− null mouse

Neurobiology of Disease 46 (2012) 476–485

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Age-related functional and structural retinal modifications in the Igf1 −/− null mouse☆ L. Rodriguez-de la Rosa a, 1, L. Fernandez-Sanchez b, 1, F. Germain c, S. Murillo-Cuesta a, I. Varela-Nieto a, P. de la Villa c,⁎, N. Cuenca b a

Instituto de Investigaciones Biomédicas ‘Alberto Sols’, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid (UAM), CIBERER Unit 761, ISCiii, IdiPAZ, Arturo Duperier 4, 28029 Madrid, Spain Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, 03690 Alicante, Spain c Departamento de Fisiología, Universidad de Alcalá, 28871 Alcalá de Henares, Spain b

a r t i c l e

i n f o

Article history: Received 7 December 2011 Revised 6 February 2012 Accepted 20 February 2012 Available online 28 February 2012 Keywords: IGF1 Retina Degeneration Deaf-blindness

a b s t r a c t Background: Mutations in the gene encoding human insulin-like growth factor-I (IGF-I) cause syndromic neurosensorial deafness. To understand the precise role of IGF-I in retinal physiology, we have studied the morphology and electrophysiology of the retina of the Igf1 −/− mice in comparison with that of the Igf1 +/− and Igf1 +/+ animals during aging. Methods: Serological concentrations of IGF-I, glycemia and body weight were determined in Igf1 +/+, Igf1 +/− and Igf1 −/− mice at different times up to 360 days of age. We have analyzed hearing by recording the auditory brainstem responses (ABR), the retinal function by electroretinographic (ERG) responses and the retinal morphology by immunohistochemical labeling on retinal preparations at different ages. Results: IGF-I levels are gradually reduced with aging in the mouse. Deaf Igf1 −/− mice had an almost flat scotopic ERG response and a photopic ERG response of very small amplitude at postnatal age 360 days (P360). At the same age, Igf1 +/− mice still showed both scotopic and photopic ERG responses, but a significant decrease in the ERG wave amplitudes was observed when compared with those of Igf1 +/+ mice. Immunohistochemical analysis showed that P360 Igf1 −/− mice suffered important structural modifications in the first synapse of the retinal pathway, that affected mainly the postsynaptic processes from horizontal and bipolar cells. A decrease in bassoon and synaptophysin staining in both rod and cone synaptic terminals suggested a reduced photoreceptor output to the inner retina. Retinal morphology of the P360 Igf1+/− mice showed only small alterations in the horizontal and bipolar cell processes, when compared with Igf1 +/+ mice of matched age. Conclusions: In the mouse, IGF-I deficit causes an age-related visual loss, besides a congenital deafness. The present results support the use of the Igf1 −/− mouse as a new model for the study of human syndromic deaf-blindness. © 2012 Elsevier Inc. All rights reserved.

Introduction Abbreviations: ABR, Auditory brainstem response; amixed, a-wave amplitude of the ERG mixed rod and cone response; bmixed, b-wave amplitude of the ERG mixed rod and cone response; bphot, b-wave amplitude of the ERG photopic cone response; bscot, b-wave amplitude of the ERG scotopic rod response; ERG, Electroretinography; IGF-I, Insulin-like growth factor-I; INL, Inner Nuclear Layer; IPL, Inner Plexiform Layer; ONL, Outer Nuclear Layer; OPL, Outer Plexiform Layer; OP, Oscillatory Potentials. ☆ LR-dlR, SM-C and IV-N carried out the ABR study and characterized the general phenotypes and the genotypes of the animal colony. FG and PdlV carried out the ERG test. LF-S and NC carried out the immunohistochemistry study. LR-dlR, SM-C and FG performed the statistical analysis. LR-dlR, IV-N, PdlV and NC designed and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript. ⁎ Corresponding author at: Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá de Henares, 28871 Alcalá de Henares, Spain. Fax: +34 918854525. E-mail address: [email protected] (P. de la Villa). 1 These authors contributed equally to this work. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2012.02.013

Patients with homozygous mutations of the human IGF1 gene causing inactivation of the IGF-I molecule present severe prenatal growth retardation, postnatal growth failure, microcephaly, mental retardation and bilateral sensorineural deafness [(Bonapace et al., 2003; Walenkamp et al., 2005; Woods et al., 1997; Woods et al., 1996) ORPHA73272, OMIM 608747]. Family members carrying a heterozygous mutation have lower weight at birth and lower height in adulthood, but no early hearing loss has been reported (Woods et al., 1996), although several reports point to a correlation between serum IGF-I levels and presbyacusis (Murillo-Cuesta et al., 2011). Mouse models are a powerful tool for the discovery and characterization of genes for sensorineural defects in humans. Mouse models

