Unidirectional Photoreceptor-to-Mu ¨ller Glia Coupling
and Unique K+Channel Expression in Caiman Retina
Astrid Zayas-Santiago1., Silke Agte2,3., Yomarie Rivera1, Jan Benedikt1, Elke Ulbricht2,5, Anett Karl2,
Jose ´ Da ´vila1, Alexey Savvinov4, Yuriy Kucheryavykh1, Mikhail Inyushin1, Luis A. Cubano1,
Thomas Pannicke2, Ru ¨diger W. Veh6, Mike Francke2,7, Alexei Verkhratsky8, Misty J. Eaton1,
Andreas Reichenbach2, Serguei N. Skatchkov1*
1Departments of Pathology, Biochemistry and Physiology, Universidad Central Del Caribe, Bayamo ´n, Puerto Rico, United States of America, 2Paul Flechsig Institute of
Brain Research, Faculty of Medicine, University of Leipzig, Leipzig, Germany, 3Division of Soft Matter Physics, Department of Physics, University of Leipzig, Leipzig,
Germany, 4Department of Physical Sciences, Universidad de Puerto Rico, Recinto de Rı ´o Piedras, Rı ´o Piedras, Puerto Rico, United States of America, 5Department of
Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom, 6Charite ´, Berlin, Germany, 7Translational Centre for Regenerative
Medicine (TRM) University of Leipzig, Leipzig, Germany, 8Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Background: Mu ¨ller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function
of neuronal cells. In most vertebrates, including humans, Mu ¨ller cells abundantly express Kir4.1 inwardly rectifying
potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium
buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and
function of potassium channels in Mu ¨ller cells from the retina of crocodiles remain, hitherto, unknown.
Methods: We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus), endowed with both diurnal and
nocturnal vision, by (i) immunohistochemistry, (ii) whole-cell voltage-clamp, and (iii) fluorescent dye tracing to investigate K+
channel distribution and glia-to-neuron communications.
Results: Immunohistochemistry revealed that caiman Mu ¨ller cells, similarly to other vertebrates, express vimentin, GFAP,
S100b, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Mu ¨ller cells but was localized in
photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Mu ¨ller cells. Electrophysiological properties of
enzymatically dissociated Mu ¨ller cells without photoreceptors and isolated Mu ¨ller cells with adhering photoreceptors were
significantly different. This suggests ion coupling between Mu ¨ller cells and photoreceptors in the caiman retina.
Sulforhodamine-B injected into cones permeated to adhering Mu ¨ller cells thus revealing a uni-directional dye coupling.
Conclusion: Our data indicate that caiman Mu ¨ller glial cells are unique among vertebrates studied so far by predominantly
expressing TASK-1 rather than Kir4.1 K+channels and by bi-directional ion and uni-directional dye coupling to
photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type
Citation: Zayas-Santiago A, Agte S, Rivera Y, Benedikt J, Ulbricht E, et al. (2014) Unidirectional Photoreceptor-to-Mu ¨ller Glia Coupling and Unique K+Channel
Expression in Caiman Retina. PLoS ONE 9(5): e97155. doi:10.1371/journal.pone.0097155
Editor: Alvaro Rendon, Institut de la Vision, France
Received February 13, 2014; Accepted April 15, 2014; Published May 15, 2014
Copyright: ? 2014 Zayas-Santiago et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This publication was made possible by grants from the National Institutes of Health: R01- NS065201-02 03 from NINDS (to SNS); Title-V PPOHA, RCMI-
SRDU from NIMHD (to LC and AZ); SC1-GM 088019-02 from NIGMS (to MJE); SC2 GM095410 from NIGMS (to YK); G12-RR03035-Project-A from NIMHD (to MI);
8G12-MD007583-27 G12-RR03035 from NIMHD (for core facilities at UCC); FIPI-NSF-UPR (to AS); From the Deutsche Forschungsgemeinschaft: FOR 748, GRK 1097,
RE 849/16-1 and SPP 1172(to AR); PA 615/2-1 (to TP). From the German Federal Ministry of Education and Research: BMBF 1315883 (to MF), From the European
Community: FP7- SPP 1172 (to AR); and EU FP 7 Program EduGlia 237956 (to AR and AV). The funding agencies had no role in study design, data collection and
analysis, decision to publish,or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Mu ¨ller glial cells  serve numerous fundamental functions in
the retina of vertebrates; many of these functions depend on
potassium channels, responsible for a high potassium conductance
of the cell membrane [2,3,4]. Although the electrophysiological
membrane properties, as well as the main functions, of Mu ¨ller cells
are similar among the vertebrates, distinct inter-specific differences
have been observed even between closely related mammals such as
monkeys and humans . To further investigate Mu ¨ller cells
functional diversity, possibly reflecting adaptations to specific
retinal circuits, it is desirable to study Mu ¨ller cells from different
groups of vertebrates. A wide variety of mammalian Mu ¨ller cells
have been investigated (e.g., ); as well as fishes (elasmobranchs
and teleosts: [7,8,9] and amphibians (salamanders and anurans:
[9,10,11,12]. In reptilians, however, only Mu ¨ller cells from the
PLOS ONE | www.plosone.org1May 2014 | Volume 9 | Issue 5 | e97155
diurnal water turtle, Pseudemys scripta elegans, were characterized
(e.g., [13,14,15,16]). Here we report a study of Mu ¨ller cells from
retinae of caiman (Caiman crocodilus fuscus), which has perfect night
vision as well as vision in the bright daylight, with a large scale of
adaptation to different light intensities. This ability is reflected by
several morphological and functional idiosyncrasies in the caiman
vision system . Incidentally, crocodiles are closer related to
birds (in which Mu ¨ller cells were never studied electrophysiolog-
ically) than to the turtles (e.g., , and references therein) which
makes the caiman an even more interesting subject of examina-
Radially oriented Mu ¨ller cells span the whole thickness of the
retina and conduct light to photoreceptors . These cells
contact all neuronal elements located within the retina. Spatial
buffering of extracellular K+ions represents another most
fundamental and extensively studied function of the Mu ¨ller cell.
In dark adapted retina, cells face large K+gradients, with K+
concentrations ranging between 6–8 mM at the photoreceptor
layer (i.e., at the distal part of Mu ¨ller cell) and 2–3 mM at the
vitreal surface where (i) Mu ¨ller cell endfeet abut the vitreous body
and (ii) complex ionic changes occur during light stimulation
[20,21,22,23]. Specific spatial distribution of K+channels 
allow Mu ¨ller cells to redistribute K+ions from sites of high
extracellular concentration to ‘buffering reservoirs’ such as the
vitreous fluid or the intraretinal blood vessels, and thus prevent
elevations of extracellular K+that may cause over-excitation of
neurons with subsequent loss of information processing.
