AN ANALYSIS OF LIGHT ADAPTATION AND INHIBITORY INPUT TO RETINAL AMACRINE CELLS

AN ANALYSIS OF LIGHT ADAPTATION AND INHIBITORY INPUT TO RETINAL AMACRINE CELLS
AN ANALYSIS OF LIGHT ADAPTATION
AND INHIBITORY INPUT TO RETINAL
AMACRINE CELLS
By
AARON JOSIAS SALAZAR
A Thesis Submitted to The Honors College
In Partial Fulfillment of the Bachelors degree
With Honors in
Physiology
THE UNIVERSITY OF ARIZONA
May 2015
Approved by:
Erika Eggers, PhD
Departments of Physiology and Biomedical Engineering
Abstract
The retina avoids signal saturation through the use of both dim-light sensing rod and bright-light
sensing cone photoreceptor circuits. Photoreceptors are responsible for converting an image into
an electrical signal, which is transmitted to bipolar cells and ganglion cells. Amacrine cells
modulate the interaction between bipolar cells and ganglion cells through inhibitory signaling,
and therefore play a large part in inner retinal processing. However, inhibitory connections
between amacrine cells are a little understood signaling component of the retina. Within this
study, amacrine cells within the mouse retina were separated into two categories: narrow-field
glycinergic and wide-field GABAergic. Amacrine cells were subjected to a light stimuli under
dark-adapted, light-adapted, and receptor isolated conditions, and the peak amplitude and charge
transfer of the L-IPSC was measured. Wide-field amacrine cells received an increase in the
percentage of inhibition from glycine after light adaptation. There is an overall decrease in
spontaneous activity with light adaptation, but an increase in the percent of glycinergic
spontaneous activity. Spatial inhibition to narrow-field amacrine cells becomes narrower with
light adaptation. L-IPSC peak amplitude decreases with light adaptation with application of a
full-field light stimulus, and also decreases as the distance between the stimulus and the cell
body increases.
Keywords: amacrine cell, gamma-aminobutyric acid (GABA), glycine, L-IPSC, sIPSC, light
adaptation
Introduction:
Images are transformed into electrical signals at the level of the rod and cone photoreceptors in
the retina. When a light stimulus is recognized by a photoreceptor, the release of glutamate onto
postsynaptic bipolar cells stops. This signal is then interpreted across various types of bipolar
cells, and is then transmitted to ganglion cells, the axons of which exit the eye through the optic
nerve and transmit retinal information to the brain. There are two major categories of
interneurons that contribute to inner retinal image processing before the information is relayed
through the ganglion cell axons - horizontal cells and amacrine cells, although amacrine cells
likely play a larger role in light adaptation of spatial signaling (Dedek et al. 2008; Mazade and
Eggers in submission). Inhibitory connections between amacrine cells, the subcategories into
which they can be organized and how inhibitory input to these cells changes between the rod and
cone pathways is the focus of this thesis.
Approximately 30 types of amacrine cells have been identified in the mammalian non-primate
retina (Lin & Masland 2006). The most recent work on this topic results from studies in the rat
and mouse retina. However, results in the mouse retina dominate because of the powerful
molecular tools that can be brought to bear in this species (Zhang & McCall 2012). This is why
we are working on the mouse retina.
Amacrine cells are generally categorized into one of two categories: wide-field GABAergic
amacrine cells or narrow-field glycinergic amacrine cells (Pourcho & Goebel 1983; Menger et al.
1998). GABAergic amacrine cells provide lateral interactions across the inner plexiform layer
(IPL), and are implicated in lateral inhibitory signaling within the IPL, typically within a single
layer (Lin and Masland 2006; Eggers 2010). This is important for determining the spatial
sensitivity of ganglion cells. Glycinergic amacrine cell dendrites are primarily involved in local
interactions between the different sublaminas of the IPL. The most prominent and bestcharacterized glycinergic narrow-field amacrine cell is the AII, involved in signal transfer from
the rod to cone pathway (Kolb and Famiglietti 1974; Famiglietti and Kolb 1975). Up to 11
further narrow-field amacrine cells have been identified in different species, of which at least 8
are glycine-positive (Pourcho and Goebel 1985; Menger et al. 1998; MacNeil and Masland 1998;
Badea and Nathans 2004).
