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Light deprivation delays morphological differentiation of bipolar cells in the rabbit retina☆

Mu-Ling Wua, Chuan-Chin Chiaoa,b,*

aInstitute of Molecular Medicine, National Tsing Hua University, Hsinchu, 30013, Taiwan bDepartment of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan

Abstract

Bipolar cells are responsible for transmitting light signals from the photoreceptors to the ganglion cells in the vertebrate retina. Their maturation process is not only important for establishing normal visual function, but may also underlie the dendritic remodeling of ganglion cells during development. It is known that light deprivation affects the synaptic connections of ganglion cells in the mammalian retina, but little is known about impact of visual experience on bipolar cell development. We used dye injection and gene gun labeling to identify bipolar cells, and characterized their morphological differentiation in normal-reared and dark-reared rabbits. Our results show that immature bipolar cells can be found as early as P1–3, and most characteristic bipolar cells can be identified during P4–6. More importantly, we found that light deprivation causes a delay rather than a permanent arrest of bipolar cell maturation in the rabbit retina. By eye opening at P10–11, both normal-reared and dark-reared rabbits possessed adult-like bipolar cells. This suggests that visual experience has a facilitating effect on the morphological differentiation of bipolar cells.

☆ This work is supported by the National Science Council (NSC-94-2311-B-007-018), the Brain Research Center of UST, and the Ministry of Education, Taiwan. * Corresponding author. Institute of Molecular Medicine, National Tsing Hua University, 101, Section 2 Kuang Fu Road, Hsinchu, 30013, Taiwan. Fax: +886 3 571 5934.

E-mail address: ccchiao@life.nthu.edu.tw (C.-C. Chiao).

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1. Introduction

Throughout the retinal development, both intrinsic and extrinsic cues are important for neural cell maturation (Livesey and Cepko, 2001; Wong and Godinho, 2003; Chalupa and Gunhan, 2004). Experiments in several vertebrate species have demonstrated that the spontaneous activities of developing ganglion cells and visual experience play significant roles in retinal network development (Sernagor and Grzywacz, 1996; Lohmann et al., 2002; Tian and Copenhagen, 2003; Zheng et al., 2004). However, the mechanisms underlying the precise morphological differentiation of bipolar cells are largely unknown (Morgan et al., 2006; Schroeter et al., 2006).

Bipolar cells are important interneurons responsible for transmitting light signal from

the photoreceptors in the outer retina to the amacrine and ganglion cells in the inner retina (Masland, 2001; Wässle, 2004). They can be classified into two functionally distinct types, namely ON and OFF bipolar cells, according to their physiological responses to light stimulation and the stratifications of their axonal terminals in the inner plexiform layer (IPL) (Kaneko, 1970; Famiglietti and Kolb, 1976; Nelson and Kolb, 1983; Saito, 1987). Precise ON/OFF segregation of bipolar cells during retinal development is crucial for ensuring correct visual signal transmission to the brain. Furthermore, excitatory inputs from the bipolar cells may modulate reorganization of the ganglion cell dendrites in the IPL (Chalupa and Gunhan, 2004).

The development of bipolar cells in the vertebrate retina has been well documented in

