European Journal of Neuroscience, Vol. 17, pp.

2084–2096, 2003 ß Federation of European Neuroscience Societies

HCN channels are expressed differentially in retinal bipolar

cells and concentrated at synaptic terminals

Frank Müller,1 Alexander Scholten,1 Elena Ivanova,1 Silke Haverkamp,2 Elisabeth Kremmer3 and U. Benjamin Kaupp1
1
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, D-52425 Jülich, Germany,
2
Max-Planck-Institut für Hirnforschung, Abteilung Neuroanatomie, Deutschordenstraße 46, D-60528 Frankfurt, Germany
3
Institut für Molekulare Immunologie, GSF-Forschungszentrum für Umwelt und Gesundheit, Marchioninistraße 25, D-81377
München, Germany

Keywords: bipolar cell, electrophysiology, HCN channels, Ih, immunocytochemistry, retina

Abstract
Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels codetermine the integrative behaviour of neurons and shape
their response to synaptic stimulation. We used immunohistochemistry and patch-clamp recording to study the composition and
distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1–4) are expressed differentially in the retina. In
particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of
heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This
suggests that HCN channels are involved in the control of transmitter release.

Introduction
In the mammalian retina, the light response of photoreceptors is
processed by an elaborate neuronal network (for review see Wässle
& Boycott, 1991; Masland, 2001). At least nine types of cone bipolar
cells and one type of rod bipolar cell provide the pathways for the
signal flow from photoreceptors to ganglion cells (for rat see Euler &
Wässle, 1995; Hartveit, 1997). Functionally, bipolar cells fall into ON-
and OFF-cells. The axons of OFF-bipolar cells, which show hyperpo-
larizing light responses, stratify in the outer half of the inner plexiform
layer (IPL). Axons of ON-bipolar cells, which depolarize in light,
stratify in the inner half of the IPL. Within the ON- and OFF-
sublamina, different bipolar cell types stratify at distinct levels.
Both ON- and OFF-bipolar cells are further subdivided into cells
which exhibit transient and sustained light responses (Awatramani &
Slaughter, 2000; Wu et al., 2000).
The output of a cone is tapped by several types of bipolar cell, each
constituting a separate ‘temporal channel’ that transfers different
characteristics of the light response. The light response of bipolar
cells is shaped by different glutamate receptors (DeVries, 2000) and by
voltage-gated channels. One class of voltage-gated cation channels
present in bipolar cells (Kaneko & Tachibana, 1985, Karschin &
Wässle, 1990) is activated by hyperpolarization and gated by cyclic
nucleotides (HCN channels; in previous studies; HCN currents were
designated If, Ih or Iq). HCN channels codetermine the resting potential
and membrane conductance and thereby play an important role in the
integrative behaviour of neurons and the sensitivity to synaptic input.
HCN channels affect the cable properties of the dendrite and shape the
time course and propagation of excitatory and inhibitory postsynaptic
potentials (for review see Pape, 1996; Kaupp & Seifert, 2001).
Correspondence: Dr Frank Müller, as above.
E-mail: f.mueller@fz-juelich.de
Received 20 December 2002, revised 28 February 2003, accepted 3 March 2003
In rod and cone photoreceptors, HCN channels shape the light
response. The channels become activated during hyperpolarization in
bright light and depolarize the cell toward the dark membrane poten-
tial, making the light response transient (Fain et al., 1978; Baylor et al.,
1984; Hestrin, 1987). Although their function in bipolar cells is not
known, HCN channels might shape the light response of OFF-bipolar
cells.
In mammals, four HCN channel genes (HCN1–4) have been iden-
tified (Ludwig et al., 1998; Santoro et al., 1998; Ludwig et al., 1999;
Seifert et al., 1999; for review see Kaupp & Seifert, 2001). When
expressed heterologously, HCN subtypes form homomeric channels
that differ in their kinetics and mid-point potential of activation (V1/2)
and in the shift of V1/2 towards more positive potentials by cAMP. The
functional diversity of HCN channels might be enhanced further either
by formation of heteromultimers from different subtypes (Chen et al.,
2001; Ulens & Tytgat, 2001) or coassembly of HCN subunits with
minimal Kþ channel-related proteins (Yu et al., 2001), previously
identified in voltage-gated Kþ channels (Abbott & Goldstein, 1998).
In this study, we address these issues concerning the function of
HCN channels in the retina: (i) which of the different bipolar cell types
express HCN channels; (ii) does a given cell type express a single HCN
channel isoform, or several; (iii) do different HCN channel subunits
colocalize  allowing for the formation of heterooligomeric chan-
nels  or are they targeted to different compartments of the cell;
and (iv), do HCN channels colocalize with neurotransmitter receptors
that might modulate their activity through changing cAMP levels?
Given the highly polarized nature of photoreceptors and bipolar cells,
the subcellular locales of HCN channels seem to be crucial for their
function. For example, spatially restricted expression of Kþ channels
was described in bipolar cells (Klumpp et al., 1995).
Our study describes for the first time the expression pattern of all
four HCN channel isoforms within the entire and well-defined
neuronal network of the retina. A detailed immunological and

