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Retinal horizontal cells reduced in a rat model of congenital stationary night blindness
Neuroscience Letters 521 (2012) 26–30
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Retinal horizontal cells reduced in a rat model of congenital stationary night blindness Lijuan Zheng a,1 , Yili Yan b,1 , Jing An a,1 , Lei Zhang a , Wei Liu a , Feng Xia a , Zuoming Zhang a,∗ a Department of Clinical Aerospace Medicine, Faculty of Aerospace Medicine, Key Laboratory of Aerospace Medicine of National Education Ministry, Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, China b Faculty of Biomedical Engineering, Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, China
h i g h l i g h t s The number and the density of retinal horizontal cells reduced in CSNB rats. Thickness of OPL and Calbindin level decreased in CSNB rats. Decrease in number and density of horizontal cells in CSNB rats and wild type rats from PND15 to PND60.
a r t i c l e
i n f o
Article history: Received 28 January 2012 Received in revised form 29 April 2012 Accepted 16 May 2012 Keywords: Retinal neurons CSNB Rat Calbindin D-28K Hereditary retinal disease
a b s t r a c t This work was conducted to determine whether congenital stationary night blindness (CSNB), which is caused by a Cacna1f mutation, could affect development of second-order neurons in the retina, such as horizontal cells (HCs). The CSNB rats and age-matched wild type rats were sacrificed at postnatal days (PND) 15, 30 and 60. Morphometric analyses of HCs, which were labeled by a primary antibody to calbindin D-28K, were performed at the light microscopic level on retinal cross sections and whole mount retinas. Calbindin D-28K was measured by western blotting in retinal samples. We found that the average number and density of HCs, Calbindin level and thickness of OPL were all decreased significantly in CSNB group compared to control group. These results indicated that second-order retinal neurons, such as horizontal cells, are affected by retinal degeneration. The relationship between the absence of HCs and the gene defect of CSNB requires further research. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by poor night vision, maintenance of photopic vision, and varied ocular symptoms of myopia, nystagmus and decreased visual acuity [7]. It is an inherited disease having three modes of genetic transmission: autosomal dominant, autosomal recessive, or X-linked recessive modes [8]. A naturally occurring rat model of X-linked CSNB was originally identified by electroretinogram (ERG) recordings obtained from a single outbred Sprague Dawley rat [29]. This rat model of CSNB is caused by a Cacna1f (which encodes the ␣1F Subunit of an L-type calcium channel) mutation, and the phenotype closely resembles the CSNB2 (which retain measurable rod function with significant impairment of cone function) [8]. Significant changes in retinal structure included the number of ribbon bodies and the connections between
∗ Corresponding author. Tel.: +86 29 8477 4817; fax: +86 29 8477 4817. E-mail addresses: [email protected], [email protected] (Z. Zhang). 1 These three authors contributed equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.049
different types of neurons in nob2 mice [2]. Our group found similar structural changes and also found the functional change in L-type calcium channels (which mediate depolarization-induced Ca2+ entry into electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells) of rod bipolar cells [27], in addition to ERG changes. Retinal horizontal cells (HCs) are the first inhibitory interneurons of the retina and stratify within the outer plexiform layer (OPL) and the inner nuclear layer (INL) [20]. HCs in mammalian retinas give rise to dendrites, which ramify in a radial pattern within the outer plexiform layer (OPL) [12,17]. Despite the similarity to axons elsewhere in the nervous system, the HCs axons do not conduct nerve impulses. Rather, they provide metabolic support for their terminal arbors, which are selectively innervated by the spherules of rod photoreceptors and serve as an independent functional entity comparable with the dendritic domain. The dendritic and the axonal domains are not only postsynaptic to the cones and the rods, respectively, but also provide inhibitory feedback to those photoreceptors [20]. HCs also play a role in modulating signals between photoreceptors and bipolar cells [19].