This paper encompasses data analysis from two separate studies within the Eggers lab on
inhibitory signaling to retinal amacrine cells. While amacrine cells modulate the excitatory
bipolar cell to ganglion cell pathway, they also inhibit other amacrine cells through lateral
inhibition. Little is known about this or how it changes between rod and cone pathways. The first
study focused on the roles that narrow-field and wide-field amacrine cells play in inner retinal
processing. This study was conducted by analyzing the sources of inhibitory input to amacrine
cells through isolated rod and cone pathways, through application of a full-field light stimulus.
Since rod and cone pathways signal at different light intensities, applying a bright background
light that causes rod saturation allows the two pathways to be investigated separately (Dacheux
and Raviola 1986; Xin and Bloomfield 1999). Application of this background light has allowed
for the investigation of inhibitory signaling to bipolar cells to be studied in the Eggers lab,
wherein they found that there was a shift from glycinergic to GABAergic inhibition with light
adaptation (Eggers 2013). Typically light adaptation causes a decreased sensitivity to light as a
result of isolation of the cone pathway, and increased sensitivity to small stimuli. Larger
GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may
be one mechanism that contributes to retinal spatial sensitivity in bright light conditions (Mazade
and Eggers 2013).
In the first study, GABAergic and glycinergic inhibition were isolated before and after light
adaptation in order to determine whether or not there is a change in the relative contributions of
inhibitory signaling from the two main inhibitory neurotransmitters within the retina. The second
study focused on the degree to which amacrine cells might play a part in this inner retinal
processing with light adaptation by studying how spatial inhibition to both narrow-field and
wide-field amacrine cells changes with light adaptation. The results of this study might be related
to how ganglion cells increase their spatial sensitivity and visual acuity after ambient
illumination is increased (Balrow et al. 1957; Dedek et al. 2008; Farrow et al. 2013).
Methods:
Mouse retinal slice preparation. Animal protocols were approved by the University of Arizona
Institutional Animal Care and Use Committee (IACUC). Male mice 35-60 days of age were
euthanized with carbon dioxide (Eggers and Lukasiewicz 2006a; Eggers et al. 2013). Their eyes
were enucleated, and the cornea and lens were removed. The eyecup was incubated for 20
minutes in cold extracellular solution (see Solutions and drugs) with 800 U/mL of hyaluronidase
to dissolve the remaining vitreous humor. The hyaluronidase solution was then replaced with icecold, oxygenated extracellular solution, and the retina was trimmed into one large rectangle by
removing the peripheral retina and leaving only the central retina in the optic disk. A
nitrocellulose membrane filter paper (0.45-µm pore size, Millipore) was placed on the retina
section, which was transferred to a hand chopper. An average of six 250-µm slices were cut,
rotated 90°, and mounted onto glass cover-slips with vacuum grease. Cells used from these slices
were never more than 700 µm away from the center of the retina, and only cells near the center
of each slice were used for recordings. In this way, much of the differential input due to the
dorsal-ventral cone opsin gradient reported in mice was mitigated (Applebury et al. 2000;
Haverkamp et al. 2005). The tissue was maintained in oxygenated extracellular solution at room
temperature. All dissection procedures were performed under infrared illumination to preserve
the light sensitivity of the preparations.
