Download Electroretinographic analysis of retinal function in mice

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).
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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).
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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,
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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).
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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,
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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;
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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. Further examination,
histological, immunocytochemical analysis of retinal and RPE cell proteins, or electron
microscope images of the retina and the RPE will shed further light on what is happening. In
the future, ERGs could be obtained from a larger sample of the Mitfmi-enu122(398) mutant mice in
order to increase the statistical power of our analysis and hence, to better represent the
population and thereby increasing both the reliability and validity of the results.
37
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