Early Retinal Neuronal Dysfunction in Diabetic Mice:

Early Retinal Neuronal Dysfunction in Diabetic Mice:
Visual Neuroscience
Early Retinal Neuronal Dysfunction in Diabetic Mice:
Reduced Light-Evoked Inhibition Increases Rod Pathway
Signaling
Johnnie M. Moore-Dotson,1 Jamie J. Beckman,2 Reece E. Mazade,2 Mrinalini Hoon,3
Adam S. Bernstein,1 Melissa J. Romero-Aleshire,1 Heddwen L. Brooks,1 and Erika D. Eggers1,4
1Department
of Physiology, University of Arizona, Tucson, Arizona, United States
Graduate Interdisciplinary Program in Physiological Sciences, University of Arizona, Tucson, Arizona, United States
3
Department of Biological Structure, University of Washington, Seattle, Washington, United States
4
Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, United States
2
Correspondence: Erika D. Eggers,
Department of Physiology, University of Arizona, P.O. Box 245051,
Tucson, AZ 85724, USA;
[email protected]
Submitted: August 19, 2015
Accepted: February 15, 2016
Citation: Moore-Dotson JM, Beckman
JJ, Mazade RE, et al. Early retinal
neuronal dysfunction in diabetic mice:
reduced light-evoked inhibition increases rod pathway signaling. Invest
Ophthalmol Vis Sci. 2016;57:1418–
1430. DOI:10.1167/iovs.15-17999
PURPOSE. Recent studies suggest that the neural retinal response to light is compromised in
diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially
susceptible to diabetic damage. The purpose of this study was to determine whether diabetes
alters rod pathway signaling.
METHODS. Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of
streptozotocin (STZ; 75 mg/kg), and confirmed by blood glucose levels > 200 mg/dL. Six
weeks after the first injection, whole-cell voltage clamp recordings of spontaneous and lightevoked inhibitory postsynaptic currents from rod bipolar cells were made in dark-adapted
retinal slices. Light-evoked excitatory currents from rod bipolar and AII amacrine cells, and
spontaneous excitatory currents from AII amacrine cells were also measured. Receptor inputs
were pharmacologically isolated. Immunohistochemistry was performed on whole mounted
retinas.
RESULTS. Rod bipolar cells had reduced light-evoked inhibitory input from amacrine cells but
no change in excitatory input from rod photoreceptors. Reduced light-evoked inhibition,
mediated by both GABAA and GABAC receptors, increased rod bipolar cell output onto AII
amacrine cells. Spontaneous release of GABA onto rod bipolar cells was increased, which may
limit GABA availability for light-evoked release. These physiological changes occurred in the
absence of retinal cell loss or changes in GABAA receptor expression levels.
CONCLUSIONS. Our results indicate that early diabetes causes deficits in the rod pathway leading
to decreased light-evoked rod bipolar cell inhibition and increased rod pathway output that
provide a basis for the development of early diabetic visual deficits.
Keywords: diabetes, GABA, bipolar cells, amacrine cells, inhibition
iabetic retinopathy is the leading cause of adult onset
blindness in the United States.1 The most notable
characteristic of diabetic retinopathy is the progressive decline
of vascular function, which ultimately causes retinal damage
and blindness.2 Additionally, there is mounting evidence from
diabetic patients and animal models that show deficits in visual
function such as contrast sensitivity, acuity, night vision, and
retinal neuronal signaling.2–4 These deficits are likely due to
direct changes in retinal signaling, as changes in the global
retinal measurement electroretinogram (ERG) are also reported
early in diabetes.2–6 Retinal signaling begins with light
detection by photoreceptors that is transduced to bipolar,
amacrine, and ganglion cells using glutamate.7 Retinal output is
modulated by GABA or glycine release from inhibitory
amacrine cells onto bipolar and ganglion cells.7 Light sensed
by rod photoreceptors triggers rod bipolar cell excitation that is
reflected in the b-wave of ERGs. Some studies have reported
reduced b-wave amplitudes in diabetes8,9 suggesting reduced
photoreceptor function, while others have shown the b-wave
to be unaffected.3,10 Electroretinogram oscillatory potentials,
D
which reflect signaling between bipolar and amacrine cells, are
consistently altered in diabetes.3,4,6,8,10 However, the ERG
technique is used to measure a population response to light and
cannot discriminate responses of individual neurons or
determine if signaling has changed due to loss of neurons.
We used the streptozotocin (STZ) mouse model of type 1
diabetes to determine what properties of retinal signaling were
altered in early (6 weeks) diabetes. We recorded the response
of single retinal neurons to light. We focused on neurons that
comprise the rod pathway because ERG studies have suggested
that they may be more susceptible to hyperglycemic damage.3,11 A recent study also found dysfunctional synaptic
transmission between neurons in the rod pathway but did
not determine whether the response to light, the natural
stimulus of the retina, was altered.12 We found that diabetes
decreases light-evoked GABAergic signaling from amacrine cells
to rod bipolar cells, which increases the output of the rod
pathway onto downstream neurons independent of rod
photoreceptor dysfunction or loss of retinal neurons.
