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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.


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Non-stratified periventricular interneurons and PVPN cell types are GABAergic. (A) Maximum Projection of a single PVPN at 5 dpf. (B,C) Single array section containing cell body of neuron in (A) labeled by GFP fluorescence in (B), anti-GABA immunofluorescence in (C). (D) Merged image of (B,C) with DAPI nuclear labeling (blue). (E) Maximum Projection of a single nsPVIN at 5 dpf. (F,G) Single array section containing cell body of neuron in (E) labeled by GFP fluorescence in (F), anti-GABA immunofluorescence in (G). (H) Merged image of (F,G) with DAPI nuclear labeling (blue). Note preservation of GFP signal and colocalization of GABA immunoreactivity (arrowheads in D,H). Scale bar, 20 μm.
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Figure 9: Non-stratified periventricular interneurons and PVPN cell types are GABAergic. (A) Maximum Projection of a single PVPN at 5 dpf. (B,C) Single array section containing cell body of neuron in (A) labeled by GFP fluorescence in (B), anti-GABA immunofluorescence in (C). (D) Merged image of (B,C) with DAPI nuclear labeling (blue). (E) Maximum Projection of a single nsPVIN at 5 dpf. (F,G) Single array section containing cell body of neuron in (E) labeled by GFP fluorescence in (F), anti-GABA immunofluorescence in (G). (H) Merged image of (F,G) with DAPI nuclear labeling (blue). Note preservation of GFP signal and colocalization of GABA immunoreactivity (arrowheads in D,H). Scale bar, 20 μm.

Mentions: To determine the neurotransmitter phenotype of dg4ii neuron types we conducted retrospective array tomography on fixed larvae following live imaging. As shown in Figure 9, retrospective analysis confirmed that both nsPVINs (four of four cells) and PVPNs (seven of seven cells) were GABAergic, although the need to identify cell types morphologically prior to both sectioning and antibody staining prohibited the analysis of large numbers of neurons. In contrast, every bsPVIN cell analyzed by immunofluorescence was found to be negative for GABA immunoreactivity (six of six cells: data not shown), indicating that this cell type is excitatory, since GABA is the only inhibitory neurotransmitter expressed in the tectum at this developmental stage (Higashijima et al., 2004). During examination of neurotransmitter phenotypes the quality of vglut2b fluorescence in situ labeling was found to be highly variable (see Materials and Methods). Combined with the need to identify cell types morphologically prior to fixation this prohibited retrospective analysis of vglut2b expression in individual labeled bsPVINs. However, based on our findings that in the SPV region of dlx5/6:gfp larvae 85.4 + 5.3% of cells were GABAergic and 15.6 + 1.5% were vglut2b-positive these neurons are most likely glutamatergic.


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

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

Non-stratified periventricular interneurons and PVPN cell types are GABAergic. (A) Maximum Projection of a single PVPN at 5 dpf. (B,C) Single array section containing cell body of neuron in (A) labeled by GFP fluorescence in (B), anti-GABA immunofluorescence in (C). (D) Merged image of (B,C) with DAPI nuclear labeling (blue). (E) Maximum Projection of a single nsPVIN at 5 dpf. (F,G) Single array section containing cell body of neuron in (E) labeled by GFP fluorescence in (F), anti-GABA immunofluorescence in (G). (H) Merged image of (F,G) with DAPI nuclear labeling (blue). Note preservation of GFP signal and colocalization of GABA immunoreactivity (arrowheads in D,H). Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Non-stratified periventricular interneurons and PVPN cell types are GABAergic. (A) Maximum Projection of a single PVPN at 5 dpf. (B,C) Single array section containing cell body of neuron in (A) labeled by GFP fluorescence in (B), anti-GABA immunofluorescence in (C). (D) Merged image of (B,C) with DAPI nuclear labeling (blue). (E) Maximum Projection of a single nsPVIN at 5 dpf. (F,G) Single array section containing cell body of neuron in (E) labeled by GFP fluorescence in (F), anti-GABA immunofluorescence in (G). (H) Merged image of (F,G) with DAPI nuclear labeling (blue). Note preservation of GFP signal and colocalization of GABA immunoreactivity (arrowheads in D,H). Scale bar, 20 μm.
Mentions: To determine the neurotransmitter phenotype of dg4ii neuron types we conducted retrospective array tomography on fixed larvae following live imaging. As shown in Figure 9, retrospective analysis confirmed that both nsPVINs (four of four cells) and PVPNs (seven of seven cells) were GABAergic, although the need to identify cell types morphologically prior to both sectioning and antibody staining prohibited the analysis of large numbers of neurons. In contrast, every bsPVIN cell analyzed by immunofluorescence was found to be negative for GABA immunoreactivity (six of six cells: data not shown), indicating that this cell type is excitatory, since GABA is the only inhibitory neurotransmitter expressed in the tectum at this developmental stage (Higashijima et al., 2004). During examination of neurotransmitter phenotypes the quality of vglut2b fluorescence in situ labeling was found to be highly variable (see Materials and Methods). Combined with the need to identify cell types morphologically prior to fixation this prohibited retrospective analysis of vglut2b expression in individual labeled bsPVINs. However, based on our findings that in the SPV region of dlx5/6:gfp larvae 85.4 + 5.3% of cells were GABAergic and 15.6 + 1.5% were vglut2b-positive these neurons are most likely 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