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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

Array tomographic visualization of bsPVIN presynaptic specializations. (A) Individual bsPVIN imaged immediately prior to fixation and processing for array tomographic analysis. (B) Section array was stained with an anti-SV2 antibody (red) and DAPI (blue) to label nuclei. (C) 5× magnification of boxed region in (B) containing a segment of the SGC targeted axonal arbor of bsPVIN shown in A. Note SV2 puncta contained within segment of GFP-labeled varicosity within this single 200 nm section. (D) 3D rendering of the section array containing the GFP-labeled neuron in (A–C). Only SV2 puncta that colocalized with areas of GFP labeling were included in rendering. Note large varicosities on the deeper SGC arbor and thin inter-varicosity neurites not detectable by laser-scanning microscopy in (A). (E) 1.75× magnification of boxed region 1 in (D). (F) Forty-five degree rotation of region in (E). Note that a majority of axonal varicosities contain distinct SV2 puncta. (G) 1.75× magnification of boxed region 2 in (D). Note that the majority of SV2 puncta are not entirely within GFP-labeled dendrite. Scale bar, 20 μm in (A), 40 μm in (B), 8 μm in (C), 3.5 μm in (D), 2 μm in (E–G).
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Figure 6: Array tomographic visualization of bsPVIN presynaptic specializations. (A) Individual bsPVIN imaged immediately prior to fixation and processing for array tomographic analysis. (B) Section array was stained with an anti-SV2 antibody (red) and DAPI (blue) to label nuclei. (C) 5× magnification of boxed region in (B) containing a segment of the SGC targeted axonal arbor of bsPVIN shown in A. Note SV2 puncta contained within segment of GFP-labeled varicosity within this single 200 nm section. (D) 3D rendering of the section array containing the GFP-labeled neuron in (A–C). Only SV2 puncta that colocalized with areas of GFP labeling were included in rendering. Note large varicosities on the deeper SGC arbor and thin inter-varicosity neurites not detectable by laser-scanning microscopy in (A). (E) 1.75× magnification of boxed region 1 in (D). (F) Forty-five degree rotation of region in (E). Note that a majority of axonal varicosities contain distinct SV2 puncta. (G) 1.75× magnification of boxed region 2 in (D). Note that the majority of SV2 puncta are not entirely within GFP-labeled dendrite. Scale bar, 20 μm in (A), 40 μm in (B), 8 μm in (C), 3.5 μm in (D), 2 μm in (E–G).

Mentions: The specific laminar targeting exhibited by the bsPVIN class of neurons suggests that they may integrate visual inputs from the SFGS neuropil layer and relay this information to the deeper SGC, which is innervated by a subset of retinal afferents and non-visual afferents from telencephalic and thalamic nuclei (Meek, 1983). To determine if the axonal and dendritic compartments are spatially segregated in bsPVINs we coinjected embryos with dg4ii and uas:dsred/uas:syp-gfp constructs to visualize the spatial distribution of presynaptic specializations. Although only a fraction of neurons labeled with this construct had adequate expression levels of both fluorescent proteins we were able to image five bsPVIN cells in which we could accurately trace neurite morphology using DsRed and monitor presynaptic specializations using Syp–GFP. In the cell body one or a few bright Syp–GFP puncta were typically observed (Figure 5A), which is consistent with previous reports of Syp fusion proteins being sorted through the Golgi complex (Pennuto et al., 2003). In the bsPVIN neurites only the arborization in the deeper SGC layer was observed to contain multiple bright Syp–GFP puncta within varicosities, which have been shown to contain presynaptic active zones (Fletcher et al., 1991). In younger larvae (3–4 dpf) the deeper SGC arbor often contained branches lacking Syp–GFP puncta (see Figure 5A), whereas these were not observed in older larvae (5–8 dpf; see Figure 6). Although this suggests that the deeper arbor is purely axonal we cannot formally exclude the possibility that it may contain both pre- and postsynaptic specializations. In contrast, we never observed bright Syp–GFP puncta in the superficial SFGS arbor. Studies in other cell types have found that Syp–GFP fusion proteins target axonal synapses and are excluded from dendritic compartments (Pennuto et al., 2003), indicating that the SFGS arborization represents the dendritic compartment of the bsPVIN cell type. In summary, the dg4ii transgene labels an interneuron type with a dendrite that targets the superficial SFGS layer of the neuropil and an axonal arbor that targets the deeper SGC.


