Introduction
Photoreceptor mitochondria are commonly found in tight clusters in the distal ellipsoid portion of photoreceptor inner segments, directly beneath the outer segment. These mitochondria were originally described as ellipsoidal in shape (Sjostrand, Reference Sjostrand1953), but later work showed that they can exhibit great morphological diversity (Ishikawa & Yamada, Reference Ishikawa and Yamada1969). Structurally distinct mitochondria are present within individual ellipsoids, within ellipsoids of different types of photoreceptors of the same species, and within ellipsoids of different species (Ishikawa & Yamada, Reference Ishikawa and Yamada1969; Kljavin, Reference Kljavin1987; Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003; Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005).
Some highly modified mitochondria are thought to function not only in energy production but also in photoreceptor optics by funneling light onto the outer segment (Hoang et al., Reference Hoang, Linsenmeier, Chung and Curcio2002), similar to the function of oil droplets in the retinas of birds, reptiles, and some fishes (Ishikawa & Yamada, Reference Ishikawa and Yamada1969; Mariani, Reference Mariani1987; Hárosi & Novales Flamarique, Reference Hárosi and Novales Flamarique2012). One example of modified photoreceptor mitochondria is the ellipsosome in the retina of some teleost fishes (MacNichol et al., Reference MacNichol, Kunz, Levine, Harosi and Collins1978; Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi2000). Giant mitochondria, also referred to as megamitochondria, are another type of atypical mitochondria that is present in the photoreceptor ellipsoids of many fish and mammalian species, including zebrafish (Collins et al., Reference Collins, Collins and Ali1996; Knabe et al., Reference Knabe, Skatchkov and Kuhn1997; Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003; Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005). Megamitochondria are large, electron dense, and located in the distal portion of inner segments (Collins et al., Reference Collins, Collins and Ali1996; Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003; Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005). They contain irregularly shaped and/or reduced numbers of cristae (Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003) and are significantly larger in size (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012) than the surrounding “regular” mitochondria.
Branchek and Bremiller (Reference Branchek and Bremiller1984) and Kljavin (Reference Kljavin1987) described the development of zebrafish photoreceptors. However, neither study reported the morphology of mitochondria in the different types of photoreceptors nor how mitochondrial morphology changed during development. Kim et al. (Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005) reported the presence of megamitochondria in the rods and cones of adult zebrafish, but their presence during development was not studied. Previously, we described the ultrastructure of the adult zebrafish retina, focusing on photoreceptor structure and their connections in the outer plexiform layer (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012). In that work, we reported the presence of megamitochondria in photoreceptor inner segments and measured their longest dimension (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012). Results from our previous studies suggested that megamitochondria may not be present in all photoreceptor types and that there may be a difference in distribution between larval and adult tissue. Consequently, in the present study, we examined the photoreceptors in developing and adult zebrafish retinas in order to (1) describe the ultrastructure of mitochondria in the photoreceptor ellipsoid during development, (2) determine whether mitochondrial morphology varied among photoreceptors, and (3) identify their absorbance using microspectrophotometry. This study contributes to our understanding of mitochondrial form and function in vertebrates.
Materials and methods
Animal care and maintenance
Eyes used for ultrastructural analysis were collected from adult (∼2.5 cm long) zebrafish (Danio rerio, Hamilton), obtained from a local supplier (Petsmart, Bowie, MD). Larval fish were obtained from in-house breeding of adult animals. Embryos were collected and maintained in fresh water aquaria at 28–29°C under a 14-h light/10-h dark photoperiod until aged 4, 8, and 15 days postfertilization (dpf). All larvae were fed from a culture of mixed Paramecia (P. aurelia, P. caudatum, and P. micronucleatum) beginning at 4 dpf. Food density in the larval containers was maintained at a minimum of 10,000 Paramecia per liter. Adult zebrafish were maintained under the same light and temperature conditions and fed Tetramin flakes daily.
All retinas used were collected approximately 3 h after the lights were turned on, and were therefore light-adapted. Fish were sacrificed in the morning by overdose with 0.02% tricane methanosulfate (MS-222; Sigma Chemical Co, St. Louis, MO). Adult fish were decapitated and the eyes enucleated and prepared for electron microscopy. Each eye was cut into hemispheres parallel to the anterior–posterior axis of the eye, with the lens and cornea anterior and the retina posterior, to facilitate penetration and epoxy resin infiltration of the tissue. In the case of larval fish, individual whole larvae were prepared for electron microscopy.
