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RESEARCH ARTICLE
Specialized photoreceptor composition in the raptor fovea
Mindaugas Mitkus
1
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Peter Olsson
1
|
Matthew B. Toomey
2
|
Joseph C. Corbo
2
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Almut Kelber
1
1
Lund Vision Group, Department of Biology,
Lund University, Lund, Sweden
2
Department of Pathology and Immunology,
Washington University School of Medicine,
St. Louis, Missouri
Correspondence
Mindaugas Mitkus, Lund Vision Group,
Department of Biology, Lund University,
S€
olvegatan 35, Lund 22362, Sweden.
Email: mindaugas.mitkus@biol.lu.se
Funding information
MM, PO, and AK were funded by the
Swedish Research Council (VR2012-2212),
the Human Frontier Science Program grant
#RGP0017/2011 and the K & A
Wallenberg Foundation (Ultimate Vision).
MT and JC were funded in part by Human
Frontier Science Program grant #RGP0017/
2011 and National Institutes of Health
grants RO1EY026672 and RO1EY024958.
MT was supported by fellowships from the
National Science Foundation (Award
#1202776), National Institutes of Health
(5T32-EY013360-12), and the McDonnell
Center for Cellular and Molecular
Neurobiology at Washington University,
St. Louis
Abstract
The retinae of many bird species contain a depression with high photoreceptor density known as
the fovea. Many species of raptors have two foveae, a deep central fovea and a shallower tempo-
ral fovea. Birds have six types of photoreceptors: rods, active in dim light, double cones that are
thought to mediate achromatic discrimination, and four types of single cones mediating color
vision. To maximize visual acuity, the fovea should only contain photoreceptors contributing to
high-resolution vision. Interestingly, it has been suggested that raptors might lack double cones in
the fovea. We used transmission electron microscopy and immunohistochemistry to evaluate this
claim in five raptor species: the common buzzard (Buteo buteo), the honey buzzard (Pernis apivorus),
the Eurasian sparrowhawk (Accipiter nisus), the red kite (Milvus milvus), and the peregrine falcon
(Falco peregrinus). We found that all species, except the Eurasian sparrowhawk, lack double cones
in the center of the central fovea. The size of the double cone-free zone differed between species.
Only the common buzzard had a double cone-free zone in the temporal fovea. In three species,
we examined opsin expression in the central fovea and found evidence that rod opsin positive
cells were absent and violet-sensitive cone and green-sensitive cone opsin positive cells were
present. We conclude that not only double cones, but also single cones may contribute to high-
resolution vision in birds, and that raptors may in fact possess high-resolution tetrachromatic
vision in the central fovea. J. Comp. Neurol. 000:000–000, 2016. V
C2016 Wiley Periodicals, Inc.
KEYWORDS
birds of prey, double cones, retina, rods, visual ecology, RRID: AB_2156055, RRID: AB_2315274,
RRID: AB_2158332, RRID: AB_2534069, RRID: AB_2534102
1
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INTRODUCTION
Visual spatial resolution (or visual acuity) defines the detail that can be
resolved in a visual scene. As in a camera, the spatial resolution of an
eye is determined by the anterior focal length (hereafter, focal length)
of the eye’s optical system and the density of the light sampling units
in the neural retina (Land & Nilsson, 2012). The larger the eye, the lon-
ger the focal length and the larger the image projected onto the retina.
The denser the receptor sampling array, the more spatial detail can be
extracted from the retinal image (Miller, 1979). Thus, in order to
achieve high spatial resolution, an animal has to have a large eye with a
dense photoreceptor array in the retina (Meyer, 1977).
Among all animals, Accipitriform and Falconiform raptors have the
most acute vision that has ever been measured (Fischer, 1969;
Reymond, 1985). Behavioral studies show that large raptors such as
Old World vultures (Fischer, 1969) and wedge-tailed eagles (Aquila
audax) (Reymond, 1985) have twice the spatial resolution of humans
(Land & Nilsson, 2012). These findings raise intriguing questions about
the basis of enhanced spatial resolution in raptorial birds.
Despite having body sizes much smaller than that of humans,
some birds of prey have eyes of equal or larger size (Martin, 1983; Rey-
mond, 1985, 1987). These large eyes allow for a long focal length that
results in a correspondingly large retinal image (Land & Nilsson, 2012).
In addition, many birds have central or temporal regions in the retina
with increased photoreceptor density (Meyer, 1977). These regions are
referred to as "areae" and may or may not contain a fovea. The fovea is
a region of the retina where photoreceptor densities are highest and
other retinal layers are fully or partially displaced, resulting in a depres-
sion, which constitutes the "fovea" proper. Unlike human eyes, which
have only one shallow central fovea, the eyes of many raptor species
J Comp Neurol. 2017;00–00 wileyonlinelibrary.com/journal/cne V
C2017 Wiley Periodicals, Inc.
