Fig 1.
Strategy for the generation of GAD65-tdTomato mice expressing the fluorescent protein tdTomato under control of the GAD65 promoter in GABAergic neurons.
A: Schematic representation of the BAC clone RPCI 23-407K8 (208,325 bp in total) containing the mouse full-length gene GAD65. Locations of PCR products for BAC verification (5’A-C; 3’A-B; Ex1) are indicated. B: Structure of the wild type GAD65 gene as well as the targeting construct. The endogenous start codon is located within exon 1. PCR reaction with primers (Ex1) flanking exon 1 results in a DNA fragment of 1012 bp in the wild type gene. The transgene consists of the ORF of tdTomato, a SV40-PolyA site as well as a FRT-flanked neomycin resistance cassette (neo) and was inserted directly after the endogenous ATG using homologous recombination provoked by homology arms (HA) indicated. C: Representation of the modified BAC containing the GAD65-tdTomato transgene after removal of the neomycin resistance cassette using Flp recombination. PCR using the same primers (Ex1) flanking exon 1 results in a product of 2746 bp in the modified BAC. D: PCR verification of the identity and integrity of the BAC using primers located 5‘ and 3‘ of the GAD65 gene (5’A-C; 3’A-B) as well as verification of the targeted modification site using primers spanning exon1 (Ex1).
Fig 2.
Overall expression pattern of tdTomato in GAD65-tdTomato transgenic mice at different developmental stages.
In sagittal brain slices, a high reporter expression was observed in the olfactory bulb and striatum as well as in cortex, hippocampus, brainstem and substantia nigra in all investigated developmental stages (A: P1, B: P3, C: P10, D: adult 2.5 months). Fluorescence intensity of cells varied highly within one brain region and between different areas of the brain as well as different developmental stages. Scale bars: 500 μm.
Fig 3.
TdTomato expression in various brain regions of GAD65-tdTomato mice at different developmental stages (P1; P3; P10; 2.5 months).
A: olfactory bulb; B: cortex; C: hippocampus; D: striatum; E: brainstem; F: cerebellum. TdTomato is expressed in numerous GABAergic neurons within these brain regions and is visible in somata, dendrites, as well as axons. The scale bar corresponds to 50 μm and applies to all panels.
Fig 4.
Expression of tdTomato in Purkinje cells in the cerebellum.
A: Overview of the cerebellum of a 2.5 month old animal showing that only a subpopulation of Purkinje cells expressed tdTomato. B, C: In these Purkinje neurons both the dendritic tree as well as the axons (arrowheads) are clearly visible. Scale bars: 500 μm (A), 50 μm (B), 20 μm (C).
Fig 5.
Cells of the rostral migratory stream (RMS) show bright red fluorescence in 2.5 month old GAD65-tdTomato mice.
A: Overview showing the RMS (arrowheads) from the ventricle (asterisk) to the olfactory bulb. Scale bar: 100 μm. B-D: Detailed images of the cells along the RMS migrating from the subventricular zone (SVZ, B) via the rostral forebrain (C) to the olfactory bulb (OB, D). Scale bar in D corresponds to 40 μm and applies to B-D. St: striatum.
Fig 6.
Expression pattern of tdTomato in the mouse retina.
A: Top view of a retinal wholemount preparation showing the distribution of tdTomato-expressing cells within the retina of a 2.5 month old animal. Scale bar: 100 μm. B-D: Transverse section of the retina stained with DAPI to stain cellular nuclei to visualize retinal layering (B). TdTomato-expressing cells (C) were found in inner nuclear layer (INL) and ganglion cell layer (GCL). D: overlay of DAPI- (blue) and tdTomato- (red) fluorescence. Scale bar in D corresponds to 40 μm and applies to B-D. IPL: inner plexiform layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. Red fluorescence within the RPE is due to autofluorescence.
Fig 7.
Immunohistochemical verification of tdTomato-expressing cells as GAD65-positive interneurons in GAD65-tdTomato mice.
An antibody raised against GAD65 (green) was used to identify tdTomato-positive cells as GABAergic neurons in 2.5 month old mice. A: cortex; B: hippocampus; C: brainstem; D: olfactory bulb. All nuclei were stained with DAPI (blue). Arrows highlight examples of cells expressing both tdTomato and GAD65. The scale bar corresponds to 50 μm and applies to all panels. E: Quantification of cells expressing tdTomato in the absence of GAD65-immunoreactivity (red); GAD65-positive cells lacking tdTomato fluorescence (green) as well as neurons simultaneously expressing tdTomato and endogenous GAD65 (yellow). While 10% to 20% of GAD65 expressing neurons do not express tdTomato, almost all cells showing tdTomato fluorescence are indeed GAD65-positive GABAergic neurons.
Fig 8.
Identification of parvalbumin-, calretinin- and somatostatin-expressing subpopulations of interneurons among tdTomato-expressing neurons in transgenic mice.
Immunohistochemical analysis showed the colocalization of the interneuron markers parvalbumin (A, B), calretinin (C, D) or somatostatin (E, F) with tdTomato fluorescence (red) in the cortex (A, C, E) and hippocampus (B, D, F) of 2.5 month old mice. Right panels show the overlay of tdTomato (red), parvalbumin, calretinin or somatostatin (green) as well as nuclei stained with DAPI (blue). Arrows highlight examples of cells expressing both tdTomato and either parvalbumin, calretinin or somatostatin. The scale bar corresponds to 50 μm and applies to all panels. G: Quantification of the relative contribution of parvalbumin-, calretinin- and somatostatin-expressing neurons to the number of cells expressing tdTomato.
Fig 9.
Identification of GABAergic and glycinergic neurons in TgN(GAD65-tdTomato) x TgN(GlyT2-EGFP) double transgenic mice by several microscopic techniques.
A: Overview of a frontal brain slice of a double transgenic 30 day old mouse showing tdTomato (red) expressing GABAergic and EGFP (green) expressing glycinergic neurons. The image was acquired using epifluorescence. Scale bar: 1 mm. B: High magnification epifluorescence image. C: GABAergic and glycinergic cells expressing tdTomato or EGFP, respectively, can also be visualized using confocal imaging. D: Also by using 2-photon laser scanning microscopy, tdTomato- and EGFP-fluorescence can be observed allowing for unequivocal identification of GABAergic and glycinergic neurons, respectively. Scale bar in D corresponds to 40 μm and applies to B-D.