Light and transmission electron microscopy
For transmission electron microscopy of adult zebrafish, a total of six eyes from three adult fish were examined. To determine the best fixative to use, two eyes were fixed in each of three glutaraldehyde-based primary fixatives (Chapman et al., Reference Chapman, Tarboush, Eagles and Connaughton2009): Millonig’s fixative (5% glutaraldehyde in double strength phosphate buffer with CaCl2, Millonig, Reference Millonig1961; Sabatini et al., Reference Sabatini, Bensch and Barrnet1963), cacodylate-containing fixative (2.5% glutaraldehyde in 0.18 M sodium cacodylate, Osinchak, Reference Osinchak1964), and 2.5% glutaraldehyde in 0.135 M sodium cacodylate and 1% tannic acid (Hayat, Reference Hayat2000). Eyes were then washed 4×, 15 min in their respective buffers (used in the primary fixatives) and then postfixed for 2 h in 1% osmium tetroxide, buffered to a pH of 7.2 at 4°C. The three primary fixatives provided adequate comparable preservation of adult retina; most micrographs were generated from the Millonig’s fixed tissue. Based on these results, two larvae from each age group were fixed in Millonig’s glutaraldehyde fixative. The specimens were then dehydrated through a graded (50, 70, 85, 95, 100, and 100%) ethanol–water series followed by two 15-min washes in propylene oxide. The tissue was infiltrated overnight at 20°C in a 1:1 mixture of epoxy resin and propylene oxide (Luft, Reference Luft1961). A 3:1 mixture of epoxy resin and propylene oxide was used to further infiltrate the tissue at 20°C for another 9 h. The tissue was then embedded in epoxy resin and placed in a polymerizing oven at 45°C for 24 h, followed by another 24 h at 60°C. Embedded tissue blocks of adult fish eyes were trimmed such that the cutting face of the block was a transverse section of the eye’s hemisphere with lens, cornea, and retina in the same cutting plane. For the larvae, the embedded tissue blocks were trimmed such that the head of the larvae with both eyes was in the cutting face. Thus, each section usually contained both eyes of a larva. Thick (0.25–1.0 µm) and thin (50 nm) sections were cut with a Porter-Blum MT2-B ultramicrotome (Ivan-Sorvall, Inc., Norwalk, CT). The thick sections were mounted on glass slides and stained with an aqueous mixture of 1% methylene blue, 1% azure A, and 1% sodium borate (Richardson et al., Reference Richardson, Jarett and Finke1960). Most photomicrographs were taken with a Bausch and Lomb research microscope equipped with Nikon lenses using Kodak Ektapan film and developed in Kodak Microdol-X. The image showing the transverse section of the adult retina (Fig. 4) was captured using an Olympus BX61 compound microscope (Olympus America, Inc., Center Valley, PA) and MetaMorph software. The ultrathin sections were collected on 300 or 400 mesh copper grids and stained with uranyl acetate in 50% ethanol for 20 min (Gibbons & Grimstone, Reference Gibbons and Grimstone1960) followed by 0.4% lead citrate for 10 min (Venable & Coggeshall, Reference Venable and Coggeshall1965). These were viewed and micrographs acquired with a JEOL-1010 transmission electron microscope (JEOL Ltd., Akashima, Japan).
Transverse section of adult zebrafish retina. Photoreceptors, located distally, have outer segments arranged in tiers. In this photomicrograph, dark ellipsoid regions (electron-dense mitochondria) in the inner segments are evident (arrows). OPL: outer plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer. Scale bar = 15 μm.
Observations were made on sagittal sections of the eye. The study focused on photoreceptors located centrally, behind the lens. Measurements of mitochondria in the inner segment of photoreceptors were collected from enlarged prints made from electron micrographs. Densitometry measurements of ellipsoidal mitochondria in photoreceptors of adult fish were performed using Image J and statistically analyzed using SPSS (ver 20) software. Densitometry values are given as mean optical density (OD) ± s.d.
Microspectrophotometry
Individual adult fish (N = 10) were dark adapted for 6–8 h. Following this adaptation period, the fish were killed by quick spinal bisection and decerebration, one eye enucleated, and the retina removed under infrared illumination. Small pieces of retina were teased apart and mounted in a drop of Ringer’s solution between two No. 1.5 glass microscope coverslips. This preparation was sealed around the edges and mounted on the gliding stage of the dichroic microspectrophotometer (DMSP) for viewing under infrared illumination using a closed circuit television system. The DMSP is a computer-controlled, wavelength-scanning, single-beam photometer that simultaneously records average and polarized transmitted light fluxes through microscopic samples (Hárosi, Reference Hárosi1987; Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi2000, Reference Novales Flamarique and Hárosi2002). The DMSP was equipped with ultrafluar (Zeiss) objectives: 32/0.4 for the condenser and 100/1.20 for the objective. With the aid of reference measurements recorded through cell-free areas, individual photoreceptor outer and inner segments were illuminated sideways with a measuring beam of rectangular cross section of ∼2 × 0.6 μm. Inner segment measurements targeted the ellipsoid area immediately adjacent to the base of the outer segment, as this is where megamitochondria had been observed by electron microscopy. Absolute absorbance spectra were computed in 2 nm increments from the obtained transmittances (each spectrum consisted of an average of eight scans). The solid spectra (fits) for the visual pigments were derived from experimental data by Fourier filtering (Hárosi, Reference Hárosi1987).