Solutions and drugs. The extracellular recording solution used for dissection and to examine
light-evoked currents contained (in mM) 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2,
20 glucose, 20 glucose, and 26 NaHCO3, and was bubbled with 95% O2-5% CO2. For voltage
clamp recordings, the intracellular solution contained (in mM) 120 CsOH, 120 gluconic acid, 1
MgCl2, 10 HEPES, 10 TEA-Cl, 10 phosphocreatine-Na2, 4 Mg-ATP, 0.5 Na-GTP, 10 EGTA
and 50 µM Alexa Fluor 488 (Invitrogen, Carlsbad, California, USA) and was adjusted to pH 7.2
with CsOH. To isolate the inhibitory receptor (R) inputs, SR-95531 (SR, 20 µM) to block
GABAARs, (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA, 50 µM) to block
GABACRs, and strychnine (1 µM) to block glycine Rs were used. All drug solutions were
washed on the slice for 5 min before recordings began using a gravity-driven superfusion system
(Cell Microcontrols, Norfolk, VA) at a rate of ~1-2 mL/minute. Unless otherwise indicated, all
chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Whole-cell recordings. Whole-cell patch-clamp recordings, sampled at 10 kHz, were made from
amacrine cells in retinal slices. Light-evoked inhibitory post synaptic currents (L-IPSCs) and
spontaneous (s)IPSCs were recorded from retinal amacrine cells voltage clamped to 0 mV, the
reversal potential of nonselective cation channel currents. Amacrine cell recordings were stable
and no rundown of the light response was observed over the recording period. Resting membrane
potentials were recorded passively in current clamp (I-clamp) mode. Liquid junction potentials
of 20 mV were corrected for at the beginning of each recording. Electrodes were pulled from
borosilicate glass (World Precision Instruments, Sarasota, FL) on a P97 Flaming/Brown puller
(Sutter Instruments, Novato, California, USA) and had resistances of 5-7 MΩ. Mice were darkadapted overnight, and all recording procedures were performed in the dark under infrared
illumination to preserve the light sensitivity of the slices. Recordings were made in extracellular
solution heated to 32°C, using thin stage and inline heaters (Cell Microcontrols, Norfolk, VA).
Responses were filtered at 6 kHz with the four-pole Bessel filter on a Multi-clamp 700B patchclamp amplifier (Molecular Devices, Sunnyvale, California, USA) and digitized with a Digidata
1140 data acquisition system (Molecular Devices, Sunnyvale, California, USA).
Morphological identification of cell subtypes. Alexa Fluor 488 included in the recording pipette
was used to label amacrine cell subtypes. The cells were imaged with Nikon Digital Sight
camera with Elements software using a Nikon Intensilight C-HGFIE Fluorescent lamp (Nikon
Instruments, Tokyo, Japan). These images were opened in Photoshop, marked and flattened in
order to produce a new image, as shown in Figure 1A and Figure 1B. They were classified as
either narrow-field or wide-field based on their axonal morphologies within the inner plexiform
layer. Cells that had a dendritic span of less than 100 µm were considered narrow-field, and
those that were wider than 100 µm were considered wide-field amacrine cells.
Full-field light stimuli. Full-field light stimuli were evoked with a light-emitting diode (LED;
Agilent HLMP-3950, λpeak=525nm, Palo Alto, CA) projected through the camera port of the
microscope, which elicited a strong response in both dark- and light-adapted conditions. We
focused our recordings on cells located within the regions of mixed green/UV cone opsin input
(Applebury et al. 2000; Haverkamp et al. 2005). Thus, 525-nm light should stimulate both rods
(peak sensitivity at 500 nm) and cones robustly. The stimulus intensity (max of 9.5 X 105
photons·µm-2/s-1), background intensity (950 photons·µm-2/s-1), and duration (30 ms) were
controlled with Clampex software by varying the current through the LED. The background
intensity was chosen as it was shown to maximally activate rods (Wang and Kefalov 2009). A
rod-saturating background light was applied for 5 minutes to light-adapt the retina slice and was
sustained throughout all light-adapted recordings.
Spontaneous (s)IPSC data was analyzed with Clampfit software. An sIPSC template was
calculated for each data file with the average of ∼10 prototypical events from the recording. The
software used this template to automatically detect spontaneous events, which were manually
accepted or rejected on the basis of strict criteria: events used to calculate the frequency were
rejected if they appeared to be noise, and events used to calculate the average peak amplitude
were rejected if they appeared to be noise or consisted of two or more overlapping events.
Frequency was calculated by dividing the number of events by the recording time. Peak
amplitude and interevent interval histogram distributions were normalized to the number of
events. The signal-to-noise ratio (SNR), which gives a measurement of the actual response
signaled over the background noise, was calculated by dividing peak amplitude of the L-IPSC by
the variance of the baseline.
