Dendritic morphology and distribution pattern of caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs).

<p>(A–E) Morphology of intracellularly injected cPAG-projecting RGCs located at different parts of the retina. Arrowheads point to axons. The distances from these cells to the optic disk are: 0.33 mm in (A), 0.62 mm in (B), 1.87 mm in (C), 3.01 mm in (D) and 4.35 mm in (E). (F) Schematic representation of the distribution pattern of Cholera toxin B subunit (CTB) retrogradely labeled cPAG-projecting RGCs from 5 animals. Coordinates of needle tips in 5 animals: •: Lambda: −3.2 mm, midline: left 1.05 mm, depth: 2.73 mm; ☆: Lambda: −3.208 mm, midline: left 0.92 mm, depth: 2.69 mm; ▴: Lambda: −3.204 mm, midline: left 1.08 mm, depth: 2.72 mm; ◊: Lambda: −3.212 mm, midline: left 1.08 mm, depth: 2.64 mm; ○: Lambda: −3.192 mm, midline: left 1.05 mm, depth: 2.82 mm; Green circled cells of A–E are correspondent to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103306#pone-0103306-g005" target="_blank">Figure 5A–5E</a>, respectively. One peripheral area (magenta arrow) and two central areas (blue arrow and cell D) have similar local densities. S: superior; I: inferior; N: nasal; T: temporal. Scale bars: 20 µm in A (applies to B–E); 1 mm in F.</p

Details in morphology of caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs).

<p>(A) A photomontage of cPAG-projecting RGC. This cell lies at 4.35 mm from the optic disk. The arrowhead points to an axon. (B–D) High power photomicrographs comparing somata among cPAG-projecting RGC (B), alpha cell (C) and melanopsin-expressing RGC (D). (E–G) High power photomicrographs comparing axons among cPAG-projecting RGC (E), alpha cell (F) and melanopsin-expressing RGC (G). The white frames highlight parts of their axons, respetively. Scale bars: 20 µm in A; 10 µm in B (applies to C and D); 5 µm in E (applies to F and G).</p

Illustration of Cholera toxin B subunit (CTB) retrogradely labeled-caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs).

<p>(A–C) Illustration of the CTB-conjugated Alexa Fluor 594 (CTB-594) retrogradely labeled RGCs in the retina. There are 4 CTB-labeled RGCs (magenta) in this field (A). These cells were not visible under blue excitation florescence filter (B). Merged photomicrograph of (A) and (B) is shown in (C). (D–F) A cPAG-projecting RGC is negative for anti-melanopsin staining. Arrow points to a CTB-594 retrogradely labeled cPAG-projecting RGC (D) that, as shown in (E), lacks melanopsin immunoreactivity. In contrast, melanopsin-positive RGCs were also seen. As shown in (E), the soma and dendrites of a melanopsin-expressing RGC (mRGC) were visible, so was an dendrite of another mRGC whose soma was not in the field (green fluorescent staining). Merged photomicrograph shows a cPAG-projecting RGC and a non-cPAG-projecting but melanopsin-positive RGC in the same field (F). Scale bars: 40 µm in C (applies to A and B); 20 µm in F (applies to D and E).</p

Variation in the sizes of dendritic fields and somata with retinal eccentricity.

<p>(A) Plot of dendritic field diameter as a function of eccentricity. (B) Plot of soma diameter as a function of eccentricity. 7 animals were used in each analysis.</p

Dendritic stratification of caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs).

<p>(A–B) These two cells are distinctly bistratified in sublamina <b><i>a</i></b> and <b><i>b</i></b> of the inner plexiform layer (IPL). Cholinergic amacrine cells were labeled with color magenta. (C–D) High-power images of upper planes in (A) and (B), illustrating that these two cPAG-projecting RGCs could distinctly bistratified in IPL. White dotted lines indicated borders of sublamina <b><i>a</i></b> and <b><i>b</i></b> of IPL. (E) Schematic summary of ramification pattern of cPAG-projecting RGCs. INL: inner nuclei layer; IPL: inner plexiform layer; GCL: retinal ganglion cell layer; a: sublamina <b><i>a</i></b> of inner plexiform layer; b: sublamina <b><i>b</i></b> of inner plexiform layer; S1–S5: stratum 1–5. Scale bars: 20 µm in A (applies to B); 20 µm in upper panel of A (applies to upper panel of B); 20 µm in C (applies to D); 5 µm in E.</p

Dendritic fields that sometimes overlap with one another among caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs).

<p>(A) Intracellularly injected cPAG-projecting RGCs in a whole-mounted retina. S: superior; I: inferior; N: nasal; T: temporal. (B) The white box in (A) viewed under high magnification. In this field three neighboring cPAG-projecting RGCs that have overlapped dendritic fields were labeled with three different colors (magenta, yellow and green). (C) Three dimensional reconstruction of overlapped dendritic fields. IPL: inner plexiform layer; a: sublamina <i>a</i> of inner plexiform layer; b: sublamina <i>b</i> of inner plexiform layer. Scale bars: 1 mm in A; 20 µm in B; 10 µm in C.</p

Innervation of melanopsin-expressing retinal ganglion cells (mRGCs) in the rostral periaqueductal gray (rPAG).

<p>(A) Coronal section of an injection site. (B–D) Arrow points to a CTB retrogradely labeled rPAG-projecting RGC. This cell was also positive for melanopsin immunoreactivity (C), but not all mRGCs could be retrogradely labeled by CTB (arrowhead in C and D). bsc: the brachium of the superior colliculus; PiRe: pineal recess; pc: posterior commissure; rPAG: the rostral periaqueductal gray. Scale bars: 300 µm in A; 20 µm in D (applies to B and C).</p

Illustration of the caudal periaqueductal gray (cPAG) injection site.

<p>(A) The schematic drawing of Cholera toxin B subunit (CTB) injection. The gray dotted line outlines the border of PAG. Note that to avoid the dye diffusion into superior colliculus (SC), the Hamilton syringe was inclined posteriorly at 30° angle from vertical and advanced 5.2 mm to target the cPAG. (B–E) A coronal section of the injection site. Trace of injection route is shown in (C) and (E). It is evident that CTB did not diffuse into the superior colliculus (SC) and dorsal raphe nuclei (DRN) (C). Large number of 5-HT+ neurons were seen in DRN (green cells in B and D). SC: superior colliculus; IC: inferior colliculus; PAG: periaqueductal gray; DRN: dorsal raphe nuclei; Aq: aqueduct. Scale bars: 300 µm in C (applies to B); 300 µm in E (applies to D).</p

Number of caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs) required to tile up the retinal surface.

<p>The calculations are used to determine the number of cPAG-projecting RGCs that are required to cover the whole retinal surface. The size of the retinal surface area is referred from Fite’s study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103306#pone.0103306-Fite1" target="_blank">[11]</a>; tiling up the whole retinal surface is generally considered as a criterion for a type of RGC, and our results indicate approximately 1300 cPAG-projecting RGCs are required to cover the whole retinal surface in this species.</p

Intact ON labelling approach is also feasible for GB filling of RGCs, with increased stability compared to ON cut approach.

<p>4A, B, and C represent ON cut approach with GB labelling at 7 days, 2 weeks and 3 weeks; 4 D, E, and F represent intact ON approach with GB labelling at 7 days, 2 weeks and 3 weeks, respectively. Scale bar represents 50 μm.</p