Results
Adults
Adult zebrafish rods had long uniform outer segments of similar width to the inner segments (Fig. 1) and contained fewer mitochondria than cones. Adult rods had both small (regular) and giant (mega) mitochondria of low electron density (E-LM, Fig. 1), as judged by the intensity of their staining. The cristae were clearly visible.
Ellipsoid region of adult rod photoreceptors. Rod ellipsoid is packed with megamitochondria, as previously reported by Kim et al. (Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005), and smaller mitochondria. Unlike in adult cones, all rod mitochondria in adult fish stain evenly. CS: ciliary stalk of the modified cilium of the rod outer segment. Double arrow: ellipsoid of the inner segment. E-LM: large electron-lucent mitochondria. Scale bar = 1 μm. (Reprinted from Tissue and Cell, vol 44, Tarboush et al., Ultrastructure of the distal retina of the adult zebrafish, Danio rerio, 264–279, 2012, with permission from Elsevier.)
The mitochondria in adult cones were morphologically diverse. Both large and small mitochondria were present in all cones (Fig. 2A and 2B). In one cone type, ellipsoid mitochondria had two different electron densities. The first mitochondrial type was electron-dense and occupied the sclerad (distal) and central position relative to the second mitochondrial type which was electron-lucent and located vitread. Some of the largest electron-dense mitochondria (E-DM) were very irregular in shape (Fig. 2A and 2B). These mitochondria resembled ellipsosomes described in the cone ellipsoids of other fish species (e.g., Ishikawa & Yamada, Reference Ishikawa and Yamada1969; Fishelson et al., Reference Fishelson, Ayalon, Zverdling and Holzman2004). Additionally, some of the large mitochondria were so electron-dense that cristae were visible (E-DM, Fig. 3).
Ellipsoid region of adult DC photoreceptors (A). The ellipsoids of DCs contain large electron-dense mitochondria (E-DM) and large electron-lucent mitochondria (E-LM). Note that both members of the DC pairs contain the two types of mitochondria. A gradation in mitochondrial density from electron-lucent (vitread) to electron-dense (sclerad) is clear in the DCs. A SC is also visible in this section and shows a similar gradation in mitochondrial electron density. N: cone nucleus. Scale bar= 1 μm. (B) N: nucleus of the DC. Scale bar = 1 μm. (Reprinted from Tissue and Cell, vol 44, Tarboush et al., Ultrastructure of the distal retina of the adult zebrafish, Danio rerio, 264–279, 2012, with permission from Elsevier.)
Morphologically different types of mitochondria are present in adult cone photoreceptor inner segments. A longitudinal section, cut at an oblique angle, showing ellipsoids at the right with only electron-lucent mitochondria (E-LM) with visible cristae, alternating with ellipsoids at the left that contain two distinct types of mitochondria: very electron-dense mitochondria (E-DM) and smaller more electron-lucent mitochondria. The increase in density seems to be due to an increase in the density of the mitochondrial matrix. Based on the location, the vitread-most ellipsoids with both electron-dense mitochondria and electron-lucent mitochondria belong to UV cones, whereas the sclerad-most ellipsoids that also contain both electron-dense mitochondria and electron-lucent mitochondria belong to DCs. Ellipsoids flanked between the DCs and UV cones are most likely those of blue-cones (B). Note that the ellipsoids of presumed blue cones contain electron-lucent mitochondria only. N: photoreceptor nucleus. Scale bar = 1 µm.
Another adult cone type examined had very electron-lucent mitochondria of variable size (Fig. 3). Mitochondria in the ellipsoids of these cones sometimes reached the size of the giant mitochondria. In oblique sections, ellipsoids containing these electron-lucent mitochondria were interspersed between other ellipsoid types (Fig. 3). Densitometry measurements indicate significant differences in mean OD between the electron-dense (1.21 ± 0.23, N = 30) and electron-lucent (0.72 ± 0.16, N = 117) in cone ellipsoids of adult retinas (t-test, P ≤ 0.01).
Adult light-adapted zebrafish photoreceptor outer segments are tiered (Fig. 4) (Suliman & Novales Flamarique, Reference Suliman and Novales Flamarique2014), with specific cone outer segments associated with each tier. Examination of micrographs from adult retinas showing cone location in the photoreceptor layer and the morphology of their outer segment allowed us to confirm cone types characterized by their mitochondria. The cone types with the electron-dense mitochondria basal to the outer segment were most likely short single (ultraviolet, UV) (SCs, Fig. 2A; UV, Fig. 3) and double (red and green) cones (DCs, Figs. 2A, 2B, and 3). Electron micrographs of DCs clearly show that both members of the pair contain very electron-dense mitochondria (E-DM, Fig. 2A and 2B). In addition, the most proximal row of photoreceptors, where UV cones are located in the light adapted retina (Raymond & Barthel, Reference Raymond and Barthel2004; Suliman & Novales Flamarique, Reference Suliman and Novales Flamarique2013), had ellipsoids with large highly electron-dense mitochondria immediately beneath the outer segment (SC, Figs. 2A and 4), and these were surrounded by less electron-dense mitochondria, characteristics of short SCs (Fig. 2A; UV, Fig. 3). The cone type with the most electron-lucent ellipsoid mitochondria was located between the UV cone inner segment and the more distal DCs, suggesting that they were long single (blue) cones (B, Fig. 3).