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for hearing deficit have allowed the study of the genetic and molecular basis of both syndromic and non-syndromic human hearing impairment (http://hereditaryhearingloss.org; Rodríguez-de la Rosa et al., 2011). IGF-I has a central role in inner ear development and function throughout evolution (Sanchez-Calderon et al., 2007). Peak expression of IGF-I in the cochlea occurs during the late embryonic and neonatal periods, with a reduced expression in the adult. IGF-I actions are exerted through the regulation of intracellular signaling pathways, and the modulation of the transcription factors FoxP3, FoxM1, and MEF2 (Magarinos et al., 2010; Sanchez-Calderon et al., 2007). Igf1 −/− mice develop a smaller cochlea and present congenital sensorineural deafness and age-related metabolic cochlear alterations (Camarero et al., 2001; Camarero et al., 2002; Cediel et al., 2006; Riquelme et al., 2010; Sanchez-Calderon et al., 2010). Interestingly, the comparative study of gene expression in the Igf1 −/− mice showed important alterations in the expression levels of genes associated with retinal development (mammalian achaete–scute homolog 1, fibroblast growth factor 15 and sine oculis-related homeobox 6) and physiopathology (tubby candidate gene, retinitis pigmentosa 1, harmonin Usher syndrome 1C and RAR-related orphan receptor beta) (SanchezCalderon et al., 2010). For the study of retinal degeneration, diverse animal models have been used, including the rd1 mice (Bowes et al., 1990; Farber and Lolley, 1974; Strettoi and Pignatelli, 2000; Strettoi et al., 2003; Strettoi et al., 2002), the rd10 mice (Barhoum et al., 2008; Chang et al., 2002; Gargini et al., 2007), RCS rats (Cuenca et al., 2005; Herron et al., 1974; Milam et al., 1998; VillegasPerez et al., 1998) and p23H rats (Cuenca et al., 2004). The spatial pattern of degeneration of the retina in these models quite resembles the human illness. Nonetheless, this does not occur with regard to the temporal pattern, since all these animals rapidly lose all their photoreceptor cells, contrarily to that described in the majority of the human retinal dystrophy. Thus, the rd1 mice completely lose the photoreceptor cells at the outer nuclear layer (ONL) in just three weeks (Bowes et al., 1990; Chang et al., 2002; Farber and Lolley, 1974), whereas the rd10 does by one month of age (Chang et al., 2002). In the RCS and P23H rats the degeneration progress is slower than in the rd1 and rd10 mice. In RCS rats the degeneration of the photoreceptors progresses during the first three months of life of the animal (Cuenca et al., 2005). In the P23H rats the degeneration begins early, so that at 20 days of age a substantial loss of rods is already observed (Cuenca et al., 2004), whereas by 40 days of age only 1–3 rows of photoreceptors are observed in the ONL of the retina and only a few photoreceptors are identified by nine months of postnatal life (Cuenca et al., 2004). With regard to the functional variations detected in these models, they do not appear to correspond to the human illness. In this way, in the rd1 mice the electroretinography responses mediated by rods do not become normal at any age (Strettoi et al., 2003), whereas in the rd10 mice responses begin to attenuate by 20 days of age and become undetectable for two months of age (Barhoum et al., 2008; Gargini et al., 2007). In this context, members of the team previously assessed the morphological and immunohistological alterations of the auditory receptor of Igf1 −/− mice (Camarero et al., 2001; Camarero et al., 2002) as well as their functional correlations (Cediel et al., 2006; Riquelme et al., 2010), but no information is currently available on their visual function. To evaluate the role of IGF-I deficit in visual function, we studied the retinal electrophysiological and immunohistochemical features of Igf1 −/− mice. Through assessment of electroretinography (ERG) and selective labeling of retinal cells, we show here that Igf1 −/− mice show age-related accelerated loss in visual function, that may enlighten the physiopathology of human IGF-I deficiency and resistance (OMIM entries 608747 and 147370).

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Materials and methods Mouse handling and genotyping Heterozygous mice with a targeted disruption of the Igf1 gene were bred and maintained on a hybrid MF1 and 129/sv mouse genetic background to increase Igf1 −/− mutant survival (Liu et al., 1993). Igf1 −/− mice mortality before adulthood is high, although between 20 and 30% survived. Igf1 +/+, Igf1 +/− and Igf1 −/− mice were studied at the time points indicated to follow their progression from young adults (1 month) to aged mice (1 year). Mouse genotypes were identified as described (Sanchez-Calderon et al., 2010). All procedures were carried out in accordance with the European Council Directive (86/609/ECC) and ARVO statement for use of animals in ophthalmic and vision research with the approval of the Bioethics Committee of the CSIC. Analytical procedures The determination of serum concentration of circulating IGF-I was measured using a standard OCTEIA Rat/Mouse IGF-I kit (sensitivity 63 ng/ml and variability 4–8%) (IDS Ltd., Boldon, UK) according to the manufacturer's recommendations as reported in Riquelme et al., 2010. ELISA data are expressed in ng/ml. Statistical comparisons of IGF-I sera levels between the different age groups were performed with a Mann–Whitney rank sum test using GraphPad InStat 3.06 (Software Inc., San Diego, CA, USA). The results were considered to be statistically significant when p ≤ 0.05. Animals were housed in a 12 h dark/light cycle and fed standard laboratory diet ad libitum with free access to tap water. Mice from 1 to 12-month-old were used for weight and blood glucose measures. For glycemia measurement, mice were fasted overnight and glucose levels in blood samples collected from the tail vein were determined using an automatic analyzer (Glucocard Gmeter; Menarini Diagnostics, Badalona, Spain). Auditory brainstem response (ABR) Mice were anesthetized by intraperitoneal administration of ketamine (Imalgene® 500, Merial, 100 mg/kg) and xylazine (Rompum © 2%, Bayer Labs, 10 mg/kg), and they were maintained at 37 °C throughout the testing period to avoid hypothermia. Both female and male mice were used; no sex-associated parameters were identified in this study. Acoustic stimulation and auditory evoked potential amplification and recording were performed with TDT System 3™ workstation and the specific software SigGenRP™ and BioSigGenRP™ (Tucker Davis Technologies, Alachua FL 32615), as described previously (Cediel et al., 2006) with the modifications reported in Riquelme et al. (2010). The following parameters were analyzed from waves registered during the ABR tests: auditory thresholds in response to click and tone burst stimuli, peak and interpeak latencies and amplitude-intensity and latency-intensity curves. Electroretinography (ERG) Mice were adapted to darkness overnight and subsequently, the whole manipulation was performed in dim red light. Mice were anesthetized with an intraperitoneal injection of a solution of ketamine (95 mg/kg) and xylazine (5 mg/kg) and maintained on a heating pad at 37 °C. The pupils were dilated by applying a topical drop of 1% tropicamide (Colircusí Tropicamida, Alcon Cusí, El Masnou, Barcelona, Spain). A topical drop of 2% Methocel (Ciba Vision, Hetlingen, Switzerland) was instilled in each eye immediately before situating the corneal electrode. Flash-induced ERG was recorded from the right eye in response to light stimuli produced with a custom made Ganzfeld stimulator. The intensity of light stimuli was measured

