Retinal distribution of Disabled-1 in a diurnal murine rodent, the Nile grass rat Arvicanthis niloticus

Experimental Eye Research 125 (2014) 236e243

Contents lists available at ScienceDirect

Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer

Retinal distribution of Disabled-1 in a diurnal murine rodent, the Nile grass rat Arvicanthis niloticus*
de
ric Gaillard a, Sharee Kuny a, Yves Sauve
a, b, * Fre a b

Department of Ophthalmology and Visual Science, University of Alberta, Edmonton, AB, Canada Department of Physiology, University of Alberta, Edmonton, AB, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 March 2014 Accepted in revised form 19 June 2014 Available online 30 June 2014

We sought to study the expression pattern of Disabled-1 (Dab1; an adaptor protein in the reelin pathway) in the cone-rich retina of a diurnal murine rodent. Expression was examined by western blotting and immunohistochemistry using well-established antibodies against Dab1 and various markers of retinal neurons. Western blots revealed the presence of Dab1 (80 kDa) in brain and retina of the Nile grass rat. Retinal immunoreactivity was predominant in soma and dendrites of horizontal cells as well as in amacrine cell bodies aligned at the INL/IPL border. Dab1þ neurons in the inner retina do not stain for parvalbumin, calbindin, protein kinase C-alpha, choline acetyltransferase, glutamic acid decarboxylase, or tyrosine hydroxylase. They express, however, the glycine transporter GlyT1. They have small ovoid cell bodies (7.1 ± 1.06 mm in diameter) and bistratified terminal plexii in laminas a and b of the IPL. Dab1þ amacrine cells are evenly distributed across the retina (2600 cells/mm2) in a fairly regular mosaic (regularity indexes z3.3e5.5). We conclude that retinal Dab1 in the adult Nile grass rat exhibits a dual cell patterning similar to that found in human. It is expressed in horizontal cells as well as in a subpopulation of glycinergic amacrine cells undetectable with antibodies against calcium-binding proteins. These amacrine cells are likely of the AII type. © 2014 Elsevier Ltd. All rights reserved.

Keywords: amacrine AII Disabled-1 Nile grass rat Arvicanthis niloticus retina horizontal cells mosaic rod pathway

1. Introduction Cell positioning during development of some layered structures of the brain (cerebral cortex, cerebellum and hippocampus) occurs partly through a signaling pathway which involves the protein reelin binding to its receptors (Apolipoprotein E Receptor 2 and Very Low Density Lipoprotein Receptor) which are expressed at the surface of several neuronal populations, and the subsequent tyrosine phosphorylation of Disabled-1, an 80 kDa cytoplasmic adaptor protein (Bar and Goffinet, 1999; Cooper and Howell, 1999; Arnaud et al., 2003). Mice lacking reelin or disabled-1 (dab1) genes display the reeler phenotype characterized by an inversion of cortical layers, various laminar defects in the cerebellum and hippocampus, severe cognitive delay, ataxia, hypotonia and seizures (Goffinet, 1984; Rice et al., 1998).

* Grants: Supported by operating funding from Canadian Institutes for Health Research (CIHR 125876). YS is an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar. * Corresponding author. Department of Physiology, 7-55 Medical Sciences Bldg, University of Alberta, Edmonton, AB, Canada, T6G 2H7. Tel.: þ1 780 492 8609.
). E-mail address: [email protected] (Y. Sauve

http://dx.doi.org/10.1016/j.exer.2014.06.019 0014-4835/© 2014 Elsevier Ltd. All rights reserved.

In addition to the brain, dab1 is expressed in the small intestine, pancreas, lens, neural retina and retinal pigment epithelium of common rodents (Schiffmann et al., 1997; BioGPS, Novartis Research Foundation, http://biogps.gnf.org). Interestingly, in species (Rodentia and other mammalian orders) that have an overall low cone density, retinal Dab1 protein is almost exclusively expressed by AII amacrine cells (Rice and Curran, 2000; Rice et al., 2001; Lee et al., 2003, 2004; Park et al., 2004; Cherry et al., 2009; Geisert et al., 2009), a key neuronal component of the rod-driven circuit (Kolb and Famiglietti, 1974). Targeting retinal Dab1 might therefore be a convenient approach to visualize AII amacrine cells when antibodies against calcium-binding proteins are ineffective (Rice and Curran, 2000). The Nile grass rat (Arvicanthis niloticus; Muridae, murinae) is a diurnal rodent closely related to Mus and Rattus, which inhabits savanna grasslands in East Africa, along the Nile River. Former studies (Gaillard et al., 2008, 2009; Gilmour et al., 2008) have shown that this animal has a larger proportion of retinal cones than mice and rats (z35e40% of all photoreceptors vs. 1e3%), better visual acuity than the latter (1.3 vs. 0.54 cycle/degree) and flicker fusion frequency similar to humans (60 Hz). Similar proportion of cones was found in Arvicanthis ansorgei (33%),

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

237

2.3. Western blotting

another species of the Arvicanthis genus (Bobu et al., 2006). These cone photoreceptors are essentially evenly distributed across the retina, segregated from rods in the two distal rows of the outer nuclear layer, and express either M- or S-opsin (UV sensitive) photopigment. Together with easy maintenance in captivity (a colony, founded by 29 Nile grass rats trapped in Kenya, has existed for almost 20 years at Michigan State University; http://rhythms. psy.msu.edu/smalelab), these characteristics make the Nile grass rat an attractive natural model to examine specific questions related to retinal cone physiology. The retinal design of this animal, however, is largely unknown. To date, only few cell types residing in the inner retina have been described, and the presence of AII amacrine cells has not been proven (Gaillard et al., 2008). We asked, therefore, whether Dab1 was expressed in the retina of the Nile grass rat, and whether this marker was as effective in identifying AII amacrine cells as in common laboratory rodents. Our results show that Dab1 is expressed in horizontal cells as well as in a subpopulation of glycinergic amacrine cells having bistratified terminal plexii in laminas a and b of the inner plexiform layer. These amacrine cells are likely of the AII type.