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1
Received: 19 October 2016
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Revised: 13 January 2017
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Accepted: 7 February 2017
DOI 10.1002/cne.24190
The Journal of
Comparative Neurology
(as well as swallows, martins, terns, kingfishers, and some other birds;
Moroney & Pettigrew, 1987; Rochon-Duvigneaud, 1943) have two
foveae: a deep central fovea that views the lateral visual field, and a
shallower temporal fovea that views the frontal visual field (e.g.,
Oehme, 1964; Reymond, 1985, 1987).
To maximize visual acuity, the fovea should only contain photore-
ceptors contributing to high-resolution vision. Rods cannot operate in
the bright-light conditions required for optimal foveal function (Snyder
& Miller, 1977) and they are accordingly absent from the foveae of
some species. For example, the primate fovea, including that of
humans, is rod-free (e.g., Finlay et al., 2008; Packer, Hendrickson, &
Curcio, 1989) and the central areae or foveae of several bird species
have been found to be rod-free (Bruhn & Cepko, 1996; Coimbra, Col-
lin, & Hart, 2015; Querubin, Lee, Provis, & O’Brien, 2009). Further-
more, to achieve highest image quality the eye needs to avoid
chromatic aberration, which is most severe for the short wavelength
light (for more detail please see Discussion). Probably for this reason
blue-sensitive cones are absent from the central-most part of some pri-
mate foveae (e.g., Martin & Gr€
unert, 1999; Wikler & Rakic, 1990) leav-
ing only green and red-sensitive cones for the tasks of highest acuity.
These observations suggest that the demands of high-acuity vision
select for a specific photoreceptor complement in the fovea.
Birds are thought to utilize different subsets of their photoreceptor
complement for specific visual tasks (Hart, 2001b). As in many other
vertebrate taxa, rod photoreceptors mediate dim light vision. Color
vision is mediated by four types of single cone photoreceptors sensi-
tive to different portions of the light spectrum: ultraviolet/violet-sensi-
tive (UV/VS cones; with maximum sensitivity in the common buzzard
[Buteo buteo] at 405 nm), blue-sensitive (S cones; 449nm), green-
sensitive (M cones; 504nm), and red-sensitive (L cones; 567nm) cones
(Lind, Mitkus, Olsson, & Kelber, 2013; €
Odeen & Håstad, 2003). The
double cones have broad spectral sensitivity, and their function is a
matter of ongoing debate. Behavioral data suggest that double cones
contribute to high-resolution achromatic vision (Jones & Osorio, 2004;
Lind & Kelber, 2011; Martin & Osorio, 2008; Osorio, Jones, & Voro-
byev, 1999). For example, in budgerigars (Melopsittacus undulatus) vis-
ual acuity for achromatic gratings was determined as 10.5 cycles/
degree, while acuity for the red-green and blue-green gratings, which
were isoluminant and had no detectable contrast for double cones,
was 4.5 and 4.3 cycles/degree, respectively (Lind & Kelber, 2011). Simi-
lar results have been found for domestic chickens (Gallus gallus domesti-
cus; P. Olsson et al., unpublished data). It has also been suggested that
double cones may contribute to color (Lind & Kelber, 2011) and motion
vision (Campenhausen & Kirschfeld, 1998). While double cones typi-
cally constitute 40–55% of all photoreceptors in the mid-periphery of
the bird retina (Hart, 2001a; Martin & Osorio, 2008), studies of the
photoreceptor complement in the central retina are rare. Braekevelt
(1993) reports that outside the foveal regions the red-tailed hawk
(Buteo jamaicensis) has a rod: single cone: double cone ratio of 2:1:5
(i.e., 62.5% double cones), suggesting that functional specializations of
double cones may be important for raptor vision renown for high spa-
tial resolution. One might therefore be inclined to hypothesize that the
raptor fovea should also contain a high density of this cell type.
Relatively few researchers have investigated the photoreceptors of
raptor foveae in any detail and only using light microscopy. Oehme
(1964) did not find any rods in foveal cross-sections of the common
buzzard or the common kestrel (Falco tinnunculus). Surprisingly, he did
not differentiate between single and double cones although he
described both cone types in an earlier study on swifts and passerines
(Oehme, 1962). Reymond (1985, 1987) carefully investigated tangential
preparations of wedge-tailed eagle and brown falcon (Falco berigora)
foveae and did not find any double cones. Although Reymond’sresults
seem to contradict the studies suggesting that double cones mediate
high-resolution achromatic vision in birds (Jones & Osorio, 2004; Lind &
Kelber, 2011; Osorio et al., 1999), they were rarely mentioned in later
reviews of avian vision (G€
unt€
urk€
un, 2000; Hart, 2001b; Jones, Pierce, &
Ward, 2007; Martin & Osorio, 2008), and have never been followed up.
In the present study, we used transmission electron microscopy
(TEM) to investigate the claim that raptor foveae lack double cones.
Furthermore, we asked whether raptor foveae, similarly to humans,
lack rods and cone types sensitive to short-wavelength light. We stud-
ied four species of the order Accipitriformes, the common buzzard, the
honey buzzard (Pernis apivorus), the Eurasian sparrowhawk (Accipiter
nisus) and the red kite (Milvus milvus), and one species of the order Fal-
coniformes, the peregrine falcon (Falco peregrinus).
