Superior colliculus connections with visual thalamus in gray squirrels (Sciurus carolinensis): evidence for four subdivisions within the pulvinar complex

Abstract

As diurnal rodents with a well-developed visual system, squirrels provide a useful comparison of visual system organization with other highly visual mammals such as tree shrews and primates. Here, we describe the projection pattern of gray squirrel superior colliculus (SC) with the large and well-differentiated pulvinar complex. Our anatomical results support the conclusion that the pulvinar complex of squirrels consists of four distinct nuclei. The caudal (C) nucleus, distinct in cytochrome oxidase (CO), acetylcholinesterase (AChE), and vesicular glutamate transporter-2 (VGluT2) preparations, received widespread projections from the ipsilateral SC, although a crude retinotopic organization was suggested. The caudal nucleus also received weaker projections from the contralateral SC. The caudal nucleus also projects back to the ipsilateral SC. Lateral (RLl) and medial (RLm) parts of the previously defined rostral lateral pulvinar (RL) were architectonically distinct, and each nucleus received its own retinotopic pattern of focused ipsilateral SC projections. The SC did not project to the rostral medial (RM) nucleus of the pulvinar. SC injections also revealed ipsilateral connections with the dorsal and ventral lateral geniculate nuclei, nuclei of the pretectum, and nucleus of the brachium of the inferior colliculus and bilateral connections with the parabigeminal nuclei. Comparisons with other rodents suggest that a variously named caudal nucleus, which relays visual inputs from the SC to temporal visual cortex, is common to all rodents and possibly most mammals. RM and RL divisions of the pulvinar complex also appear to have homologues in other rodents.

Figure 1

Western blot characterization of VGluT2 antibody. The VGluT2 antibody recognizes a 56-kDa protein in gray squirrel cerebellar lysate, which is the expected molecular weight of the VGluT2 protein.

Figure 2

Photomicrographs of coronal sections through the superior colliculus of the gray squirrel after staining with Nissl (A), cytochrome oxidize (CO; B), vesicular glutamate transporter-2 (VGluT2; C), and acetylcholinesterase (AChE; D). Seven layers can be distinguished from one another by using each of these stains. SZ, stratum zonale; SGS, stratum griseum superficiale; SO, stratum opticum; SGI, stratum griseum intermedium; SAI, stratum album intermedium; SGP, stratum griseum profundum; SAP, stratum album profundum. Note the presence of three possible layers within the SGI. Images were taken from two squirrels: Nissl, CO, VGluT2 are from the same squirrel; the AChE photomicrograph is from a second squirrel. Scale bar = 1 mm.

Figure 3

Architectonic characteristics of subdivisions within the gray squirrel pulvinar complex. Coronal sections through various stages of the pulvinar complex were stained for Nissl substance (A–D), acetylcholinesterase (AChE; E–H), cytochrome oxidase (CO; I–L), and vesicular glutamate transporter-2 (VGluT2; M–P). Sections on the left are more caudal and progress to more rostral sections on the right. The borders of proposed subdivisions within the pulvinar complex are shown with dashed lines. Images were taken from two squirrels: Nissl, AChE, and VGluT2 are taken from the same squirrel, whereas the CO images are taken from a second squirrel. Scale bar = 1 mm.

Figure 4

Architectonic characteristics of subdivisions within the gray squirrel pulvinar complex. Horizontal sections through various stages of the pulvinar complex were stained for Nissl substance (A–C) and acetylcholinesterase (AChE; D–F). Sections on the left are more dorsal and progress to more ventral sections on the right. The borders of proposed subdivisions within the pulvinar complex are shown with dashed lines. Scale bar = 1 mm.

Figure 5

Photomicrographs of a coronal section of the pulvinar complex in gray squirrel stained for AChE. B is a higher magnification image of the boxed area in A. Note the difference in the direction of fibers between rostral lateral medial (RLm) and rostral lateral lateral (RLl). C is an image of a Nissl-stained section; D is an image of a cytochrome oxidase-stained section. Scale bars = 1 mm in A; 250 μm in B; 0.5 mm in C,D.

Figure 6

Photomicrographs of coronal sections of the ventral lateral geniculate nucleus (LGNv) in gray squirrels stained for Nissl substance (A), cytochrome oxidase (CO; B), acetylcholinesterase (AChE; C), and vesicular glutamate transporter-2 (VGluT2; D). The dashed line indicates the border between the medial and lateral subdivisions of the LGNv as well as the border between the intergeniculate leaflet (IGL) and the LGNv. Images were taken from two squirrels. Scale bar = 1 mm.

Figure 7

Photomicrographs of coronal sections of the parabigeminal (PB) and the nucleus of the brachium of the inferior colliculus (NBIC) in gray squirrels stained for Nissl substance (A), acetylcholinesterase (AChE; B), vesicular glutamate transporter-2 (VGluT2; C), and cytochrome oxidase (CO; D). A–C are taking from one squirrel; D was taken from a second squirrel. Scale bar = 1 mm.

Figure 8

Superior colliculus (SC) connections with visual thalamus in squirrel 04-15. A: The extent and estimated retinotopic position of the fluoro-ruby (FR) injection site on a reconstructed dorsal view of the SC. B: Coronal thalamus sections are arranged in caudal to rostral progression, with the most caudal section in the upper left and the most rostral section located in the lower right. Locations of labeled axon terminals are shown with dots, whereas the locations of retrogradely labeled cell bodies are represented with triangles. C: High-power photomicrograph of terminal label within the pulvinar complex. The magnified photograph corresponds to the box located in section 136 of B. D: High-power photomicrograph of terminal label within the pulvinar complex corresponding to the box located in section 128 of B. Scale bars = 1 mm in B; 0.5 mm in C,D.

