GABA-glycine cotransmitting neurons in the ventrolateral medulla: development and functional relevance for breathing

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type

Strategy for the generation of GAD65-tdTomato mice expressing the fluorescent protein tdTomato under control of the GAD65 promoter in GABAergic neurons.

<p><b>A:</b> 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>B:</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. <b>C:</b> 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. <b>D:</b> 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).</p

Cells of the rostral migratory stream (RMS) show bright red fluorescence in 2.5 month old GAD65-tdTomato mice.

<p><b>A:</b> Overview showing the RMS (arrowheads) from the ventricle (asterisk) to the olfactory bulb. Scale bar: 100 μm. <b>B-D:</b> 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.</p

Strategy for the generation of GAD65-tdTomato mice expressing the fluorescent protein tdTomato under control of the GAD65 promoter in GABAergic neurons.

<p><b>A:</b> 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>B:</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. <b>C:</b> 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. <b>D:</b> 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).</p

Identification of parvalbumin-, calretinin- and somatostatin-expressing subpopulations of interneurons among tdTomato-expressing neurons in transgenic mice.

<p>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. <b>G:</b> Quantification of the relative contribution of parvalbumin-, calretinin- and somatostatin-expressing neurons to the number of cells expressing tdTomato.</p

Expression pattern of tdTomato in the mouse retina.

<p><b>A:</b> 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>B-D:</b> 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). <b>D:</b> 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.</p

Identification of GABAergic and glycinergic neurons in TgN(GAD65-tdTomato) x TgN(GlyT2-EGFP) double transgenic mice by several microscopic techniques.

<p><b>A:</b> 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>B:</b> High magnification epifluorescence image. <b>C:</b> GABAergic and glycinergic cells expressing tdTomato or EGFP, respectively, can also be visualized using confocal imaging. <b>D:</b> 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.</p

Expression of tdTomato in Purkinje cells in the cerebellum.

<p><b>A:</b> Overview of the cerebellum of a 2.5 month old animal showing that only a subpopulation of Purkinje cells expressed tdTomato. <b>B, C:</b> 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).</p

Overall expression pattern of tdTomato in GAD65-tdTomato transgenic mice at different developmental stages.

<p>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.</p