2
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MATERIALS AND METHODS
2.1
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Biological samples
We used eyes from common buzzards (n52specimens,oneadultand
one juvenile), honey buzzard (n51, juvenile), Eurasian sparrowhawks
(n52, one adult female and one juvenile), red kites (n53, two adults
and one juvenile), and peregrine falcon (n51, juvenile) shortly after the
birds had been euthanized by cervical dislocation. All birds were
severely injured wild specimens of unknown sex (except the adult
female Eurasian sparrowhawk) cared for by a bird rescue station in
southern Sweden. The birds were euthanized for reasons unrelated to
this study. Juvenile birds were fully grown flying individuals still in their
first set of plumage. The collection of the eyes from these specimens
was approved by the Swedish Environmental Protection Agency (per-
mit no. NV-00160-12). Some of the eyes were also used in an earlier
study on ocular media transmittance (Lind et al., 2013) or in other stud-
ies currently in preparation.
We used light microscopy (LM), TEM, and immunohistochemistry
to study retinal samples from four defined locations: the central fovea
(CF), the central retina (CR), the temporal fovea (TF), and the temporal
retina (TF). Approximate locations, with respect to each other and to
the pecten of the eye, are indicated in Figure 1. The central and tempo-
ral retinal pieces were taken approximately 3–6 mm from the central
and temporal retinae, respectively.
2.2
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Light and transmission electron microscopy
One eye of common buzzard (juvenile), one eye of honey buzzard, one
eye of Eurasian sparrowhawk (juvenile), three eyes of three red kites
2
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The Journal of
Comparative Neurology
MITKUS ET AL.
(adult and juvenile), and one eye of the peregrine falcon were used for
LM and TEM studies. Only retinal samples from the central fovea were
analyzed for the honey buzzard. The eyes were removed immediately
after euthanasia, hemisected and placed in fixative (2.5% glutaralde-
hyde in 0.1M sodium cacodylate buffer) for 24hr. The eyes were then
transferred into fresh 0.1 M sodium cacodylate buffer and stored at
48C until further processing. Small retinal samples (ca. 535 mm) were
cut and post-fixed in 1% osmium tetroxide, dehydrated in a graded
series of ethanol and acetone, and embedded in Epon. Thin retinal
cross-sections (2 lm) were made with a microtome (11800 Pyrami-
tome, LKB AB, Bromma, Sweden), stained with Azur II–Methylene
Blue, coverslipped with Entellan New (Merck, Darmstadt, Germany),
and visualized using a light microscope (Zeiss Axiophot equipped with
a Nikon DC-Fi1c digital camera). The orientation of the samples was
lost during the embedding procedure, and thus we cannot determine
whether samples were sectioned along the nasotemporal or dorsoven-
tral retinal axis. Ultrathin retinal cross-sections (50nm) were made with
an ultramicrotome (Ultracut UCT, Leica), stained with 2% uranyl ace-
tate and lead citrate in a LKB ultrastainer (LKB AB, Bromma, Sweden),
and visualized by TEM (120kV Jeol 1230 equipped with a Gatan Multi-
scan CCD camera, JEOL USA, MA).
To obtain tangential sections of the foveal region we used two dif-
ferent technical approaches. The first sample obtained in the study (the
central fovea of the red kite) was embedded in such a way that it could
be sectioned at a tangent to the photoreceptor axis and used directly
for TEM studies. All other foveal samples were first sectioned parallel
to the photoreceptor axis for inspection by LM until we found the
deepest part of the fovea, then the samples were rotated 90 degrees
and sectioned at a tangent to the receptor axis for inspection of cell
cross-sections by TEM. Because of this procedure, the sections for
TEM (except the central fovea of the red kite prepared with the first
method) contained only half of the foveal region. We measured the
extent of the double cone-free zones by scanning the samples directly
in the TEM using high magnification, which allowed us to identify the
cell profiles. Due to the difficulty in aligning the samples exactly parallel
to the external limiting membrane and because the photoreceptor layer
is not perfectly flat in the fovea (see Figure 2), photoreceptors could
not always be visualized at exactly the same level even in the same pic-
ture. The LM and TEM images were imported into Adobe Photoshop
CS6, where brightness, contrast, and image dimensions were adjusted.
To evaluate the relative proportion of the double cones we
counted photoreceptor cross-sectional profiles in the 25 325 lm
counting frames in the TEM or LM micrographs of the CF, CR, and TF
retinal regions, and in 50 350 lmcountingframesintheLMmicro-
graphs of the TR regions. All cells inside the counting frame and those
intersecting the acceptance lines, but not intersecting the rejection
lines, were included in the counts (Gundersen, 1977). Because tissue
shrinkage was not evaluated in this study, we did not measure photore-
ceptor dimensions or estimate the density.
