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Sensitivity and dynamic range of rod pathways in the mammalian retina
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Sensitivity and dynamic range of rod pathways in the mammalian retina
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Content
SENSITIVITY AND DYNAMIC RANGE OF ROD PATHWAYS IN THE
MAMMALIAN RETINA
by
Arvin Cyrus Arman
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
August 2010
Copyright 2010 Arvin Cyrus Arman
ii
Epigraph
“A man should look for what is, and not what he thinks should be.”
-- Albert Einstein
I have found these words to be inspirational and guiding
throughout my time as a graduate student. They remind me
that our goal as investigators of nature is to faithfully report
our observations, and not to predicate findings based on our
expectations.
iii
Dedication
This thesis is dedicated to my mother, whose emphasis on education taught me
to never stop learning; and to my father, whose own fascination with science
taught me to see the beauty in the mysteries of nature.
And to my wife who has never stopped believing in me.
iv
Acknowledgments
The experiments and work described here-in were all made possible
through the support of my advisor, Dr. Alapakkam Sampath. It is through his
guidance and support (both academic and financial) over the years that I am able
to complete this thesis; and for that I will always be grateful. Dr. Sampath has
been an excellent academic role model, as well as an excellent role model for life
in general. To him I express my deepest appreciation.
I would also like to recognize my thesis/dissertation committee, Dr. Bosco
Tjan, Dr. Judith Hirsch, Dr. Norberto Gryzwacz, and Dr. James Weiland for their
valuable insight and direction over the years.
I would also like to thank the Neuroscience Graduate Program (NGP) for
accepting me into the program and for providing my first year’s support.
Specifically, I would like to acknowledge the NGP administrators, Linda Bazilian,
Vanessa Clark, and Gloria Wan for their help navigating the bureaucratic
landscape of the university.
I also extend my appreciation to my colleagues and friends at the Zilkha
Neurogenetic Institute for sharing in the trials and tribulations of graduate school,
including: Dr. Haruhisa Okawa, Dr. Kiyoharu Joshua Miyagishima, Dr. Alan
Horsager, Dr. Johan Pahlberg, and Alice Cho. Thank you for making my
graduate experience memorable.
v
Most importantly I want to thank my wife, Melissa, who has been with me
through this entire process. When I thought I couldn’t possibly continue, she
taught me not to live with any regrets.
vi
Table of Contents
Epigraph ii
Dedication iii
Acknowledgments iv
List of Figures vii
Abstract viii
Preface xii
Chapter 1: Introduction 1
Chapter 2: OFF cone bipolar cells do not contribute to OFF 19
signals near visual threshold
Chapter 2 Introduction 19
Chapter 2 Materials and Methods 21
Chapter 2 Results 26
Chapter 2 Discussion 38
Chapter 3: Bleached rod photoresponses persist and traverse 44
the retina
Chapter 3 Introduction 44
Chapter 3 Materials and Methods 46
Chapter 3 Results 51
Chapter 3 Discussion 61
Chapter 4: Conclusion 66
References 70
vii
List of Figures
Figure 2.1s: Supplemental data 22
Figure 2.1: Ganglion cell current-clamp recordings 29
Figure 2.2: AII amacrine cell current-clamp recordings 31
Figure 2.3: OFF bipolar cell current-clamp recordings 33
Figure 2.4: Compiled data 35
Figure 2.5: Voltage-clamp recordings 37
Figure 3.1: ERG and MSP data 53
Figure 3.2: ERG Weber-Fechner Relation 55
Figure 3.3: Bipolar cell patch-clamp recordings and images 58
Figure 3.4: Rod bipolar versus cone bipolar cells comparisons 60
viii
Abstract
Nearly all sensory systems must find a way to represent a wide range of
input signals and translate them into meaningful neural responses. The human
visual system is able to operate effectively from starlight to bright sunlight, a
range that spans about twelve orders of magnitude of light intensity. To reliably
transmit changes in light stimuli over this range the mammalian retina has
evolved several specializations to report changes in the light environment that
include: 1) the evolution of two photoreceptor types, the rods and cones, that
operate at different light levels, 2) several neural pathways with which to encode
the output of these photoreceptors, and 3) adaptive mechanisms at all levels of
retinal processing to modulate light sensitivity based on light history. To address
questions regarding the sensitivity and the operating range of rod pathways in
the mammalian retina, I will present data from two projects designed to
investigate how rod-generated signals traverse the retinal circuitry under various
lighting conditions.
In the first study titled “OFF cone bipolar cells do not contribute to OFF
signals near visual threshold”, I examined how signals traverse the retinal
circuitry under fully dark-adapted conditions. Under these conditions a
specialized retinal pathway conserved across mammalian species, called the
Rod Bipolar Pathway, carries visual signals near absolute threshold. In this
pathway rod signals converge downstream on depolarizing (AII) amacrine cells,
ix
but the route for signal flow from AII amacrine cells to OFF ganglion cells near
visual threshold remains unclear; signals can be relayed directly to OFF ganglion
cells, or to OFF ganglion cells through OFF cone bipolar cell synaptic terminals. I
show in physiological recordings from dark-adapted retinas that glycinergic
synapses between AII amacrine cells and OFF ganglion cells conveyed sensitive
rod signals, whose response thresholds were elevated by strychnine.
Strychnine, surprisingly, did not affect response threshold in any subtype of OFF
cone bipolar cell, but produced a mild depolarization of the resting membrane
potential, indicating a tonic glycinergic inhibition under dark-adapted conditions.
These studies indicate that near visual threshold that rod signals are conveyed to
OFF ganglion cells from AII amacrine cells, without contributions from OFF cone
bipolar cells. This argues against the notion that the rod pathways must
ultimately impinge on traditional cone pathways before reaching ganglion cells.
In the second study, titled ”Bleached rod photoresponses persist and
traverse the retina”, alongside Dr. Kiyoharu Joshua Miyagishima, I examined the
mechanism by which rods remain responsive despite the bleaching of a majority
of their visual pigment. Exposure to bright bleaching light results in adaptation in
a manner similar to exposures to background light that desensitize the cell. In
rods, which are more sensitive, this adaptation is long-lasting since they are slow
to regain their sensitivity. The loss in sensitivity can be explained by two
phenomena; the first resulting from the loss in available photopigment for photon
absorption, and the second resulting from residual catalytic activity of the
x
photoproducts of bleaching. We measured the steady-state sensitivity following
defined extents of pigment bleaching in the mouse retina. In single cell
recordings from bleached retina we show that alternative rod pathways preserve
and pool rod signals to improve overall sensitivity in the mesopic range.
The experiments described in this dissertation focus on the functional
properties of rod vision by using transgenic mice where cone photoreceptors are
unable to generate light activated responses (Gnat2
-/-
). These mice allow the
study of the lower and upper limits of rod vision. Studying scotopic signaling in
dark-adapted retinas from Gnat2
-/-
mice has implications for understanding
specific forms of nyctalopia, such as: congenital stationary night blindness and
retinitis pigmentosa (RP), which are classified as deficiencies in seeing under
dim lighting conditions. The experiments described in Chapter 2 have
contributed to our understanding how visual signals generated by the rod
photoreceptors traverse the retinal pathways en route to more central structures
within the brain. These pathways are likely to be the therapeutic targets for
nyctalopic diseases.
Conversely studying the functional properties of light-adapted retinas
using the same animal models can give insight into hemeralopia, which is
defined as any deficiency in seeing under very bright lighting conditions.
Hemeralopic individuals typically present with achromatopsia and an inability to
form visual signals interpretable by the nervous system under bright lighting
conditions. The experiments in Chapter 3 have contributed to our understanding
xi
of how individuals with no functional cones (rod monochromats) are able
functionally navigate under bright lighting conditions.
xii
Preface
This thesis is based upon studies conducted between June 2006 and May
2010 at the Zilkha Neurogenetic Institute, Keck School of Medicine, at the
University of Southern California. This thesis encompasses only part of my work
on rod mediated signaling in the mammalian retina; other collaborative projects
using similar methods as the ones described below, have been completed with
investigators at other universities.
Chapter 2 represents the vast majority of the experimentation I performed
during my time as a graduate student. I used a combination of slice and whole-
mount preparations to determine the sensitivity of many of the cells in dark-
adapted retina of mice lacking cone photoresponses (Gnat2
-/-
), ultimately finding
that there exists a pathway for scotopic signaling which is independent of
canonical cone pathways.
Chapter 3 represents a collaborative effort with Dr. Kiyoharu Joshua
Miyagishima in which we recorded electroretinogram (ERG) responses from
isolated retina and used pharmacological reagents to separate the a-wave and
the b-wave to look at the changes in the rod response in the Gnat2
-/-
retinae. Dr.
Miyagishima and I worked together to make single cell recordings from cells of
the inner nuclear layer (INL) of bleached retinal slices to look independently at
how the alternative rod pathways contributed to rod vision following bleaching.
Dr. Soile Nymark contributed microspectrophotometry data, which further
xiii
validated the estimates of the fraction of visual pigment bleached in our
experiments.
1
Chapter 1: Introduction
Nearly all sensory systems must find a way to represent a wide range of
input signals and translate them into meaningful neural responses. The human
visual system is able to operate effectively from starlight to bright sunlight, a
range that spans roughly twelve orders of magnitude of light intensity (Rodieck,
1998). The pupil serves as the first stage of sensitivity control by changing the
amount of the light reaching the photoreceptors. The diameter of the pupil can
change by a factor of four, allowing light intensity to change by a factor of sixteen
(Atchison and Smith, 2000). However, this alone cannot account for the
complete range of light to which the mammalian retina is sensitive. To reliably
transmit changes in light stimuli over this range the mammalian retina has
evolved several specializations to report changes in the light environment that
include: 1) the evolution of two photoreceptor types, the rods and cones, that
operate at different light levels, 2) several neural pathways with which to encode
the output of these photoreceptors, and 3) adaptive mechanisms at all levels of
retinal processing to modulate light sensitivity based on light history.
Rods vs. Cones
The evolution of the duplex retina in vertebrates with two classes of ciliary
photoreceptors, the rods and cones, marks a departure from the single type of
photoreceptor present in invertebrates. The use of two photoreceptor subtypes
2
with different sensitivities to light allows the vertebrate retina to respond over a
greater range of light intensities. By switching between rods and cones, the
vertebrate retina is thus able to maximize visual sensitivity depending on the
ambient light level. It is now believed that such an arrangement allows the
vertebrate retina to reduce energy consumption in daylight, when the rods are
not responsive (Okawa et al., 2008).
Rods mediate vision when photons are scarce; their design and
cytoarchitecture are optimized for maximal sensitivity to incoming photons. Rods
are capable of generating a reproducible response to a single absorbed photon
(Barlow, 1964), which is critical for setting the sensitivity of scotopic vision when
the retinal circuitry pools thousands of rods (see below). As the mean
background light level increases, rods themselves are able to adapt, which
allows them to signal light intensities up to ~ 100,000 R* or more per second (Yin
et al., 2006).
Cone photoreceptors are ~ 100-fold less sensitive than rods, and are
critical for our daytime vision under conditions when the exquisitely sensitive rod
photoreceptors are saturated. The reduced sensitivity of cones arises from
reduced amplification within cone phototransduction, and mechanisms designed
to shut off phototransduction more quickly than rods. Furthermore, even in the
brightest light the cone photocurrent does not remain saturated, which leaves
open the ability to adapt and signal changes in light intensity even under
conditions where a majority of the photopigment is bleached. The optimizations
3
of cones to function in bright light with virtually little overlap with rod light levels
allows for a smooth transition from rod to cone vision, in what is called the
mesopic range.
The Pathways Concept
The adaptive features of the rod and cone light response allow these
photoreceptors to remain responsive over a larger range of light intensities by
preventing response saturation. However, the dynamic range of the range of the
rods and cones themselves cannot account for the ~ 12 orders of magnitude in
light intensity we experience. Another strategy used by the mammalian retina to
extend further the dynamic range of vision is to utilize multiple neural pathways to
carry light-evoked signals. The functional properties of these pathways (e.g.
convergence, gain, and adaptation) can then be adjusted to maximize the visual
system’s ability to remain responsive over the largest range of light intensities.
To date the most studied retinal pathways in mammals are those that carry
signals from rod photoreceptors to ganglion cells (Nusinowitz et al., 2007).
