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Electroretinographic analysis of retinal function in mice with various mutations in the Microphthalmia transcription factor (Mitf) gene Snæfríður Guðmundsdóttir Aspelund Lokaverkefni til BS-gráðu Sálfræðideild Heilbrigðisvísindasvið Electroretinographic analysis of retinal function in mice with various mutations in the Microphthalmia transcription factor (Mitf) gene Snæfríður Guðmundsdóttir Aspelund Lokaverkefni til BS-gráðu í sálfræði Leiðbeinandi: Þór Eysteinsson Sálfræðideild Heilbrigðisvísindasvið Háskóla Íslands Febrúar 2016 Ritgerð þessi er 16 eininga lokaverkefni til BS gráðu í sálfræði og er óheimilt að afrita ritgerðina á nokkurn hátt nema með leyfi rétthafa. © Snæfríður Guðmundsdóttir Aspelund 2016 Prentun: Prentmet Reykjavík, Ísland 2016 Abstract The Mitf (microphthalmia-associated transcription factor) gene has proven to be crucial for the correct RPE development, which is essential for the proper eye morphogenesis and for the proper function of the photoreceptors. Loss of function mutations in this gene can cause ocular hypopigmentation, microphthalmia and blindness. The purpose of this study was to analyze the retinal function of mice with various mutations in the Micropthtalmia-associated transcription factor (Mitf) gene. Electroretinography (ERG) was used for the evaluation and hence to determine the role of the MITF protein in retinal and visual function. In order to do so, Mitf mutations ranging in severity were selected, from a weak phenotypic effect (Mitfmi-enu122(398)) to the most severe (Mitfmi-wh/Mitfmi). Mitfmi-wh/+ and Mitfmi/+ mutants were also examined and compared to wild type mice (C57BL/6J). It was hypothesized that the impact of the mutations on the Mitf gene function would be based on its gene location. If that would have been the case, the only blind mutant would have been the Mitfmi-wh/Mitfmi heterozygotes. Instead, ERG recordings revealed that only one of the four mutant genotypes had any retinal function, the Mitfmi-enu122(398). Independent samples t-tests with 95% confidence intervals were performed on the data to test for the mean differences between the comparison groups, wild-type mice and the Mitfmi-enu122(398). The only significant comparisons (α= 0,05) were that the wild-type mice had significantly higher mean amplitudes of the photopic a-waves and scotopic oscillatory potentials. Furthermore, the Mitfmi-enu122(398) had significantly shorter implicit times for the photopic b-waves and scotopic c-waves. These results suggest that the MITF protein has a minor effect on the retinal function of the Mitfmi-enu122(398) mutant mice. Overall, this study further demonstrates the large impact that the Mitf gene has on retinal function. Acknowledgments This study is submitted for the Bachelor of Science degree in Psychology at the University of Iceland. First and foremost I would like to thank my great supervisor, Dr. Þór Eysteinsson, for invaluable assistance and guidance throughout the whole study and for all the time and work he gave this project. I would also like to thank Andrea García Llorca for her assistance and for letting me use the light microscope pictures she took. I would also like to thank Dr. Eiríkur Steingrímsson for the help and everybody else at the Biomedical Center who assisted me. I would also like to express my gratitude to the repair team at Landspítalinn for fixing one crucial research instrument in a desperate time of need. I am also deeply grateful for all the help and motivation from my boyfriend, Hlynur Freyr Jónsson. Furthermore, I am thankful for all the support from all my family and friends, especially my wonderful parents. Last but not least, I would also like to thank Dr. Birgir Hrafnkelsson and Valdís Björk Þorgeirsdóttir for their suggestions. Thank you all! Table of Contents Abstract ..................................................................................................................................... 2 Acknowledgments..................................................................................................................... 2 List of tables and figures .......................................................................................................... 5 About the eye ............................................................................................................................ 6 Vision................................................................................................................................................... 6 Eye structure ....................................................................................................................................... 6 The Retina ................................................................................................................................. 7 The Structure of the Retina.................................................................................................................. 7 The Mouse Retina ................................................................................................................................ 8 The Function of the Retina .................................................................................................................. 8 MITF ....................................................................................................................................... 10 The Role of Mitf in the Development and Function of the Retina ..................................................... 11 Mitf mutations and pathology............................................................................................................ 12 The mutations used in this study........................................................................................................ 13 Electroretinogram (ERG) ...................................................................................................... 14 Aims ......................................................................................................................................... 17 Method..................................................................................................................................... 18 Animals .............................................................................................................................................. 18 Instruments ........................................................................................................................................ 18 Research Design ................................................................................................................................ 19 Procedure .......................................................................................................................................... 19 Data Analysis .................................................................................................................................... 21 Results ..................................................................................................................................... 22 Discussion ................................................................................................................................ 34 References ............................................................................................................................... 38 4 List of tables and figures Figure 1. The Mitf mutant phenotypes used in this study ............................................... 14 Figure 2. Scotopic ERGs from all the mutant mice ........................................................ 23 Figure 3. Scotopic ERGs overlapped and compared between the groups ...................... 24 Figure 4. Recordings of dark-adapted oscillatory potentials .......................................... 25 Figure 5. The relationship between the mean amplitudes of the a- and b-waves ± standard error of the means, in response to all the light stimuli intensities .... 27 Figure 6. Recordings of dark-adapted c-waves ............................................................... 28 Figure 7. Dark-adapted flicker responses ....................................................................... 29 Figure 8. Photopic ERG recordings ................................................................................ 30 Figure 9. Photopic ERGs overlapped and compared between the groups ...................... 31 Figure 10. Light micrograph images of segments from the retinae and the RPE ............. 32 Table 1. Means and standard deviations for the amplitudes and implicit times of the scotopic ERGs ................................................................................................. 26 Table 2. Means and standard deviations for the amplitudes and implicit times of the photopic ERGs ................................................................................................ 