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with a photometer (Mavo Monitor USB, Gossen, Nürenberg, Germany) at the level of the eye. For each light intensity stimulus, 4–64 consecutive stimuli were averaged, with an interval between light flashes in scotopic conditions of 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). Electrical signals were digitized at 20 kHz with a Power Lab data acquisition board (AD Instruments, Chalgrove, UK). Bipolar recording was performed between an electrode fixed on a corneal lens (Burian-Allen electrode, Hansen Ophthalmic Development Lab, Coralville, IA) and a reference electrode located in the mouth, with a ground electrode located in the tail. Rod mediated responses were recorded under dark adaptation to light flashes of − 2 log cd·s·m − 2. Mixed rod and cone mediated responses were recorded in response to light flashes of 1.5 log cd·s·m − 2. Oscillatory potentials (OP) were isolated using white flashes of 1.5 log cd·s·m− 2 in a recording frequency range of 100–10,000 Hz. Cone mediated responses were recorded in response to light flashes of 2 log cd·s·m − 2 on a rod saturating background of 30 cd·m − 2. The amplitudes of the a-wave and b-wave and peak to peak OP were measured off-line and the results averaged (n = 4–8 per group). Measurements were performed by an observer blind to the experimental condition of the animal. Statistical analysis Statistical analysis of ABR and ERG data was performed using SPSS v19.0 software, and each group of age was considered independent. A general linear model procedure with analysis of the variance (ANOVA) was carried out. Post hoc multiple comparisons included Bonferroni test when equal variances are assumed and Tamhane test when equal variances are not assumed. Data are expressed as mean ± SD or SEM. The results were considered significant at p b 0.05. Immunohistochemistry Animals were killed with a lethal dose of pentobarbital, and the eyes were enucleated and immersion-fixed with 4% paraformaldehyde for two hours at 4 °C, and then washed with 0.1 M Naphosphate buffer, pH 7.4. The eye cups were cryoprotected sequentially in 15, 20 and 30% sucrose. The lens were removed and eyes cups were embedded in Tissue-Tek OCT (Sakura Finetek, Zoeterwouden, Netherlands) and frozen in liquid N2. Sixteen μm-thick sections were obtained in a cryostat (Leica CM 1900, Leica Microsystems) mounted on Plus glass slides. (Thermo Scientific, Madrid, Spain) and air dried. For blocking non-specific staining, sections were incubated in 10% normal donkey serum for 1 h (Jackson, West Grove, PA, USA) with 0.5% Triton X-100 and then incubated overnight at room temperature with combinations of different primary antibodies diluted in PBS containing 0.5% Triton X-100. All primary antibodies used in

Table 1 Primary antibodies used in this work. Molecular marker

Antibody

Source

Working dilution

Bassoon

Mouse, clone SAP7F407

1:1000

Calbindin D-28K

Rabbit polyclonal Rabbit polyclonal

Stressgen (Ann Arbor, MI), VAM-PS003 SWant (Bellinzona, Switzerland), CB-38 Santa Cruz Biotechnology, sc-10800 Chemicon, MAB5258

Protein kinase C (PKC), α isoform Synaptophysin

Mouse, clone SY38

1:500 1:100

1:500

this study had been previously shown to be useful in the mouse retina (Table 1). Subsequently, the sections were washed in PBS and incubated in 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 nuclear dye TO-PRO-3 iodide (Molecular Probes) was added at 1 μM simultaneously to secondary antibodies and slides were incubated at room temperature. 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. Unless otherwise indicated, images were obtained from central retinal sections. TIFF images were enhanced using Adobe Photoshop CS3. Results Igf1 gene-dosage determines differences in the mouse phenotype The body weight of Igf1 +/+, Igf1 +/− and Igf1 −/− mice was measured at 1, 3, 6, 9 and 12 months of age. All genotypes showed an age-related increase in body weight, although it was lower for the Igf1 −/− mouse (Fig. 1A). The greatest body weight gain occurred during the first trimester of life in all cases, while in the following months (3–9) the body weight was maintained (Igf1 −/−) or showed a small increase (Igf1 +/+ and Igf1 +/−). Finally, from 9 to 12 months of age there was a small decrease in the weight of Igf1 +/+ and Igf1 −/− mice. The body weight of Igf1 +/+ and Igf1 +/− mice was compared with that of Igf1 −/− mice and the correlation was statistically significant at all ages, while the comparison between Igf1 +/+ and Igf1 +/− mice was only significant between the ages of 3 and 9-month-old. The fasting blood glucose levels in each of the three genotypes were measured between one month and one year of age (Fig. 1B). Igf1 +/+ and Igf1 +/− mice showed similar values of glucose that were maintained throughout the study. However, Igf1 −/− mice showed an average value of glucose level greater than those of Igf1 +/+ and Igf1 +/− mice during the first month of life and then decreased gradually from 6 to 12 months of age. Comparisons between different genotypes and ages did not show significant differences. Sera from Igf1 +/+, Igf1 +/− and Igf1 −/− mice were collected at several times between 1 and 12 months of age to analyze the circulating IGF-I levels using a specific ELISA assay. As expected, the Igf1 −/− mice had no detectable levels of IGF-I in any of the ages studied (Fig. 1C) and secondly, Igf1 +/− mice presented mean values of IGF-I levels lower than those of Igf1 +/+ mice. Moreover, Igf1 +/− mice showed a statistically significant progressive age-related decrease in serum IGF-I levels, measured between 1 and 12 months of age (data not shown). To study the hearing phenotype, ABR recordings of Igf1 +/+, Igf1 +/− and Igf1 −/− mice were obtained at 1, 3, 6, 9 and 12 months of age, at least 6 mice per group were tested. ABR thresholds in response to click stimulus in Igf1 +/+ and Igf1 +/− mice indicated normal hearing from one month up to six months of age, whereas Igf1 −/− mice showed a profound deafness (Figs. 1D, E). Statistically significant differences were found when Igf1 −/− mice were compared with the other genotypes at 1, 3 and 6 months of age. Accordingly, the audiogram of Igf1 −/− mice presented elevated thresholds in response to pure frequencies (8–28 kHz). The comparison of the audiograms of 1-month-old Igf1 −/− mice with those of Igf1 +/+ and Igf1 +/− mice showed highly statistically significant differences for all the frequencies studied (Fig. 1F). However, the evolution of ABR thresholds from 6 to 12 months of age differed between Igf1 +/+ and Igf1 +/− mice. Igf1 +/− mice exhibited an earlier increase of the thresholds, especially for the high frequencies, although no statistical differences were found compared to Igf1 +/+