2.3
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Morphological identification of the double cones
We identified double cones in transmission electron micrographs by
their typical paired inner segment cross-sectional profile, in which the
principal and accessory members are directly apposed to each other,
without intervening M€
uller cell processes (Braekevelt, 1993; Nishimura,
Smith, & Shimai, 1981). In retinal regions outside the foveae, these pro-
files could also reliably be detected in light micrographs. We did not
use other methods to discriminate double cones from the other recep-
tor types of the bird retina, such as the size and coloration of the oil
droplet (Kram, Mantey, & Corbo, 2010), the larger diameter of inner
segments visible in the whole-mounted retina (Coimbra et al., 2015) or
the position of the nuclei visible in cross-sectioned retina (Braekevelt,
1993, 1998), because all of these alternative methods turned out to be
less reliable in the foveal region of the retina where photoreceptors are
very narrow.
2.4
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Immunohistochemical identification of
photoreceptor subtypes in the fovea
Because of the limited number of samples we obtained, only one eye
of the common buzzard (adult), one eye of the Eurasian sparrowhawk
(adult), and one eye of the peregrine falcon (juvenile) were used for
immunohistochemical studies. Only CF retinal samples were available
for the Eurasian sparrowhawk and the peregrine falcon. The TF sam-
ples of these species were accidentally damaged during the following
preparation procedure. The eyes were removed shortly after the eutha-
nasia, hemisected, and placed in fixative (4% paraformaldehyde in
0.1 M phosphate buffered saline [PBS] with 3% sucrose) for 1hr at
room temperature. After rinsing in PBS (twice for 5 min), small retinal
samples (ca. 4 34 mm) were cut, placed in 30% sucrose solution in PBS
and incubated at 4 8C. Once the retinal samples had equilibrated, an
equal volume of cryostat freezing section medium (Neg-50
TM
, Richard-
Allan Scientific
TM
)wasadded.After2–3 hr the sucrose/freezing
medium mixture was replaced with 100% freezing medium and incu-
bated overnight at 4 8C. Semi-thin retinal cross-sections (10 lm) were
obtained using a cryostat (Microm HM 560, Thermo Scientific), col-
lected on gelatin-chrome alum coated glass slides and stored at 280 8C
until further processing. The orientation of the samples was lost during
the procedures and thus we cannot determine whether the samples
were sectioned along the nasotemporal or dorsoventral retinal axis.
FIGURE 1 Schematic drawing of the eyecup in the skull with
approximate positions, where the retina samples were taken.
CF 5central fovea; CR 5ce ntral retina; TF 5temporal fovea;
TR 5temporal retina. Redrawn and modified from “Oehme (1964)”.
MITKUS ET AL. The Journal of
Comparative Neurology
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3
For immunohistochemical staining, retina sections were thawed at
room temperature and washed three times in PBS. To facilitate the vis-
ualization of the photoreceptor outer segments that were hidden by
the intercalated processes of the retinal pigment epithelium we
bleached the sections with 10% hydrogen peroxide solution in PBS for
16 hr at 4 8C following the methods of Manicam et al. (2014). After
rinsing and bleaching, we blocked the retinal sections in PBS with 0.5%
Triton X-100, and 2% normal goat (A-11001, Life Technologies, RRID:
AB_2534069; for rod opsin and green opsin primary antibodies) or 2%
normal donkey serum (A-11055, Life Technologies, RRID:
AB_2534102; for UV/Violet opsin antibody) for 1hr at room tempera-
ture. We then applied the primary antibodies diluted in blocking buffer
described above and incubated the sections for 16hr at 4 8C. We again
rinsed the sections three times in PBS, applied the secondary antibod-
ies diluted in blocking buffer, and incubated them in the dark for 1hr at
room temperature. Details of the antibodies and working dilutions are
given below and in Table 1. Finally, the sections were rinsed three
times in PBS, counterstained with 40,6-diamidino-2-phenylindole
FIGURE 2 Raptor foveae. LM images of retinal cross-sections through the central (left column) and temporal (right column) foveae of the
common buzzard (a, b), Eurasian sparrowhawk (c, d), peregrine falcon (e, f), red kite (g, h, j), and honey buzzard (i). Magnified image of the
red kite temporal fovea (j) showing tilted retinal columns in the inner nuclear layer on the sides of the center of the fovea (indicated by
arrows) that can also be found on all central and temporal foveae. There was no temporal fovea in the honey buzzard retina. The dark blue
band in the center of the common buzzard central fovea (a) is a sectioning/staining artifact. PRL 5photoreceptor layer; ONL 5outer nuclear
layer; OPL 5outer plexiform layer; INL 5inner nuclear layer; IPL 5inner plexiform layer; GCL 5ganglion cell layer. Scale bars: 100 lm
4
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The Journal of
Comparative Neurology
MITKUS ET AL.
(DAPI; D9542, Sigma Aldrich, St. Louis, MO) to label nuclei, and cover-
slipped with Vectashield mounting media (H-1200, Vector Laborato-
ries, Burlingame, CA).