32 5 About the eye Vision Vision is the process through which reflected light from objects in the environment is translated into a mental image. The visual information flow begins when light enters the eye and is focused on the retina by the lens. There the light gets absorbed by the photoreceptors where it produces a detachment between photopigment molecules, opsin and retinal. This results in a cascade of biochemical reactions that leads to a reduction in the dark current, and thus hyperpolarizes the photoreceptors. This process, termed phototransduction, transduces the light energy of the stimulus into neural signals and transmits them through intervening networks of neurons forming the neuroretina. Then the neural signals travel along the optic nerve via the visual pathways to the primary visual cortex of the brain. The brain then interprets the information, creating our perception of the visual world (Dowling, 1987; Carlson, 2013; Leibovic, 1990; Silverthorn, 2009). Eye structure The first tissue that light encounters is the cornea, a transparent external surface that covers both the iris and the pupil (Kolb, 2012). The pupil is a dark circular opening at the center of the iris where light enters the eye. The iris is a colored circular muscle, controlled by the ciliary muscle, which regulates the amount of light entering the eye by means of controlling the size of the pupil. The aqueous humor fills the space behind the cornea whilst supplying oxygen and nutrients to, and removing waste from, the cornea and the crystalline lens (Wolfe, Kluender and Levi, 2012). Light enters through the cornea which is the first and most powerful lens of the optical system. It focuses the incoming light, allowing together with the crystalline lens the production of a sharp image at the photoreceptor level (Kolb, 2012). After passing through the lens, the light enters the vitreous chamber, the space between the lens and the retina filled with the vitreous humor. After travelling through the vitreous chamber, the light is brought into focus at the retina, where seeing really begins (Wolfe et al., 2012). 6 The Retina The Structure of the Retina The retina derives from the neural tube which evaginates early in embryonic life to form two optic vesicles that form the optic cup. The neural epithelium on the inner wall of the optic cup eventually becomes the retina. The cells of the inner wall divide to form a neuroepithelial layer called neuroblasts, which then differentiate into all of the retinal cells (Dowling, 1987). All vertebrate retinas are organized according to the same basic plan, with two synaptic layers intercalated between three cellular layers. The retina is divided into three main layers. The most distal retinal cells are the photoreceptors, whose neurons transduces photic energy into electrical potentials. Proximal to that is the bipolar cell layer, which conveys information from the photoreceptors to the ganglion cells in the ganglion cell layer (Leibovic, 1990). The combined refractive power of the cornea, lens and the aqueous and vitreous humor focus light images onto the retina. The mammal retina is duplex, meaning that it consists of two different kinds of photoreceptors, namely cones and rods, which operate under different conditions. The cones are specialized for day vision, fine visual acuity and color, whilst rods are specialized for low levels of light intensity, such as those found at night (Leibovic, 1990; Ward, 2015; Wolfe et al., 2012). In addition, the retina contains interplexiform cells and horizontal cells that interconnects adjacent photoreceptors and the outer processes of the bipolar cells. The retina also contains amacrine cells that interconnects adjacent ganglion cells and the inner processes of the bipolar cells (Carlson, 2013; Dowling, 1987). Behind the retina, interdigitating with the photoreceptors, lies the retinal pigment epithelium cell layer (RPE). The RPE cells contain melanin, a black pigment that absorbs stray light and gives the pupil its dark appearance, which is located on melanin granules in these cells. The RPE cells have several very important functions, such as providing metabolic support for photoreceptors, light reflection and absorption, and has a role in adaptation to darkness and light (Leibovic, 1990). The primary glial cells in the retina are the Müller cells whose main function is to maintain homeostasis in the retina (Kolb, 2013). The retina can be described as an accessible part of the brain and is, in many ways, an ideal part of the brain to study. The retina lines the back of the eye and is hence very approachable (Dowling, 1987). 7 The Mouse Retina The difference between the retinae of different vertebrate species lies for the most part in the sizes and numbers of the cells (Dowling, 1987). All vertebrate retinas have analogous gross structure and the same cell types, although the detailed cellular morphology, classes, abundance, chemical messengers and synaptic connections of cells may differ (Leibovic, 1990). The mouse retinal inner nuclear layers contain cell populations that are, overall, quite similar to other mammals. The major difference between the photoreceptor mosaics of other mammals and mice is the higher density of very small rods in the latter, that along with all the other retinal cells are in accordance to their overall small eye size. The cells are distributed in the following proportions: 3.1% horizontal cells, 41% bipolars, 16% Müller cells and 39% amacrine cells, proving the proportions to be remarkably constant between mammals (Jeon, Strettoi and Masland, 1998). The advantage in using mice in visual research is that their retinas are high in homology with humans and the fact that they are also mammals. They are fully grown two months after birth, so they grow up quite fast. It is also easy to examine their phenotype in appearance, behaviour, cellular function, and to examine the effects of genetic engineering on their phenotype, including retinal function. The Function of the Retina Vision begins at the retina which is a very thin layer of tissue lining the back of the eyes. The retina contains specialized photoreceptors that transduce light into neural signals as described earlier. Light striking a photoreceptor produces a hyperpolarization of the cell, so the photoreceptor releases less glutamate which, in turn, depolarizes the membrane in some of the bipolar cells and hyperpolarizes others. This depolarization causes the bipolar cell to release more glutamate, which excites the ganglion cell, the output cells of the retina. They convert neural signals into series of action potentials that are transmitted, via axonal projections, to the visual areas of the brain (Carlson, 2013; Leibovic, 1990; McCall and Gregg, 2008). The retinal neurons convey information with two kinds of voltage change. Most of them pass their information via graded potentials, an electrical potential that can vary continuously in amplitude, meaning that photoreceptors do not respond in all-or-nothing fashion in order to communicate the number of photons being absorbed by them. Ganglion cells on the other hand, 8 convey information via action potentials, since their axons form the optic nerve and thus communicates with the brain (Wolfe et al., 2012). Both types of photoreceptors consist of an inner and an outer segment. The inner segment manufactures molecules called visual pigments which are then stored in the outer segment, whose tip touches the retinal pigment epithelium. Each photoreceptor has one of the four types of visual pigments found in the human retina. The visual pigment known as rhodopsin is found in the rods but each cone has one of the other three pigments, one of which responds to short wavelengths, one which responds to medium and one to long wavelengths (Wolfe et al., 2012). The visual pigment consists of a protein known as opsin, which determines which wavelenghts of light they absorb and a chromophore called retinal which captures light photons (Kolb, 2013; Wolfe et al., 2012). In total darkness, retinal binds into a binding site of opsin. When activated, retinal changes shape to a new configuration, no longer binds with opsin and is released from the pigment in a process known as bleaching. When light bleaches rhodopsin, retinal is released and transducing begins a second-messenger cascade that hyperpolarizes the rod which in turn, releases less glutamate onto the bipolar neurons. This leads to action potentials traveling through bipolar to ganglion cells, with modifications from horizontal and amacrine cells (Silverthorn, 2009). The bipolar cells can be subdivided based on their response polarity, which is regulated by the type of glutamatergic postsynaptic receptor that is expressed on their dendritic terminals that can be either depolarizing or hyperpolarizing. These two types of bipolar cells have been described in all species, referred to as on-center and off-center bipolar cells. On-center bipolar cells depolarize in response to center illumination and express the metabotropic glutamate receptor 6 (mGluR6). Off-center bipolar cells hyperpolarize in response to illumination of the center of its receptive field and express AMPA/kainate receptors. Thus, the bipolar cell receptive field is concentrically organized into two antagonistic zones, such that illumination at the center decreases the response to illumination at the surround of the receptive field and vice versa. Synaptic transmission to all types of ON bipolar cells from photoreceptors is primarily mediated by the mGluR6 receptor, resulting in a sign reversal at photoreceptor/ON bipolar cell synapse, which is essential for establishing parallel ON and OFF retinal pathways (Dowling, 1987; McCall and Gregg, 2008; Nakajima et al., 1993; Snellman, Kaur, Shen and Nawy, 2008; Werblin and Dowling, 1969). 