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Light-Evoked Inhibition Is Reduced In Diabetes
RESEARCH DESIGN
AND
METHODS
Animals
Animal protocols conformed with the ARVO Statement for the
Use of Animals in Ophthalmic and Visual Research and were
approved by the University of Arizona Institutional Animal Care
and Use Committee. Experiments used C57BL/6J male mice
(Jackson Laboratories, Bar Harbor, ME, USA) that were housed
in the University of Arizona animal facility and given the
National Institutes of Health-31 rodent diet food and water ad
libitum. Five-week-old mice were fasted for 4 hours and
injected intraperitoneally with either STZ (Sigma-Aldrich Corp.,
St. Louis, MO, USA; 75 mg/kg body weight) dissolved in 0.01 M
pH 4.5 citrate buffer or citrate buffer vehicle for three
consecutive days.13 Body weight and urine glucose were
monitored weekly. Six weeks after the first injection, mice
were fasted for 4 hours and blood glucose was measured
(OneTouch UltraMini; LifeScan, Milpitas, CA, USA). Streptozotocin-injected animals with blood glucose 200 mg/dL were
eliminated from the study. Fasting blood glucose was 361 6
9.5 mg/dL (n ¼ 50 mice) for STZ-treated mice and 137 6 5 mg/
dL (n ¼ 36 mice; P < 0.001 unpaired Student’s t-test) for
control mice. Body weights of diabetic and control mice were
21.6 6 0.3 and 24.5 6 0.4 g (P < 0.001), respectively.
Solutions and Drugs
Extracellular solution (bubbled with 95%/5% O2/CO2) contained (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 20
glucose, 26 NaHCO3, 2 CaCl2. Extracellular solution for
spontaneous GABAC receptor (R) recordings contained (in
mM): 120 NaCl, 15 KCl, 1 MgCl2, 1.25 NaH2PO4, 5 glucose, 26
NaHCO3, 2 CaCl2. The recording pipette intracellular solution
contained (in mM): 120 CsOH, 120 gluconic acid, 1 MgCl2, 10
HEPES, 10 EGTA, 10 TEA-Cl, 10 phosphocreatine-Na2, 4 MgATP, 0.5 Na-GTP, 50 lM Alexa Fluor-488 (Invitrogen, Carlsbad,
CA, USA) adjusted to pH 7.2 with CsOH. Strychnine (500 nM–1
lM), SR95531 (20 lM), TPMPA (1,2,5,6-Tetrahydropyridin-4yl)methylphosphinic acid hydrate, 50 lM) were used to block
glycine, GABAA, and GABAC receptors, respectively. Tetrodotoxin (TTX; 500 nm) and CdCl2 (100 lM) were used to block
voltage-gated Naþ and Ca2þ channels. All solutions were
applied (~1 mL/minute) via gravity-driven superfusion system
(Cell Microcontrols, Norfolk, VA, USA). Chemicals were
purchased from Sigma-Aldrich Corp.
Preparation and Recordings
Six weeks after injections, retinal slices were prepared from
mice dark-adapted overnight. Infrared illumination was used
for dissections to preserve the light sensitivity.14 Briefly, eyes
were enucleated from mice killed using carbon dioxide,
corneas and lenses removed, eyecups incubated in extracellular solution with hyaluronidase (800 units/mL) for 20 minutes,
and retinas removed. The retina was trimmed, mounted onto
filter paper, and sliced into 250-lm slices. Whole-cell voltageclamp recordings in dark-adapted retinal slices were made
under infrared illumination at 328C.14 Light-evoked inhibitory
postsynaptic currents (L-IPSCs) and spontaneous (s)IPSCs were
recorded at 0 mV (reversal potential for nonselective cation
channels). Light-evoked excitatory postsynaptic currents (LEPSCs), spontaneous (s)EPSCs, and miniature (m)EPSCs were
recorded at 60 mV (reversal potential for Cl). Borosilicate
glass electrodes (World Precision Instruments, Sarasota, FL,
USA) had resistances of 5 to 7 MX and the series resistance
during recordings was typically 10 to 20 MX. Liquid junction
potentials of 20 mV were corrected prior to recording.
Responses were filtered at 5 kHz on an Axopatch 200B
amplifier and digitized at 10 kHz using a Digidata 1440A A/D
board and Clampex software (Molecular Devices, Sunnyvale,
CA, USA). Alexa fluorescence was imaged at the end of each
recording using an Intensilight fluorescence lamp and Digitalsight camera controlled by Elements software (Nikon Instruments, Tokyo, Japan) to confirm rod bipolar cell15 and AII
amacrine cell16 morphology. Full-field light stimuli were
generated by a light emitting diode (LED, kpeak ¼ 525 nm)
projected through the microscope camera port onto the retina.
The light stimuli used were calibrated as photons/lm2/s and
converted to rhodopsin isomerizations per second using a
collecting area of 0.5 lm2.17 Light intensity and duration (30
ms) were controlled by varying the current through the LED.
Electrophysiology Analysis and Statistics
Light-evoked inhibitory postsynaptic currents and L-EPSCs for
each condition were averaged, and the peak amplitude and
charge transfer (Q - over the time of the response for each cell)
were measured using Clampfit (Molecular Devices). Because
the data were not normally distributed, log two-way repeated
measures ANOVAs were used to compare L-IPSCs and L-EPSCs
between conditions across light intensities, using the StudentNewman-Keuls (SNK) for pairwise comparisons. P-values
reflect the main treatment effect of STZ unless otherwise
indicated. Mini-Analysis software (Synaptosoft, Fort Lee, NJ,
USA) was used to analyze the peak amplitude, tdecay, interevent
interval and frequency of sIPSCs, sEPSCs, and mEPSCs.18–20
The threshold was set from 2 to 5 pA depending on the
baseline noise, and events were subjected to visual confirmation. Events separated by an interval ‡ 5 ms were fit with a
single exponential to measure the decay time constant. Values
were compared using the Kolmogorov–Smirnov test (K–S).
Average GABAA and GABAC receptor sIPSCs from rod bipolar
cells and AII amacrine cell mEPSCs were generated using MiniAnalysis software. Differences were considered significant
when P < 0.05.