The sections were imaged with a spinning disk confocal micro-
scope (BX61WI; Olympus, Tokyo, Japan). The raw images from the
confocal microscope were imported into Adobe Photoshop CS6, where
the intensity profiles were adjusted and images from opsin staining
were merged with DAPI images. For a given species, the exposure
times and image intensity adjustments were done the same way for all
images.
2.5
|
Antibody characterization
To identify rod photoreceptors we used a monoclonal anti-rhodopsin
antibody (Ret-P1, MAB5316, Millipore, RRID: AB_2156055), which
was raised against amino acids 4–10 (TEGPNFY) at the N-terminus of
rhodopsin of the membrane fraction from adult rat (Rattus norvegicus)
retina (Barnstable, 1980; Coimbra et al., 2015; Silver et al., 1988). This
antibody has been shown to specifically label rod photoreceptors in
other vertebrates including fish, salamander, pigeon, and mice (Barnsta-
ble, 1980; Querubin et al., 2009; Taylor, Loew, & Grace, 2011). In sev-
eral passerine species it has been shown to strongly label rods, but it
also appears to have some cross-reactivity with Rh2 opsin and there-
fore weakly label green-sensitive cones (Coimbra et al., 2015).
To further parse the distribution of rods and green-sensitive cones
we also stained retinae with a second monoclonal anti-rhodopsin anti-
body (Rho4D2, ab98887, Abcam, RRID: AB_2315274), which was
raised against amino acids 2–39 at the N-terminus of bovine rhodopsin
(NGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSM, Hicks &
Molday, 1986). This antibody has been shown to reliably label rods in
fish, amphibians and mammals (New, Hemmi, Kerr, & Bull, 2012), and
strongly label both rods and green-sensitive cones in chickens (Fischer,
Stanke, Aloisio, Hoy, & Stell, 2007). The labeling of green-sensitive
cones with anti-rhodopsin antibodies reflects the fact that green-
sensitive cone opsin and rhodopsin share a high degree of amino acid
homology (Okano, Kojima, Fukada, Shichida, & Yoshizawa, 1992). We
interpreted the presence of Rho4D2 positive cells in regions where
Ret-P1 staining was weak or absent as indicating the presence of
green-sensitive cones and the absence of rods.
Finally, we labeled violet-sensitive cones using a goat polyclonal
antibody (OPN1SW, SC14363, Santa Cruz, RRID: AB_2158332), which
was raised against amino acids 8–27 at the N-terminus of human blue
cone opsin (EFYLFKNISSVGPWDGPQYH, Schiviz, Ruf, Kuebber-Heiss,
Schubert, & Ahnelt, 2008) This antibody has been shown to be ultra-
violet/violet cone-specific in birds (Nießner et al., 2011), and reflects
the fact that human blue cone opsin (SWS1) is orthologous to the avian
ultraviolet/violet-sensitive cone opsin (Hunt & Peichl, 2014).
3
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RESULTS
3.1
|
General observations about raptor foveae
The retinae of the common buzzard, the Eurasian sparrowhawk, the
red kite and the peregrine falcon all had a deep central fovea and a
shallower temporal fovea (Figure 2). The temporal fovea of the red kite
appeared as a very shallow indentation (Figure 2h), but it was still pos-
sible to visually locate it in the unfixed and later in the fixed unstained
retina. In the stained cross-sections the presence of the fovea was dis-
tinguished from artifactual indentation by the thickening of the retinal
layers and tilting of the cell columns in the inner nuclear layer, on both
sides of the fovea (Figure 2j). We could not identify a temporal fovea
TABLE 1 The primary and secondary antibodies used in this study
Antibody Immunogen
Source, host and clonality,
cat #, RRID Dilution
Primary antibody Primary
antibody
dilution
Rho4D2 Bovine (Bos taurus) rhodopsin:
NGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSM
Abcam, mouse monoclonal,
cat # ab98887, RRID: AB_2315274
1:500
Ret-P1 Rat (Rattus norvegicus) retina membrane fraction,
epitope - rhodopsin: TEGPNFY
Millipore, mouse monoclonal,
cat # MAB5316, RRID: AB_2156055
1:300
OPN1SW Human (Homo sapiens) blue-sensitive opsin:
EFYLFKNISSVGPWDGPQYH
Santa Cruz, goat polyclonal,
cat # SC14363, RRID: AB_2158332
1:300
Secondary antibody Secondary
antibody
dilution
Goat-anti-mouse IgG (H1L)
Secondary Antibody, Alexa
Fluor 488 conjugated
Gamma Immunoglobins Heavy and Light chains Life Technologies, goat polyclonal,
cat # A-11001, RRID: AB_2534069
1:1,000
Donkey-anti-goat IgG (H1L)
Secondary Antibody, Alexa
Fluor 488 conjugated
Gamma Immunoglobins Heavy and Light chains Life Technologies, donkey polyclonal,
cat # A-11055, RRID: AB_2534102
1:1,000
MITKUS ET AL. The Journal of
Comparative Neurology
|
5
of the honey buzzard either in fresh or fixed retina, suggesting that this
species may instead have a temporal area without a foveal depression.