9 The retinal pigment epithelium (RPE) cells are a multifunctional and indispensable component of the vertebrate retina. RPE cells support some functions of the retina such as supplying nutrients and maintaining the retinal structural integrity (Becerra, 2006; Simo, Villarroel, Corraliza, Hernandez and Garcia-Ramriez, 2010; Tombran-Tink, Shivaram, Chader and Johnson, 1995). The RPE layer abuts the photoreceptors and the choroidal layer and furthermore plays an adhesive role that aids in the attachment of the retina to the choroidal layer. One of its many functions is to absorb stray light to prevent it from refracting inside the eye and distorting the visual image. RPE also manages the uptake, processing and transport of retinoids that subserve vision. The RPE cell layer is strategically placed between the photoreceptors and the choroidal layer and by virtue of this location, forms a blood-retinal barrier via its tight junctions. This epithelial highly selective transport serves to supply nutrients to the photoreceptors and to regulate the ion homeostasis in the subretinal space. Another important function of the RPE is the renewal of the photoreceptor‘s outer segments with phagocytosis by shedding the destroyed tips (Bok, 1993; Strauss, 2005). Research on hereditary types of retinal degeneration shows a strong dependence of the photoreceptors on the RPE and vice versa. The presence of the RPE is moreover essential for the correct morphogenesis of the neural retina and is still required after the beginning of the retinal differentiation, either indirectly or directly, to sustain the retinal lamina organization (Raymond and Jackson, 1995; Strauss, 2005). The activities of the RPE begin early in life and continues to do so until lost through disorder or death of the host. Once differentiated the RPE does not renew itself by cell division under normal circumstances. Thus, it must remain viable and functional, throughout the lifetime of the individual, a period that can exceed 100 years in humans (Bok, 1993). MITF The microphthalmia locus (mi) encodes a basic-helix-loop-helix-zipper (bHLH-Zip) transcription factor called MITF (microphthalmia-associated transcription factor). Mitf and its gene locus have been extensively studied. Mutations at the microphthalmia locus are associated with a defective Mitf. Mutations in the Mitf gene have been reported in numerous and distinct species, ranging from zebrafish to humans (Hodgkinson et al., 1993; Hughes, Lingrel, Krakowsky and Anderson, 1993; Steingrímsson, Copeland and Jenkins, 2004). Mitf is of critical importance for the correct physiology and pathology of many diverse organs, such as the eye, ear, immune system, bone and skin (Arnheiter, 2010). Therefore, 10 microphthalmia mutant mice can have defects in some or all of the various organs, for instance, reduced eye size or microphthalmia, early onset of deafness, a failure of secondary bone resorption and loss of pigmentation (Hallsson et al., 2004; Hodgkinson et al., 1993). Common to all the mutations are defective neural-crest-derived melanocytes, resulting in a reduction or lack of pigmentation in the coat, inner ear and eye (Steingrímsson et al., 2003). Mitf mutations affect the development of the eye and the development of several different cell types, including both kinds of pigment cells in the human body, melanocytes and RPE cells (Moore, 1995; Nakayama et al., 1998). Mitf is not only substantial for making pigment since mice homozygous for Mitf null mutations entirely lack melanocytes and their RPE fails to develop regularly. All Mitf mutations affect melanocytes in various degrees, in addition with others also affecting the RPE resulting in hypopigmented or unpigmented eyes. Thus, Mitf has a significant role in the function, structure, differentiation and survival of pigment cells (Arnheiter, 2010; Bharti, Nguyen, Skuntz, Bertuzzi and Arnheiter, 2006; Hou and Pavan, 2008; Steingrímsson et al., 2004). Moreover, mutational studies in mice have shown that Mitf is a master regulator for the development and survival of melanocytes. It is also a key transcription factor in controlling the expression of major melanogenic proteins and in melanocyte differentiation (Lekmine and Salti, 2007; Levy, Khaled and Fisher, 2006). Mitf controls melanocytes at the level of specification, proliferation, migration, replenishment, malignant transformation, differentiation and survival. Mitf promotes RPE differentiation, regulates the proliferation of the RPE during development and consequently their retinal function (Adijanto et al., 2012; Arnheiter, 2010; Hodgkinson et al., 1993; Nakayama et al., 1998). The Role of Mitf in the Development and Function of the Retina The Mitf gene is expressed in the mouse Retinal Pigment Epithelium during the optic vesicle and optic cup stages of eye development and plays critical roles in the RPE establishment within the eye region. The function of the photoreceptors depend vitally on RPE normality because they influence the development and maintenance of normal retinal morphology by properly patterning the retina. Defects in any of those fundamental RPE activities can cause photoreceptor dysfunction, primary photoreceptor degeneration, retinal degeneration and blindness. These aforementioned abnormalities in the structure or function of the RPE can be traced back to a dysfunctional Mitf (Gaur, Yao Liua and Turnera, 1992; Goding, 2006; 11 Hodgkinson et al., 1993; Jablonski, Tombran-Tink, Mrazek, and Iannaccone, 2000; Longbottom, Fruttiger, Douglas, Martinez-Barbera, Greenwood and Moss, 2009; Moore, 1995; Raymond and Jackson, 1995; Smith, 1992; Strauss, 2005). The lack of Mitf function during ocular development in Mitf mutant embryos results in a microphthalmic phenotype. In other Mitf mice mutations, the partitioning of the eye tissue into neural retina and RPE is disturbed, leading to the transformation of RPE into a stratified neural retina. Mutant RPE cells hyperproliferate, thicken, lose expression of a number of RPE transcription factors and gain instead expression of neuroretinal transcription factors, consequently leading to a transdifferentiation into a laminated second retina. Thus, Mitf appears to play a role in the establishment of the RPE within the presumptive eye region and is involved in the separation of the mammalian optic vesicle into the neural retina and RPE (Nguyen and Arnheiter, 2000; Goding, 2006; Hallsson et al., 2004). Möller, Eysteinsson and Steingrímsson (2004) found that the Mitf protein is required for the continued function of the neural retina throughout the lifespan of the mouse by studying their retinal function in a set of microphthalmia mutant mice using electroretinography. Mitf mutations and pathology In mice, there are over 30 distinct alleles of Mitf that have different effects on the phenotype and can be arranged in allelic series according to their phenotypic severity, from normal to microphthalmic animals with osteopetrosis (Steingrímsson et al., 2003; Steingrímsson et al., 2004). The Mitfmi mutation is the most severe allele as it results in premature death, when in fact the Mitfmi-spotted (Mitfmi-sp), the mildest allele, does not even lead to a visible phenotype in homozygous mice (Pogenberg et al., 2012). The general phenotype of microphthalmic mice includes abnormal eye development, fur pigment deficiency, osteopetrosis tracked to osteoclasts and a deformity in mast cell capacity (Hughes et al., 1993). Most of the Mitf mutations affects the regulation of the development and function in mast cells while only a few mutations affect the osteoclasts in their bone remodeling role (Arnheiter, 2010). Clearly, the requirement for Mitf function varies greatly between different cell types in which the lowest requirement is observed in osteoclasts and the highest in melanocytes (Steingrímsson et al., 2003). Mitf is a master regulator of melanocyte development and also for melanoma oncogene (Levy et al., 2006). Mitf regulates 12 also skeletal muscle formation, where it is significantly expressed during myogenesis