The estimated amount of transmitter release mediating rod
bipolar cell GABAA and GABAC receptor-mediated L-IPSCs, and
AII amacrine cell L-EPSCs were calculated using custom Matlab
software. Release functions were calculated by deconvolution
analysis21 using the relationship20:
L I=EPSCðtÞ ¼ releaseðtÞ˜s=m I=EPSCðtÞ;
ð1Þ
such that
releaseðtÞ ¼ F 1
F ½L I=EPSCðtÞ
;
F ½s=m I=EPSCðtÞ
ð2Þ
Data are reported as mean 6 standard error of the mean (SEM)
unless otherwise indicated.
Immunohistochemistry, Imaging, and Analysis
Whole-mounted retinas were fixed in 4% paraformaldehyde for
15 (GABAA a1 subunit and protein kinase C [PKC]) or 30
minutes (PKC and TO-PRO-3) at room temperature, and rinsed
with 0.1 M phosphate-buffered saline (PBS; pH 7.4). The tissue
was incubated overnight in blocking solution (5% Donkey
serum and 0.5% Triton X-100 in 0.1 M PBS) then incubated
with primary antibodies for 3 (GABAA a1 subunit and PKC) or
5 days (PKC) at 48C. Anti-PKC (mouse monoclonal antibody,
1:1000; Sigma-Aldrich Corp.) primary antibody labelled rod
bipolar cells. Anti-GABAA a1 subunit antibody (guinea pig
polyclonal, 1:5000, provided by J.M. Fritschy) labelled GABAA
receptors. Retinas were incubated overnight with secondary
antibodies in PBS, then with TO-PRO-3 (Life Technologies,
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Light-Evoked Inhibition Is Reduced In Diabetes
Carlsbad, CA, USA) nuclear stain (retinas labeled for only PKC),
washed with PBS, and mounted in Vectashield (Vector Labs,
Burlingame, CA, USA). Secondary antibodies utilized were antimouse Alexa 488 (1:1000; Invitrogen), anti-guinea pig Alexa
568 (1:1000; Invitrogen) or anti-mouse Alexa 649 (1:500;
Jackson ImmunoResearch, West Grove, PA, USA).
To determine the numbers of cells per each layer, retinas
were imaged using a Zeiss (Oberkochen, Germany) LSM 510
Meta confocal microscope with a 403 1.2 NA objective.
Stacked images (1-lm thick) were acquired from four regions
500 lm from the optic nerve head (142.9 3 142.9 lm2). Cells
were counted using the ImageJ Cell Counter plug-in (http://
rsbweb.nih.gov/ij/plugins/cell-counter.html; provided in the
public domain by the National Institutes of Health, Bethesda,
MD, USA). A different marker was used to mark unique cells in
each vertical (Z-axis) layer to prevent double counting. Only
cells with nuclei more than 50% within the borders of the
region of interest were counted. The cell numbers were
averaged among the four locations in each retina, compared
between groups using an unpaired Student’s t-test, and
considered significant if P < 0.05.
To analyze GABAA receptor labeling, images were acquired
on a FV1000 Olympus (Center Valley, PA, USA) laser scanning
confocal microscope with a 603 1.35 NA objective at a voxel
size of 0.051, 0.051, 0.3 lm. Images were processed with
MetaMorph (Molecular Devices) and Amira (FEI Visualization
Sciences Group, Hillsboro, OR, USA) software programs. To
isolate rod bipolar cell terminals, the PKC signal was masked in
3D using the Labelfield function in Amira. The mask was
multiplied with the receptor channel using the Arithmetic
function in Amira to exclude receptor signal outside the mask
and isolate the receptor signal exclusively within the PKC
labeled rod bipolar cell terminals. A constant threshold was
applied on the receptor channel to exclude any background
nonclustered signal and determine the receptor pixels within
the mask. The volume of detected receptor pixels was
expressed as a percentage of the total volume occupied by
the pixels within the mask and compared between groups
using an unpaired Student’s t-test.22–24
RESULTS
Early Diabetes Decreases Amacrine Cell Signaling
to Rod Bipolar Cells
To determine if early diabetes (6 weeks post-STZ) reduces rod
bipolar cell activation by rod photoreceptors, we recorded LEPSCs from rod bipolar cells after 30 ms light stimuli of varying
light intensities that cover both rod and cone activation (Fig.
1A).25 There were no significant differences in either the peak
amplitudes (P ¼ 0.2; Fig. 1B) or Q (P ¼ 0.8; Fig. 1C) of the LEPSCs. These results show that 6 weeks of diabetes does not
reduce light-evoked activation of rod bipolar cells by rod
photoreceptors and agree with ERG studies that show no
difference in the b-wave.
To determine if amacrine cell-mediated inhibition to rod
bipolar cells is changed in early diabetes, we recorded L-IPSCs
from rod bipolar cells (Fig. 1D). Rod bipolar cell L-IPSCs from
diabetic mice had lower peak amplitudes (P ¼ 0.002; Fig. 1E)
and Q’s (P ¼ 0.02; Fig. 1F) compared to control. Pairwise
comparisons determined that peak amplitudes were reduced at
dim and bright intensities (P < 0.05, SNK post hoc), and Q was
reduced at a dim intensity (P ¼ 0.03). Thus, light-evoked
inhibitory input from amacrine cells to rod bipolar cells is
reduced after 6 weeks of diabetes, independent of excitatory
input from rod photoreceptors, suggesting an imbalance in
excitatory and inhibitory inputs.
Early Diabetes Decreases Light-Evoked GABAergic
Inhibition to Rod Bipolar Cells
Inhibition to rod bipolar cells is composed of glycinergic
inputs onto glycine receptors and GABAergic inputs onto
GABAA and GABAC receptors.26 However, proportionally
GABAergic inputs contribute most of the inhibition to rod
bipolar cells.19 Changes in GABA production and GABA
receptor properties have been reported in diabetes,10,27,28
which may underlie our measured inhibition decrease.