L. Zheng et al. / Neuroscience Letters 521 (2012) 26–30
In the present study, we tried to confirm change in the number of HCs [8], observe the changes of HCs in the developmental stage of CSNB rats and provide further experimental evidence for the mechanism of signal transmission in the retina. 2. Methods 2.1. Animals and tissue preparation Wild-type Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Research Center of the Fourth Military Medical University. CSNB rat models that were developed by the Fourth Military Medical University [8,29] and were maintained under a 12 h light-dark cycle with an average ambient light of 25–37 W/cm2 (170–250 lx; measured with IL1400 photometer, International Light Technologies, Peabody, USA). The day of birth was designated postnatal day (PND) 0. The pups were randomly assigned to different groups, which were sacrificed at different ages (PND 15, 30 and 60). Animals were anesthetized with a lethal dose of sodium pentobarbital (1,20,000 ng/g, i.p.) or decapitated [10,21]. All procedures that involved animals adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of the Fourth Military Medical University. 2.2. Histology Both of the eyes from CSNB rats and wild type rats were examined at PND 15, 30 and 60. Following deep anesthesia, the eyes were enucleated and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h. The anterior portions of the eyes were then removed, and the posterior portions were fixed for an additional 12 h. The eyecups were dehydrated in an alcohol series and then embedded in paraffin. Sections 5 m thick were cut along the vertical meridian through the optic nerve head and stained with hematoxylin and eosin. 2.3. Immunocytochemistry The retinal sections were incubated overnight with a primary antibody against Calbindin D-28K (rabbit monoclonal; 1:2000, Chemicon, Southampton, UK) in 5% normal bovine serum albumin (BSA), 0.5% Triton X-100, and 0.02% sodiumazide in phosphatebuffered saline (PBS). The sections were then washed in PBS and incubated with the biotinylated secondary antibody, followed by the avidin–biotin–peroxidase detection system (Universal Vectastain SABC Elite Kit, Boster, WuHan, China) for 2 h. Using 3, 3-diaminobenzidine (DAB) as the chromogen, the progress of the immunoreaction was monitored under a light microscope. The reaction was stopped by the removal of the DAB followed by buffer washes. Control sections were treated similarly, except that the primary antibody was omitted from the procedure. The number of calbindin D-28K-labeled cells was counted at a magnification of 400×. For each animal, the results obtained from six separate sections were averaged. 2.4. Wholemount immunocytochemistry Wholemount immunocytochemistry was performed as previously described [9,21,22,28]. 6 rats in each group were used and for each rat, left eyes were enucleated after an anesthetic overdose and fixed in 4% paraformaldehyde for 1 h. The dorsal pole of each eye was marked with a suture before enucleation. The anterior parts of the eyes were then removed, and the eyecups were fixed overnight. The neural retinas were separated from the pigment epithelium and were thoroughly washed. Retinal wholemounts were rinsed
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in phosphate buffer, preincubated in 10% normal donkey serum and 1% Triton-X100 in PBS for 3 h and rinsed with PBS. The tissues were then incubated with agitation for 3 days at 4 ◦ C in a cocktail of primary antibodies and 1% Triton-X100 in PBS. Following incubation, the tissues were rinsed in PBS and incubated in the FITC-labeled secondary antibody overnight and then rinsed in PBS. Finally, nuclei were stained with 4 ,6-diamidino-2-phenylindole for 1 h. Four incisions were then made in the retina, thus dividing it into dorsal, ventral, temporal and nasal quadrants. Retinas were then mounted on clean slides under a coverslip. All reagents and washing buffers contained 0.1% Triton X-100 to permeabilize membranes. The number of calbindin D-28K-labeled cells was counted at a location centered 1 mm from the optic nerve head at a magnification of 400×. Wholemounts from CSNB rats and wild type rats were sampled in four quadrants at central and peripheral locations (i.e., eight fields per retina) to determine the average densities of calbindinimmunopositive horizontal cells. Fields were selected randomly at low magnification, without respect to cell density, at comparable eccentricity in each quadrant. Sampled fields were 0.225802 mm2 for the horizontal cells [21].
2.5. Western blotting The retinas were lysed with 100 l RIPA (Beyotime, BeiJing, China) and placed on ice. The extracts were then sonicated and centrifuged at 15 ◦ C for 10 min at 12,000 × g. The supernatant was collected, and the protein concentration was measured. A 100 g aliquot of protein per sample was fractionated by SDS–polyacrylamide gel electrophoresis and electroblotted to activated polyvinylidenefluoride membrane. The blot was incubated at room temperature for 1 h in a blocking solution (5% BSA prepared in Tris-buffered saline with 0.1% Tween 20, pH 7.4), followed by overnight incubation at 4 ◦ C in the primary antibody diluted in the blocking solution. The primary antibodies included rabbit anti-calbindin (1:1000; Chemicon, Temecula, CA), and mouse anti-actin (1:2000; Sigma–Aldrich, St. Louis, MO). The blot was then probed with a horseradish peroxidaseconjugated secondary antibody and developed by the enhanced chemiluminescence method. The immunoreactive bands were detected with a Chemi Genius Bioimaging system. Densitometric analysis was performed with Image software. Following background subtraction, the signal for each protein was normalized to the loading control, -actin. The data (integrated densities) were expressed as the change in protein levels of CSNB rats relative to the controls [16].
2.6. Quantitative analysis Quantitative studies were performed in 6 animals (males and females) per time point (PND 15, 30 and 60) and per treatment (SD and CSNB), selected randomly from different litters. All measurements were performed relying on a proven system of random sampling. Photomicrographs were coded and analyzed blindly to avoid experimental bias. Statistical analyses of the measure of number of HCs at different groups and times were made using independent samples of nonparamentric test and Nemeyi test, and statistical analyses of the thickness of OPL, density of HCs and Calbindin levels were made using two-way ANOVA and Tukey test of post hoc test in the SPSS statistical software program (version 14.0).