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Fig. 1. Temporal evolution of body weight, glycemia, IGF-I levels and hearing loss. (A) Body weight of Igf1+/+ (open circles; WT), Igf1+/− (open triangles; Hz) and Igf1−/− (closed circles; KO) mice was measured. Weight data of WT and Hz mice were statistically significant at all ages studied compared with that of KO (***, p b 0.001), while significant differences between Igf1+/+ and Igf1+/− were shown only in mice from 3 to 9 month-old (§§§, p b 0.001; §, p = 0.014). At least 80 mice were studied per genotype. (B) Glycemia levels were measured in Igf1+/+, Igf1+/− and Igf1−/− fasted mice from blood samples. Glucose levels in Igf1+/+ and Igf1+/− mice were similar throughout the study, but Igf1−/− null mouse glucose values were not steady. Comparisons did not show significant differences. At least 28 mice/genotype were measured. (C) Serum IGF-I levels. Igf1−/− mice showed no detectable levels of IGF-I during the study. Taken together all the ages studied, the mean values of IGF-I levels were lower in Igf1+/− mice than those of Igf1+/+ mice (*, p = 0.026). Circulating levels of IGF-I were analyzed in at least 8 mice/genotype. (D) Representative ABR recordings in response to click stimulus of Igf1+/+ (left column), Igf1+/− (middle column) and Igf1−/− (right column) mice, at 1, 6, 9 and 12 months of age. Wild type and heterozygous mice showed a normal pattern of ABR waves up to six months, whereas null mice exhibited a congenital profound deafness. (E) ABR thresholds in response to click stimulus in Igf1+/+ (open bars), Igf1+/− (closed bars) and Igf1−/− mice (gray bars) at different ages. Igf1+/+ and Igf1+/− mice showed an age-related increase in ABR thresholds, whereas the Igf1−/− mice were deaf from the youngest age studied. ***, p b 0.001; ###, p b 0.001; †, p = 0.033. (F) ABR thresholds in response to tone burst stimuli (8–28 kHz) in Igf1+/+, Igf1+/− and Igf1−/− mice. The audiogram in the Igf1−/− mice was always elevated. ***, p b 0.001; ###, p b 0.001; ##, p = 0.001 (6 months, 16 and 28 kHz; 9 months, 8 and 28 kHz) or p = 0.003 (6 months, 20 kHz); #, p = 0.046; ††, p = 0.006; †, p = 0.024 (9 months, 8 kHz), p = 0.013 (9 months, 28 kHz) or p = 0.032 (12 months, 28 kHz). (G) Peak I latency-intensity curves after click stimulation in Igf1+/+, Igf1+/− and Igf1−/− mice. The null mutant mice showed an elevated curve compared to wild type and heterozygous mice. Latency-intensity curves at 12 months of age were similar in all the genotypes. Statistical analysis was performed with ANOVA and post hoc Bonferroni test. Data are presented as the mean ± SEM. * indicates comparison between Igf1−/− and the other genotypes; # between Igf1−/− and Igf1+/+ mice; † between Igf1−/− and Igf1+/− mice and §§§ between Igf1+/− and Igf1+/+ mice. Degrees of freedom were 2 and F values were > 1 for all the ANOVAs.

mice (Figs. 1D–F). On the other hand, Igf1 −/− mice maintained high ABR thresholds, with significant differences when compared to either Igf1 +/+ or Igf1 +/− mice at 9 months of age. Finally, mice from the three genotypes showed similar increased ABR thresholds in response to click and tone bursts at 12 months of age, without any relevant statistical differences (Figs. 1E, F). Fig. 1G shows the latency-sound intensity curve for peak I in the three genotypes at different ages, indicating that, at the same sound level, Igf1 −/− mice presented an increased peak I latency, compared to Igf1 +/+ and Igf1 +/− mice. Electroretinography reveals altered retinal function in homozygous Igf1 −/− mice To determine possible effects of IGF-I deficiency on retinal function, a series of ERG experiments was performed on Igf1 −/− mice, and on their corresponding control littermates Igf1 +/− and Igf1 +/+, respectively at different times of animal development (Fig. 2). Initially, visual function was tested in 2-month-old mice (p60). Fig. 2A shows the retinal responses to light stimuli on Igf1 −/−, Igf1 +/− and Igf1 +/+ mice obtained under dark and light adaptation conditions. The rod-driven circuitry was tested under scotopic conditions. At p60, the amplitude of the scotopic b-wave (bscot), did not