These include the circuits that carry light information near scotopic threshold,
known as the Rod Bipolar Pathway or ‘Classical’ Rod Pathway (Dacheux and
Raviola, 1986; Strettoi et al., 1992), and those that operate at higher rod light
levels that may provide a seamless mesopic transition to photopic vision: the
Rod-Cone and Rod-Off Pathways (Protti et al., 2005; Smith et al., 1989; Smith et
al., 1986; Soucy et al., 1998; Volgyi, 2004). Much less is known about the
4
functional properties of the cone pathways that maximize dynamic range (see
below). As described below, each of the rod pathways is thought to ultimately
impinge on traditional cone circuitry before relaying signals to the retinal ganglion
cells.
Rod-Bipolar Pathway
Psychophysical studies have demonstrated at the limits of scotopic vision
that the human visual system is capable of detecting the absorption of few
photons of light (Bouman and Van Der Velden, 1947; Hecht et al., 1942; Sakitt,
1972). This remarkable sensitivity arises from the fact that individual rods can
reliably signal the absorption of a single photon (Baylor et al., 1979a;
Schneeweis and Schnapf, 1995), and that a specialized retinal circuitry referred
to as the Rod Bipolar Pathway can combine these signals such that a ganglion
cell projecting centrally can pool thousands of rod signals (Sterling et al., 1988).
The discovery of a dedicated depolarizing bipolar cell and amacrine cell that
carries rod signals in the rabbit retina (Dacheux and Raviola, 1986) lead to the
finding that this circuit appears conserved across all mammalian species
(Nusinowitz et al., 2007; Wässle, 2004). A hallmark of this pathway is the
convergence of rod-mediated signals at many stages of processing that is critical
for our scotopic sensitivity. For instance, as many as 20-100 rods converge on a
rod (ON) bipolar cell, and 20-30 rod bipolar cells converge on an AII amacrine
cell (Sterling et al., 1988). Thus a ganglion cell that sums the output of many AII
5
amacrine cells can pool upwards of 10,000 rods (and perhaps 20,000 rods in
cats). The AII cells signal to ON cone BPCs via sign-conserving gap-junctions
and to OFF cone BPCs via sign-inverting glycinergic synapses. As the name
implies cone BPCs primarily carry cone signals, but under low lighting conditions
the signals from the Rod-Rod Bipolar pathway get shunted into their circuitry.
Ultimately by pooling rod signals and eliminating rod noise to preserve best the
single photon response from individual rods, the Rod Bipolar Pathway can
extend the dynamic range of vision down to light levels where a small fraction of
the rods absorb a photon.
Signal transfer from rods to rod bipolar cells
At scotopic threshold, vision relies on a sparse number of photons at the
retina, which produces few photon absorptions per thousands of rods within the
0.2 s integration time of the rod photoresponse (Walraven et al., 1990). Under
these conditions the transmission of a small graded hyperpolarization upon
photon absorption requires that rod synapse is appropriately optimized. The
transmission of small graded single photon responses at the rod synaptic
terminal is aided by two specializations. First, the resting dark membrane
potential, or voltage, sits at ~ -40 mV (Cao et al., 2008; Schneeweis and
Schnapf, 1995), near the steepest point in the relationship between voltage and
L-type Ca
2+
channel opening. Thus small changes in membrane potential
produce substantial changes in the number of open channels thereby altering
6
glutamate release. Second, if the rod bipolar cell is sensing reductions in
glutamate release due to photon absorption, then statistical lapses of glutamate
release in darkness would mimic light absorption (Rao-Mirotznik et al., 1995).
Thus the high rate of glutamate release generated in darkness by the specialized
synaptic ribbon in the rod spherule reduces the probability of these lapses.
Together these synaptic properties allow the small light-evoked signals from rods
to be reproducibly transferred to downstream neurons.
Despite the rod synaptic specializations for the transmission of single
photon absorptions, the depolarization in darkness due to open cGMP-gated
channels is also a complicating factor in the detection of these sparse signals.
Open cGMP-gated channels in turn will report internal fluctuations in cGMP
produced by the phototransduction mechanism, which are commonly referred to
as dark noise (Baylor et al., 1979a, b; Baylor et al., 1980). Since rods generate a
small (~1 mV) graded hyperpolarization upon photon absorption (Baylor et al.,
1984b; Schneeweis and Schnapf, 1995; Tamura et al., 1989), the downstream
convergence of thousands of rod signals would cause the light-evoked response
from a single rod to be overwhelmed by the dark noise of the majority. Given the
magnitude of dark noise in individual rods, it has been proposed that some type
of nonlinear combination of rod signals would be required to increase the
detection of the single photon responses in downstream cells (Baylor et al.,
1984a; Baylor et al., 1984b). Since rod photoreceptors are relatively depolarized
in darkness, the steady release of glutamate from the synapse (Trifonov, 1968)
7
provides some insights into potential mechanisms. Van Rossum and Smith
(1998) suggested that postsynaptic saturation at the rod-to-rod bipolar synapse
would allow noise generated by open cGMP-gated channels in the rod outer
segment to be eliminated. They proposed that the saturation of postsynaptic
glutamate receptors would provide a nonlinear way to eliminate the rod noise,
since the synapse would not be able to relay small changes in membrane
potential that reflect rod noise. Later work suggested that such ‘thresholding’ is
critical for maximizing the detection of the single photon response in retinal
neurons downstream of the rods (Berntson et al., 2004a; Field and Rieke, 2002).
In particular, the extent of nonlinear signaling appears to be set to separate
optimally the rod single photon response from rod noise, allowing scotopic vision
to reach the highest possible sensitivity (Field and Rieke, 2002).
The mechanism that underlies the nonlinear threshold at the rod synapse
has been studied to some extent, but is hindered by a lack of identification of the
components of the signaling pathway. Light-evoked signaling between rod
photoreceptors and rod (ON) bipolar cells results in a membrane depolarization,
effectively inverting the sign of the rod’s hyperpolarizing light response. The
postsynaptic mechanism underlying this sign inversion is a G-protein signaling
pathway initiated by the metabotropic glutamate receptor, mGluR6. mGluR6 in
turn activates a G
oα
G-protein (Dhingra et al., 2002; Dhingra et al., 2000; Nawy,
1999), which leads to a series of unidentified events that close a TRPM1 cationic
transduction channel (Chen et al., 2010; Koike et al., 2010; Morgans et al.,
8
2009). Thus, upon light-absorption glutamate release from rods is reduced,
thereby reducing the activity of the mGluR6 signaling pathway and allowing
transduction channels to open and depolarize the cell.
In the context of the mGluR6 signaling cascade, it now appears that the
nonlinear threshold that eliminates rod noise is due to saturation within the
signaling cascade, and not at the level of the glutamate receptors (Sampath and
Rieke, 2004). Furthermore, evidence from axotomized rod bipolar cells indicates
that nonlinear signal transfer does not arise due to feedback in the inner
plexiform layer (Euler and Masland, 2000). Saturation of the mGluR6 signaling
cascade allows the elimination of noise by making the rod bipolar cell insensitive
to small fluctuations in glutamate driven by noise in the rod photoreceptor (van
Rossum and Smith, 1998). Only when the rod’s membrane potential is
hyperpolarized sufficiently does the glutamate concentration in the synaptic cleft
reduce enough to relieve the synapse from saturation. Such an operation thus
allows larger hyperpolarization due to light absorption to cross the rod synapse,
while masking smaller fluctuations that are more likely due to noise in the rod
photocurrent. Near absolute visual threshold such synaptic processing is
necessary to maximize the detectability of rod signals (Field and Rieke, 2002).
Signal transfer from rod bipolar cells to AII amacrine cells
The convergence of the Rod Bipolar Pathway moving from rods to rod
bipolar cells and finally AII amacrine cells requires the further accentuation of the
9
single photon response. Two main specializations between these cells appear
well tuned to improve further the detection of the single photon response, and
thus push the dynamic range of vision to lower light intensities. First, a
specialized ribbon synapse between the rod bipolar cell and the AII amacrine
cells allow the coordinated release of multiple vesicles upon stimulation (Singer
et al., 2004). Such multivesicular release increases the amplitude of the AII
amacrine cell response, allowing it to be distinguished from vesicular release due
to noise in the rod bipolar cell. Second, the electrical coupling of AII amacrine
cells by Connexin-36 (Deans et al., 2002) appears to reduce noise in the network
(Dunn et al., 2006), allowing an improved signal-to-noise ratio (Vardi and Smith,
1996).
Signal transfer from AII amacrine cells to OFF cone bipolar cells
The earliest electrophysiological studies of bipolar cell responses
demonstrated the center-surround organization of retinal bipolar cells and were
the first to distinguish between two classes of cone bipolar cells: those which
depolarize to the onset of light (ON bipolar cells) and those which hyperpolarize
to the onset of light (OFF bipolar cells) (Boycott and Dowling, 1969; Dowling and
Werblin, 1969; Kaneko, 1970; Kaneko, 1973; Kolb and Nelson, 1983; Matsumoto
and Naka, 1972; Nelson and Kolb, 1983; Schwartz, 1974; Toyoda, 1973). This
functional distinction between ON and OFF bipolar cells arises because ON
bipolar cells express the metabotropic glutamate receptor mGluR6 (Masu et al.,
10
1995; Nomura et al., 1994; Vardi et al., 2000) at their synapses with
photoreceptors, while the OFF bipolar cells express various isoforms of
ionotropic glutamate receptors (Brandstatter et al., 1997; Devries, 2000; Hack et
al., 2001). In the case of the OFF bipolar cells they also express GlyRs on their
axon terminals (Euler and Wassle, 1995; Grunert et al., 1994; Grunert and
Wassle, 1996; Haverkamp et al., 2003; Haverkamp et al., 2004; Heinze et al.,
2007; Ivanova et al., 2006; Milam et al., 1993; Sassoe-Pognetto et al., 1994;
Suzuki et al., 1990).
There is currently limited data in the literature regarding the physiological
input to OFF bipolar cells (Eggers and Lukasiewicz, 2006a, b, 2010; Eggers et
al., 2007; Ivanova et al., 2006). A study by Ivanova and colleagues (2006) used
“puffed” glycine to elicit responses in OFF bipolar cells; while this technique has
confirmed the results of previous immunocytological studies demonstrating that
there are indeed GlyRs on the axonal terminals of OFF bipolar cells (Euler and
Wassle, 1995; Grunert et al., 1994; Grunert and Wassle, 1996; Haverkamp et al.,
2003; Haverkamp et al., 2004; Heinze et al., 2007; Ivanova et al., 2006; Milam et
al., 1993; Sassoe-Pognetto et al., 1994; Suzuki et al., 1990), they are unable to
address the physiological activation thresholds of these receptors/channels.
Subsequent studies by Eggers and colleagues (Eggers and Lukasiewicz, 2006a,
2010; Eggers et al., 2007) have looked at how inhibitory input shapes the
temporal decay of the response in various bipolar cell types. However, to my
knowledge, the experiments in Chapter 2 are the first to look directly at the
11
threshold of light-evoked excitatory and inhibitory responses in OFF bipolar cells
under fully dark-adapted conditions.
Ganglion cell sensitivity
Retinal ganglion cells must also relay single-photon response generated in
individual rods to higher visual centers, a requirement for the high sensitivity of
rod vision (Bouman and Van Der Velden, 1947; Hecht et al., 1942). Through the
process of neural convergence, as well as the mechanisms described above,
ganglion cells, and AII amacrine cells have about the same flash sensitivity
(Dunn et al., 2006).
Recordings of many groups from dark-adapted cat retinal ganglion cells
indicate bursts of ~ 2-3 action potentials occurred with a frequency consistent
with an upstream origin that may be the rods (Barlow et al., 1971; Levick et al.,
1983; Mastronarde, 1983a, b, c). When recording in absolute darkness,
spontaneous bursts of ~3 spikes occurred with the same frequency (Poisson
statistics) as spontaneous isomerizations within photoreceptors themselves. In
these same experiments, when small amounts of light were flashed at the retina,
it was determined that single photon absorption also generated ~3 spikes.
Similar conclusions were drawn by Mastonarde and colleagues using a cross-
correlation analysis from paired ganglion cell recordings in the presence and
absence of background lights.
12
Rod-Cone Pathway
A consequence of the high sensitivity of the Rod Bipolar Pathway is that it
saturates at modest light levels where the rods themselves are not saturated. To
capture light-evoked signals from rods until they themselves saturate additional
pathways are required. Studies of the ultrastructure of the outer plexiform layer
reveal that gap junctions exist between rods and rods, and rods and cones
(Hornstein et al., 2005; Schneeweis and Schnapf, 2000). The nature of the rod-
to-rod gap junction is unclear; but has been proposed to dissipate small rod
signals into the network allowing signal averaging at the cost of a 2-fold elevation
in visual threshold (Hornstein et al., 2005). The rod-to-cone gap junctions have
been studied in more detail and are found in a majority of rods (Smith et al.,
1986). Evidence points to the expression of the gap junction subunit connexin 36
in cones (Deans et al., 2001), but not in rods. Thus the electrical gap junctions
between rods and cones must be heterologous. Given the lower input
impedance of cones, signal transfer will preferably travel from rods → cones
(Smith et al., 1986), which would allow these signals to be relayed to ganglion
cells via the cone circuitry (Nelson, 1977).
