Functional and structural modifications during retinal degeneration in the rd10 mouse

Neuroscience 155 (2008) 698 –713

FUNCTIONAL AND STRUCTURAL MODIFICATIONS DURING RETINAL DEGENERATION IN THE rd10 MOUSE R. BARHOUM,a1 G. MARTÍNEZ-NAVARRETE,b1 S. CORROCHANO,c F. GERMAIN,a L. FERNANDEZ-SANCHEZ,b E. J. DE LA ROSA,c P. DE LA VILLAa1 AND N. CUENCAb1*

half of RP patients have a mutation in genes that encode proteins involved in the phototransduction cascade. Mutations in the gene encoding the ␤ subunit of the cGMP phosphodiesterase (␤-PDE) have been identified in patients with autosomal recessive RP (McLaughlin et al., 1993), which account for approximately 5% of such cases in the world. The identification of these mutations has facilitated the development of mouse models of retinal degeneration, which have been used to study the cellular and molecular mechanisms of photoreceptor degeneration (reviewed in Chang et al., 2002). The rd1 mouse is one of the best known models of retinal degeneration (Pdebrd1⫺/rd1⫺, for review see Farber et al., 1994). In this model, a point mutation in exon 7 of the ␤-PDE gene leads to the rapid, large-scale loss of rod photoreceptors within 15–20 days of postnatal life (P15– 20, Portera-Cailliau et al., 1994). Despite this rapid degeneration, the rd1 model has been widely used to study the mechanisms underlying retinal degeneration (Doonan et al., 2003; Hart et al., 2005). The structural modifications to retinal neurons following photoreceptor degeneration have been characterized in rd1 animals (Marc et al., 2003; Jones and Marc, 2005; Strettoi and Pignatelli, 2000; Strettoi et al., 2003), and the sensitivity of degenerated rod postsynaptic retinal cells to retinal neurotransmitters has also been addressed in this model (Varela et al., 2003). Accordingly, the rd1 mouse model of retinal degeneration has been used to test therapeutic approaches that may inhibit the process of photoreceptor apoptosis and degeneration (Frasson et al., 1999a,b; Takano et al., 2004; Komeima et al., 2006; MacLaren et al., 2006). Other animal models used to study how progressive photoreceptor degeneration affects rod and cone relay pathways include a rat model with a recessive mutation generated by the Royal College of Surgeons (RCS), in which the gene encoding for the Mertk tyrosine kinase receptor is disrupted (D’Cruz et al., 2000; Dufour et al., 2000). In this rat model, retinal pigment epithelial cells fail to phagocytose the rod outer segments shed, leading to the generation of a zone of debris of outer segment material and progressive photoreceptor degeneration (Cuenca et al., 2005). The P23H transgenic rat is a model of autosomal dominant RP, in which a mutation in the rhodopsin gene leads to a loss of rods and to a more protracted loss of cones (Cuenca et al., 2004). In both animal models, there is substantial loss of rods and a reduction of rod bipolar dendrites by P21. During the course of photoreceptor loss, the progressive changes mainly affect cells that are involved in the rod relay pathway. By P60, a few cells still remain in the outer nuclear layer, whereas in the rd1

a

Departamento de Fisiología, Universidad de Alcalá, Alcalá de Henares 28871, Spain

b

Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante E-03080, Spain

c

3D Lab (Development, Differentiation and Degeneration), Departamento de Fisiopatología Celular y Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas CSIC, Madrid 28040, Spain

Abstract—Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm2. In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with agematched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: retina, rod, cone, bipolar cells, phosphodiesterase, apoptosis.

Retinitis pigmentosa (RP) includes a large group of inherited retinal disorders that cause progressive loss of retinal function. Indeed, it represents one of the main causes of blindness in the world, with an incidence of approximately one in 4000 humans (Berson, 1993). Approximately one 1

These authors contributed equally to this work. *Corresponding author. Tel: ⫹96-5903400; fax: ⫹96-5909569. E-mail address: [email protected] (N. Cuenca). Abbreviations: ERG, electroretinogram; IPL, inner plexiform layer; mGluR6, metabotropic glutamate receptor 6; P, postnatal day; RCS, Royal College of Surgeons; RP, retinitis pigmentosa; ␤-PDE, ␤ subunit of the cGMP phosphodiesterase.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.06.042

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mouse the rods begin to degenerate around P8 –P10, and by P20 only a few scattered photoreceptors are left (Strettoi et al., 2002). Recently, another mouse model with a spontaneous mutation was described, the rd10 strain (Chang et al., 2007; Gargini et al., 2007). The rd10 mouse strain is a newly described model of retinal degeneration that bears a point mutation in exon 13 of the ␤-PDE gene (Chang et al., 2002, 2007). In the rd10 mouse, the specific mutation in ␤-PDE also leads to a massive loss of rod photoreceptors in the first weeks of postnatal life, although the time course of retinal degeneration seems to be slower than in the rd1 mouse. Morphological and physiological studies (Gargini et al., 2007) have shown that the rd10 mouse model may be a good model to assess rescue approaches, since photoreceptor degeneration starts around P18. The delay of rod death in the rd10 mouse means that retinal degeneration does not coincide with the period of photoreceptor maturation, as is the case in the rd1 model. The time course of retinal degeneration in rd10 has been described (Gargini et al., 2007) and it starts at P18 peaking around P25. Furthermore, the secondary degeneration experienced by bipolar and horizontal cells postsynaptic to degenerated rods was also studied. Indeed, electroretinogram (ERG) studies revealed that the alteration in the physiology of the inner retina parallels the structural degeneration. To gain insight into the progress of photoreceptor degeneration and the associated secondary events, we carried out structural and electrophysiological experiments in the rd10 mouse model of rod degeneration (Pdebrd10⫺/rd10⫺). Here, we have characterized several aspects of rd10 retinal degeneration that have not yet been addressed. We have counted the degenerating photoreceptors, which allowed us to estimate the number of photoreceptors undergoing degeneration at different times. Moreover, we studied cone and rod morphology during retinal degeneration to gain an idea of the ability of cone cells to survive rod degeneration. During this period, we also studied modifications in the connectivity between rods, cones and their postsynaptic bipolar and horizontal cells. Indeed, our structural study looked further into the inner retina to describe the morphological alterations experienced by cells in the rod pathway. Finally, to test whether rod degeneration determines functional modifications in inner retinal cells, we studied the effect of retinal neurotransmitters on bipolar cells postsynaptic to degenerated rods. These studies complete the information regarding the rd10 mouse model of retinal degeneration (Chang et al., 2007; Gargini et al., 2007), providing a more complete morphological and functional characterization of this rd10 model and highlight the benefits of using this model in future research.