3.2
|
The raptor central fovea lacks rods
Anti-rhodopsin immunolabeling showed little or no reactivity in the cen-
tral region of the Eurasian sparrowhawk and peregrine falcon central
fovea (Figure 3c,e). We detected Ret-P1 positive cells in the common
buzzard central fovea (Figure 3a); however, the intensity of labeling was
much weaker than in nonfoveal regions (Figure 3b) suggesting that this
was most likely green-sensitive cone opsin cross-reacting with the Ret-
P1 antibody (Coimbra et al., 2015). Therefore, we conclude that rods
are absent from the central most portion of the central fovea of these
three species. We also did not detect Ret-P1-positive cells in the tem-
poral fovea of the common buzzard (Figure 3d). It was not possible to
precisely measure the area of these rod-free zones in the retinal prepa-
rations. However, evaluation of the available images suggests that the
size of the rod-free zone differs between species (Figure 3).
3.3
|
The raptor fovea lacks double cones
We did not observe double cones in the central fovea of the common
buzzard, honey buzzard, red kite or peregrine falcon by TEM
(Figures 4a,i,m and 5). However, double cones were present in the cen-
tral fovea of the Eurasian sparrowhawk (Figure 4e). In the red kite cen-
tral fovea, the double cone-free zone had a diameter of approximately
200 lm. The fovea was smaller in the common buzzard and the honey
buzzard (approximately 100 lm), and smallest in the peregrine falcon
(approximately 30 lm). Outside the double cone-free zone of the fal-
con (Figure 4m), double cones were present, but rare. In the temporal
fovea of the common buzzard we found a small zone (only approxi-
mately 25 lm in diameter) without double cones (Figure 4c), but we
did not detect a double cone-free zone in the temporal fovea of any of
the other species (Figure 4g,k,o). The proportion of double cones
among all photoreceptors varied widely across the species (Table 2). As
we only had one sample per species and per retinal area, we cannot
determine the extent to which these differences are species-specific or
due to interindividual variation.
FIGURE 3 Rhodopsin expression in the rods of the raptor retina. Confocal images of retinal cross-sections labeled with antibodies directed
to rhodopsin (Ret-P1; green) of the central fovea (a), the temporal retina (b), and the temporal fovea (d) of the common buzzard, the central
fovea of the peregrine falcon (c) and the central fovea of the Eurasian sparrowhawk (e). DAPI used to counter-stain nuclei is shown in pur-
ple. For labeling of retinal layers see legend of Figure 2. Scale bars: 50 lm
6
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The Journal of
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MITKUS ET AL.
3.4
|
Violet- and green-sensitive cones are present in
raptor central foveae
We were not able to discriminate between single cone types morpho-
logically, but we did observe OPN1SW-positive cones in the common
buzzard, the Eurasian sparrowhawk and the peregrine falcon retinae
(Eurasian sparrowhawk and peregrine falcon temporal foveae were not
available), including the central fovea of each species (Figure 6). This
indicates that violet-sensitive single cones are present in the central
foveae of these species. We also observed Rho4D2 positive cells in
the retinae of these three species (Eurasian sparrowhawk and pere-
grine falcon temporal foveae were not available). In the central fovea
of the Eurasian sparrowhawk, the staining of outer segments is less
obvious and only visible as dots (Figure 6c,d), most likely because the
outer segments were sectioned at an oblique angle in this sample. In
the central foveae of these species we observed clear staining with
Rho4D2, a marker of both rods and green-sensitive cones (Figure 6).
However, in these regions, staining with the more specific rod marker
FIGURE 4 Tangential retinal sections through the photoreceptor inner segments of the common buzzard (a–d), Eurasian sparrowhawk (e–
h), red kite (i–l), and peregrine falcon (m–p) retina. TEM (a–g, i, j, l–o) and LM (h, k, p) images of the central fovea (a, e, i, m), the central
retina close to the fovea (b, f, j, n), the temporal fovea (c, g, k, o), and the temporal retina (d, h, l, p). Arrows point to the double cones. At
the edge of the double cone-free zone of the common buzzard temporal fovea some double cones are also visible (arrow in c). Scale bars:
5lm
MITKUS ET AL. The Journal of
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7
Ret-P1 was weak or absent (Figure 3). We interpreted this pattern to
indicate that the central fovea of these species lacks rods, but does
contain green-sensitive cones.
4
|
DISCUSSION
The goal of our study was to examine the photoreceptor complement
of the raptor fovea to better understand the adaptations underlying
the exceptional visual acuity of these birds. We had hypothesized that
the fovea would be specifically populated with double cones, a class of
photoreceptors that has been ascribed a range of functions including
the mediation of high-resolution achromatic vision in birds (Jones &
Osorio, 2004; Lind & Kelber, 2011; Osorio et al., 1999). We also
hypothesized that dim light-sensitive rods and violet-sensitive cones
would be excluded from the fovea. We found that rods and double
cones were absent from the foveae of several raptor species while
violet-sensitive cones were present, suggesting that not only double
cones, but also single cones may contribute to high-resolution vision in
birds, and that raptors may in fact possess high-resolution tetrachro-
matic vision in the central fovea.
