vglut2 and gad expression reveal distinct patterns of dual GABAergic versus glutamatergic cotransmitter phenotypes of dopaminergic and noradrenergic neurons in the zebrafish brain

Abstract

Throughout the vertebrate lineage, dopaminergic neurons form important neuromodulatory systems that influence motor behavior, mood, cognition, and physiology. Studies in mammals have established that dopaminergic neurons often use γ-aminobutyric acid (GABA) or glutamatergic cotransmission during development and physiological function. Here, we analyze vglut2, gad1b and gad2 expression in combination with tyrosine hydroxylase immunoreactivity in 4-day-old larval and 30-day-old juvenile zebrafish brains to determine which dopaminergic and noradrenergic groups may use GABA or glutamate as a second transmitter. 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. Thus, the majority of catecholaminergic neurons are gad1b/2-positive and coexpress GABA. A very few gad1/2-negative dopaminergic groups, however, express vglut2 instead and use glutamate as a second transmitter. These glutamatergic dual transmitter phenotypes are the Orthopedia transcription factor-dependent, A11-type dopaminergic neurons of the posterior tuberculum. All together, our results demonstrate that all catecholaminergic groups in zebrafish are either GABAergic or glutamatergic. Thus, cotransmission of dopamine and noradrenaline with either GABA or glutamate appears to be a regular feature of zebrafish catecholaminergic systems. 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.

Keywords: A11 dopaminergic neurons; hypothalamus; noradrenergic; posterior tuberculum; preoptic region; subpallium.

Figure 1

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).

Figure 2

Expression of the glutamatergic marker vglut2 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,B2: overviews indicating planes of histological sections (for details, see Fig. 1B1,B2). C–J: Fluorescent in situ hybridization to vglut2 (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 (D1–D3 through J2) show enlargements of the areas framed by white boxes in the overviews. The only groups that appear to express vglut2 are the Otp-dependent groups 2,4–6 in the posterior tuberculum/hypothalamus (E,E2, G,G2). The pretectal group (C), the prethalamic group 1 (E,E2), the hypothalamic groups 3 and 7 (G,G2, I,I2), and the preoptic group (J,J2) do not express vglut2. The noradrenergic neurons of the locus coeruleus (D–D3) are intermingled with vglut2-expressing cells, but they do not appear to express vglut2. The telencephalic groups in the olfactory bulb and in the subpallium (F,F2 and H,H2, respectively) do not express vglut2 but are located in areas adjacent to vglut2-expressing neurons. All images in C–J are 4–13-μm confocal z-projections, except for those in D1–D3, which show a single confocal plane. For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S2. Scale bar = 50 μm in B1 (applies to B1,B2), C, D, D3 (applies to D1–D3), E, E2 (applies to E1,E2), F, G, G2 (applies to G1,G2), I, I2 (applies to I2,I2), J.

Figure 3

Localization of gad1b/2- or vglut2-expressing cells and TH-ir neurons in the telencephalon of the juvenile brain. A: Lateral overview of a juvenile brain stained for th by in situ hybridization (image taken from Filippi et al., 2010), which serves as anatomical reference to indicate the levels of the sections shown in B–G. B–G: Fluorescent in situ hybridization to detect gad1b/2 (C,F; green) or vglut2 (D,G; green) expression combined with anti-TH immunofluorescence (red). In each row, the image at the left shows an overview of the considered section and displays only TH immunostaining. Higher magnifications of the approximate area framed by the white square are shown to the right, together with either gad1b/2 (C,F) or vglut2 (D,G) staining. All images are 6–10-μm confocal z-projections of areas comprising TH-ir cells in the olfactory bulb (OB; B–D) and in the subpallium (SP; E-G). All telencephalic THir neurons express gad1b/2 (C,F) but not vglut2 (D,G), indicating that they are GABAergic. To better display the colocalization of the markers, C1–C3 and F1–F3 show the individual channels of the areas framed in C and F, respectively. For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S3. Scale bar = 50 μm in B–G.