show significant differences between wild type and mutant mice (Fig. 2B). Under scotopic conditions, light stimuli of high intensity evoked mixed responses. The a-wave amplitude measured in these conditions (amixed) reflects the functionality of rod and cone photoreceptors. Averaged data for the amixed amplitude did not show significant differences between the three genotypes by p60 (Fig. 2B). The bwave amplitudes measured in response to high-intensity stimuli (bmixed) did not show any significant differences among Igf1 −/−, Igf1 +/− and Igf1 +/+ mice (Fig. 2B). To determine the contribution of third order neurons to light-induced ERG, oscillatory potentials (OP) were isolated from the electrophysiological recordings. The OP recorded in response to high-intensity light stimuli under scotopic conditions showed no significant differences among Igf1 −/−, Igf1 +/− and Igf1 +/+ mice (Fig. 2B). The cone driven circuitry was tested in p60 old mice under photopic conditions by measuring the ERG response to intense flashes of light in the presence of rod-saturating light stimulation. Photopic b-wave responses recorded from p60 old mice to light stimuli of very high intensity (bphot) did not show significant modifications among Igf1 −/−, Igf1 +/− and Igf1 +/+ mice (Fig. 2B). Visual function was tested in 4-month-old mice (p120) (Figs. 2A, B). The amplitude of the scotopic b-wave, did not show significant differences between wild type Igf1 +/+ and Igf1 +/− mice, although a

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Fig. 2. Temporal evolution of visual loss in Igf1+/+, Igf1+/− and Igf1−/− mice by electroretinography recordings. (A) Representative ERG recordings of Igf1+/+ (left column, WT), Igf1+/− (middle column, Hz) and Igf1−/− (right column, KO) mice, at 2, 4, and 12 months of age. Rod responses, mixed responses and oscillatory potentials (OP) were recorded under dark adapted conditions; cone responses were recorded under light adapted conditions. Igf1+/+, Igf1+/− and Igf1−/− mice showed normal ERG responses up to two months of age. Igf1+/+ and Igf1+/− mice showed normal ERG responses by 4 months of age, whereas Igf1−/− mice exhibited a significant decrease of ERG wave amplitudes. Igf1+/+ mice showed normal ERG responses by 12 months of age, whereas Igf1+/− mice exhibited a significant decrease of ERG wave amplitudes and Igf1−/− mice showed almost no ERG light responses. Vertical calibration corresponds to 100 μV for rod, mixed and OP responses, and 50 μV for cone responses. Horizontal calibration corresponds to 150 ms for rod, mixed and cone responses, and 50 ms for OP responses. (B) Histogram representation of the ERG wave amplitudes averaged from Igf1+/+ (WT, n = 8), Igf1+/− (Hz, n = 8) and Igf1−/− mice (KO, n = 5) at different ages (2, 4, and 12 months). The ERG wave amplitudes (bscot, amixed, bmixed, OP and bphot) of the light responses were measured as shown on traces in A. Statistical analysis was performed with ANOVA and post hoc Bonferroni tests. Data are presented as the mean ± SD. * indicates comparison between Igf1−/− and Igf1+/+ mice; # indicates comparison between Igf1−/− and Igf1+/− mice; † indicates comparison between Igf1+/− and Igf1+/+ mice. The ERG response amplitudes of the Igf1+/+, Igf1+/− and Igf1−/− mice showed no statistical differences by p60. The ERG response amplitudes of the Igf1−/− mice showed a significant decrease by p120 and p360 when compared with Igf1+/+ mice (***, p b 0.001). The ERG response amplitudes of the Igf1+/− mice also showed a significant decrease by p360 when compared with Igf1+/+ mice (†††, p b 0.001). Comparison between of the Igf1+/− and Igf1−/− ERG response amplitudes also showed significant differences by p120 (##, p = 0.001 [bscot], p = 0.003 [amixed, bmixed] or p = 0.008 [OP and bphot]) and p360 (###, p b 0.001, #, p = 0.022 [bmixed] or p = 0.031 [OP]). F values were >10, degrees of freedom were 2 and for all the ANOVAs. (C) Age dependent reduction in the ERG mediated responses in the Igf1+/+ (WT), Igf1+/− (Hz) and Igf1−/− (KO) mice. Averaged bscot, amixed, bmixed, OP and bphot amplitudes from ERG recordings shown in B for p60, p120 and p360, and those averaged by p180 and p270 are represented as a function of postnatal age. Data are presented as the mean ± SD.

statistically significant reduction was observed in Igf1 −/− mice when compared with Igf1 +/+ or Igf1 +/− mice. The a- and b-wave amplitudes from mixed responses neither showed significant differences among Igf1 +/− and Igf1 +/+ mice genotypes by p120, but again, a statistically significant reduction in the amixed wave was consistently observed in Igf1 −/− mice when compared with Igf1 +/+ or Igf1 +/− mice. The amplitude of the bmixed wave in the in Igf1 −/− mice also showed statistically significant differences between the Igf1 −/− and Igf1 +/+ mice. Oscillatory showed no significant differences between Igf1 +/− and Igf1 +/+ mice by p120, but a statistically significant decrease in OP amplitude was observed in p120 Igf1 −/− mice when compared with Igf1 +/+ (p b 0.001) or Igf1 +/− mice. Photopic b-wave responses recorded from p120 old mice showed no significant differences between Igf1 +/− and Igf1 +/+ mice by p120, but again, a significant decrease in bphot amplitude was observed in Igf1 −/− mice by p120 when compared with Igf1 +/+ mice. Visual function was also tested in 6 and 9-month-old mice (p180 and p270) of Igf1 −/−, Igf1 +/− and Igf1 +/+ genotypes. As a rule, a decrease in the ERG wave amplitudes was observed for all mice genotypes, being differences statistically significant between Igf1 −/− and Igf1 +/+ genotypes (data not shown). Visual function was finally tested in 1-year-old mice (p360) of the three genotypes (Figs. 2A,B). The