In the Mu ¨ller cells and astrocytes of humans and of most
animals studied, inwardly rectifying K+(Kir) channels, specifically
Kir4.1 (Kcnj10), play a key role for glia-neuron interactions (for
recent reviews, see [3,25,26,27]), being fundamental for example
for glutamate clearance [28,29]. Genetic variations of Kir4.1
channels in humans and animals underlie severe disorders in the
brain and in the retina, such as epilepsy, disruption of
electroretinogram, glaucoma, stroke, ataxia, hypokalemia, hypo-
magnesemia, and metabolic alkalosis [27,30,31,32,33]. In addi-
tion, recently identified Kir4.1 mutations were found to result in
autoimmune inhibition, contributing to pathogenesis of multiple
sclerosis , hearing loss , autism  and seizures [30,33].
The mutated Kir4.1 protein is not inserted in the membrane or
the channels are blocked, as revealed by the absence of
representative potassium currents . A loss of Kir4.1 channels
function was also found in the retina and in the brain in diabetes
, transient ischemia , and in trauma ; with deficit in
Kir4.1 mediated permeability being linked to failures in neuronal
function and neuronal cell death .
In Mu ¨ller glia, Kir4.1 channels operate in concert with other
types of K+channels, which include (i) ATP-sensitive potassium
channels (K(ATP)) assembled from Kir6.1 and SUR1 subunits
[41,42,43,44,45], (ii) Kir2.1 inwardly rectifying channels [3,24]
and (iii) two pore domain (2P) potassium channels of the TASK
family [43,46,47]. Among these channels the Kir2.x family
displays strong inward rectification, and thus cannot mediate the
K+outward currents essential for spatial buffering . The ATP-
sensitive Kir6.x family, on the other hand, requires two
preconditions to function [48,49]. First, a functional K(ATP)
channel can only be assembled from two structurally diverse
proteins: the Kir6.x (Kir6.1 or Kir6.2) subunits and a sulfonylurea
receptor (SUR) of the ATP-binding cassette superfamily (SUR1,
SUR2A or SUR2B) [50,51,52]. Second, Kir6.1/SUR1 channels
are inactivated at physiological ATP concentrations ; rather, a
depletion of ATP is necessary to open these channels [53,54],
unless phospholipids such as PIP3, PIP2, PIP and PI partially
remove the ATP block [54,55]. In contrast, Kir4.1 channels are
functional under normal conditions, and ATP is even necessary for
their opening . Finally, the TASK (KCNK family) channels
contribute only minutely to the whole-cell currents recorded from
frog and mammalian Mu ¨ller cells [3,47]. Thus, it is difficult to
contemplate how these K+channels may maintain the functions of
Mu ¨ller cells in the absence of Kir4.1 channels.
Surprisingly in the present study we found that Kir4.1 channels
are absent in caiman Mu ¨ller glial cells, which, however, express
TASK channels. Furthermore, we observed a unique communi-
cation between photoreceptors and attached Mu ¨ller cells, with uni-
directional permeation of the fluorescent dye, sulforhodamine,
from cones to Mu ¨ller glial cells. We hypothesize that in the caiman
retina, spatial K+buffering may involve both Mu ¨ller cells and
photoreceptors via trans-cellular K+fluxes. Preliminary results
were reported in abstract form [57,58].
Materials and Methods
Experiments were carried out with IACUC approval and in
accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research and according to institutional
animal care and use guidelines. Adequate measures were taken to
minimize pain or discomfort to experimental animals. Mu ¨ller cells
were isolated from retinae of adult caiman (Caiman crocodilus fuscus)
as previously described [12,45,47].
Retinal sections from 8 caimans were used for the immunohis-
tochemical studies. Eyes were perfusion-fixed in situ. Caimans (with
a length of 50 cm to 120 cm on average) were immobilized on ice
before being anesthetized. Animals were subsequently i.p.
anesthetized with tiletamine HCl/Zolazepam HCl, 5 mg/kg
before intraventricular perfusion of fixatives. The thorax was
opened and a catheter was placed in the left ventricular chamber
of the heart for vascular perfusion. Perfusion for immunohisto-
chemistry contained 4% paraformaldehyde (as the only fixative) in
phosphate buffered solution (PBS): NaCl 136.9 mM, KCl
2.7 mM, Na2HPO4 10.1 mM, KH2PO4 1.8 mM with pH 7.4
for 30 minutes. The right atrium was cut to allow outflow of
perfusate. After perfusion, eyes were enucleated and pieces (0.5 x
0.5 mm) of the retina were cut and rinsed in fresh PBS.
For agar-embedding we used the isolated retinal pieces
embedded in 3% agarose (w/v) in PBS as previously described
[39,47] and agar cubes with tissue inside were cut by 80 mm
sections. For cryo-embedding, after fixative perfusion the tissues
were cryoprotected by immersion in 0.15 M sucrose in 0.1 M
phosphate buffer, pH 7.4 (for 24 hrs), 0.5 M sucrose (for 24 hrs)
and 0.8 M sucrose (for 48 hrs) and subsequently frozen at 260uC
in liquid pentane and then stored in a 280uC freezer until next
use. Cryostat sections of 25 mm cut using a vibratome (Leica
VT1000S, Leica, Germany) were used.
For immunofluorescence analysis, retinal sections were incu-
bated in 5% normal serum with 0.3% Triton X-100 and 1%
dimethyl sulfoxide (DMSO) in PBS for 1 hr and subsequently
overnight at 4uC with primary antibodies. After the washing of
retinal slices from primary antibody in 1% bovine serum albumin
in saline, the secondary antibodies were applied for 4 hrs at room
temperature. Texas Red-conjugated donkey anti-goat IgG (1:200),
carbocyanin Cy5-coupled donkey anti-rabbit IgG (1:200), Cy3-
coupled goat anti-rabbit IgG (1:200), Cy5-coupled goat anti-
mouse IgG (1:200), Cy2-coupled donkey anti-rabbit IgG (1:200),
and Cy2-coupled goat anti-rabbit IgG (1:500; Dianova, Hamburg,
Germany) were used. Controls were obtained by omitting the
Unique K+ Channel Expression in Caiman Retina
PLOS ONE | www.plosone.org2May 2014 | Volume 9 | Issue 5 | e97155
primary antibody. The images were obtained using confocal
scanning microscopes (LSM 510 META, Zeiss, Oberkochen,
Germany and Olympus Fluoview FV1200, Olympus, Japan).
