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Characterization of genetically targeted neuron types in the zebrafish optic tectum.

Robles E, Smith SJ, Baier H - Front Neural Circuits (2011)

Bottom Line: The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS.Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale.These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California San Francisco San Francisco, CA, USA.

ABSTRACT
The optically transparent larval zebrafish is ideally suited for in vivo analyses of neural circuitry controlling visually guided behaviors. However, there is a lack of information regarding specific cell types in the major retinorecipient brain region of the fish, the optic tectum. Here we report the characterization of three previously unidentified tectal cell types that are specifically labeled by dlx5/6 enhancer elements. In vivo laser-scanning microscopy in conjunction with ex vivo array tomography revealed that these neurons differ in their morphologies, synaptic connectivity, and neurotransmitter phenotypes. The first type is an excitatory bistratified periventricular interneuron that forms a dendritic arbor in the retinorecipient stratum fibrosum et griseum superficiale (SFGS) and an axonal arbor in the stratum griseum centrale (SGC). The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS. The third type is a GABAergic periventricular projection neuron that extends a dendritic arbor into the SGC and a long axon to the torus semicircularis, medulla oblongata, and anterior hindbrain. Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale. This approach revealed several novel aspects of tectal circuitry, including: (1) a glutamatergic mode of transmission from the superficial, retinorecipient neuropil layers to the deeper, output layers, (2) the presence of interneurons with mixed dendrite/axon arbors likely involved in local processing, and (3) a heretofore unknown GABAergic tectofugal projection to midbrain and hindbrain. These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

No MeSH data available.


Related in: MedlinePlus

The dlx5/6:GFP transgene labels a subpopulation of glutamatergic neurons. (A) Confocal image of vglut2b fluorescence in situ hybridization in a 5-dpf dlx5/6:GFP larvae. (B) 2× magnification of regions indicated by boxes in (A). Merged images of GFP immuofluorescence and vglut2b in situ labeling (left) and vglut2b in situ labeling alone (right). GFP fluorescence was used to trace cell body outlines. Note strong, punctate vglut2b in situ signal in the perinuclear region of cell body in B2. In contrast, the majority of cells in the SPV (B1) and all cells in the superficial NP (B3) lacked this punctate vglut2b mRNA signal. (C) Quantification of glutamatergic phenotypic analysis in dlx5/6:GFP. Scale bar, 20 μm in (A), 10 μm in (B).
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Figure 3: The dlx5/6:GFP transgene labels a subpopulation of glutamatergic neurons. (A) Confocal image of vglut2b fluorescence in situ hybridization in a 5-dpf dlx5/6:GFP larvae. (B) 2× magnification of regions indicated by boxes in (A). Merged images of GFP immuofluorescence and vglut2b in situ labeling (left) and vglut2b in situ labeling alone (right). GFP fluorescence was used to trace cell body outlines. Note strong, punctate vglut2b in situ signal in the perinuclear region of cell body in B2. In contrast, the majority of cells in the SPV (B1) and all cells in the superficial NP (B3) lacked this punctate vglut2b mRNA signal. (C) Quantification of glutamatergic phenotypic analysis in dlx5/6:GFP. Scale bar, 20 μm in (A), 10 μm in (B).

Mentions: Several studies have previously reported the presence of glutamatergic, GABAergic, and cholinergic neurons in the larval tectum. Notably, studies have also reported an absence of aminergic (noradrenergic and serotonergic) and glycinergic neurons at this developmental stage (Higashijima et al., 2004; McLean and Fetcho, 2004). Immunostaining and in situ hybridization data suggest that a large percentage of tectal neurons are glutamatergic or GABAergic (Higashijima et al., 2004; Sato et al., 2007; Smear et al., 2007), whereas cholinergic cells are relatively rare (Arenzana et al., 2005). To determine if the non-GABAergic population of neurons labeled in dlx5/6:GFP larvae are glutamatergic we conducted vglut2b fluorescence in situ hybridization in conjunction with retrospective GFP antibody staining. vglut2b in situ analysis revealed that a large percentage of cells in the SPV layer of the tectum are glutamatergic (Figure 3A). Subregion analysis of fluorescence in situ labeling confirmed that GFP-positive neurons with cell bodies in the superficial neuropil, which are GABA-positive, do not express vglut2b (Figure 3B). In contrast, a small percentage of dlx5/6:GFP-positive neurons in the SPV layer were positive for vglut2b cDNA (Figure 3C; 15.6 + 1.5%; data obtained from 555 cells from nine different larva). Closer examination of vglut2b-positive neurons confirmed the presence of discrete perinuclear puncta within the cell body (Figure 3B). These results indicate that the non-GABAergic population of tectal neurons labeled in dlx5/6:GFP larvae is glutamatergic.


Characterization of genetically targeted neuron types in the zebrafish optic tectum.