Tissues were homogenized in SDS buffer with protease inhibitor cocktail (cØmplete®, Roche Applied Science, Laval, QC, Canada). Protein levels were determined using a protein assay kit (Pierce® BCA, Thermo Fisher Scientific, Ottawa, ON, Canada). After addition of 2% (v/v) 2-mercaptoethanol and 1% (v/v) saturated bromophenol blue, protein extracts were boiled for 5 min and resolved on a 10% SDS-PAGE gel. Proteins were transferred to polyvinylidene fluoride membranes, blocked for 1 h with 5% non-fat milk diluted in TBS0.1% Tween-20, incubated overnight with anti-Dab1-B3 antibody diluted 1:1000 in the blocking solution. Alpha-tubulin (TU-02, Santa Cruz Biotech, Santa Cruz, CA; 1:500) was used as a loading control. Immunoblots were washed 3  10 min in TBS-T and reacted for 1 h with anti-rabbit or anti-mouse IgG, HRP-conjugated antibodies (NA934 and NA931, respectively; 1:5000 in the blocking solution; GE Healthcare, Little Chalfont, UK). After final extensive washes and addition of chemiluminescent reagent (NEL 103; Perkin Elmer, Wellesley, MA), protein bands were visualized on a Kodak Image Station 440 (Eastman Kodak, Rochester, NY). Net intensity of bands was calculated using imaging software (Kodak, v.4.0.3).

2. Methods

2.4. Immunohistology

2.1. Animals

Cryosections (20 mm thick) parallel to the naso-temporal retinal axis were collected from 3 eyes (each from separate animals: NR74, NR125, NR156) fixed in 4% paraformaldehyde, and mounted on glass slides. After washing, the sections were blocked for 2 h in a medium containing 0.05% Tween 20, 0.1% Triton X-100 and 5% non-fat milk (in PBS) and reacted overnight with primary antibodies diluted appropriately in a 1:10 solution of the previous blocking medium. On the following day, sections were washed, blocked again for 1 h and processed either for immunohistofluorescence or immunohistochemistry. In the first case, sections were exposed for 2 h to goat antirabbit Alexa488, donkey anti-rabbit-Alexa488 (Molecular Probes Inc., Eugene, OR) or donkey anti-goat-Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies diluted to 1:500. In the second case, sections were treated with biotinylated goat antirabbit IgG (1:200; Dako, Glostrup, Denmark), exposed for 2 h to avidin-biotin-HRP complex (Vectastain ABC Elite kit, Vector Labs, Burlingame, CA) and reacted for 5e10 min with a solution containing 0.05% diaminobenzidine (DAB), 1.5% nickel ammonium sulfate and 0.03% H2O2 in acetate buffer 0.1M (pH 6). After a final washing in PBS, sections were covered with an antifade solution (Fluoromount, Sigma), and coverslipped. Reactions were run at room temperature. Three additional retinas (also from separate subjects: NR76, NR79, NR139) were prepared as whole mounts. Following euthanasia with Euthanyl (Bimeda-MTC Animal Health Inc., Cambridge,

Experiments were performed on young Nile grass rats (1.5e3 months, unless otherwise stated) and adult C57BL/6 wild-type mice (as controls). Both species were maintained in the Animal Care Facility at the University of Alberta under a 12:12 lightedark cycle, and supplied with water and standard rodent chow. All procedures were approved by the Institutional Animal Care and Use Committee, and conformed to the EU Directive 2010/63/EU (http:// ec.europa.eu/environment/chemicals/lab_animals/legislation_en. htm). 2.2. Primary antibodies Primary antibodies, hosts and sources are listed in Table 1. All of these antibodies are commonly used to identify retinal cell types in mammals including Nile grass rats (Gaillard et al., 2008; Haverkamp and W€ assle, 200; Cuenca et al., 2002). The affinity-purified Dab1-B3 polyclonal antibody (a gift from B. Howell) raised against residues 107-243 at the N-terminus of the Dab1 protein has been fully characterized (Howell et al., 1997) and used efficiently in mouse (Rice and Curran, 2000; Rice et al., 2001; Claes et al., 2004). Immunostaining with this antibody is absent in dab1 knock-out mice (Rice and Curran, 2000). Table 1 List of the antibodies used in present study. Antigen

Abbreviation

Host

Source

Catalog/clone#

Dilution

Disabled 1, affinity purified* Protein kinase Ca, monoclonal Calbindin D-28K, monoclonal Calcium binding protein 5, affinity purified Calretinin, affinity purified Parvalbumin, polyclonal Glycine transporter1, polyclonal Tyrosine hydroxylase, monoclonal g-aminobutyric acid decarboxylase, monoclonal Heavy (200 KDa) neurofilament, monoclonal Choline acetyltransferase, affinity purified Glutamine synthetase, monoclonal

Dab1 PKCa CaB CaBP5 CaR PV GlyT1 TH GAD67 RT97 ChAT GS

Rabbit Mouse Mouse Rabbit Goat Rabbit Goat Mouse Mouse Mouse Goat Mouse

B. Howell, Upstate, NY Santa Cruz Biotech., Santa Cruz CA Sigma, St Louis, MO F. Haeseleer R&D Systems, Minneapolis, MN Swant, Bellinzona, Switzerland Chemicon, Temecula, CA Immunostar, Hudson, WI Chemicon, Temecula, CA DSHB, Univ. Iowa, IO Chemicon, Temecula, CA BD Biosciences, San Jose, CA

Dab1-B3 SC8393; clone H7 C9848; clone CB-955 UW89 AF5065 PV28 AB 1770 22941; clone LNC1 MAB5406 RT97 AB144P 610517; clone 6

1: 500 1:250 1:500 1:1000 1:500 1:1000 1:1000 1:500 1:1000 1:50 1:250 1:1000

* Note: Rabbit polyclonal (AB5840; Chemicon, Temecula, CA) and mouse monoclonal (H3 and D4, from A. Goffinet, Univ. Louvain, Belgium) were also assayed, but were ineffective.