We used the following primary antibodies: (i) for glial markers:
mouse anti-S100b (1:1000; Sigma-Aldrich, S2532), mouse anti-
glial fibrillary acidic protein (GFAP; 1:300; Sigma-Aldrich,
G6171), mouse anti-glutamine synthetase (GS; 1:500; Millipore,
MAB302), mouse anti-vimentin (1:300; PROGEN Biotechnik
GmbH, Heidelberg, Germany, VIM 3B4), (ii) for channel
proteins: rabbit anti-TASK-1 (1:200; Santa Cruz Biotechnology,
SC-28635), rabbit anti TREK-2 (1:500; Alomone), rabbit anti
TASK-3 (1:300; Alomone), mouse anti-Kir4.1 (1:300; Sigma-
Aldrich, WH0003766M1), rabbit anti-Kir4.1 (1:200; Alomone,
APC-035), rabbit anti-Kir6.1 (1:200; Alomone, APC-105), rabbit
anti-Kir6.1, -Kir6.2, and -SUR1 (1:200; 1:150; 1:200 respectively;
R.W. Veh, Charite ´, Berlin), rabbit anti-aquaporin-4 (AQP4;
1:200; Sigma-Aldrich, A5971), rabbit anti-aquaporin-4 (AQP4;
1:200; Santa Cruz Biotechnology, SC-20812) and (iii) rabbit anti-
Gat1(Garod transducin; 1:200; Santa Cruz Biotechnology, SC-
389) for rod photoreceptor labeling.
For the detection of the gap junction protein connexin 43
(Cx43) we used rabbit anti-Cx43 (1:100; Sigma-Aldrich, C6219),
whereas for labelling glutamine synthetase mouse anti-glutamine
synthetase (1:1000; Millipore, MAB302) followed by the secondary
antibodies Cy3-conjugated donkey anti-rabbit IgG (1:200) and
Cy5-conjugated donkey anti-mouse IgG (1:200). To visualize the
cone photoreceptors, i.e., their segments and their synaptic
pedicles, we used the biotin-conjugated lectin peanut agglutinin
(1:200; PNA, Sigma-Aldrich, Germany) which specifically binds to
cone photoreceptor cells . During the incubation with the
secondary antibodies, streptavidin labelled with Cy2 (1:200;
Sigma-Aldrich) was applied to bind to PNA. The cell nuclei were
stained with Hoechst 33258.
In summary, the above described retinal markers were
visualized using either the DAB procedure with nickel contrast
(Kir4.1, Kir6.1, Kir6.2, SUR1, TASK-1, TASK-3, TREK-2) or a
fluorescence method for laser scanning confocal microscopy
(Kir4.1, GFAP, Vimentin, S100b, GS, AQP4, DAPI, Hoechst,
PNA, Gat1and Cx43). The sections were processed for immuno-
histochemical detection as previously described [44,46,47] or for
Kir4.1, Kir6.1 and Kir6.2 channels [41,45,46,60,61].
Electrophysiology in retinal wholemounts
Caimans were immobilized on ice and dark adapted for 1 hr
before being anesthetized as described above. Anesthetized
caimans were sacrificed by decapitation followed by removal of
eyes. Retinae isolated from eyes were treated with a mix of
collagenase/dispase (2 mg/mL) and DNase-1 (0.1 mg/mL) in
PBS (pH 7.4) for 30 min at room temperature. After treatment,
retinae were washed in PBS and placed in a recording chamber for
electrophysiological recording of the vitreal endfeet of Mu ¨ller cells.
The extracellular solution (ECS) to perfuse retinal tissue contained
(in mM): NaCl 110, CaCl22, MgCl21, NaH2PO41.25, NaHCO3
25, D-glucose 25, KCl varied from 2.5 to 10 mM (substituted by
NaCl to adjust osmolarity); pH 7.4, after aeration by carbogen
(95% O2+5% CO2). Electrodes were filled with intracellular
solution (ICS) containing (in mM): K-gluconate 130, Na-gluconate
10, NaCl 4, HEPES 10, MgATP 4, phosphocreatine 4, NaGTP
0.3, pH adjusted to 7.2 with KOH/HCl. Spermine (0.25 mM;
Sigma-Aldrich) was added to ICS in accordance with the finding
that free spermine levels in Mu ¨ller cells are in the submillimolar
concentration range . After filling with ICS, the micropipette
resistance was ,8 MV. Voltage and current clamp recordings in
whole-cell patch-clamp mode were performed using a MultiClamp
700A patch-clamp amplifier with a DigiData 1322A interface
(Molecular Devices, Inc., Sunnyvale, CA, USA). The cells were
kept at holding potential equal to equilibrium resting potential (to
keep membrane current at zero level) and the cells were stimulated
by a step to 2150 mV (for 120 ms) with following rising voltage
ramp to +150 mV during 80 ms and then a step back to resting
voltage. The pClamp 10 software package (Molecular Devices,
Inc., CA) was used for data acquisition and analysis.
Note: retinal tissue stored for longer than 1 hr was not used. To
study the coupling between Mu ¨ller cells in retinal wholemounts,
the fluorescent dye Lucifer Yellow (LY) 2 mM (Sigma-Aldrich)
was added to the ICS as in .
Electrophysiology in isolated cells
Retinae were cut into pieces (0.5 x 0.5 mm) and rinsed in fresh
309 mOsm which corresponds to the osmolarity measured for
vitreal liquid sampled from caiman eyes), then were transferred
into Ca2+-Mg2+-free PBS containing papain (24 unit/ml) for
30 min, at 37uC. After washing and trituration in PBS and then in
Dulbecco’s Modified Eagle medium (DMEM), isolated cells were
briefly rinsed in DMEM containing 0.001 mg/ml DNase-1
(Sigma-Aldrich, D-4263, from bovine pancreas). The cells were
then washed in DNase-free DMEM medium, stored for 10 min on
ice for sedimentation; then the supernatant was exchanged for
fresh DMEM and the cells stored on ice. This procedure yielded
Mu ¨ller cells with characteristic morphology and preserved fine
processes. We investigated two types of isolated cells: single Mu ¨ller
cells (SM) and Mu ¨ller cells with attached photoreceptors (SMP).
Cells were placed in the recording chamber, allowed to settle on
the bottom of the chamber mounted on the stage of an inverted
microscope (Nikon DIAPHOT 300, Nikon, Japan) with attached
three-axis water hydraulic fine micromanipulator (MHW-3,
Narishige, Japan) or an upright microscope (BX51WI, Olympus,
Japan) with piezoelectric micromanipulators (MX7500 with MC-
1000 drive, Siskiyou, Inc., Grants Pass, OR) used for positioning
micropipettes during voltage-clamp and current-clamp recordings.
The electrophysiological recordings were performed at room
temperature. Electrodes for whole cell recording were pulled in
four steps (using Sutter P-97 puller, USA) from hard glass (GC-
150-10 glass tubing, Clark Electromedical Instruments, England).