Full-field inhibitory input data analysis and statistics. L-IPSC traces from a given response
condition were averaged with Clampfit software (Molecular Devices), and the charge transfer
(Q) and peak amplitude measured in each condition. Because of the significant amount of
spontaneous activity, it was difficult to measure a peak from amacrine cell L-IPSCs. Therefore,
to estimate the peak, average traces were decimated (50 fold) and each point was replaced with
the average of those data points to limit variations due to spontaneous activity. To determine
changes in total current, the Q was measured, which represents the magnitude of the response. Q
was measured in Clampfit over the length of the roughly 1 second response, using the same time
parameters in each condition for the same cell. For intensity-response curves, light-evoked
responses were normalized to the maximal response in the dark-adapted condition. The
normalized data were plotted against the log10 of the stimulus intensity.
Bar light stimuli. Bar stimuli (25 µm wide) were used to determine spatial inhibition, and were
presented using a white organic light-emitting diode (OLED Microdisplay, eMagin EMA100503 SXGA Monochrome White XL, Bellevue, WA) projected through the camera port of the
microscope, which elicited strong responses in both dark- and light-adapted conditions.
Recordings were from cells located within the regions of mixed green/UV cone opsin input
(Applebury et al. 2000; Haverkamp et al. 2005) which ensured that all possible pathways were
present. The stimulus intensity (7.83 x 104 photons/µm2/sec) and the background rod-saturating
light (1150 photons/µm2/sec) were controlled with custom MatLab software by controlling the
intensity, size, location, and duration of the bar stimuli through the OLED screen. The
background intensity was chosen as it was shown to maximally activate rods (Wang and
Kefalov, 2009) and produced significant changes in inner retinal inhibition (Eggers and Mazade,
2013). A long light stimulus (1 s) was used to determine the type of inhibition and excitation to
all recorded bipolar cells as well as to illicit robust responses with a very small stimulus. An
adapting background light was applied for 5 min to light-adapt the retina slice and was sustained
throughout all light-adapted recordings.
Spatial inhibitory input data analysis and statistics. L-IPSC traces from a given response
condition were averaged using Clampfit software (Molecular Devices, Sunnyvale, California,
USA) and the charge transfer (Q) and peak amplitude were measured in each condition. Due to
the significant amount of spontaneous activity, it was difficult to measure a peak from amacrine
cell L-IPSCs. Therefore, to estimate the peak, average traces were decimated (50 fold) and each
point was replaced with the average of those data points to limit variations due to spontaneous
activity. To determine changes in total current, the Q was measured, which represents the
magnitude of the response. Q was measured in Clampfit over the length of the response,
typically 1-2 seconds, using the same time parameters in each condition for the same cell. The
baseline Q was added to the Q standard deviation which was subtracted from all raw Q
measurements to negate any current due to baseline or spontaneous events. For spatial
distribution curves, light-evoked Q’s were normalized to the maximal response in the darkadapted condition and peak amplitude raw data was used. The normalized and raw data were
plotted against the distance the stimulus was from the cell. In order to compare between the dark
and light-adapted conditions at any given stimulus distance, both sides of the spatial distributions
at equal distances were averaged and plotted as bar graphs.
2-way Analysis of Variance (ANOVA) with Student-Newman-Keuls (SNK) posthoc test was
used to compare spatial distributions before and after light adaptation as well as between
response characteristics at each stimulus distance. Student’s t-tests (2-tailed, paired) were used to
compare resting membrane potentials and timing parameters before and after light adaptation.
Differences were considered significant when p < 0.05 (*). All averaged data are reported as
mean ± SEM.
Results:
Identification of narrow-field and wide-field amacrine cells
Within the mouse retina, narrow-field, radially spanning amacrine cells (Fig 1A) were found to
have an average dendritic spread of 58 µm, and highly stratified wide-field amacrine cells (Fig
1B) were found to have an average dendritic spread of 124 µm. A Student’s t-Test was
conducted to determine whether or not there was a clear difference in dendritic field of the
narrow- and wide-field amacrine cells. The calculated p-value was less than 0.05, meaning that
the difference in dendritic field for narrow- and wide-field amacrine cells is statistically
significant (Fig 1C).