and required for efficient myotube formation (Ooishi, Shirai, Funaba and Murakami, 2011). Waardenburg syndrome is a dominantly inherited syndrome of hearing loss and pigmentary disturbances which are caused by mutations in the human Mitf gene (Tassabehji, Newton and Read, 1994). Tietz syndrome is also an inherited syndrome caused by a mutation in Mitf and is characterized by congenital profound deafness and generalized hypopigmentation (Smith, Kelley, Kenyon and Hoover, 2000). Hence, melanocytes are required for the normal development and function of the cochlea and moreover, Mitf activity appears to be important both for sufficient pigmentation and for normal hearing function in humans. Coat colour mutant mice have therefore been valuable for defining the genetic requirements of melanocyte development and furthermore its influence on the auditory system development (Ni et al., 2012; Tachibana et al., 1992; Tachibana, 1999). The mutations used in this study In this study, the following Mitf mutant phenotypes were used: Mitfmi-wh/+ and Mitfmi/+ heterozygotes, Mitfmi-wh/Mitfmi heterozygotes and Mitfmi-enu122(398) homozygotes. In none of these mutations has retinal function or morphology been previously examined. The Mitfmi-wh/+ heterozygotes have a grayish-brown coloured coat and a white ventral spot (Figure 1). They have a profound hearing deficit, characterized by loss of outer hair cells and stria vascularis abnormalities (Ni et al., 2012). This hearing dysfunction in heterozygous Mitfmi-wh/+ mice exhibits a model for the aforementioned dominant Waardenburg type 2 and Tietz syndromes. During embryonic development, the Mitfmi-wh/+ embryos have fewer melanoblasts than normal mice. Their cochlear melanocytes are present at birth, but they fail to survive in the postnatal cochlea, due to a deficiency in melanocyte survival (Ni et al., 2012). Mitfmi/+ heterozygotes display a weak phenotype with an occasional small head blaze or belly streaks. Homozygotes are, however, microphthalmic with a white fur and osteopetrosis (Steingrímsson et al., 2003). The Mitfmi-wh/Mitfmi mutation affects the DNA-binding domain or the transcriptional activation domains of the Mitf protein, which can consequently result in little or no Mitf production. The phenotype of the Mitfmi-wh mutant animals is unusual in that the heterozygotes, which are used in this study, is the most severe phenotype associated with any mutation at the Mitf locus compared to its homozygotes, that only have an intermediate phenotype. In the 13 animals with the heterozygous phenotype, the coat colour is severely affected while eye development is not, suggesting that the Mitfmi-wh mutation has much more serious effects on melanocytes than RPE cells. The reason is that the threshold is lower for the Mitf requirement in RPE cells than in melanocytes and as a result, the eye phenotype is more efficiently complemented than the coat colour phenotype. The coat colour of the Mitfmi-wh/Mitf mi mutant mice is white and their eyes dark ruby (Figure 1) (Steingrímsson et al., 2003). Mitfmi-enu122(398) homozygotes, which are used in this study, have regularly developed eyes, a white abdomen and large unpigmented marks over the rest of the normally pigmented fur (Figure 1). These point mutations are induced by N-ethyl-N-nitrosourea (ENU) and support the molecular and phenotypic traits of the deletion mutations. They moreover indicate that the transgenic method reflects the function of Mitf accurately both during melanocyte and eye development (Bauer et al., 2009; Justice, Noveroske, Weber, Zheng and Bradley, 1999). Figure 1. The Figure shows the Mitf mutant phenotypes used in this study, in the following order: Mitfmi-wh/+, Mitfmi-wh/Mitf mi, Mitfmi-enu122(398). The Mitfmi-wh/+ have a grayish-brown coat color and a white ventral spot. The coat colour of the Mitfmi-wh/Mitf mi mutants is lighter than dilute. Mitfmi-enu122(398 ) mice have large unpigmented marks over the rest of the normally pigmented coat. Electroretinogram (ERG) The electroretinogram (ERG) is a non-invasive technique used to record the overall electrical activity in the retina by measuring the retinal field potentials. It provides an objective measure of retinal function and is furthermore one of the most widely used diagnostic tools in the study of visual disorders (Dowling, 1987; Qian, Shah, Alexander and Ripps, 2008). When a low-resistance electrode is positioned on the ocular corneal surface and a reference electrode is placed elsewhere on the head, voltage changes can be recorded each time 14 the eye is stimulated with a flash of light projected onto the retina. The responses of retinal neurons are manifested in a gross potential which is then amplified and observed on an oscilloscope or in an appropriate computer software for analyzing electrophysiological recordings (Dowling, 1987; Leibovic, 1990). The ERG response is generated by various types of cells in the retina and originates from synchronized electrical activity consequential of serial processing within the retina (Leibovic, 1990). The ERG has a few distinctive components that are separated in time and can be divided into several distinct waves. The most prominent of these are the three major ERG waves, a-wave, b-wave and oscillatory potentials. At low flash intensities, the ERG response is dominated by a slow b-wave of positive polarity, growing in amplitude along with increasing flash intensity. When the flash intensity reaches to approximately -2 log cd*s/m2, the a-wave anticipates the b-wave emerging as a negative polarity (Peachey and Ball, 2003). Studies have demonstrated that the a-wave is primarily a reflection of photoreceptor activity (Miura, Wang, Ivers and Frishman, 2009; Möller et al., 2004) and furthermore that the b-wave requires the depolarizing bipolar cells (Miura et al., 2009; Pardue and Peachey, 2014). However, the precise source of the b-wave is still controversial. The b-wave has the largest amplitude component and follows the a-wave as a large positive potential. It has been shown that the b-wave accounts for light-induced electrical activity in retinal cells post-synaptic to the photoreceptors, and furthermore that the primary input comes from light-induced activity in ON-center bipolar cells (Lei and Perlman, 1999; Stockton and Slaughter 1989; Perlman, 1995). Declaring that the b-wave either originates precisely in these cells or as a result of the lightinduced extracellular potassium concentration changes of the Müller cells adjacent to them (Kline, Ripps and Dowling, 1978; Newman and Odette 1984; Wen and Oakley, 1990). Studies have shown that neither bipolar cells nor the K+-mediated potential of the Müller cells are the sole source of the b-wave (Wurziger, Lichtenberger and Hanitzsch, 2001; Yanagida and Tomita, 1982). Therefore, the current leading hypothesis is that the b-wave reflects mainly the light-induced activity of both on-center bipolar cells and Müller cells. Studies have moreover shown that both the a- and b-waves are partly modified by amacrine cells (Dong and Hare, 2000, 2002; Möller and Eysteinsson, 2003). Mutant mice with the absence of, or significant reduction of the ERG b-wave when the a-wave is present have been referred to as no b-wave (nob) (Pardue, McCall, LaVail, Gregg and Peachey, 1998). Human patients with similar ERG phenotype have the disorder congenital stationary night blindness, which is characterised by a 15 profound loss of rod-mediated vision (Miyake, Yagasaki, Horiguchi, Kawase and Kanda, 1986). Therefore, the nob mutants seem to provide an animal model for CSNB and have been influential in the discovery of the underlying genes of this disorder. Nob is consequential from when there is a signal transmission failure within the vertical excitatory pathways or from the photoreceptors to the depolarizing bipolar cells (McCall and Gregg, 2008; Pardue et al., 1998; Pardue and Peachey, 2014). The relationship between the a- and b-waves is a useful clinical assessment tool and a useful index for evaluating the ERG. The a-wave is dependent on the photoreceptor activity and thus depends on their integrity and also on flash intensities. However, the b-wave depends on the a-wave and thus the retinal signal transmission, from photoreceptors to bipolar cells. So the a-wave displays the input to the proximal retina while the b-wave displays its output. Therefore, it is deduced that the b/a ratio depends only on retinal function and thus, a normal b/a ratio should be attained from a normal retina (Perlman, 1983). A prominent c-wave, representing the activity of the Müller cells, rods and mainly the RPE cells, follows the b-wave at the termination of illumination (Dowling, 1987; Leibovic, 1990; Noell, 1954; Oakley and Green, 1976; Steinberg, Schmidt and Brown, 1970). The c-wave can be used to assess the functional integrity of the RPE, the photoreceptors and the interactions between them (Perlman, 1995). A d-wave, which can