amplitude of the scotopic b-wave was significant reduced in the Igf1 +/− mice, when compared with Igf1 +/+ mice and was almost null in the Igf1 −/− mice. Mixed responses were also significantly reduced in the Igf1 +/− mice. The a- and b-wave amplitudes of the mixed response showed statistically significant differences with Igf1 +/+ mice. Mixed responses were also almost null in the Igf1 −/− mice. Similarly, OP showed significant differences among Igf1 −/−, Igf1 +/− and Igf1 +/+ mice by p360. Photopic b-wave responses recorded from p360 old mice also showed significant differences among Igf1 −/−, Igf1 +/− and Igf1 +/+ mice. Average data on ERG wave amplitudes and statistically significant analyses are shown in Fig. 2B for 2, 4 and 12-month-old mice. Plot representation of averaged ERG wave amplitudes as a function of animal ages showed the time course of retinal dysfunction in all three genotypes (Fig. 2C). As it has been previously shown, wild type animals suffered a slight decrease in ERG wave amplitudes along life (Li et al., 2001); for one year old animals, amplitudes of the ERG waves decreased one third of their initial values. Our work in the Igf1 −/− and the Igf1 +/− mice showed significant differences with the Igf1 +/+ animals. From our analysis we appreciated that Igf1 −/−, Igf1 +/− and Igf1 +/+ mice showed equally normal function at young ages (p60). By p120, rod and cone mediated responses

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were decreased in the Igf1 −/− mice, when compared with Igf1 +/− and Igf1 +/+ mice. From this age, a linear steep reduction in the rod and cone mediated responses was clearly observed in the Igf1 −/− mice with age, showing an almost absent light response for 1-yearold animals. The Igf1 +/− mice also showed a linear reduction of ERG wave amplitudes from p120, although detectable ERG light responses may be still observed at one year of animal age. Our electrophysiological results indicated that retinal function in the Igf1 −/− mutants was severely altered by p360, and that the dysfunction may mainly affect the cells postsynaptic to rod and cone photoreceptors. Immunohistochemistry reveals altered retinal structure in homozygous Igf1 −/− mice To study changes in retinal morphology in the Igf1 −/− animals we used TOPRO to stain the nucleus of all cell types (Fig. 3). Vertical section of Igf1 +/+ and Igf1 −/− retinas at four months of age showed no differences in retinal thickness of any retinal nuclear layer (Figs. 3A and B). At 12 months of age, only the thickness of the outer plexiform layer (OPL) in the Igf1 −/− was reduced (Fig. 3D) when compared with Igf1 +/+ animals (Fig. 3C). Since we have found a decrease in the ERG b-wave, we decided to explore whether connectivity markers associated with rods, cones and their postsynaptic cells may be altered in the transgenic Igf1 −/− mice. In the mouse retina, there is a single type of ON rod bipolar cell, which is immunoreactive for PKC (protein kinase C alpha). The rod bipolar cell bodies are mostly

Fig. 3. General view of the Igf1+/+ and Igf1−/− retinas stained with TOPRO 3 showing all retinal cells layers. No differences in layer thickness were found at 4 (A,B) and 12 months (C,D) between Igf1+/+ and Igf1−/− mice. Only the thickness of the OPL in the Igf1−/− mouse at 12 months was thinner that in wild type mouse. Scale bar represents 20 μm.

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aligned in the outermost part of the inner nuclear layer (INL). Each rod bipolar cell has a single primary dendrite, and a tuft of dendritic terminals establishes connections with rod spherules through a large dendritic arbor in the OPL. Their single axon runs perpendicularly through the IPL, ending in its innermost stratum as large axon terminal end-bulbs, together with some lateral terminal varicosities, close to the ganglion cell layer. In order to identify synaptic contacts between photoreceptors and ON rod bipolar cells in the OPL during aging, antibodies against bassoon and PKC were used. Bassoon is a presynaptic protein located at the synaptic ribbon in cone and rod terminals in the OPL. In the inner plexiform layer (IPL), bassoon is concentrated at conventional GABAergic amacrine synapses but is absent from the bipolar cell ribbon synapses (Brandstatter et al., 1999). We used antibodies against PKC to identify the dendritic terminals of rod bipolar cells that represent one of the postsynaptic elements to rod photoreceptors. Double labeling with bassoon and PKC showed the relationship between photoreceptors and dendritic terminals of rod bipolar cells (Fig. 4). At 2 months of age, no differences in the paired bassoon-PKC staining were found in the Igf1 +/− and Igf1 −/− compared with Igf1 +/+ (Figs. 4A, B and C). At 4 months of age, Igf1 −/− retinas showed a retraction of rod bipolar dendrites and a decrease in the synaptic contacts between rod spherules and ON rod bipolar dendrites compared with Igf1 +/− and Igf1 +/+ retinas (Figs. 4D, E and F). The most remarkable differences in connectivity between photoreceptors and ON rod bipolar cells were found in Igf1 −/− retinas at 12 months of age, where a loss of ON rod bipolar cell dendrites was evident and a few paired contacts could be observed (Fig. 4I). Also, bassoon immunostaining was mislocated, moving from the OPL to the outer nuclear layer, around photoreceptor cell bodies (Fig. 4I). Although the loss of contact in the Igf1 +/− (Fig. 4H) was not as dramatic as in the Igf1 −/−, differences with Igf1 +/+ (Fig. 4G) could be observed, with less contacts between rod terminal tips and bassoon immunoreactivity. We studied further cell connections between horizontal cells and axon terminals of photoreceptors. Horizontal cells can be identified using antibodies against calbindin (Fig. 5). In the mouse retina, dendritic tips of horizontal cells make contacts with the cone pedicle while horizontal cell axon terminal tips contact rod photoreceptors in the OPL, at the rod spherules. A continuous plexus in the OPL and tip terminal of horizontal cells may be easily identified in the 2month-old Igf1 +/+ mouse retina (Fig. 5A). No differences were found in the horizontal morphology and tip terminal in Igf1 +/− and Igf1 −/− (Figs. 5A, B and C). At 4 months of age, the regular and dense plexus of horizontal cell processes and tip terminals of Igf1 +/− and Igf1 −/− mice were different from Igf1 +/+, whereas a clear reduction of dendrites and axon terminal tips was observed in Igf1 −/− mice (Figs. 5D, E and F). At 12 months of age, horizontal cell processes in Igf1 +/− mice were diminished and a decrease of terminal tips could be observed compared with Igf1 +/+. In Igf1 −/− mice, calbindin-immunoreactive terminal tips were difficult to recognize, and the horizontal cell plexus showed gaps in the OPL (Figs. 5G, H and I). To identify if photoreceptor axon terminals were lost with aging in Igf1 −/− mice, synaptophysin immunostaining was analyzed. The 2month-old Igf1 +/+ mouse showed a continuous and thick immunoreactivity band of synaptophysin located on top of the horizontal cell terminals. At this age, retinas of the Igf1 +/− and Igf1 −/− mice did not show significant differences in synaptophysin immunoreactivity when compared with Igf1 +/+ (Figs. 6A, B and C). At 4 months of age, a decrease of photoreceptor axon terminals immunolabeling was observed in the Igf1 −/− compared with Igf1 +/− and Igf1 +/+ (Figs. 6D, E and F). Finally, at 12 months of age (Figs. 6G, H and I), immunostaining for the synaptophysin in the Igf1 −/− mouse retina was no longer distributed as a continuous layer, and it showed labeling gaps corresponding to the lack of horizontal cells processes in the OPL. Also a thin band of photoreceptor axon terminals was located on