Robles E, Smith SJ, Baier H - Front Neural Circuits (2011)

The dlx5/6:GFP transgene labels a subpopulation of glutamatergic neurons. (A) Confocal image of vglut2b fluorescence in situ hybridization in a 5-dpf dlx5/6:GFP larvae. (B) 2× magnification of regions indicated by boxes in (A). Merged images of GFP immuofluorescence and vglut2b in situ labeling (left) and vglut2b in situ labeling alone (right). GFP fluorescence was used to trace cell body outlines. Note strong, punctate vglut2b in situ signal in the perinuclear region of cell body in B2. In contrast, the majority of cells in the SPV (B1) and all cells in the superficial NP (B3) lacked this punctate vglut2b mRNA signal. (C) Quantification of glutamatergic phenotypic analysis in dlx5/6:GFP. Scale bar, 20 μm in (A), 10 μm in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046383&req=5

Figure 3: The dlx5/6:GFP transgene labels a subpopulation of glutamatergic neurons. (A) Confocal image of vglut2b fluorescence in situ hybridization in a 5-dpf dlx5/6:GFP larvae. (B) 2× magnification of regions indicated by boxes in (A). Merged images of GFP immuofluorescence and vglut2b in situ labeling (left) and vglut2b in situ labeling alone (right). GFP fluorescence was used to trace cell body outlines. Note strong, punctate vglut2b in situ signal in the perinuclear region of cell body in B2. In contrast, the majority of cells in the SPV (B1) and all cells in the superficial NP (B3) lacked this punctate vglut2b mRNA signal. (C) Quantification of glutamatergic phenotypic analysis in dlx5/6:GFP. Scale bar, 20 μm in (A), 10 μm in (B).
Mentions: Several studies have previously reported the presence of glutamatergic, GABAergic, and cholinergic neurons in the larval tectum. Notably, studies have also reported an absence of aminergic (noradrenergic and serotonergic) and glycinergic neurons at this developmental stage (Higashijima et al., 2004; McLean and Fetcho, 2004). Immunostaining and in situ hybridization data suggest that a large percentage of tectal neurons are glutamatergic or GABAergic (Higashijima et al., 2004; Sato et al., 2007; Smear et al., 2007), whereas cholinergic cells are relatively rare (Arenzana et al., 2005). To determine if the non-GABAergic population of neurons labeled in dlx5/6:GFP larvae are glutamatergic we conducted vglut2b fluorescence in situ hybridization in conjunction with retrospective GFP antibody staining. vglut2b in situ analysis revealed that a large percentage of cells in the SPV layer of the tectum are glutamatergic (Figure 3A). Subregion analysis of fluorescence in situ labeling confirmed that GFP-positive neurons with cell bodies in the superficial neuropil, which are GABA-positive, do not express vglut2b (Figure 3B). In contrast, a small percentage of dlx5/6:GFP-positive neurons in the SPV layer were positive for vglut2b cDNA (Figure 3C; 15.6 + 1.5%; data obtained from 555 cells from nine different larva). Closer examination of vglut2b-positive neurons confirmed the presence of discrete perinuclear puncta within the cell body (Figure 3B). These results indicate that the non-GABAergic population of tectal neurons labeled in dlx5/6:GFP larvae is glutamatergic.

Bottom Line: The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS.Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale.These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California San Francisco San Francisco, CA, USA.

ABSTRACT
The optically transparent larval zebrafish is ideally suited for in vivo analyses of neural circuitry controlling visually guided behaviors. However, there is a lack of information regarding specific cell types in the major retinorecipient brain region of the fish, the optic tectum. Here we report the characterization of three previously unidentified tectal cell types that are specifically labeled by dlx5/6 enhancer elements. In vivo laser-scanning microscopy in conjunction with ex vivo array tomography revealed that these neurons differ in their morphologies, synaptic connectivity, and neurotransmitter phenotypes. The first type is an excitatory bistratified periventricular interneuron that forms a dendritic arbor in the retinorecipient stratum fibrosum et griseum superficiale (SFGS) and an axonal arbor in the stratum griseum centrale (SGC). The second type, a GABAergic non-stratified periventricular interneuron, extends a bushy arbor containing both dendrites and axons into the SGC and the deepest sublayers of the SFGS. The third type is a GABAergic periventricular projection neuron that extends a dendritic arbor into the SGC and a long axon to the torus semicircularis, medulla oblongata, and anterior hindbrain. Interestingly, the same axons form en passant synapses within the deepest neuropil layer of the tectum, the stratum album centrale. This approach revealed several novel aspects of tectal circuitry, including: (1) a glutamatergic mode of transmission from the superficial, retinorecipient neuropil layers to the deeper, output layers, (2) the presence of interneurons with mixed dendrite/axon arbors likely involved in local processing, and (3) a heretofore unknown GABAergic tectofugal projection to midbrain and hindbrain. These observations establish a framework for studying the morphological and functional differentiation of neural circuits in the zebrafish visual system.

No MeSH data available.


Related in: MedlinePlus