EXPERIMENTAL PROCEDURES Animals C57BL/6j (wild type) and C56BL/6jrd10/rd10 (rd10) mice were used in the experiments at P20, P25, P30, P35, P40 and P60. All animals were obtained from the Jackson Laboratory (Bar Harbor,

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ME, USA), and they were maintained and bred at the University of Alcalá on a 12-h light/dark cycle. Light cycle illumination of 30 cd/m2 was measured with a photometer (Mavo Monitor USB, Gossen, Nürnberg, GE). All animals were handled in accordance with the European Union guidelines for the use of laboratory animals (European Directive 86/609/EEC), minimizing animal suffering and numbers used for experiments.

ERG recordings Prior to ERG recording, mice were adapted to the dark overnight. The mice were then anesthetized under dim red light by i.p. injecting a solution of ketamine (95 mg/kg) and xylazine (5 mg/kg) and they were then maintained on a heated pad at 37 °C. The pupils were dilated by topical application of 1% tropicamide (Colircusí Tropicamida, Alcon Cusí, SA, El Masnou, Barcelona, Spain) and to optimize electrical recording, a topical drop of 2% Methocel (Ciba Vision AG, 8442 Hetlingen, Switzerland) was instilled in each eye immediately before the corneal electrode was put in place. Anesthetized animals were placed on a Faraday cage and all experiments were performed in absolute darkness. The eyelids of the mice were separated to optimize electrode application and light stimulation. Flash-induced ERG responses were recorded from the right eye in response to light stimuli produced with a Ganzfeld stimulator. The intensity of the light stimulus was measured with a photometer (Mavo Monitor USB) at the level of the eye and for each intensity of light, an average of 4 to 64 consecutive light stimuli was presented. The interval between light flashes applied in scotopic conditions was 10 s for dim flashes and up to 60 s for the highest intensity. Under photopic conditions, the interval between light flashes was fixed at 1 s. The ERG signals were amplified and band filtered between 0.3 and 1000 Hz with a Grass amplifier (CP511 AC amplifier, Grass Instruments, Quincy, MA, USA). Electrical signals were digitized at 10 kHz with a Power Laboratory data acquisition board (ADI Instruments, CA, USA). Recordings were saved on a PC and analyzed off line. Bipolar recordings were obtained using an Ag:AgCl mouse electrode fixed on a corneal lens (Burian-Allen electrode, Hansen Ophthalmic Development Laboratory, Coralville, IA, USA), a reference electrode located in the mouth, and with a ground electrode located on the tail. The electrode was mounted on a coarse micromanipulator for easy positioning over the mouse eye. Rod-mediated responses were recorded under dark adaptation following light flashes ranging from ⫺4 to ⫺1.52 log cd·s·m⫺2. Mixed rod- and cone-mediated responses were recorded following light flashes ranging from ⫺1.52– 0.48 log cd·s·m⫺2. Cone-mediated responses were recorded following light flashes ranging from ⫺0.52–2 log cd·s·m⫺2 on a rod saturating background of 30 cd/m2. The amplitude of the a-wave was measured from the baseline to the trough of the a-wave and the results were averaged. Likewise, the amplitudes of the b-wave were measured from the trough of the a-wave to the peak of the b-wave and averaged. Measurements were recorded by an observer who was blind to the experimental condition of the animal and the statistical analysis was performed using the Student’s t-test.

TUNEL-labeling of whole-mounted retinas Animals were decapitated at P20, P25, P30, P35 and P40, their eyes were removed and the retinas were dissected out and flatmounted on nitrocellulose filter (Sartorius, Göttingen, Germany) with the photoreceptor layer side up. The retinas were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 at 4 °C overnight. To detect apoptotic cells, whole mounted retinas were processed for TUNEL (Promega, Madison, WI, USA) according to the manufacturer recommendations. After labeling, the retinas were counterstained with DAPI and mounted in vectashield (Vector Laboratories, Burlingame, CA, USA) for microscopical observation. A TCS SP2 Laser-confocal microscope (Leica, Microsystems, Wetzlar,

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Germany) was used to acquire images at 10⫻ around the optic nerve head, the total TUNEL-labeled cells were then quantified per mm2 in the central retina and analyzed statistically.

Immunohistochemistry For immunohistochemistry, at least three animals were studied at each time point. Animals were killed with a lethal dose of pentobarbital, and the eyes were enucleated and fixed in 4% paraformaldehyde in PBS for 2 h at 4 °C. The eyecups were then washed in PBS, cryoprotected in 30% sucrose overnight, embedded in OCT and 16 ␮m thick cryostat sections (Leica CM 1900, Leica Microsystems) were mounted on glass slides. Sections were incubated in 10% normal donkey serum for 1 h (Jackson, West Grove, PA, USA) to avoid non-specific staining and they were then single or double immunostained overnight at room temperature with combinations of different primary antibodies diluted in PBS containing 0.5% Triton X-100. All primary antibodies used in this study had been previously shown to be useful in the mouse retina (Table 1). Subsequently, the sections were washed in PBS and exposed to the secondary antibodies, Alexa Fluor 488 – conjugated donkey anti-rabbit IgG (green) and Alexa 546 – conjugated donkey anti mouse IgG (red, Molecular Probes, Eugene, OR, USA) at a 1:100 dilution for 1 h. The sections were finally washed in PBS, mounted in fluoromount Vectashield (Vector Laboratories) and coverslipped for viewing by laser-confocal microscopy (Leica TCS SP2 Leica Microsystems). Immunohistochemical controls were performed by omission of either the primary or secondary antibodies and to identify retinal cells, we used TO-PRO three iodide 1:1000 (Molecular Probes). Unless otherwise indicated, images were obtained from central retinal sections and all images were obtained from the projections of four to six single frames. TIFF images were enhanced using Adobe Photoshop 7.0 software.