238

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

ON, Canada), ocular globes were marked dorsally for orientation and removed. The retinas were then carefully dissected from the eyecup, marked dorsally for orientation, cut into four distinct quadrants, fixed overnight at 4  C in 4% paraformaldehyde and processed with the anti-Dab1-B3 antibody as described above, with the exception that incubations with the primary and secondary antibodies were performed at 4  C and lasted up to 3 days. 2.5. Imaging and quantification Images were captured either on a Leica DM6000B fluorescence microscope equipped with a computer controlled motorized stage, or an Olympus FluoView 1000 confocal microscope using x40 or x63 oil immersion objectives. Routine quantification was performed with ImageJ software (v1.44c; National Institute of Health, NIH, Bethesda, MD; available at http://rsb.info.nih.gov/ij/) and appropriate plugins (available on the ImageJ website). Topographic analysis [packing density, nearest-neighbor distance and Voronoi domain; (Gaillard et al., 2009)] was examined using the “Delaunay/Voronoi” plugin. Only Dab1-positive cells that did not touch the borders of frames (area ¼ 0.0376 mm2) taken in the center (from 0 to 1250 mm from the optic nerve head, ONH) and the far periphery (>2500 mm from the ONH) of each quadrant of retina whole mounts were considered for analysis. Dual expression of proteins by single elements in frames was assessed using the “Colocalization” plugin (implemented by Pierre Bourdoncle, Institut Jacques Monod, Paris). Labeling density in structures of interest was estimated from diaminobenzidine-processed sections with a dedicated subroutine of Image-Pro software (v4.0; Media Cybernetics, SilverSpring, MD; see Gaillard et al., 2013 for procedure). Pictures for illustrations were imported into Photoshop software (v6.0, Adobe, San Jose, CA) to adjust brightness and contrast, and to create plates. Statistics were performed using Statview® software (SAS Institute Inc., Cary, NC). Values in text are given as mean ± SD. 3. Results 3.1. Expression of Dab1 protein in mouse and Nile grass rat Immunoblots (Fig. 1A) of hippocampal, cerebellar and cortical extracts from adult wild-type mouse show a major product at z80 kDa as well as minor bands at z72, z55 and z36 kDa. All these products are present in the mouse retina, but at a much lower level. Differences in p80 expression between the four neural tissues examined agree with the known levels of Dab1 expression in these regions (BioGPS; Novartis Research Foundation; http://biogps.gnf. org). Immunoblots with CNS tissues from adult Nile grass rats reveal comparable banding patterns. In addition to p80, p72, p55 and p36 products, the Nile grass rat retina clearly displays a heavy band at z130 kDa. Liver extract shows 72, 55 and 36 kDa products, but no band at z80 kDa suggesting that, as with the mouse (Smalheiser et al., 2000), the Dab1 protein is not expressed in the liver of adult Nile grass rats. Replicas (n ¼ 2) of this experiment, performed at 6 months interval with separate subjects of various ages (1, 3 and 5 months) and different batches of Dab1-B3 antibody, provided identical results. No change in the expression of p80 in mouse and Nile grass rat retinas was observed with aging. 3.2. Immunolocalization of Dab1 in the mouse retina In agreement with a previous study (Rice and Curran, 2000), the Dab1-B3 antibody labels only AII amacrine cells in the mouse retina. Reactive neurons display a slightly oval soma (longest dimension: 7.7 ± 1.0 mm; n ¼ 50), thick lobular appendages (size: 1.65 ± 0.38 mm; n ¼ 47) in lamina a of the inner plexiform layer

Fig. 1. A. Western blotting for Dab1 expression in various tissues extracted from a 10month-old wild-type (WT) mouse and a 5-month-old Nile grass rat (50 mg protein per lane; 20 s film exposure). The Dab1-B3 antibody recognizes the same products in mouse and Nile grass rat brain tissues. The major band at 80 kDa (arrows) corresponds to the full-length Dab1 protein. Bands at 72, 54 and 36 kDa are likely non-specific given their presence in the liver, which does not express Dab1 protein. A p130 product (asterisk) of unknown origin is present in the Nile rat retina. B. Examples of AII amacrine cells labeled with Dab1-B3 antibody in two different WT mice aged 1 month. Labeling is present in cell bodies at the vitreal border of the INL and in large appendages (arrowheads) in the OFF lamina of the IPL. Labeling of processes (see arrow for example) and terminals in the ON lamina (vertical bars) is weak. C. Regular arrangement of Dab1þ AII amacrine cells at the INL/IPL border. Abbreviations: INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer.

(IPL), and a descending process with terminal profiles in subjacent lamina b of the IPL (Fig. 1B). Labeling localizes mainly to the cell body cytoplasm and soma-process junction; it is weak in lamina b. These cells are regularly arranged (inter-cell distance: 13.3 ± 3.7 mm; n ¼ 50; Fig. 1C) at the vitreal border of the inner nuclear layer (INL). 3.3. Distribution of Dab1 in the Nile grass rat retina In addition to diffuse labeling located at photoreceptor inner segments, Dab1 immunoreactivity in the retina of Nile grass rats

