vglut2 and gad expression reveal distinct patterns of dual GABAergic versus glutamatergic cotransmitter phenotypes of dopaminergic and noradrenergic neurons in the zebrafish brain.
Our results show that most dopaminergic neurons also express GABAergic markers, including the dopaminergic groups of the olfactory bulb (homologous to mammalian A16) and the subpallium, the hypothalamic groups (A12, A14), the prethalamic zona incerta group (A13), the preoptic groups (A15), and the pretectal group.All together, our results demonstrate that all catecholaminergic groups in zebrafish are either GABAergic or glutamatergic.We compare our results with those that have been described for mammalian systems, discuss the phenomenon of transmitter dualism in the context of developmental specification of GABAergic and glutamatergic regions in the brain, and put this phenomenon in an evolutionary perspective.
Affiliation: Developmental Biology, Institute of Biology I, Faculty of Biology, Albert-Ludwigs-University Freiburg, 79104, Freiburg, Germany.
- Brain/growth & development*/metabolism
- Glutamate Decarboxylase/metabolism*
- Vesicular Glutamate Transport Protein 2/metabolism*
- Zebrafish Proteins/metabolism*
- Adrenergic Neurons/metabolism
- Dopaminergic Neurons/metabolism
- Glutamic Acid/metabolism
- In Situ Hybridization, Fluorescence
- Microscopy, Confocal
- gamma-Aminobutyric Acid/metabolism
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fig01: Expression of GABAergic markers gad1b/2 in relation to TH immunoreactivity at 96 hpf. A: Schematic lateral (top) and dorsal (bottom) views of the zebrafish larval brain showing the distribution of catecholaminergic groups detectable by immunohistochemistry at 4 dpf (modified from Tay et al., 2011). B1: Lateral overview of a larval brain immunostained for TH (red), showing the distribution of DA neurons in the olfactory bulb (OB), subpallium (SP), preoptic area (PO), pretectum (Pr), prethalamus (group 1), posterior tuberculum/medial hypothalamus (altogether labeled here vDC and comprising DA groups 2–6), and posterior hypothalamus (group 7), as well as the noradrenergic group in the locus coeruleus (LC). B2 Same image, indicating the dorsoventral level of the confocal planes shown in C–J. C–J: Fluorescent in situ hybridization to gad1b (C,D,F,H,J, green) or gad2 (E,G,I, green) combined with anti-TH immunofluorescence (red). Left panels (C–J) show overviews of dorsal views for planes encompassing different TH-ir clusters. Right panels (C1–C3 through J2) show enlargements of the areas framed by white boxes in the overviews. Double labeling revealed that the majority of DA groups expressed both gad isoforms, which had mostly overlapping expression patterns. gad expression and TH immunoreactivity were codetected for the pretectal group (Pr; C–C3), the prethalamic group (group 1; E,E2), the telencephalic groups (SP and OB; F,F2 and H,H2, respectively), and the preoptic group (PO; J center, J1,J2). The noradrenergic neurons of the locus coeruleus expressed only gad1b (D–D3, arrowheads), although not all of them did so (arrows in D1–D3). The liquor-contacting neurons in the medial hypothalamus (group 3; G–G3) and a subset in the posterior hypothalamus (group 7; I–I3) appear to weakly express only gad2. The arrowheads point at a gad2-expressing DA neuron, whereas a cell immunoreactive for TH but apparently free from gad2 expression is indicated by arrows. Neither gad1b nor gad2 expression could be detected in the posterior tubercular Otp-dependent groups 2,4–6 (E,E2, G–G3). All images in C–J2 are confocal z-projections (5–20-μm depth), except for those in G1–G3, and the top and bottom parts of J, which are separated by white lines and display the retina section, in single confocal planes only. For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S1. Scale bar = 50 μm in B1 (applies to B1,B2), C–E, E2 (applies to E1,E2),F, G, G3 (applies to G1–G3), I, J, J3 (applies to J1–J3).
For microscopic observation, larvae were mounted in 80% glycerol/PBS containing 1% agarose. Confocal z-stacks of whole-mount larvae and sectioned brains were recorded by using a Zeiss LSM 5 DUO laser scanning confocal microscope. Then z-projections from subsets of focal planes were generated with the Zeiss LSM software and exported as image format files. The images were assembled into figures and processed with the Adobe (San Jose, CA) Photoshop CS2 9.0 software. Adjustments to contrast and brightness were made by using the Photoshop Levels functions. All adjustments were made to whole images including the enlarged sections, except for Figures 1I1–I3, 3C1–C3, 3F1–F3, and 4F1–F3, for which in the whole enlarged section red and green levels were adjusted to better visualize red and green channel colocalization.