Preparation of retinal slices Retinal slices were prepared from the P60 mice as described elsewhere (De la Villa et al., 1998). Briefly, mice were deeply anesthetized with diethyl-ether (Katayama Chemical Industries or Sigma Chemical Co.) and after verifying the disappearance of corneal reflexes, animals were killed by cervical dislocation. The animals’ eyes were enucleated and hemisected, and the retinas were detached from the pigment epithelium with fine forceps. Throughout the procedure, the isolated retina was kept in an

ice-cold standard solution containing (in mM), NaCl 135, KCl 5, MgCl2 1, CaCl2 2, glucose 10 and Hepes 10, and with 0.01% bovine serum albumin and Phenol Red 0.001% adjusted to pH 7.4 with NaOH. The isolated retina was attached to nitrocellulose filter paper (Advantec Tokyo, JP, pore size 3.0 ␮m), photoreceptor side up, and negative pressure was applied to the back of the filter paper in order to improve the attachment of the retina to the filter. The retina on the filter paper was then transferred to the recording chamber and it was fixed to the bottom with silicon grease. The chamber was filled with standard solution, placed on the stage of a handmade mechanical slicer and cut vertically in 100 –150 ␮m thick sections together with the filter paper. Several slices were then transferred to the neighboring recording chamber (200 ␮l capacity) and placed cut-side down with both ends fixed with silicon grease. On average, 15 slices were obtained from the central portion of the retina.

Single cell recording procedures Retinal slices were initially continuously perfused at a rate of 0.6 ml/min with standard solution and membrane currents were recorded by patch pipette in the whole-cell configuration (Hamill et al., 1981). The pipette was connected to a current–voltage converter (Axoclamp 200A, Axon Instruments) and an Ag–AgCl indifferent electrode was connected via an agarose-bridge to the superfusate. Holding (Vh) and command (Vp) voltages were generated by a personal computer connected to a Power Laboratory data acquisition board using the Scope® software (ADInstruments, Chalgrove, UK). The time and voltage resolution of the pulse generator were 0.1 ms and 1 mV, and the data were sampled and digitized with a 12-bit A/D converter after passing through an eight-pole Bessel filter. The sampling rate was set at a value between 0.2 and 20 ms depending on the type of analysis, and the sampled data were analyzed off-line using the Scope® software. Peak ion current magnitudes induced by glutamate application were measured and the current amplitudes were averaged from three consecutive drug applications. The pipette solutions contained (in mM) KCl 110, NaCl 10, MgCl2 1, EGTA 5, CaCl2, 0.5, Hepes 10, GTP 1, cGMP 0.1, ATP 1, cAMP 0.01 (adjusted to pH 7.2 with KOH). Patch pipettes were made of Pyrex tubing (1.2 mm o.d.) and they were pulled in two steps (Narishige Scientific Instruments pipette puller, P-83, Tokyo). After heat polishing, the inner diameter of the pipette was about 0.5–1 ␮m. The capacitance of the pipette was compen-

Table 1. Primary antibodies Molecular marker

Antibody (reference)

Source

Working dilution

mGluR6

Rabbit anti-mGluR6 (Nakajima et al., 1993; Cagiano et al., 2007) Rabbit polyclonal (Oh et al., 2007) Mouse, clone MC5 (Johnson et al., 2003; Brand et al., 2005) Rabbit polyclonal (Gong et al., 2007)

Neuromics (Edina, MN, USA)

1:3000

SWant (Bellinzona, Switzerland) Santa Cruz Biotechnology (Santa Cruz, CA, USA) Santa Cruz Biotechnology (Santa Cruz, CA, USA) R.S. Molday, University of British Columbia (Vancouver, Canada) B. Howell, NIH, Bethesda, (MD, USA)

1:500 1:100

J. F. McGinnis, University of Oklahoma (Oklahoma City, OK, USA) Chemicon (Temecula, CA, USA)

1:2000

Cytosignal (Irvine, CA, USA)

1:200

Calbindin D-28K Protein kinase C, ␣ isoform (PKC␣) Protein kinase C, ␣ isoform (PKC␣) Rhodopsin Dab1 Recoverin Synaptophysin Transducin, G␣c subunit

Mouse, clone 4D2 (McNally et al., 1999; Dentchev et al., 2003) Rabbit polyclonal (Rice and Curran, 2000; Lee et al., 2006, 2007) Mouse monoclonal (McGinnis et al., 1999; Chen et al., 1999) Mouse, clone SY38 (Zhang et al., 2006; Martínez-Navarrete et al., 2007) Rabbit polyclonal (Kraft et al., 2005; Zhang et al., 2007)

1:100 1:100 1:300

1:500

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sated electrically and the resistance of the pipette measured in the bath was usually about 5–20 M⍀. After obtaining the gigaseal, the membrane patch under the pipette was ruptured by gentle suction and the voltage clamp condition was established. Electrode series resistance ranged from 10 to 50 M⍀ and it was compensated by up to 80%. The leak current was not subtracted from the data.

Identification of recorded bipolar cells All recorded cells were filled with Alexa 488 (Molecular Probes) during recording and they were morphologically identified under the fluorescence microscope after completing electrical recording. In a series of experiments, the retinal slices were fixed in paraformaldehyde in PBS for 4 h at 4 °C after the electrophysiological experiments were terminated, and they were further processed for laser-confocal analysis. Most cells were identified as bipolar cells as they had the typical round soma and a long axon with terminals expanding in the inner plexiform layer (IPL). Most of bipolar cells recorded had axons ramifying in the innermost part of the IPL, thereby corresponding to on-type bipolar cells (Euler and Wässle, 1995; Euler et al., 1996). Rod bipolar cells were easily identified according to the classification of Ghosh et al. (2004). On occasion, other types of bipolar and amacrine cells were recorded and they were identified by their typical morphology, shape and stratification when viewed under the fluorescence microscope. The present work includes the data from a total of 28 rod bipolar cells from C57BL/6J mice and 38 rod bipolar cells from rd10 mice. Data obtained from other types of bipolar cells and amacrine cells were not included here. All data were averaged from the ionic currents and they are expressed as the mean⫾S.D.