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

was found in horizontal cells, in amacrine cells aligned at the vitreal border of the INL, in scant neurons residing in the ganglion cell layer (GCL), and finally, in both the outer and inner plexiform layers where dendrites of horizontal and amacrine cells, respectively, spread (Fig. 2A). This labeling pattern, currently observed in the three retinas studied regardless of the immunohistochemical procedure, was confirmed by densitometry measures (n ¼ 15; Fig. 2B). Labeling density at photoreceptor inner segments was z3.0-fold the background value, whereas levels in the outer plexiform layer, at the somato-dendritic junction in inner retina neurons as well as in terminals (located within sublayers s4-s5 of the IPL; Fig. 2A,C) were much higher (8e11-fold the background value). According to double labeling studies (data not illustrated), Dab1 was never expressed in rod bipolar cells (reacted for PKCa), in OFF cone bipolar cells (reacted for CaBP5), in glial Müller cells (reacted for glutamine synthetase, GS) and in the nerve fiber layer (reacted either for CaR or the heavy 200-kDa isoform of the neurofilament triplet). Dab1 is expressed by horizontal cells in the human retina (Lee et al., 2004). Single and double labeling experiments demonstrate a similar occurrence in the Nile grass rat (Fig. 2DeE, 3A). Firstly, Dab1 colocalized in the dendritic network and, to a lesser degree, in the cell body cytoplasm with calbindin (CaB; Fig. 2D), a robust marker for horizontal cells in Nile grass rats (Gaillard et al., 2008). Secondly, Dab1 was present in some large horizontal cell processes stained for the heavy 200-kDa isoform of the neurofilament triplet (Fig. 2E). Finally, Dab1 colocalized with GAD67 (data not shown) that is expressed within horizontal cell bodies at an extremely low level (Gaillard et al., 2008). Dab1þ cells at the vitreal border of the INL have slightly pyriform, vertically oriented cell bodies (major axis ¼ 7.1 ± 1.06 mm; minor axis ¼ 5.45 ± 0.7 mm; n ¼ 58) and are distributed quite regularly (inter-cell distance: 17.6 ± 5.7 mm; n ¼ 57) along a single row located at the INL/IPL boundary (Fig. 3A). In addition to the soma, Dab1 labeling often demonstrates one faintly reactive dendritic process directed to the ganglion cell layer as well as, in the best preparations, punctiform figures likely representative of terminal profiles in the IPL (Fig. 3B, C). These terminal profiles are localized mainly in lamina a (sublaminas s1-s2, above the median calretinin stratum) and in lamina b (sublaminas s4-s5, below the inner calretinin stratum). Labeling is nearly absent in sublamina s3 (Fig. 3B). The presence of terminal profiles in both laminas a (OFF inputs) and b (ON inputs) of the IPL is a distinctive feature of bistratified AII type amacrine cells in mammals (see Fig. 1B, right panel). As previously observed (Gaillard et al., 2008), labeling retinal sections for CaR revealed various amacrine cell profiles within the INL and the GCL. None of these neurons, however, had a morphology obviously resembling the AII category. Most frequently, CaRþ cells in the INL had monostratified terminals in the middle of the IPL (Fig. 3D). Processing sections (n ¼ 13) for both Dab1 and CaR demonstrated no CaR expression in the soma of the majority (86%) of the Dab1þ amacrine cells (n ¼ 234). Dab1þ amacrine cells also did not stain for PKCa (a marker for an amacrine cell population with still unknown function), ChAT (a marker for “starburst” amacrine cells) and GAD67 (a marker for GABAergic amacrine cells; data not shown) as well as for CaB (a marker weakly expressed by some amacrine cells; Fig. 3E) and TH (the rate limiting enzyme in catecholamine biosynthesis; Fig. 3F,G). Dopaminergic neurons in the Nile grass rat are relatively sparse (average density: z28 cells/mm2) and extend long processes (z300 mm) at the INL/ IPL border (Gaillard et al., 2008). As found in mouse (Rice and Curran, 2000), dopaminergic varicosities surround, and likely contact (Fig. 3F,G), Dab1þ cell bodies. Double labeling for parvalbumin was not assayed because this calcium-binding protein is

239

Fig. 2. Dab1 expression in the Nile grass rat retina. A. Dab1 is mainly expressed by horizontal cells and amacrine cells at the INL/IPL boundary. Immunoperoxidase labeling. Contrast enhanced using a “shadow” filter in ImageJ. B. Densitometric profile of Dab1 expression as measured along the vertical line drawn in the previous panel. Peaks correspond to horizontal cell dendritic network, amacrine cell soma and terminals in the IPL. C. Subsequent section in the same animal labeled for calretinin to define the sublayers of the IPL indicated in A. Scale bar for A and B ¼ 25 mm. D. Double staining for Dab1 (green) and calbindin (red) in horizontal cells. White arrows indicate punctate Dab1 reaction in a horizontal cell soma. Single confocal plane. E. Horizontal cell processes expressing the heavy 200 kDa neurofilament (red) also stained for Dab1 (green). Single confocal plane. Scale bar for D and E ¼ 20 mm. Horizontal white bar on the bottom right of panels in D and E indicates the INL/IPL boundary. Abbreviations: IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; HOR, horizontal cells; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; BG, average background level.

restricted to horizontal cells in the Nile grass rat retina (Gaillard et al., 2008). In contrast, all Dab1þ amacrine cells were immunoreactive to an antibody against the glycine transporter-1 (Fig. 3HeK), the preferred marker for glycinergic amacrine cells (Haverkamp and W€ assle, 2000). Dab1 and GlyT1 labels at the soma were clearly distinct (cytoplasm vs. outer membrane, respectively), in agreement with the known cellular location of these two proteins.