chicks (Quesada et al., 1981; Quesada and Genis-Galvez, 1985), rats (Morest, 1970), ferrets (Miller et al., 1999), and recently in mice (Morgan et al., 2006) and zebrafish (Schroeter et al., 2006). The intrinsic factors regulating bipolar cell development have been extensively studied (Burmeister et al., 1996; Bramblett et al., 2004; Chow et al., 2004; Cheng et al., 2005; Rowan and Cepko, 2005); however, the extrinsic factors that determine the stratification pattern of bipolar cells have not been conclusively identified. Ablation of ganglion cells (Gunhan-Agar et al., 2000) and depletion of cholinergic amacrine cells (Gunhan et al., 2002) at birth in mouse retinas do not disturb the axonal stratifications of bipolar cells in the IPL. Blocking AII amacrine cells during development has no significant effect on the axonal ramification of rod bipolar cells (Rice et al., 2001). This evidence indicates that bipolar cell development is not sensitive to perturbations of the inner retinal neurons, although perturbed amacrine cell lamination in the lakritz zebrafish mutant does affect bipolar cell axonal stratification (Kay et al., 2004). In contrast, the stratification patterns of cat retinal ganglion cells in the IPL are affected by intraocular injection at birth of the glutamate analog 2-amino-4-phosphonobutyrate (APB) (Bodnarenko and Chalupa, 1993). This result is commonly interpreted as suggesting that glutamate-mediated afferent activity regulates the formation of the ON and OFF pathways. Light deprivation, which alters glutamate-mediated afferent activity, thus may affect ganglion cell development ( Vistamehr and Tian, 2004; Mehta and Sernagor, 2006) and perhaps bipolar cell development as well. Although a recent study has shown that rod bipolar cells are able to develop normally without rod photoreceptors (Strettoi et al., 2004), these mutant mice are still able to detect light using their cone photoreceptors, thus this glutamate-mediated afferent activity is still a possible factor that may regulate bipolar cell development.

In this study, we specifically tested whether light deprivation at birth will affect the

axonal stratification of bipolar cells in the rabbit retina. Using a gene gun technique and dye

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injection, we labeled 419 bipolar cells in normal-reared and dark-reared rabbits at different postnatal stages. To our surprise, we found that light deprivation causes a delay rather than a permanent arrest of bipolar cell maturation.

2. Results

Cells identified in the different retinas were grouped together according to the postnatal time in days (P0–1, P2–3, P4–5, P6–7, P8–9, and P10–P14) and the rearing conditions (normal and dark). Consistent with previous morphological studies of bipolar cell development in chicks (Quesada et al., 1981), ferrets (Miller et al., 1999), mice (Morgan et al., 2006), and zebrafish (Schroeter et al., 2006), our results show a similar morphological differentiation pattern (Fig. 1a). There were no distinguishable bipolar cells present at birth in the rabbit retina. The cell soma was elliptical or elongated. Their processes were branched and extended significantly into both the neuroblastic layer (NBL) and the ganglion cell layer (GCL). In contrast to an early immunohistochemical study of bipolar cell development in the rabbit retina, which stated that rod bipolar cells could not be identified until P6 (Casini et al., 1996), the first recognizable bipolar cells could be detected as early as P1 in this study, and most likely were cone bipolar cells. The development of bipolar cells in the rabbit retina typically takes 4–6 days for them to fully mature in the light rearing condition and by P8 most bipolar cells have reached their adult-like morphology.

Fig. 1. Morphological differentiation of bipolar cells throughout their developmental stages. All cells in both (a) normal-reared and (b) dark-reared rabbits were labeled by tungsten particles coated with DiI. Images are collapsed confocal z-stacks and superimposed on the phase contrast image to show the retinal lamination. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar = 20 μm.

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In contrast to morphological differentiation of bipolar cells in the normal-reared rabbits, maturation of bipolar cells in the dark-reared condition showed a different temporal pattern (Fig. 1b). The development of the bipolar cells was delayed rather than arrested under the light deprived condition. Initially, the cell morphology in the normal- and dark-reared rabbits was similar. However, cells in the dark-reared rabbits retarded their morphological differentiation from P2 to P5. After P5, most cells appeared to resume their developmental process, and reached a similar maturation level at P10 as their normal-reared counterparts. More selected images of bipolar cells for the different age groups in the normal-reared and dark-reared rabbits are shown in the supplementary material (Figs. S1 and S2). To quantify the dark rearing effect on bipolar cell development described above, we analyzed several morphological parameters of the labeled cells. Although the distinct bipolar cell types of the rabbit retina have been extensively studied (McGillem and Dacheux, 2001; MacNeil et al., 2004), immature bipolar cells are extremely difficult to classify into their types, except into the ON and OFF categories. Thus, we grouped all labeled cells into four classes at each developmental stage. Since we could not confidently separate bipolar cells from Muller cells in earlier age groups, all unseparated cells with “bipolar-like” appearance were put into the “B/M” category. We used the percentage of the B/M cells among the total labeled cells as an indication of bipolar cell differentiation. All labeled cells with recognizable bipolar cell characteristics were separately placed into either the ON or OFF type category, depending on their axonal stratifications in the IPL. However, some cells with distinct bipolar cell characteristics but with their axonal terminals either ramified in both ON and OFF layers or diffusely stratified across the ON/OFF border of the IPL were put into another category, called “undefined BP” cells.