Drug application In most experiments, glutamate (Sigma Chemical Co, St. Louis, WA, USA) was applied under pressure from a puff pipette positioned in the proximity of the recorded cell (De la Villa et al., 1995), using a fine tip pipette (ca. 2 ␮m). The pipette-tip was positioned close to the cell soma for recording.

RESULTS In a series of preliminary experiments, ERG recordings were obtained from a group of control and rd10 animals, and the time course of functional degeneration of rd10 mice was studied. As in previous reports (Chang et al., 2002; Gargini et al., 2007), electroretinographic responses were completely absent at a P40 in the rd10 animals. However, ERG performed at P20 showed that the scotopic and photopic a- and b-waves were already impaired in amplitude when compared with the age-matched control mice (Fig. 1). Time course of photoreceptor death Because the rd10 mouse model is relatively new and it could be especially useful for therapeutic purposes, it is important to fully describe the time course of photoreceptor death in this model (Otani et al., 2004; Boatright et al., 2006). We initially studied the time course of retinal degeneration in the rd10 mouse retina by examining recoverin immunoreactivity, which labels rod and cone photoreceptors, as well as certain types of bipolar cells in rodents (Fig. 2, McGinnis et al., 1999). In the C57BL/6J wild type mouse, antibodies against recoverin showed that the ONL contained 10 –14 rows of photoreceptors with well-organized outer segments aligned in parallel (Fig. 2a). How-

Fig. 1. Electroretinographic responses from control C57BL/6J and rd10 mice at P20. (A) Examples of ERG recordings from a C57BL/6J and a rd10 mice. Rod responses to light flashes of ⫺2.09 log cd·s·m⫺2 and mixed responses (rod and cone) to light flashes of 1.57 log cd·s·m⫺2 were recorded under dark adaptation. Cone responses were recorded under light adaptation to light flashes of 1.57 log cd·s·m⫺2. Horizontal calibration⫽100 ms; vertical calibration⫽50 ␮V. (B) Histogram representing the ERG a- and b-wave amplitudes (mean⫾S.D.) of the rod, mixed and cone responses measured from a group of C57BL/6J (n⫽5) and rd10 (n⫽5) animals.

ever, in the rd10 mouse recoverin staining was distinct from as early as 20 days of age. In these animals the ONL was thinner, containing only eight to nine rows of photoreceptors, and the outer segments were disorganized (Fig. 2b). By P30, there was a drastic reduction in cell number in the ONL and only about three cell rows of recoverinstained photoreceptors could be observed (Fig. 2c). By P40, the ONL was reduced to two to three photoreceptor cell rows, and no inner and outer segments could be identified (Fig. 2d and e). Thus, photoreceptor degeneration in the rd10 mouse model was clearly evident at P20, although we wondered whether any signature of retinal degeneration could be detected at earlier postnatal ages (see below). To determine whether degeneration affected the central and peripheral retina indistinctly in the rd10 mouse, we stained the nuclei of retinal cells with TO-PRO at different ages (Fig. 3). While there were no remarkable differences in the number of photoreceptor rows at P20 (Fig. 3a), by P30 the central retina had degenerated faster than the peripheral retina, only two to three rows of photoreceptor cell nuclei remaining in the central area compared with seven to eight rows in the peripheral retina (inset in Fig. 3b). Thus, retinal degeneration in the rd10 mouse model advances more rapidly in the central retina that in the

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Fig. 2. Cryostat sections of C57BL/6J and rd10 mice retina immunostained with antibodies against recoverin and transducin. Recoverin immunostained cones and rods (red) and transducin immunostained cones (green). (a) Photoreceptor rows labeled in the C57BL/6J mouse. (b–f) The decrease in photoreceptor rows from P20 to P40 in rd10 mice. At P30, cones had a small and short outer segment (d, arrowheads). The photoreceptors that remained at P40 corresponded to cones (f) and pedicles from cones (f, arrows). Scale bar⫽20 ␮m.

periphery. This observation led us to focus our attention on degeneration in the central retina, and the quantification of photoreceptor loss and the specific morphological changes experienced by different retinal cells was henceforth studied in the central retina. To quantify the degree of photoreceptor degeneration in the rd10 mouse retina, TUNEL labeling was performed at different postnatal ages from P20 to P40. A comparison of retinas at different stages showed a temporal pattern of TUNEL-labeling (Fig. 4) as quantified in the central area of the retina (2.25 mm2; comprising around 7.5% of the total retinal surface). The number of dying cells at P20 was already high and it had increased threefold by P25 when the maximum level of cell death was observed. Afterward, cell death diminished progressively until P40, the last age studied here. TUNEL labeling of degenerating cells was also studied in retinal slices (data not shown) although no systematic comparisons were made between the central and peripheral retina. As in other rd mouse models of retinal degeneration, these experiments confirm that intense photoreceptor cell death occurs by apoptosis and that it peaks around P25. To further characterize the changes in photoreceptors and their postsynaptic cells, we further studied retinal structure by dual immunocytochemistry.

Cones and rods It is important to know how cones might be affected by the progression of rod cell degeneration. Antibodies against ␥-transducin specifically label cones and some types of cone bipolar cells. In the C57BL/6J wild type mouse, ␥-transducin immunoreactivity was particularly prominent in the cone outer segments, whereas cone inner segments, cell bodies and pedicles were only lightly stained (Fig. 5a). Cone cell bodies were confined to the outermost layer of the ONL. By contrast, rhodopsin antibodies specifically label rod cells and in the control mouse retina rhodopsin immunoreactivity was exclusively confined to the outer segments (Fig. 5b). While in rd10 mice at P20 the number of cones was similar to that in control mice, their cell bodies were distributed at different levels of the ONL and they had undergone morphological changes (Fig. 5c). Dual immunostaining with antibodies against transducin and rhodopsin showed that in the dystrophic rd10 mouse, even as young as 20 days old, the outer segment staining was different from that in age-matched controls. Although rhodopsin-stained outer segments were evident, there was also rhodopsin staining of the cell bodies (Fig. 5d; arrowheads), as well as in some axons and spherules of the rods (Fig. 5d; arrows). Their outer segments were swollen and fragmented, and normal outer segments were no longer