240

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

Fig. 3. Dab1-expressing amacrine cells in the Nile grass rat. A. Horizontal cell labeling (arrowheads) and alignment of Dab1þ amacrine cells (stars) at the INL/IPL border. Of note, labeled processes in the IPL are localized at the s3-s4 border. B, C. Terminal profiles of Dab1þ amacrine cells in sublaminas s1-s2 and s4-s5 (vertical bars) of the IPL in the center and far periphery (close to the ora serrata) of the retina, respectively. Pictures treated with the “Shadows” filter (ImageJ software) to improve morphology and contrast. D. Cross-section stained with CaR only, revealing a non-AII amacrine cell with terminals at the s2-s3 border. E. Retinal whole mount (NR139; ventral quadrant; ganglion cells upward) treated for Dab1 (green) and calbindin (red; over-exposed in this frame). Dab1þ cells do not stain for CaB. Actual densities for Dab1þ and CaBþ cells in this frame are 2581/mm2 and 1780/mm2, respectively. F, G. Double staining for Dab1 (green) and TH (red) at two different locations in the same cross section. THþ varicosities (red) accumulate closely (arrowheads) around Dab1þ cell bodies (green). H. Glycinergic amacrine cells stained for GlyT1. I. The same cells labeled for both GlyT1 and Dab1. All Dab1þ cells express GlyT1, but in a different cellular compartment. Stars indicate a pure glycinergic neuron. J, K. Same processing, but in another animal. L. Example of a Dab1þ cell (of unknown type) in the GCL having large soma (vertical axis ¼ 14.5 mm) and dendrites running to sublamina s3. Picture treated with the “Shadows” filter (ImageJ software) as in B. Arrowheads point to faintly labeled amacrine cells. Except where indicated, scale bars correspond to 20 mm. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Finally, in addition to horizontal and amacrine cells, Dab1 immunoreactivity was observed, but infrequently, in INL processes found at the s3-s4 boundary (arrows Fig. 3A,L), as well as in very few cell bodies of the GCL. This monostratified layer contains, as in other mammals, both displaced amacrine (mainly ChATþ and CaRþ) and retinal ganglion cells (Gaillard et al., 2008). Counts performed in frames (n ¼ 16) taken randomly over the three retinas studied returned 290 Dab1þ cells in the INL, but only 9 Dab1þ cells in the GCL. The latter cells were found exclusively in the middle of the retina; none were present in the periphery (5 frames) or center (2 frames). They had characteristically large pear-shaped somas (vertical axis: 9.0e15.2 mm; median value: 11.9 mm), and displayed (on cross sections) 1e2 primary processes directed to the s3-s4 boundary (Fig. 3L; stratification: z 69.4% of

the IPL thickness). However, neither the full morphology of the dendritic field nor the presence of an axon could be determined in these cells. 3.4. Retinal mosaic of Dab1þ amacrine cells Dab1þ amacrine cells were evenly distributed across the Nile grass rat retina and peaked centrally (Table 2). Cell density increases z1.4-fold from periphery (2160 ± 257/mm2; n ¼ 20) to center (3056 ± 395/mm2; n ¼ 18). The lowest density (2086 ± 310/ mm2; n ¼ 6) was found at the dorsal periphery. The maximum density observed at the optic disc peaked at 3956 cells/mm2. Given an average cell density of 2600 ± 560 cells/mm2 [similar to that in mice; (Rice and Curran, 2000)], there would be approximately

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

241

Table 2 Mean density, nearest neighbor distances (NND), Voronoi domains (VD) and regularity indexes (RI) of Dab1þ amacrine cells in the Nile grass rat retina. Counts performed on frames (area ¼ 0.0376 mm2) taken from 3 separate animals. Values correspond to mean ± SD. Ranges are in brackets. Location

# of fields

# of cells in fields

Mean density (cells/mm2)

NND (average value; mm)

RI (NND)

VD (average value; mm2)

RI (VD)

Periphery

20

Center

18

81 ± 10 (62e95) 114 ± 13 (97e139)

2160 ± 257 (1648e2533) 3056 ± 395 (2580e3960)

14.90 ± 3.53 (13.5e16.6) 13.6 ± 2.9 (12e14.5)

4.27 ± 0.46 (3.66e5.22) 4.8 ± 0.5 (4.0e5.5)

412.0 ± 104.4 (346e474) 302.4 ± 73.2 (251e350)

4.0 ± 0.48 (3.26e4.95) 4.2 ± 0.4 (3.53e4.66)

112,000 Dab1þ amacrine cells in the Nile rat retina [average area ¼ 43 mm2; (Gaillard et al., 2008)]. In addition to density measures, we examined the spatial organization of the Dab1þ amacrine cells by computing, for each cell in the previous frames (Fig. 4A), the Voronoi domain (VD; defining points in the plane that were closer to that cell than to any other cell in the field; Fig. 4B) and the distance with its nearest neighbor (NND; Fig. 4C,D). Average values are given in Table 2. Note that both VD and NND values tend to decrease significantly (P ¼ 0.0005 in both cases; one-tailed t-test) from the periphery to the center of the retina. Regardless of the eccentricity, regularity indexes (RI; ratio of the mean area value to the standard deviation) exceed 3.3, which denotes a non-random distribution of the Dab1þ amacrine cells across the retinal surface. This assumption is supported by the close correspondence (P ¼ 0.96; Chi-square test of normality) between the experimentally observed distribution of NNDs and the theoretical Gaussian curve as calculated with cell density and mean NND in a particular field (Fig. 4C,D). 4. Discussion Translation of the Dab1 gene in mouse generates p120, p80, p60, p45 and p36 products with a common N-terminal amino-acid

sequence recognizable by the Dab1-B3 antibody (Howell et al., 1997). The occurrence of these products in the CNS is, however, highly dependent on the age of the animal: while most products are expressed in embryos, only the major one (p80) is present in the brain, and two (p100/80 and p60) are detected in the retina and the pituitary pars posterior of adult specimens (Arnaud et al., 2003; Rice et al., 2001; Howell et al., 1997; Smalheiser et al., 2000; Herrick and Cooper, 2002). Immunoblots shown in Fig. 1 might suggest that all Dab1 isoforms are present in the brain and retina of adult Nile grass rats. Such a statement is questionable, however, because products of 72, 54 and 36 kDa are very likely non-specific given their expression in liver, which lacks the 80 kDa (Dab1) product (Howell et al., 1997; Smalheiser et al., 2000). Moreover, the p130 product has never been observed in western blotting experiments using protein extracts from brain or retina of adult mice (Rice et al., 2001; Howell et al., 1997; data presented here). One possibility is that this product might be another adaptor protein, p130Cas, which displays the consensus amino acid sequence found in Dab1 around Y232 (a key tyrosine kinase recognition site) that is recognized by the Dab1-B3 antibody (B. Howell, personal communication; see below for function). Regardless of the exact nature of the extra bands revealed by western blotting, it is clear that the Dab1-B3 antibody is equally efficient in mouse and Nile