Physiological recordings have indicated robust rod signals in the cones
photoreceptors of several species (Hornstein et al., 2005; Nelson, 1977;
Schneeweis and Schnapf, 1995). In particular, measurements from dark-adapted
retina (Hornstein et al., 2005) indicate that rod-to-cone coupling must exist, going
against the idea that to maximize visual sensitivity that the rods and cones
13
should be uncoupled in darkness (Smith et al., 1986). Recordings from mouse
retinal whole mounts suggest that the signals through this pathway are 10-fold
less sensitive than for the Rod Bipolar pathway (Volgyi, 2004). This physiological
data corresponds well with psychophysical experiments indicating a secondary
rod pathway with 10-fold lower sensitivity than the primary pathway (Sharpe and
Stockman, 1999a).
Rod-Off Pathway
A third rod pathway has been identified in some species of mammals.
Under conditions where the Rod-Bipolar and Rod-Cone Pathways are blocked or
eliminated, OFF signals with rod sensitivity have been shown to persist in the
mouse retina (Soucy et al., 1998). Direct synaptic contacts have been identified
anatomically in mice (Tsukamoto et al., 2001), rats (Hack et al., 1999; Muller et
al., 1993), and rabbits (Li et al., 2004). However, direct contacts between rods
and OFF bipolar cells have not yet been reported in higher mammals (Boycott
and Dowling, 1969). It is possible that an OFF rod pathway evolved as a
specialization in nocturnal rodents to sense dark objects against a light sky
(Tsukamoto et al., 2001).
The Rod-OFF pathway is believed to be active at 5-10 fold higher light
intensities than the Rod Bipolar pathway (Volgyi, 2004). Its prominence as a
signaling circuit in rodents appears to vary based on the percentage of the OFF
cone bipolar cell population that sees direct signals from rods (Protti et al., 2005).
14
It is now believed that between 5% and 25% of cone OFF bipolar cells may see
direct contacts from rods (Mataruga et al., 2007), the upper end suggesting that
this pathway may play an integral role in visual processing.
Despite attempts by various groups to characterize the physiological
response properties of the rod pathways (Protti et al., 2005; Volgyi, 2004), the
exact sensitivities and operating ranges of each pathway remain unclear. The
lack of physiological evidence elucidating the role of these pathways in rod vision
arises from common signaling mechanisms used by each. Thus genetic (Soucy
et al., 1998; Volgyi, 2004) or pharmacological (Soucy et al., 1998; Volgyi, 2004)
manipulation of each rod signaling pathway will also influence other pathways,
making it impossible to unambiguously identify the properties of each.
Nevertheless, the evidence now points the Rod Bipolar Pathway as the primary
carrier of single photon responses near absolute visual threshold, with an ~ 5-10
fold reduction in the sensitivity of the Rod-Cone and Rod-Off Pathways that may
carry signals at higher light levels where cone function is merged.
Cone Pathways
Our understanding of the cone pathways is far less complete than our
understanding of rod pathways in the mammalian retina. The physiological
properties of the cones and cone pathways remains one of the frontiers in retinal
neurobiology, especially as cone function dominates our visual experience.
15
Depending on the species, between 9 and 12 types of cone bipolar cells have
been identified in mammalian retinas (Ghosh et al., 2004; MacNeil et al., 2004;
Wassle, 2004), which may connect to as many as 10-15 types of retinal ganglion
cells (Dacey, 1994; Field and Chichilnisky, 2007; Mangrum et al., 2002; Peterson
and Dacey, 1998). Other than the specific pathway for S-cones (Dacey, 1993;
Kolb et al., 1997), there is presently little understanding of specific circuits that
carry cone responses to ganglion cells.
Adaptation to Mean Background Light
A common feature of all sensory systems is the ability to adapt to
increases in the mean level of a stimulus by reducing the gain of the system.
Such sensitivity adjustments allow the sensory system to remain maximally
responsive as the stimulus intensity changes. Both rods and cones in the retina,
as well as their circuitry, exhibit adaptive mechanisms that are designed to
increase the dynamic range of the receptor. However, in the context of the
dynamic range of the visual system, the influences of adaptation on the lower
limit of scotopic and upper limit of photopic vision are opposite. To retain
maximal sensitivity near absolute visual threshold, the retina must maximize its
gain for the single photon response and any adaptive mechanism would allow
the rod circuitry to aid in the transition from scotopic to mesopic vision.
Conversely, the upper light limits of our visual experience are ultimately defined
16
by adaptation in the cone photoreceptors, which continue to operate even when
a majority of the photopigment is bleached (Burkhardt and Fahey, 1999).
Adaptation - Rod Pathways
Adaptation within a neural circuit with considerable convergence will begin
centrally and move peripherally. Under these circumstances downstream cells
will be the first to detect sufficient signal to adapt for the weakest stimuli, and the
rod pathways are no exception. In the Rod Bipolar Pathway, weak background
light begins to reduce the gain of ganglion cells and AII amacrine cells before
adaptation is detectable in rod bipolar cells or rod photoreceptors (Dunn et al.,
2006). Some mechanisms that provide this gain reduction have been identified,
particularly at the rod-to-rod bipolar, and the rod bipolar-to-AII amacrine
synapses. For instance, at the rod-to-rod bipolar synapse the influx of Ca
2+
through mGluR6 transduction channels reduces the bipolar cell gain for
subsequent stimulation (Berntson et al., 2004a; Berntson et al., 2004b; Nawy,
2004). In addition, at the rod bipolar-to-AII amacrine synapse depression
mediated by depletion of the available pool of vesicles (Singer and Diamond,
2006) can be evoked at individual synapses by single photon responses (Dunn
and Rieke, 2008). These adaptive mechanisms allow the Rod Bipolar Pathway
to extend its range to higher light levels where they merge with the other rod
pathways. However, the extension of the dynamic range of vision to lower light
17
levels requires that the retina remain maximally responsive to single photons,
and thus these adaptive mechanisms would impair absolute threshold.
Adaptation - Cone Pathways
It has been well documented that adaptation of ganglion cell responses in
the cone pathways occurs at lower light levels than where adaptive features of
cone pathways have been documented (Boynton and Whitten, 1970; Schnapf et
al., 1990; Schneeweis and Schnapf, 1999). Cone and Horizontal cell recordings
have demonstrated that cone adaptation is only observed for lighting conditions
exceeding 1000 R*s
-1
(Lee et al., 1999; Smith et al., 2001). However, in-vivo
recordings of ganglion cell spiking exhibit adaptation at lighting conditions much
less than 1000 R*s
-1
(Lee et al., 1990; Purpura et al., 1990). This adaptation of
the post-synaptic cone circuitry in turn prevents these pathways from saturating,
thereby allowing the extension of the cone operating range through mesopic to
photopic vision. It would seem that adaptation in the cones and adaptation in the
post-receptoral circuitry are in fact mutually exclusive (Dunn et al., 2007),
ultimately though, the upper limits of cone vision are directly imposed by
receptoral adaptation of the cones themselves and not by the post-synaptic
circuitry.
Summary
18
The vertebrate retina has developed many strategies to maximize the
dynamic range of vision. At the initial stages of light detection the evolution of
two photoreceptor types, the rods and cones, allow the visual system to signal a
wide range of light intensities. By dividing the output of these receptors across
many retinal pathways, each which is subject to its own optimization and
adaptation, the human eye is capable of providing the brain with information that
extends the range vision to encompass approximately twelve orders of
magnitude of light intensity.
In Chapter 2 I present evidence that near the absolute threshold for vision,
OFF signals are relayed to OFF ganglion cells directly through a glycinergic
amacrine cell. To further validate this result I demonstrate that under completely
dark-adapted conditions, the predominant input to each of the classified subtype
of OFF cone bipolar cells is excitatory. Conversely, the predominant input to the
OFF ganglion cells is inhibitory.
In Chapter 3 I present the results of our studies relating the dependence of
adaptation with the concentration of bleached pigment in the mouse retina. We
tested the hypothesis that rod photoresponses continue to travel through the
retina despite bleaching, relying on the alternative rod pathways to reach the
retinal output. Although the rod photoresponses are greatly desensitized, the
persistence of visual sensitivity in classical psychophysical studies on the rod
monochromat suggest that rod information may play an important role at light
intensities thought to be dominated by cone photoresponses.
19
Chapter 2: OFF cone bipolar cells do not contribute to OFF
signals near visual threshold
Chapter 2 Introduction
The mammalian visual system is capable of responding to light stimuli that
vary in intensity by 12 orders of magnitude, a feature that originates in two
classes of photoreceptor cells (rods and cones) and several parallel pathways
from photoreceptors to ganglion cells that function at different mean light levels
((Volgyi, 2004); also see Chapter 1). Near the absolute threshold for seeing,
when photons are scarce, all mammals use a specialized rod pathway, called the
Rod Bipolar Pathway (Dacheux and Raviola, 1986; Strettoi et al., 1990), which
pools the output of thousands of rods and provides ganglion cells with a highly
sensitive input. In this pathway many rods converge onto rod ON bipolar cells,
which subsequently converge on narrow-field depolarizing AII amacrine cells
(Dacheux and Raviola, 1986; Strettoi et al., 1990). It is thought that the AII
amacrine cell in turn relays signals to ON cone bipolar cells through sign-
conserving gap junctions, and to OFF cone bipolar cells via sign-inverting
glycinergic synapses (Dacheux and Raviola, 1986; Famiglietti and Kolb, 1975;
Strettoi et al., 1990) allowing these signals to reach the retinal output, the ON
and OFF ganglion cells respectively (Devries and Baylor, 1995; Hack et al.,
1999; Soucy et al., 1998; Tsukamoto et al., 2001).
This classical scheme is complicated by the presence of glycinergic
synapses between AII amacrine cells and OFF ganglion cells (Chun et al., 1993;
20
Famiglietti and Kolb, 1975; Kolb, 1979; Kolb and Nelson, 1993; Owczarzak and
Pourcho, 1999; Strettoi et al., 1992) providing a route by which the Rod Bipolar
Pathway can signal to ganglion cells independently of the cone circuitry. Thus
there are two possible routes by which glycinergic AII amacrine cells can provide
inhibitory signals to OFF ganglion cells: indirectly via synapses on OFF cone
bipolar cell synaptic terminals which in turn synapse onto OFF ganglion cells, or
directly through synapses on the OFF ganglion cell dendrites. In the mouse
retina, inhibitory synapses from AII amacrine cells directly to OFF ganglion cells
may be the predominant input to these cells (Murphy and Rieke, 2006, 2008;
VAN Wyk et al., 2009), leaving in question the role played by the OFF cone
bipolar synaptic terminals to signaling near absolute visual threshold.
We sought to determine the functional contributions near visual threshold
of these two routes from AII amacrine cells to OFF ganglion cells using murine
retinas lacking cone light responses. Electrophysiological recordings made from
both OFF ganglion cells and OFF cone bipolar cells in the dark-adapted retina
reveal that glycinergic transmission from AII amacrine cells is more sensitive in
OFF ganglion cells; pharmacological blockade of glycine receptors by strychnine
dramatically impairs the threshold for light responses in OFF ganglion cells, but
surprisingly does not influence response threshold in OFF cone bipolar cells.
Furthermore, OFF cone bipolar cells exhibit little inhibitory current under dark-
adapted conditions. Thus low response thresholds in OFF ganglion cells cannot
be explained by glycinergic transmission between AII amacrine cells and OFF
21
cone bipolar synaptic terminals. Recordings from each known subtype of OFF
cone bipolar cell show instead that the application of strychnine depolarizes the
resting membrane potential, but does not reduce the signal-to-noise ratio of the
light response. While recent evidence suggests that AII amacrine cells may be
operational at brighter light levels (Liang and Freed, 2010; Manookin et al.,
2008), our results suggest that the predominant pathway carrying rod responses
near absolute visual threshold appears to be the glycinergic output of AII
amacrine cells to OFF ganglion cells.
Chapter 2 Materials and Methods
Mice and Preparation
Gnat2
-/-
mice (bred for > 5 generations into a C57/Bl6 background and obtained
from Dr. Bo Chang, The Jackson Laboratory) were dark-adapted overnight and
sacrificed according to protocols approved by the IACUC of the University of
Southern California (Protocol #10890). These mice lack the α subunit of the
heterotrimeric G-protein cone transducin, and thus lack cone light responses.