Figure 4

Localization of gad1b/2- or vglut2-expressing cells and TH-ir neurons in the anterior diencephalon of the juvenile brain. A: Overview of section planes shown in B–M (for details, see Fig. 3A legend). B–M: Fluorescent in situ hybridization to detect gad1b/2 (C,F,I,L; green) or vglut2 (D,G,J,M; green) expression combined with anti-TH immunofluorescence (red). The DA clusters in the magnocellular preoptic nucleus (PM; B–D), in the prethalamus and suprachiasmatic nucleus (VL, SC; E–G), in the pretectum (Pr; H–J), and in the posterior tuberculum (PT; K–M) are shown. In each row, the image at the left shows an overview of the considered section and displays only TH immunostaining. The pretectal, prethalamic, and suprachiasmatic clusters are included in the same overview, shown twice in E and H. Higher magnifications of the approximate area framed by the white square in the overviews are shown at their right, together with either gad1b/2 (C,F,I,L) or vglut2 (D,G,J,M) staining. The majority of the images are 4–15-μm confocal z-projections, except for C1–C3, G, and I1–I3, which are single planes. All DA clusters considered in this figure are also GABAergic, as demonstrated by coexpression of TH with gad1b/2, but not with vglut2. The only exception is represented by the suprachiasmatic nucleus, in which some DA neurons express gad1b/2 (arrowhead in F1–F3) and some do not (arrow in F1–F3). For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S4. Scale bar = 50 μm in B–M.

Figure 5

Localization of gad1b/2 or vglut2 expression and TH immunoreactivity in Otp-dependent DA neurons of the juvenile brain. A: Overview of section planes shown in B–J (for details, see Fig. 3A legend). B–J: Fluorescent in situ hybridization to detect gad1b/2 (C,F,I; green) or vglut2 (D,G,J; green) expression combined with anti-TH immunofluorescence (red). The posterior tubercular and hypothalamic Otp-dependent DA neurons are shown. The overviews in B, E, and H are 2–10-μm confocal z-projections encompassing the large pear-shaped DA neurons in the posterior tuberculum (PT; B,E) and the group in the posterior tuberal nucleus (PTN; H). Higher magnifications of the approximate areas framed by the white squares in the overviews are shown at their right, together with either gad1b/2 (C,F,I) or vglut2 (D,G,J) staining (3–16-μm z-projections). All Otp-dependent DA neurons express vglut2 (D,G,J) but not gad1b/2 (C,F,I), indicating that they are also glutamatergic. Individual channels demonstrating coexpression of TH with vglut2 are shown for each group in D1–D3, G1–G3, and J1–J3. For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S5. Scale bar = 50 μm in B–J.

Figure 6

Localization of gad1b/2 or vglut2 expression and TH immunoreactivity in hypothalamus and hindbrain. A: Overview of section planes shown in B–J (for details, see Fig. 3A legend). B–J: Fluorescent in situ hybridization to detect gad1b/2 (C,F,I; green) or vglut2 (D,G,J; green) expression coupled with anti-TH immunofluorescence (red). The overviews in B, E, and H are 5–10-μm confocal z-projections encompassing the hypothalamic groups of liquor-contacting DA neurons located in the paraventricular organ (PVO; B) and in the posterior recess (PR; E), as well as the noradrenergic neurons of the locus coeruleus in the hindbrain (LC; H). All these groups appear to be GABAergic by coexpression of TH with gad1b/2 (C,F,I; 5–8-μm projections), but not with vglut2 (D,G,J; 3–9-μm projections). C1–C3, F1–F3, and I1–I3 are single-channel enlarged views of the framed areas in C, F, and I, respectively. For abbreviations, see list. A magenta–green copy of this figure is available as Supporting Information Figure S6. Scale bar = 50 μm in B–J.

Figure 7

Localization of gad1b/2 or vglut2 expression and TH immunoreactivity in the medulla oblongata at 30 dpf. A: Overview of section plane shown in B–D (for details, see Fig. 3A legend). B–D3: Fluorescent in situ hybridization to detect gad1b/2 (C; green) or vglut2 (D; green) expression combined with anti-TH immunofluorescence (red). The overview in B is a 10-μm confocal z-projection showing TH-ir noradrenergic neurons of the medulla oblongata (MO) in the hindbrain. These neurons were intermingled but not colocalizing with gad-expressing cells (C; 3-μm projection). Instead, we detected coexpression of TH with vglut2 (D–D3; 6-μm projection). D1–D3 are single-channel enlarged views of the framed area in D. The arrowheads point at examples of coexpression. A magenta–green copy of this figure is available as Supporting Information Figure S7. Scale bar = 50 μm in B–D.