Therefore, we investigated whether light-evoked inhibition
mediated by both GABA receptor types are decreased in early
diabetes by recording L-IPSCs mediated by isolated GABAA
(strychnine þ TPMPA) or GABAC receptors (strychnine þ
SR95531; Figs. 2A, 2D). Diabetes reduced both the peak
amplitudes and Q of GABAA receptor input to rod bipolar cells
(peak: P ¼ 0.05; Q: P ¼ 0.003; Figs. 2B, 2C). Pairwise
comparisons showed that the average peak amplitude was
reduced at dim intensities (P ¼ 0.05; Fig. 2B). The average Q
was reduced at dim and bright intensities (P 0.01; Fig. 2C).
The peak amplitudes (Figs. 2D, 2E) of GABAC receptor L-IPSCs
were also significantly reduced (P ¼ 0.006) at multiple
intensities. Q was reduced overall, but pairwise comparisons
did not determine a significant decrease at any one intensity (P
¼ 0.02; Fig. 2F). Thus, reduced light-evoked rod bipolar cell
inhibition after 6 weeks of diabetes may reflect reduced
GABAergic inhibitory input from amacrine cells.
Early Diabetes Increases Spontaneous GABAergic
Rod Bipolar Cell Activity
Reduced light-evoked GABAergic signaling may be explained
by changes in presynaptic amacrine cell function, or changes
in the postsynaptic GABAA and GABAC receptors on rod
bipolar cell terminals. To distinguish between these possibilities, we measured spontaneous (s)IPSCs mediated by GABAA
and GABAC receptors in the absence of a stimulus to determine
if receptor and transmitter release properties, reflected by
differences in sIPSC peak amplitude and sdecay values versus
frequency, are altered18–20 (Fig. 3). GABAA receptor sIPSC peak
amplitudes and frequencies were increased in diabetic mice
(Figs. 3B, 3C; Table), with no difference in the sdecay (P ¼ 0.8).
In contrast, although the frequency of GABAC receptor sIPSCs
increased (Figs. 3D, 3F; Table), the peak amplitude was
unchanged (Fig. 3E) and there was also no difference in the
sdecay (P ¼ 0.2). These results suggest that spontaneous GABA
release from amacrine cells and GABAA receptor cluster size or
activity on rod bipolar cell terminals are increased after 6
weeks of diabetes.
Early Diabetes Increases Rod Bipolar Cell Output
to AII Amacrine Cells
The decrease in rod bipolar cell inhibition in diabetic mice
predicts an increase in rod bipolar cell output onto downstream targets. Unlike other retinal bipolar cell types that
contact ganglion cells, the excitatory output of rod bipolar
cells goes to AII amacrine cells.29,30 To determine if the
reduction in light-evoked inhibition to rod bipolar cells causes
an increase in light-evoked rod bipolar cell activation of AII
amacrine cells in diabetes, we recorded L-EPSCs from AII
amacrine cells (Fig. 4A). L-EPSC peak amplitudes of AII
amacrine cells from diabetic mice increased compared to
control (Fig. 4B), and pairwise analysis found increases at dim
intensities (P 0.05). In contrast, there was no difference in
the Q between control and diabetic mice (P ¼ 0.2; Fig. 4C).
Our data showing that spontaneous GABAergic inhibition to
the rod bipolar cell increases in diabetes predict reduced
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Light-Evoked Inhibition Is Reduced In Diabetes
FIGURE 1. Light-evoked inhibition to rod bipolar cells is reduced after 6 weeks of diabetes. (A) Representative traces of L-EPSCs averaged from two
responses at the maximum light intensity recorded from rod bipolar cells in response to a 30-ms light stimulus onto rod photoreceptors (R, inset)
are shown. Light-evoked excitatory postsynaptic currents are not different between control (n ¼ 7 cells from 5 mice) and diabetic (n ¼ 8 cells from 7
mice) rod bipolar cells. (B, C) The peak amplitude (B) and magnitude (Q, C) of L-EPSCs at increasing intensities are similar between both groups (P
¼ 0.2, 2-way ANOVA). (D) Representative traces of L-IPSCs averaged from two responses at the maximum light intensity recorded from rod bipolar
cells in response to a 30-ms light stimulus (inset) are shown. (E, F) The light-evoked inhibitory postsynaptic currents peak amplitude (E) and Q (F)
of diabetic (n ¼ 18 cells from 12 mice) treated rod bipolar cells are reduced compared to control cells (n ¼ 16 cells from 12 mice) at multiple
intensities. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant; black bar ¼ light stimulus.
spontaneous excitatory output onto AII amacrine cells in
diabetic animals. Surprisingly, we found that the peak
amplitude (Fig. 4F, control: 25 6 0.3 pA; diabetic: 33 6 0.2
pA) and the frequency (Fig. 4G, control: 88 6 15 Hz, diabetic:
111 6 16 Hz) of sEPSCs of AII amacrine cells were increased in
diabetic mice. These results could be explained by changes in
either presynaptic glutamate release from rod bipolar cells or
the postsynaptic AII amacrine cell receptors that mediate the
EPSCs. Since spontaneous and evoked glutamate release at the
rod bipolar-AII amacrine cell synapse occurs by multivesicular
release, or the simultaneous fusion of multiple vesicles,31 it is
possible that increased spontaneous GABAergic inhibition does
not affect spontaneous release from rod bipolar cells because
of a high amount of multivesicular release.