Despite the lack of phototransduction cone cells remain, with little retinal
degeneration up to 15 weeks of age (Chang et al., 2006). We further
characterized morphological changes from 36-week-old wild-type (WT) and
Gnat2
-/-
mice in epoxy resin sections (Chen et al., 2006) and do not document
retinal degeneration up to this age (Supplemental Figure 2.1). Physiological
22
recordings were performed in mice that were typically 8-12 weeks old, and
displayed robust rod-driven light responses with properties similar to WT in all of
the recorded retinal cell types (Supplemental Figure 2.1).
Supplemental Figure 2.1
Fixed tissue sections reveal that the loss of cone α-transducin in mice does not result in
significant retinal degeneration up to 36 weeks of age. Gnat2
-/-
mice in this study were younger
than 16 weeks. Patch-clamp (I
clamp
) recordings were made from second-order retinal neurons
(horizontal cells, rod bipolar cells, ON and OFF cone bipolar cells) of dark-adapted WT and
Gnat2
-/-
retinae under similar conditions. Families of light-evoked responses to flashes of
increasing strength were recorded, and estimates of threshold (defined as where SNR = 1),
resting membrane potential, and time-to-peak were calculated from these flash families. No
significant differences were found between WT and Gnat2
-/-
mice for the rod driven signals in any
of the second-order retinal neurons of the inner nuclear layer. Together, these data suggest that
there are no gross anatomical or physiological abnormalities due to the absence of cone-driven
signals in the Gnat2
-/-
retinae.
23
Electrophysiological Recordings
Procedures used for the handling of retinal tissue, and electrophysiological
recordings from bipolar cells, amacrine cells, and ganglion cells were similar to
those in (Cao et al., 2008). Briefly, animals were dark-adapted overnight, eyes
were enucleated under infrared illumination, the lens and cornea removed, and
the resulting eyecups were stored at 32°C in Ames’ media equilibrated with 5%
CO
2
/95% O
2
. All subsequent manipulations of the retinal tissue were done under
infrared illumination (> 950 nm) using infrared image converting goggles. For
electrophysiological recordings the retinas were prepared as described below
and superfused with Ames’ media heated to 35-37°C. To block glycinergic
transmission, the GlyR receptor antagonist strychnine was used (Bolz et al.,
1985). Strychnine-HCl (Sigma) was dissolved in dH
2
0 to prepare a single stock
solution of 1 mM that was used for all subsequent experiments. The stock
solution was diluted in Ames’ media to produce a final concentration of 20 µM.
Whole-cell recordings were made from ON and OFF ganglion cells in
dark-adapted whole-mount preparations, and from OFF cone bipolar cells and AII
amacrine cells in dark-adapted retinal slices, and as described previously (Dunn
et al., 2006; Sampath et al., 2005). The internal solution for the ganglion cell
recordings consisted of (in mM): 110 CsCH
3
SO
3
, 12 TEA-Cl, 10 HEPES, 10
EGTA, 2 QX-314, 4 ATP-Mg, 0.5 GTP-Tris, 0.5 MgCl
2
; pH was adjusted to ~7.3
with CsOH, and osmolarity was adjusted to ~280mOsm. This solution prevented
ganglion cells from firing action potentials thereby allowing the synaptic currents
24
to be isolated. OFF ganglion cells with highly sensitive responses tended to
have large oval shaped cell bodies, while ON ganglion cells had large polygonal
shaped cell bodies, consistent with anatomical characterizations of α ganglion
cells (Peichl and Wassle, 1981; Wassle et al., 1981). The internal solution for the
OFF bipolar cell and AII amacrine cell recordings consisted of (in mM): 125 K-
Aspartate, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0.5 CaCl
2
, 1 ATP-Mg, 0.2 GTP-
Mg; pH was adjusted to ~7.3 with NMG-OH, and osmolarity was adjusted to
~280mOsm. Identification of OFF bipolar cells was performed by the location of
their cell bodies and their hyperpolarizing response to the onset of the light
stimulus. In some recordings the morphology of the OFF bipolar cell was
document to determine its subtype (Ghosh et al., 2004; Pignatelli and Strettoi,
2004). In these whole-cell recordings Lucifer Yellow (Invitrogen) was included in
the electrode internal solution and pictures were taken following the recordings,
or the tissue section was fixed overnight in 4% paraformaldehyde, mounted on a
microscope slide and imaged using a Zeiss Axio Imager Z1 with an Apotome
slider module.
Flash families were measured in response to a 10ms flash from a blue
LED (λ
max
~ 470 nm, FWHM ~ 30 nm) whose strength varied from generating a
just-measurable response and increased by factors of 2. Membrane currents
were low-pass filtered at 300 Hz by an 8-pole Bessel filter, and digitized at 1 kHz.
Series and input resistances from these cells were monitored at regular intervals
to ensure stability.
25
Light Calibration
In all recordings light was focused on the retinal tissue with a 20X 0.75NA
(Nikon) objective lens. Light intensities were calculated for all recordings as an
effective photon flux at the peak wavelength of spectral sensitivity for mouse
rhodopsin (λ
max
~ 501 nm) to facilitate comparison across cell types. In each
instance the effective number of activated rhodopsins per rod (R*/Rod) was
determined by convolving the power-scaled spectral output of the light source
with the normalized spectral sensitivity curve for mouse rhodopsin, correcting for
added neutral density in the light path. The R*/Rod was calculated based on the
effective collecting area our recording setup (Cao et al., 2008; Okawa et al.,
2008). For whole mount experiments light calibrations were also corrected for
the loss of light through the filter paper on which the retina was mounted.
Analysis and Estimating Response Threshold
We define response threshold as the flash strength where the signal-to-
noise ratio (SNR) is equal to 1, the point at which the signal becomes
distinguishable from the noise. To determine this flash strength we generated
distributions of noise and response amplitudes from flash families. The noise
variance was determined from the baseline membrane potential in the 0.2 s prior
to the flash, and the size of the light evoked potential was determined from the
best fit of each individual trial to the mean response at each flash strength. From
26
these distributions we estimated the SNR for each cell at each flash strength
using the relation:
Where µ
s
and µ
n
are the mean of the distributions of the extracted signals and
the noise, respectively, and σ
s
and σ
n
are the standard deviations of distributions
of the signal and the noise. This definition of SNR is typically referred to as the
unequal variance model of d’ or d
a
(Swets et al., 1961; Swets, 1961a, b).
Calculating the SNR at each flash strength generates a profile for each cell,
which was well described by a saturating exponential of the form:
Threshold was defined as the flash-strength where this function equals 1.
Two-tailed Student T-tests were used to determine statistical significance for the
difference between distributions; all averaged data is presented as a mean ±
standard error of the mean (SEM), with p-values listed.
Chapter 2 Results
Strychnine dramatically increases the threshold for light responses in OFF
ganglion cells
!
y ="a1"e
"bx
( )
!
SNR =
µ
s
" µ
n
#
s
2
+#
n
2
2
27
Previous work demonstrated that glycinergic transmission is a primary
contributor to the scotopic light responses of mammalian OFF ganglion cells
(Muller et al., 1988). To determine how the glycine receptors (GlyRs) set
response threshold in these cells, we assessed the ability of the GlyR antagonist,
strychnine, to alter the threshold for rod signals in the retinas of mice lacking
cone light responses (see Chapter 2 Materials and Methods). As shown in
Figure 2.1A, current clamp recordings from OFF ganglion cells in the presence of
20 µM of the GlyR antagonist, strychnine, reduced the amplitude of light-evoked
hyperpolarization. We quantified the influence of strychnine on the threshold for
light-evoked signals by plotting the signal-to-noise ratio (SNR) of the light
response as a function of flash strength used (Figure 2.1B; see Materials and
Methods). Collected results from 7 OFF ganglion cells (Figure 2.1C) demonstrate
that the application of strychnine resulted in an ~ 5-fold increase in the threshold
for rod responses, with threshold increasing from 0.0052 ± 0.0006 R*/Rod in
Ames’ media to 0.0233 ± 0.0045 R*/Rod in the presence of strychnine (p =
0.005).
The observed 5-fold shift in the rod response threshold when blocking
glycinergic transmission supports previous studies indicating that the alternative
Rod-Cone and Rod-OFF pathways operate at light levels ~ 5 times greater than
the Rod Bipolar pathway (Protti et al., 2005; Volgyi, 2004). Similar effects of
strychnine were not observed in current clamp recordings from ON ganglion cells
(Muller et al., 1988), where light-evoked potentials remained unaltered at all flash
28
strengths (Figure 2.1D-2.1F). Figure 2.1F shows that the application of 20 µM
strychnine in 4 ON ganglion cells did not alter the threshold for rod responses
(0.011 ± 0.0058 R*/Rod in Ames’ media to 0.013 ± 0.007 R*/Rod with strychnine;
p = 0.624).
The specificity of strychnine’s action to OFF ganglion cells, but not ON
ganglion cells, has classically been attributed to the blockade of glycinergic
synapses between AII amacrine cells and OFF cone bipolar synaptic terminals
(Dacheux and Miller, 1976; Famiglietti and Kolb, 1975; Strettoi et al., 1990),
which ultimately feed onto OFF ganglion cell dendrites closing cation channels.
29
Figure 2.1
A) Current clamp flash-response families from a dark-adapted Gnat2
-/-
OFF ganglion cell in the
absence (black) and presence (red) of 20 µM strychnine. Flashes delivered at the time indicated
by the downward arrow yielded 0.002, 0.007, 0.017, 0.036, 0.075, 0.15, and 0.31 R*/Rod. B)
Signal-to-Noise ratio (SNR) of flash responses are plotted versus the flash strength for the cell
shown in (A), and were fit with a saturating exponential function from which response threshold
was determined (see Experimental Procedures). C) Collected data from 7 OFF ganglion cells,
indicating that response threshold shifted ~5-fold in the presence of strychnine, from 0.0052 ±
0.0006 R*/Rod in Ames’ media to 0.023 ± 0.0045 R*/Rod in Ames’ media with strychnine (p =
0.005). D) Current clamp flash-response families from a dark-adapted Gnat2
-/-
ON ganglion cell
in the absence (black) and presence (red) of 20 µM strychnine. Flashes delivered at the time
indicated by the downward arrow also yielded 0.002, 0.007, 0.017, 0.036, 0.075, 0.15, and 0.31
R*/Rod. E) SNR of flash responses are plotted versus the flash strength for the cell shown in (D),
and were fit with a saturating exponential function from which response threshold was determined
(see Experimental Procedures). F) Collected data from 4 ON ganglion cells indicate that
response threshold was unaffected by the presence of strychnine, and was 0.011 ± 0.0058
R*/Rod in Ames’ media and 0.013 ± 0.007 R*/Rod in Ames’ media with strychnine (p = 0.624).
However, strychnine’s effect on OFF ganglion cells may also originate from direct
glycinergic synapses with AII amacrine cells (Chun et al., 1993; Famiglietti and
Kolb, 1975; Kolb, 1979; Kolb and Nelson, 1993; Owczarzak and Pourcho, 1999;
30
Strettoi et al., 1992), or within AII amacrine cells themselves, which are known to
express GlyRs (Haverkamp et al., 2004). Thus in principle, strychnine’s effect on
OFF ganglion cells may result from any upstream action in their afferent
connections originating in AII amacrine cells or OFF cone bipolar cells.
Strychnine does not affect rod response threshold in AII amacrine cells
We assessed the effect of strychnine on the light responses of AII
amacrine cells. Current clamp recordings from AII amacrine cells reveal that the
addition of 20 µM strychnine produced little change in light responses (Figure
2.2A) and did not alter the threshold for rod responses (Figure 2.2B). In 9 AII
amacrine cells threshold was 0.012 ± 0.002 R*/Rod in Ames’ media and 0.0153 ±
0.0022 R*/Rod in strychnine (Figure 2.2C; p = 0.063). Strychnine’s lack of effect
on the AII amacrine cell light response rules out both the AII amacrine cells and
their upstream rod bipolar cells, which also express functional GlyRs on their
axon terminals (Chávez and Diamond, 2008; Eggers and Lukasiewicz, 2006a;
Gillette and Dacheux, 1995; Suzuki et al., 1990) and receive recursive inhibitory
input via amacrine cells (Kolb and Nelson, 1981; Kolb and Nelson, 1983; Kolb et
al., 1981; Nelson and Kolb, 1985), as the source of the ~5-fold shift in threshold
observed in the OFF ganglion cells. Furthermore, these results are consistent
with the lack of strychnine’s effect in ON ganglion cells (Figure 2.1D through
2.1F), which receive input from ON cone bipolar synaptic terminals that are
coupled by gap junctions with AII amacrine cells (Kolb, 1979; Strettoi et al., 1990;
31
Strettoi et al., 1992; Veruki and Hartveit, 2002). Thus the strychnine-mediated
shift in the threshold for rod responses in OFF ganglion cells must be attributable
to the AII amacrine cell’s efferent connections to either the OFF bipolar synaptic
terminal, or directly to OFF ganglion cells.