As expected, the percentage of the B/M cells was high at P1–3 among the normal-reared rabbits, fell significantly at P4–7, and all cells with bipolar-like appearance could be easily identified as either bipolar cells or Muller cells after P8 (Fig. 2a). In contrast, while the percentage of the B/M cells fell gradually throughout the developmental stages in the dark-reared rabbits, some proportion of B/M cells remained even at P8–9 (Fig. 2b). Nevertheless, all cells could be confidently recognized after P10. This indicates that light deprivation delays morphological differentiation in bipolar cells. Another way of looking at this result is to compare the percentage of all recognizable bipolar cells among the total labeled cells under both normal and dark rearing conditions. Fig. 2c shows this comparison. At birth, both normal-reared and dark-reared rabbits had low percentages of identified bipolar cells. However, from P2–3 to P4–5, the percentage increased significantly in the normal-reared rabbits, and leveled off after P4–5. By P8–9, all bipolar cells were adult-like. In contrast, the percentage increased steadily in the dark-reared rabbits, but showed a significant delay over P2–7. Nevertheless, all bipolar cells were adult-like by eye opening at around P10–11.

In addition to the light deprivation effect on morphological differentiation of the bipolar cells, both rearing conditions showed a pattern of ON/OFF development asymmetry. The recognizable OFF bipolar cells appeared to exist in a higher proportion than the ON bipolar cells during the earlier developmental stages (Figs. 2a and b). This difference diminished after P6–7 in the normal-reared rabbits and P8–9 in the dark-reared rabbits. By eye opening, ON and OFF bipolar cells had reached roughly equal numbers. This observation is consistent with a previous study showing that the OFF bipolar cells develop earlier than ON bipolar cells (Sherry et al., 2003).

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Fig. 2. Light deprivation delays the number of recognizable bipolar cells in early developmental stages. The proportion of cell types changes throughout the developmental stages in both (a) normal-reared and (b) dark-reared rabbits. (c) The percentage of bipolar cells (including ON, OFF, and undefined BP cells) increases as the retina development proceeds in both normal-reared and dark-reared rabbits.

Previous studies of bipolar cell development in other animals have shown that the immature bipolar cell soma is more elliptical than that of mature bipolar cells (Quesada et al., 1981; Miller et al., 1999; Morgan et al., 2006), thus we sought to quantify the soma aspect ratio as an index of bipolar cell maturation. The length and width of the soma of individual cells were measured and used to calculate the soma aspect ratio (the soma length divided by the soma width). The lower the soma aspect ratio is, the maturer the cell is. Our results show that both normal- and dark-reared rabbits displayed a decreasing soma aspect ratio from birth to eye opening (Fig. 3). However, the dark-reared animals had a more elongated soma than their normal-reared counterparts between P2 and P5, but the difference diminished after P6–7.

Fig. 3. Light deprivation delays the decrease in the soma aspect ratio. The soma aspect ratio (defined as the soma length divided by the soma width) decreases throughout developmental stages in both normal-reared and dark-reared rabbits.

The soma location of bipolar cells in the adult retina is typically in the upper two-third of the INL. However, the soma of developing bipolar cells could range across all levels of the INL (Quesada et al., 1981; Miller et al., 1999; Morgan et al., 2006). We divided the INL into three equal layers (N1, N2, and N3), and estimated the cell location of all labeled cells. It appears that around 20% of labeled cells had their soma in the lower one-third of the INL (N3)

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between P0 and P3 in the normal-reared rabbits (Fig. 4a). This percentage dropped in P4–5 and P6–7, and disappeared after P8. By contrast, a significant portion of soma remained in the lower one-third of the INL (N3) from birth to P8–9 in the dark-reared rabbits (Fig. 4b). This result indicates that immature bipolar cells are more abundant in the dark-reared condition than in the normal-reared condition, especially between P4 and P9.