be detected at the cessation of illumination in cone-dominated retinas, can express either horizontal or off-bipolar cell depolarization, or both (Dowling, 1987; Stockton and Slaughter 1989). D-waves were not recorded for this research since the light stimulus required to evoke the d-wave must be more than 10 µs in duration. The ERG response to flickering light stimuli manifests the transition from rod to cone-mediated vision. Studies show that ON bipolar cells primarily influence the generation of the flicker response (Perlman, 1995; Qian et al., 2008). Oscillatory wavelets are superimposed on the b-wave when using relatively bright flashes. The origins of oscillatory potentials (OPs) are currently unknown, but evidence suggests that it arises at different levels within the retina. The probable generators of the OPs are bipolar or interplexiform cells. However, OPs seem to indicate neuronal synaptic activity in inhibitory feedback pathways, which are at least partly generated by amacrine cells or other dopaminergic neurons, like interplexiform cells (Dowling, 1987; Wachtmeister, 1998). The aforementioned ERG components are measured in two primary ways, amplitude (µV) and time (ms). The time is measured by means of implicit times, from flash onset to the 16 base or apex of the ERG elements. Implicit times can illustrate the progression of retinal degenerative disorders, in which they are prolonged (Creel, 2015). Non-measurable ERG responses display „flat“ recordings and are often recorded from loss of function mutations and in other abnormal cases, such as when human patients have retinitis pigmentosa (e.g. Hartong, Berson and Dryja, 2006). Comparative studies show that the mouse ERG, which is used in this study, is very similar to that of other mammals (e.g. Pinto, Invergo, Shimomura, Takahashi and Troy, 2007). Aims Mitf has been widely studied, mainly due to its wide-ranging importance for various bodily functions and organs. However, hardly any studies have analyzed the effect Mitf has on retinal and visual function. The results could be clinically interesting because of its relevance to retinal degeneration and other ophthalmic disorders, for treatment purposes in the future. This study is an electroretinographic analysis of the retinal function in mice with various Mitf mutations. The purpose of this study is to examine what role Mitf may play in the generation of the ERG and hence, its role in both retinal and visual function. Mitf is operationalized with five differing and hardly examined Mitf mutations, ranging in severity. As a result, each mutant genotype should impact the retinal function and in accordance with the mutation severity. 17 Method Animals A total of 10 mice where selected at random and used in this experiment. The mice were both males and females. The following genotypes were tested: three Mitfmi-wh/+ heterozygotes, three Mitfmi-wh/Mitfmi heterozygotes, three Mitfmi-enu122(398) homozygotes, one Mitfmi/+ heterozygote and three wild type mice (C57BL/6J) were used as controls. All the mouse strains used in this research were obtained from Professor Eiríkur Steingrímsson (Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, Reykjavik, Iceland). The wild type mice were originally bred at the Jackson Laboratory, but the others were bred and raised at the animal facilities in the Biomedical Center at the University of Iceland, were they were kept under standard conditions. Mice from approximately 4 to 14 weeks of age were examined, with the median age of 10 weeks. The mice weight ranged from 24 - 42g. Although each mouse was only tested once, three animals of each genotype were tested, except only one of the Mitfmi/+ genotype. The mice were kept in transparent polypropylene cages with wire tops and wood chip bottom. They were maintained on a 12 hour light and dark cycle. The temperature in the room was 23˚C and the humidity level was 60%. The animals had free access to water and rat food RMI (E) from Special Diet Services (Essex, England). Experimental procedures and all the animals were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for Use of Animals in Ophthalmic and Vision Research. The license number for these experiments is 0312-0102. Instruments For electroretinographic (ERG) recordings a coiled urethanecoated stainless steel wire (0·2 mm in diameter) was used as the corneal electrode (Goto, 1996). A platinum wire was placed in the chin of the mouse for reference (Peachey and Ball, 2003) and another thin platinum electrode was placed in the outer ear, serving as ground. Signals were amplified differentially 1000 times with a MacLab BioAmp, model ML131 amplifier (AD Instruments, Australia). Then the recordings went through a 50 Hz notch filter to remove mains interference. Bandwidths (high and low pass filters) were set at 0,3–500 Hz. The recordings were then converted into a computer by an analog/digital converter, PowerLab 8SP (ADInstruments Ltd., Australia) and stored on the computer using the program 18 LabChart 7 Pro (ADInstruments, Australia). A 50 – 500 Hz digital LabChart filter in a separate channel further isolated the oscillatory potentials and the noise level monitored with proper settings. The sampling rate was set at 40 kHz. Grass PS-33 photic stimulator (Astro-Med/Grass Inc., West Warwick, USA) was used to generate 10 µs white xenon light flashes to stimulate the retina during the ERGs. When activating the trigger switch on the photic stimulator it signals the A/D device and the lamp, so the ERG data acquisition can start 50 ms before the signal and last for 2000 ms after the light stimulation. The photic stimulator has five light intensity settings with each setting twice as bright as the one preceding it, all of which were used in these experiments, ranging from the lowest luminance at the retina 0,67 log cd sec/m2 to the highest at 1.87 log cd sec/m2. Research Design This study is an experiment with a between series design. The purpose of this study was to examine the effect Mitf has on retinal function. The ERG is a measurement of retinal function, so ERG is the dependent variable in this study while Mitf is the independent variable. The Mitf is operationalized with the five different mutations, ranging in severity and thus, impacting the retinal function differently. However, measurements disclosed that only one of these mutations was not completely sightless, the Mitfmi-enu122(398) homozygotes. So the independent variable consists of two categorical independent groups, the Mitfmi-enu122(398) which are the experimental group and this study compares their ERG values with the ones of the control group, the wildtype mice (C57BL/6J). The experiments were conducted in a closed room to minimize extraneous disturbances. For that same reason, the cables were wrapped in tin foil and the electrodes always kept clean, both to minimize extraneous noise during measurements and to maximize their effectiveness. The experiments always followed a strict protocol, using the best equipment available. The technique and therefore also the protocol were always updated with new information, both from researches and through trial and error learning, thereby always using the best technique available. Procedure The mouse was given an intraperitoneal injection of 40 mg/kg-1 Ketamine (Pfizer, Denmark) and 4 mg/kg-1 Xylazine (Chanelle Pharmaceuticals, Ireland) prior to each experiment. The 19 anesthesia was maintained by half of the original dose, intraperitoneal injection of 20 mg/kg-1 Ketamine and 2mg/kg-1 Xylazine. The pupil of the examination eye was dilated with topical application of Mydriacil® (1% tropicamide) eye drops (Alcon Inc., U.S.A) or cyclopentolate (Chauvin Pharmaceuticals Ltd., U.K) drops. Alcaine (Alcon, Inc., U.S.A) eye drops were used as a corneal local anesthetic. Saline solution drops were systematically applied to the eye as a tear supplementation both to prevent dehydration and to provide adequate electrical conduction for the corneal electrode. Preventing the cornea from dryness also reduces the likelihood of cataract formation. In order to further cataract prevention, an Ocuscience LLC contact lens (Kansas city, MO, USA) completely covered the cornea through a layer of 1% methyl cellulose gel, during dark adaptation (Ridder, Nusinowitz and Heckenlively, 2002). After the dark adaptation the contact lens was removed under dim red illumination and the corneal electrode placed on the cornea again, which always contacted the cornea through a layer of 1% methyl cellulose (Goto, 1996). After the mouse was anaesthetized the whiskers were cut away with scissors, limiting their interfering. The mouse was placed laterally on a heating pad in order to preserve its natural body temperature, monitored with a rectal thermos sensor placed beneath the animal. Both the thermos sensor and the heating pad were in turn connected to a thermal regulator. The mouse head was moved in a straight line with the body