Chemogenetic ablation of dopaminergic neurons leads to transient locomotor impairments in zebrafish larvae

Abstract

To determine the impact of a controlled loss of dopaminergic neurons on locomotor function, we generated transgenic zebrafish, Tg(dat:CFP-NTR), expressing a cyan fluorescent protein-nitroreductase fusion protein (CFP-NTR) under the control of dopamine transporter (dat) cis-regulatory elements. Embryonic and larval zebrafish express the transgene in several groups of dopaminergic neurons, notably in the olfactory bulb, telencephalon, diencephalon and caudal hypothalamus. Administration of the pro-drug metronidazole (Mtz) resulted in activation of caspase 3 in CFP-positive neurons and in a reduction in dat-positive cells by 5 days postfertilization (dpf). Loss of neurons coincided with impairments in global locomotor parameters such as swimming distance, percentage of time spent moving, as well as changes in tail bend parameters such as time to maximal bend and angular velocity. Dopamine levels were transiently decreased following Mtz administration. Recovery of some of the locomotor parameters was observed by 7 dpf. However, the total numbers of dat-expressing neurons were still decreased at 7, 12, or 14 dpf, even though there was evidence for production of new dat-expressing cells. Tg(dat:CFP-NTR) zebrafish provide a model to correlate altered dopaminergic neuron numbers with locomotor function and to investigate factors influencing regeneration of dopaminergic neurons. Behavioral changes and neurochemical deficiencies have been previously described in the zebrafish following administration of neurotoxins such as 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which target the catecholaminergic neuron populations. Although dopaminergic neurons have been shown to regenerate in both the planarian (Nishimura et al. 2011) and salamander (Parish et al. 2007) neurotoxin-based models, this has not been demonstrated in the zebrafish brain. This lack of information may be attributed, in part, to the fact that although 6-hydroxydopamine and MPTP induce both neurochemical and behavioral changes, no dopaminergic neuron loss and cell death are detected in zebrafish (Anichtchik et al. 2004). Indeed, in larval zebrafish, MPTP treatments results in a short-lived reduction in tyrosine hydroxylase (TH) immunoreactivity at 5 and 6 dpf which is recovered by 7 dpf and does not lead to signs of apoptosis or cell death.To address the events that take place following DA neuron loss and to determine whether the ablated neuronal population can be re-established in the brain of larval zebrafish, we have Received March 25, 2015; revised manuscript received May 20, 2015; accepted May 26, 2015. Address correspondence and reprint requests to Dr Marc Ekker, Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa Ottawa, K1N-6N5, Canada. E-mail: mekker@uottawa.ca Abbreviations used: BrdU, 5-bromo-2 0 -deoxyuridine; CFP-NTR, cyan fluorescent protein-nitroreductase fusion protein; EGFP, -enhanced green fluorescent protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydro...

Nifurpirinol: A more potent and reliable substrate compared to metronidazole for nitroreductase‐mediated cell ablations

“…A range of 2.5 to 10 µM of effective concentrations also means that NFP offers the choice to determine the optimal dose for ablation which might be cell‐dependent. Our study shows that the NFP/NTR combination is effective not only for pancreatic beta cells but is extendable to other ablation models like osteoblasts and, most importantly, for dopaminergic neurons for which ablation appears tricky and harsher MTZ regimens proved to be mandatory …”

“…To determine if NFP could improve ablations of cell types that are difficult to ablate, we used the Tg(dat:CFP‐NTR) which expresses NTR and CFP in dopaminergic neurons under the control of the dat ( slc3a6 ) promoter. With these fish, even a 72‐hours MTZ regimen only lead to moderate ablation (50–60%) . To compare MTZ and NFP efficiencies in inducing dopaminergic neuron ablation, we treated Tg(dat:CFP‐NTR) larvae at 3 dpf for 48 hours with 7.5 mM MTZ or 5 µM NFP (Figure B).…”

“…10 Tg(ins:dsRed) m101811 was provided by Didier Stainier. Tg(dat:CFP-NTR) 12 (Zfin reference Tg(slc6a3:CFP-NTR)) was given by Marc Ekker.…”

Role of branchiomotor neurons in controlling food intake of zebrafish larvae

“…In NTR-expressing cells, the prodrug metronidazole (Mtz) is converted into cytotoxic metabolites leading to cell death within 24-48 hours of treatment (Drabek et al, 1997). NTR-mediated cell ablation has been successfully employed in zebrafish, and demonstrated to be both effective and specific, resulting in the ablation of NTR-expressing cells while leaving neighboring cells unharmed (Curado et al, 2008; Godoy et al, 2015; Pisharath et al, 2007; Pope and Voigt, 2014). …”

Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons

“…These cells of the jaw, branchial arches (BA), and reticular astrocytes of the optic nerve were seen in the Tg(dat:EGFP) line, 8 as well as a third transgenic line using the same genomic sequences from the dat locus. 13 Global dorsal and lateral views of the overall mCherry expression in live larvae were taken at 3 dpf ( Fig. 2A).…”