Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish

Aim We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. Methods A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. Results The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. Conclusion Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.Peer reviewe

Cellulär dopaminlagring i hjärnan hos zebrafisk embryon: betydelsen av vmat2

VMAT2 is an integral membrane protein with a crucial role in the monoaminergic system. It transports cytosolic dopamine and other neurotransmitters into synaptic vesicles for storage and later exocytotic release. VMAT2 has been found to have a neuroprotective effect, for instance by preventing oxidation of free cytosolic dopamine. Research has suggested that increased VMAT2 expression or function could have a protective effect against Parkinson’s disease. Rapid embryonic development, transparency during larval stage, and high homology with the human genome are some of the factors that makes the zebrafish a popular model organism. The aim of this research was to optimize the protocol for immunohistochemistry with an anti-dopamine antibody, and to find out how the absence of a functioning vmat2 gene in zebrafish mutants (vmat2-/-) affects dopamine immunostaining, compared to embryos with normal vmat2 function (vmat2+/+). Zebrafish larvae were fixed in a fixation agent, whereafter brains were dissected and immunostaining was performed. Individual methods in the protocol were adjusted for each experiment until the result of the dopamine staining was considered optimal. We found that the choice of fixative affected dopamine staining strongly. A clear reduction in dopamine immunoreactive cells was observed in zebrafish larvae lacking a functioning vmat2 gene compared to zebrafish with normal vmat2 function. The results also suggested that the localization of dopamine-storing cells in zebrafish larvae resembles more tyrosine hydroxylase 2 than tyrosine hydroxylase 1 expression pattern, especially in the hypothalamus. Vmat2-/- zebrafish could be useful when investigating how monoamine transport affects different diseases and their pharmacological treatment.VMAT2 är ett membranprotein med avgörande roll i det monoaminerga systemet. Det transporterar bland annat cytosoliskt dopamin och övriga neurotransmittorer in i synaptiska vesiklar, där de lagras för senare synaptisk utsöndring. VMAT2 har visat sig ha en nervskyddande effekt, bland annat genom att förhindra oxidering av fritt cytosoliskt dopamin. Forskning har antytt att ökad expression eller funktion av VMAT2 kunde ha en skyddande effekt mot Parkinsons sjukdom. Zebrafisken är en populär modellorganism, bland annat tack vare snabb embryonal utveckling, genomskinlighet i larvstadie samt hög grad av likhet med det mänskliga genomet. Syftet med forskningen var att optimera protokollet för immunohistokemi med anti-dopamin antikropp, samt att undersöka hur frånvaron av en fungerande vmat2 gen i genmodifierade zebrafisk embryon (vmat2-/-) påverkar dopaminfärgningen, jämfört med embryon med normal förekomst av vmat2 (vmat2+/+). Embryon har fixerats i fixeringsmedel och hjärnorna har därefter dissekerats, varefter immunofärgning utförts. För varje experiment justerades enskilda metoder i protokollet tills resultatet av dopaminfärgningen ansågs optimalt. I studien framkom att valet av fixeringsmedlet inverkade starkt på dopaminfärgningen. En tydlig minskning i dopaminpositiva celler kunde observeras hos embryon som saknade fungerande vmat2 gen i relation till embryon med normal förekomst av vmat2. Resultaten antydde också att lokalisationen av dopaminlagrande celler i zebrafisk embryon mer liknar tyrosinhydroxylas 2 än tyrosinhydroxylas 1 expressionen, framförallt i hypothalamus. Vmat2-/- zebrafisken kan ha potential som modellorganism då man vill undersöka hur transporten av monoaminer inverkar på olika sjukdomar och på behandlingen av dem