Electrodes were filled with intracellular solution (ICS) containing
(in mM): KCl 130, MgCl21, CaCl21, EGTA 10, HEPES 10,
Na2ATP 3, spermine HCl 0.25, pH adjusted to 7.2 with KOH/
HCl. They had resistances of 4-6 MOhm; after cell penetration,
the access resistance was 10–15 MOhm, compensated by at least
75%. The extracellular solution (ECS) contained (in mM): NaCl
145, CaCl22.5, MgCl22, and HEPES 10; KCl varied from 2.5 to
10 mM (substituted by NaCl to adjust osmolarity). For recording
from single isolated cells we used an Axopatch-200B amplifier with
a CV-203BU headstage and a MultiClamp 700A amplifier with
CV-7A headstage. DigiData 1200A and DigiData 1322A inter-
faces were used respectively for data acquisition and analysis
(Axon Instruments, Molecular Devices, USA). High frequencies .
1 kHz were cut off and signals were digitized at 5 kHz. The
pCLAMP-9 (Axon Instrument, USA) and pCLAMP-10 (Molec-
ular Devices, USA) software packages were used for data
acquisition and analysis.
To isolate conductances mediated by different potassium
channels we used established pharmacological procedures: chan-
nels of TASK family were blocked by bupivacaine 0.1–1 mM
[47,64], whereas Kir channels were blocked by 0.1–0.3 mM of
barium [12,65,66]. ATP (3 mM) was used in the ICS to inhibit Kir
6.1/SUR1 (K(ATP)) channels . Spermine (250 mM) was added
(osmolarity wasadjusted to
Unique K+ Channel Expression in Caiman Retina
PLOS ONE | www.plosone.org3May 2014 | Volume 9 | Issue 5 | e97155
to the ICS to block the outward component of Kir channels
[67,68]. Addition of spermine in the ICS also helped to separate
TASK-mediated currents from Kir currents . At the
concentrations used, barium has little effect on TASK channel
currents [69,70,71]. The patched cells were kept at holding
potential equal to equilibrium resting potential and then a step to
2150 mV (for 10 ms) with following rising voltage ramp to +
150 mV (for 16 ms) and back to resting voltage. To ensure the
ionic nature of whole cell currents, we performed standard control
experiments where KCl was substituted with CsCl in the ICS and
in ECS; under these conditions, no currents were recorded (not
shown). Inhibitors of Kir and 2P-domain channels, barium
chloride and bupivacaine were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA and Taufkirchen, Germany).
Fluorescent tracer diffusion studies
We analyzed the diffusion of the gap-junction channel-
permeable dyes LY (see above) and sulforhodamine-B, dialyzed
in whole-cell configuration in current clamp (zero current) mode.
The patch pipettes were filled with ICS described above and
containing 2 mM of LY or sulforhodamine-B. LY (Sigma-Aldrich,
MW 457.25), is a green- to yellow-fluorescent negatively charged (-
2 charges) dye, while sulforhodamine-B (Molecular Probes, MW
558.66) is an orange- to red-fluorescent non-charged water-soluble
(polar) sulfonic acid tracer with strong absorption and good
Data were analyzed using pCLAMP-10 (Molecular Devices,
USA), Origin 8 software (OriginLab, Northampton, MA, USA)
and are reported as mean 6 standard error of the mean.
Significant differences between groups of data were evaluated
using Student’s t-test.
Glial markers in caiman Mu ¨ller cells
First, we analyzed the presence of the established glial marker
proteins, S100b (glial specific calcium binding protein), GS
(glutamine synthetase), vimentin, and GFAP (glial fibrillary acidic
protein) in caiman retinal Mu ¨ller cells. In addition, we examined
the localization of the alpha subunit of G-protein transducin
(Gat1), a G-protein involved in phototransduction, in caiman
photoreceptor cells. Immunoreactivity for all four glial proteins
was identified in caiman Mu ¨ller cells, and Gat1 was detected in
caiman photoreceptor cells (Figs. 1, 2).
We found a robust expression of GFAP in all Mu ¨ller cells, in
somata, in processes and even in very fine distal branches (Fig. 1A).
S100b (Fig. 1B) and vimentin (Fig. 2) were expressed throughout
the length of all Mu ¨ller cells, in all retinal areas. Prominent S100b
and vimentin staining was observed in somata, distal processes and
endfeet. Similarly, GS immunoreactivity was observed in all
Mu ¨ller cell compartments including somata and distal processes
(Fig. 1C). In many vertebrate species, strong GFAP expression is
only observed in cases of reactive gliosis whereas normal Mu ¨ller
cells are devoid of GFAP immunolabeling (e.g., ). However,
exceptions have been described; for instance, in the retina of
goldfish  and horse , the Mu ¨ller cells are GFAP
immunopositive under normal conditions. Thus, the GFAP
expression of caiman Mu ¨ller cells is suggested here to represent
a species-specific feature, rather than a pathological event.
It has been previously demonstrated that the water channel
AQP4 is mainly localized in the endfeet of Mu ¨ller cells within the
rat retina . Using different primary antibodies, AQP4 staining
was found basically at the inner limiting membrane which abuts to
the vitreal Mu ¨ller cell endfeet (figure S1).
Expression of Kir4.1, Kir6.1, Kir6.2 and SUR1
In all vertebrate species studied so far, Kir4.1 channels were
found to be robustly expressed by Mu ¨ller cell endfeet at the inner
limiting membrane, and in ‘en-passant endfeet’ around blood
vessels [3,24,44,77,78,79,80]. Most strikingly, Kir4.1 immunolabel
was not located to caiman Mu ¨ller cells (Fig. 2A). Rather, the
channel protein was located to photoreceptor cells (Fig. 2A) similar
as Gat1 (Fig. 2B). Using different antibodies for Kir4.1 (and DAB
versus fluorescent techniques) we found similar localization of
Kir4.1 protein (DAB images not shown). Thus, immunoreactivity
for Kir4.1 was co-localized with the photoreceptor marker, Gat1
Immunoreactivity for Kir6.1 was found in caiman Mu ¨ller cells
(Fig. 2C) whereas Kir6.2 was located to neurons (not shown). This
differential localization has also been observed in amphibians and
in guinea pig [43,44,45]. However, it has been shown that Kir6.1
expression alone is not sufficient to mediate K+currents; the
sulfonylurea receptor subunit SUR1 is necessary as a partner of
Kir6.1 to form functional K(ATP) channels in Mu ¨ller glial cells
[42,45], in other glial cells  and in non-glial cells . The
SUR1 immunolabelling was not detected in caiman Mu ¨ller cells
(figure S2). Together, these data imply that, unlike in other
vertebrate species studied so far, in caiman Mu ¨ller cells the
inwardly rectifying K+channels, Kir6.1 and Kir4.1, are not
available to mediate K+currents, and to maintain the membrane
potential of the cells, therefore, we studied other K+-channel
Localization of two pore domain K+(2P) channels TASK-1,
TASK-3, and TREK-2
To examine the putative presence of other potassium channels
necessary to maintain the membrane potential of the cells  we
used antibodies against three 2P-domain channels (TASK-1,
TASK-3, TREK-2). Both TASK-1 and TASK-3 immunolabelling
was detected in Mu ¨ller cells, albeit with different intensity.
