrafish lateral habenula. Mechanisms governing release of GABA from the neurons in the posterior hypothalamus, which are likely to be in the tuberomammillary nucleus, remain to be defined.Ministry of Education (MOE)Nanyang Technological UniversityPublished versionThis research was supported by a Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant and the Singapore Ministry of Education under its Academic Research Fund Tier 2 (MOE2017-T2-058) and Tier 1 (MOE2016-T1-001-152) awards

Neural correlates of state transitions elicited by a chemosensory danger cue

Detection of predator cues changes the brain state in prey species and helps them avoid danger. Dysfunctionality in changing the central state appropriately in stressful situations is proposed to be an underlying cause of multiple psychiatric disorders in humans.Ministry of Education (MOE)Nanyang Technological UniversityThis research was supported by a Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Start-Up Grant and by the Singapore Ministry of Education under its Academic Research Fund Tier 2 Award (MOE2017-T2-058) to SJ, and by Yale-NUS College grants R-607-265-225-121 and IG16-LR003 to ASM