Fig. 4. Light deprivation delays the disappearance of soma located in the lower one-third of the INL. The proportion of cells with soma located in the INL changes throughout the developmental stages in both (a) normal-reared and (b) dark-reared rabbits.

In summary, light deprivation has a significant effect on the morphological maturation of bipolar cells in the rabbit retina. Our results suggest that visual stimulation is a facilitating factor for the normal development of bipolar cells, but visual deprivation does not permanent arrest the maturation of bipolar cells.

3. Discussion

Earlier studies indicate that axonal arbors of bipolar cells diffusely ramify in the IPL initially, and then become narrowly stratified throughout as their development progresses (Quesada et al., 1981; Miller et al., 1999). However, recent studies seem to support the idea that axonal terminals of developing bipolar cells are preferentially determined to enter either the ON or OFF layer of the IPL (Morgan et al., 2006; Schroeter et al., 2006). Our results also show that bipolar cells have preferred ON or OFF arborizations once they are recognizable as bipolar cells.

Previous electron-microscopic studies in mammalian retinas have shown that ribbon

synapses appeared first in the OFF layer and later in the ON layer of the IPL during development (Olney, 1968a,b;McArdle et al., 1977, Crooks and Morrison, 1989; Hendrickson, 1996). However, a study of ferret bipolar cell development did not find any temporal asymmetry between ON and OFF layer stratification (Miller et al., 1999). In contrast, Sherry et al. (2003) reported that the expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in the development of ON vs. OFF circuits. In this study, we also observed this temporal asymmetry between ON and OFF layers, whereby the OFF bipolar cells apparently developed earlier than the ON bipolar cells.

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Disruption of glutamate-mediated interactions during development prevents patterning of ganglion cell's dendritic stratification (Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995; Bodnarenko et al., 1999). It is possible that bipolar cells facilitate remodeling of ganglion cell dendrites by providing spatially localized signals. Our results show that morphologically identified bipolar cells can be detected as early as P1–2, suggesting a potential role for bipolar cells in ganglion cell development.

Bipolar cells have been considered the last neuronal cells to develop in the retina, and

thus are thought to be influenced by other early-developed retinal neurons (Wong and Godinho, 2003). The delayed bipolar cell development under the dark rearing conditions observed in this study suggests that light may promote the maturation of bipolar cells. APB treatments of developing cat retina caused a delay in the ON/OFF segregation of ganglion cell dendrites in the IPL but did not permanently arrest it (Bodnarenko et al., 1995). In the dark-reared environment, continuous release of glutamate by depolarization of photoreceptors is analogous to treatment with the mGluR6 agonist APB (Bodnarenko and Chalupa, 1993). Thus, this result implies that disruption of glutamate release by APB or light has a similar effect on control of normal retinal circuit formation.

How light deprivation causes a delay in bipolar cell maturation found in this study is

uncertain. It has been demonstrated that light deprivation affects IPL circuitry during development in turtles (Sernagor and Grzywacz, 1996; Mehta and Sernagor, 2006) and mice (Tian and Copenhagen, 2003; Vistamehr and Tian, 2004; Zhang et al., 2005). It is possible that fine tuning of the amacrine and ganglion cells' dendrites may be refined by the axonal terminals of the developing bipolar cells. Our findings imply that light deprivation makes photoreceptors release glutamate in a constant manner and this delays the refining of their target cells (i.e., amacrine and ganglion cells) by the bipolar cells and as a consequence of this, there are alternations in certain properties of the inner retinal circuitry.