Wide field amacrine cells receive more glycinergic inhibition under light adapted conditions
In the first study, the L-IPSC in the retinal amacrine cell was measured first after dark
adaptation. Strychnine was applied to the cells to blockade inhibition from glycine before light
adaptation in order to isolate for GABAergic inhibition in the dark-adapted cell. This was
repeated for amacrine cells after light adaptation in order to determine whether or not the
percentage of glycinergic and GABAergic inhibition changed under the different conditions.
There was no observable difference in the ratio of GABAergic to glycinergic inhibition to
narrow-field amacrine cells (Figure 2A). However, after light adaptation of wide field amacrine
cells there was a significant decrease in the ratio of GABAergic to glycinergic inhibition. Figures
2E and 2F demonstrate the light response in narrow- and wide-field amacrine cells with isolation
of GABAergic inhibition both before and after light adaptation. The difference in inhibitory
signaling to wide field amacrine cells before and after light adaptation is statistically significant
according to the Student's T-Test, which might suggest that the contribution of glycinergic
inhibitoin to amacrine cells increases with light adaptation.
There is an increase in sIPSC activity due to glycine receptors with light adaptation
As can be seen in Figure 2E and 2F, there is a significant amount of background spontaneous
activity, which may affect signal transmission of light-evoked responses. Spontaneous activity
was measured under dark-adapted and light-adapted conditions and also after application of
sequential receptor antagonists in order to determine the source of spontaneous activity and how
it changed under the described conditions.
The frequency and peak amplitude of spontaneous inhibitory post synaptic currents (sIPSCs)
were measured for both narrow- and wide-field amacrine cells before and after light adaptation
with the application of strychnine, as shown in Figure 3. There was no statistically significant
difference in either peak amplitude or spontaneous frequency with light adaptation alone (Figure
3A, 3B). However, there was a statistically significant difference in spontaneous peak amplitude
with the application of strychnine in the dark for both narrow- and wide-field cells (Figure 3D).
This might suggest more glycinergic spontaneous activity in the light-adapted retina in both
categories.
In addition to that, there was a significant decrease in spontaneous frequency after the
application of strychnine to light adapted cells in both subcategories. This suggests that
sIPSCs are primarily caused by the activation of glycine receptors through various cone
pathways (Figure 3E). Histograms for spontaneous event peak amplitudes and interevent
intervals, along with example traces for spontaneous activity were shown in Figures 4, 5, and 6
for the three different methods of treatment: with strychnine before light-adaptation, with light
adaptation and with strychnine after light-adaptation. Spatial inhibition to narrow-field amacrine
cells becomes narrower with light adaptation
In the second study, the spatial extent of inhibition was measured before and after light
adaptation. A 25 µm bar of light was applied in 100 µm increments from 600 µm to the left of
the cell body to 800 µm to the right. The spatial inhibitory graphs for charge transfer were
normalized to the center L-IPSC in the light and in the dark. The normalized charge transfer was
plotted before and after light adaptation, and an ANOVA with repeated measures test conducted
(Figure 7A). The ANOVA test yielded a p value of 0.002, meaning that the difference in charge
transfer before and after light adaptation was statistically significant when paired with increasing
distance of the light stimulus from the center of the amacrine cell for narrow-field amacrine cells.
One sided analysis of charge transfer for narrow-field amacrine cells with spatial inhibition was
conducted by averaging the charge transfer at equal distances from the center of the cell. This is
shown in Figure 7C. This confirmed that there was a statistically significant distance in charge
transfer at distances greater than or equal to 300 µm from the center of the cell, and suggests that
spatial inhibition to narrow-field amacrine cells narrows significantly after light adaptation.
Sufficient data was not collected to create a similar graph for wide-field amacrine cells as in
Figure 7A.
The values for charge transfer when the light stimulus was directly above the cells were averaged
over all cells, normalized to the charge transfer in the dark and shown in Figure 7D, in order to
show how charge transfer changed with light adaptation and after the application of receptor
antagonists. This location was used because the greatest value for charge transfer was observed
here. This was repeated before and after light adaptation, after isolation for GABAa receptors and
after isolation for glycine receptors (Figure 7D) in both narrow-field and wide-field amacrine
cells. A student t-Test showed that there was a significant difference in charge transfer for
narrow field amacrine cells with light adaptation and after isolation for GABAa across all
amacrine cells. From this, we can conclude that glycinergic inhibition plays a critical role in
inhibitory signaling in the rod pathway.