4 days postfertilization
After 24 h posthatching (4 dpf), the retina had two rows of photoreceptor outer segments (a proximal and a distal row) and three different types of photoreceptors could be distinguished by the morphology of mitochondria in the ellipsoids. The first photoreceptor type contained small moderately electron-dense mitochondria (white arrow) that were somewhat loosely packed, that is, had variable intermitochondrial spaces (Fig. 5). The cristae of these mitochondria formed ridges, with parallel closely packed membranes. Based mainly on mitochondrial shape, the large obliquely oriented outer segment, and the infrequent occurrence of these photoreceptor cells at this age, this photoreceptor type was most likely the rod, which first appears between 2 and 3 dpf (Kljavin, Reference Kljavin1987).
Developing rods and cones have morphologically distinct mitochondria at 4 dpf. Rod mitochondria (white arrow) are smaller than cone mitochondria (black arrow). Both have visible cristae derived from the inner mitochondrial membrane. Developing rods have wider outer segments (ROS) than cones (COS). Note that rod nuclei (RN) often extend to a more vitread position in the outer nuclear layer than cone nuclei (CN). PG: retinal pigment granule. GA: Golgi body. Scale bar = 1 μm.
The second photoreceptor type encountered in 4 dpf retinas contained very closely packed mitochondria, some of which were megamitochondria (black arrow, Fig. 5). Photoreceptors of this second type had columnar nuclei in the outer nuclear layer, confirming their identity as cones. Cristae ridges were very prominent in the megamitochondria of these cones, and the smaller mitochondria (white arrow) were more electron-lucent than the mitochondria of the presumed rods. Additionally, the small mitochondria in these cones were less electron dense than the megamitochondria, similar to the mitochondria in cones of adult zebrafish. It is tempting to suggest that, based on positioning of this cone type, they are UV or short SCs. However, this would be highly speculative without any other supporting evidence as the tiering of cones is still not as in the adult fish at this stage (Alison et al., Reference Alison, Barthel, Skebo, Takechi, Kawamura and Raymond2010).
The third photoreceptor type (Fig. 6) identified at 4 dpf, also a cone, contained both giant and small mitochondria. However, these giant mitochondria were more electron-lucent than those in the other cone type and lacked the consistently linearly arrayed cristae of the first cone type. Because of the reduced number of cristae, the mitochondria were more electron-lucent in comparison with those found in the first cone type (shown in Fig. 5). These mitochondria contained both longitudinal and circular profiles within the outer mitochondrial membrane, suggesting that the inner mitochondrial membrane ramified as either cristae ridges or tubules. This cone type was encountered less frequently than the first cone type.
Electron-lucent mitochondria at 4 dpf. Cone mitochondria (black arrows) of the third type of photoreceptor identified at 4 dpf were electron-lucent. No differences in electron density were observed in the mitochondria within the ellipsoids of these cells. Ellipsoids of the second type of photoreceptor with mitochondria of moderate electron-density had clearly visible cristae (inset). N: cone nucleus. Scale bar= 1 μm.
8 days postfertilization
By 8 dpf, the two rows of photoreceptor outer segments noted at 4 dpf were more distinct (Fig. 7) and the radial distance between them had increased. Many of the photoreceptors in the inner proximal row had shorter more conical outer segments than those in the distal row. Presumed rods, with small mitochondria in their ellipsoids, were located at the very edge of the distal row of photoreceptors (not shown). Cones of the first type, with giant and small electron-dense mitochondria packed with cristae, were found in both the distal (presumed DCs) and proximal (presumed SC) rows. The third photoreceptor type (also a SC), with the electron-lucent ellipsoid mitochondria, was observed only in the proximal row (Fig. 7). This cone type had an outer segment that was wider at the base than the one characteristic of cones in the distal row.
Mitochondria in photoreceptor ellipsoids at 8 dpf. Photoreceptor outer and inner segments form two rows. Mitochondria in the proximal (more vitread) row of inner segment ellipsoids (black arrows) are more electron-lucent than those in the distal row (white arrows) (see inset also). Cones in the proximal row also have wider outer segments than those in the distal row. Scale bar = 1 μm.
15 days postfertilization
There were no changes in the morphology or arrangement of mitochondria in cone ellipsoids between one and two weeks of age. The electron-lucent mitochondria (black arrow, Fig. 8) had more cristae within the mitochondrial membrane; however, they were less electron-dense than the mitochondria in the other cone type that contained only electron-dense mitochondria (white arrow, Fig. 8).