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L. Zheng et al. / Neuroscience Letters 521 (2012) 26–30
Fig. 2. Immunocytochemistry pictures of wild type SD and CSNB rat retinas at PND15, 30 and 60. A,A1,C,C1,E,E1: SD rat retina; B,B1,D,D1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 20 m in length. The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length. Fig. 1. Representative histological sections of thickness of OPL at SD and CSNB rat retinas. A,A1,C,C1,E,E1: SD rat retina; B,B1,D,D1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 20 m in length. The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length.
3. Results 3.1. Results of histology and immunochemistry To analyze the thickness of the OPL, hematoxylin and eosin (HE) stained retinal sections from CSNB rats were compared with their respective control rats (Fig. 1). The results showed that the thickness of the OPL was reduced (P < 0.01) in CSNB rats when compared with age-matched wild type rats (Fig. 4A). To determine whether there were any changes in the number of HCs stained retinal sections from CSNB rats were compared with wild type rats. HCs were labeled with calbindin, a calcium-binding protein and a known marker for HCs and their processes, particularly their proximal dendrites [20]. Control retinas showed the staining of bodies and processes in the OPL (Fig. 2A, C and E). In contrast, labeling for HCs in affected retinas was obviously reduced (Fig. 2B, D and F; Fig. 4B). To observe the change at different ages, we compared data at different ages in each group. There were decreases at the number of HCs (P < 0.01) in PND60 compared to the other two time points in CSNB rats and wild type rats (Fig. 4B). The mean number of HCs was compared from the center to the periphery of the retina as in previous studies [3,23] and no obvious differences between central and peripheral retina were found in either wild type or CSNB rats at PND 15, 30 and 60 (P > 0.05).
Fig. 3. Wholemount immunocytochemistry of the retinas from SD and CSNB rats at 15, 30 and 60 days. A,A1,B,B1,C,C1: SD rat retina; D,D1,E,E1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 50 m in length.The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length.
L. Zheng et al. / Neuroscience Letters 521 (2012) 26–30
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Fig. 4. Statistical of wild type rat and CSNB rat retinas at PND15, 30 and 60. (A) OPL thickness. *: difference between CSNB group and SD group, ** p < 0.01. (B) Mean horizontal cells numbers. *: difference between CSNB group and SD group, ** p < 0.01; #: difference among three time points within each group, ## p < 0.01. (C) Mean horizontal cells densities of SD and CSNB rats. *: difference between CSNB group and SD group, * p < 0.05, ** p < 0.01; #: difference among three time points within each group, ## p < 0.01.
3.2. Results of wholemount immunocytochemistry HCs were marked with Calbindin in both wild type and CSNB rats (Fig. 3). All the cells were counted that fell within the area of graticule including cells overlapping the lower and left borders, but excluding those overlapping the other borders according to the Chu method [3]. Measurements showed that the eyepiece graticule covered an area of per 40× visual field (0.096 mm2 ) which were used to calculate the density of HCs [3]. Statistics showed that there was a decrease in the average density of HCs in CSNB rats at the three ages tested when compared with wild type rats (Fig. 4C). There were no obvious differences in the distribution of average densities of HCs either in wild type or CSNB rats (P > 0.05). To look for changes at different ages, we compared the data at the different time points in each groups, and showed that both in CSNB and wild type rats, there was significant difference between PND15 and PND 60 (P < 0.01). 3.3. Results of western blotting Retinal Calbindin protein levels in wild type and CSNB rats were observed by Western blotting (WB). The results showed that the Calbindin levels in the retina were lower in CSNB rats than in wild type rats at PND15 and PND60 (P < 0.05), but there was no obvious differences at PND30 (P > 0.05) (Fig. 5). We compared the data at the different time points within each group, and found that no obvious difference in the level of Calbindin protein was observed both in CSNB and wild type rats (P > 0.05). 4. Discussion Some retinal degeneration can cause loss of photoreceptors accompanied by significantly morphological changes in the inner second-order retinal neurons, including dendritic retraction and neuritic sprouting in horizontal and bipolar cells [4–6,11,14–16,18,25,26]. The morphology of HCs is plastic, and the morphology of the cell soma and synapse changes with the alteration of environment or disease state [1,13,16,20,21,24], but there are few reports on the number of HCs lost. In this study, we found that there was a decrease in the number of HCs in CSNB (one of retinal degeneration diseases) rats compared to wild type rats. In adult CSNB rats, around 43.25% of HCs were lost compared to wild type rats. Meanwhile, the outer plexiform layer became thinner in the retinas of CSNB rats. It was previously reported in nob2 mice, which is also caused by a Cacna1f gene mutation, that the dendritic morphology of HCs was relatively skeletal at PND 10 and mature stages, but it did not mention any change in the number of HCs [21].