4.1
|
Rod-free zones in the foveae
Foveae of primates (Finlay et al., 2008), at least some fish (Collin & Col-
lin, 1999) and some birds (Bruhn & Cepko, 1996; Coimbra et al., 2015;
Querubin et al., 2009) have been observed to be rod-free. We found
rod-free zones in and around the central fovea of the common buzzard,
the Eurasian sparrowhawk and the peregrine falcon as well as the tem-
poral fovea of the common buzzard (Figure 3; red kite and honey buz-
zard were not investigated in this respect). While the absence of rods
in the central fovea of the Eurasian sparrowhawk and the peregrine fal-
con is clear, there was some staining in the common buzzard foveae.
The Ret-P1 antibody is known to have weak cross-reactivity with the
Rh2 opsin expressed in green-sensitive cones (Coimbra et al., 2015).
The stained outer segments in the common buzzard foveae appear to
be much narrower and have a fainter signal than in the temporal retina
(Figure 3b) or in the regions farther away from the temporal fovea (Fig-
ure 3d). As Oehme (1964) also reported the absence of rods from the
central and temporal foveae of the common buzzard and common kes-
trel, we consider it most likely that Ret-P1 cross-reacts with the Rh2
opsin in these regions.
Even though we could not precisely quantify the area of the rod-
free zones in our histological preparations, the apparent cross-sectional
diameter of these zones suggests that there might be large variation in
the extent of the rod-free zone among raptor species, similar to what
has recently been reported in some passerine birds (Coimbra et al.,
2015). Rod-free zones have also been reported in the area centralis of
chickens (Bruhn & Cepko, 1996) and in the fovea of pigeons (Columba
livia; Querubin et al., 2009). The absence of rods in and around the
fovea allows for a higher density of cones, which is needed for optimal
high-acuity vision in bright light.
4.2
|
Double-cone free zones in foveae
In this study, we confirm earlier suggestions that double cones are
absent from the fovea of some raptor species (Reymond, 1985, 1987).
We found large differences in the extent of the double cone-free zone
in the central foveae among the species we investigated. Unlike the
foveae of the other raptors examined, the central fovea of the Eurasian
sparrowhawk contained double cones. With the exception of the tem-
poral foveae of the common buzzard, which had a small double cone-
FIGURE 5 TEM micrograph of tangential retinal section through
the photoreceptor inner segments of the honey buzzard central
fovea. S cale b ar: 5 lm
TABLE 2 Summary of the double cone and rod presence/absence in the retina of five raptor species investigated in this study
Species
Common buzzard Honey buzzard Eurasian sparrowhawk Red kite Peregrine falcon
Retinal region
Double
cones Rods
Double
cones Rods
Double
cones Rods
Double
cones Rods
Double
cones Rods
Central fovea No No No n.a. Yes (50.0%) No No n.a. No No
Central retina Yes (48.4%) n.a. n.a. n.a. Yes (37.0%) n.a. Yes (20.6%) n.a. Yes (12.3%) n.a.
Temporal fovea No No n.a. n.a. Yes (58.0%) n.a. Yes (24.1%) n.a. Yes (35.6%) n.a.
Temporal retina Yes (39.6%) Yes n.a. n.a. Yes (51.4%) n.a. Yes (21.3%) n.a. Yes (33.7%) n.a.
n.a. 5not applicable (the sample was not available or not analysed with a certain method)
8
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The Journal of
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MITKUS ET AL.
free zone, the temporal foveae of the red kite, the Eurasian sparrow-
hawk and the peregrine falcon all contained double cones.
In previous studies Reymond (1985, 1987) did not find double
cones in the central and temporal foveae of the brown falcon. She also
did not find double cones in the central fovea of the wedge-tailed
eagle, but did not note the presence or absence of the double cones in
the temporal fovea of this species. In neither of the studies does she
mention the extent of the double cone-free zones. Reymond’s and our
findings indicate variation in the double cone distribution among raptor
foveae. Whether this is the case for other avian clades is currently
unknown. However, variation in cone type proportions between differ-
ent nonfoveal retinal regions is well documented in many bird species
(Hart, 2001a).