Immunoreactivity for TASK-1 was strong; it was localized
throughout the length of Mu ¨ller cells, in endfeet, inner stem
processes, somata, and distal processes (Fig. 2D); in contrast
TASK-3 was rather weakly expressed only in distal processes of
Mu ¨ller cells (unpublished data). This pattern of TASK-1
expression throughout the Mu ¨ller cells corresponds to what was
reported for amphibian  and mammalian retinal Mu ¨ller glia
. TREK-2 immunoreactivity was not detectable in caiman
Mu ¨ller cells (not shown). In summary, TASK-1 appears to be the
dominant K+channel type in caiman Mu ¨ller cells. Further
electrophysiological recordings were made to prove this assump-
Electrophysiological recordings from Mu ¨ller cell endfeet
in retinal wholemounts
In wholemount preparations of the caiman retina (Fig. 3A),
Mu ¨ller cells were patched at their endfeet at the top of the retina
with photoreceptor side down. The average membrane potential
(n =23 cells) was 269.861.3 mV. Membrane potential was
highly sensitive to extracellular K+, with an average depolarization
of 26.562.0 mV (n=15) in response to [K+]o changes from
2.5 mM to 10 mM, close to the Nernstian shift for K+(,29 mV)
(Fig. 3B), whereas the current-voltage curves in 2.5 mM as well as
10 mM [K+]oshowed linear properties.
Unique K+ Channel Expression in Caiman Retina
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Neither barium (Ba2+), an efficient Kir channel blocker
[12,16,65,66], nor bupivacaine (a 2P-domain channel blocker,
specific for TASK-1: [47,64]) had a strong effect on glial
membrane potential in the wholemount preparations; the depo-
larization caused by barium was 0.7360.14 mV (n=7) and the
depolarization by bupivacaine was 0.5660.32 mV (n=7) (not
Mu ¨ller cells in the wholemount preparations can retain inter-
cellular coupling thus affecting the space clamp . To access the
coupling in the wholemount preparation we injected the
fluorescent dye, Lucifer Yellow, into the endfeet of the patch-
clamped cells. After removing the pipette the dye was found also in
adjacent Mu ¨ller cells (Fig. 3C, D), indicating cell-cell coupling.
This coupling was not extensive, as in most of the cases only one
additional cell was labeled; a similar weak coupling has been
described to occur between rabbit retinal Mu ¨ller cells . When
the gap junction blocker carbenoxolone (200 mM) was applied, the
dye remained in the injected cell (Fig. 3E) and no dye-coupling was
observed. Together, these data show that whole-cell currents
cannot be reliably recorded from the cells in the wholemount
Figure 1. Expression of retinal glial and photoreceptor markers in the caiman retina. Immunostaining for glial fibrillary acidic protein
(GFAP), glial specific calcium binding protein (S100b), glutamate-to-glutamine converting enzyme glutamine synthetase (GS), and DAPI (blue) which
shows nuclei of retinal cells. (A) Confocal (left) and Nomarski (right) images showing the expression of GFAP, (B) S100b, and (C) glutamine synthetase.
The markers delineated the overall structure of Mu ¨ller cells: white arrowheads point to the inner limiting membranes (ILM) in the vitreal endfeet
areas. White arrows point to stalks of Mu ¨ller cells. Blue arrowheads show nuclei of pigment epithelium cells. Scale bar =20 mm. ILM-inner limiting
membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, ONL-outer nuclear layer, OPL-outer plexiform layer, IS-inner
segments of photoreceptors, OS-outer segments of photoreceptors, RPL-retinal pigment epithelium layer.
Figure 2. Differential localization of potassium channels and glial markers in the caiman retina. (A) Inwardly rectifying potassium
channels Kir4.1 (green) are localized in the area of the photoreceptors and in outer nuclear layer (red arrowhead), but not in Mu ¨ller cell processes such
as stalks or endfeet. This is different from most of vertebrates where Kir4.1 channels were found in Mu ¨ller cells . (B) Glial Mu ¨ller cell specific marker
vimentin, V (green); photoreceptor specific alpha-1 subunit of transducin, Gat1 (red); nucleus-specific marker, DAPI (blue). Vimentin staining is
observed in endfeet, somata, stalks and in distal processes (yellow arrowhead points somata, white arrow shows stalk). DAPI staining (blue) shows
nuclei of retinal cells. Alpha-1 subunit of transducin (Gat1, red) is a marker of the second messenger G-protein cascade and it is found mostly in
photoreceptors. (C) ATP-dependent K+channel, Kir6.1 (black), is localized in stalks (white arrow) and in endfeet, while (D) two pore domain acid
sensitive K+channels, TASK-1 (black) are found in whole Mu ¨ller cell compartments: in endfeet (black arrowhead), stalks (white arrow), somata (yellow
arrowhead) and in distal processes (white arrowhead). Scale bar =20 mm. ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform
layer, INL-inner nuclear layer, ONL-outer nuclear layer, IS- inner segments of photoreceptors.
Unique K+ Channel Expression in Caiman Retina
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condition. To overcome this problem, we studied enzymatically
dissociated single cells.
Electrophysiological recordings from isolated caiman
Mu ¨ller cells
Enzymatic dissociation yielded either completely or incomplete-
ly isolated (Fig. 4A, B, C) Mu ¨ller cells. In the latter case, small
numbers of cones (Fig. 4A) or rods (Fig. 4B) remained attached to
a Mu ¨ller cell. Whole-cell patch clamp recordings were made from
completely isolated Mu ¨ller cells (SM) as well as from Mu ¨ller cells
with adhering photoreceptor cells (SMP). The patch pipette was
placed at the soma of the Mu ¨ller cell if not stated otherwise.
Resting membrane potential in isolated Mu ¨ller cells was more
hyperpolarized than when recorded from endfeet in the retinal
wholemounts. The average membrane potential recorded from
SM in control ECS (K+2.5 mM) was 282.5061.93 mV (n=8)
and SMP cells showed 284.2062.03 mV (n=10). Under control
conditions membrane potentials were very similar between the SM
and SMPs (Fig. 4). Thus, the measured resting membrane
potentials were close to the ideal (Nernstian) K+equilibrium
potential, similar as reported previously from fish, amphibian,
reptilian and mammalian Mu ¨ller cells [3,12,16,65,66,83,84]. In
addition, we tested cell responses to elevation of extracellular K+
from 2.5 mM to 10 mM; SM depolarized from 282.5060.31 mV
(control) to 254.7561.11 mV (high K+; n=8), (Fig. 4G). Similar
depolarizations occurred in SMP, from 284.2062.03 mV (con-
trol) to 257.9061.46 mV (n=10). If the Mu ¨ller cells were
patched at their endfeet, nearly identical values were measured.