Fig. 5. Representative immunoblots for Calbindin and -action for SD and CSNB rats. Immunoblot analysis showing a reduction in calbindin levels of CSNB rats. *: difference between CSNB group and SD group, * p < 0.05.
In CSNB rat retinas, the rod system is damaged mainly [8]. Some studies proposed that the HC axon does not conduct nerve impulses but rather provides metabolic support for its terminal arbor, which is selectively innervated by the spherules of rod photoreceptors and serves as an independent functional entity comparable with the dendritic domain [20]. If it is true that part of HCs are lost in CSNB rats, it suggests that the lost HCs might be CACNA1F gene dependent mediately. The mutation in Cacna1f gene might disturb the metabolism or function in some kind of neurons and caused the HCs lost during development process. This study hints that the change of inherited retinal disease may influence the retinal HCs indirectly, which will need to be addressed in future studies. References [1] P.R. Bayley, C.W. Morgans, Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina, Journal of Comparative Neurology 500 (2007) 286–298. [2] B. Chang, J.R. Heckenlively, P.R. Bayley, N.C. Brecha, M.T. Davisson, N.L. Hawes, A.A. Hirano, R.E. Hurd, A. Ikeda, B.A. Johnson, M.A. McCall, C.W. Morgans, S. Nusinowitz, N.S. Peachey, D.S. Rice, K.A. Vessey, R.G. Gregg, The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses, Visual Neuroscience 23 (2006) 11–24.
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[3] Y. Chu, M.F. Humphrey, I.J. Constable, Horizontal cells of the normal and dystrophic rat retina: a wholemount study using immunolabelling for the 28-kDa calcium-binding protein, Experimental Eye Research 57 (1993) 141–148. [4] N. Cuenca, I. Pinilla, Y. Sauve, R. Lund, Early changes in synaptic connectivity following progressive photoreceptor degeneration in RCS rats, European Journal of Neuroscience 22 (2005) 1057–1072. [5] R.N. Fariss, Z.Y. Li, A.H. Milam, Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigmentosa, American Journal of Ophthalmology 129 (2000) 215–223. [6] C. Gargini, E. Terzibasi, F. Mazzoni, E. Strettoi, Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study, Journal of Comparative Neurology 500 (2007) 222–238. [7] P. Goodwin, Hereditary retinal disease, Current Opinion in Ophthalmology 19 (2008) 255–262. [8] Y. Gu, L. Wang, J. Zhou, Q. Guo, N. Liu, Z. Ding, L. Li, X. Liu, J. An, G. Yan, L. Yao, Z. Zhang, A naturally-occurring mutation in Cacna1f in a rat model of congenital stationary night blindness, Molecular Vision 14 (2008) 20–28. [9] Y.H. Gu, Z.M. Zhang, T. Long, L. Li, B.K. Hou, Q. Guo, A naturally occurring rat model of X-linked cone dysfunction, Investigative Ophthalmology and Visual Science 44 (2003) 5321–5326. [10] R.M. Huckfeldt, T. Schubert, J.L. Morgan, L. Godinho, G. Di Cristo, Z.J. Huang, R.O. Wong, Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions, Nature Neuroscience 12 (2009) 35–43. [11] B.W. Jones, C.B. Watt, J.M. Frederick, W. Baehr, C.K. Chen, E.M. Levine, A.H. Milam, M.M. Lavail, R.E. Marc, Retinal remodeling triggered by photoreceptor degenerations, Journal of Comparative Neurology 464 (2003) 1–16. [12] H. Kolb, The connections between horizontal cells and photoreceptors in the retina of the cat: electron microscopy of Golgi preparations, Journal of Comparative Neurology 155 (1974) 1–14. [13] K.A. Linberg, G.P. Lewis, B. Matsumoto, S.K. Fisher, Immunocytochemical evidence that rod-connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat, Molecular Vision 12 (2006) 1674–1686. [14] R.E. Marc, B.W. Jones, C.B. Watt, E. Strettoi, Neural remodeling in retinal degeneration, Progress in Retinal and Eye Research 22 (2003) 607–655. [15] A.H. Milam, Z.Y. Li, R.N. Fariss, Histopathology of the human retina in retinitis pigmentosa, Progress in Retinal and Eye Research 17 (1998) 175–205. [16] S. Nagar, V. Krishnamoorthy, P. Cherukuri, V. Jain, N.K. Dhingra, Early remodeling in an inducible animal model of retinal degeneration, Neuroscience 160 (2009) 517–529.