One of the possible explanations for the absence of double cones
in the fovea may be adaptation to maximal receptor density. Two
members of a double cone together have greater inner segment
FIGURE 6 Opsin expression in the single cones of the raptor central fovea. Confocal images of retinal cross-sections labeled with antibod-
ies directed to two opsins: SWS1 (violet-sensitive cones; OPN1SW, blue, left column) and Rh2 (green-sensitive cones; Rho4D2, green, right
column) of the common buzzard (a, b), Eurasian sparrowhawk (c, d), and peregrine falcon (e, f). DAPI used to counter-stain nuclei is shown
in purple. For labeling of retinal layers see legend of Figure 2. Scale bars: 50 lm
MITKUS ET AL. The Journal of
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9
diameter than a single cone, and contribute two outer segments to this
layer. However, there are indications that both members function as a
single receptive unit, because they are optically and electrically
coupled. Two outer segments are optically isolated from other photo-
receptors by pigmented apical processes of the retinal pigment epithe-
lium cells, but not from each other (Hart, 2001b; Young & Martin,
1984). In addition, calculations indicate that due to the properties of
the inner segments "optical cross-talk" occurs between two members
of the double cone (Wilby, 2014). Furthermore, the presence of gap
junctions between the inner segments of the principle and accessory
members suggests that they are sharing, at least partly, their electrical
signals (Smith, Nishimura, & Raviola, 1985). Therefore, double cone
inclusion in the fovea would limit the maximum possible receptive unit
density essential for detailed spatial and chromatic tasks in bright light.
The exclusion of double cones from the region of highest acuity in spe-
cies renowned for their high-resolution vision suggests that not only
double cones, but also single cones contribute to achromatic high-
resolution vision in birds.
4.3
|
Single cones and the problem of
chromatic aberration
To achieve highest image quality, the optical system needs to focus
light of all wavelengths onto the same focal plane. However, light of
different wavelengths is refracted to varying extents when passing
through a lens making it impossible to focus all wavelengths on a single
focal plane without additional optical adaptations. This phenomenon,
known as chromatic aberration, is particularly pronounced for short
wavelengths of light and large lenses, and can compromise visual acu-
ity. To reduce this problem, the primate visual system filters out shorter
wavelengths in the lens, thereby limiting the spectral range of light
reaching the retina (Douglas & Jeffery, 2014). In addition, blue-
sensitive cones (that are homologous to the violet-sensitive cones of
raptors) are absent from the central-most part of some primate foveae
(e.g., Martin & Gr€
unert, 1999; Wikler & Rakic, 1990). Thus, in these
species, high-resolution vision is restricted to longer wavelengths. The
size of the blue-sensitive cone-free zone is smaller in smaller eyes and
absent in the common marmoset (Callithrix jacchus), which has the
smallest eye of the primate species so far investigated (Martin &
Gr€
unert, 1999). These examples indicate that smaller primate eyes with
lower spatial resolution can cope with the degree of chromatic aberra-
tion they experience, however, larger eyes may not. Spatial resolution
in the foveae of raptors is similar to or even higher than in primates
(Fischer, 1969; Reymond, 1985, 1987). As the ocular media of raptors
transmit more short-wavelength light than those of primate eyes (Lind
et al., 2013), chromatic aberration should be a more serious problem
for these species than for primates. Therefore, we were surprised to
find violet-sensitive cones throughout the fovea. We consider it likely
that the other two cone types, blue-sensitive (S) cones and red-
sensitive (L) cones, are present in the raptor fovea as well, possibly
allowing for tetrachromatic color vision in the central fovea.
How do raptors avoid the problem of chromatic aberration that is
predicted to occur in a fovea containing cones with sensitivities ranging
from below 400 nm (violet) to almost 700 nm (red)? Chromatic aberra-
tion is a serious problem only if the optical system has a wide pupil
aperture as compared to the focal length of the eye. Thus, in order to
avoid chromatic aberration raptors could close the pupil when they
need a well-focused color image. Although Miller (1979) reports that
raptors with shorter focal lengths than humans have larger pupil diame-
ters even in the bright daylight, falconers report that their birds narrow
the pupil when fixating on an object before taking flight (Simon Potier,
personal communication). It has also been suggested that the multifocal
lenses found in various vertebrates solve this problem by providing the
retina with a well-focused image for all wavelengths (Kr€
oger, Campbell,
Fernald, & Wagner, 1999). Two accipitriform species have been found
to have bifocal lenses (Lind, Kelber, & Kr€
oger, 2008), but so far, no
functional evidence supporting this hypothesis has been published.
Therefore, it remains unclear, which mechanisms allow raptors to use
the highly resolved tetrachromatic image created in the central fovea.
ACKNOWLEDGMENTS
We are very grateful to Kenneth Bengtsson for help with collecting
samples, and to Eva Landgren, Carina Rasmussen and Ola Gustafs-
son for expert help with tissue processing. We thank Jo~
ao Paulo
Coimbra for useful comments and suggestions on an earlier version,
which improved the manuscript.
CONFLICT OF INTEREST
The authors have no known conflicts of interest that could inap-
propriately influence this work.
ROLE OF AUTHORS
MM conceived the project and planned it in collaboration with PO,
MT, JC and AK. MM and PO did the retinal sampling and TEM anal-
ysis. MT did the immunohistochemistry analysis. MM, PO, MT, JC
and AK analyzed the data and wrote the paper.
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How to cite this article:MitkusM,OlssonP,ToomeyMB,
Corbo JC, Kelber A. Specialized photoreceptor composition in
the raptor fovea. JCompNeurol.2017;00:000–000. https://doi.
org/10.1002/cne.24190
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MITKUS ET AL.