Fig. 4. Topographic arrangement of Dab1 immunoreactive amacrine cells. A. Flat-mounted retina. Cell distribution in the temporal periphery. B. Corresponding Voronoi domains. Of the 95 neurons present in this field (density ¼ 2527/mm2), 50 were retained for quantification. Voronoi domains are on average 404.4 ± 100 mm2 wide and the regularity index is 4.1. C. Distribution of NNDs in one field, at the ventral periphery. D. Distribution of NNDs in another field at the center (ventral quadrant) of the retina. Bin width ¼ 3.5 mm (half the soma diameter). Values for these fields are NND ¼ 15.1 ± 3.8 mm; RI ¼ 4.0 (panel C) and NND ¼ 13.3 ± 2.6 mm; RI ¼ 5.2 (panel D), respectively. Bell-shaped curve in each histogram corresponds to the Gaussian fit.

242

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243

grass rat, and detects in the brain and retina of both species a p80 product corresponding to the full-length Dab1 protein (mDab555). Dab1 immunoreactivity in the Nile grass rat retina is present at the photoreceptor level, in horizontal cells, in very few neurons in the ganglion cell layer and in a large population of amacrine cells. The origin of the faint photoreceptor labeling (never seen before in rodents) is unclear. However, for reasons discussed above, it may reflect the ability of the Dab1-B3 antibody to target p130Cas. This adaptor protein has important functions in the retina, mainly in cell motility, as a component of the integrin signaling machinery. For example, p130Cas is activated by the avb5 integrins (Albert et al., 2000), which are involved in the phagocytosis of photoreceptor outer segments (Finnemann and Nandrot, 2006). Labeling of horizontal cells has been already observed in humans using the antimDab1-C antibody (raised against a peptide at the C-terminus of the protein; Howell et al., 1997) that otherwise labels solely AII amacrine cells in mouse, rat, rabbit and guinea pig (Lee et al., 2003, 2004, 2006). Co-labeling with markers specific to horizontal cells supports such an assertion. Labeling in the ganglion cell layer is very scarce, and the labeled cell type is unknown. With respect to soma diameter and suspected terminal location, these neurons might tentatively be retinal ganglion cells classified as RGCC1 (Sun et al., 2002), PV7 (Kim and Jeon, 2006) or cluster2 cells (Kong et al., 2005) in mouse. An extensive description of the retinal ganglion cell types in the Nile grass rat is however needed to validate this conjecture. Labeling of an unknown cell type in the GCL has also been reported in cat (Lee et al., 2004). Finally, Dab1 labeling in horizontal, amacrine and ganglion cells has been observed in the cone-dominated (6 cones per rod) retina of posthatching chicks (Gao et al., 2010). In all species investigated so far, Dab1þ amacrine cells have been classified as AII amacrine cells based on standard morphological and biochemical criteria. AII amacrine cells account for almost 10% of the amacrine cell population in mammals. They are typically bistratified interneurons with cell bodies arranged as a single row at the vitreal part of the INL and dendritic fields in laminas a and b of the IPL (Vaney et al., 1991; Strettoi and Masland, 1996). In general, these cells are detected immunohistologically by targeting glycine (or glycine transporter-1) and the calcium-binding proteins CaR and PV (W€ assle et al., 1993, 1995; Jeon and Jeon, 1998; Massey and Mills, 1999; Kolb et al., 2002; Scher et al., 2003; Jeon et al., 2007). The occurrence of these calcium-binding proteins in AII amacrine cells is, however, species-dependent. In rodents, for example, AII amacrine cells in guinea pigs express CaR (Lee et al., €ssle et al., 1993); and those 2003); those in rats stain for PV (Wa €ssle, in mice are negative for both CaR and PV (Haverkamp and Wa 2000). The existence of AII amacrine cells in the ground squirrel is a subject of debate (Cuenca et al., 2002; Li et al., 2010). Dab1þ amacrine cells in the Nile grass rat retina have small cell bodies and a bistratified arborization pattern with dendritic terminals in laminas a and b of the IPL. They are regularly distributed at the INL/IPL border, tiling the retina in a non-random fashion with regularity index values formerly reported for Dab1þ/AII amacrine cell mosaics in bat, cat, guinea pig, human, rabbit and rat (Lee et al., 2003, 2004; Jeon et al., 2007; Casini et al., 1998). Although all are clearly labeled for the high affinity glycine transporter-1, none of these Dab1þ amacrine cells express CaB or PV, and a vast majority of them are also negative for CaR. Only few (14%) Dab1þ neurons show CaR immunoreactivity. This observation might be a false positive result (detection of single protein expression in separate cells) due to the thickness (20 mm) of the confocal stacks used for calculation. Alternatively, CaR might be expressed at different levels in Dab1þ cells. A recent genetic study (Siegert, 2010) in retinal cells of the mouse showed that Fam81a and Fbxo genes (two markers of AII amacrine cells) are expressed in 98% and 66% of the