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Light-Evoked Inhibition Is Reduced In Diabetes
FIGURE 2. GABAergic inhibition from amacrine cells to rod bipolar cells is reduced in early diabetes. (A) GABAA receptor L-IPSCs are reduced in
diabetic rod bipolar cells as shown by representative traces averaged from two responses at the maximum light intensity. (B, C) The peak
amplitudes and Q of GABAA receptor L-IPSCs are decreased at increasing intensities in diabetic rod bipolar cells compared to control (n ¼ 7 cells
from 6 mice for both groups). (D) Light-evoked inhibitory postsynaptic currents mediated by GABAC receptors are reduced in diabetic rod bipolar
cells as shown by representative traces averaged from two responses at the maximum light intensity. (E, F) GABAC receptor L-IPSCs peak amplitudes
(E) and Q (F) are decreased at multiple intensities in diabetic rod bipolar cells compared to control (n ¼ 5 cells from 3 mice for both groups). *P <
0.05, **P < 0.01; black bar ¼ light stimulus.
To determine if increased multivesicular release underlies
the diabetes-induced increase in AII amacrine cell sEPSC
amplitude, we recorded miniature (m) EPSCs evoked by the
release of single vesicles. mEPSCs were isolated from dark-
adapted AII amacrine cells by recording in the presence of TTX
and Cd2þ to block action potentials and synaptic input, and
limit multivesicular release. If increased spontaneous excitatory activity is due to multivesicular release, we would expect
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Light-Evoked Inhibition Is Reduced In Diabetes
FIGURE 3. Spontaneous GABAergic input to rod bipolar cells from amacrine cells is increased after 6 weeks of diabetes. (A) Representative traces of
spontaneous GABAA receptor activity recorded from rod bipolar cells are shown. There were fewer GABAA receptor sIPSCs detected in control
(black trace) compared to diabetic (gray trace) rod bipolar cells. (B) Distribution of GABAA receptor sIPSCs normalized to the number of events
shows increased peak amplitudes in diabetic (n ¼ 19 cells from 12 mice, 3382 events) rod bipolar cells compared to control (n ¼ 19 cells from 11
mice, 1768 events; P < 0.0001 K-S test). (C) Distribution of GABAA receptor sIPSCs normalized to the number of events shows decreased interevent
intervals (IEI) in diabetic rod bipolar cells (P < 0.0001). (D) Representative traces of spontaneous GABAC receptor activity recorded from rod
bipolar cells are shown. More GABAC receptor sIPSCs were detected in diabetic (gray trace) rod bipolar cells than in control (black trace). (E) The
peak amplitudes of GABAC receptor-mediated sIPSCs are similar (P ¼ 0.7). (F) Distribution of GABACR sIPSCs normalized to the number of events
shows decreased IEI in diabetic (n ¼ 12 cells from 7 mice, 9355 events) rod bipolar cells compared to control (n ¼ 20 cells from 12 mice, 2167
events; P < 0.0001). Black and gray filled circles represent the median values for control and diabetic, respectively.
the peak amplitude of diabetic mEPSCs to be similar to control
mEPSCs. We found that the average AII amacrine cell mEPSC
peak amplitude was reduced in diabetic cells (Figs. 4D, 4H;
Table) compared to control. There was no significant
difference in the average frequency (P ¼ 0.3) or sdecay (P ¼
0.4). These results suggest that increased AII amacrine cell
spontaneous activity may be due to augmented multivesicular
release from the rod bipolar cell in diabetes and that there may
even be a small decrease in glutamate receptor expression on
AII amacrine cells in diabetes.
Diabetes Leads to Decreased GABA Release From
Amacrine Cells to Rod Bipolar Cells and Increased
Rod Bipolar Cell Glutamate Release
Our results of decreased light-evoked and increased spontaneous inhibitory input to rod bipolar cells in diabetes suggest that
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Light-Evoked Inhibition Is Reduced In Diabetes
TABLE. Values for sIPSC Properties in Control and Diabetic Rod Bipolar Cells, and mEPSC Properties in Control and Diabetic AII Amacrine Cells
Condition
GABAA receptor control
GABAA receptor diabetic
GABAC receptor control
GABAC receptor diabetic
AII glutamate receptor control
AII glutamate receptor diabetic
Peak Amplitude, pA
10.2
15
6.4
6.7
15.8
9.9
6
6
6
6
6
6
0.5
3 pA
0.3
0.6
0.2
0.1
Frequency, Hz
0.375
0.834
0.467
1.34
4.3
8.3
6
6
6
6
6
6
0.117
0.334
0.15
0.22
2.0
3.3
sdecay, ms
Number of Cells
6
6
6
6
6
6
19
19
12
20
4
4
3.1
3.2
29.8
22.3
1.2
1.1
0.6
0.8
4.5
3.1
0.1
0.1
Data are average values of peak amplitude, frequency, and sdecay of sIPSCs.
increased spontaneous release of GABA may lead to decreased
L-IPSCs by reducing the amount of GABA available to be
released after a stimulus. To determine whether there was a
decrease in light-evoked GABA release, we used deconvolution
analysis,20,21 which assumes that the release of individual
vesicles that evoke sIPSCs sum linearly to evoke a L-IPSC.
Therefore, this analysis reflects the minimum amount of
quantal release required to evoke a response. Diabetes reduced
light-evoked GABA release onto both GABAA (P < 0.001) and
GABAC (P < 0.01) receptors (Fig. 5). The peak of GABA release
onto GABAA receptors, as well as the amount of GABA released
over time (Q, vesicles*ms, P < 0.001, data not shown), were
reduced in diabetic cells. Pairwise comparisons showed that
the peak of release (Fig. 5B) was reduced at each light intensity.
Similarly, the peak (Fig. 5E) and total amount (P < 0.05) of
release onto GABAC receptors were reduced. We conclude that
reduced rod bipolar cell light-evoked GABAergic inhibition is
due to decreased GABA release from amacrine cells onto rod
bipolar cell terminals.
Our analysis of AII amacrine cell L-EPSCs and mEPSCs (Fig.