doi:10.1046/j.1460-9568.2003.02634.x

electrophysiological analysis on bipolar cells shows that the four
isoforms are expressed differentially and suggests that the vast major-
ity of HCN channels are expressed as homo-oligomers. Strikingly,
HCN channels are highly concentrated at synaptic terminals in photo-
receptors and bipolar cells.
Materials and methods
Antibodies against HCN channels
Peptides used to produce anti-HCN antibodies consisted of seven N-
terminal amino acids (aa) (CGSSHHH), followed by 35 aa specific for
a given HCN channelsubtype [peptides were named RTQ (HCN1; aa
R650 to P685 in rat), QQA (HCN2; aa Q676 to F710 in rat), TLL
(HCN3; aa T640 to S675 in rat), and SHG (HCN4; aa S1046 to L1083
in rat)]. Rabbit polyclonal antibody PPc73K (against HCN4) was
obtained after immunization with peptide SHG and was purified by
affinity chromatography using the peptide coupled on Activated Thiol
Sepharose (Amersham Pharmacia Biotech., Freiburg, Germany). To
produce the rat monoclonal antibodies RTQ-7C3, QQA-3G7, QQA-
4A6, TLL-6C5, TLL-7H7 and SHG-1E5, immunization of rats and
fusion of immune spleen cells were performed according to standard
procedures. A polyclonal antibody against HCN1 was obtained from
Alomone (Jerusalem, Israel).
Membrane proteins from HEK293 cells and rat retina
Adult rats were deeply anesthetized with isoflurane and decapitated.
Retinae were dissected free and frozen in liquid nitrogen. Rat retina was
homogenized in a glass/Teflon homogenizer in ice-cold 20 mM HEPES-
NaOH at pH 7.4, 20 mM NaCl, 1 mM EDTA, 0.1 mM EGTA and 1 mM
DTT. Mammalian protease inhibitor cocktail (Sigma, Deisenhofen,
Germany, 1 : 500) was present in all steps. The suspension was washed
twice by centrifugation at 100 g for 10 min (4 8C) to separate the
membranes from nuclei. Membranes were collected by centrifugation
at 21 000 g for 15 min (4 8C). The membrane pellet was resuspended in
20 mM HEPES-NaOH at pH 7.4, 500 mM NaCl, 1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, washed by centrifugation, and resuspended in 20 mM
HEPES-NaOH at pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA,
1 mM DTT. Membrane proteins from transfected and mock-transfected
HEK293 cells were isolated by the same procedure.
SDS–PAGE and Western blot analysis
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-
PAGE) was carried out by standard procedures (Laemmli, 1970).
Western blot analysis was carried out using Immobilon-P membranes
(Millipore, Schwalbach, Germany) as recommended by the manufac-
turer. Membrane proteins were electrotransferred on a 7.5% SDS-
polyacrylamide gel, electroblotted onto Immobilon-P membranes,
which were blocked with 0.5% milk powder in phosphate-buffered
saline (PBS) and probed with the following primary antibodies: RTQ-
7C3 1 : 250; QQA-3G7 1 : 5; TLL-6C5 1 : 5; and SHG-1E5 1 : 5. The
secondary antibody goat anti-rat IgG þ M (Jackson/Dianova, Ham-
burg, Germany) was conjugated to horseradish peroxidase and used at
1 : 7000 dilution. The immunoblots were developed with Super-
Signal1 (Pierce, Perbio Science, Bonn, Germany).
For deglycosylation, membrane proteins were first denatured in the
presence of 0.5% SDS/1% 2-mercaptoethanol for 10 min at 37 8C and
then incubated in 50 mM PBS/1% NP-40 and 500 U of PNGase F
(NEB, Frankfurt, Germany) for 4 h at 37 8C.
Light microscopy
All procedures were approved by the local animal care committee.
Adult rats were deeply anaesthetized with isoflurane and decapitated.
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 2084–2096
HCN channels in bipolar cells 2085
Eyes were enucleated and opened by an encircling cut at the limbus.
The retinae in the eyecup were immersion-fixed for 10–15 min in 4%
paraformaldehyde (PA) in 0.1 M phosphate buffer (PB) at room
temperature and washed in PB several times. Tissue was incubated
in 10% sucrose in PB for 30 min, followed by 30% sucrose for 3 h. The
retina was flat embedded and frozen in OCT compound. Vertical
sections (20-mm thick) were cut on a cryostat and collected on gelati-
nized slides. Sections were incubated in 5% chemiblocker (Chemicon,
Hofheim, Germany), 0.5% Triton-X100 in PB for 1 h. Sections were
incubated overnight with primary antibodies diluted in the same solu-
tion with 0.05% NaN3 added. Sections were washed in PB and
incubated in secondary antibodies diluted in 5% chemiblocker in
PB for 1 h, washed in PB and coverslipped with Prolong antifade
kit (Molecular Probes, Göttingen, Germany) or Aqua Polymount
(Polysciences, Eppelheim, Germany). In some cases, the staining
was intensified by using a secondary antibody raised in goat, followed
by a tertiary antibody donkey anti-goat that carried the same fluor-
ophore. Sections were examined with a Leica TCS confocal laser
scanning microscope (Leica Microsystems, Heidelberg, Germany)
with 40 /1.0 and 63 /1.32 oil immersion lenses. Images were
processed and printed with Adobe Photoshop or Corel Draw. For
double or triple labelling, primary antibodies were mixed and applied
simultaneously. All secondary antibodies were highly cross-absorbed
and were carefully tested to exclude reactions with the wrong primary
antibody. Concentration of the antibodies, laser intensity and filter
settings were carefully controlled and the sequential scanning mode
was employed to completely rule out cross-talk between the fluores-
cence detection channels. Band pass filters of 500–530 nm for green
fluorescence (Alexa488), 580–650 nm for red fluorescence (Alexa568
and Cy3) and 680–750 nm for infrared fluororescence (Cy5) were used.
Electron microscopy
Adult rats were killed and retinae were prepared as described above.
Retinae were fixed in 4% PA for 40 min. Retinae were dissected out,
cryoprotected in 30% sucrose overnight and shock-frozen in liquid
nitrogen. Vibratome sections were cut and processed as described
previously using the ABC detection kit (Vector Laboratories, Alexis
Germany, Grünberg, Germany) with diaminobenzidine as chromogen,
followed by silver intensification (Sassoè-Pognetto et al., 1994).
Primary antibodies
RTQ-7C3 (rat, HCN1) 1 : 50; Anti-HCN1 (rabbit, Alomone, Jerusa-
lem, Israel) 1 : 400; QQA-4A6, QQA-3G7 (rat, HCN2) 1 : 5 to 1 : 20;
TLL-6C5 (rat, HCN3) 1 : 5 to 1 : 20; SHG-1E5 (rat, HCN4) 1 : 5 to
1 : 20; PPc73K (rabbit, HCN4) 1 : 400; PKCa (rabbit; Santa Cruz,
Heidelberg, Germany) 1 : 40 000; Recoverin (rabbit; gift of Dr Koch,
Jülich) 1 : 40 000; ChAT (choline acetyltransferase, mouse; Chemicon,
Hofheim, Germany) 1 : 400; Dopamine D1 (rat; Sigma, Deisenhofen,
Germany) 1 : 500; mGluR6 (rabbit; gift of Dr Nakanishi, Kyoto)
1 : 7500; Kinesin II (mouse; Berkeley Antibody Company, Richmond,
USA) 1 : 50; and CabP (mouse; Sigma) 1 : 300 were used.
Secondary antibodies
Anti-rabbit Alexa488, anti-rat Alexa488, anti-mouse Alexa488, anti-
rat Alexa568 (all goat; Molecular Probes, 1 : 500); anti-rat Cy3
(1 : 500), anti-goat FITC (1 : 100), anti-rabbit Cy3 (1 : 1000) and
anti-mouse Cy5 (1 : 100) (all donkey; Dianova, Hamburg, Germany)
were used.
Electrophysiology
Procedures were approved by the local animal care committee. Adult
rats were deeply anaesthetized with isoflurane and decapitated.
2086 F. Müller et al.