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

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

Array tomographic visualization of bsPVIN presynaptic specializations. (A) Individual bsPVIN imaged immediately prior to fixation and processing for array tomographic analysis. (B) Section array was stained with an anti-SV2 antibody (red) and DAPI (blue) to label nuclei. (C) 5× magnification of boxed region in (B) containing a segment of the SGC targeted axonal arbor of bsPVIN shown in A. Note SV2 puncta contained within segment of GFP-labeled varicosity within this single 200 nm section. (D) 3D rendering of the section array containing the GFP-labeled neuron in (A–C). Only SV2 puncta that colocalized with areas of GFP labeling were included in rendering. Note large varicosities on the deeper SGC arbor and thin inter-varicosity neurites not detectable by laser-scanning microscopy in (A). (E) 1.75× magnification of boxed region 1 in (D). (F) Forty-five degree rotation of region in (E). Note that a majority of axonal varicosities contain distinct SV2 puncta. (G) 1.75× magnification of boxed region 2 in (D). Note that the majority of SV2 puncta are not entirely within GFP-labeled dendrite. Scale bar, 20 μm in (A), 40 μm in (B), 8 μm in (C), 3.5 μm in (D), 2 μm in (E–G).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Array tomographic visualization of bsPVIN presynaptic specializations. (A) Individual bsPVIN imaged immediately prior to fixation and processing for array tomographic analysis. (B) Section array was stained with an anti-SV2 antibody (red) and DAPI (blue) to label nuclei. (C) 5× magnification of boxed region in (B) containing a segment of the SGC targeted axonal arbor of bsPVIN shown in A. Note SV2 puncta contained within segment of GFP-labeled varicosity within this single 200 nm section. (D) 3D rendering of the section array containing the GFP-labeled neuron in (A–C). Only SV2 puncta that colocalized with areas of GFP labeling were included in rendering. Note large varicosities on the deeper SGC arbor and thin inter-varicosity neurites not detectable by laser-scanning microscopy in (A). (E) 1.75× magnification of boxed region 1 in (D). (F) Forty-five degree rotation of region in (E). Note that a majority of axonal varicosities contain distinct SV2 puncta. (G) 1.75× magnification of boxed region 2 in (D). Note that the majority of SV2 puncta are not entirely within GFP-labeled dendrite. Scale bar, 20 μm in (A), 40 μm in (B), 8 μm in (C), 3.5 μm in (D), 2 μm in (E–G).
Mentions: The specific laminar targeting exhibited by the bsPVIN class of neurons suggests that they may integrate visual inputs from the SFGS neuropil layer and relay this information to the deeper SGC, which is innervated by a subset of retinal afferents and non-visual afferents from telencephalic and thalamic nuclei (Meek, 1983). To determine if the axonal and dendritic compartments are spatially segregated in bsPVINs we coinjected embryos with dg4ii and uas:dsred/uas:syp-gfp constructs to visualize the spatial distribution of presynaptic specializations. Although only a fraction of neurons labeled with this construct had adequate expression levels of both fluorescent proteins we were able to image five bsPVIN cells in which we could accurately trace neurite morphology using DsRed and monitor presynaptic specializations using Syp–GFP. In the cell body one or a few bright Syp–GFP puncta were typically observed (Figure 5A), which is consistent with previous reports of Syp fusion proteins being sorted through the Golgi complex (Pennuto et al., 2003). In the bsPVIN neurites only the arborization in the deeper SGC layer was observed to contain multiple bright Syp–GFP puncta within varicosities, which have been shown to contain presynaptic active zones (Fletcher et al., 1991). In younger larvae (3–4 dpf) the deeper SGC arbor often contained branches lacking Syp–GFP puncta (see Figure 5A), whereas these were not observed in older larvae (5–8 dpf; see Figure 6). Although this suggests that the deeper arbor is purely axonal we cannot formally exclude the possibility that it may contain both pre- and postsynaptic specializations. In contrast, we never observed bright Syp–GFP puncta in the superficial SFGS arbor. Studies in other cell types have found that Syp–GFP fusion proteins target axonal synapses and are excluded from dendritic compartments (Pennuto et al., 2003), indicating that the SFGS arborization represents the dendritic compartment of the bsPVIN cell type. In summary, the dg4ii transgene labels an interneuron type with a dendrite that targets the superficial SFGS layer of the neuropil and an axonal arbor that targets the deeper SGC.

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