Dab1þ neurons, respectively. Finally, part of the neurons co-labeled for CaR and Dab1 might be either non-AII amacrine cells or a subpopulation of AII amacrine cells. These possibilities are supported by findings that almost 3.4% of Dab1þ cells in mouse contain the ler5 gene usually present in non-AII glycinergic amacrine cells (Siegert, 2010) and that small populations of the PVþ AII amacrine € lgyi et al., 1997) or the neucells in rabbit and cat express CaR (Vo
briel and Straznicky, 1992), respectively. rofilament triplet (Ga 5. Conclusion The present study shows that Disabled-1 is expressed in the retina of the Nile grass rat, a diurnal murine species in which 35e40% of all photoreceptors are cones. Dab1 expression is mainly present in horizontal cells and a fraction of glycinergic amacrine cells that are reminiscent of the AII type described in various mammalian species using antibodies against calcium-binding proteins. This dual expression pattern is not observed in common laboratory rodents, but resembles that found in the human retina. These results, firstly suggest that targeting retinal Dab-1 might be decisive in mammalian species in which the presence of AII amacrine cells is controversial (for instance, in the cone-dominated retina of the ground squirrel); and secondly, further strengthen the use of the Nile grass rat as a model to investigate human retina function in health, disease and associated therapies. Acknowledgments Authors are grateful to Drs. B. Howell and A. Goffinet for generously donating various Dab1 (B3, D4 and H3) antibodies and for their constructive comments. References Albert, M.L., Kim, J.I., Birge, R.B., 2000. alphavbeta5 integrin recruits the CrkIIDock180-rac1 complex for phagocytosis of apoptotic cells. Nat. Cell Biol. 2, 899e905. Arnaud, L., Ballif, B.A., Forster, E., Cooper, J.A., 2003. Fyn tyrosine kinase is a critical regulator of Disabled-1 during brain development. Curr. Biol. 13, 9e17. Bar, I., Goffinet, A.M., 1999. Decoding the Reelin signal. Nature 399, 645e646. Bobu, C., Craft, C.M., Masson-Pevet, M., Hicks, D., 2006. Photoreceptor organization and rhythmic phagocytosis in the Nile rat Arvicanthis ansorgei: a novel diurnal rodent model to study cone pathophysiology. Invest. Ophthalmol. Vis. Sci. 47, 3109e3118. Casini, G., Rickman, D.W., Trasarti, L., Brecha, N.C., 1998. Postnatal development of parvalbumin immunoreactive amacrine cells in the rabbit retina. Dev. Brain Res. 111, 107e117. Cherry, T.J., Trimarchia, J.M., Stadlerb, M.B., Cepko, C.L., 2009. Development and diversification of retinal amacrine interneurons at single cell resolution. Proc. Natl. Acad. Sci. U. S. A. 106, 9495e9500. Claes, E., Seeliger, M., Michalakis, S., Biel, M., Humphries, P., Haverkamp, S., 2004. Morphological characterization of the retina of the CNGA3(e/e)Rho(e/e) mutant mouse lacking functional cones and rods. Invest. Ophthalmol. Vis. Sci. 45, 2039e2048. Cooper, J.A., Howell, B.W., 1999. Lipoprotein receptors: signaling functions in the brain? Cell 97, 671e674. Cuenca, N., Deng, P., Linberg, K.A., Lewis, G.P., Fisher, S.K., Kolb, H., 2002. The neurons of the ground squirrel retina as revealed by immunostains for calcium binding proteins and neurotransmitters. J. Neurocytol. 31, 649e666. Finnemann, S.C., Nandrot, E.F., 2006. MerTK activation during RPE phagocytosis in vivo requires alphaVbeta5 integrin. Adv. Exp. Med. Biol. 572, 499e503.
briel, R., Straznicky, C., 1992. Immunocytochemical localization of parvalbuminGa and neurofilament triplet protein immunoreactivity in the cat retina: colocalization in a subpopulation of AII amacrine cells. Brain Res. 595, 133e1336. Gaillard, F., Bonfield, S., Gilmour, G.S., Kuny, S., Mema, S.C., Martin, B.T., Smale, L.,
, Y., 2008. Retinal anatomy and visual perforCrowder, N., Stell, W.K., Sauve mance in a diurnal cone-rich laboratory rodent, the Nile grass rat (Arvicanthis niloticus). J. Comp. Neurol. 510, 525e538.
, Y., 2009. Topographic arrangement of S-cone photoreGaillard, F., Kuny, S., Sauve ceptors in the retina of the diurnal Nile grass rat (Arvicanthis niloticus). Invest. Ophthalmol. Vis. Sci. 50, 5426e5434.
, Y., 2013. Retinorecipient areas in the diurnal murine Gaillard, F., Karten, H.J., Sauve rodent Arvicanthis niloticus: a disproportionally large superior colliculus. J. Comp. Neurol. 521, 1699e1726.