Figure 2.2
A) Current clamp flash-response families from a dark-adapted Gnat2
-/-
AII amacrine cell in the
absence (black) and presence (red) of 20 µM strychnine. Flashes delivered at the time indicated
by the downward arrow yielded 0.02, 0.06, 0.13, 0.29, 0.59, 1.2, and 2.4 R*/Rod. B) SNR of flash
responses are plotted versus the flash strength for the cell shown in (A), and were fit with a
saturating exponential function from which response threshold was determined (see Experimental
Procedures). C) Collected data from 9 AII amacrine cells indicate that response threshold was
unaffected by the presence of strychnine, and was 0.012 ± 0.002 R*/Rod in Ames’ media and
0.015 ± 0.0022 R*/Rod in Ames’ media with strychnine (p = 0.624).
Strychnine does not influence response threshold in OFF cone bipolar cells
In the classically described Rod Bipolar Pathway rod-driven signals from
the AII amacrine cells pass to OFF cone bipolar synaptic terminals through sign-
inverting glycinergic synapses (Dacheux and Raviola, 1986; Famiglietti and Kolb,
1975; Strettoi et al., 1990). This idea is consistent with anatomical studies in the
rabbit retina that indicate these connections represent the majority of the output
of the lobular appendages of AII amacrine cells (Strettoi et al., 1992). However,
anatomical studies from murine retinae estimate that perhaps as much as 50% of
32
the output from the AII lobular appendages is directed to OFF ganglion cells
(Chun et al., 1993). To determine the extent to which the synapses from AII
amacrine cells ==> OFF bipolar cells play a role in setting the rod threshold of
OFF ganglion cells, we recorded light-evoked potentials from every OFF cone
bipolar cells subtype in dark-adapted retinal slices (as document by (Ghosh et
al., 2004)).
33
Figure 2.3
A-D) Type 1 – 4 OFF cone bipolar cells in Gnat2
-/-
retinas were identified based on the
stratification of the axon terminal in the inner plexiform layer and the width of the dendritic arbor
(top). White lines denote the limits of the inner plexiform layer, with the mid-point separating OFF
from ON sublamina marked with a gray line. Current clamp recordings of flash families in the
absence (black) and presence (red) of 20 µM strychnine for the cells photographed (top) are
shown (middle). The application of strychnine in all 27 OFF cone bipolar cells led to a
depolarization of the cell’s resting membrane potential (V
m
), from 42.8 ± 1.3 mV in Ames’ media
to -37.6 ± 1.2 mV in Ames’ media with strychnine (p=0.0013). SNR of flash responses are plotted
versus the flash strength (bottom) for the cell shown in the middle, and were fit with a saturating
exponential function from which response threshold was determined (see Experimental
Procedures). E) Collected data from 27 OFF cone bipolar cells across all subtypes indicate that
despite the depolarization of the resting membrane potential that response threshold was
unaffected by the presence of strychnine, and was 0.012 ± 0.002 R*/Rod in Ames’ media and
0.015 ± 0.0022 R*/Rod in Ames’ media with strychnine (p = 0.624).
While previous anatomical indicated that the output of AII amacrine cells
may be limited to certain OFF cone bipolar cell subtypes (Tsukamoto et al.,
2001), Figures 2.3A-2.3D surprisingly show that light responses were not
attenuated during the application of 20 µM strychnine in any of the 4 subtypes.
Response families in the absence (black) and presence (red) of strychnine
34
instead reveal an increase in magnitude of the light-evoked hyperpolarization to
the brighter flashes. The increase in response size results from a significant
depolarization in the resting membrane potential of the OFF cone bipolar cells,
shifting from -42.8 ± 1.3 mV in Ames’ media to -37.6 ± 1.2 mV with strychnine (n
= 27; p = 0.0013). Despite the increase in size of light responses, strychnine did
not influence the threshold for rod responses in OFF cone bipolar cells (Figure
2.3E; threshold was 0.4578 ± 0.0427 R*/Rod in Ames’ media and 0.3916 ±
0.0388 R*/Rod with strychnine; n = 27; p = 0.0549). This finding suggests a tonic
glycinergic input to OFF cone bipolar cells independent of light exposure is
continually present under dark-adapted conditions.
OFF ganglion cell threshold is selectively elevated by strychnine
We compared the influence of 20 µM strychnine on the threshold for rod
responses in AII amacrine cells, cone OFF bipolar cells, and OFF ganglion cells.
Figure 2.4A plots on a cell-by-cell basis the response threshold for light-evoked
signals for cells bathed Ames’ media versus threshold when moved to Ames’
containing strychnine. A line with unity slope is included that reflects no change
in response threshold under these conditions. Figure 2.4A highlights two
important conclusions: (1) Threshold for rod-driven signals in OFF cone bipolar
cells is ~ 35-fold higher than in AII amacrine cells, and ~ 80-fold higher than OFF
ganglion cells, suggesting that OFF bipolar cells cannot receive a significant
contribution from AII amacrine cells near their response threshold. Otherwise,
35
OFF bipolar cell thresholds would be comparable to the AII amacrine cell
thresholds. (2) OFF ganglion cell response thresholds, but not response
thresholds in either AII amacrine cells or OFF cone bipolar cells, are increased
significantly in the presence of strychnine as indicated by the clustering of cells
below the line of unity slope.
Figure 2-4
A) Collected data from current clamp recordings of OFF ganglion cells, AII amacrine cells, and
OFF cone bipolar cells (independent of subtype) is shown on a cell-by-cell bases as the threshold
in Ames’ media plotted versus the threshold in Ames’ media with 20 mM strychnine. A line with
unity slope is included to compare changes in response threshold between recording conditions.
B) Indentified pathways in which rod generated signals can traverse the retinal circuitry en route
to ganglion cells. Glycinergic AII amacrine cells make inhibitory (-) synapses with both OFF
ganglion cells and OFF cone bipolar cells.
Such differences between OFF cone bipolar cells and OFF ganglion cells
are also evident in the dimmest flash response where an influence of strychnine
can be seen; in OFF bipolar cells an effect of strychnine can be seen for flashes
that generate 6.8 R*/rod (Figure 2.3E), but in OFF ganglion cells differences are
seen for flashes that generate 0.018 R*/rod (Figure 2.1C).
36
In sum, the strychnine-dependence of response threshold must therefore
be set by the properties of glycinergic transmission to OFF ganglion cells, and do
not originate in the light response of AII amacrine cells or OFF cone bipolar cells.
Input to OFF bipolar cells in darkness is primarily excitatory
The lack of influence of strychnine on the response threshold of OFF cone
bipolar cells is peculiar, especially given the large number of anatomically
defined synapses between AII amacrine cells and OFF cone bipolar cells in
many mammalian species (Chun et al., 1993; Hendrickson et al., 1988; Marc and
Liu, 1985; Pourcho and Owczarzak, 1991a, b; Strettoi et al., 1992; Tsukamoto et
al., 2001). We sought to characterize the relative contributions of inhibitory
versus excitatory input to OFF cone bipolar cells in darkness to establish the
physiological process that dominates their light response under dark-adapted
conditions. Figure 2.5A-2.5D shows families of light responses in every
anatomically identified class of OFF cone bipolar cell when the membrane
potential is held at either -60 mV (isolates excitatory nonselective cationic current
while holding the membrane potential near E
Cl
) or at +10 mV (isolates the
inhibitory anionic current while holding the membrane potential near E
Cat
). The
excitatory current under these conditions was always greater than the inhibitory
current, indicating that near the resting membrane potential of OFF cone bipolar
cells (~ -40mV; Figure 2-3) that ~ 75% of the input is excitatory (assuming E
Cl
= -
60 mV, E
Cat
= +10 mV, and that I
max
,Cat/I
max
,Cl = 1.7 + 0.26, n = 22).
37
Figure 2-5
A-D) Type 1 – 4 OFF cone bipolar cells in Gnat2
-/-
retinas were identified based on the
stratification of the axon terminal in the inner plexiform layer and the width of the dendritic arbor
(top). Unlike the fixed sections photographed in Figure 2.3, these photographs were taken
immediately following physiological recordings. Voltage clamp flash families are shown for each
subtype of OFF cone bipolar cell (below) at V
m
= -60 mV (black) and V
m
= +10 mV (blue). E)
Voltage clamp recording from an OFF ganglion cell (Vm = + 10 mV) in the absence (blue) and
presence (red) of strychnine. Inset shows the response of the OFF ganglion cell to steps of light
that generated ~ 3 Rh*/rod/ s while the cell’s membrane potential was held at -60 mV (black) and
+10 mV (blue). F) Collected data from 7 OFF ganglion cells (V
m
= -60 mV), indicating that
response threshold shifted ~6-fold in the presence of strychnine, from 0.0028 ± 0.0006 R*/Rod in
Ames’ media to 0.0165 ± 0.0045 R*/Rod in Ames’ media with strychnine (p = 0.015).
Consistent with the lack of contribution of OFF cone bipolar cells to signals
near visual threshold, we observe largely inhibitory input to OFF ganglion cells
under these conditions. Figure 2.5E (inset) demonstrates the light-evoked
38
currents generated in OFF ganglion cells to a step of light when the membrane
potential is held at +10 mV and -60 mV. Consistent with previous studies
(Murphy and Rieke, 2006, 2008; VAN Wyk et al., 2009) a strong inhibitory input
is seen at the onset of the light step when the membrane potential is held at +10
mV, and a weaker excitatory input is seen at the offset of the light step when the
membrane potential is held at -60 mV. We measured the threshold for light-
evoked inhibition while the OFF ganglion cell membrane potential was held at
+10 mV in the absence and presence of 20 µM strychnine (Figure 2.5E). As
shown in Figure 2.5F, threshold for light-evoked inhibition was increased by ~ 6-
fold in the presence of strychnine (threshold was 0.0028 ± 0.0006 R*/Rod in
Ames’ media and 0.0165 ± 0.0045 R*/Rod in Ames’ media with strychnine; n = 7;
p = 0.015). Thus direct glycinergic inhibition can account largely for the ~ 5-fold
shift in response threshold observed for current-clamped OFF ganglion cells
(Figure 2.1C).
Chapter 2 Discussion
Anatomical convergence and synaptic specializations have been
recognized as the hallmarks of the Rod Bipolar Pathway that underlie its high
sensitivity (reviewed by (Field et al., 2005; Masland, 2001a, b; Singer, 2007)).
AII amacrine cells provide a key junction point in this circuit where rod driven
signals move into the cone circuitry and can be subsequently relayed to both ON
39
and OFF ganglion cells. Anatomically-defined synapses between AII amacrine
cells and OFF cone bipolar cells are widely believed to be the principal pathway
for relaying the most sensitive rod signals to OFF ganglion cells, but little is
known about the functional properties of these connections near visual threshold.
Here we have characterized the properties of rod-driven signals as they move
from AII amacrine cells to OFF ganglion cells in the dark-adapted retina of mice
lacking cone photoresponses (see Chapter 2 Materials and Methods).
Consistent with previous studies we find that the dark-adapted
responsiveness of OFF ganglion cells is attenuated during the application of
strychnine (Muller et al., 1988), but surprisingly we find that this sensitivity to
strychnine does not originate in the synapses between AII amacrine cells and
OFF cone bipolar cells. Instead strychnine’s effect appears to manifest in the
efferent connections of the AII amacrine cell, and provides evidence against the
idea provided by many anatomical studies (Chun et al., 1993; Hendrickson et al.,
1988; Marc and Liu, 1985; Pourcho and Owczarzak, 1991a, b; Strettoi et al.,
1992; Tsukamoto et al., 2001) that AII amacrine cell to OFF cone bipolar
synapses carry rod signals near visual threshold.