Retinae were dissected free in oxygenized Hanks balanced salt solu-
tions (HBSS) and mounted onto a millipore filter (Schleicher and
Schuell, NC10, Dassel, Germany). Slices (150 mm) were cut in a
custom-made device by rolling over the retina with a curved scalpel
blade (Boos et al., 1993). Before recording, slices were treated with
DNase I (Roche, Molecular Biochemicals, Mannheim, Germany;
50 mg/mL in HBSS) for 10 min. Recordings were carried out at room
temperature with an Axopatch-200 amplifier and Pclamp6 software
(Axon Instruments, Foster City, USA). The recording chamber was
perfused with an oxygenized solution containing (in mM): NaCl, 137;
KCl, 5.4; CaCl2, 1.25; MgSO4, 0.81; KH2PO4, 0.44; Na2HPO4, 0.34;
Hepes, 10; Glucose, 5.6; Phenol red, 0.031; pH 7.4. Intracellular
solution contained (in mM): K-gluconate, 126 KCl, 4; NaCl, 10;
CaCl2, 1; MgCl2, 1; EGTA, 10; Hepes, 10; MgATP, 1; Na3GTP, 1;
Lucifer Yellow, 0.008% (Sigma); and Biocytin, 0.4% (Sigma); pH 7.3.
Measured liquid junction potential was 10 mV. All measurements were
corrected for the liquid junction potential. Pipette resistances ranged
from 11 to 17 MO, seal resistances were 4–15 GO and input resistances
of bipolar cells were at least 1 GO. Cells were voltage-clamped with
the patch-clamp technique in the whole-cell mode. The membrane
potential (Vm) of the cells was clamped to a holding value of 50 mV
(which was close to the resting potential of most bipolar cells and did
not activate other voltage-gated channels) and HCN currents were
recorded during hyperpolarizing steps (55 mV to 115 mV, 15 mV
increments). At Vm ¼ 50 mV, some HCN channel isoforms have a
finite open probabality; moreover, when Vm is stepped back to 50 mV
after a hyperpolarization, closure of HCN channels is rather slow.
Therefore, after each hyperpolarization step, a short depolarizing pulse
to 10 mV was performed, which rapidly and completely closed HCN
channels (data not shown). After recording, slices were fixed in 4%
paraformaldehyde for 15 min and stained for HCN channel immunor-
eactivity as described above.
Heterologous expression in HEK293 cells
Whole cell recordings and Western blot analysis were performed on
FLP-INTM.-293 cells that stably expressed human HCN1 (hHCN1) or
hHCN4, respectively (Stevens et al., 2001) or on HEK293 cells that
were transiently transfected with cDNA encoding rat HCN1 (rHCN1),
rHCN2, and rHCN3, respectively, as described previously (Baumann
et al., 1994). Recording conditions were identical to those in retinal
slices.
Results
All HCN channel isoforms are expressed in the rat retina
Monoclonal and polyclonal antibodies were used to study the four
HCN channel isoforms in the retina by Western blotting and immu-
nohistochemistry. The specificity of each antibody was tested on HCN
channels expressed in HEK293 cells. Antibody RTQ-7C3, directed
against the C-terminus of HCN1, recognized a protein with an apparent
molecular weight (Mw) of 102 kDa in a membrane preparation of
HCN1-transfected HEK293 cells (Fig. 1A, lane 2), but not of mock-
transfected cells (lane 1). This value is identical to the predicted Mw;
treatment of membranes with the deglycosidase PNGase F did not
change the Mw (lane 3), suggesting that HCN1 is not glycosylated in
HEK293 cells. In a retinal membrane preparation, the antibody
labelled a protein of 110 kDa (lane 4). The higher Mw is because of
glycosylation; treatment with PNGase F lowered the Mw to a value
identical to that in HEK293 cells (lane 5). A commercially available
polyclonal antibody against the N-terminus stained the same bands

Fig. 1. Monoclonal antibodies RTQ-7C3 (A, HCN1), QQA-3G7 (B, HCN2), TLL-6C5 (C, HCN3) and SHG-1E5 (D, HCN4) recognize the HCN channel subtypes in
their glycosylated and deglycosylated forms.(A) Lane 1, mock-transfected HEK293 cells; lane 2, HEK293 cells, transfected with rHCN1; lane 3, HEK293 cells,
transfected with rHCN1 and treated with PNGase F; lane 4, rat retina; lane 5, rat retina, treated with PNGase F. (B) Conditions as in A, except that HEK293 cells were
transfected with rHCN2. Lane 6 (rat retina, treated with PNGase F) was incubated with secondary antibody only. (C) Conditions as in A, except that HEK293 cells
were transfected with rHCN3. (D) Lane 1, HEK293 FlipIn-wild-type; lane 2, HEK293 FlipIn-hHCN4; lane 3, HEK293 FlipIn-hHCN4, treated with PNGase F; lane 4,
rat retina. Within each panel, lanes 1–3, and 4–6, respectively, were loaded with the same amount of protein: lane 1–3 (A) 0.75 mg; (B and C) 2 mg; (D) 3 mg; lanes 4–6
(A and B) 50 mg; (C) 120 mg; (D) 50 mg.