Under bupivacaine, SM cells were depolarized from 2
79.8662.27 mV to 257.5064.72 mV (n=8), whereas SMP cells
were depolarized significantly less, from 284.2062.03 mV to 2
71.0063.76 mV (n=10) (Fig. 4H). This difference in sensitivity to
the TASK-1 blocker between SM and SMP suggests that the
attached photoreceptors (Fig. 4A, B) may modify the Mu ¨ller cell
electrophysiology via cell-cell coupling. Bupivacaine (1 mM)
Figure 3. Mu ¨ller cells in retinal wholemounts. (A) Recordings from vitreal endfeet of Mu ¨ller cells. A patch pipette (MP) at the vitreal surface
penetrating a single endfoot while many round- shaped endfeet with different diameters are visible. The patch clamp technique was used for
recording the electrical currents and voltages as well as for fluorescent dye injection. The insert shows the differential interference contrast (DIC)
image of a retinal axial section with a patch pipette positioned at the endfoot membrane. (B) Representative currents from caiman Mu ¨ller cell endfeet
in response to a voltage ramp of 80 ms duration from 2150 mV to +150 mV (I/V-curves). The curves show the I/V relationship in different
extracellular [K+], 2.5 mM and 10 mM, and the washout in 2.5 mM demonstrating classical glial sensitivity to potassium. (C) and (D) Two
representative examples for Lucifer Yellow diffusion from injected endfoot (white arrowhead) to neighbor endfoot (white arrow) under control
conditions. (E) Lucifer Yellow injection into a single endfoot in a retinal wholemount perfused with the gap junction blocker carbenoxolone (200 mM).
The dye is not propagating to the neighboring cells.
Unique K+ Channel Expression in Caiman Retina
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Figure 4. Cellular tandem between Mu ¨ller cells and photoreceptors: involvement of 2P domain K+channels. (A) Mu ¨ller cell soma (MS)
with attached cones approached by a patch pipette (MP). In A, B, and C, white arrowheads and white dotted lines show the area of contact between
the inner segment (IS) of photoreceptors and MS. S points to the soma of cones. (B) Rods with outer segments (black circle), inner segments (black
diamond) and somata (open black square) attached to MS. In B and C, thick white arrow shows MS. (C) Isolated Mu ¨ller cell without photoreceptors.
Curved arrows show endfeet. Thin white arrow shows stalk. (D) Average currents recorded from MS of isolated Mu ¨ller cells (solid line, n=5) and
recorded from MS of isolated Mu ¨ller cells with photoreceptors attached (dotted line, n=5). The I/V-curves were obtained in response to a linear
voltage ramp from 2150 mV to +150 mV in control ECS K+=2.5 mM. Mu ¨ller cells have linear outward currents near K+-equilibrium potential. (E)
Effect of 2-P domain channel blocker bupivacaine (BUPI 1 mM) on isolated Mu ¨ller cells with (dotted line) and without photoreceptors attached (solid
line). After BUPI, residual currents are reduced ,15 times. (F) Adding the Kir channel blocker barium (Ba2+, 100 mM) to BUPI, caused a near complete
block of residual current (from 2100 mV to +50 mV). (G) Membrane potentials of cells in 2.5 and 10 mM K+ECS. [K+]o=10 mM induces
depolarization. (H) Application of BUPI 1 mM and BUPI 1 mM with barium 100 mM were used to test for 2P-domain and Kir channels respectively.
Single Mu ¨ller cells (solid black column, n=8) and cells with photoreceptors attached (grey column, n=10) show different sensitivity to BUPI. Addition
of Ba2+further depressed membrane potentials in both cells, but with no significant difference. Error bars represent standard errors of the mean
(SEM), where p,0.05 was considered significant (*).
Unique K+ Channel Expression in Caiman Retina
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reduced the membrane currents evoked by voltage ramps in
voltage-clamp mode (Fig. 4D, E). I/V-curves after the bupivacaine
block show a minor remnant of bupivacaine-insensitive Kir
currents. The addition of Ba2+, a K+channel blocker particularly
effective for Kir channels, should thus reduce the difference in
depolarization between SM and SMP cells. In fact, if Ba2+
(0.1 mM) was applied together with bupivacaine (1 mM) the cells
were furtherdepolarized (Fig.
30.8865.83 mV (n=8) and SMP cells to 238.8063.90 mV
Taken together, isolated Mu ¨ller cells showed a nearly perfect
K+-sensitivity and also a sensitivity to blockers, unlike cells patched
in retinal wholemounts (cf. Fig. 3); this is ascribed to imperfect
space clamp control in the wholemount situation. The electro-
physiological data from isolated cells support the immunohisto-
chemical data by revealing bupivacaine-sensitive currents that
may be ascribed to functional TASK-1 channels. Noteworthy,
however, in cells with adhering photoreceptors the depolarization
induced by bupivacaine was smaller than in single Mu ¨ller cells,
suggesting that Kir channels contribute to the membrane potential
(Fig. 4H). This could be explained if Mu ¨ller cells and photore-
ceptor cells were electrically coupled. Thus, we tested the isolated
cell groups (Fig. 4A, B) for a possible dye coupling.
4F, H); SMcells to
Coupling between cone and Mu ¨ller cell
The electrophysiological and pharmacological data (Fig. 4D-H)
suggest that Mu ¨ller cells can transfer K+from and to adhering
photoreceptors, via a coupling between the Mu ¨ller cell cytoplasm
and that of the photoreceptor cells. We, therefore, analyzed the
diffusion of the gap-junction channel-permeable dye sulforhoda-
mine-B. The dye was applied intracellularly in whole-cell
configuration when holding the cell in the current clamp mode.
We detected the dye-coupling in SMPs; however, the spread of the
tracer is unidirectional, occurring only in the direction from the
cone to the Mu ¨ller cell (Fig. 5A, B). When we patched the soma of
a Mu ¨ller cell the tracer easily filled the entire Mu ¨ller cell body, but
never spread to the photoreceptors (Fig. 5B). When patching the
inner segment of one of the adhering cones, the tracer diffused
throughout the photoreceptor and through the entire adjacent
Mu ¨ller cell (Fig. 5A). We also used several other dyes such as
Lucifer yellow, Alexa Fluor-488, Alexa Fluor-568, but these
compounds were not permeable from one cell to another in any
direction (data not shown). This difference may be caused by the
fact that Lucifer yellow and both Alexa dyes are negatively
charged molecules whereas sulforhodamine-B is a neutral mole-
cule. When a cone inner segment was patched, the tracer spread
throughout the entire photoreceptor and the attached Mu ¨ller cell
(Fig. 5A), but not into neighboring photoreceptors (Fig. 5A, insert).