4. Experimental procedures 4.1. Tissue preparation

The normal-reared rabbit group was made up of New Zealand White rabbits at the various postnatal stages and these were purchased individually from a local breeder. The dark-reared rabbit group was also made up of New Zealand White rabbits at various postnatal stages, in this case the pregnant rabbits and their kits were kept in a completely dark room in our animal facility from before the kits birth. Before tissue preparation, the animals were anesthetized using a 1:1 mixture of ketamine (75–100 mg/kg) and xylazine (15–200 mg/kg). After enucleation and hemisection, the vitreous humor was removed and the retina was carefully detached from the retinal pigment epithelium. The animal was then euthanized with an overdose of ketamine. All procedures were approved by the institutional animal care and use committee and were in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. The isolated retina was fixed in 4% paraformaldehyde in 0.1 M PB for 20 min. After rinsing, a small piece of retina (8 mm × 5 mm) was embedded in 2% low melting point agarose (Sigma, St. Louis, MO) at 37 °C for vertical section. It is known that retinal neurons develop at different rates at different eccentricities (Robinson, 1990). To ensure similar sampling locations, an area 2 mm away from the visual streak in the ventral side of the retina was chosen. The retinal slices (200–300 μm thick) were subjected to either DiI gene gun labeling or Lucifer Yellow injection.

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4.2. Dye injection

The retina slice was prepared as described above but without fixing. The sample was placed in a perfusion chamber with oxygenated Ames medium on the stage of the fluorescence microscope (Axioskop 2 FS, Zeiss, Germany). An aluminosilicate glass pipette (pulled from a P97 puller, Shutter Instrument) was filled with 4% Lucifer Yellow (in 0.1 M Tris buffer) for microinjection. The soma of cells located in the upper two-third of the INL was targeted randomly for dye injection at a fixed location within a reference grid. To ensure complete injection, the soma of cells 50 μm deep in the retinal slice was selected. The dye injection was delivered using an AM1600 intracellular amplifier (A-M Systems, Inc., Carlsborg, WA) with a negative current of 0.3–3 nA for 10–60 s. The injected cells were imaged immediately using a cooled CCD camera (Roper Scientific, Tucson, AZ) for analysis. 4.3. DiI gene gun labeling

A Tefzel tube (Bio-Rad, Hercules, CA) was coated with 0.1 mg/ml polyvinyl

pyrrolidone (PVP) solution in pure ethanol and dried completely in the Tubing Prep Station (Bio-Rad). A total of 4 mg DiI (Molecular Probes #D-288) was dissolved in 200 μl methylene chloride (0.02 M), and mixed with 50 mg of tungsten particles (diameter 1.7 μm). After these were spread out on a clean glass slide, the tungsten particles were scraped with a razor blade onto weighing paper, and transferred into the PVP-precoated tubing. Fine particle dispersion was achieved by holding the tubing into an ultrasonic bath for several minutes.

DiI particles were delivered using a Bio-Rad Helious gene gun device, and the helium

pressure was set at 75–85 psi. A piece of nylon filter (Small Part, Inc. Miami Lakes, FL) with 5 μm pore size and another piece of the same nylon filter with 100 μm pore size were interposed between the gene gun and the specimen. The gene gun was positioned perpendicularly to these nylon filters and 2 cm above the retinal surface. After shooting, the specimen was incubated in Ames medium for 10 min and mounted for confocal microscopy. 4.4. Confocal microscopy and data analysis

Images of the gene gun labeled cells were collected using a Laser Scanning Confocal Microscope (LSM 5 Pascal, Zeiss, Germany). Either a 40× objective lens (Plan-Neofluar, 0.75 NA, Zeiss) or a 63× objective lens (C-Apochromat, 1.2 NA, Zeiss) was used, depending on size of the cell.

Only labeled interneurons that met the following two criteria were examined in this

study: (1) the cell is clearly filled or labeled, and can be distinguished from neighboring cells; (2) the cell has “bipolar-like” processes, that is, one process extends to the outer plexiform layer (OPL), and the other process goes in the opposite direction and reaches to the IPL. The second criterion is unable to separate bipolar cells from Muller cells, especially in earlier postnatal stages when both cell types are developing. A total of 84 injected cells and 335 gene gun labeled cells from 58 normal-reared and dark-reared rabbits were included in this study.

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Acknowledgments We thank Ms. Hui-Ju Yang, Ya-Chien Chan, and Chi-Cheng Wat for technical

assistance with the dark-rearing condition, and Dr. Ralph Kirby for improving the English of the manuscript. This work is supported by the National Science Council of Taiwan (NSC-94-2311-B-007-018).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2007.06.091.

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