Peak amplitude decreases as the distance between the light stimulus and the center of the cell
increases, and also decreases with light adaptation
Peak amplitude of the L-IPSC was measured as the light stimulus was moved in 100 µm
increments across the retinal slice for narrow-field amacrine cells, in order to determine whether
or not there was a noticeable change with light adaptation. A one sided analysis bar graph was
created, and the difference in peak amplitude was found to be statisically significant according to
the Student-Newman-Keuls Method at 700 and 800 µm from the center of the cell. The peak
amplitude decreased significantly at long distances away from the cell body with light adaptation
(Figure 8A). While the charge transfer and peak amplitude decreased with increasing distance of
the light stimulus from the cell body after light adaptation, the signal to noise ratio increased
significantly (Figure 8B). This was most likely due to a decrease in sIPSC frequency and peak
amplitude with light adaptation.
The peak amplitude difference with light adaptation at the center bar for narrow field amacrine
cells was also graphed. Application of the student t-Test showed that there was no statistical
difference in peak amplitude of the L-IPSC with light adaptation (p>0.05).
Discussion
In this study we found that there was an increase in the percent of inhibition that is received from
GABAergic input after light adaptation in wide-field amacrine cells. There is also a narrowing in
spatial inhibition with light adaptation. We also decided to redefine what constituted “narrowfield” and “wide-field” in the mouse retina.
Traditionally, amacrine cells have been divided into broad categories of narrow field (30-150
µm), small-field (150-300 µm, medium field (300-500 µm), and wide-field (>500 µm) cells,
based on their dendritic span across the IPL (Kolb et al. 1981). Division by size is useful because
dendritic spread is an important determinant of a cell’s function in visual processing (Masland,
2012). However, these values were determined in the cat retina. The size might differ due to the
difference in retinal size between the mouse and the cat.
When measurements were made in the mouse retina, amacrine cells that go between multiple
sublamina of the IPL did not normally exceed a dendritic span of more than 100 µm. Similarly,
amacrine cells with dendritic spans that exceeded 100 µm had axonal processes that were highly
stratified, as shown in Figure 1A. A previous study found that most narrow-field amacrine cells
were approximately 40 µm in diameter, although the dendritic tree diameters never exceeded 100
µm (Menger 1998). This study was also conducted in the rat retina, not the mouse. This is why a
new classification system for mouse retinal amacrine cells was proposed, where narrow-field
was defined as having a dendritic span of less than 100 µm, and wide-field as having a dendritic
span of more than 100 µm. It is understood that there are multiple subcategories into which
amacrine cells can be further classified, but for the purpose of this paper only the two
subcategories were used.
In the first study with a full-field light stimulus, a decrease in both the charge transfer and peak
amplitude of the L-IPSC was observed with light adaptation. Narrow-field and wide-field amacrtine
cells had both GABA and glycine inputs in the dark and light in all cellsAn increase in the proportion
of glycinergic signaling contributing to the L-IPSC after light adaptation could have occurred as
a result of an increase in glycinergic inhibition with light adaptation, or as a result of a decrease
in GABAergic inhibition, or both (Figure 2B). In both cases, there was a clear decrease in charge
transfer after light adaptation, application of strychnine, and the application of SR, but not after
the application of TPMPA. Therefore, the data would suggest that GABAc receptors do not play
a large role in modulating narrow-field or wide-field amacrine cell inhibitory signaling (Figure
2C, 2D). This is expected, since in the mammalian retina GABAC receptors have been described
on bipolar cells only (Enz et al. 1996; Euler et al. 1996; Feigenspan and Bormann 1994a;
Feigenspan et al. 1993; Pan and Lipton 1995; Yeh et al. 1996).