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 2084–2096

(data not shown); however, a second band with a Mw of approximately
60 kDa was also labelled. It remains to be determined whether this
band represents a truncated form of HCN1.
Antibody QQA-3G7 (anti-HCN2) recognized a strong band at
98 kDa (similar to the predicted Mw of 95 kDa) and a weaker band
at 120 kDa in membranes of HCN2-transfected cells (Fig. 1B, lane 2).
Deglycosylation abolished the 120 kDa band and enhanced the 98 kDa
band (lane 3). In retinal membranes, a weak band of 98 kDa and a
stronger band of 108 kDa were detected (lane 4). After deglycosyla-
tion, the 108 kDa band disappeared, whereas the 98 kDa band was
strongly enhanced (lane 5). Another anti-HCN2 antibody (QQA-4A6)
gave similar results (data not shown). In the retina preparation used for
detection of HCN2, but not in the preparation used for detection of
HCN1, HCN3 and HCN4, an additional band of 120 kDa became
visible after deglycosylation. This band was also observed in a control
experiment (secondary antibody alone, lane 6), and most likely reflects
an unspecific cross-reactivity of the secondary antibody.
In HCN3-transfected cells (Fig. 1C), antibody TLL-6C5 (anti-
HCN3) labelled proteins of 88 kDa (similar to the predicted Mw of
86 kDa) and 92 kDa (Fig. 1C, lane 2). Neither of the two bands was
observed in mock-transfected cells (lane 1). The upper band was not
because of glycosylation, because it was not abolished by treatment
with PNGase F (lane 3). In the retina, the anti-HCN3 antibody
recognized a 90 kDa band (lane 4). Upon deglycosylation, the band
shifted to the predicted Mw of 86 kDa (lane 5). Another anti-HCN3
antibody (TLL-7H7) yielded identical results (data not shown).
In HCN4-transfected cells, antibody SHG-1E5 (anti-HCN4)
labelled two bands of 132 kDa and 162 kDa (Fig. 1D, lane 2). After
deglycosylation, the upper band was abolished and the lower band was
enhanced (lane 3); the Mw of the lower band agrees with the predicted
Mw of 129 kDa. In retinal membranes, a single band of 132 kDa was
detected (lane 4), indicating that the majority of HCN4 channels is not
glycosylated. The rabbit antibody PPc73K (anti-HCN4) yielded simi-
lar results (data not shown). A recent study using in situ hybridization
suggested that HCN4 is not expressed in mouse retina (Moosmang
et al., 2001). However, immunological as well as electrophysiological
evidence demonstrates that HCN4 is expressed in bipolar cells of both
rat (see below) and mouse (data not shown).
Figure 2 gives an overview of the cellular localization of HCN
channel isoforms in the rat retina. HCN1 (labelled with the polyclonal
antibody) was expressed throughout the retina (Fig. 2A). In the outer
retina, rod and cone photoreceptors were stained. In the inner nuclear
layer (INL), cone bipolar cells (long arrow) were positive that sent
dendrites to the outer plexiform layer (OPL) and axons to a dense band
in the middle of the inner plexiform layer (IPL). Several thinner bands
in the IPL were also marked. One band in the OFF-sublamina of the
IPL (arrowhead) seems to originate from amacrine cells rather than
bipolar cells, because the band is much thinner than bipolar cell axon
terminals stratifying in this region (compare Euler & Wässle, 1995)
and because processes were not labelled by an antibody against the
vesicular glutamate transporter 1, which is a good marker for bipolar
axon terminals (data not shown). In fact, many labelled amacrine cell
bodies (short arrow) were found at the border of the INL and IPL. In the
ganglion cell layer (GCL), some somata, presumably ganglion cells,
were strongly labelled. Axon bundles were labelled in the nerve fibre
layer (NFL). At high power magnification, HCN immunofluorescence
was found in a thin rim, presumably the plasma membrane, surround-
ing processes and somata, as expected for a channel protein. This is
shown for two ganglion cell somata in Fig. 2E and for a cone soma and
inner segment (arrows) in Fig. 2F. The monoclonal antibody RTQ-7C3
yielded identical results in the inner retina; however, it only labelled
rods and cones weakly. Despite this difference, we think that the label
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 2084–2096
HCN channels in bipolar cells 2087
in photoreceptors reflects true HCN1 immunoreactivity, because: (i)
RTQ-7C3 stained rods and cones in the mouse retina (data not shown);
(ii) message for HCN1 was found in photoreceptors of the mouse
(Moosmang et al., 2001); and (iii), rods and cones of the rat retina show
currents with the typical activation kinetics of HCN1 (data not shown).
HCN4 (antibody PPc73K) was detected in two different bipolar
cells (long arrow), the axons of which projected to two different strata
in the IPL (Fig. 2B). A strongly immunoreactive stratum was observed
in the OFF-sublamina of the IPL (short arrow); a weaker stratum was
detected in the middle of the IPL (arrowhead). Few amacrine cells and
somata in the GCL were also immunoreactive.
HCN2 (antibody QQA-4A6) was detected in several bands in the
IPL, in small, lobular structures that were reminiscent of axon terminal
systems of bipolar cells (Fig. 2C, short arrows). Few amacrine cell
bodies (arrow) and, very rarely, some bipolar cell bodies and cone
photoreceptors (Fig. 2G, arrow) were faintly labelled. Blood vessels
(bv) were labelled unspecifically by the secondary antibody.
The expression of HCN3 (antibody TLL-6C5) was restricted to the
plexiform layers. Figure 2D was exposed strongly to visualize the
weak label of HCN3 in the IPL. Strong HCN3 label was observed in
the OPL, associated with cone pedicle synapses (arrow). Blood vessels
were labelled unspecifically by the secondary antibody.
In summary, the four HCN channel genes are differentially
expressed in the retina. In the following, we will show that at least
for the class of bipolar cells there is little overlap that would allow for
the formation of heterooligomeric channels.
Type 3 bipolar cells express HCN4
In the following, we will classify bipolar cells according to the
schemes by Euler & Wässle (1995) and Hartveit (1997). Nine types
of cone bipolar cells and one type of rod bipolar cell can be distin-
guished according to their level of axonal stratification in the IPL. Type
1–4 bipolar cells stratify on the OFF-sublamina, i.e. in the outer half of
the IPL. Type 5 bipolar cells terminate in the middle of the IPL, and
type 6–9 bipolar cells and the rod bipolar cells stratify in the ON-
sublamina, i.e. in the inner half of the IPL. We observed strong HCN4
staining in type 3 cone bipolar cells, the bifurcated axons of which
stratify in the OFF-sublamina of the IPL (Figs 2B and 3A). Type 2
bipolar cells exhibit a similar stratification pattern, but cannot be
mistaken for the HCN4-positive cells. Figure 3B shows a double
staining with antibodies against HCN4 (red) and against recoverin
(green), that is expressed in type 2 bipolar cells (short arrows) and type
8 bipolar cells (long arrows) (Euler & Wässle, 1995). Clearly, HCN4-
positive axon terminals stratify mostly below those of type 2 cells.
Yellow spots occur only when the two kinds of processes cross each
other in the section.
We compared hyperpolarization-activated currents recorded from
type 3 cells in retinal slices with currents recorded from HEK293 cells
that express HCN4 (Fig. 3C and D). In HEK293 cells, hyperpolarizing
steps produced inward currents that activated with a slow time course
(Fig. 3C). Currents reached a steady-state only during hyperpolariza-
tion for several seconds (lower panel, Vm ¼ 100 mV). Slowly decay-
ing tail currents were observed after stepping Vm back to the holding
potential. At Vm ¼ 100 mV, current activation was best fitted by two
exponentials (t1 ¼ 550  116 ms, t2 ¼ 2394  222 ms; n ¼ 6). Hyper-
polarization-activated currents in type 3 bipolar cells showed a
similarly slow time course (Fig. 3D; t1 ¼ 430  32 ms, t2 ¼ 2654 
410 ms; n ¼ 8), and were reversibly blocked by 1 mM extracellular
Csþ, which is regarded as a hallmark of HCN currents (Fig. 3E). HCN
immunofluorescence and, hence, channel density, is higher at the axon
terminal than at the soma. In the whole-cell recording, voltage attenu-
ation along the bipolar cell axon might be expected to shift the
2088 F. Müller et al.