using a short string threaded behind its upper front teeth, maintaining normal breathing by keeping the airway safely open. The animal was prepped as previously described for at least ten minutes in order to fully anesthetize the animal. The a-, b- and c-waves were recorded in addition to oscillatory potentials (OPs) and 10 Hz flicker responses. In order to elicit the ERG recordings, light stimuli at five increasing intensities were used, 0.67, 0.97, 1.27, 1.57 and 1.87 log cd s/m2. Stimuli were presented twice in a row in the order of increasing luminosity. Flicker ERGs were recorded at a 10 Hz flicker rate in response to a luminant stimuli of 1.87 log cd s/m2. After the animal preperation all the light sources were covered or turned off. The mice were dark adapted for 30 minutes before testing the rod responses. Rod responses in mice have been shown to require 30 min to stabilize at maximum amplitude (Nusinowitz, Azimi and Heckenlively, 2000). At least one minute elapsed in darkness before the next stimulus in order to allow the photoreceptors to recover from any photopigment bleaching and to maintain the dark adaptation state between flashes (Peachey and Ball, 2003). After the dark adaptation 20 recordings the mouse was light adapted and prepared for ten minutes. The light adaptation ERG protocol was the same as the one during the dark adaptation. After the experiments finished, the anaesthetized animals were sacrificed via cervical dislocation and then bled by femoral artery sectioning. Afterwards, the mice were enucleated and prepared for further examination, either histological, immunocytochemical or for electron microscope images. Data Analysis The a-wave amplitude was measured from baseline to the lowest peak of the cornea-negative deflection. The b-wave was measured from baseline, or lowest point of the a-wave if it was present, to its peak within 200 ms after the stimulus onset. The light adapted c-waves were only measured in response to a luminant stimuli of 1.87 log cd s/m2 but at all luminous intensities during dark adaptation. The c-wave amplitude was measured as the difference between the PIII component tail and the apex voltage value at 800 ms afterwards (Dornstauder et al., 2012). Implicit times of the a-, b- and c-waves were measured from flash onset to their apex. The amplitudes of the oscillatory potentials were measured with the oscillatory index, by obtaining the mean amplitude of the first three wavelets from peak to peak. The mean implicit time was measured from flash onset to each wavelet peak. The mean amplitude and implicit times of the 10 Hz flicker responses were measured as the difference between the apex and base of the first three peaks. The b/a wave ratio was only measured from the dark adaptation recordings, on account of the light adapted a-wave usually being tiny or absent, as found in most studies of the mouse ERG. Independent samples t-test was performed on the ERG a-waves, b-waves, b/a wave ratio, c-waves, oscillatory potentials, 10 Hz flicker amplitudes and implicit times to test for the mean differences between the groups. The alpha level of significance for the t-test was α = 0,05. IBM SPSS Statistics 20 (IBM SPSS, U.S.A) was used in the data analysis. All the recordings were made in LabChart 7 (ADInstruments, Australia) and then the data entered into Microsoft Excel (Microsoft, U.S.A). SigmaPlot 13 (SYSTAT, U.S.A) was used to plot the graphs. 21 Results The implementation of the independent samples t-test requires approximately normally distributed values and that they fulfill the homogenity of variance within the comparison groups. The normality assumption was found reasonable within both groups based on Q-Q plots. The homogenity of variance was tested using the Levene‘s Test for Equality of Variances. The test demonstrated which group variances could be treated equally and if not, they were properly handled. Lastly, significant outliers were screened for using boxplots because they can reduce the validity of the results. No significant outliers were found. In order to ascertain the effects of the differing Mitf mutations on retinal function, ERGs were recorded from all the mutants and compared to the ERGs of wild type mice. 22 Figure 2. Scotopic ERGs recorded from one wild type mouse (top recording) and from a mouse with each of the Mitf mutations, in response to a white light stimuli of 1.87 log cd s/m2. This Figure shows the lack of ERG components in all the mutants except the Mitfmi-enu122(398) genotype. 23 Figure 2 shows an example of dark adapted ERGs, from a wild-type mouse and one of each mutation, in response to the brightest stimulus light intensity. This Figure shows that no measurable ERGs were recorded from Mitfmi-wh/+, Mitfmi-wh/Mitfmi nor the Mitfmi/+ mutants. The Mitfmi-enu122(398) mutants are therefore the only mutant genotype with sight and thereby the only mutant with measurable retinal function. Henceforth, the Mitfmi-enu122(398) will be the only mutant genotype compared with the wild type mice. An independent samples t-test and 95% confidence intervals (CI) was run on the data for the mean differences between the comparison groups, wild-type mice and the Mitfmienu122(398) . Figure 3. Scotopic ERGs recorded from one wild-type mouse (coloured black) and the only mutant genotype with sight, Mitfmi-enu122(398) (coloured blue). These records are the same as in Figure 2 but overlapped and on a much faster and shorter time scale to show the a- and b-waves distinctly. The scotopic a-wave amplitude had the mean difference of 43,4 µV (95% CI, -17.5 to 104.3) which was not significant, t(8) = 1.6, p = 0.14 (Figure 3). Neither was the comparison for the implicit times, with the mean difference 1 ms (95% CI, -2.9 to 4.9), t(8) = 0.59, p = 0.58 ms. 24 The difference between the scotopic b-wave ERGs was also not significant, with the mean amplitude difference of 14.8 µV (95% CI, -52.3 to 81.9), t(8) = 0.5, p = 0.6 µV. The mean difference between implicit times was 0.2 ms (95% CI, -3 to 3.4), t(4.2) = 0.17, p = 0.87 ms. Figure 4. The oscillatory potentials of the same recordings that are showed in Figure 2, but with the amplifier bandwidth set at 100-1000 Hz, to filter out the a-, b- and c-waves. This Figure shows recordings from one wild type mouse (top recording) and from the only mutant genotype with sight, the Mitfmi-enu122(398). The mean amplitude for the scotopic wild-type mice oscillatory potentials (Figure 4) was significantly higher than for the Mitfmi-enu122(398) (Table 1), t(8) = 8.7, p = 0.00, with the mean difference being 29.6 µV (95% CI, 21.7 to 37.5). However, the group difference in implicit times was non-significant, with the mean difference of 0.4 ms (95% CI, -2.5 to 3.3), t(8) = 0.32, p = 0.76. The narrow CI around the zero for the a-, b-wave and the oscillatory potentials implicit times reveals that the group difference is potentially zero, or at best very small. Furthermore, the standard deviation of the scotopic wild-type means are high, indicating a lot of variance within their measurements. 25 The mean amplitude and implicit times in response to the brightest light stimuli intensity for scotopic a-waves, b-waves and oscillatory potentials, along with the t-test results, are shown in Table 1. Table 1. Means and standard deviations (x̅ ± SD) for scotopic ERGs, both amplitudes and implicit times. Group n A-wave A-wave B-wave B-wave OPs OPs (µV) (ms) (µV) (ms) 4 229 ± 47 11 ± 3 416 ± 55 30 ± 3 66 ± 6 26 ± 2 Mitfmi-enu122(398) 3 186 ± 36 10 ± 2 401 ± 35 30 ± 0,45 37 ± 4 25 ± 2 T-test P = 0.14 P = 0.58 P = 0.63 P = 0.87 Wild-type (µV) (ms) P = 0.00 P = 0.76 The difference between the groups in the amplitude of the photopic oscillatory potentials was not significant at α = 0.05 level, with the mean amplitude for the wild-type mice being 5.2 ± 3.3 µV in comparison to the 4.2 ± 1.3 µV for the Mitfmi-enu122(398). The mean difference between the groups was 1 µV (95% CI, -2.7 to 4.7), t(8) = 0.6, p = 0.55. Mitfmi-enu122(398) had longer implicit times than the wild type, 22.4 ± 3.6 versus the 21.6 ± 4.3 µV. The tests for did not yield significant results, with the mean implicit time difference being -0.8 ms (95% CI, -6.6 to 4.9), t(8) = -0.3, p = 0.76. 