F. Gaillard et al. / Experimental Eye Research 125 (2014) 236e243 Gao, Z., Monckton, E.A., Glubrecht, D.D., Logan, C., Godbout, R., 2010. The early isoform of Disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina. Mol. Cell. Biol. 30, 4339e4353. Geisert, E.E., Lu, L., Freeman-Anderson, N.E., Templeton, J.P., Nassr, M., Wang, X., Gu, W., Jiao, Y., Williams, R.W., 2009. Gene expression in the mouse eye: an online resource for genetics using 103 strains of mice. Mol. Vis. 15, 1730e1763. Gilmour, G.S., Gaillard, F., Watson, J., Kuny, S., Mema, S.C., Bonfield, S.,
, Y., 2008. The electroretinogram (ERG) of a diurnal coneStell, W.K., Sauve rich laboratory rodent, the Nile grass rat (Arvicanthis niloticus). Vis. Res. 48, 2723e2731. Goffinet, A.M., 1984. Events governing organization of postmigratory neurons: studies on brain development in normal and reeler mice. Brain Res. 319, 261e296. €ssle, H., 2000. Immunocytochemical analysis of the mouse retina. Haverkamp, S., Wa J. Comp. Neurol. 424, 1e23. Herrick, T.A., Cooper, J.A., 2002. A hypomorphic allele of dab1 reveals regional differences in reelin-Dab1 signaling during brain development. Development 129, 787e796. Howell, B.W., Gertler, F.B., Cooper, J.A., 1997. Mouse disabled (mDab1): a Src binding protein implicated in neuronal development. EMBO J. 16, 121e132. Jeon, M.H., Jeon, C.J., 1998. Immunocytochemical localization of calretinin containing neurons in retina from rabbit, cat, and dog. Neurosci. Res. 32, 75e84. Jeon, Y.K., Kim, T.J., Lee, J.Y., Choi, J.S., Jeon, C.J., 2007. AII amacrine cells in the inner nuclear layer of bat retina: identification by parvalbumin immunoreactivity. NeuroReport 18, 1095e1099. Kim, T.J., Jeon, C.J., 2006. Morphological classification of parvalbumin-containing retinal ganglion cells in mouse: single-cell injection after immunocytochemistry. Invest. Ophthalmol. Vis. Sci. 47, 2757e2764. Kolb, H., Famiglietti, E.V., 1974. Rod and cone pathways in the inner plexiform layer of cat retina. Science 186, 47e49. Kolb, H., Zhang, L., Dekorver, L., Cuenca, N., 2002. A new look at calretininimmunoreactive amacrine cell types in the monkey retina. J. Comp. Neurol. 453, 168e184. Kong, J.H., Fish, D.R., Rockhill, R.L., Masland, R.H., 2005. Diversity of ganglion cells in the mouse retina: unsupervised morphological classification and its limits. J. Comp. Neurol. 489, 293e310. Lee, E.J., Kim, H.J., Kim, I.B., Park, J.H., Oh, S.J., Rickman, D.W., Chun, M.H., 2003. Morphological analysis of Disabled-1-immunoreactive amacrine cells in the guinea pig retina. J. Comp. Neurol. 466, 240e250. Lee, E.J., Kim, H.J., Lim, E.J., Kim, I.B., Kang, W.S., Oh, S.J., Rickman, D.W., Chung, J.W., Chun, M.H., 2004. AII amacrine cells in the mammalian retina show Disabled-1 immunoreactivity. J. Comp. Neurol. 470, 372e381.

243

Lee, E.J., Mann, L.B., Rickman, D.W., Lim, E.J., Chun, M.H., Grzywacz, N.M., 2006. AII amacrine cells in the distal inner nuclear layer of the mouse retina. J. Comp. Neurol. 494, 651e662. Li, W., Chen, S., DeVries, S.H., 2010. A fast rod photoreceptor signaling pathway in the mammalian retina. Nat. Neurosci. 13, 414e416. Massey, S.C., Mills, S.L., 1999. An antibody to calretinin stains AII amacrine cells in the rabbit retina: double-label and confocal analysis. J. Comp. Neurol. 411, 3e18. Park, S.J., Lim, E.J., Oh, S.J., Chung, J.W., Rickman, D.W., Moon, J.I., Chun, M.H., 2004. Ectopic localization of putative AII amacrine cells in the outer plexiform layer of the developing FVB/N mouse retina. Cell Tissue Res. 315, 407e412. Rice, D.S., Sheldon, M., D’Arcangelo, G., Nakajima, K., Goldowitz, D., Curran, T., 1998. Disabled-1 acts downstream of Reelin in a signaling pathway that controls laminar organization in the mammalian brain. Development 125, 3719e3729. Rice, D.S., Curran, T., 2000. Disabled-1 is expressed in type AII amacrine cells in the mouse retina. J. Comp. Neurol. 424, 327e338. Rice, D.S., Nusinowitz, S., Azimi, A.M., Martinez, A., Soriano, E., Curran, T., 2001. The Reelin pathway modulates the structure and function of retinal synaptic circuitry. Neuron 31, 929e941. Scher, J., Wankiewicz, E., Brown, G.M., Fujieda, H., 2003. AII amacrine cells express the MT1 melatonin receptor in human and macaque retina. Exp. Eye Res. 77, 375e382. Schiffmann, S.N., Bernier, B., Goffinet, A.M., 1997. Reelin mRNA expression during mouse brain development. Eur. J. Neurosci. 9, 1055e1071. Siegert, S., 2010. Molecular Logic of Retinal Cell Types (PhD Thesis). University of Basel, Faculty of Science. available at. edoc.unibas.ch. Smalheiser, N.R., Costa, E., Guidotti, A., Impagnatiello, F., Auta, J., Lacor, P., Kriho, V., Pappas, G.D., 2000. Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells. Proc. Natl. Acad. Sci. U. S. A. 97, 1281e1286. Strettoi, E., Masland, R.H., 1996. The number of unidentified amacrine cells in the mammalian retina. Proc. Natl. Acad. Sci. U. S. A. 93, 14906e14911. Sun, W., Li, N., He, S., 2002. Large-scale morophological survey of rat retinal ganglion cells. Vis. Neurosci. 19, 483e493. Vaney, D.I., Gynther, I.C., Young, H.M., 1991. Rod-signal interneurons in the rabbit retina: 2. AII amacrine cells. J. Comp. Neurol. 310, 154e169. €lgyi, B., Pollak, E., Buza
s, P., G
Vo abriel, R., 1997. Calretinin in neurochemically welldefined cell populations of rabbit retina. Brain Res. 763, 79e86. €ssle, H., Grünert, U., Ro €hrenbeck, J., 1993. Immunocytochemical staining of AIIWa amacrine cells in the rat retina with antibodies against parvalbumin. J. Comp. Neurol. 332, 407e420. €ssle, H., Grünert, U., Chun, M.-H., Boycott, B.B., 1995. The rod pathway of the Wa macaque monkey retina: identification of AII-amacrine cells with antibodies against calretinin. J. Comp. Neurol. 361, 537e551.