[17] F. Pan, S.C. Massey, Rod and cone input to horizontal cells in the rabbit retina, Journal of Comparative Neurology 500 (2007) 815–831. [18] S.J. Park, I.B. Kim, K.R. Choi, J.I. Moon, S.J. Oh, J.W. Chung, M.H. Chun, Reorganization of horizontal cell processes in the developing FVB/N mouse retina, Cell and Tissue Research 306 (2001) 341–346. [19] R.A. Poche, B.E. Reese, Retinal horizontal cells: challenging paradigms of neural development and cancer biology, Development 136 (2009) 2141–2151. [20] M.A. Raven, E.C. Oh, A. Swaroop, B.E. Reese, Afferent control of horizontal cell morphology revealed by genetic respecification of rods and cones, Journal of Neuroscience 27 (2007) 3540–3547. [21] M.A. Raven, N.C. Orton, H. Nassar, G.A. Williams, W.K. Stell, G.H. Jacobs, N.T. Bech-Hansen, B.E. Reese, Early afferent signaling in the outer plexiform layer regulates development of horizontal cell morphology, Journal of Comparative Neurology 506 (2008) 745–758. [22] M.A. Raven, B.E. Reese, Mosaic regularity of horizontal cells in the mouse retina is independent of cone photoreceptor innervation, Investigative Ophthalmology and Visual Science 44 (2003) 965–973. [23] B.E. Reese, M.A. Raven, S.B. Stagg, Afferents and homotypic neighbors regulate horizontal cell morphology, connectivity, and retinal coverage, Journal of Neuroscience 25 (2005) 2167–2175. [24] C. Ribelayga, S.C. Mangel, Tracer coupling between fish rod horizontal cells: modulation by light and dopamine but not the retinal circadian clock, Visual Neuroscience 24 (2007) 333–344. [25] E. Strettoi, V. Pignatelli, C. Rossi, V. Porciatti, B. Falsini, Remodeling of secondorder neurons in the retina of rd/rd mutant mice, Vision Research 43 (2003) 867–877. [26] E. Strettoi, V. Porciatti, B. Falsini, V. Pignatelli, C. Rossi, Morphological and functional abnormalities in the inner retina of the rd/rd mouse, Journal of Neuroscience 22 (2002) 5492–5504. [27] Y. Tao, Q. Guo, R. Wang, J. An, F.L. Cao, B. Xie, L. Zhang, Z.M. Zhang, Electrophysiology properities of L-type calcium channel in rod bipolar cells of CSNB rat, Chinese Ophthalmic Research 28 (2010) 535–539. [28] B. Xie, S. Nakanishi, Q. Guo, F. Xia, G. Yan, J. An, L. Li, T. Serikawa, T. Kuramoto, Z. Zhang, A novel middle-wavelength opsin (M-opsin) nullmutation in the retinal cone dysfunction rat, Experimental Eye Research 91 (2010) 26–33. [29] Z. Zhang, Y. Gu, L. Li, T. Long, Q. Guo, L. Shi, A potential spontaneous rat model of X-linked congenital stationary night blindness, Documenta Ophthalmologica 107 (2003) 53–57.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Retinal horizontal cells reduced in a rat model of congenital stationary night blindness Lijuan Zheng a,1 , Yili Yan b,1 , Jing An a,1 , Lei Zhang a , Wei Liu a , Feng Xia a , Zuoming Zhang a,∗ a Department of Clinical Aerospace Medicine, Faculty of Aerospace Medicine, Key Laboratory of Aerospace Medicine of National Education Ministry, Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, China b Faculty of Biomedical Engineering, Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, China
h i g h l i g h t s The number and the density of retinal horizontal cells reduced in CSNB rats. Thickness of OPL and Calbindin level decreased in CSNB rats. Decrease in number and density of horizontal cells in CSNB rats and wild type rats from PND15 to PND60.