Figure 9

Superior colliculus (SC) connections with visual thalamus in squirrel 09-02. A: Photomicrograph of fluoro-ruby (FR) injection site on the left and an image of the adjacent cytochrome oxidase (CO) section on the right. B: Dorsal view of the extent and estimated retinotopic location of the FR injection site. C: Coronal sections of thalamus with the locations of axon terminals (small black dots) and retrogradely labeled cell bodies (black triangles). Sections are arranged in a caudal (top left) to rostral (bottom right) direction. D,E,G are high-power images of axon terminal label within the pulvinar and ventral lateral geniculate nucleus. F shows labeled cell bodies and axon terminals within the caudal pulvinar from section 336 in B. D corresponds to the boxed area in section 331; E corresponds to the large boxed area in section 376; G corresponds to the smaller boxed area in section 376. Scale bars = 1 mm in A,C; 250 lm in D; 0.5 mm in E; 100 lm in F; 0.25 mm in G.

Figure 10

Superior colliculus (SC) connections with visual thalamus in squirrel 09-23. A: The extent and estimated retinotopic location of the fluoro-ruby (FR) injection site are indicated on a reconstructed dorsal view of the SC. B: Coronal sections of thalamus with the reconstructed locations of axon terminals (dots) and labeled cells (triangles) within each section. C: High-power photomicrograph of the FR injection site. D: Photomicrograph of the adjacent cytochrome oxidase (CO) section to C. E: High-power image of the axon terminals within the box in section 334 shown in B. F: High-power image of the axon terminals within the box in section 299 in B. Scale bars 1 mm in B–D; 0.5 mm in E; 250 μm for F.

Figure 11

Superior colliculus (SC) connections with visual thalamus in squirrel 09-44. A: Reconstructed dorsal view of the SC with the extent and location of the fluoro-ruby (FR; dark gray) and choleratoxin subunit B (CTB; light gray) injection sites. B: Coronal sections of thalamus arranged in a caudal, top left, to rostral, bottom right, manner, with the location of labeled axon terminals for CTB (light gray dots) and FR (dark gray dots) as well as retrogradely labeled CTB (squares) and FR (triangles) in each section. C: Magnified photomicrographs of the CTB injection site in a coronal section. D: Magnified photomicrograph of the FR injection site in a coronal section. E: High-power image of CTB anterograde label within the large box in section 86. F: High-power image of FR anterograde label within the small box in section 86. Scale bars = 1 mm in B–D; 0.5 mm in E; 250 μm F.

Figure 12

Superior colliculus (SC) connections with visual thalamus in squirrel 09-50. A: Dorsal view of the SC with the location and extent of choleratoxin subunit B (CTB; light gray) and fluoro-ruby (FR; dark gray) injection sites. B: Coronal sections of thalamus arranged in a caudal (top left) to rostral (bottom right) progression. The location of CTB-labeled axon terminals (dots) and CTB-labeled cells (squares), as well as FR labeled axon terminals (dots) and FR labeled cells (triangles) are presented for each section. C: Magnified photomicrograph of FR label in section 233 of B. D: High-power images of the FR injection site in coronal section. E: Photomicrograph of CTB injection site in coronal sections. Scale bars = 1 mm in B; 250 μm in C; 0.5 mm in D,E.

Figure 13

Superior colliculus (SC) connections with the parabigeminal nucleus in squirrel 09-50. A: Coronal sections of the midbrain arranged in a caudal (top) to rostral (bottom) progession. B: Dorsal view of the SC with the location and extent of CTB (light gray) and FR (dark gray) injections sites. C: Magnified photomicrograph of CTB label in the contralateral parabigeminal nucleus in section 98. D: Magnified photomicrograph of CTB label in the ipsilateral parabigeminal nucleus in section 98. E: Magnified photomicrograph of FR label in the contralateral parabigeminal nucleus in section 103. F: Magnified photomicrograph of FR label in the ipsilateral parabigeminal nucleus in section 113. Scale bars = 1 mm in A; 0.25 mm in C,D,F; 0.1 mm in E.

Figure 14

Current proposal of gray squirrel pulvinar organization. A: Summary of connections between the superior colliculus and pulvinar complex as well as connections between the pulvinar complex and visual cortex in gray squirrels. A, 1970 is Abplanalp (1970); K et al., 1972b is Kaas et al. (1972b); R&H, 1977 is Robson and Hall (1977); W et al., 2008 is Wong et al. (2008). B: Topographic organization of RLl and RLm based on upper and lower field representations. C: Topographic organization within RLl based on central and peripheral visual field representations. D: Lateral view of the right hemisphere of a gray squirrel with occipital, and temporal visual areas highlighted in white (adapted from Wong and Kaas, 2008).

Figure 15

Possible pulvinar/lateral posterior complex organization schemes for gray squirrel (A), degu (B), hamster (C), and rat (D). Subdivisions of the pulvinar that receive SC projections are highlighted in gray. Asterisk symbols represent bilateral SC input. Information for A is based on descriptions from Robson and Hall (1977), Wong et al. (2008), and the current study. B is based on descriptions from Kuljis and Fernandez (1982). C is based on descriptions from Crain and Hall (1980) and Ling et al. (1997). The gray lined areas represent discrepancies between Crain and Hall (1980) and Ling et al. (1997) with respect to SC projections. D is based on descriptions of Takahashi (1985).