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Fig. 3. Low magnification cross section of retinas labeled with TO-PRO. (a) Rd10 mouse retina at P20 showing no differences in photoreceptor rows between the central and peripheral retina. (b) Rd10 mouse retina at P30 showing two to three remaining rows of photoreceptors in the central area compared with several rows in peripheral retina. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Insets show magnified areas of the retina (as indicated by the arrows). Scale bar⫽200 ␮m.

evident. At P30, double labeling showed that the ONL was reduced to two to three photoreceptor cell body rows (Fig. 5e), where one row of the remaining photoreceptors was transducin immunoreactive and corresponded to the cones. At this age, there were striking differences in cone morphology (Fig. 5e) and the shortening of the cones, from the pedicle to outer segment, was coupled with short inner and outer segments that appeared to be disrupted and that had lost their normal parallel alignment (Fig. 5e). By P40, the co-localization of recoverin and transducin indicated that the few persisting photoreceptors were mostly cones (Fig. 5 g). These cells were no longer aligned in the ONL and they had a highly abnormal morphology. Indeed, there were no longer any normal outer segments distinguishable

in the cones and they had intense ␥-transducin labeling in their cell bodies, as well as smaller and shorter processes. Most of the photoreceptor axon terminals in the OPL at this age were pedicles of cones and their alignment in the OPL was lost (Fig. 5 g). By P30, double labeling with antibodies against rhodopsin and transducin showed that only one row of photoreceptors was composed of rods (Fig. 5e). The rhodopsin immunoreactivity in the remaining rod cell bodies increased at this age and their outer segments had a distorted morphology (Fig. 5e and 5f). At P40, only a few rod photoreceptors remained and they were distributed sparsely across the thin ONL. All remaining rods exhibited no outer segments and had an abnormal morphology (Fig. 5 g and 5h).

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Fig. 4. Density of TUNEL-labeled cells (means⫾S.D., cells/mm2) around the optic nerve head in the rd10 mouse retina at different degenerative states (P20, P25, P30, P35 and P40). Confocal images of TUNEL-labeled whole-mounted retinas of rd10 mice with a 10⫻ objective around the optic nerve head (✹) is shown for each specific age. Scale bar⫽200 ␮m.

Photoreceptor axon terminals and horizontal cells Horizontal cell bodies are located in the outer part of the INL and make connections with both rod and cone photoreceptors. This sole horizontal cell type in the mouse retina can be identified using antibodies against calbindin. In the C57BL/6J wild type mouse retina, calbindin-labeling produced punctate staining of dendritic arbors protruding from the cell body and thin tangential axonal elongations in the OPL that ended in an extensive arborization (Fig. 6a). Horizontal cell dendrites contacted cone pedicles and their axon terminals connected with rod spherules. To determine how axon terminals change during the course of photoreceptor degeneration we studied the labeling of synaptophysin antibodies as a presynaptic marker of the whole presynaptic photoreceptor profile. When used in conjunction with calbindin, a continuous layer of presynaptic label was juxtaposed to the dendritic tips of the horizontal cells in the OPL (Fig. 6b). In the rd10 mouse at P20, double labeling for synaptophysin and calbindin highlighted the loss of axon terminals in photoreceptors and a retraction of the horizontal cell processes in the OPL (Fig. 6c). In some areas, horizontal cells sent processes deep into the INL around blood vessels, and the laminar array of the OPL was distorted by abnormal blood vessels, indicative of retinal degeneration (Fig. 6c; arrows). By P30, synaptophysin immunoreactivity was restricted to scattered profiles distributed discontinuously in the OPL (Fig. 6d arrowheads). The dendritic processes of horizontal cells were very flat and narrow, and the punctate calbindin staining was greatly diminished, indicating the complete retraction of the dendritic terminals of the horizontal cells (Fig. 6d). As degeneration progressed, at P40 the horizontal cell dendrites and axonal processes were reduced in size and complexity (Fig. 6e). No dendritic terminals were

observed and in most of the retina, the lamination of the OPL was distorted (Fig. 6e). Synaptophysin immunoreactivity was clearly diminished and was only associated with horizontal cell processes, which was quite infrequent (Fig. 6e; arrowheads). Hence, their appeared to be a decrease of synaptic pairing between the photoreceptors and horizontal cells in the OPL. Rod bipolar cells The antibody against the protein kinase C alpha isoform (PKC-␣) labels rod bipolar cells and a subclass of amacrine cells in the mouse retina (Grëferath et al., 1990). In the C57BL/6J mouse retina, the rod bipolar cells have a normal morphology and PKC-stained somata are located in the outermost part of the INL (Fig. 7a). The dendritic terminals of the rod bipolar cells established connections with rod spherules through the large dendritic arbor at the end of a single dendritic trunk in the OPL. The dendritic trees of rod bipolar cells were bushy and erect structures, and they penetrated into the OPL (Fig. 7a; arrows). These cells also develop a single cell axon that runs perpendicular through the IPL and ends at the innermost stratum of the IPL where it forms large terminal end-bulbs with lateral varicosities (Fig. 7a; arrowheads). By P20, rod bipolar cells in the rd10 mouse retina began to display the changes that would become more evident as development proceeded. These included a loss of PKC-immunoreactivity, less profuse dendritic terminals in the OPL (Fig. 7b; arrows), and smaller terminal end-bulbs and varicosities in the proximal sublamina of the IPL (Fig. 7b). By P30, the progression of the degeneration was manifested by the retraction of rod bipolar cell dendrites. These dendrites became flatter and the loss of dendritic terminals above the cell bodies was more evident (Fig. 7c). Indeed, only a few dendritic tips

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Fig. 5. Cryostat sections of retinas stained with antibodies against transducin (red) and rhodopsin (green) showing normal cone and rod outer segments in C57BL/6J (a, b) compared with the fragmented outer segments in rd10 at P20 (c, d) and the short outer segments at P30 and P40 (e, f, arrowheads and g, h). From P20 to P40 (d, f and h), immunoreactivity in rod cell bodies (d, arrowheads) and axons (d and f, arrows) increased. Scale bar⫽20 ␮m.

could be observed in these animals (Fig. 7c; arrows), and the end-bulbs of axonal terminals were reduced in size and complexity (Fig. 7c). At P40, when there were at most two layers of photoreceptors left, the rod bipolar cells had lost their dendrites (Fig. 7d). The loss of PKC-immunoreactivity in the axons and axon terminals of bipolar cells was also evident and their terminals were further reduced in size and density (Fig. 7d).