Both light adaptation and increasing distance from the light stimulus to the amacrine cell body
caused a decrease in both the total charge transfer and peak amplitude in narrow- and wide-field
amacrine cells. The spatial inhibitory response of narrow-field amacrine cells narrowed with
light adaptation, which indicates an increase in spatial sensitivity through the cone photoreceptor
pathway in comparison to the rod pathway. Similar activity was observed in bipolar cells in a
previous study. Total inhibition became significantly narrower and smaller under light adapted
conditions, with little to no inhibition remaining at the furthest distances (Mazade and Eggers in
submission). This shows that spatial inhibitory input to the inner retina becomes narrower and
smaller with increasing background light.
Spatial inhibition was studied in order to determine the effect of light adaptatation might have on
center surround inhibition modulated by retinal amacrine cells. Both narrow- and wide-field
amacrine cells measured in this study had dendritic spans that never exceeded 300 µm across the
IPL. However, when a bar of light was applied over 300 µm away from the center of the
amacrine cell being measured, a decrease in the inhibitory response was still observed. This may
be due to gap junctions beween amacrine cells (Mazade and Eggers in submission). Previous
studies have shown that gap junctions become uncoupled between AII amacrine cells, and AII
amacrine cell activation is decreased (Mazade and Eggers 2013, Dacheux and Raviola 1986)
after light adaptation, which would explain the narrowing in spatial inhibition after the
application of a rod saturating background. In addition to this, horizontal cells in the outer retina
become uncoupled with light adaptation (Xin and Bloomfield 1999), although amacrine cells
likely play a larger role in light adaptation of spatial signaling (Dedek et al. 2008; Mazade and
Eggers in submission). Upon application of strychnine in the dark and light, it was found that the
percent of glycinergic inhibition to wide-field amacrine cells increased after light adaptation.
There is also a decrease in spontaneous frequency after light adaptation and receptor isolation
with strychnine. Spontaneous currents are modulated by the various receptors for glycine and
GABA. A decrease in spontaneous frequency from might indicate a decrease in activity at
GABA receptors, or an increase in activity at glycine receptors. This could be from other
amacrine cells and also bipolar cells. This decrease in spontaneous activity could have also
contributed to the increase in the signal to noise ratio after light-adaptation in narrow-field
amacrine cells. The signal to noise ratio is calculated by dividing the peak amplitude by the
variance of the baseline. By decreasing spontaneous activity, the variance decreased, which
caused an overall increase in the signal to noise ratio, shown in Figure 8B.
Acknowledgements
I would like to thank Reece Mazade for providing the data necessary for this study. I would also
like to thank Erika Eggers, Reece Mazade and Johnnie Moore-Dotson for guidance on the
creation of this thesis, technical assistance, and for providing the tools required to make this
research project possible.
Grants:
-
NIH grant EY018131 (EDE)
-
University of Arizona NIH Graduate Training in Systems and Integrative Physiology
grant 5T32GM008400 (REM)
-
ARCS Foundation (REM)
References
Applebury ML, Antoch MP, Baxter LC, Chun LL, Falk JD, Farhangfar F, Kage K,
Krzystolik MG, Lyass LA, Robbins JT. The murine cone photoreceptor: a single cone
type expresses both S and M opsins with retinal spatial patterning. Neuron 27: 513–523,
2000.
Badea, T.C., Nathans, J. Quantitative analysis of neuronal morphologies in the mouse retina
visualized by using a genetically directed reporter. J. Comp. Neurol 480: 331–351, 2004.
Barlow HB, Fitzhugh R, Kuffler SW. Change of organization in the receptive fields of the cat's
retina during dark adaptation. The Journal of Physiology 137:338-354. 1957.
Chen X, Hsueh HA, Werblin FS. Amacrine-to-amacrine cell inhibition: Spatiotemporal
properties of GABA and glycine pathways. Visual Neuroscience 28: 193-204, 2011.
Dacheux RF, Raviola E. The rod pathway in the rabbit retina: a depolarizing bipolar and
amacrine cell. J Neurosci 6: 331–345, 1986.
Dedek K, Pandarinath C, Alam NM, Wellershaus K, Schubert T, Willecke K, Prusky GT, Weiler
R, Nirenberg S. Ganglion cell adaptability: does the coupling of horizontal cells play a
role? PLoS One 3:e1714, 2008.