Thus, the observed uni-directional tracer diffusion from a cone to
a Mu ¨ller cell but not to other adjacent cones, and not from the
Mu ¨ller cell to a cone represents a unique cell-to-cell communi-
cation for relatively large, uncharged molecules. By contrast, small
cations such as K+can probably permeate in both directions, as
indicated by the electrophysiological data. We assumed that the
movement of ions and sulforhodamine-B might be mediated by
gap junctional coupling. To clarify this point, we performed an
immunohistochemical staining for connexin 43 (Cx43).
In a study on rabbit retina, Mu ¨ller cells were found to express
Cx43 immunoreactivity . Antibodies directed to Cx43
revealed robust, punctate labeling mainly in the outer caiman
retina (Fig. 6B). Double-labeling of Mu ¨ller cells by antibodies
against glutamine synthetase (GS) and of cone photoreceptors by
the lectin peanut agglutinin (PNA) showed that much of the Cx43
immunoreactivity can be ascribed to Mu ¨ller cells and cone
pedicles (Fig. 6).
Unique distribution of glial K+channels in caiman Mu ¨ller
Here we provide the first morphological and functional
characterization of Mu ¨ller glial cells of the Spectacled caiman
(Caiman crocodilus fuscus). Mu ¨ller cells from crocodiles have been
studied morphologically [85,86,87,88,89] but studies of the
physiological characteristics of these cells were not yet performed.
We show here that caiman Mu ¨ller cells resemble Mu ¨ller cells in
some other vertebrate species by expressing classical glial markers,
such as S100b, glutamine synthetase, GFAP (Fig. 1), and vimentin
(Fig. 2B). Caiman Mu ¨ller cells display a very negative membrane
potential, close to the equilibrium potential for K+, similarly to
that found in Mu ¨ller cells of all other vertebrates studied so far (e.g,
[3,4]), and their membrane potential was sensitive to small
changes of K+concentration (Fig. 4G). However, we detected a
striking peculiarity in the type(s) of K+channels mediating this K+
High K+conductance of Mu ¨ller cells of amphibian, reptilian
[3,12,16,66,90,91,92]. The expression of Kir4.1 (Kcnj10) in
Mu ¨ller cells seems to be much conserved among adult vertebrates
. There are two instances when Kir channels in Mu ¨ller cells and
other glia are not functional: (i) during very early development up
to 10–15 postnatal days in Mu ¨ller cells [91,93,94] and in astrocytes
[95,96], or (ii) in adulthood under pathological conditions when
[39,60,61,84,37,97,98]. In this study we used healthy adult
We provide several lines of evidence suggesting that unlike in
Mu ¨ller cells of other healthy vertebrates, functional Kir-type K+
channels are not predominantly expressed in normal adult caiman
Mu ¨ller cells. First, Kir4.1 and Kir6.2 immunoreactivities were not
detectable in caiman Mu ¨ller cells. Second, the glial K(ATP)
channel subunit, Kir6.1, was immunolabeled in the Mu ¨ller cells
but was not accompanied by its functionally essential SUR1
satellite subunit, [41,44,45]. Moreover, in the presence of ATP (in
cytoplasm as well as in recording pipettes) the opening probability
of Kir6.1 channels is extremely low [45,54]. In addition, the I/V-
curves recorded from enzymatically isolated single Mu ¨ller cells
(Fig. 4D, E) failed to display a Kir-like pattern (i.e., strong inward
rectification near K+-equilibrium potential ). Therefore, Kir-
type K+channels known to occur in Mu ¨ller cells (and other glial
cells) of other vertebrates are not available in caiman Mu ¨ller cells;
this suggests that other types of K+channels are responsible for
maintaining the hyperpolarized membrane potential of these cells.
We found that TASK-1 2P channels were immunolocalized in
Mu ¨ller cells but not in other cells of the caiman retina (Fig. 2D).
Membrane currents mediated by these channels are characterized
by linear (or slightly rectified due to internal sodium block ) I/
V-curves typical for TASK channels [64,70,99] that are expressed
specifically in Mu ¨ller glia of different animals . This fits to our
observation that the I/V curves in caiman Mu ¨ller cells were linear
near equilibrium potential (Fig. 4D). Furthermore, a TASK
blocker bupivacaine [47,64] inhibited outward currents almost
completely (from 2.3 nA to ,0.15 nA at +75 mV (Fig. 4D, E,
dotted lines)). This indicates that TASK channels generate most of
the K+currents in caiman Mu ¨ller cells. It cannot be excluded that
there are other (hitherto unknown) K+channels which are
and functionally inhibited
Unique K+ Channel Expression in Caiman Retina
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Figure 5. Unidirectional flow of sulforhodamine-B dye from cone to caiman Mu ¨ller cell. (A) Upper panel: whole-cell patch clamp
technique using micropipette (MP) filled with sulforhodamine-B penetrating the inner segment of a cone attached to a Mu ¨ller cell. The dye filled the
cone and the Mu ¨ller cell: tracing of the photoreceptor cell body and inner segment (IS) and spreading throughout the whole Mu ¨ller cell from the
soma (MS) to the endfeet. Lower panel: combined (DIC and fluorescent) image showing that sulforhodamine-B is not permeable to neighboring
photoreceptors, but filled only the cone which was patched. Insert shows an enlarged image of fine attachments of three cones to a single glial cell
where two cones are not showing fluorescent dye. (B) Upper panel: whole-cell patch clamp of a Mu ¨ller cell resulted in the dye-tracing of the cell body
and endfeet, while no spreading of the dye occurred to the photoreceptors attached to this Mu ¨ller cell. Lower panel: patch of the Mu ¨ller cell soma
only traced the Mu ¨ller cell; the dye did not spread to the attached photoreceptors.
Figure 6. Localization of connexin 43 (Cx43) in the caiman retina. (A) Immunolocalization of the Mu ¨ller cell-specific protein glutamine
synthetase (GS, red) and staining of the nuclei by Hoechst 33258 (blue). The micrograph shows the overlay of the fluorescence image and the
transmitted light with visible retinal layers. (B) Immunostaining of Cx43 (green), yellow arrowheads point to Mu ¨ller cell-like structures. (C)-(E) Overlay
of Cx43 and GS staining clearly demonstrates co-localization in Mu ¨ller cell processes (yellow arrowheads). Moreover, cone outer segments and cone
pedicles are stained by the lectin peanut agglutinin (PNA, purple). Localization of Cx43 in cone pedicles is demonstrated at higher magnification in (E)
(white arrowheads). ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, OPL-outer plexiform
layer, ONL-outer nuclear layer, OLM-outer limiting membrane, IS-inner segments of photoreceptors, OS-outer segments of photoreceptors.