To evaluate the concomitant loss of postsynaptic receptor molecules at the rod-cone/bipolar synapse we used antibodies against metabotropic glutamate receptor 6 (mGluR6). The mGluR6 is a synaptic marker of rod and cone ON-bipolar cell terminals, and mGluR6 labeling is confined to the tips of bipolar cell dendrites (Masu et al., 1995). By P20, mGluR6 is present in dendritic bipolar cell terminals, producing a layer of continuous punctuate stain-

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Fig. 6. Morphological changes in the outer plexiform layer during retinal degeneration. (a– e) Micrographs of horizontal cells visualized by calbindin immunostaining (green) and photoreceptor axon terminals detected by synaptophysin immunoreactivity (red), showing their paired synaptic contacts in the C56BL6j mouse retina (a, b). In the P20 rd10 retina (c) horizontal cell processes show a decrease in paired immunoreactive contacts with synaptophysin labeled profiles. Horizontal cell processes around vascular tracts were found at P20 (c, arrows). There was a loss of photoreceptor axon terminals and horizontal cell processes during the course of the degeneration, a few pairs remained at P30 and P40 (d, e, arrowheads). (f– h) Double immunostaining with PKC-␣ and mGluR6 at P20 (f), P40 (g) and P60 (h) in rd10 mouse shows a loss of mGluR6 immunoreactive puncta in the dendritic tips of rod ON-bipolar cells (arrowheads) and in disk-like formations corresponding to ON cone bipolar cell dendrites contacting cone pedicles (f, arrows). Ectopic immunoreactive puncta in the cell bodies of rod bipolar cells were found at P20 (g, arrows). (i, j) Morphological changes in type 4 cone bipolar cells immunostained with recoverin antibodies at P20 (i) and P40 (j). Arrowheads show the dendrites of cone bipolar cells connected with the remaining cone axon terminals at P40. Scale bar⫽20 ␮m.

ing in the outer OPL (Fig. 6f) that reflects the dendritic tips of the rod ON-bipolar cells associated with rod spherules. Also, disk-like formations more proximal to the OPL can be observed, which represent the contacts of cone ON-bipolar

cells with cone pedicles (Fig. 6f; arrowheads). By P40, little mgluR6 receptor immunoreactivity was detected in the OPL, indicating the loss of connectivity between photoreceptors and bipolar cells (Fig. 6g). Some immunoreactivity

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Fig. 7. Sections of rod bipolar cells during the course of degeneration in the rd10 mouse retina immunostained with antibodies against PKC-␣. (a) Rod bipolar cell morphology in the C57BL/6J mouse. Arrows indicate the vertical dendrites toward rod spherules and arrowheads indicate well developed axon terminals of rod bipolar cells. (b– d) Morphological changes in rod bipolar cells in rd10 P20, P30 and P40. Arrows in (b), (c) and (d) indicate the loss of dendrites during degeneration. Note the decrease in immunoreactivity and the loss of axon terminals during retinal degeneration. Scale bar⫽20 ␮m.

in the OPL appeared to be associated with the remaining cone ON-bipolar cell dendrites since they were not associated with PKC-␣ immunoreactive rod bipolar cells (Fig. 6g, arrowheads). Other displaced immunoreactive puncta were present at the cell bodies and axons of rod bipolar cells (Fig. 6g, arrows). At P60, some remaining mGluR6 immunoreactivity was still evident, associated with rod and cone ON-bipolar cells (Fig. 6h, arrowheads). As postulated above, to clearly define the time at which retinal degeneration commences in the rd10 mice, a series of double immunolabeling experiments were performed at P14, P16 and P18 using antibodies against recoverin to identify photoreceptors and PKC-␣ to identify rod bipolar cells (Fig. 8). At P18, photoreceptors have completed their development (see for comparison Fig. 8 g with Fig. 2a) as reflected by the gradient of rod bipolar cell dendrite devel-

opment from P14 to P18, with more profuse dendritic branches at P18 (Fig. 8b, 8e, 8h). At 18 days of age, rod bipolar cells have the same morphology as in the adult retina (compare Fig. 8h– 8i with Fig. 6a). Cone bipolar cells In addition to photoreceptors, recoverin antibodies also label a type of cone bipolar cell in the mouse retina. This cell type has a diffuse axon terminal with varicosities in the distal part of the IPL, corresponding to the type 4 OFF cone bipolar cells described previously (Ghosh et al., 2004). By P20, this cell type displayed no appreciable differences in rd10 mice when compared with those in C57BL/6J mice (Fig. 6i). By P40, the general morphology of this cone bipolar cell was not drastically modified, and only an apparent decrease in cell body

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Fig. 8. Sections of retinas from rd10 mice at P14 (a– c), P16 (d–f) and P18 (g–i). Recoverin staining of photoreceptors (red; a, d, g) and PKC-␣ staining of rod bipolar cells (green; b, e, h) are shown. Double labeling for PKC-␣ and recoverin is shown (c, f, i). Rod bipolar dendrites develop until they develop profuse dendritic branches at P18. OS and IS of photoreceptors at P18 have a normal morphology. OS: outer segments, IS: inner segments, ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bar⫽20 ␮m.

size and shorten of axons was observed. At P40, connections of cone bipolar dendrites with the axon terminals of the remaining cones were still evident (Fig. 6j; arrowheads), indicating that the cone pathway was not completely disrupted at this age in the rd10 mouse. AII amacrine cells Because AII amacrine cells represent the major output of rod bipolar cells and changes in their axon terminals were found (see above Fig. 9), we studied how the AII amacrine cells were influenced by the impaired excitation from rod

bipolar cells following rod photoreceptor degeneration. Dab-1 antibodies label AII amacrine cells in the mouse retina (Rice and Curran, 2000) and in the C57BL/6J mouse retina, AII-amacrine cell bodies were located at the border between the inner nuclear and IPLs (Fig. 9a). These cells were bistratified with lobular appendages in sublamina a of the IPL and fine processes stratifying in the sublamina b, displaying the typical morphology of AII amacrine cells in the mammalian retina. In the sublamina b of the IPL, axon terminals of rod bipolar cells make chemical synapses with AII amacrine cells (Fig. 9a, arrowheads). At P20, some