Mitochondria in photoreceptor ellipsoids at 15 dpf. Electron-lucent mitochondria in the proximal row of inner segment ellipsoids (black arrows) have more visible cristae at this age. Electron-dense mitochondria (white arrows) are predominantly found in the distal row of inner segments. Note the narrower outer segments of cones in the distal row. CN: cone nucleus; COS: cone outer segment. Inset to emphasize the mitochondrial differences shown in the lower right corner of the figure. Scale bars in both = 1 μm.
Photoreceptor photopigments in adult zebrafish
In accordance with previous microspectroscopy studies (Robinson et al., Reference Robinson, Schmitt, Harosi, Reece and Dowling1993; Cameron, Reference Cameron2002), we found four cone visual pigments and one rod visual pigment in the retina of adult zebrafish (Fig. 9). The short SCs had an UV visual pigment with maximum wavelength of absorbance (λ max) mean at 361 nm (Fig. 9A), whereas the long SCs had a short wavelength (S) visual pigment with mean λ max at 417 nm (Fig. 9C). Each DC had one member possessing a middle wavelength (M) visual pigment with mean λ max at 478 nm (Fig. 9E), whereas the other member housed a long wavelength (L) visual pigment with mean λ max at 566 nm (Fig. 9G). Rods had a visual pigment with mean λ max at 500 nm (Fig. 9I). These λ max values denote visual pigments based primarily on vitamin A1 chromophore (Hárosi, Reference Hárosi1994).
Absorbance spectra of isolated photoreceptors from adult zebrafish. Short SCs have an UV visual pigment in the outer segment (A), and the ellipsoid shows a small peak of absorbance in the UV region (B). Long SCs have a short wavelength (S) visual pigment in the outer segment (C), and exhibit either a small peak of absorbance around 420 m (upper trace) or no appreciable absorbance (lower trace) in the ellipsoid (D). Note: For clarity purposes, in panels D, F, and H, 0.02 OD units were added to the upper trace to separate it from the bottom trace. DCs have a middle wavelength (M) visual pigment in one member (E) and a long wavelength (L) visual pigment in the other member (G). Either spectral cone type can have a pinkish-looking round body in the ellipsoid under infrared illumination which, if present, has a characteristic absorbance peak at 420 nm (upper traces, F, H). The other member does not have a round body in the ellipsoid and its absorbance is low (lower traces, F, H). Rods have a typical vertebrate rhodopsin visual pigment peaking in the middle wavelengths (500 nm, I) and the ellipsoid has no appreciable absorbance (J).
Under infrared illumination and with the camera output viewed through a long wavelength barrier (>730 nm) filter, some of the short SCs and members of DCs exhibited pinkish looking round bodies in the ellipsoid region immediately proximal to the base of the outer segment. The region of these round bodies corresponded to the location of the large electron-dense mitochondria observed in electron micrographs. When measurements were acquired from the ellipsoid of UV cones, a small absorbance (∼0.015 OD units, Fig. 9B) peaking around 354 nm was observed. By contrast, the majority of ellipsoids of long SCs either had no significant absorbance (Fig. 9D, lower trace) or showed a small peak (∼0.01 OD units, Fig. 9D upper trace) around 420 nm. The latter absorbance profile was found in a minority of long cones measured (2 of 20). DCs often had one member (containing either L or M visual pigment) with a visible round body in the ellipsoid, while the other member lacked it. The member with the round body had an ellipsoid absorbance peak (∼0.02–0.03 OD units, Fig. 9F and 9H, upper traces) around 420 nm, whereas the other member’s ellipsoid showed very low absorbance (<0.01 OD units, Fig. 9F and 9H, lower traces). Rod ellipsoids had very low absorbance across the measured spectrum (Fig. 9J). None of the photoreceptor ellipsoids showed any signs of dichroism, that is, differential absorbance as a function of the polarization of incident light.
Discussion
Previously, we identified different morphological types of mitochondria in the ellipsoid region of adult zebrafish cone photoreceptors (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012). Giant electron-dense mitochondria in the distal inner segment of some cones were centrally located, closer to the outer segment, and more densely packed than the smaller electron-lucent mitochondria in this region. These two populations of mitochondria differed significantly in size (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012).
Adult cone ellipsoids of UV and DCs contained both electron-dense and electron-lucent mitochondria in their inner segments (Table 1). The nonrandom distribution of these large electron-dense and small electron-lucent mitochondria, with the electron-dense mitochondria located more sclerad, is also found in photoreceptor inner segments of several teleost species, including tilapia (Grun, Reference Grun1975), cutlips minnow (Collins et al., Reference Collins, Collins and Ali1996), and carp (Ishikawa & Yamada, Reference Ishikawa and Yamada1969). A similar arrangement has also been reported in some mammals (Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003). Giant mitochondria have been reported in both rod and cone photoreceptors of fish and mammals (Lluch et al., Reference Lluch, Lopez-Fuster and Ventura2003; Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005), including zebrafish (Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005). Interestingly, though all photoreceptors (rods and cones) at all ages examined contained mitochondria of different sizes (i.e., large and small), only a subset of cone types contained large electron-dense mitochondria with few altered cristae. These were not observed in the inner segments of blue cones from adult fish, whose inner segments contained large and small mitochondria of the same electron density nor were they found in larval photoreceptors.