a r t i c l e
i n f o
Article history: Received 28 January 2012 Received in revised form 29 April 2012 Accepted 16 May 2012 Keywords: Retinal neurons CSNB Rat Calbindin D-28K Hereditary retinal disease
a b s t r a c t This work was conducted to determine whether congenital stationary night blindness (CSNB), which is caused by a Cacna1f mutation, could affect development of second-order neurons in the retina, such as horizontal cells (HCs). The CSNB rats and age-matched wild type rats were sacrificed at postnatal days (PND) 15, 30 and 60. Morphometric analyses of HCs, which were labeled by a primary antibody to calbindin D-28K, were performed at the light microscopic level on retinal cross sections and whole mount retinas. Calbindin D-28K was measured by western blotting in retinal samples. We found that the average number and density of HCs, Calbindin level and thickness of OPL were all decreased significantly in CSNB group compared to control group. These results indicated that second-order retinal neurons, such as horizontal cells, are affected by retinal degeneration. The relationship between the absence of HCs and the gene defect of CSNB requires further research. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by poor night vision, maintenance of photopic vision, and varied ocular symptoms of myopia, nystagmus and decreased visual acuity [7]. It is an inherited disease having three modes of genetic transmission: autosomal dominant, autosomal recessive, or X-linked recessive modes [8]. A naturally occurring rat model of X-linked CSNB was originally identified by electroretinogram (ERG) recordings obtained from a single outbred Sprague Dawley rat [29]. This rat model of CSNB is caused by a Cacna1f (which encodes the ␣1F Subunit of an L-type calcium channel) mutation, and the phenotype closely resembles the CSNB2 (which retain measurable rod function with significant impairment of cone function) [8]. Significant changes in retinal structure included the number of ribbon bodies and the connections between
∗ Corresponding author. Tel.: +86 29 8477 4817; fax: +86 29 8477 4817. E-mail addresses: [email protected], [email protected] (Z. Zhang). 1 These three authors contributed equally to this work. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.049
different types of neurons in nob2 mice [2]. Our group found similar structural changes and also found the functional change in L-type calcium channels (which mediate depolarization-induced Ca2+ entry into electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells) of rod bipolar cells [27], in addition to ERG changes. Retinal horizontal cells (HCs) are the first inhibitory interneurons of the retina and stratify within the outer plexiform layer (OPL) and the inner nuclear layer (INL) [20]. HCs in mammalian retinas give rise to dendrites, which ramify in a radial pattern within the outer plexiform layer (OPL) [12,17]. Despite the similarity to axons elsewhere in the nervous system, the HCs axons do not conduct nerve impulses. Rather, they provide metabolic support for their terminal arbors, which are selectively innervated by the spherules of rod photoreceptors and serve as an independent functional entity comparable with the dendritic domain. The dendritic and the axonal domains are not only postsynaptic to the cones and the rods, respectively, but also provide inhibitory feedback to those photoreceptors [20]. HCs also play a role in modulating signals between photoreceptors and bipolar cells [19].
L. Zheng et al. / Neuroscience Letters 521 (2012) 26–30
In the present study, we tried to confirm change in the number of HCs [8], observe the changes of HCs in the developmental stage of CSNB rats and provide further experimental evidence for the mechanism of signal transmission in the retina. 2. Methods 2.1. Animals and tissue preparation Wild-type Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Research Center of the Fourth Military Medical University. CSNB rat models that were developed by the Fourth Military Medical University [8,29] and were maintained under a 12 h light-dark cycle with an average ambient light of 25–37 W/cm2 (170–250 lx; measured with IL1400 photometer, International Light Technologies, Peabody, USA). The day of birth was designated postnatal day (PND) 0. The pups were randomly assigned to different groups, which were sacrificed at different ages (PND 15, 30 and 60). Animals were anesthetized with a lethal dose of sodium pentobarbital (1,20,000 ng/g, i.p.) or decapitated [10,21]. All procedures that involved animals adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of the Fourth Military Medical University. 2.2. Histology Both of the eyes from CSNB rats and wild type rats were examined at PND 15, 30 and 60. Following deep anesthesia, the eyes were enucleated and immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h. The anterior portions of the eyes were then removed, and the posterior portions were fixed for an additional 12 h. The eyecups were dehydrated in an alcohol series and then embedded in paraffin. Sections 5 m thick were cut along the vertical meridian through the optic nerve head and stained with hematoxylin and eosin. 2.3. Immunocytochemistry The retinal sections were incubated overnight with a primary antibody against Calbindin D-28K (rabbit monoclonal; 1:2000, Chemicon, Southampton, UK) in 5% normal bovine serum albumin (BSA), 0.5% Triton X-100, and 0.02% sodiumazide in phosphatebuffered saline (PBS). The sections were then washed in PBS and incubated with the biotinylated secondary antibody, followed by the avidin–biotin–peroxidase detection system (Universal Vectastain SABC Elite Kit, Boster, WuHan, China) for 2 h. Using 3, 3-diaminobenzidine (DAB) as the chromogen, the progress of the immunoreaction was monitored under a light microscope. The reaction was stopped by the removal of the DAB followed by buffer washes. Control sections were treated similarly, except that the primary antibody was omitted from the procedure. The number of calbindin D-28K-labeled cells was counted at a magnification of 400×. For each animal, the results obtained from six separate sections were averaged. 2.4. Wholemount immunocytochemistry Wholemount immunocytochemistry was performed as previously described [9,21,22,28]. 6 rats in each group were used and for each rat, left eyes were enucleated after an anesthetic overdose and fixed in 4% paraformaldehyde for 1 h. The dorsal pole of each eye was marked with a suture before enucleation. The anterior parts of the eyes were then removed, and the eyecups were fixed overnight. The neural retinas were separated from the pigment epithelium and were thoroughly washed. Retinal wholemounts were rinsed
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in phosphate buffer, preincubated in 10% normal donkey serum and 1% Triton-X100 in PBS for 3 h and rinsed with PBS. The tissues were then incubated with agitation for 3 days at 4 ◦ C in a cocktail of primary antibodies and 1% Triton-X100 in PBS. Following incubation, the tissues were rinsed in PBS and incubated in the FITC-labeled secondary antibody overnight and then rinsed in PBS. Finally, nuclei were stained with 4 ,6-diamidino-2-phenylindole for 1 h. Four incisions were then made in the retina, thus dividing it into dorsal, ventral, temporal and nasal quadrants. Retinas were then mounted on clean slides under a coverslip. All reagents and washing buffers contained 0.1% Triton X-100 to permeabilize membranes. The number of calbindin D-28K-labeled cells was counted at a location centered 1 mm from the optic nerve head at a magnification of 400×. Wholemounts from CSNB rats and wild type rats were sampled in four quadrants at central and peripheral locations (i.e., eight fields per retina) to determine the average densities of calbindinimmunopositive horizontal cells. Fields were selected randomly at low magnification, without respect to cell density, at comparable eccentricity in each quadrant. Sampled fields were 0.225802 mm2 for the horizontal cells [21].