Fig. 2. Immunohistochemical localization of HCN1–4. (A) HCN1-specific antibody labelled rod and cone inner segments, somata and axons. Cone bipolar cells
(long arrow) whose axons terminate in the middle of the IPL and amacrine cells (short arrow), some somata in the GCL and ganglion cell axons in the NFL were also
labelled. (B) Two bipolar cell types (long arrow) that terminate in the OFF-sublamina (short arrow) or in the middle of the IPL (arrowhead) and some amacrine cells
were labelled for HCN4. (C) HCN2 staining was found in several bands in the IPL (short arrows) and in some amacrine cell bodies (long arrow). (D) Antibodies
against HCN3 strongly labelled cone pedicle synapses (arrow) in the OPL and, although much weaker, processes in the IPL. (E) Two ganglion cell somata show clear
HCN1 staining in the plasma membrane (arrow). (F) HCN1 is found in the plasma membrane of cone somata and inner segments (arrows) and of rod inner segments.
(G) Weak HCN2 staining was found in somata and processes of cones (arrow). Scale bars, 50 mm (A–D); 10 mm (E–G). OS, outer segments; IS, inner segments; OPL,
outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fibre layer; bv, blood vessel.

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 2084–2096
 HCN channels in bipolar cells 2089

Fig. 3. Type 3 bipolar cells express HCN4. (A) HCN4 immunoreactivity in the inner retina. Strongest staining is found in axon terminals of type 3 bipolar cells
(arrow). (B) Double labelling for recoverin (left, green) and HCN4 (middle, red). The right panel shows the merged image. Short arrows mark type 2 bipolar cells,
long arrows type 8 bipolar cells. Type 2 bipolar cells are not HCN4-immunoreactive. bv, blood vessel. (C) Currents mediated by HCN4 channels in HEK293 cells.
Upper panel: Vm was stepped from 50 mV to hyperpolarizing levels (55 to 115 mV, 15 mV increments) for 500 ms. HCN4 currents activated with a slow time
course. Lower panel: Vm was stepped from 50 mV to 100 mV for 14 s. (D) HCN currents recorded in a type 3 bipolar cell. Currents were remarkably similar to
those of HCN4 homomeric channels. Conditions as in C. (E) HCN currents (1; Vm ¼ 100 mV) in type 3 bipolar cells were blocked by 1 mM extracellular Csþ (2); (3)
recovery. (F) The slice with the Lucifer Yellow-filled cell (green) was counterstained with HCN4 antibodies (red). The cell stratified exactly in the HCN4-positive
stratum. Borders of the IPL are marked by white bars. Scale bars, 15 mm.

apparent activation range of HCN channels. We did not observe
significant changes, probably because bipolar cells are electrically
quite compact. After recording, counterstaining with HCN4 antibodies
demonstrated that the Lucifer Yellow-filled cell (green) stratified
exactly in the HCN4-positive layer (red) (Fig. 3F). These observations
suggest that, in type 3 bipolar cells, HCN4 forms homo-oligomeric
channels.
Type 5 bipolar cells express both HCN1 and HCN4
HCN1 and HCN4 are both present in bipolar cells that terminate in the
middle of the IPL (see Figs 2A and B, and 4A), where bipolar cells type
5 and type 6 stratify (Euler & Wässle, 1995; Hartveit, 1997). The axon
terminals of type 5 and 6 cells are separated by the dendritic stratum of
the ON-cholinergic amacrine cells (Euler et al., 1996). To distinguish
between type 5 and 6 cells, we double-labelled sections for ChAT
(Fig. 4B, red) and HCN1 (green). Cholinergic ON- and OFF-amacrine
cells form two dendritic strata. The lower stratum is formed by the
dendrites of the ON-cholinergic cells. HCN1-positive axon terminals
(Fig. 4B, green) were observed preferentially above the ON-choliner-
gic stratum (arrow), indicating that they originated from type 5 bipolar
cells. Occassionally, weakly stained axon terminals were detected
below the ON-cholinergic band (arrowhead), suggesting that type 6
cells are also HCN1-positive. Double labelling with antibodies against
HCN1 (red) and HCN4 (green) revealed that type 5 bipolar cells
express both HCN isoforms (Fig. 4C). In the HCN1 staining, the most
prominent label is found in axon terminals of type 5 bipolar cells
(arrows). Type 6 bipolar cells are barely visible in this section. Weakly
labelled processes, presumably from amacrine cells, are found in the
upper part of the IPL. HCN4 is prominent in axon terminals of type 3
bipolar cells in the upper part of the IPL. Here, HCN1 and HCN4 label
do not colocalize. In contrast, in the stratum formed by type 5 bipolar
cells HCN1 and HCN4 are colocalized. Processes have the same shape
in both stainings and appear yellowish in the overlay (arrows).
The colocalization is consistent with the idea that HCN1 and HCN4
form hetero-oligomeric channels, but does not necessarily prove this
point. Alternatively, homo-oligomeric channels consisting of either

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2090 F. Müller et al.

Fig. 4. Type 5 bipolar cells express both HCN1 and HCN4. (A) HCN1-positive bipolar cells terminate in the middle of the IPL. (B) Double labelling of the IPL for
ChAT (red, left) and HCN1 (green, middle). The right panel shows the merged image. In the ChAT staining, an amacrine cell and the OFF-cholinergic (upper) and the
ON-cholinergic (lower) stratum in the IPL can be distinguished. HCN1-positive axon terminal systems from type 5 bipolar cells were found above (arrow), weakly
labelled type 6 axon terminals below the ON-cholinergic stratum (arrowhead). (C) Double labelling of the IPL for HCN1 (red) and HCN4 (green). In the axon
terminals of type 5 cells, HCN1 and HCN4 were colocalized (arrows). Scale bars, 10 mm. (D) HCN1 currents in a HEK293 cell. Upper panel: Vm was stepped from
50 mV to hyperpolarizing levels for 500 ms. Currents at Vm ¼ 100 mV and 115 mV reached steady-state after 400 ms. Lower panel: Vm was stepped from
50 mV to 100 mV for 14 s. (E) HCN currents recorded in a type 5 bipolar cell. Conditions as in D. Currents activated with a time course similar to HCN1, but
reached steady-state levels only during long steps. (F) HCN currents (1) were blocked by Csþ (2); (3) recovery. A transient component in the tail currents, probably a
T-type Ca2þ current persisted blockage. (G) The Lucifer Yellow-filled cell stratified in the HCN1-positive stratum. Scale bar, 15 mm.