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Fig. 9. Changes in AII amacrine cells revealed by Dab1 immunoreactivity. (a) Dual immunoreactivity for PKC-␣ and Dab1 showing the typical lobular appendages of AII amacrine cells in sublamina a, and synaptic contacts with axon terminals of rod bipolar cells in sublamina b of the IPL (arrowheads). The decrease of Dab1 immunoreactivity is evident, along with the disrupted morphology of AII amacrine cells in P20 (c), P30 (d) and P40 (e) rd10 mouse retina. Arrowheads in (b– e) indicate the loss of lobular appendages in sublamina a. Scale bar⫽40 ␮m.

changes in AII amacrine cell morphology were observed in the rd10 mouse. The AII lobular appendages in sublamina a were smaller than in the C56BL6j mouse retina (compare Figs. 9 b and 9c; arrowheads), whereas the dendrites in sublamina b looked normal. At P30, fewer and smaller lateral lobular appendages were identified in sublamina a of the IPL (Fig. 9d), however, a dramatic loss of Dab1 immunoreactivity was observed by P40 in AII amacrine cells. Lobular appendages were difficult to find and the distortion of both AII amacrine cell bodies and their processes was evident (Fig. 9e). Glutamate sensitivity in rod bipolar cells Due to the structural modifications observed in the cells of the rd10 retina involved in the rod pathway, we further tested the functionality of rod bipolar cells. In a series of experiments,

we studied the glutamate sensitivity of rod bipolar cells using the whole cell patch clamp technique in retinal slices (see Experimental Procedures). These experiments were carried out in 60-day-old rd10 mice in which complete degeneration of rod photoreceptors has occurred. Local application of glutamate onto rod bipolar cells in retinal slices induces a decrease in membrane conductance that may be observed as an upward deflection in the current traces recorded from cells clamped at negative potentials. These currents are mediated by the mGluR6 metabotropic glutamate receptors in the mammalian rod bipolar cells (De la Villa et al., 1995). We then studied if the currents recorded upon activation of glutamate receptors were modified in the rd10 dystrophic animals. Rod bipolar cells were recorded from the dystrophic animals and the glutamate-induced conductance was tested in these cells.

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Rod bipolar cells were readily identified by structural criteria (soma location, axon length and axon terminal shape) although a complete absence of cell dendrites was noted in rod bipolar cells from dystrophic animals at P60. Glutamate (100 ␮M) was repeatedly puffed every 10 s over the dendrites/soma of rod bipolar cells and a change in membrane conductance was observed. We tested the effect of glutamate in a total of 32 cells and 16 responded to glutamate in retinal slices of C57BL/6J control mice, whereas 22 of 38 rod bipolar cells responded to glutamate in retinal slices of rd10 dystrophic mice. The response to local application of 100 ␮M glutamate on a single rod bipolar cell in the retinal slice from a control C57BL/6J mouse (Fig. 10a) and from a dystrophic rd10 mouse (Fig. 10b) was compared. The recorded cells were labeled with Alexa 488 and cell images were obtained by laser-confocal microscopy after finishing patch clamp recordings. As previously noted, most rod bipolar cells lack dendrites at 60 days of

age (Fig. 10c). During the patch clamp experiments, ionic currents activated by glutamate were tested at different membrane potentials and thus, the change in membrane conductance induced by glutamate may be calculated from the current/voltage relationship (Fig. 10d). The change in membrane conductance induced by glutamate in rod bipolar cells of wild type C57BL/6J mice and rd10 mice was ⫺1.77⫾0.23 nS and ⫺1.61 nS, respectively. Hence, it appears that rod bipolar cells in rd10 mice maintain their glutamate sensitivity at 60 days of age. Indeed, as shown above, most dendrites of rod bipolar cells are retracted in the rd10 mice by p60 but the expression of mgluR6 is still visible at the cell membrane (see Fig. 6h).

DISCUSSION In this study, we have characterized the rd10 mouse model of retinal degeneration from both a morphological and

Fig. 10. Ionic currents induced by local application of glutamate to rod bipolar cells recorded in the retinal slice preparation from control and rd10 mice at p60. (A) Superimposed current recordings obtained at different membrane potentials (Vh) in response to puff application of 100 ␮M L-glutamate (Glu) to a rod bipolar cell from a control mouse. (B) Superimposed current recordings obtained at different membrane potentials in response to puff application of 100 ␮M Glu on a rod bipolar cell from a rd10 mouse. (C) Confocal image of a recorded rod bipolar cell from a P60 rd10 mouse labeled with Alexa 488 through the patch pipette. Note the loss of dendrites at this age. (D) Current–voltage relationship of the Glu induced currents in rod bipolar cells from control (
) and rd10 (Œ) mice. Values of maximum current amplitude at each membrane potential were averaged (mean⫾S.D.) from 16 rod bipolar cells in the retina of control mice and 21 rod bipolar cells in the retina of the rd10 mice.