2.5. Western blotting The retinas were lysed with 100 l RIPA (Beyotime, BeiJing, China) and placed on ice. The extracts were then sonicated and centrifuged at 15 ◦ C for 10 min at 12,000 × g. The supernatant was collected, and the protein concentration was measured. A 100 g aliquot of protein per sample was fractionated by SDS–polyacrylamide gel electrophoresis and electroblotted to activated polyvinylidenefluoride membrane. The blot was incubated at room temperature for 1 h in a blocking solution (5% BSA prepared in Tris-buffered saline with 0.1% Tween 20, pH 7.4), followed by overnight incubation at 4 ◦ C in the primary antibody diluted in the blocking solution. The primary antibodies included rabbit anti-calbindin (1:1000; Chemicon, Temecula, CA), and mouse anti-actin (1:2000; Sigma–Aldrich, St. Louis, MO). The blot was then probed with a horseradish peroxidaseconjugated secondary antibody and developed by the enhanced chemiluminescence method. The immunoreactive bands were detected with a Chemi Genius Bioimaging system. Densitometric analysis was performed with Image software. Following background subtraction, the signal for each protein was normalized to the loading control, -actin. The data (integrated densities) were expressed as the change in protein levels of CSNB rats relative to the controls [16].
2.6. Quantitative analysis Quantitative studies were performed in 6 animals (males and females) per time point (PND 15, 30 and 60) and per treatment (SD and CSNB), selected randomly from different litters. All measurements were performed relying on a proven system of random sampling. Photomicrographs were coded and analyzed blindly to avoid experimental bias. Statistical analyses of the measure of number of HCs at different groups and times were made using independent samples of nonparamentric test and Nemeyi test, and statistical analyses of the thickness of OPL, density of HCs and Calbindin levels were made using two-way ANOVA and Tukey test of post hoc test in the SPSS statistical software program (version 14.0).
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Fig. 2. Immunocytochemistry pictures of wild type SD and CSNB rat retinas at PND15, 30 and 60. A,A1,C,C1,E,E1: SD rat retina; B,B1,D,D1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 20 m in length. The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length. Fig. 1. Representative histological sections of thickness of OPL at SD and CSNB rat retinas. A,A1,C,C1,E,E1: SD rat retina; B,B1,D,D1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 20 m in length. The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length.
3. Results 3.1. Results of histology and immunochemistry To analyze the thickness of the OPL, hematoxylin and eosin (HE) stained retinal sections from CSNB rats were compared with their respective control rats (Fig. 1). The results showed that the thickness of the OPL was reduced (P < 0.01) in CSNB rats when compared with age-matched wild type rats (Fig. 4A). To determine whether there were any changes in the number of HCs stained retinal sections from CSNB rats were compared with wild type rats. HCs were labeled with calbindin, a calcium-binding protein and a known marker for HCs and their processes, particularly their proximal dendrites [20]. Control retinas showed the staining of bodies and processes in the OPL (Fig. 2A, C and E). In contrast, labeling for HCs in affected retinas was obviously reduced (Fig. 2B, D and F; Fig. 4B). To observe the change at different ages, we compared data at different ages in each group. There were decreases at the number of HCs (P < 0.01) in PND60 compared to the other two time points in CSNB rats and wild type rats (Fig. 4B). The mean number of HCs was compared from the center to the periphery of the retina as in previous studies [3,23] and no obvious differences between central and peripheral retina were found in either wild type or CSNB rats at PND 15, 30 and 60 (P > 0.05).