HCN1 or HCN4 might be located in close proximity. To test these
hypotheses, we compared HCN currents recorded from type 5 cells and
from HCN1 or HCN4 in HEK293 cells.
Figure 4D shows currents in a HEK293 cell expressing HCN1.
HCN1 currents activated much faster than HCN4 currents (compare
Figs 3C and 4D). Current activation was best described by two
time constants t1 ¼ 54  7 ms and t2 ¼ 242  56 ms (n ¼ 16; Vm ¼
100 mV). Hence, HCN1 and HCN4 currents can be distinguished
readily by their different time course of activation. In both channels
two time constants are needed to describe current activation. Assuming
that type 5 cells are furnished with homo-oligomers of HCN1 and
HCN4, then HCN currents in these cells can be described by the
algebraic sum of HCN1 and HCN4 currents. Hence, currents should
show a very fast kinetic component (t1 of HCN1), a very slow
component (t2 of HCN4) and an intermediate component that reflects
the arithmetic mixture of t2 of HCN1 and t1 of HCN4, which are too
similar to be fitted independently. These expectations are born out by
experiments (Fig. 4E). Type 5 cells exhibited HCN currents that
developed with a time course similar to HCN1; however, steady-state
was only reached during long steps. A fit of HCN currents in type 5
bipolar cells (n ¼ 27) required three exponentials: t1 ¼ 63  5 ms,
which is close to t1 of HCN1; t2 ¼ 379  32 ms, and t3 ¼ 2945 
592 ms, which is close to t2 of HCN4 (Vm ¼ 100 mV). Our data are
therefore consistent with the hypothesis that HCN1 and HCN4 form

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 HCN channels in bipolar cells 2091

independent homo-oligomeric channel populations in type 5 bipolar
cells, and there is no need to invoke the existence of hetero-oligomeric
channels to explain the electrophysiological data. HCN1 channels
account for roughly 80% and HCN4 for 20% of the total current in type
5 bipolar cells. However, we cannot rule out that a fraction of hetero-
oligomeric channels exist that activate with the intermediate time
constant t2. When Vm was stepped back to the holding potential, a
transient and a sustained current component could be distinguished.
The transient component was blocked by 4 mM Co2þ (data not shown)
but not by Csþ (Fig. 4F). This most likely originates from T-type Ca2þ
channels that exist in a variety of bipolar cells (Pan, 2000). Therefore,
deactivation properties of HCN channels were not included into our
analysis. The axon of the type 5 bipolar cells stratified exactly in the
HCN1-immunoreactive stratum of the IPL (Fig. 4G).
HCN2 is densely packed at axon terminals of ON-bipolar cells
HCN2 immunoreactivity was prominent in axon terminals in the inner
part of the IPL. It appears that most, if not all bipolar cells that stratify
in this sublamina are HCN2-positive. Double labelling revealed that
HCN2 is localized in axon terminals of type 8 bipolar cells and of rod
bipolar cells. A single confocal section through the IPL (Fig. 5A)
shows axons of type 8 bipolar cells labelled for recoverin (red) and
HCN2 (green). Recoverin antibodies label individual axon terminal
swellings (arrows). HCN2-immunoreactive puncta are distributed
throughout the ON-sublamina, indicating expression of HCN2 in
several cell types. Many of the HCN2 puncta are found along the
border, i.e. at the plasma membrane, of the terminal swellings. In
Fig. 5B, rod bipolar cells labelled for PKCa (red) (Greferath et al.,
1990) and HCN2 (green), terminate close to the GCL. At the axonal
swellings, HCN2 staining was prominent at restricted patches of the
plasma membrane (arrows). Therefore, in a given plane of section, not
all rod bipolar terminals showed HCN2 staining.
Studies in crayfish and hippocampal neurons suggest that HCN
channels modulate synaptic transmission either directly by some
unknown mechanisms or by cAMP-dependent depolarization (Beau-
mont & Zucker, 2000; Mellor et al., 2002). The triple staining in
Fig. 5C suggests that HCN2 channels might subserve such a function in
bipolar cells. The bipolar cell synapse displays a characteristic elec-
tron-dense structure, called the synaptic ribbon (Dowling & Boycott,
1966), which can be visualized by antibodies against kinesin II
(Muresan et al., 1999; Ghosh et al., 2001). Rod bipolar terminals
were identified by protein kinase C (PKC) staining (Fig. 5C; left panel,

Fig. 5. HCN2 is highly localized on the axon terminal systems of ON-bipolar cells. (A) Confocal section through the ON-sublamina of the IPL showing individual
recoverin-positive axon terminal swellings of type 8 bipolar cells (left, REC, arrows) and numerous HCN2-immunoreactive puncta (middle). Many puncta are
localized on the axonal swellings, close to their border. (B) Confocal section labelled for PKC (red) and HCN2 (green). HCN2 staining was found on the membrane of
rod bipolar axon terminals (arrows). (C) Confocal section through the IPL triple labelled for PKC (blue), kinesin II (KIN, red) and HCN2 (green). In the left panel,
kinesin II-positive red ribbons were found on rod bipolar axon terminals (arrows). In the right panel, HCN2 was often found in close proximity of ribbon synapses
(double arrows). Scale bars, 10 mm (A and B); 5 mm (C).

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2092 F. Müller et al.

Fig. 6. HCN2 currents in HEK293 cells and rod bipolar cells. (A) HCN2 currents in a HEK293 cell. Upper panel: Currents activated with a pronounced delay and did
not reach steady-state level within the 500 ms step. Lower panel: Vm was stepped from 50 mV to 100 mV for 7 s. (B) HCN currents recorded in a rod bipolar cell.
Currents were small and therefore appeared noisy. Conditions as in A. Currents activated with a time course similar to HCN2, but the delay was less pronounced. (C)
HCN currents (1) were blocked by Csþ (2); (3) recovery. (D) The Lucifer Yellow-filled rod bipolar cell. Scale bar, 15 mm.

blue). Numerous synapses, appearing as red dots by the kinesin II
labelling, exist on the rod bipolar terminals (KIN; arrows). A few
ribbons do not colocalize with the PKC staining; presumably they
originate from terminals of other types of bipolar cells. The overlay of
images of the ribbon staining (red) and the HCN2 staining (green)
demonstrates that HCN2 channels are found in close proximity to
ribbon synapses (right panel).
Figure 6A and B compare HCN2 currents in HEK293 cells (panel
A) with HCN currents recorded from a rod bipolar cell (panel B).
The current in HEK293 cells developed with a pronounced sigmoi-
dal onset (upper panel) and reached steady-state only after pro-
longed hyperpolarization (lower panel; Vm ¼ 100 mV; t1 ¼ 157 
14 ms, t2 ¼ 553  131 ms, n ¼ 12). The sigmoidal onset of currents
is much less pronounced in the bipolar cell. Whereas currents in rod
bipolar cells and HEK cells can be described by two exponentials of
similar values (rod bipolar cell: t1 ¼ 137  14 ms, t2 ¼ 696 
119 ms, n ¼ 28), the kinetic components have different proportions
in the two cell types. The fast and slow components account for
41  8% and 59  8% (n ¼ 12), respectively, of the total current in
HCN2 channels, and for 68  13% and 32  13% (n ¼ 28) in rod
bipolar cells. HCN currents in rod bipolar cells were blocked by
1 mM Csþ (Fig. 6C). Thus, immunohistochemistry argues that rod
bipolar cells contain only the HCN2 isoform, whereas the properties
of the current argue that channel properties are modified either by
additional subunits or by other mechanisms. In preliminary experi-
ments similar currents were recor- ded from type 8 and from
putative type 7 and 9 bipolar cells (data not shown). Very small
spots of HCN2 immunoreactivity (data not shown) were also
observed on the axon terminals of type 3 cells, which predomi-
nantly express HCN4 (see Fig. 3) and type 5 cells, which predo-
minantly express HCN1 and HCN4 (see Fig. 4). In these cells,
HCN2 constitutes a minute fraction of HCN channels that is too
small to be identified in the whole-cell recording.
HCN3 is highly concentrated at cone pedicles
HCN3 staining was most prominent at cone pedicle synapses
(Fig. 7A), where the cone contacts up to 10 different cell types (for
review see Haverkamp et al., 2000). HCN3 channels might be
expressed either presynaptically in cones or in any one of the post-
synaptic processes. To address this question, we double-labelled
sections for HCN3 and for markers of dendritic tips of either ON-
bipolar cells (mGluR6), or horizontal cells (CabP), or some types of
OFF-bipolar cells (GluR1). Figure 7B shows staining of a whole-
mounted retina for HCN3 and mGluR6; the plane of focus was in
the OPL. HCN3 (red) was localized in circular or oval structures
(diameter  5 mm). The green dots represent mGluR6 in dendritic tips
of ON-bipolar cells. The dots clustered at high density within the area
encircled by HCN3. However, no colocalization of mGluR6 and HCN3
was observed. Similarly, no colocalization was observed between
HCN3 and either CabP or GluR1 (data not shown), indicating that
HCN3 might be localized presynaptically in cones rather than in
postsynaptic processes. Two cone pedicles (CP) are shown in electron
micrographs in Fig. 7C. In both cases, ribbons (arrows) and invaginat-
ing processes (arrowheads) can be identified. HCN3 immunoreactivity
was observed as silver grains presynaptically at the cone membrane
that surrounds invaginating processes and in finger-like extensions at
the base of the cone pedicle. Interestingly, similar staining was
observed with antibodies against dystrophin and b-dystroglycan (Kou-
len et al., 1998; Blank et al., 1999) and with antibodies against the N-
methyl-D-aspartate (NMDA) receptor subunit NR1C2 (Fletcher et al.,
2000).
HCN channels in the retina may be modulated differentially
cAMP modulates both the threshold and kinetics of HCN channel
activation. The effects are particularly pronounced for HCN4
(Ludwig et al., 1999; Seifert et al., 1999). In the retina, dopamine
is a prominent neuromodulator that is coupled to cAMP meta-
bolism. Figure 8 shows a double staining for dopamine D1 receptors
(green) and HCN4 (red). D1 immunoreactivity was observed in the
OPL, in some somata in the INL and in several bands in the IPL,
confirming results of a previous study (Veruki & Wässle, 1996). The
staining of blood vessels (bv) was because of the secondary anti-
body. Two somata of HCN4-positive bipolar cells were labelled for
D1 receptors (arrows). D1 receptors and HCN4 were colocalized in
axons of type 5 cells in the middle of the IPL (arrowhead) but not in
axons of type 3 cells in the upper stratum. We conclude that by
binding to D1 receptors, dopamine might modulate HCN channels
in some, but not all, bipolar cell types.

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 HCN channels in bipolar cells 2093

Fig. 7. HCN3 is localized presynaptically in cone pedicle synapses. (A) HCN3 at cone pedicle synapses in the OPL. (B) Whole-mount preparation labelled for HCN3
(red) and mGluR6 (green) with the focus in the OPL. The lower panel shows the merged image. mGluR6-positive ON-bipolar cell dendrites were clustered at cone
pedicles, but they did not overlap with HCN3 staining. (C) Electron microscopic localization of HCN3 at the cone pedicle base and at finger-like extensions. Arrows
mark synaptic ribbons, arrowheads invaginating processes. CP, cone pedicle. Scale bars, 5 mm (A and B); 0.5 mm (C).

Fig. 8. Dopamine D1 receptors are colocalized with HCN4 on type 5 bipolar cells. Sections through the inner retina labelled for D1 receptors (green) and HCN4 (red).
D1 receptors were found on HCN4-positive bipolar cell somata (arrows) and on axon terminals of type 5 cells (arrowhead) but not on type 3 cells. Scale bar, 25 mm.

Discussion
Do HCN channels exist as homo- or hetero-oligomers?
Overlapping expression patterns of HCN isoforms revealed by in situ
hybridization (Ludwig et al., 1998; Santoro et al., 1998; Moosmang
et al., 1999) as well as results from single-cell polymerase chain
reactions (Franz et al., 2000) are consistent with (but do not prove) the
hypothesis that HCN subunits form heteromultimers. Moreover, coex-
pression of HCN1 and HCN2 produced channels with kinetic proper-
ties that cannot be reproduced by the algebraic sum of the kinetics of
HCN1 and HCN2 homomeric channels (Chen et al., 2001; Ulens &
Tytgat, 2001). In the following we argue that, in bipolar cells of the
mammalian retina, HCN isoforms for the most part do not coassemble.
Type 3 bipolar cells strongly express HCN4, and HCN currents in these
cells are very similar to those of HCN4 in a cell line. HCN currents in
type 5 cells can be reproduced by the algebraic sum of two independent
populations of homomeric HCN1 (which accounts for roughly 80% of
the current) and HCN4 channels (roughly 20%). Thus, there is no

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2094 F. Müller et al.
evidence for the formation of heteromeric channels in type 5 cells. In
both type 3 and type 5 cells, HCN2 immunoreactivity is observed in
very few, small spots at the axon terminals (data not shown), suggest-
ing that the contribution of HCN2 to the total currents is too small to be
identified in the recordings. Although we cannot rule out that hetero-
oligomeric channels are formed at these few sites, they certainly would
account for only a minute fraction of HCN channels.
Immunocytochemical data suggest that HCN2 is the only isoform
expressed in rod bipolar cells. Whereas HCN currents in rod bipolar
cells and HCN2 currents in HEK293 cells can be described by similar
exponentials, the kinetic components have different proportions in the
two cell types. The HCN2 channels in rod bipolar cells might include
as yet unknown subunits. Coexpression of MIRP1 with HCN1 in
Xenopus oocytes was reported to accelerate the kinetics of current
activation (Yu et al., 2001). Whether rod bipolar cells in fact express
MIRP subunits needs to be investigated in the future.
Rods seem to express HCN1 only; cones express HCN1, HCN2 and
HCN3. The immunoreactivity for HCN1 is strong in the inner segment,
the soma and axon of cones, but much weaker in the pedicle. The
distribution of HCN2 is similar, although HCN2 immunoreactivity was
very weak and barely above background. HCN3 is restricted to cone
pedicles. In most cases, HCN3 and HCN1 staining did not overlap
(data not shown). In conclusion, our results provide no evidence for the
formation of hetero-oligomeric HCN channels in bipolar cells and
photoreceptors.
Functional roles for HCN channels in the retina
HCN currents were first detected in rod photoreceptors of the retina
(Fain et al., 1978), where they counteract the hyperpolarizing response
at high light intensities by allowing an inward current that depolarizes
the cell. In order to resist the hyperpolarization quickly, rapidly
activating HCN isoforms might be most suited. Our result that
HCN1, which shows the fastest activation, is the predominant isoform
in both rods and cones, confirms this expectation and agrees with
previous results (Moosmang et al., 2001; Demontis et al., 2002).
HCN channels might have a similar function in OFF-bipolar cells
and shape the light response. Of the four OFF-bipolar cells (type 1–4;
Euler et al., 1996; Hartveit, 1997), only type 3 cells showed clear HCN
immunoreactivity. A transient OFF-bipolar cell, classified as either
type 2 or type 3, was described recently (Euler & Masland, 2000).
However, it is worth debating whether a channel activating as slow as
HCN4 can substantially truncate the light responses of bipolar cells (at
body temperature, and in the physiological voltage range, t is a few
hundred milliseconds (Ishii et al., 2001). A recent study suggested that
sustained and transient OFF responses are generated by different types
of glutamate receptors at the OFF-bipolar dendrites (DeVries, 2000).
All ON-bipolar cells express HCN channels. ON-bipolar cells
depolarize during illumination but hyperpolarize in darkness, when
the photoreceptors release glutamate. During prolonged darkness,
activation of HCN channels might help to set a suitable range of
membrane voltage in ON-bipolar cells.
How far does the voltage range of activation of HCN channels
overlap with the physiological voltage range of bipolar cells?
Under our experimental conditions, HCN channels started to activate
at about 50 to 55 mV. Bipolar cell resting potentials in our slice
preparation ranged from 35 to 55 mV, therefore we found overlap
of the two voltage ranges in some, but not in all, bipolar cells. In dark-
adapted rat retinal slices, Euler & Masland (2000) described average
resting potentials of 45 mV for rod bipolar cells and 39 to 36 mV
for cone ON- and OFF-bipolar cells. Therefore, it seems as if the
average membrane voltage of bipolar cells is too depolarized to allow
ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 17, 2084–2096
significant HCN channel activation. It seems odd that bipolar cells
express an ion channel type that should have no physiological function.
There are two solutions for this conundrum. First, the membrane
voltage of bipolar cells in the intact retina might be more negative than
the average values reported from slice recordings. In fact, Euler &
Masland (2000) showed individual bipolar cells with resting potentials
as negative as 61 mV. Moreover, the standard deviations given for the
average values of 39 and 36 mV are large (10–14 mV). This could
reflect different strengths of hyperpolarizing inputs to different bipolar
cells in the slice. It is probable that bipolar cells tend to be more
depolarized in slices than in intact retinae, because long-range inhi-
bitory input coming from the wide-field GABAergic amacrine cells
might be reduced during the slicing procedure. Second, binding of
cAMP to HCN channels shifts the threshold of activation by up to
15 mV towards more depolarized values (DiFrancesco & Tortora,
1991; Gauss et al., 1998; Ludwig et al., 1998, 1999; Santoro et al.,
1998; Seifert et al., 1999). A variety of neurotransmitters and neuro-
peptides that control cAMP levels, such as dopamine, somatostatin,
vasoactive intestinal peptide and enkephalin are present in amacrine
cells and were shown to modulate ion channels in photoreceptors,
bipolar cells and amacrine cells (Feigenspan & Bormann, 1994; Veruki
& Yeh, 1994; Akopian & Witkovsky, 1996; Petrucci et al., 2001;
Yazulla et al., 2001). In preliminary experiments (data not shown), we
indeed found strong shifts of HCN channel activation after internal
dialysis of bipolar cells with cAMP. We propose that the impact of
HCN channels on the bipolar cell function depends strongly on the
activation of modulatory amacrine cell circuits. This hypothesis needs
to be tested in future experiments.
HCN channels are densely packed on synaptic terminals
The most striking feature of retinal HCN channels is their high density
on synaptic terminals. HCN1 and HCN4, although present in the
bipolar cell bodies, are localized preferentially at the axon terminals.
HCN2 is localized in a punctate fashion on bipolar cell axon terminals,
which are densely covered with synaptic input and output sites. Finally,
HCN3 was restricted to the base of the cone pedicle. What might be the
functional significance of this channel distribution? Recent studies
show that HCN channels might directly modulate synaptic transmis-
sion (Beaumont & Zucker, 2000; Mellor et al., 2002; but see Cheva-
leyre & Castillo, 2002). The activity of HCN channels is modulated by
cAMP. At their axon terminal system, bipolar cells receive strong input
from amacrine cells. The neuromodulators might control the gain of
synaptic transmission at bipolar cell synapses through modulation of
HCN channel activity by cAMP-signalling pathways. The effective
coupling between cAMP synthesis and HCN channel modulation
could require that receptors, adenylyl cyclase and HCN channels
are in close proximity at the synaptic terminal. In fact, we show here
that dopamine D1 receptors are colocalized with HCN4 channels in
some, but not all, bipolar cell types.
An increase in cAMP and subsequent modulation of HCN
channels could also occur by means of a Ca2þ-dependent adenylate
cyclase (Lüthi & McCormick, 1999). Ca2þ entry through synaptic
L-type Ca
2þ
channels could occur in close proximity to HCN
channels. For cones and bipolar cells, it is tempting to speculate
that these mechanisms are involved in tuning the synaptic gain
during sustained illumination.
Acknowledgements
We thank Sarah Theissen, Petra Hoffmann, Mechthilde Bruns, the late Martina
Dumbsky, Gong-Sum Nam and Walter Hofer for technical assistance, Michael
Beyermann for the synthesis of HCN peptides, Heinz Wässle and Reinhard

Seifert for critically reading the manuscript and Anita Eckert for preparing the
manuscript. This work was supported by the Strategy Fund of the Helmholtz
Association of Research Centers (O1SF9904).
Abbreviations
aa, amino acids; bv, blood vessel; ChAT, choline acetyltransferase; CP, cone
pedicle; GCL, ganglion cell layer; HBSS, Hanks balanced salt solution; HCN,
hyperpolarization-activated and cyclic nucleotide-gated; INL, inner nuclear
layer; IPL, inner plexiform layer; IS, inner segment layer; NFL, nerve fibre
layer; PA, paraformaldehyde; PB, phosphate buffer; PBS, phosphate-buffered
saline; PK, protein kinase; ONL, outer nuclear layer; OPL, outer plexiform
layer; OS, outer segment layer; Vm, membrane potential.
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