Comparison of mitochondrial characteristics in rod and cone photoreceptor ellipsoids. For each age and photoreceptor type, the characteristics of mitochondria (including electron density, relative number, and size) are given
Differences in ellipsoid mitochondria were identified in larval photoreceptors and can be used to distinguish among photoreceptor types
Here, we extend our findings in photoreceptors of adult fish to the developing retina and report that morphological differences in mitochondria can also be identified in larval cone photoreceptors as early as 4 dpf (Table 1). Zebrafish have five types of retinal photoreceptors: one rod and four cone types (Branchek & Bremiller, Reference Branchek and Bremiller1984). Other studies have shown that by ∼55 hours postfertilization (hpf), all photoreceptor opsins are present in the retina (Larison & Bremiller, Reference Larison and Bremiller1990; Robinson et al., Reference Robinson, Schmitt and Dowling1995; Schmitt et al., Reference Schmitt, Hyatt and Dowling1999) and that the first cones become morphologically distinct at this time. These are the UV cones. Subsequent differentiation of the remaining photoreceptor types results in functional electroretinogram (ERG) recordings (Saszik et al., Reference Saszik, Bilotta and Givin1999) and optokinetic responses (Haug et al., Reference Haug, Biehlmaier, Mueller and Neuhauss2010) in larvae as young as 4–5 dpf, indicating that the retina is functional at these ages. Based on our morphological data, in 4-dpf larvae, we were able to distinguish two tiers of developing photoreceptor outer segments, and three types of photoreceptors based on mitochondrial characteristics. A presumptive SC type was identified in the proximal photoreceptor tier, as were rods. Another cone photoreceptor type, with a distinct ellipsoid mitochondrial morphology was also identified. These mitochondrial differences among cone types were also evident at 8 and 15 dpf, with increasing numbers of photoreceptors within each type observed at later ages. The increase in number of photoreceptors, particularly those with darkly staining, that is, electron-dense ellipsoid mitochondria, likely reflects the maturation of DCs, which were observed to have a similar mitochondrial morphology as presumptive SCs.
Though all photoreceptor types observed in adult retinas were present at 15 dpf, cone ellipsoids in larvae at this age were not adult-like yet (Table 1), as evidenced by the continued growth and extensive changes in mitochondrial morphology and EM staining properties observed between 15 dpf and adulthood. As the eye grows, ellipsosomes differentiate from existing mitochondria in the ellipsoid region (Nag & Bhattacharjee, Reference Nag and Bhattacharjee1995; Fishelson et al., Reference Fishelson, Ayalon, Zverdling and Holzman2004). Our results indicate that by 8 dpf, some ellipsoid mitochondria start to change into large irregularly-shaped structures (Fig. 7). However, none of these mitochondria have the extensive staining density, and reduction of cristae is seen in the megamitochondria of adult cones. This suggests that transformation of mitochondria into the large ellipsosome-like megamitochondria continues to occur with age in zebrafish, as reported in other teleosts (Nag & Bhattacharjee, Reference Nag and Bhattacharjee1995), and may indicate why ERG recordings made from larval zebrafish at the ages used in this study are not similar to those from adults (Saszik et al., Reference Saszik, Bilotta and Givin1999; Tarboush et al., Reference Tarboush, Chapman and Connaughton2012). Larval ERG recordings have a prominent a-wave, whereas recordings from adults reveal the b-wave as the dominant waveform (Saszik et al., Reference Saszik, Bilotta and Givin1999).