Dunn FA, Doan T, Sampath AP, Rieke F. Controlling the gain of rod-mediated signals in the
Mammalian retina. The Journal of neuroscience : the official journal of the Society for
Neuroscience 26:3959-3970, 2006.
Eggers ED, Lukasiewicz PD. GABAA, GABAC and glycine receptor-mediated inhibition
differentially affects light-evoked signalling from mouse retinal rod bipolar cells. J
Physiol 572: 215–225, 2006a.
Eggers ED, Lukasiewicz PD. Multiple pathways of inhibition shape bipolar cell responses in
the retina. Visual Neuroscience 28: 95-108, 2011.
Eggers ED, Mazade RE, Klein JS. Inhibition to retinal rod bipolar cells is regulated by light
levels. J Neurophysiol 110: 153–161, 2013.
Enz R., Brandstätter J. H., Wässle H., Bormann J.Immunocytochemical localization of the
GABAC receptor rho subunits in the mammalian retina. J. Neurosci. 16:4479–4490,
1996.
Euler T., Schneider H., Wässle H. Glutamate responses of bipolar cells in a slice preparation of
the rat retina. J. Neurosci.16:2934–2944, 1996.
Famiglietti, E.V., Kolb, H. A bistratified amacrine cell and synaptic circuitry in the inner
plexiform layer of the retina. Brain Res. 84, 293–300. 1975.
Farrow K, Teixeira M, Szikra T, Viney TJ, Balint K, Yonehara K, Roska B. Ambient
illumination toggles a neuronal circuit switch in the retina and visual perception at cone
threshold. Neuron 78:325-338. 2013.
Feigenspan A., Bormann J. Differential pharmacology of GABAA and GABAC receptors on rat
retinal bipolar cells. Eur. J. Pharmacol. 288:97–104, 1994a.
Feigenspan A., Wässle H., Bormann J. Pharmacology of GABA receptor Cl− channels in rat
retinal bipolar cells. Nature361:159–162, 1993.
Haverkamp S, Wassle H, Duebel J, Kuner T, Augustine GJ, Feng G, Euler T. The
primordial, blue-cone color system of the mouse retina. J Neurosci 25: 5438–5445, 2005.
Kolb H, Nelson R, Mariani A. Amacrine cells, bipolar cells and ganglion cells of the cat retina:
a Golgi study. Vision Res 21:1081–1114. 1981.
Lin, B. & Masland, R.H. Populations of wide-field amacrine cells in the mouse retina.
The Journal of Comparative Neurology 499, 797–809, 2006.
MacNeil, M.A., Masland, R.H. Extreme diversity among amacrine cells: implications for
function. Neuron 20, 971–982. 1998.
Menger, N., Pow, D.V., Wässle, H. Glycinergic amacrine cells of the rat retina. J. Comp.
Neurol. 401, 34–46. 1998.
Masland, RH. The tasks of amacrine cells. Visual Neuroscience 29, 3-9; 2012.
Pan Z. H., Lipton S. A. Multiple GABA receptor subtypes mediate inhibition of calcium influx at
rat retinal bipolar cell terminals. J. Neurosci. 15:2668–2679. 1995.
Pourcho RG, Goebel DJ. Neuronal subpopulations in cat retina which accumulate the GABA
agonist, (3H)muscimol: a combined Golgi and autoradiographic study. J Comp Neurol
219: 25–35, 1983.
Wang JS, Kefalov VJ. An alternative pathway mediates the mouse and human cone visual
cycle. Curr Biol 19: 1665–1669, 2009.
Wu, S.M. and Yang, X.L. Modulation of synaptic gain by light. Proc. Nat'l Acad. Sci.,
89, 11755-11758, 1992.
Xin D, Bloomfield SA. Comparison of the responses of AII amacrine cells in the dark- and lightadapted rabbit retina. Vis Neurosci 16: 653–665, 1999.
Yeh H. H., Grigorenko E. V., Veruki M. L. Correlation between a bicuculline-resistant response
to GABA and GABAA receptor rho-1 subunit expression in single rat retinal bipolar
cells. Vis. Neurosci. 13:283–292, 1996.
Zhang, C. & McCall, M.A. Receptor targets of amacrine cells. Visual Neuroscience 29: 11-29,
2012.
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement