ARVO 2016 Annual Meeting Abstracts
309 Bipolar, Horizontal and Amacrine cells
Tuesday, May 03, 2016 8:30 AM–10:15 AM
Tahoma 5, TCC Paper Session
Program #/Board # Range: 2829–2835
Organizing Section: Visual Neuroscience
Program Number: 2829
Presentation Time: 8:30 AM–8:45 AM
Quantification of the neurons in the rod pathway of human retina
Sammy C. Lee, Rhian J. Aghajani, Paul R. Martin, Ulrike Grunert.
Save Sight Institute - Department of Ophthalmology, University of
Sydney, Sydney, NSW, Australia.
Purpose: The rod pathway serves scotopic vision, and involves
multiple rod photoreceptors converging onto rod bipolar cells, which
in turn converge onto AII amacrine cells. AII amacrine cells are
proposed as a target for optogenetic restoration of vision in retinal
disease, therefore it is important to understand the distribution and
density of AII cells and their connections in human retina.
Methods: Post mortem human eye tissue was obtained within 3.5 hours
of death from the Lions NSW Eye Bank at the Sydney Eye Hospital with
ethical approval by The University of Sydney Human Research Ethics
committee. Retinal pieces of defined eccentricities were embedded
in Agarose. Vertical sections were cut at 100 mm thickness along the
horizontal meridian using a Vibratome. Sections were then processed
for immunofluorescence to label rod photoreceptors, rod bipolar cells
(protein kinase C alpha), and AII amacrine cells (calretinin). Sections
were imaged with a Zeiss confocal microscope and z-stacks were taken
along the entire section. The densities of rods, rod bipolar cells, and
AII amacrine cells was determined along the horizontal meridian for
eccentricities 0 to ~14 mm.
Results: Rods first appear at 300 mm from the center of the fovea.
The rod peak density (at 3 mm) is ~200,000 cells/mm2; density then
gradually declines to ~72,000 cells/mm2 at 13 mm eccentricity. Rod
bipolar cell density follows a similar pattern with a first appearance
at about 300 mm eccentricity and peak at 3 mm eccentricity with
~22,000 cells/mm2. Rod bipolar density falls to ~8500 cells/mm2 at
8 mm. Calretinin positive (presumed AII) amacrine cells appear at
about 200 mm, have peak density at 1 mm with ~12,000 cells/mm2
and decline to ~6,000 cells/mm2 at eccentricities of 6 mm and above.
Therefore at 3 mm there is an average convergence of 9:4:1 between
rods, rod bipolar and AII amacrine cells. At 1 mm, where the AII
density is highest, the convergence is 10:1:1.
Conclusions: We found convergence from rods to RBCs to AII
amacrine cells throughout the retina. The spatial resolution of the rod
pathway is limited by the peak density of AII amacrine cells. Our
results indicate rod pathway resolution would peak outside the fovea
at 1 mm (~4 degrees eccentricity).
Commercial Relationships: Sammy C. Lee; Rhian J. Aghajani,
None; Paul R. Martin, None; Ulrike Grunert, None
Support: NH&MRC Project grant 1042609; Australian Research
Council Centre of Excellence for Integrative Brain Function; Grant
number: CE140100007
Program Number: 2830
Presentation Time: 8:45 AM–9:00 AM
Cone photoreceptors generate inhibitory ON and OFF responses
in dopamingergic amacrine cells via distinct OFF pathways
Sheng-Nan Qiao1, 2, Yong-Mei Zhong2, Dao-Qi Zhang1. 1Eye
Research Institute, Oakland University, Rochester, MI; 2Institute of
Neurobiology, Fudan University, Shanghai, China.
Purpose: Dopaminergic amacrine cells (DACs) are stimulated by
photoreceptors to release dopamine, which plays a critical role in
retinal light adaptation. Cone photoreceptors transmit light signals
to amacrine cells via parallel ON and OFF pathways. Previous
studies reported that DACs are excited by ON bipolar cells, but it is
unclear whether DACs receive input from OFF bipolar cells. Here we
determine how the OFF pathway regulates the DAC activity.
Methods: We utilized a transgenic mouse model lacking rod and
melanopsin function in which retinal DACs were labeled with red
fluorescence protein. Light responses of DACs were recorded under
voltage clamp.
Results: With 470-nm light stimuli, DACs (Vh= -66 mV) displayed
three classes of light responses: an initial ON response, a delayed
ON (d-ON) response and an OFF response. When L-AP4 was used
to block ON pathways, the OFF and d-ON responses persisted while
the ON response was blocked, indicating that the OFF and d-ON
responses are mediated by the OFF pathway. The current-voltage
relations showed that reverse potentials of the OFF and d-ON
responses were around -50 mV (close to the equilibrium potential
of Cl-) suggesting that OFF bipolar cells mediate inhibitory OFF
and d-ON responses indirectly. Additionally, the OFF response was
blocked by ACET, a kainate (KA) receptor antagonist, indicating that
the OFF response is mediated by OFF bipolar cells expressing the KA
receptor. In contrast, the d-ON response was not affected by ACET
but blocked by additional CNQX, a KA/AMPA receptor antagonist,
indicating that the d-ON response is mediated by OFF bipolar cells
expressing the AMPA receptor. Furthermore, the OFF response was
inhibited by both GABAzine and strychnine, indicating that this
response is mediated by GABAergic and glycinergic amacrines. The
d-ON response was eliminated by strychnine but not GABAzine,
suggesting that glycinergic amacrines mediate the d-ON response.
Conclusions: OFF bipolar cells are reported to express different
combinations of glutamate receptors: type 1 express the AMPA
receptor, type 2 and 3a express the KA receptor and type 3b and 4
express both receptors. Therefore, our data suggests that the OFF
response is mediated by type 2 and 3a OFF bipolar cells through
GABAergic and glycinergic amacrine cells, whereas the d-ON
response is mediated by type 1 OFF bipolar cells through glycinergic
amacrine cells.
Commercial Relationships: Sheng-Nan Qiao; Yong-Mei Zhong,
None; Dao-Qi Zhang, None
Support: China Scholarship Council; NIH R01 EY022640
Program Number: 2831
Presentation Time: 9:00 AM–9:15 AM
Rod photoreceptors mediate light-evoked activity of
dopaminergic amacrine cells across a wide range of light
intensities
Xiwu Zhao2, 1, Sheng-Nan Qiao2, 3, Yong-Mei Zhong3,
Kwoon Y. Wong1, Dao-Qi Zhang2. 1Ophthalmology & Visual
Sciences, University of Michigan, Ann Arbor, MI; 2Eye Research
Institute, Oakland University, Rochester, MI; 3Institute of
Neurobiology, Fudan University, Shanghai, China.
Purpose: Dopamine plays a critical role in the retinal dark-light
switch during the night-day transition. Yet, the way in which
dopamine release is regulated in scotopic and mesopic lighting
conditions remains unknown. Here we examine rod-mediated
responses of dopaminergic amacrine cells (DACs) at a wide range of
light intensities in order to understand the contribution of rods to the
regulation of DAC activity.
Methods: A transgenic mouse model in which DACs are
genetically labeled with red fluorescent protein was used. The cone
phototransduction pathway was genetically eliminated in this model.
Possible inputs from intrinsically photosensitive retinal ganglion cells
to DACs were blocked by TTX (Prigge and Zhang, 2015). In most
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
experiments, fluorescent labeling was visualized using a multi-photon
laser to minimize photopigment bleaching.
Results: At a holding potential of -70 mV, DACs exhibited inward
currents at light onset (white light, 1-s duration) with a threshold
light intensity of 7.5 log quanta cm-2 s-1 (near the rod sensitivity
threshold). The peak amplitude of these light-evoked currents
gradually increased as the light intensity increased and reached its
maximum value (55.8±25.6 pA, n=3) at 9.5 log quanta cm-2 s-1.
As light intensity exceeded 9.5 log quanta cm-2 s-1, peak response
amplitude remained unchanged although the responses became more
prolonged. The responses lasted for ~15 s (n=3) after light off at 13.5
log quanta cm-2 s-1. In addition, rod-mediated responses of DACs
were persistent during 120-s steady background illumination at both
lower (8.5 log quanta cm-2 s-1, n=3) and higher (11.5 log quanta cm-2
s-1, n=3) intensities. DACs also responded to flashing lights (8.5 log
quanta cm-2 s-1 at 1 Hz and 11.5 log quanta cm-2 s-1 at 0.25 Hz, n=3).
Furthermore, DACs exhibited a transient outward current at light
onset when the holding potential was at 0 mV. Strychnine (a glycine
receptor antagonist) increased the light-evoked inward current
recorded at -70 mV but attenuated the outward current measured at 0
mV, suggesting that rod-mediated responses of DACs consist of both
excitatory and inhibitory components.
Conclusions: Our results suggest that rods can mediate light-evoked
activity of DACs at a wide range of light intensities, thus potentially
triggering dopamine release in scotopic, mesopic or even photopic
lighting conditions.
Commercial Relationships: Xiwu Zhao, None; Sheng-Nan Qiao,
None; Yong-Mei Zhong, None; Kwoon Y. Wong, None;
Dao-Qi Zhang, None
Support: NIH R01 EY022640
Program Number: 2832
Presentation Time: 9:15 AM–9:30 AM
The absence of complexin 3 alters rod bipolar cell pathway
function
Joshua H. Singer1, Lena S. Mortensen4, Silvia J. Park2, Jiang-bin Ke1,
Lei Zhang1, Nils Brose4, Jeong-Seop Rhee4, Jonathan B. Demb2, 3.
1
Biology, University of Maryland, College Park, MD;
2
Ophthalmology, Yale University, New Haven, CT; 3Cellular and
Molecular Physiology, Yale University, New Haven, CT; 4AG
Synapse Physiology, Max-Planck-Institute of Experimental Medicine,
Göttingen, Germany.
Purpose: Complexins (cplx) regulate exocytosis by modulating the
SNARE complex. A retina-specific cplx isoform, cplx3, is found in
two neurons—rod bipolar (RB) and AII amacrine cells—in the circuit
mediating night vision. We investigated the role of cplx3 in scotopic
signal encoding by RB→AII synapses in the mouse retina.
Methods: Cplx3 knockout (cplx3-/-) mice and detailed methods are
published (Xue et al. 2008; Ke et al. 2014). Retinas from littermate
cplx3-/- and cplx3+/+ mice (either sex, P35-50) were used. We
assessed transmission at RB→AII synapses by paired voltage-clamp
recording of RBs and AIIs in light-adapted slices (200 µm) prepared
from all areas of the retina. We made voltage- and current-clamp
recordings from AIIs and ON α ganglion cells (GCs) in dark-adapted
whole-mount preparations of the ventral retina to assess neural
responses to light stimulation.
Results: Cplx3-/- attenuated phasic Ca2+-dependent transmission
(~3-fold reduction; P <<0.01) and moderately enhanced Ca2+independent spontaneous exocytosis (~2-fold increase; P << 0.01)
without affecting vesicle recycling and, surprisingly, coordinated
multivesicular release (MVR). We evaluated retinal circuit function
in rod-mediated vision and found that cplx3-/- slowed both the rise
and decay of responses to changes in full-field contrast at RB→AII
synapses. Consequently, contrast-evoked changes in AII VM were
slowed and reduced in amplitude, and propagation of RB output to
ON α GCs via cone bipolar (CB) cells was inhibited dramatically:
contrast-evoked excitatory currents recorded in ON α GCs were
reduced in amplitude and slowed by cplx3-/-. Control experiments
demonstrated that ON α GCs’ responses to cone stimulation in
cplx3-/- were fast and transient, similar to cplx3+/+, indicating that
transmission at ON CB→ON α GC synapses was unaffected in
cplx3-/-.
Conclusions: Inhibition of phasic release at RB→AII synapses in
cplx3-/- resulted from the absence of a facilitatory effect of cplx3
on Ca2+-dependent exocytosis rather than from a depletion of the
RRP by enhanced spontaneous release. The persistence of MVR
in cplx3-/- provided insight into underlying mechanisms. Cplx3-/prevented RB→AII synapses from encoding contrast in rapid changes
in release rate; this defect altered signaling to downstream ON α GCs.
Our study links changes in synapse and circuit function and reveals a
role for spontaneous release in the regulation of circuit gain control.
Commercial Relationships: Joshua H. Singer, None;
Lena S. Mortensen, None; Silvia J. Park, None; Jiang-bin Ke,
None; Lei Zhang, None; Nils Brose, None; Jeong-Seop Rhee,
None; Jonathan B. Demb, None
Support: NIH Grant EY107836 to JHS; NIH Grant EY021372 to
JBD and JHS; DFG Grant SFB889 to LSM, NB and JSR
Program Number: 2833
Presentation Time: 9:30 AM–9:45 AM
Calcium signaling at Cx36 gap junctions in the mouse retina
John O’Brien1, Ya-Ping Lin1, Wade Kothmann2, 3, Keith B. Moore1,
Cheryl K. Mitchell1, Jeffrey S. Diamond2. 1Ophthalmology, Univ of
Texas Houston Med Sch, Houston, TX; 2NINDS, Bethesda, MD;
3
American University, Washington, DC.
Purpose: Electrical synapses made of Cx36 are capable of
potentiation via a Ca2+-Calmodulin-CaM kinase II signaling pathway.
This has been observed to date in goldfish Mauthner neurons,
mammalian AII amacrine cells and inferior olive neurons. We
hypothesize that such signaling is widespread.
Methods: To study Ca2+ signaling we developed a Cx36-GCaMP3
fusion protein and transgenic mice expressing Cx36-GCaMP
driven by a 5 kb Cx36 promoter. We examined Ca2+ signaling by
optical imaging and functionality by tracer coupling in transfected
mammalian cells. We examined Ca2+ signals in response to glutamate
puffs in retina slice preparations from Cx36-GCaMP transgenic mice
by optical imaging.
Results: Cx36-GCaMP transfected in mammalian cells formed
junctional plaques and generated strong, transient fluorescence
increases upon addition of ionomycin. Cx36-GCaMP supported
tracer coupling in HeLa cells that was regulated by PKA activity in
the same manner as wild type Cx36. In HEK cells co-transfected
with NMDA receptor subunits NR1 plus either NR2A, NR2B or
NR2C, Cx36-GCaMP produced transient increases in fluorescence
upon perfusion of glutamate. In slices of retina from Cx36-GCaMP
transgenic mice, approximately half of fluorescent gap junctions
imaged in the inner plexiform layer responded to 2 sec puffs of
glutamate with fluorescence increases lasting 10-15 sec. Responses
varied greatly in magnitude with many individual gap junctions
responding with peak F/Fo > 3. Co-application of the NMDA
receptor antagonist CPP blocked or strongly attenuated some of those
responses, significantly reducing the average peak response.
Conclusions: Many gap junctions in the retina experience transient
Ca2+ increases dependent on NMDA receptors. Some also experience
Ca2+ increases of a different origin. This Ca2+ may regulate coupling
via CaMKII signaling.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: John O'Brien, None; Ya-Ping Lin,
None; Wade Kothmann, None; Keith B. Moore, None;
Cheryl K. Mitchell, None; Jeffrey S. Diamond, None
Support: NIH grant EY012857 (JO), Frederic B Asche endowment
(JO), NIH intramural award NS003039 (JSD)
Program Number: 2834
Presentation Time: 9:45 AM–10:00 AM
Connectomics of irradiance-encoding ON bipolar-cell inputs to
ipRGCs
Shai Sabbah1, Min Tae Kim1, Gabrielle Manoff1, Ananya Bhatia-Lin1,
Carin Papendorp1, Kevin Briggman2, David M. Berson1.
1
Neuroscience, Brown University, Providence, RI; 2National Institute
for Neurological Disorders and Stroke, National Institute of Health,
Bethesda, MD.
Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs)
are unique among ganglion cells in their capacity to encode
environmental light intensity (irradiance). They do so even at light
intensities sufficient to activate rods and cones, but too low to activate
melanopsin. Which bipolar cell (BC) types transmit irradiance data
from photoreceptors to ipRGCs and how do they do so?
Methods: We analyzed BC-to-ipRGC circuits in a serial blockface
electron-microscopic dataset (k0725) from adult mouse retina
(200x250x70 um2) stained to reveal organelles. We reconstructed
several examples of most types of ipRGCs, recognizable from their
branching patterns and levels of dendritic stratification. Ribbon
contacts onto their dendrites were identified, the presynaptic BCs
reconstructed, and other synaptic associations of these BCs mapped.
Results: We confirmed earlier reports that ipRGCs receive ectopic
en passant ribbon synapses from the shafts of ON cone BC axons
near the outer margin of the inner plexiform layer (IPL). These were
monads, usually with several ribbons each. All ipRGC types with
dendrites in this layer (M1, M3, M6) were targets of such synapses,
as were several amacrine-cell types. Overall, neurons postsynaptic
to ectopic synapses restricted their dendrites to the melanopsin
immunoreactive bands (accessory ON and inner half of conventional
ON sublayers). Thus, these ectopic synapses appear closely linked to
ipRGC networks. Type 6 ON cone BCs (n=52) were the main source
of ectopic ribbons, but Type 8 and 9 BCs also contributed (n=35).
Most Type 6, 8 and 9 BCs possessed ectopic ribbons, whereas Types
5 and 7 and rod BCs almost never did. Ectopic ribbons (n=233) had
a spatial density of ~6000/mm2. BCs with ectopic ribbons made
conventional dyad ribbon contacts in the ON sublayer of the IPL.
Postsynaptic targets of these cells (n=18) included at least four
ipRGC types (M2, M3, M5, M6); M3 and M6 cells received both
ectopic and conventional ribbon inputs. However, neurons other than
ipRGCs must receive input from these BCs because some stratified in
the mid-IPL, which ipRGCs never do.
Conclusions: ipRGCs get input from three cone BC types (6, 8 and
9) through mixed conventional and ectopic ribbon synapses; the
ectopic pathway may be unique to ipRGCs. Thus, both specific BCs
and specialized synapses underlie the irradiance-encoding synaptic
light responses of ipRGCs.
Commercial Relationships: Shai Sabbah, None; Min Tae Kim,
None; Gabrielle Manoff, None; Ananya Bhatia-Lin,
None; Carin Papendorp, None; Kevin Briggman, None;
David M. Berson, None
Support: NIH R01 EY12793
Program Number: 2835
Presentation Time: 10:00 AM–10:15 AM
Postnatal development of rod bipolar cell dendrites in mouse
retina
Ivan Anastassov, Felice Dunn. Ophthalmology, University of
California San Francisco, San Francisco, CA.
Purpose: Rod bipolar cells (RBC) receive glutamate from rods
through fine dendritic tips, which contain molecular machinery
necessary to convey visual information. Establishment of
convergence between rods and RBCs, and localization of synaptic
proteins are critical to correct function. However, the development
of the fine dendritic trees and the temporal expression patterns of
dendritic transduction proteins have not been examined on the level
of individual RBCs. Thus, it is unknown how dendritic branches
develop the appropriate number of rod contacts.
Methods: Retinas were taken from a Grm6-TdTomato transgenic
mouse line (Kerschensteiner et al. 2009) at different stages of
postnatal development, ranging from P7 to P82. In these animals,
tdTomato is expressed under the control of the Grm6 promoter
in a subset of ON bipolar cells, allowing for easy morphological
identification of RBCs. Isolated retinas were flat-mounted, fixed,
and stained with antibodies against pre- and post-synaptic proteins
(Ribeye, mGluR6, TRPM1). The tissue was imaged by confocal
microscopy and RBC dendrites and somas were masked using Amira.
The number of RBC dendritic tips, the expression of post-synaptic
proteins within the tips of individual RBCs, and the expression of
pre-synaptic markers were examined as a function of age.
Results: At postnatal day (P)7, RBCs in the periphery have immature
neurites while centrally located RBCs have ~12 dendritic tips (SD
1.73). mGluR6 staining at P7 is diffuse throughout the soma and
processes and does not appear punctate at the tips. At P13-14,
neurites in the periphery are gone and the average number of RBC
dendritic tips increases to ~24 (SD 7.76); mGluR6 staining starts
to appear more punctate in the dendritic tips. At P21-30, dendritic
tip number increases to ~31 (SD 5.28; SD 5.94), and mGluR6
expression is limited to the tips of dendritic processes. At P69-70, tip
number peaks at ~37 (SD 4.91; SD 2.91) and reaches a plateau. Each
dendritic tip contains punctate mGluR6.
Conclusions: The number of dendritic tips increases with age and
begins to reach an asymptote after P21. Punctate expression of
mGluR6 lags several days behind tip differentiation, which starts at
P7. Previous studies show dendritic processes of RBCs invaginating
into rod terminals starting at P10, which could coincide with punctate
mGluR6 expression in RBC tips. Studies on TRPM1 and Ribeye
expression during these developmental stages are ongoing.
Commercial Relationships: Ivan Anastassov, None; Felice Dunn,
None
Support: NIH Grant EY024815
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.