4) suggest that diabetes may cause an increase in glutamate
release from rod bipolar cells. To determine if the increased LEPSC peak amplitude we measured was due to increased
glutamate release from rod bipolar cells, we used deconvolution analysis to estimate the amount of glutamate release at
each light-intensity (Figs. 5C, 5F). Due to the high rate of
multivesicular release at the rod bipolar-AII amacrine cell
synapse, we used an averaged mEPSC in place of an averaged
sEPSC to deconvolve with the L-EPSC and determine the
estimated glutamate release. The peak (Fig. 5F) of light-evoked
glutamatergic vesicle release was increased in diabetic mice (P
< 0.05). Pairwise comparisons showed significant increases at
dim intensities (P < 0.05). Thus, rod bipolar cell glutamate
release is increased in diabetes as a result of reduced rod
bipolar cell GABAergic inhibition.
Early Diabetes Does Not Cause Cell or Receptor
Loss
Although the above results suggest a change in the physiological function of rod bipolar and amacrine cells, they do not
exclude the possibility of changes in retinal morphology. To
test whether there was loss of retinal neurons after 6 weeks of
diabetes, we used TO-PRO-3 to label nuclei in whole-mounted
retinas (Fig. 6A). We found no difference between control and
diabetic mice in the numbers of cells in the outer nuclear layer
(P ¼ 0.9 unpaired Student’s t-test), inner nuclear layer (P ¼ 0.5),
or ganglion cell layer (P ¼ 0.5; Fig. 6B). It is possible that the
loss of retinal neurons is limited to specific types of cells, as
previously reported,32 since the loss of only one cell-type
might not be detected in our analysis of total retinal neurons.
Since ERGs are population measurements, loss of rod bipolar
cells could explain decreases in rod pathway signaling. We
labeled rod bipolar cells with an antibody against PKC,33 (Fig.
6A) and found similar numbers of rod bipolar cells in control
and diabetic mice (P ¼ 0.4; Fig. 6B).
Analysis of the spontaneous GABAA receptor input to rod
bipolar cells suggested an increase in either rod bipolar cell
GABAA receptor clusters or activity (Fig. 3B). Therefore, we
labelled the a1 subunit of GABAA receptors on rod bipolar
cells34 (Fig. 7). Since GABAA a1 receptor expression is
abundant and not limited to rod bipolar cell terminals, GABAA
a1 receptor labeling in locations other than rod bipolar cell
terminals was excluded digitally (see Research Design and
Methods).22–24 The volume of rod bipolar cell terminals
occupied by GABAA a1 receptors was not different between
control and diabetic mice (Fig. 7B, P ¼ 0.3). Thus, decreased
amacrine cell-mediated rod bipolar cell inhibition in early
diabetes does not reflect changes in the amount of GABAA
receptors that occupy the rod bipolar cell terminals.
DISCUSSION
We found that light-evoked amacrine cell-mediated inhibition
to rod bipolar cells is reduced after 6 weeks of diabetes
without changes in excitatory drive from rod photoreceptors
or large scale loss of retinal neurons. Our results indicate that
compromised GABAergic inhibition from amacrine cells due to
reduced GABA release underlies the decreased inhibition that
leads to increased light-evoked excitatory output from the rod
pathway. These results are in agreement with a recent study
that showed signaling at the rod bipolar-AII amacrine cell
synapse is compromised; however, light-evoked responses of
rod bipolar and amacrine cells were not measured.12 Here we
have shown that the processing of a light signal by the inner
retinal circuitry is compromised in early diabetes by directly
measuring light-evoked inhibition from amacrine cells to rod
bipolar cells. Dysfunctional retinal signaling in the rod pathway
was detected after just 6 weeks of diabetes. This imbalance in
retinal excitatory and inhibitory signaling likely contributes to
visual deficits reported in diabetes.
Although it is not currently possible to measure the
response of individual neurons or determine what mechanisms
have changed in vivo, our results do support previous results
from diabetic humans and animal models. Our results showing
reduced inhibition are consistent with a commonly reported
consequence of diabetes, reduced amplitudes, and increased
latency of oscillatory potentials that reflect amacrine cell
activity measured by ERGs.2,3,6,9,10,35 Differences in the
oscillatory potentials in diabetes correlate directly between
humans and animal models.5 Our results showing no deficits in
photoreceptor inputs to rod bipolar cells also agree with ERG
studies that show no differences in the in vivo ERG a- or bwaves in early diabetes,3,10 although there remains some
controversy.8,9,11
Another advantage of recording from neural retinal tissue
directly is that we can differentiate loss of neurons from
dysfunctional neural activity as the cause of visual changes.
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Light-Evoked Inhibition Is Reduced In Diabetes
IOVS j March 2016 j Vol. 57 j No. 3 j 1425
FIGURE 4. Excitatory input from rod bipolar cells to downstream AII amacrine cells is increased in early diabetes. (A) Representative traces of LEPSCs averaged from two responses at the 4750 Rh*/rod/sec light intensity recorded from AII amacrine cells after a 30-ms light stimulus (inset) are
increased in diabetes. (B) The peak amplitudes are increased at multiple intensities in diabetic (n ¼ 9 cells from 8 mice) AII amacrine cells compared
to control (n ¼ 7 cells from 5 mice, P ¼ 0.05, 2-way ANOVA). (C) The magnitude (Q) of the L-EPSCS are similar (P ¼ 0.2). (D, E) Spontaneous
excitatory input from rod bipolar cells recorded from AII amacrine cells is increased in diabetic (gray trace) cells compared to control (black trace)
determine by measuring mEPSCs (D) and sEPSCs (E). (F, G) Distribution of sEPSCs normalized to the number of events shows increased peak
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Light-Evoked Inhibition Is Reduced In Diabetes
IOVS j March 2016 j Vol. 57 j No. 3 j 1426
amplitudes (F) and reduced interevent intervals (G) in diabetic (n ¼ 9 cells from 8 mice, 9859 events) cells compared to control (n ¼ 7 cells from 5
mice, 6140 events; P < 0.0001 K-S test). (H) Distribution of mEPSCs show increased peak amplitudes in diabetic (n ¼ 4 cells, 3995 events) cells
compared to control (n ¼ 4 cells, 2049 events; P < 0.0001 K-S test). Black and gray filled circles represent the median values for control and
diabetic, respectively. *P < 0.05, n.s., not significant; black bar ¼ light stimulus.