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Fig. 4. Synaptic contacts between rod photoreceptors and rod bipolar cell dendrites in the Igf1+/+, Igf1+/− and Igf1−/− mice. Immunostaining for rod bipolar cells (PKC, green) and synaptic ribbon (bassoon, red). (A) The interaction of ON rod bipolar cell dendrites with paired photoreceptor synaptic ribbons could be observed in the Igf1+/+ mouse at 2 months. (B) The picture shows the synaptic contacts of ON rod bipolar cells with paired photoreceptors in the Igf1+/− mouse at 2 months. (C) At two months of age, the Igf1−/− mouse showed no apparent changes in connectivity between ON rod bipolar cells and rod axon terminals compared with Igf1+/− and Igf1+/+ mice. Retraction of bipolar cell dendrites and loss of photoreceptor synaptic ribbon were observed in Igf1−/− mouse by 4 months of age (F), compared with Igf1+/− (E) and Igf1+/+ mice (D). At 12 months of age, loss of synaptic contacts between ON rod bipolar and photoreceptors (arrowheads) was evident in the Igf1−/− (I) compared with Igf1+/− (H) and Igf1+/+ mice (G). Scale bar represents 20 μm.

top of the horizontal cells compared with Igf1 +/+, this indicating that axon terminals were lost with aging in the Igf1 −/− mouse. At this age, Igf1 +/− retinas showed intermediate synaptophysin immunoreactivity between Igf1 +/+ and Igf1 −/− mice. Discussion The present work on the IGF-I deficient mouse supports the use of new mouse models for the study of human syndromic deaf-blindness. The study on Igf1 −/− mice, Igf1 +/− mice and Igf1 +/+ mice allowed us to compare the serological levels of IGF-I with phenotype manifestations in body weight and glycemia and a good correlation was observed. Moreover, we showed that Igf1 −/− mice suffer a profound deafness from birth, while the Igf1 +/− mice start to loose auditory capacity by 6 months of age. Although auditory function is also affected in the Igf1 +/+ mice, the process seems to be slower in Igf1 +/+. All three mice genotypes become equally deaf by 12 months of age. Contrary to the auditory function, visual function in the Igf1 −/− mice is normal until four months of age. From then, a progressive loss in visual function is observed in the Igf1 −/− mice becoming almost blind at

twelve months of age. The evolution of the visual function in the Igf1 +/− mice is also affected, and a decrease in retinal responses is observed by 6 months of age, although no complete blindness is reached by 12 months of age. Visual impairment in the IGF-I deficient mouse is parallel to a decrease in cell contacts at the first synapse of the visual pathway, and no clear decrease of retinal cell numbering is observed in this mouse model of syndromic blindness. Patients that suffer homozygous mutations of the human IGF1 gene present severe bilateral sensorineural deafness (Walenkamp and Wit, 2007; Murillo-Cuesta et al., 2011). Accordingly, the Igf1 −/− mice experience a profound deafness from birth, due to an abnormal development of the auditory nervous system, where IGF-I plays a critical role (Sanchez-Calderon et al., 2010). On the contrary, development of the retina in the Igf1 −/− mice seems not to be compromised by the deficiency in IGF-I, which seems to be critical for the normal vascularization of the mammalian retina, but not for its development (Calvaruso et al., 1996; Hellstrom et al., 2002). Therefore, normal retinal function and structure are observed in the Igf1 −/− mice at birth. Human heterozygous mutation in the IGF-I gene has lower weight at birth and lower height in adulthood, but