OFF cone bipolar cells do not contribute to OFF ganglion cell photoresponses
near visual threshold
Perhaps the most surprising finding of this work is that the response
threshold of dark-adapted OFF cone bipolar cells was unaffected during the
40
application of strychnine (leading to our increase in strychnine concentration up
to 20 µM; see Figure 2.3). Anatomical studies of mammalian retina all share in
common that a large fraction of the output of the lobular appendages of AII
amacrine cells are to OFF cone bipolar cells, with the fraction varying from 50%
of the output in cat (Kolb and Nelson, 1993; Nelson et al., 1993) and rat (Chun et
al., 1993), to as much as 95% of the output in rabbit (Strettoi et al., 1992). The
latter of these studies has lead to the assumption that these synapses must be
important for the processing of light-evoked signals for the Rod Bipolar pathway,
whose function as a low light level detection pathway has been long appreciated.
Physiological studies of inhibition in OFF cone bipolar cells have focused
on characterizing the postsynaptic receptors responsible. However, studies
which puff glycine directly onto OFF bipolar axon terminals (Ivanova et al., 2006),
making it difficult to draw inferences about their functional properties. More
recent studies in the mouse retina have characterized the inhibitory light
response of OFF cone bipolar cells in dark-adapted retinas to bright flashes
(Eggers and Lukasiewicz, 2010; Eggers et al., 2007), but do not define the
features of the signal or noise in the light response or synaptic transmission that
would be critical for defining the detection threshold. In sum, these studies
unequivocally identify GlyRs on OFF cone bipolar synaptic terminals, but don’t
necessarily provide insights into signaling near visual threshold.
We characterized the role of GlyRs on setting the response threshold of
dark-adapted OFF cone bipolar cells by measuring how threshold changes
41
during application of strychnine. Given the heterogeneity of potential contacts
between AII amacrine cells and the various OFF cone bipolar cell subtypes
(Tsukamoto et al., 2001), we endeavored to measure the influence of strychnine
on light responses of all identified classes of cells (Ghosh et al., 2004). The
common theme among all OFF cone bipolar subtypes (Figure 2.3) was that
responses to flashes in darkness were not reduced by strychnine, as might be
expected, but instead they were frequently larger in amplitude. This increase
could be attributed to a strychnine-dependent depolarization of the resting
membrane potential, which appears to result from the block of a tonic inhibition of
OFF cone bipolar cells that is normally present in darkness. Thus the properties
of signal transmission between AII amacrine cells and OFF cone bipolar cells
cannot explain the high sensitivity of glycinergic signaling to OFF ganglion cells
under dark-adapted conditions.
On the origin of low sensitivity glycinergic synapses in OFF cone bipolar cells
The apparent lack of sensitivity of glycinergic synapses between AII
amacrine cells and OFF cone bipolar cells may ultimately result from a lack of
sensitivity in either the presynaptic release or in the postsynaptic response to
glycine. At present there is little information about the voltage-dependence of
glycine release from the lobular appendages of AII amacrine cells, but the
synaptic output is believed to require L-type Ca
2+
channels (Habermann et al.,
2003). Given that the membrane potential of AII amacrine cells in darkness is
42
relatively hyperpolarized, these low threshold Ca
2+
channels may require a larger
depolarization to open compared to those that yield the response to very dim
light (Figure 2.2). It is interesting to note that OFF ganglion cells also receive
input from the lobular appendages of AII amacrine cells (Chun et al., 1993; Kolb
and Nelson, 1983; Strettoi et al., 1992) and their response threshold is impaired
significantly by strychnine (Figure 2.1A-2.1C). Thus if there are presynaptic
differences in the ability of AII amacrine cells to release glycine to OFF cone
bipolar cells versus OFF ganglion cells, they must result from differences in the
properties of their contacts with these cells.
Perhaps a more straightforward explanation for the lack of sensitivity of
glycinergic synapses may be that OFF cone bipolar cells lack the expression of
the GlyR anchoring protein, gephyrin. Immunohistochemistry and electron
microscopy indicate that gephyrin does not colocalize to the synapse between
OFF bipolar cells and AII amacrine cells (Fischer et al., 2000; Sassoe-Pognetto
et al., 1994), unless there exists an isoform not recognized by the antibody used
in the previous studies (Heinze et al., 2007). Since gephyrin serves to cluster
and anchor GlyRs to the post-synaptic membrane, the density of GlyRs at the
synapse may be diminished in its absence (Fischer et al., 2000; Legendre, 2001;
Lynch, 2004; Vannier and Triller, 1997) resulting in a reduced efficiency of
released glycine in evoking inhibitory currents. In any case, the properties of
glycinergic synapses between AII amacrine cells and OFF cone bipolar cells
43
requires further characterization to determine the lighting conditions where they
become active.
44
Chapter 3: Bleached rod photoresponses persist and
traverse the retina
Chapter 3 Introduction
Our visual experience is mediated by two photoreceptor cell types in the
retina, the rods and cones. Rod photoreceptors are exquisitely sensitive and
mediate our visual experience near absolute threshold (Hecht et al., 1942). In
addition, rods continue to signal light absorption at brighter light intensities where
their signals begin to mix with the photoresponses of cones, referred to as the
mesopic range (Krizaj, 2000; Stockman and Sharpe, 2006). The upper limit of
mesopic vision, where rods no longer contribute to visual sensitivity (Aguilar and
Stiles, 1954), is difficult to define because cones are able to retain
responsiveness in very bright bleaching light. The conventional wisdom is that
cones predominantly mediate vision during the day. However, classical
psychophysical evidence indicates that rod monochromats surprisingly retain
sensitivity and can function efficiently in daylight despite their low acuity and
inability to see color (Blakemore and Rushton, 1965a, b; Rushton, 1961). In fact,
these early studies show that the upper limits of rod vision are often masked by
cone photoresponses in normal subjects.
The persistence of rod vision under conditions when a large fraction of the
visual pigment is bleached indicates rods are capable of encoding visual
information even though they are desensitized, and that pathways exists to
transmit this information to the retinal output, the ganglion cells.
45
Furthermore, several neural pathways are responsible for relaying rod
photoresponses through the retina. The rod bipolar pathway is traditionally
recognized for being the most sensitive thereby providing our vision in dim light
(Dacheux and Raviola, 1986; Smith et al., 1986), with the secondary rod
pathways relaying rod signals to retinal ganglion cells at higher light levels where
the rod bipolar pathway may be saturated (Daw et al., 1990; Smith et al., 1986).
How these retinal pathways read out signals from partially bleached rods remains
unclear.
To determine the upper light limits of rod photoreceptor function, we
combined electrophysiological recordings from whole retina and single cells to
determine how visual pigment bleaching influences sensitivity in Gnat2
-/-
mice.
We found that pigment bleaching resulted in a persistent reduction in rod
sensitivity that is explained by a combination of the loss of the visual pigment,
and desensitization of rod phototransduction proportional to the concentration of
the bleached pigment. However, rods retain considerable responsiveness at
steady-state after 80-90% of the visual pigment is bleached. Patch-clamp
recordings from retinal slices in which 70% of the visual pigment was bleached
revealed that ON and OFF cone bipolar cells exhibit ~ 4-fold greater responsivity
than neighboring rod bipolar cells, contrary to the ~ 2-fold greater responsivity of
rod bipolar cells in dark-adapted slices. Thus, following substantial pigment
bleaching the retina may preferentially use secondary rod pathways to convey
rod photoresponses to retinal ganglion cells.
46
Chapter 3 Materials and Methods
Mouse lines and preparation
Transgenic mice were crucial in these experiments for isolating the
functional properties of the rod photoreceptors, and the circuits that carry their
output to retinal ganglion cells. While some wild-type mice (WT; C57BL6,
Jackson Laboratory) were used for control experiments, most experiments were
performed on mice lacking cone transducin and thus lacking cone
photoresponses (Gnat2
-/-
bred for >5 generations in a C57BL6 background;
(Chang et al., 2006)). The retina of Gnat2
-/-
mice do not show degeneration up to
36 weeks of age, and display robust rod driven responses in rod bipolar cells
(See Supplemental Figure 2.1).
Mice were maintained on a 12 hour day-night cycle and were dark-
adapted overnight prior to experimentation. Animals were euthanized in
accordance with protocols approved by the Institutional Animal Care and
Use
Committee of the University of Southern California, following the Guide for the
Care and Use of Laboratory
Animals and the Animal Welfare Act. All
experimental manipulations were performed in darkness with infrared illumination
and were visualized with infrared image converters (BE Meyers, WA). Following
euthanasia, eyes were enucleated, the lens and cornea were removed,
and
eyecups were stored in darkness at 32°C in Ames’ media buffered with sodium
bicarbonate (Sigma, Cat# A1420), and equilibrated with 5% CO
2
/ 95% O
2
.
47
Before recordings the retina was isolated from the retinal pigment epithelium to
prevent regeneration of the visual pigment, rhodopsin.
Electroretinogram recordings
The sensitivity of rod phototransduction was assessed in
electroretinogram (ERG) recordings from whole-mount retina. ERGs were used
to record the rod-driven a-wave from isolated retinas, similar to procedures
described in (Wang et al., 2009). Briefly, a piece of dark-adapted retina was
mounted photoreceptor side-up over a machined 1 mm hole in a Plexiglass
recording chamber, and was gently flattened using forceps. A slice anchor was
used to hold the retina flat, and the tissue was superfused in darkness at a rate
of 8 ml/min with 35-37
o
C Ames’ media buffered with sodium bicarbonate and
equilibrated with 5% CO
2
/ 95% O
2
resulting in pH ~ 7.4. The trans-retinal
potential change to flashes of light was measured using Ag/AgCl half-cells with a
differential amplifier (Model DP-311; Warner Instruments), and was sampled at
10 kHz and low-pass filtered at 3 kHz. Further 30 Hz digital low-pass filtering
was done offline in MATLAB. Flashes of light, as well as bleaching illumination,
were delivered from a traditional light bench as described previously
(Miyagishima et al., 2009). ERG a-waves were isolated pharmacologically by
superfusing the retina with Ames’ media containing 16 µM APB, 10 µM CNQX,
100 µM Picrotoxin, and 20 µM Strychnine to eliminate downstream contributions
from all bipolar and horizontal cells.
48
Single cell patch-clamp recordings
To determine the sensitivity of cells downstream of rods following pigment
bleaching, whole-cell patch clamp recordings were made from bleached retinal
slices. Briefly, isolated retinas were first bleached (70%) using the light bench
described above for ERG recordings, and were stored in darkness for 30 minutes
to allow the retina to reach a steady-state. Retinal slices were then prepared and
light-stimuli were delivered as described previously (Cao et al., 2008; Okawa et
al., 2010; Okawa et al., 2008). Whole-cell voltage clamp recordings (V
m
= -60
mV) were made from rod and cone bipolar cells, whose identity was confirmed by
both the properties of the response to light and their retinal morphology, which
was visualized and photographed after the addition of Lucifer Yellow (Sigma) to
the internal solution. The internal solution for patch clamp recordings consisted
of (in mM): 125 KAsp, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0.5 CaCl
2
, 0.2 MgCl
2
,
1 ATP-Mg, 0.2 GTP-Tris, pH to 7.2 w/ NMG-OH.
Responsivity will be determined by recording responses to families of
increasing flash strength. The responsivity can be derived from the response (R)
flash-strength (I) relationship, which can be fit with a Hill curve, defined as:
€
R
R
max
=
1
1 +
1
kI
( )
n
49
Where R
max
is the maximum response amplitude, k is the reciprocal of I
1/2
(the half maximal flash-strength), and n is the Hill exponent. Fitting of recorded
response flash-strength relationships with Hill curves was done using a
constrained non-linear optimization routine using MATLAB.
Microspectrophotometry
The extent of photopigment bleaching in rods was assessed using a
microspectrophotometer described previously (Cornwall et al., 1984; Jones et al.,
1996). Briefly, the dark-adapted retina was isolated from the retinal pigment
epithelium as described above and a piece of retina was then placed
photoreceptor side up on a glass cover-slip window located in the bottom of a 2
mm-deep Plexiglass recording chamber. The retina was gently flattened on this
window using forceps and a slice anchor similar to that described above for
electrophysiological recordings. The tissue was superfused at a rate of 2.5
ml/min with 35-37
o
C Ames’ media buffered with sodium bicarbonate, and
equilibrated with 5% CO
2
/ 95% O
2
.
The recording chamber was placed on a microscope stage located in the
beam path of the microspectrophotometer and visual pigment spectra (optical
density vs. wavelength) were obtained from a region of the retina along its edge
where isolated outer segments were visually identified. That this area contained
predominantly rod photoreceptor outer segments was confirmed by absorption
spectra that peaked on average at about 500nm (see Results, Fig 3.1).