Unique K+ Channel Expression in Caiman Retina
PLOS ONE | www.plosone.org9 May 2014 | Volume 9 | Issue 5 | e97155
maintaining a (depolarized but substantial) membrane potential
after Ba2+and bupivacaine block (Fig. 4H) but certainly they
cannot mediate large K+currents (Fig. 4F). The location of
aquaporin-4 (AQP4) in the caiman retina can be sufficient for
water transport. AQP4 has been found in the endfeet processes at
the (i) ILM and (ii) around blood vessels in the vascular retina
. However, the caiman has an avascular retina with no
vessels in the retinal parenchyma, and as we demonstrated with
two different antibodies, AQP4 is localized only in the endfeet area
of the ILM, where TASK1 is located as well (Fig. S1). This opens
the questions: Is an AQP4/TASK1 assembly functional for water
transport as has been shown for AQP4/Kir4.1 [100,101], or is the
water transport due solely to K+-channel function ?
Regardless, this is a separate avenue of research and does not
fulfill the scope of the present study. Taken together, caiman
Mu ¨ller glial cells appear to be unique among the vertebrates
studied so far, as their hyperpolarized membrane potential and
high K+conductance, both essential for a wealth of glia-neuron
interactions in the retina [3,4], rely upon TASK 2P rather than
Coupling of Mu ¨ller glial cells
Here we show that caiman Mu ¨ller cells, similar to Mu ¨ller cells of
other species [9,82,103,104], display somewhat limited coupling to
their immediate cellular neighbors (Fig. 3C-F) and express the gap-
junction protein, connexin 43 (Fig. 6). The Mu ¨ller cell-Mu ¨ller cell
coupling may also contribute to the ‘passive’ I/V characteristic of
the currents recorded in retinal wholemount preparations (Fig. 3B).
More strikingly, however, we observed a coupling between
Mu ¨ller cells and photoreceptor cells. This coupling appears to
allow for a free bi-directional flux of small cations such as K+, as
the electrophysiological properties recorded from enzymatically
dissociated Mu ¨ller cells were changed if the dissociation process
failed to detach all photoreceptor cells from the Mu ¨ller cell (Fig. 4).
This coupling may be mediated by gap junctions formed by
connexin 43 (Fig. 6). An expression of connexin 43 by Mu ¨ller cells
as well as neurons has been demonstrated before  but to the
best of our knowledge, electrical coupling between Mu ¨ller cells and
photoreceptor cells is a novel finding. At present the functional
role can only be speculated upon; however, it is tempting to
suggest that spatial buffering of excess K+, one of the most
fundamental functions of Mu ¨ller cells  may involve direct K+
fluxes between Mu ¨ller cells and photoreceptor cells in the caiman
We also found evidence for an uni-directional coupling between
cone photoreceptors and Mu ¨ller cells, allowing for the transfer of
the non-charged tracer, sulforhodamine-B, from cones to Mu ¨ller
cells but not vice versa (and not between adjacent cones) (Fig. 5). A
uni-directional coupling has earlier been described between
different types of glial cells [106,107,108], but not between glia
and neurons in retina. Now it remains to be clarified how such
uni-directional coupling is established. The observed heterologous
cone-to-Mu ¨ller cell coupling appears to be a unique novel finding,
particularly as it does not involve homologous cone-to-cone
coupling (Fig. 5A). This might be indicative of a hierarchy of
signaling between neighboring cells, useful, for instance, for
molecular filtering of large size molecules but not of small cations.
This is the first functional study of retinal glial Mu ¨ller cells from
a representative of the crocodiles, the Spectacled caiman. The cells
share many properties with Mu ¨ller cells in other vertebrates but
display several particular features, (1) a unique endowment of
caiman Mu ¨ller cells with K+channels, substituting Kir-like K+
channels by TASK 2P channels; (2) electrical coupling of Mu ¨ller
glial cells with photoreceptors and (3) unidirectional tracer
propagation from cones to Mu ¨ller cells. It may be speculated that
heterologous coupling between Mu ¨ller cells and photoreceptor
cells may allow for a specific cell-to-cell molecular signaling and
modification of spatial buffering of K+ions.
caiman retina. (A) Immuno-staining for AQP4 (1:200, Sigma-
Aldrich A5971-primary antibody) followed by the secondary
antibody carbocyanine Cy2-coupled donkey anti-rabbit IgG
(green, 1:200) revealed a strong signal in the inner limiting
membrane (ILM). (B) Omitting the AQP4 antibody demonstrated
autofluorescence in the outer retina. Cell nuclei were stained by
Hoechst 33258 (blue). (C) Additional immuno-staining for AQP4
made in another caiman retina using a different AQP4 primary
antibody (1:200, Santa Cruz Biotechnology SC-20812) followed
by the secondary antibody carbocyanine Cy3-coupled goat anti-
rabbit IgG (red, 1:200) revealed a strong labeling in the ILM, as
well. GCL, ganglion cell layer; INL, inner nuclear layer; IPL,
inner plexiform layer; ONL, outer nuclear layer; OPL, outer
Localization of the water channel AQP4 in the
KATP channels compared with reference markers in the
caiman retinae. (A) Nomarski image: no expression for SUR1
was found in Mu ¨ller cells or in other caiman retinal cells. B and C
show no SUR1 staining (bright contrast images) in different
caiman retinae. (D) Other markers of Mu ¨ller cells are shown to
allow assessment of the cellular organization: lectin peanut
agglutinin that is specific for cones (PNA, blue), glutamine
synthetase in Mu ¨ller cells (GS, red) and connexin-43 (Cx43,
green) also specific for Mu ¨ller cells. Scale bar =20 mm. ILM-inner
limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform
layer, INL-inner nuclear layer, ONL-outer nuclear layer, IS-
photoreceptor inner segments.
Sulfonylurea-1 receptor (SUR1) subunit of
The authors thank Betzaida Torres, Paola Lo ´pez Pieraldi, Natalia
Skachkova, Dr. Priscila Sanabria, Dr. Felix Makarov and Dr. Lidia Zueva
for their superior technical and scientific assistance. The work is dedicated
to the memory of Dr. Richard K. Orkand.
Conceived and designed the experiments: SNS AZS AR MF RWV TP
LAC AV MJE. Performed the experiments: AZS SA YR JB EU AK JD
YK MI AS RWV MF SNS. Analyzed the data: AZS SA YR JB AS MF AR
LAC MJE SNS. Contributed reagents/materials/analysis tools: RWV.
Wrote the paper: AZS SNS MJE TP AR MF RWV LAC YK MI AS SA
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