FIGURE 5. Amacrine cell GABA release is reduced and rod bipolar cell glutamate release is increased in early diabetes. (A, D) Representative traces
of the time course of GABA release onto rod bipolar cell GABAA (A) and GABAC (D) receptors estimated from deconvolution analysis of GABA
receptor-mediated sIPSCs and L-IPSCs are shown. (B, E) The peak of GABAergic vesicle release is decreased at multiple intensities in diabetic rod
bipolar cells compared to control (GABAA: n ¼ 7 cells from 6 mice for both groups; GABAC: n ¼ 5 cells from 3 mice for both groups). (C, F)
Representative traces (C) of the time course of glutamate release shows that release is increased (F) from rod bipolar cells to AII amacrine cells in
diabetes. Deconvolution analysis of glutamate receptor mEPSCs and L-EPSCs shows that the peak of glutamate vesicle release is higher in diabetic (n
¼ 9 cells from 8 mice) cells to control (n ¼ 7 cells from 5 mice, P < 0.05, 2-way ANOVA). Representative traces are deconvolved from L-IPSCs
recorded at the maximum intensity (GABA) or L-EPSCs recorded at the 4750 Rh*/rod/sec light intensity (glutamate). *P < 0.05, **P < 0.01, ***P <
0.001. q/ms, quanta/millisecond.
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Light-Evoked Inhibition Is Reduced In Diabetes
FIGURE 6. No loss of retinal neurons is detected in early diabetes. (A) Representative images of whole mounted retinas stained with PKCa (green)
to label rod bipolar cells (RBCs) or the nuclei stain TO-PRO-3 (red) from control (top panels) and diabetic (bottom panels) mice are shown. The
outer nuclear layer (ONL) contains photoreceptors. The inner nuclear layer (INL) contains horizontal, bipolar, and amacrine cells. The ganglion cell
layer (GCL) contains ganglion cells and displaced amacrine cells. (B) Quantification of the number of cells per layer is shown. There is no significant
difference in the average number of cells per layer between control and diabetic treated retinas (n ¼ 7 mice for both groups). n.s., not significant.
Scale bars: 25 lm.
Previous reports have shown loss of retinal neurons in humans
with diabetes36,37 and in animal models as early as 4 weeks
after the onset of experimental diabetes,32,38 which could
explain the observed physiological changes. For example, loss
of retinal neurons leads to changes in retinal neuronal signaling
in the rd1 mouse model of retinitis pigmentosa.39,40 Given that
we did not observe loss of cells in any retinal layer or rod
bipolar cells from diabetic mice (Fig. 6), we conclude that
there is no change in gross retinal morphology in early
diabetes. Because we could only make recordings from live
neurons, our results distinguish between changes due to
dysfunctional neural signaling and changes due to cell loss,
although it is still possible that subpopulations of neurons
could be lost. Thus, unlike in the rd1 mouse, our results show
changes in retinal neuronal signaling without significant loss of
retinal neurons.
Although we did not observe changes in neuronal
morphology, we cannot rule out the possibility that changes
in the blood retinal barrier contribute to retinal neuronal
dysfunction.41 In diabetes, retinal blood vessels exhibit deficits
in light-induced dilation suggesting that neurovascular coupling is compromised.42 Also, ERG deficits are predictive of
vascular growth.43 The connection between neuronal signaling
and retinal vasculature is not well understood, but this
neurovascular unit is likely disrupted in diabetes.
We found that diabetes reduced GABAergic light-evoked
inhibition to rod bipolar cells (Fig. 2), which may be a common
characteristic of neuronal diabetic damage as diabetes also
decreases GABAergic inhibition in other neural systems,44 but
the mechanisms for this dysfunction are not known. Reduced
expression of hippocampal GABAA receptor mRNA occurs in
diabetic rats.45 However, our results suggest that GABAA
receptor expression is not decreased at 6 weeks of diabetes
since we see an increase in GABAA receptor spontaneous
activity (Fig. 3) and no change in GABAA a1 receptor
expression on rod bipolar cell terminals (Fig. 7). Altered
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Light-Evoked Inhibition Is Reduced In Diabetes
FIGURE 7. GABAA receptor expression on rod bipolar cell terminals is not changed in early diabetes. (A) GABAA a1 receptors (cyan) colabeled with
PKC positive rod bipolar cell terminals (red) in control and diabetic retinas. Maximum intensity projection of a confocal image stack representing a
thickness of 9 lm. A 3D mask was created for the PKC immunolabeled terminals to isolate the GABAA a1 signal exclusively within the rod bipolar
cell terminals. After application of a threshold to eliminate background fluorescence, GABAA a1 receptor pixels could be detected (Mask panels,
white). (B) There is no difference in the volume of the detected GABAA a1 pixels expressed as % occupancy of the PKC labeled rod bipolar
terminals (RBCs) in control and diabetic retinas (n ¼ 4 mice for both groups). P ¼ 0.3 Student’s t-test; n.s., not significant.