Potential functions of inner segment mitochondria
In other species where two types of ellipsoid mitochondria are observed, it has been suggested that these mitochondria function to (1) provide energy and/or (2) alter incoming light prior to reaching the outer segments (Ishikawa & Yamada, Reference Ishikawa and Yamada1969; Knabe et al., Reference Knabe, Skatchkov and Kuhn1997; Kim et al., Reference Kim, Lee, Chang, Oh, Mun, Chung and Shin2005). In mammals with avascular retinas, mitochondria are suggested to play a role in oxygen availability (Stone et al., Reference Stone, van Driel, Valter, Rees and Provis2008) and energy production (Linton et al., Reference Linton, Holzhausen, Babai, Song, Miyagishima, Stearns, Lindsay, Wei, Chertov, Peters, Caffe, Pluk, Seeliger, Tanimoto, Fong, Bolton, Kuok, Sweet, Bartoletti, Radu, Travis, Zagotta, Townes-Anderson, Parker, Van der Zee, Sampath, Sokolov, Thoreson and Hurley2010). In zebrafish, photoreceptor mitochondria were found only in the inner segment. These findings are in agreement with those of Stone et al. (Reference Stone, van Driel, Valter, Rees and Provis2008), who reported the absence of mitochondria in photoreceptor axons or somata of avascular mammalian retinas. Stone et al. (Reference Stone, van Driel, Valter, Rees and Provis2008) explained the location of mitochondria in photoreceptors in terms of proximity to oxygen sources. The photoreceptor layer in zebrafish receives oxygen from choroidal vasculature which is adjacent to the retinal pigment epithelium. Blood vessels in the choroid were readily visible in both larval and adult retinas in this study (not shown, but see Chapman et al., Reference Chapman, Tarboush, Eagles and Connaughton2009). Thus, the restriction of mitochondria to the inner segment of zebrafish suggests a metabolic role, aiding oxygen consumption by these cells (Stone et al., Reference Stone, van Driel, Valter, Rees and Provis2008). In addition, inner segment mitochondria serve as the only source of energy for photoreceptor cells in avascular retinas (Linton et al., Reference Linton, Holzhausen, Babai, Song, Miyagishima, Stearns, Lindsay, Wei, Chertov, Peters, Caffe, Pluk, Seeliger, Tanimoto, Fong, Bolton, Kuok, Sweet, Bartoletti, Radu, Travis, Zagotta, Townes-Anderson, Parker, Van der Zee, Sampath, Sokolov, Thoreson and Hurley2010). Photoreceptors have high metabolic activity and demand for ATP (reviewed in Wong-Riley, Reference Wong-Riley2010). In dark-adapted conditions, the dark current depolarizes photoreceptors. In light-adapted conditions, the dark current is reduced, neurotransmitter release is altered, and metabolic demands change (Bui et al., Reference Bui, Kalloniatis and Vingrys2003). Cones are reported to be more metabolically active than rods, based on the greater number of mitochondria in cone inner segments and the increased crystal surface area within each mitochondrion (Perkins et al., Reference Perkins, Ellisman and Fox2004). We suggest that these metabolic functions are performed by the small mitochondria arranged in a U-shape around the electron-dense megamitochondria (Fig. 6A and 6B). These “peripherally located” mitochondria are adjacent to the inner segment membrane, where Na+/K+ ATPase pumps are located (Stone et al., Reference Stone, van Driel, Valter, Rees and Provis2008).
Some authors have suggested that the electron-dense mitochondria in cones function to improve their waveguide characteristics by acting as micro-lenses that help focus light waves onto the narrow outer segment (Ishikawa & Yamada, Reference Ishikawa and Yamada1969; Knabe et al., Reference Knabe, Skatchkov and Kuhn1997; Hoang et al., Reference Hoang, Linsenmeier, Chung and Curcio2002), much like oil droplets do in the retinas of birds and some reptiles (Young & Martin, Reference Young and Martin1984; Vorobyev, Reference Vorobyev2003). Oil droplets are typically found apically in the ellipsoid and are believed to enhance color discrimination and protect cone outer segments from UV damage by filtering light in the near UV range (Pedler & Boyle, Reference Pedler and Boyle1969; Goldsmith et al., Reference Goldsmith, Collins and Licht1984; Vorobyev, Reference Vorobyev2003). Retinal cones in some fish species, such as the guppy (Poecilia reticulata), contain large organelles called ellipsosomes that are derived from mitochondria (Kunz, Reference Kunz2006). The very large, dense irregular mitochondria identified in presumed UV and DCs in adult zebrafish retinas have a morphology and occupy a position similar to that reported for ellipsosomes, suggesting that these mitochondria may be involved in altering visual stimuli before it reaches the outer segment (Tarboush et al., Reference Tarboush, Chapman and Connaughton2012).
To test whether large mitochondria could significantly alter physiological illumination, we conducted microspectrophotometric measurements of adult zebrafish outer segments and of the ellipsoid region of the associated inner segments. Absorbance spectra measured from the round bodies in the ellipsoids of zebrafish photoreceptors, corresponding to the electron-dense mitochondria and resembling the ellipsosomes and pseudo-ellipsosomes in other fish (MacNichol et al., Reference MacNichol, Kunz, Levine, Harosi and Collins1978; Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi1999, Reference Novales Flamarique and Hárosi2000), did not correspond to cytochrome-c, which is the pigment identified in ellipsosomes (Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi2000). Instead, the round bodies in zebrafish inner segments had absorbance spectra resembling that of crocetin, a carotenoid found in the ellipsoids of some old world monkeys including the tree shrew (Bowmaker et al., Reference Bowmaker, Astell, Hunt and Mollon1991). Interestingly, the tree shrew also has megamitochondria in the cone ellipsoids, and these have been hypothesized to concentrate light onto the outer segments (Knabe et al., Reference Knabe, Skatchkov and Kuhn1997), improving signal detection (Hárosi & Novales Flamarique, Reference Hárosi and Novales Flamarique2012). It is unlikely that the megamitochondria in zebrafish have a light filtering role since the peak absorbance measured (∼0.03 OD units) would change light transmittance by a mere 1–10−0.03 = 0.067. This peak absorbance is, in fact, 10 to 23 times smaller than typical absorbances reported for ellipsosomes, for which a filtering role has been proposed (MacNichol et al., Reference MacNichol, Kunz, Levine, Harosi and Collins1978; Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi1999, Reference Novales Flamarique and Hárosi2000).