26 Figure 5. Mean scotopic ERGs from all the wild-type mice and from the only mutant genotype with sight, all the Mitfmi-enu122(398) mice. This Figure shows the relationship between the mean amplitudes of the a- and b-waves (± SEM), in the response to all of the light stimuli intensities used. Figure 5 depics the numerical data from above and shows the relationship between the mean amplitudes of the a- and b-waves of both groups, and their responses to different light stimuli. The Figure shows that the a-wave amplitude means are on average higher for the wildtype mice except for luminance 1.27 log cd s/m2 where the difference is either tiny or none. The amplitude means for the wild-type mice and Mitfmi-enu122(398) b-waves appear to be the same for the first two light intensities, a little bit higher for the Mitfmi-enu122(398) in the light intensity 1.27 log cd s/m2 and then higher for the wild-type mice in the two last highest light intensities, 1.57 and 1.87 log cd s/m2. The standard errors of the means are all rather large for each mean of the wild-type mice, likely due to the small sample size and thus creating uncertainty about the true population means. The mean b/a ratio (± standard deviations) was calculated for the scotopic ERGs and compared between the groups, with non-significant results. The mean difference between the 27 groups was -0.46 µV (95% CI, -1.1 to 0.21), t(4.5) = -1.8, p = 0.14, with the mean amplitude being little higher for the Mitfmi-enu122(398) or 2.4 ± 0.55 µV compared to the 1.9 ± 0.13 µV of the wild-type mice. Figure 6. Scotopic C-waves from one wild-type mouse (top) and from the only seeing mutants, the Mitfmi-enu122(398). The recordings are responses to a white light stimuli of 1.87 log cd s/m2, as is in Figure 2, but in a much longer time scale to show the c-wave. Figure 6 shows the electroretinographic recordings of the scotopic c-wave from a wild type mouse and the Mitfmi-enu122(398) in response to the highest light stimulus intensity and on a longer time scale than the recordings in Figure 2. The difference between the groups was nonsignificant, t(7) = 0.3, p = 0.77, but wild-type mice had higher mean amplitudes, 237.6 ± 27.4 µV compared with the 231 ± 38.6 µV of the Mitfmi-enu122(398), with the mean difference being 6.6 (95% CI, -45.2 to 58.4) µV. However, the difference between mean implicit times for the scotopic c-wave were significant, t(8) = 2.4, p = 0.04 with longer implicit times for wild-type mice, or 591 ± 21.6 ms in comparison to the 542.4 ± 39.9 ms of the Mitfmi-enu122(398). The mean difference between the groups was 48.6 (95% CI, 1.8 – 95.4) ms. The CI is wide which indicates the uncertainty that the mean difference in the population could be small, 1.8 ms or large, 95.4 ms. 28 The tests for the photopic c-wave amplitude did not yield significant results, t(8) = -8.9, p = 0.4. The mean difference was -52.8 µV (95% CI, -190.5 to 84.9) µV, with the higher amplitude mean of the Mitfmi-enu122(398), 243 ± 126.5 µV compared to the 190.2 ± 42.7 µV of the wild-type mice. The mean photopic implicit time for the c-wave was also longer for the wildtype mice, with the mean implicit time being 402 ± 105 ms for wild-type and 360.6 ± 133.4 ms. The difference was not significant, with the mean implicit time being 41.2 ms (95% CI, -133.8 to 216.2) ms, t(8) = 0.54, p = 0.6. However, the CI for the scotopic c-wave implicit times, the photopic c-wave amplitudes and implicit times were all enormous, leading to much uncertainty about the true population mean, with the probability of a really small difference or a really big one. Figure 7. Dark adapted flicker ERG recordings from wild-type mouse (left) and Mitfmi-enu122(398), the only mutant with sight, respondent to 10 Hz flicker stimuli of 1.87 log cd s/m2. The mean amplitudes for the scotopic 10 Hz flicker stimulation (Figure 7) did not yield significant results, or t(5) = 0.19, p = 0.86 with the mean amplitudes for the wild-type mice being 134.5 ± 35.5 µV and 130 ± 21.2 µV for the Mitfmi-enu122(398). The mean difference between the comparison groups was 4.5 µV (95% CI, -55.6 to 64.6). The mean implicit times for the 10 Hz flicker responses was not significant either, t(5) = 0.66, p = 0.54 with the mean difference 20 (95% CI, -58.3 to 98.3) ms. Both those confidence intervals (CI) are wide and contain zero, so they bring uncertainty because the population differences could be big or nonexistent. Table 2 shows the mean amplitude and implicit times for the photopic 10 Hz flicker stimulation, with the mean amplitude difference being -43.3 µV (95% CI, -136.7 to 50.1), t(4) = -1.3, p = 29 0.27 and with the mean difference for the implicit times 23 ms (95% CI, -85.9 to 131.9), t(2) = 0.9, p = 0.46 ms. Figure 8. Photopic ERGs recorded from one wild-type mouse (top) and from the only mutant genotype with sight, the Mitfmi-enu122(398). The recordings are responses to a white light stimuli of 1.87 log cd s/m2. Each mouse was tested both for light-adapted and dark-adapted ERGs. Figure 8 exhibits the response of a wild-type mouse and Mitfmi-enu122(398) in response to a stimulus of the highest luminance. As under scotopic conditions, the photopic ERGs yielded no measurable responses from the other mutant genotypes, Mitfmi-wh/+, Mitfmi-wh/Mitfmi nor the Mitfmi/+ and hence the data is not shown. 30 Figure 9. Photopic ERGs recorded from one wild-type mouse (black) and one from the only seeing mutant phenotype, Mitfmi-enu122(398) (blue). These are the same recordings as in Figure 7, only overlapped. The mean amplitude for the photopic a-wave was significantly higher for the wild-type mice, or t(8) = 3.5, p = 0.01 with the mean amplitude 16.2 ± 3.5 µV compared to the 8.6 ± 3.3 µV of the Mitfmi-enu122(398), with the mean difference 7.6 µV (95% CI, -2.7 to 12.5). For the photopic a-wave implicit time the difference did not yield signifcant results, t(4.6) = -0.3, p = 0.75, with the mean difference between the groups -0.4 (95% CI, -3.2 to 2.4). The implicit time for the wild type mice was a little shorter, 15 ± 0.7 than the 15.4 ± 2.6 for the Mitfmi-enu122(398). As Table 2 lists, there was no significant difference between the comparison groups in the photopic b-wave amplitude, with the mean difference -8.2 µV (95% CI, -73.8 to 57.4) (Figure 7). For the implicit times on the other hand, the t-test yielded significant results, t(8) = 2.7, p = 0.03. The mean difference between the groups was 6.4, with 95% confidence that the wild-type mice have 1 to 12 ms longer implicit time than the Mitfmi-enu122(398) (95% CI, 0.9 to 11.9) ms (Figure 8). 31 Table 2. Mean and standard deviation (x̅ ± SD) for light adapted ERGs, both amplitudes and implicit times. Group n 10 Hz 10 Hz B-wave B-wave (µV) (ms) (µV) (ms) 81 ± 16 250 ± 44 87 ± 43 40 ± 4 Mitfmi-enu122(398) 3 124 ± 56 227 ± 3 95 ± 47 34 ± 4 T-test P = 0,27 P = 0,46 P = 0,78 P = 0,03 Wild-type 3 Figure 10. Light micrograph images (with 40x magnification) of a segment from the retina and the RPE from both a wild-type mouse (left) and from the only non-blind mutant, Mitfmi-enu122(398) (right), using Toluidine blue staining. So far, histological examination has been carried out on only wild type and Mitfmienu122(398) mice. Figure 10 shows light microscopic micrographs of retinas and the RPE from one wild type mouse and one with the Mitfmi-enu122(398) mutation, stained with Toluidine blue stain. The main differences between the light micrograph pictures of the wild-type mouse and the Mitfmi-enu122(398) is the morphology of the latter. The Mitfmi-enu122(398) retinal pigment epithelium cell layer (seen as a dark band of cells above the photoreceptors at the top of each micrograph) is white spotted and shows localized thinning of melanin-containing areas of the layer. The choroid in the Mitfmi-enu122(398) was removed before taking the micrograph, due to the preparation of the samples for the electron microscope. It should be noted that these histological results are preliminary, and do not include the other mutations examined, and the samples were not prepared specifically for light microscopy, but for electron microscopy and then Toluidine blue stained. 32 The hearing function of each mouse was operationalized by coarsely measuring their behavioral responses to a loud clapping sound. The wild-type mice showed evidence of normal hearing function and were accordingly the most reactive to the clap. The reaction of the mutant mice were then compared to that of the wild-type mice. The Mitfmi-enu122(398) mice were less responsive than the wild-type mice. The blind mutants had the least reaction, thus probably having a hearing deficiency. 