50
Measurements were made over the wavelength range 400 nm to 800 nm in 80
steps of 5 nm step width, with each individual spectral scan completed in 1
second. The absorbance spectrum was calculated according to Beers Law
according to the relation:
€
OD =log
10
I
o
I
t
⎛
⎝
⎜
⎞
⎠
⎟
Where OD is optical density or absorbance, I
o
is the light transmitted through a
cell free space adjacent to the retinal patch, and I
t
is the light transmitted through
the tissue. The measuring beam was adjusted to be an area ~6 µm
2
. The
intensity of the measuring beam bleached less than 0.1% of the visual pigment
per scan. Generally, five complete sample and five baseline scans were
averaged to increase the signal-to-noise ratio of a measured spectrum. Dark-
adapted spectra of the visual pigment measured in this way were then compared
with spectra measured immediately after and at increasing times following
exposure to a 520 nm light from a xenon flash gun calibrated to bleach either
50% or 80% of the visual pigment (Fig. 3.1). Such recordings of absorption
spectra were continued for 30-90 min after pigment bleaching, after which the
retina was exposed again to a light calculated to bleach in excess of 99%. This
latter measurement was used to calculate the total bleaching fraction that
occurred under the earlier bleaching conditions.
51
Chapter 3 Results
Pigment bleaching desensitizes the rod-driven ERG a-wave
In amphibian photoreceptors bleached visual pigment is known to activate
weakly the phototransduction cascade and desensitize the photoresponse
(Cornwall and Fain, 1994; Cornwall et al., 1995; Jones et al., 1996), a process
known as bleaching adaptation (reviewed by (Fain, 2001)). While similar
mechanisms appear to control the sensitivity of rod photoresponses in the mouse
retina (Fan et al., 2005; Woodruff et al., 2002), the dependence of rod sensitivity
on the extent of pigment bleaching is unknown. We measured the responsivity of
the rod-driven ERG a-wave in mice lacking cone photoresponses (Gnat2
-/-
; see
Chapter 2 Materials and Methods) following 10% - 90% bleaching of the visual
pigment, rhodopsin. Figure 3.1A plots representative a-wave response families
collected for a dark-adapted retinal whole mount and after steady-state was
reached following either 50% or 80% bleaching of the visual pigment,
respectively. Bleach-adapted responses exhibited the characteristic changes
expected following exposure to bright light, including accelerated response
kinetics and a reduction in response amplitude per incident photon, which scaled
with the fraction of bleached pigment (Leibovic et al., 1987).
The fraction of bleached visual pigment generated in ERG experiments
was also determined independently using microspectrophotometry. Figure 3.1B
plots the visual pigment absorbance spectra obtained from a retina under the
same conditions of bleaching light exposure. Spectra were measured in the
52
dark-adapted state (black), following a defined pigment bleaching light exposure
(red, either 50% or 80%), and after exhaustive bleaching the visual pigment
(gray). Difference spectra calculated under these conditions (Figure 3.1C)
allowed the quantification of fractional visual pigment bleaching, which was
calculated by subtracting the spectra obtained following exhaustive bleaching
from both dark-adapted and fractionally bleached spectra (Figure 3.1C). The
amounts of pigment bleaching shown here were consistent with calculations of
pigment bleaching expected, based on the duration, intensity, and wavelength of
the incident light, and the photosensitivity of the visual pigment (Jones et al.,
1996). Thus, rod-driven photoresponses remain in intact retinas under conditions
where a large fraction of the visual pigment is bleached.
53
Figure 3.1
Rod photoresponses in darkness and following pigment bleaching. (A) Pharmacologically
isolated ERG a-waves measured from whole-mount retinas of Gnat2
-/-
mice in darkness and 30
min after 50% and 80% bleaching of the visual pigment (see Materials and Methods). Flashes at
the time indicated by the upward arrow delivered 0.30, 21, 74, 160, 250, 440, 1500, 5400, 13000,
28000, 76000 photons µm
-2
in darkness, 160, 440, 2000, 5400, 13000, 28000, 76000, 100000
photons µm
-2
after the 50% bleach, and 80, 360, 970, 2300, 5000, 14000, 39000, 110000,
490000 photons µm
-2
after the 80% bleach. (B) Calibration of the bleaching light in situ using
microspectrophotometry (see Materials and Methods). Absorbance spectra for whole retina were
measured in darkness (black), following 50% (top) or 80% (bottom) bleaching of the visual
pigment (red), and following a total bleach of the visual pigment (grey) (C) Relative pigment
concentrations were determined in difference spectra, by subtracting the total bleach spectra from
spectra collected in darkness and after 50% or 80% bleaching. Dashed lines at values of 0.5
(top) and 0.2 (bottom) define the expectation for 50% and 80% bleaching, respectively.
We quantified the extent of desensitization of the a-wave by plotting the
responsivity (R), normalized to the responsivity in darkness (R
D
), as a function of
the fraction of bleached pigment (Figure 3.2). Responsivity was determined as
the flash strength that yielded a half-maximal response from response families
like those in Fig 3.1A. In every retina tested, the reduction in responsivity
0.6
0.4
0.2
0.0
0.4
0.2
0.0
700 600 500 400
Wavelength (nm)
1.0
0.5
0.0
1.0
0.5
0.0
700 600 500 400
Wavelength (nm)
0.2
0.1
0.0
Dark
0.2
0.1
0.0
Photoresponse (mV)
0.2
0.1
0.0
1.0 0.5 0.0
Time (s)
50% Bleach
80% Bleach
A.
B. C.
Optical Density
Normalized Optical Density
Figure 1
54
exceeded that expected based on the loss of visual pigment (Figure 3.2, dashed
trace). Instead, the relative reduction in responsivity was well fitted by a Weber-
Fechner function that describes a logarithmic relationship between the
responsivity and the fraction of visual pigment (R/R
D
= (1 – bleach)/(1 +
k*bleach); see Figure 3.2). The best fit for the Weber-Fechner function was
achieved with a value of the scaling factor k = 9, indicating that a 11% bleach
results in a decrease in responsivity of the rod-driven a-wave by a factor of 2.
This is in contrast to the value obtained from salamander rods of k ~ 16,
indicating that ~ 6% bleach reduces responsivity by a factor of 2 (Jones et al.,
1996). Thus, while quantitative differences appear to exist between amphibian
and mammalian rods (perhaps due to temperature differences), these results
indicate that for mouse rods, bleached visual pigment also produces an
equivalent light that desensitizes the rod photoresponse (Fan et al., 2005;
Woodruff et al., 2002).
We also measured the responsivity of the a-wave as a function of the
fraction of bleached pigment in WT mice (Figure 3.2, upward triangles), and
found consistently that the responsivity always exceeded the responsivity of a-
waves generated by rods alone in Gnat2
-/-
retinas. Cones represent ~ 3% of the
photoreceptor cells in the mouse retina (Carter-Dawson and LaVail, 1979a, b),
but appear to improve substantially visual responsivity under conditions where a
large fraction of the visual pigment is bleached. Such cone responsivity will
normally prevent a determination of the upper limits of rod vision.
55
Figure 3.2
Reduction in responsivity of rod photoresponses versus the extent of pigment bleaching.
The responsivity of rod photoresponses was determined from the flash strength that
yielded a half-maximal response. This responsivity following pigment bleaching (R),
relative to the responsivity in darkness (R
D
), is plotted versus the fraction of the visual
pigment bleached for Gnat2
-/-
retinas (n = 19). The dashed line reflects the expected
loss of responsivity due to the loss of quantum catch. The fit line is a Weber-Fechner
relationship scaled by the fraction of bleached pigment, where R/R
D
= (1 – bleach)/(1 +
k*bleach), and k = 9 (see also Jones et al., 1996). The 95% confidence intervals for the
fit are denoted by dotted lines.
0.001
0.01
0.1
1
1.0 0.8 0.6 0.4 0.2 0.0
Fraction Bleached Pigment
Gnat2
-/-
WT
56
ON and OFF Cone bipolar cells are more sensitive than rod bipolar cells in
bleached retina
In the outer retina rod photoresponses enter several identified retinal
circuits that relay these signals to the retinal output, the ganglion cells (reviewed
in Chapter 1). The parsing of the rod photoresponse to bipolar cells is the first
step in this process. We measured responsivity changes using patch clamp
recordings from identified rod and cone bipolar cells in slices of bleached retina.
In these experiments, we first bleached 70% of the visual pigment in a piece of
retina, waited ~ 30 min to allow sensitivity to recover to a steady-state, and
prepared retinal slices for single cell recording. Light-evoked currents were then
made from bipolar cells in voltage clamp (V
m
= -60 mV), which were later
identified morphologically through the incorporation of Lucifer Yellow in the
electrode internal solution. Recordings were typically made from rod and cone
bipolar cells in the same region of the retinal slice (see Figure 3.3C), within 150
µm of each other, ensuring that measured signals originated from a common
pool of rods that are not influenced by inhomogeneities in the bleaching light.
Figure 3.3A displays a typical recording from a rod bipolar cell, and an ON
and OFF cone bipolar cell recording in a bleached Gnat2
-/-
retina. Pigment
bleaching resulted in a reduction in responsivity of the response, as evidenced by
the higher flash strengths required to elicit a full flash family. Figure 3.3B plots
the normalized response versus flash strength for the three cells shown in Figure
3.3A. Figure 3.3C illustrates how cells recorded in this study were imaged
57
subsequently to identify cell type based on their dendritic morphology and axonal
stratification. The three cells pictured from left to right include two rod bipolar
cells and one OFF cone bipolar cell.
58
Figure 3.3
Light evoked currents in neighboring rod bipolar, OFF cone bipolar cells, and ON cone bipolar
cells in the Gnat2
-/-
following pigment bleaching. Single cell recordings from a bleached retina
slice in excess of 30 minutes after a 70% bleach (see Materials and Methods). A) Rod bipolar
responses to increasing flashes of light delivered at the time indicated by the upward arrow
yielding: 14, 28, 56, 110, 230, 450, 900, 1800 photons µm
-2
. For the ON and OFF cone bipolar
cell, flashes delivered at the time indicated by the arrow yielded 4, 8, 16, 32, 63, 130, 250, 500
photons µm
-2
. B) Normalized response maxima are plotted versus flash strength for the rod
bipolar cell (filled circles), ON cone bipolar cell (open circles), and OFF cone bipolar cell (open
squares) shown in (A), and were fit with a Hill Equation from which the half maximal flash
strengths was determined. The estimated half-maximal flash strengths for the rod bipolar cell,
and ON and OFF cone bipolar cell were 110 photons µm
-2
, 16 photons µm
-2
, and 41 photons µm
-2
respectively. C) An example composite fluorescence image of two rod bipolar cells and an OFF
cone bipolar cell filled with Lucifer Yellow. From left to right: rod bipolar cell, rod bipolar cell, and
OFF cone bipolar cell.
59
Figure 3.4 compares the responsivities of ON and OFF cone bipolar cells
with neighboring rod bipolar cells after 70% pigment bleaching by plotting the
log
10
values of their half-maximal flash strength against one another. Each point
on the plot therefore represents one cone bipolar cell/rod bipolar cell pair,
however the averages across the population are shown in Table 1.
Table 3.1: I
1/2
of Gnat2
-/-
bipolar cells.
n I
1/2
(ϕ mm
-2
) n I
1/2
(ϕ mm
-2
) Fold Increase
Rod Bipolar 33 13 + 0.7 16 210 + 37 16
ON cone bipolar 8 14 + 2.9 7 71 + 32 5
OFF cone bipolar 20 21 + 1.7 8 68 + 18 3.2
Horizontal cell 5 40 + 3.1 4 609 + 37 15
The mean half maximal flash strength for each cell type is reported. The
mice were randomized in order to minimize circadian effects from complicating
the analysis.
Figure 3.4 includes lines with slopes of 1, 2, 3, 4, 5, and 6 to reflect the
fold increases for the responsivity of ON or OFF cone bipolar cells with respect to
neighboring rod bipolar cells. Among 7 ON cone bipolar cells we observed that
the responsivity following the 70% bleach was ~ 4.45-fold higher than
neighboring rod bipolar cells (4.5 ± 0.89 fold; n = 7). Among 8 OFF cone bipolar
cells we observed that the post-bleach responsivity was ~ 3-fold higher than
Dark Adapted 70% Bleach
60
neighboring rod bipolar cells (3.28 ± 0.58 fold; n = 8). In contrast, for dark-
adapted retinas rod bipolar cells are typically ~ 2-fold more responsive than
either ON or OFF bipolar cells for rod signals.
Figure 3.4
Rod BPC and ON/OFF cone BPC comparison. Half-maximal flash strengths for ON and OFF
cone bipolar cells are plotted against the half-maximal flash strength of a neighboring rod bipolar
cell. Filled symbols represent data collected under dark-adapted conditions, open symbols
represent data collected after a 70% bleach. Rod and cone bipolar cells were always within 150
mm of each other. ON cone bipolar cells (circles) and OFF cone bipolar cells (squares) in the
Gnat2
-/-
are shown on a log scale.