Horizontal cell spectral responses in zebrafish indicate that these cells receive inputs from all cone types, which underlie monophasic, biphasic, triphasic, and tetraphasic responses (Connaughton & Nelson, Reference Connaughton and Nelson2010). Generation of bi-, tri-, and tetraphasic responses occurs via feedback circuits from horizontal cells onto the different spectral cone types. This spectral feedback has been shown to fine tune cone responses (Connaughton & Nelson, Reference Connaughton and Nelson2010). Thus, zebrafish photoreceptors may not need a filtering pigment in the ellipsosome to enhance cone sensitivity because horizontal cell feedback circuits most likely fulfill this role by narrowing the photoreceptor spectral sensitivity curves. A light funneling function of megamitochondria, assumed to take place by concentrating light into the center of the cone due to the presumed higher refractive index provided by large, or densely packed, mitochondria (Novales Flamarique & Hárosi, Reference Novales Flamarique and Hárosi2000), may take place in zebrafish.
Nonoptical functions of the pigment crocetin include enhancing oxygen diffusion (see Bowmaker et al., Reference Bowmaker, Astell, Hunt and Mollon1991) and preventing photoreceptor degeneration by inhibiting oxidative- and stress-related cellular pathways involving caspase-3 (Yamauchi et al., Reference Yamauchi, Tsuruma, Imai, Nakanishi, Umigai, Shimazawa and Hara2011) and stabilizing mitochondrial membranes (as in the lung, Venkatraman et al., Reference Venkatraman, Konga, Peramaiyan, Ganapathy and Dhanapal2008). Thus, electron-dense mitochondria present in zebrafish photoreceptors may exert a protective role by decreasing apoptosis via oxidative stress. Rods and cones are differentially sensitive to apoptosis, with cones more susceptible to oxidative stress (Perkins et al., Reference Perkins, Ellisman and Fox2004). This differential sensitivity may be reflected in the location of the electron-dense ellipsosome-like megamitochondria in the cones of zebrafish.
If zebrafish cones are more metabolically active (and therefore require more energy) than rods, and if the electron-dense megamitochondria are protective against apoptosis, one might suspect that megamitochondria should be present in all cone types. However, our data indicate that this is not the case. It is not known why electron-dense megamitochondria are absent from blue cone ellipsoids in adult zebrafish, though habitat/environmental conditions have been suggested for other species (Nag & Bhattacharjee, Reference Nag and Bhattacharjee1995). The four different spectral types of cones are present in approximately equal numbers throughout the zebrafish retina (Robinson et al., Reference Robinson, Schmitt, Harosi, Reece and Dowling1993), and second-order bipolar and horizontal cells respond to inputs from all four cone types (Saszik et al., Reference Saszik, Bilotta and Givin1999; Nelson & Singla, Reference Nelson and Singla2009; Connaughton & Nelson, Reference Connaughton and Nelson2010). Bipolar cell ON-responses, recorded as the ERG b-wave, show nonopponent (Hughes et al., Reference Hughes, Saszik, Bilotta, DeMarco and Patterson1998) dominant UV and blue cone inputs (Nelson & Singla, Reference Nelson and Singla2009). Horizontal cell spectral responses include UV-preferring and blue-preferring triphasic responses as well as a tetraphasic response (Connaughton & Nelson, Reference Connaughton and Nelson2010). Retinotectal ON-responses are similar to ERG recordings, with maximal sensitivity to UV and blue stimuli (McDowell et al., Reference McDowell, Dixon, Houchins and Bilotta2004). These short-wavelength responses occur in addition to responses elicited by stimulation of long-wavelength (red) sensitive and middle-wavelength (green) sensitive cones (Saszik et al., Reference Saszik, Bilotta and Givin1999; Nelson & Singla, Reference Nelson and Singla2009; Connaughton & Nelson, Reference Connaughton and Nelson2010). Thus, physiological data clearly indicate that all cones are active in the zebrafish retina and stimulation of the different cone types, either individually or in combination, initiates signal transduction.
Conclusion
Our data suggest that electron-dense megamitochondria present in some adult zebrafish photoreceptors do not play a role in filtering incoming light. Rather, we propose that electron-dense megamitochondria may serve to enhance energy production for photoreceptor function and/or exert a protective role by decreasing apoptosis via oxidative stress. The fact that these electron-dense mitochondria are differentially distributed among the different photoreceptor types suggests different energy production and/or use by the various photoreceptors.
Acknowledgments
American University Mellon Fund (to V.P.C. and R.T.), the Helmlinge Award (to R.T.), and Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant [238886] (to I.N.F.) are acknowledged.