33 Discussion The purpose of this study was to examine the retinal function of mice with various mutations in the Micropthtalmia-associated transcription factor (Mitf) gene, using electroretinography. The Mitf gene is operationalized with five different mutations, ranging in severity and thus, with each genotype impacting retinal function differently. In this study, the following Mitf mutations were examined: three Mitfmi-wh/+ heterozygotes, one Mitfmi/+ heterozygotes due to breeding difficulties, three Mitfmi-wh/Mitfmi heterozygotes and three Mitfmi-enu122(398) homozygotes. The number of animals tested was determined by the availability of animals with each mutation at the time. The results from the mutants were then compared to those of the controls, wild-type mice (C57BL/6J). Interestingly, the ERG measurements revealed that only one genotype of the mutations had any evidence of retinal function, and thus not blind, the Mitfmi-enu122(398). These results indicate that the Mitf gene has a strong impact on retinal function, on account of the fact that three out of the four mutant genotypes used in this study were without any retinal function and thus blind. It was hypothesized that the retinal function was impacted in accordance with the location of the mutation on the gene and hence its severity. If that would have been the case, the only blind mutant genotype would have been the Mitfmi-wh/Mitfmi heterozygotes. Furthermore, the hearing function of each mouse was crudely assessed by their behavioral reaction to a loud clapping sound. The results indicated that their auditory function really affiliated with their retinal function, in that the Mitfmi-enu122(398) mice reacted less than their wild-type counterparts to auditory stimuli, and the blind mutants reacted the least. These results further demonstrate that dysfunctional Mitf has wide-ranging consequences that are of crucial importance for sensory function. Furthermore, the more dysfunctional the Mitf gene is, the more damage it causes, proven by examining these severity ranging mutations. The Mitfmienu122(398) had the least severe change in the gene and had accordingly the best sensory responses of all the mutants. These results are in accordance with previous studies about the importance of the Mitf gene for various bodily functions and organs (Arnheiter, 2010; Moore, 1995; Möller et al., 2004; Ni et al, 2012; Steingrímsson et al., 2004). Prior studies had already shown the Mitfmi-wh/+ heterozygotes to be deaf (Ni et al, 2012), due to dysfunctional Mitf. No previous study has been carried out that examined the hearing function of the other mutations. Above all, these mutant genotypes have hardly been examined at all, at least not with regards to their retinal or visual function, or any other neural function. 34 Out of 17 measurements from wild-type mice, four dark-adapted sessions were used but only three light-adapted, due to too much noise in the ERG recordings, as well as other technical difficulties during these experiments. The mutant sample size could have been larger to increase the statistical power, but it is more ethical to sacrifice as few laboratory animals as possible, especially in this case were the blind mutants did not show any measurable ERG responses. Furthermore, the mutant samples were large enough to clearly show that the ERG responses to light was unmeasurable from animals with three of the Mitf mutations. It is of interest that the „flat“ non-detectable ERG responses from these animal are comparable to what is found in human patients with retinal degenerative diseases like retinitis pigmentosa (e.g. Hartong et al., 2006). The Mitfmi-enu122(398) mice had significantly lower light adapted a-wave amplitudes, which derive from the photoreceptors, than the wild-type mice. However, the b/a ratio was unaffected and almost identical between the two groups, indicating a localized photoreceptor disorder since the b/a ratio reflects signal transmission from photoreceptors to retinal bipolar cells, which were apparently unaffected by the mutations (Möller et al., 2004; Perlman, 1995). In addition to the significantly shorter implicit time of the light adapted b-wave, the Mitfmienu122(398) had a significantly shorter scotopic c-wave implicit time. These results are intriguing, since previous studies report abnormal implicit times as prolonged, in comparison to the control groups, wild-type mice (e.g. Creel, 2015). No previous study was found that reported the opposite, abnormally significantly shorter implicit times in mice. Shorter implicit times may indicate faster signal transmission along a neural network like the retina, but at this point it is not possible to determine if that is the case, and then why, so clearly further studies of the histology and function of synapses in these retinae are needed. But this and other evidence suggests that the Mitf gene may be affecting neuronal functions to greater extent than we know at this point. The mean amplitude for the oscillatory potentials in the wild-type mice were almost twice as high as the ones of the Mitfmi-enu122(398). The oscillatory potentials are believed to be generated by neuronal activity in the inner retinal layers, probably bipolar cells, interplexiform cells or most likely by a subpopulation of amacrine cells (Dowling, 1987; Möller et al., 2004; Wachtmeister, 1998). Amacrine cells are also believed to partly modify a- and b-waves, both of which were significantly altered in the Mitfmi-enu122(398), in comparison to wild-type mice (Dong and Hare, 2000, 2002; Möller and Eysteinsson, 2003). The light adapted a-wave was 35 significantly lower in the Mitfmi-enu122(398) whereas its photopic b-wave implicit time was significantly shorter. Together, these results suggest that there may be defective amacrine cell function in the Mitfmi-enu122(398) mice. Looking at the majority of the statistically non-significant comparisons, the wild-type mice had non-significantly slightly higher mean amplitudes than the Mitfmi-enu122(398). However, this was not the case with regard to the photopic b- and c-waves, light adapted flicker, and the b/a ratio. As for the implicit times of the Mitfmi-enu122(398), they had mostly shorter implicit times than the wild-type mice, except for the photopic oscillatory potentials. In those exceptions, the difference was small and non-significant, plus the enormous zero containing CI. Interestingly, the many higher Mitfmi-enu122(398) amplitude means for the photopic ERGs could suggest that they have a greater quantity of cones than wild-type mice. This can only be researched with a larger Mitfmi-enu122(398) sample, and would require confirmation by histological examination of retinal specimen from these mice. Such specimens, using Toluidine blue staining are available, and are currently being processed and examined in our laboratory. The light microscophic analysis, performed on the animals in continuation of the electroretinography, revealed that the retinal pigment epithelium layer in the Mitfmi-enu122(398) shows localized thinning of melanin-containing areas of the layer. This is probably due to the localized degeneration of the retinal pigment epithelium in these animals, since their retina looks normal. The retina and the RPE of the other mutations are currently being examined, but dysfunction and/or degeneration of the RPE is very common in animals with mutations in the Mitf gene, due to its important role in pigment cells function (Arnheiter, 2010; Bharti et al., 2006; Hou and Pavan, 2008; Möller et al, 2004; Steingrímsson et al., 2004). Such degeneration is the most likely cause for the blindness of the other mutant genotypes used in this study, the Mitfmi-wh/+ heterozygotes, Mitfmi/+ heterozygotes and Mitfmi-wh/Mitfmi, but the histological studies of these retinae need to be completed before this can be determined. However, preliminary histological results of the Mitfmi-wh/+ and the Mitfmi-wh/Mitfmi indicate immense retinal degeneration, lacking both the photoreceptor cell layer and the outer plexiform layer. In addition, the retina of the Mitfmi/+ appearing to be much attenuated. Functional retinal pigment epithelium is very important for normal functioning photoreceptors and vice versa (Raymond and Jackson, 1995; Strauss, 2005). So if either one is degenerating, it could have a mirroring effect on the other. Such interactions between these types of cells could therefore explain the abnormal a-wave and especially the abnormal c-wave, 36 with the a-wave expressing photoreceptor function and the c-wave reflecting the physiological interaction of photoreceptors, rods, Müller cells and mainly retinal pigment epithelium (Oakley and Green, 1976; Steinberg et al., 1970). This could also possibly explain the shorter photopic b-wave and c-wave implicit times, or that the thinning photoreceptor or RPE layer, transmits impulses faster with less cells to go through. However, the amplitude of the c-wave was not significantly lower in the Mitfmi-enu122(398), only the a-wave amplitude, so a photoreceptor deficit can not be excluded. Furthermore, abnormal and degenerative cells in the retina, as the RPE cell layer in the Mitfmi-enu122(398) proved to be, could cause a chain reaction, progressively damaging more and more cells. That could possibly explain all the retinal abnormality in the Mitfmi-enu122(398). Combined, these results suggest that the Mitfmi-enu122(398) genotype has generally a minor effect on the activity of the Mitf gene, at least both in relation to visual and hearing function. It was hypothesized that the impact of the mutation on the gene function would be based on its location on the gene. If that would have been true, it would have only impacted the c-wave and no other components of the ERG. It would be of interest to examine these animals in earlier and later stages of life, to see if they are born blind or if retinal degeneration occurs gradually over time. 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