Fig. 3. Wholemount immunocytochemistry of the retinas from SD and CSNB rats at 15, 30 and 60 days. A,A1,B,B1,C,C1: SD rat retina; D,D1,E,E1,F,F1: CSNB rat retina. A,B,C,D,E,F: 400×, the bar denotes 50 m in length.The image of the area marked by the box in A,B,C,D,E,F is shown at a higher magnification (1000×) in A1,B1,C1,D1,E1,F1, the bar denotes 10 m in length.
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Fig. 4. Statistical of wild type rat and CSNB rat retinas at PND15, 30 and 60. (A) OPL thickness. *: difference between CSNB group and SD group, ** p < 0.01. (B) Mean horizontal cells numbers. *: difference between CSNB group and SD group, ** p < 0.01; #: difference among three time points within each group, ## p < 0.01. (C) Mean horizontal cells densities of SD and CSNB rats. *: difference between CSNB group and SD group, * p < 0.05, ** p < 0.01; #: difference among three time points within each group, ## p < 0.01.
3.2. Results of wholemount immunocytochemistry HCs were marked with Calbindin in both wild type and CSNB rats (Fig. 3). All the cells were counted that fell within the area of graticule including cells overlapping the lower and left borders, but excluding those overlapping the other borders according to the Chu method [3]. Measurements showed that the eyepiece graticule covered an area of per 40× visual field (0.096 mm2 ) which were used to calculate the density of HCs [3]. Statistics showed that there was a decrease in the average density of HCs in CSNB rats at the three ages tested when compared with wild type rats (Fig. 4C). There were no obvious differences in the distribution of average densities of HCs either in wild type or CSNB rats (P > 0.05). To look for changes at different ages, we compared the data at the different time points in each groups, and showed that both in CSNB and wild type rats, there was significant difference between PND15 and PND 60 (P < 0.01). 3.3. Results of western blotting Retinal Calbindin protein levels in wild type and CSNB rats were observed by Western blotting (WB). The results showed that the Calbindin levels in the retina were lower in CSNB rats than in wild type rats at PND15 and PND60 (P < 0.05), but there was no obvious differences at PND30 (P > 0.05) (Fig. 5). We compared the data at the different time points within each group, and found that no obvious difference in the level of Calbindin protein was observed both in CSNB and wild type rats (P > 0.05). 4. Discussion Some retinal degeneration can cause loss of photoreceptors accompanied by significantly morphological changes in the inner second-order retinal neurons, including dendritic retraction and neuritic sprouting in horizontal and bipolar cells [4–6,11,14–16,18,25,26]. The morphology of HCs is plastic, and the morphology of the cell soma and synapse changes with the alteration of environment or disease state [1,13,16,20,21,24], but there are few reports on the number of HCs lost. In this study, we found that there was a decrease in the number of HCs in CSNB (one of retinal degeneration diseases) rats compared to wild type rats. In adult CSNB rats, around 43.25% of HCs were lost compared to wild type rats. Meanwhile, the outer plexiform layer became thinner in the retinas of CSNB rats. It was previously reported in nob2 mice, which is also caused by a Cacna1f gene mutation, that the dendritic morphology of HCs was relatively skeletal at PND 10 and mature stages, but it did not mention any change in the number of HCs [21].
Fig. 5. Representative immunoblots for Calbindin and -action for SD and CSNB rats. Immunoblot analysis showing a reduction in calbindin levels of CSNB rats. *: difference between CSNB group and SD group, * p < 0.05.
In CSNB rat retinas, the rod system is damaged mainly [8]. Some studies proposed that the HC axon does not conduct nerve impulses but rather provides metabolic support for its terminal arbor, which is selectively innervated by the spherules of rod photoreceptors and serves as an independent functional entity comparable with the dendritic domain [20]. If it is true that part of HCs are lost in CSNB rats, it suggests that the lost HCs might be CACNA1F gene dependent mediately. The mutation in Cacna1f gene might disturb the metabolism or function in some kind of neurons and caused the HCs lost during development process. This study hints that the change of inherited retinal disease may influence the retinal HCs indirectly, which will need to be addressed in future studies. References [1] P.R. Bayley, C.W. Morgans, Rod bipolar cells and horizontal cells form displaced synaptic contacts with rods in the outer nuclear layer of the nob2 retina, Journal of Comparative Neurology 500 (2007) 286–298. [2] B. Chang, J.R. Heckenlively, P.R. Bayley, N.C. Brecha, M.T. Davisson, N.L. Hawes, A.A. Hirano, R.E. Hurd, A. Ikeda, B.A. Johnson, M.A. McCall, C.W. Morgans, S. Nusinowitz, N.S. Peachey, D.S. Rice, K.A. Vessey, R.G. Gregg, The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses, Visual Neuroscience 23 (2006) 11–24.
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