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Fig. 5. Horizontal cell modifications in the Igf1+/+, Igf1+/− and Igf1−/− mice. Confocal fluorescence micrographs of retinal cross sections showing horizontal cell using antibodies against calbindin. (A) In the 2-month-old Igf1+/+ mouse, horizontal cell terminal tips were easily identified (arrows). At this age, no loss of horizontal plexus in the OPL and terminal tips was found in Igf1+/− compared with Igf1−/− mice retina (B,C respectively). At 4 months of age, the regular and dense plexus of horizontal cell processes and tip terminals in the OPL in Igf1+/− (E) and Igf1−/− (F) were different from Igf1+/+ mice retinas (D), and there was a clear reduction of dendrites and axon and terminal tips (arrows) compared with Igf1+/+ mice. At 12 months of age, (I), the horizontal cell terminal tips showed a clear retraction where sparse calbindin immunoreactivity was shown no longer distributed as a continuous layer in the OPL, compared with Igf1+/− (H) and Igf1+/+ mice (G). Scale bar represents 10 μm.

no early hearing loss (Woods et al., 1996). Accordingly, these mutations do not present a phenotype strikingly different to that found in the wild type mouse. In the Igf1 +/− mouse, the hearing loss and the degeneration of the visual system progress in parallel with aging. From the present experiments, no clear relationship between partial IGF1 gene deficiency and visual progressive loss may be established. Sensorineural visual defects have not been addressed thoroughly in patients that suffer homozygous mutations of the human IGF1 gene. To our knowledge, no visual tests have been shown in these patients to date. IGF-I has been associated with the pathogenesis of diabetic retinopathy, although its role is not fully understood (Gerhardinger et al., 2001; Hellstrom et al., 2002; Ruberte et al., 2004). The synthesis of IGF-I is decreased in both human and experimental diabetes. However, no changes in the activation of its receptor and downstream antiapoptotic effector, nor retinal microvascular cell apoptosis, were found (Gerhardinger et al., 2001). In our experiments, the Igf1 −/− mouse experiences a decrease in wave amplitude of the retinal electrical responses that become almost flat at 12 months of age. Absence of ERG responses has been addressed in several mouse models of retinal degeneration (Chang et al., 2002). In most dystrophic mice, decrease in the number of retinal photoreceptors parallels the decrease in ERG wave amplitudes (Barhoum et al., 2008; Cuenca et al., 2004; Cuenca et al., 2005; Gargini et al.,

2007; Strettoi and Pignatelli, 2000; Strettoi et al., 2003; Strettoi et al., 2002). However, the current Igf1 −/− mouse model does not experience a significant loss of retinal photoreceptors, but a significant loss of cell contacts in the OPL between photoreceptors and their postsynaptic cells, bipolar and horizontal cells was found. In a transgenic mice model overexpressing IGF-I no changes in the retinal layer thickness were found and interestingly the specific expression of IGF-I was detected in the OPL and the inner segment of photoreceptors (Ruberte et al., 2004). These transgenic mice showed altered retinal vascularization at 6 months of age and older (Ruberte et al., 2004). Since IGF-I has been shown to be involved in synaptic plasticity (Torres-Aleman, 2009), the decrease in ONL synaptic contacts may be related to the absence of IGF-I and thus may have an effect on retinal responses; the decrease of rod photoreceptor-ON rod bipolar cell contacts would result in a decrease of the ERG b-wave, but should not completely compromise the ERG a-wave generated at the photoreceptor cells. From the present work, we can confirm the structural and functional alteration of retinal connectivity in the Igf1 −/− mouse that parallels deficit in visual function, but we cannot completely explain the absence of a-wave in the mixed ERG responses. It is possible that the lack of IGF-I could affect photoreceptor responses but not their morphology. Therefore, we cannot discard the possibility that photoreceptor function may be also affected in this

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Fig. 6. Synaptic contacts between photoreceptors and horizontal cell terminals in the Igf1+/+, Igf1+/− and Igf1−/− mice. Confocal fluorescence micrographs of retinal cross sections showing the degeneration of horizontal cell terminals contacting photoreceptor axon terminals, using antibodies against synaptophysin to label photoreceptor terminals (red channel) and calbindin to label horizontal cells (green). (A) The Igf1+/+ mouse retina showed a continuous and thick immunoreactivity band of synaptophysin located on top of the horizontal cell terminals. At 2 months of age, the Igf1+/− (B) and Igf1−/− (C) mice showed no synaptophysin-IR differences with Igf1+/+ mice. At 4 months of age, a decrease of axon terminals immunostained with antibodies against synaptophysin was found in the Igf1−/− (F) compared with Igf1+/− (E) and Igf1+/+ mice (D). At 12 months of age, synaptophysin immunoreactivity in the Igf1−/− mouse retina (I) was no longer distributed as a continuous layer, and a thin band of axon terminals was located on top of horizontal cells compared with Igf1+/+. At 12 months of age, Igf1+/− mouse retinas showed intermediate synaptophysin immunoreactivity between Igf1+/+ and Igf1−/− retinas. Scale bar represents 10 μm.

mouse model and we suggest that the functional role of pigment epithelial cells may be compromised due to the lack of IGF-I (Takagi et al., 1994). It has been previously shown that disruptions of the IGF-I gene results in hypomyelination and interneuronal loss of different brain regions (Beck et al., 1995), but to our knowledge, this work shows for the first time that disruptions of the Igf1 gene results in a decrease of the number of cell contacts in a specific brain region such as the retina. We conclude that the maintenance of normal levels of IGF-I is required for normal visual function and its lack leads to a loss of vision over time. Finally, we propose the Igf1 −/− and Igf1 +/− mice as new models of progressive deaf-blindness of potential use to test novel IGF-I-based therapies aimed at the protection and repair of sensory systems. Acknowledgments This research was funded by grants from the Spanish Ministry of Science and Innovation SAF2010-21879 and RETICS RD07/0062/ 0008 to PdlV; BFU2009-07793/BFI, Fundaluce, ONCE and RETICS RD07/0062/0012 to NC; and SAF2008-0064, SAF2011-24391 and Intra-CIBERER programs to IV-N. We kindly acknowledge the

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