subunit expression and increased open time of GABAC
receptors was reported in isolated rod bipolar cells from
diabetic rats,46 but changes in synaptic or physiological
properties of the rod bipolar cells were not investigated. Our
results of increases in GABAC sIPSC frequency without a
change in timing or peak amplitude suggest that the properties
of synaptic GABAC receptors on rod bipolar cell terminals are
not changed in early diabetes. Thus, changes to GABAergic
input to rod bipolar cells are likely occurring presynaptically in
amacrine cells. In agreement with this, we found that reduced
light-evoked GABAergic inhibition was due to decreased GABA
release from amacrine cells, but the mechanism is unclear. A
recent study in diabetic mice found that A17 amacrine cells,
which form reciprocal connections with rod bipolar cells and
comprise ~50% of rod bipolar cell inhibition,14,47,48 have
reduced Ca2þ-permeability when activated by exogenous
glutamate.12 If A17 amacrine cell Ca2þ influx in response to a
light stimulus is reduced, then GABA release from A17
amacrine cells onto rod bipolar cells would be limited, as
blocking the accumulation of Ca2þ in amacrine cells attenuates
release under nondiseased conditions.49 Additionally, a previous study showed a downregulation of synaptic proteins in
early diabetes that would also limit transmitter release.50
Since loss of inhibition to rod bipolar cells increases the
output of the rod pathway,20 we predicted that the reduced
light-evoked inhibition in diabetes would increase the output
of the rod pathway onto AII amacrine cells, the output neurons
of the rod pathway. As there is a strong relationship between a
single rod bipolar cell depolarization and AII amacrine cell
excitation, and AII amacrine cells receive inputs from multiple
rod bipolar cells, even a small increase in depolarization of the
rod bipolar cell membrane potential due to loss of inhibition
could result in a large change in AII excitatory currents.51 Our
results show that disinhibition of the rod bipolar cell in
diabetes increased the glutamate release (Fig. 5F) and
consequently increased L-EPSC peak amplitude (Fig. 4B), but
not the charge transfer of AII amacrine cells (Fig. 4C). A recent
study found that the transient (peak) and sustained (charge
transfer) components of AII amacrine cell responses have
distinct functions with the transient component encoding
contrast and the sustained component encoding luminance.52
Although the properties that underlie these distinct AII
amacrine cell responses remain unclear, the peak and transient
components may be governed by different mechanisms. It is
possible that diabetes affects the mechanisms that control the
peak of the response more than those that mediate the charge
transfer.
We expected that the increased spontaneous GABAergic
inhibitory inputs to rod bipolar cells (Fig. 3) might decrease
spontaneous excitatory activity in AII amacrine cells, but
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Light-Evoked Inhibition Is Reduced In Diabetes
actually observed an increase. Consistent with this, a recent
study showed increased spontaneous excitatory activity of AII
amacrine cells in diabetic rats, thought to be due to reduced
tonic GABAC receptor input to rod bipolar cells.12 However,
they did not measure spontaneous inhibition to rod bipolar
cells directly, and experiments were performed in light-adapted
conditions that significantly decrease spontaneous inhibition.14
We hypothesized that increased spontaneous excitatory
activity of AII amacrine cells reflected increased multivesicular
release of glutamate from rod bipolar cells. Our results
showing decreased mEPSC peak amplitude in AII amacrine
cells from diabetic mice support this hypothesis and suggest
that the high rate of rod bipolar cell release is able to overcome
increased spontaneous inhibition. This may be due to the
properties inherent to rod bipolar cell ribbon synapses, which
are designed to support graded potential changes with
sustained multivesicular release,53 unlike amacrine cell synapses, which are more similar to conventional synapses. Thus,
amacrine cells are likely to be more sensitive to vesicle
depletion by increased spontaneous release than rod bipolar
cells. Our present study is the first to investigate light-evoked
responses of rod bipolar cells in diabetes that may be regulated
differently than spontaneous activity. Increased output in
diabetes is not limited to the rod pathway, as a different study
has shown that ON ganglion cells, which likely receive ON
cone bipolar cell inputs, have increased spontaneous bursting
activity due to loss of inhibition.54
How does an imbalance between excitatory-inhibitory
signaling translate into deficits in visual function? Amacrine cell
inhibition significantly contributes to center-surround organization that refines spatial and temporal tuning in the inner
retina.55–57 In the rod pathway, the AII amacrine cell centersurround is governed by excitatory rod bipolar cell input (center)
and amacrine cell inhibition to rod bipolar cells (surround).57
Blocking GABAergic inhibition reduces the sensitivity of ganglion
cells to stimuli size58 and may reduce contrast sensitivity. Thus,
our results indicate that in the early stages of diabetes in a model
system, the retinal circuitry has a reduced capacity to refine
spatial and temporal tuning prior to sending it to the brain. This
may underlie the deficits in visual acuity and contrast sensitivity
detected in diabetic patients and animal models.
Acknowledgments
The authors thank Ronald Lynch, Ralph Fregosi, and Richard
Levine of the University of Arizona, and Nii Addy of Yale University
for helpful discussion and comments on the manuscript.
Supported by the Juvenile Diabetes Research Foundation, Innovative Grant Award 5-2013-163 (EDE) and Postdoctoral Fellowship 3PDF-2014-105-A-N (JMM); the National Institutes of Health (NIH;
R01-EY026027, EDE) and Cardiovascular Training Grant
2T32HL7249-36A1 appointment (JMM); a New Investigator Award
from the Alcon Research Institute (EDE); and a grant from the
International Retinal Research Foundation (EDE). MH was supported by NIH Grant EY10699 to R. Wong.
Disclosure: J.M. Moore-Dotson, None; J.J. Beckman, None; R.E.
Mazade, None; M. Hoon, None; A.S. Bernstein, None; M.J.
Romero-Aleshire, None; H.L. Brooks, None; E.D. Eggers, None
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