10
2
3
4
5
6
7
8
9
100
2
3
4
5
6
7
8
9
1000
Rod Bipolar I
1/2
(! µm
-2
)
10
2 3 4 5 6 7 8 9
100
2 3 4 5 6 7 8 9
1000
Cone Bipolar I
1/2
(! µm
-2
)
, OFF cone bipolar
, ON cone bipolar
2 3 4 5 6
1
1/2
1/3
1/4
61
Chapter 3 Discussion
The control of sensitivity by bleached visual pigment, and the retinoid
byproducts of phototransduction, have been studied extensively in the amphibian
retina (Ala-Laurila et al., 2006; Cornwall and Fain, 1994; Kolesnikov et al., 2007;
Miyagishima et al., 2009), but relatively little is known in mammals about how
bleaching adaptation influences the sensitivity of rod photoreceptors or cells
within the retinal network. In particular, while it is appreciated that cones
dominate our daytime vision, little is known about the upper limit of light intensity
where rods contribute to visual processing. Here we have characterized the
effects of bleached visual pigment on the responsivity of rod photoreceptors and
downstream bipolar cells in the retina of mice lacking cone photoresponses. Two
main conclusions arise from this work: 1) Rods continue to signal light
absorption following substantial bleaching of the visual pigment. In the steady-
state following bleaching of 80-90% of the visual pigment, robust rod-driven a-
waves are observed. 2) Following substantial bleaching of the visual pigment,
the output of rod photoreceptors is relayed to the retinal output with higher
responsivity by ON and OFF cone bipolar cells rather than rod bipolar cells.
Rod photoresponses persist despite substantial bleaching of the visual pigment
Most of our visual experience occurs during the daytime when a
substantial fraction of the visual pigment in our photoreceptor cells is bleached.
Bleaching results in a persistent reduction in rod photoreceptor sensitivity due in
62
part to the loss in quantum catch and a proportional decline in phototransduction
sensitivity. Under normal conditions the pigment, rhodopsin, is constantly
regenerated to maintain available photon capture as the background light levels
increase, eventually reaching equilibrium with the rate of bleaching. The
advantage of these experiments is that they occur independent of the RPE,
allowing us to hold the pigment concentration at different levels and measure the
steady-state dependence on responsivity. Under normal conditions, the upper
limits of rod vision and the retinal pathways responsible for transmitting this
information cannot be determined because rod signals are often masked by the
presence of cone photoresponses (see Fig 3.2). Cones are fundamentally less
sensitive than rods thereby allowing their responses at higher light intensities.
Furthermore, cones are able to recovery fully their dark current during the
exposure to bright light (Kenkre et al., 2005) and can regenerate their visual
pigment more rapidly (Arshavsky, 2002; Mata et al., 2002). The Gnat2
-/-
retina
therefore provides a unique opportunity to study the pathways involved in
carrying rod signals to the retinal output in the absence of cone photoresponses.
Here we provide experimental evidence that mammalian rod
photoresponses can maintain robust photoresponses even when 80-90% of their
pigment is bleached; responsivity scales with the fraction of bleached pigment
according to Weber’s law consistent with bleached pigment activating
phototransduction at a low level. This modulation of responsivity will reduce the
gain of the phototransduction cascade allowing rods to respond at previously
63
unappreciated light levels. Ultimately the absolute light levels defining the upper
limit of rod function will require the quantification of the size of the available pool
of 11-cis retinal, and the time course of pigment synthesis and decay.
Switching from the rod bipolar pathway to alternative pathways following pigment
bleaching
Our current understanding of the retinal processing of rod signals suggest
that there are several pathways through which rod visual information is faithfully
transmitted to ganglion cells. These include the Rod-Bipolar pathway, the Rod-
Cone pathway, and the Rod-OFF pathway. In darkness, the Rod-Bipolar
pathway is traditionally recognized for being the most sensitive of the three and is
credited for absolute behavioral threshold when there is light detection from only
a few photon absorptions (Dacheux and Raviola, 1986; Smith et al., 1986). In
darkness, the properties of the rod-to-rod bipolar cell synapse (see Chapter 1)
enable rod bipolar cells to be ~ 2 fold more sensitive than either ON or OFF cone
bipolar cells.
The alternative rod pathways include the Rod-Cone pathway and the Rod-
OFF pathway and have been implicated to promote vision at higher light levels
(Sharpe and Stockman, 1999b). Whereas the Rod-OFF pathway supplies a
direct connection between rods and a subset of OFF cone bipolar cells (Soucy et
al., 1998), the Rod-Cone pathway relies on gap junctions that electrically couple
rods and cones and participates in distributing and encoding this visual
64
information in the retina (Mills et al., 2001). Our results indicate that following
steady state after a 70% bleach there is an ~ 8-fold relative shift in the
responsivity of the pathways for rod signals, with ON and OFF cone bipolar cells
becoming ~ 3 times more responsivity than neighboring rod bipolar cells.
These findings suggest that the alternative pathways may play an
increasingly important role as the amount of bleach increases. Interestingly,
horizontal cells (n=4) recorded from the Gnat2
-/-
are even less responsive than
nearby rod bipolar cells following a 70% bleach, and therefore they cannot
account for the observed 8-fold relative shift in the responsivity of the pathways.
Thus given the similar extent of convergence of rods on rod bipolar cells versus
ON and OFF cone bipolar cells, the dramatic change in the response properties
of the rod bipolar and ON and OFF cone bipolar cells in darkness and following
bleaching most likely follow from desensitized signal transmission at the rod-to-
rod bipolar synapse. We find that ON and OFF cone bipolar cells possess
greater responsivity to rod signals than nearby rod bipolar cells allowing the
continued signaling of changes in light intensity even under bleaches where the
rod and rod bipolar cell responses are greatly desensitized.
The persistence of rod signals following substantial pigment bleaching
suggests there is a significant rod contribution to visual responsivity over a wider
range of light intensities than previously appreciated. In support of this idea, rod
signals can be measured from ganglion cells in a whole mount preparation in
guinea pig retinas even at light intensities of 5.14 log
10
Rh*/rod/s, (equivalent to
65
direct sunlight reflected from the ground) (Yin et al., 2006). The desensitization
of rod photoresponses in the rod bipolar cell under conditions of substantial
pigment bleaching might allow the retina to tradeoff smoothly the high sensitivity
of the rod bipolar pathway in darkness for a larger dynamic range of intensities
that can be accurately signaled through secondary rod pathways. In mammals,
we therefore predict that secondary rod pathways will allow the faster recovery of
visual responsivity following exposures to bright bleaching light.
66
Chapter 4: Conclusion
The means by which the retina is able to signal faithfully changes in light
intensity over a broad operating range probes a question which is at the heart of
perceptual neuroscience: how do biological systems capture information from the
external environment and transform it into meaningful neural signals? The retina
presents an excellent system in which to address such questions. As one of the
most studied neural tissues, there exists a wealth of anatomical and physiological
information regarding the individual neuronal subtypes that comprise the defined
retinal circuits, making it possible to pose questions regarding the sensitivity and
operating range of neural pathways with tractable answers.
The remarkable ability of the retina to sense light at the very limits of
intensities present in the physical environment is a testament to the adaptive
capabilities of the nervous system. This work presented in this dissertation has
advanced our understanding of how rod-mediated signaling impacts neural
responses under dark-adapted and photo-bleached conditions. The findings in
Chapter 2 challenge the conventional wisdom that OFF responses near absolute
visual threshold are mediated through the OFF cone bipolar cells terminals
before reaching OFF ganglion cells. I present data indicating that the OFF
signals near threshold are not dependant at all on OFF bipolar cells, suggesting
instead that the pathway for these signals is routed directly from the AII amacrine
cell. This result questions the role of OFF cone bipolar cells to vision when only
67
a small fraction of the rods are even receiving photonic stimulation. Based on my
results, OFF α-ganglion cells are at least as sensitive (d
a1
≈ 1 photoisomerization
in every 200 rods; see Figure 2.1) as the ON α-ganglion cells, and they exhibit
robust signals even under completely dark-adapted conditions. I found that
under dark-adapted conditions the primary input to OFF bipolar cells is excitatory
(Figure 2.5), presumably originating from a decrease in cations resulting from a
reduction in the release of glutamate from the cone pedicle. My work
demonstrates (Figure 2.5) that under dark-adapted conditions the primary input
to OFF α-ganglion cells is through direct inhibition, presumably the AII amacrine
cell, based on the known effective connectivity of the circuit (Chun et al., 1993;
Famiglietti and Kolb, 1975; Kolb, 1979; Kolb and Nelson, 1983; Owczarzak and
Pourcho, 1999; Strettoi et al., 1992). Recent studies have suggested that the AII
amacrine cells are functional even at light levels when the rod bipolar cells are
saturated (Liang and Freed, 2010; Manookin et al., 2008), suggesting that their
efferent connections are relaying signals beyond the scotopic range. Future
studies should attempt to determine the operating conditions in which the
glycinergic synapses between AII amacrine cell lobular appendages and OFF
cone bipolar cell terminals become active. Based on the anatomical data
available, it seems that the relative proportion of efferent connections from the AII
amacrine cell varies from species to species (Chun et al., 1993; Kolb and Nelson,
1993; Strettoi et al., 1992), perhaps optimizing retinal signaling based on the light
environment to which these animals are exposed. Although it is tempting to
68
generalize these results from the murine retina to all mammals, it will be
important to replicate these findings in other animal models.
It is generally accepted that the rods are the phylogenically younger
photoreceptors (Bowmaker, 1998); this is true for all species. Thus a variety of
cone pathways, used to generate chromatic information about the environment,
linking cones to second and third order neurons, already existed before the
development of the rod photoreceptors. In mammals it appears as though the
rods integrated with the already existing cone circuitry, using those cone
pathways which would be inactive in dim light, to feed their signals to the
ganglion cells (Smith et al., 1986; Soucy et al., 1998; Strettoi et al., 1990; Strettoi
et al., 1994; Strettoi et al., 1992; Tsukamoto et al., 2001; Volgyi, 2004). In
Chapter 3 I present data that demonstrates that the existence of rod signals at
cone light levels is dependant on these alternative rod pathways; suggesting that
the alternative rod pathways become increasingly important as the system
adjusts to brighter bleaching light intensities. These data further validate the
existence of secondary rod pathways mediating signals through the cones.
Although it remains to be seen how these remaining rod signals contribute
to visually-guided behavior, recent evidence suggests that they could have
significant implications to understanding how our internal biological clock is
regulated. Recent studies have suggested that gap junction coupling is under
circadian control (Ribelayga et al., 2008; Ribelayga and Mangel, 2003, 2005;
Ribelayga et al., 2002, 2004; Yang and Wu, 1989), regulating the suppressive
69
effect of rod transduction on the cone pathways in steady background light and
vice versa (Cameron and Lucas, 2009; Manglapus et al., 1998). Thus, a
feedback mechanism may exist whereby rod signals contribute to synchronizing
the circadian clock, which in turn regulates gap junction coupling, redistributing
rod and cone signals and altering pathway convergence in the retina. Future
studies in this field will provide insights into how this light information could
impact our physiological and behavioral function and overall human health
(Zisapel, 2001a, b).
70
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Abstract (if available)
Abstract
Nearly all sensory systems must find a way to represent a wide range of input signals and translate them into meaningful neural responses. The human visual system is able to operate effectively from starlight to bright sunlight, a range that spans about twelve orders of magnitude of light intensity. To reliably transmit changes in light stimuli over this range the mammalian retina has evolved several specializations to report changes in the light environment that include: 1) the evolution of two photoreceptor types, the rods and cones, that operate at different light levels, 2) several neural pathways with which to encode the output of these photoreceptors, and 3) adaptive mechanisms at all levels of retinal processing to modulate light sensitivity based on light history. To address questions regarding the sensitivity and the operating range of rod pathways in the mammalian retina, I will present data from two projects designed to investigate how rod-generated signals traverse the retinal circuitry under various lighting conditions.
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Arman, Arvin Cyrus
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Core Title
Sensitivity and dynamic range of rod pathways in the mammalian retina
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College of Letters, Arts and Sciences
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Doctor of Philosophy
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Neuroscience
Publication Date
08/03/2010
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05/28/2010
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), Grzywacz, Norberto M. (
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), Hirsch, Judith A. (
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), Tjan, Bosco S. (
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), Weiland, James (
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)
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