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physiological perspective, revealing new data regarding the time course of retinal degeneration in this model. Our work shows that the functional activity of retinal cells postsynaptic to degenerated rods is clearly maintained after photoreceptor degeneration. To define the time course of photoreceptor degeneration, we quantified the degenerating photoreceptors in the central retina at different times and we found that this process peaks around P25 in rd10 mice. Through immunocytochemical studies, we examined the course of cone and rod degeneration and its effect on their postsynaptic cells. Furthermore, we describe significant alterations to the inner retina that mainly affect the AII amacrine cells of the rod pathway. Finally, we assessed the notion that due to its slower time course, this model of retinal degeneration maintains the functional input of rod bipolar cells for longer periods than models in which retinal degeneration progresses more rapidly, such as the rd1 (Varela et al., 2003). The rod Pde6b gene, located on chromosome 5 in the mouse, encodes the beta subunit of cGMP-PDE and it has 22 exons. Retinal degeneration in the rd10 mouse model is caused by a missense mutation in exon 13 of this gene (Chang et al., 2007). The onset of cell death we observed involves a pattern of photoreceptor degeneration similar to that observed previously in the rd1 mouse model (PorteraCailliau et al., 1994). In the rd1 mouse, cell death in the central retina commences at P10 and it is completed by P20. Here, we also observed a more rapid onset of cell death in the central retina than in the periphery in the rd10 mice. In this model degeneration starts just before P20 and it finishes sometime after P40. The maximum peak of degeneration is at P25 rather than at P14 in the rd1 mouse. The number of photoreceptors in the mature mouse retina represents 70% of the retinal cells in the wild type C57BL/6J strain. In the ONL, where photoreceptors are located, 97% of the cells are rods and only 3% are cones. Ours results show that at P20, when the normal mouse retina can be considered mature and functional, the rd10 mouse retina has already started to degenerate and there is a reduction in the number of photoreceptors in the ONL in histological sections. At P40 only a few photoreceptor nuclei are found and they are mainly cones. This was confirmed after the specific staining of rod photoreceptors with the rhodopsin antibody, which showed that almost all the rods had disappeared by P40, whereas transducin staining in cones persisted. The time course of degeneration in the rd10 mouse model of retinal degeneration seems to offer some advantages over other models of rd. In the well-known rd1 model, rod death overlaps the late phase of retinal synaptogenesis, establishing certain limitations for the use of such a model in rescue studies. Indeed, even in the case of effective therapeutic use of some drugs, no complete recovery of degeneration has been observed (Frasson et al., 1999b; Komeima et al., 2006). However, in models in which degeneration progresses more slowly than in the rd10 mouse, greater sprouting of cell processes is observed, mainly from bipolar or horizontal cells (Claes et al., 2004; Cuenca et al., 2004; Haverkamp et al., 2006), which

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may difficult the complete recovery of the normal retina structure even if the therapy were effective. In addition to these arguments, the rd10 model of retinal degeneration has recently been used with success to test the effects of different therapeutic approaches (Otani et al., 2004; Rex et al., 2004; Boatright et al., 2006; Komeima et al., 2007; Phillips et al., 2008). Axon terminals in sublamina b of the IPL, rod bipolar cells make chemical synapses with AII amacrine cells (Fig. 7a, arrowheads). AII amacrine cells established homologous gap junctions with neighboring AII amacrines in the inner most region of the IPL, as well as heterologous junctions with axon terminals of cone bipolar cells in sublamina b of the IPL (Kolb and Famiglietti, 1974; Strettoi et al., 1992). Our results suggest that rod death during rd10 retinal degeneration induces structural changes at the rod bipolar cell axon terminals and probably, impaired chemical synaptic contact with AII amacrine cells in sublamina b of the IPL. Therefore, AII morphology and the gap junctions between AII cells and cone bipolar cells might also be affected. It is interesting to note that not only the first postsynaptic neurons (horizontal and bipolar cells) are affected by rod photoreceptor degeneration, but second order postsynaptic neurons are also impaired, AII amacrine cells. In P23H and RCS animals (Cuenca et al., 2004; Wang et al., 2005) similar morphological changes were found in this type of amacrine cells, although no changes in AII amacrine cells have been documented in the rd1 mouse model, even at P90 (Strettoi et al., 2000). Another significant difference between the rd1 and the rd10 models is the lack of process sprouting in rd10 animals (Strettoi et al., 2002; Cuenca et al., 2005). A common feature of all models of retinal degeneration is that rod degeneration promotes the retraction of the dendrites from rod bipolar and horizontal cells, indicating that rod cells support the maintenance of secondary retinal neurons. Moreover, cones became smaller during the course of degeneration, probably due to the lack of structural support from surrounding rods and probably through the deprivation of neurotrophic factors. Our results show that the scotopic pathway that involves rod bipolar cells and AII amacrine cells is impaired during rod degeneration in rd10 animals. However, dendrites of recoverin-labeled bipolar cells were connected with remaining cone axon terminals indicating that the structure of the cone pathway was not completely disrupted by P40: However, no significant cone response has been addressed in ERG experiments at this age. Finally, a functional decrease in glutamate sensitivity was observed in rod bipolar cells from rd1 mouse after photoreceptor degeneration (Varela et al., 2003). Here, we show that rod bipolar cells in the rd10 mouse model still respond to local application of glutamate even after complete degeneration of rod photoreceptors. We argue that the slower time course of rod degeneration in the rd10 model permits the virtually complete functional organization of the rod–rod bipolar synapse to be established by P20, as demonstrated by the presence of b-wave ERG response. Accordingly, we argue that the molecular cas-

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cade that mediates glutamate-induced responses coupled to mGluR6 receptor activation is fully developed in the rd10 but not in the rd1 mouse model. This phenomenon might justify why after rod degeneration in the rd10 mice, rod bipolar cells may maintain the molecular mechanisms underlying the glutamate response. Moreover, it has been shown that rod bipolar cells can make synaptic contacts with cones in the absence of rod development (Strettoi et al., 2004). It would be interesting to test whether rod bipolar cells retain their glutamate sensitivity in models of retinal degeneration that experience slower degeneration (Strettoi et al., 2004; Claes et al., 2004; Cuenca et al., 2004, 2005). Finally, it would also be interesting to test whether the sensitivity to retinal neurotransmitters such as GABA or glycine is preserved in rod bipolar cells, since structural modifications between AII cells and rod bipolar cell axon terminals have been observed here and in other mouse models of retinal degeneration (Claes et al., 2004). Indeed, we are currently testing the functionality of the inner retina in the rd10 mouse model of retinal degeneration. Acknowledgments—We are extremely grateful to Dr. B. Chang for kindly supplying the rd10 animals. We thank A. Robles, M. T. Seisdedos and S. Hernández for technical support and M. Sefton for English corrections. This research was supported by grants from the Spanish Ministerio de Educación y Ciencia (SAF200405870 and SAF2007-66175 to E.J.d.l.R. and P.d.l.V., and BFI2003-01404 and BFU2006-00957 to N.C.), the Ministerio de Sanidad (RETICS RD07/0062 to P.d.l.V. and N.C.), the Comunidad de Madrid (08.5-0019.1/2001 to E.J.d.l.R. and 08.5– 0049/ 2003 to P.d.l.V.), Fundaluce to P.d.l.V., and the ONCE and Fundaluce to N.C. S.C. was supported by a postgraduate fellowship from the Ministerio de Educación y Ciencia.

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(Accepted 11 June 2008) (Available online 3 July 2008)