Visual Neuroscience (2014), 31, 11–23. Copyright © Cambridge University Press, 2014 0952-5238/14 $25.00 doi:10.1017/S095252381300059X
Variability in mitochondria of zebrafish photoreceptor ellipsoids
R. TARBOUSH,1,* I. NOVALES FLAMARIQUE,2 G.B. CHAPMAN,3 and V.P. CONNAUGHTON1 1Department
of Biology, American University, Washington DC of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada 3Department of Biology, Georgetown University, Washington DC *Present address: Department of Neurotrauma, Navy Medical Research Center, Silver Spring, Maryland 20910 2Department
(Received June 25, 2013; Accepted November 25, 2013)
Abstract Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter. Keywords: Development, Retina, Inner segment
are another type of atypical mitochondria that is present in the photoreceptor ellipsoids of many fish and mammalian species, including zebrafish (Collins et al., 1996; Knabe et al., 1997; Lluch et al., 2003; Kim et al., 2005). Megamitochondria are large, electron dense, and located in the distal portion of inner segments (Collins et al., 1996; Lluch et al., 2003; Kim et al., 2005). They contain irregularly shaped and/or reduced numbers of cristae (Lluch et al., 2003) and are significantly larger in size (Tarboush et al., 2012) than the surrounding “regular” mitochondria. Branchek and Bremiller (1984) and Kljavin (1987) 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. (2005) 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., 2012). In that work, we reported the presence of megamitochondria in photoreceptor inner segments and measured their longest dimension (Tarboush et al., 2012). 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
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, 1953), but later work showed that they can exhibit great morphological diversity (Ishikawa & Yamada, 1969). 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, 1969; Kljavin, 1987; Lluch et al., 2003; Kim et al., 2005). 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., 2002), similar to the function of oil droplets in the retinas of birds, reptiles, and some fishes (Ishikawa & Yamada, 1969; Mariani, 1987; Hárosi & Novales Flamarique, 2012). One example of modified photoreceptor mitochondria is the ellipsosome in the retina of some teleost fishes (MacNichol et al., 1978; Novales Flamarique & Hárosi, 2000). Giant mitochondria, also referred to as megamitochondria,
Address correspondence to: V.P. Connaughton, Department of Biology, Hurst Hall, Rm. 101, American University, 4400 Massachusetts Ave, NW, Washington DC 20016. E-mail: [email protected]
11
12 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 glutaraldehydebased primary fixatives (Chapman et al., 2009): Millonig’s fixative (5% glutaraldehyde in double strength phosphate buffer with CaCl2, Millonig, 1961; Sabatini et al., 1963), cacodylate-containing fixative (2.5% glutaraldehyde in 0.18 M sodium cacodylate, Osinchak, 1964), and 2.5% glutaraldehyde in 0.135 M sodium cacodylate and 1% tannic acid (Hayat, 2000). 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, 1961). 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
Tarboush et al. 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., 1960). 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, 1960) followed by 0.4% lead citrate for 10 min (Venable & Coggeshall, 1965). These were viewed and micrographs acquired with a JEOL-1010 transmission electron microscope (JEOL Ltd., Akashima, Japan). 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, 1987; Novales Flamarique & Hárosi, 2000, 2002). 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, 1987).
Results Adults Adult zebrafish rods had long uniform outer segments of similar width to the inner segments (Fig. 1) and contained fewer mitochondria
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Mitochondria of zebrafish cone ellipsoids
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 electronlucent (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, 2014), 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 electrondense 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, 2004; Suliman & Novales Flamarique, 2013), had ellipsoids with large highly electrondense mitochondria immediately beneath the outer segment (SC, Figs. 2A and 4), and these were surrounded by less electrondense 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
Fig. 1. Ellipsoid region of adult rod photoreceptors. Rod ellipsoid is packed with megamitochondria, as previously reported by Kim et al. (2005), 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 electronlucent 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.)
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. 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, 1969; Fishelson et al., 2004). Additionally, some of the large mitochondria were so electron-dense that cristae were visible (E-DM, Fig. 3).
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, 1987). 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., 2010).
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Fig. 2. 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.)
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.
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 electrondense 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
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Mitochondria of zebrafish cone ellipsoids
Fig. 3. 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.
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.
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).
15 days postfertilization
Photoreceptor photopigments in adult zebrafish
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
In accordance with previous microspectroscopy studies (Robinson et al., 1993; Cameron, 2002), we found four cone visual pigments and one rod visual pigment in the retina of adult zebrafish (Fig. 9).
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Fig. 4. 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.
The short SCs had an UV visual pigment with maximum wavelength of absorbance (λmax) mean at 361 nm (Fig. 9A), whereas the long SCs had a short wavelength (S) visual pigment with mean λmax at 417 nm (Fig. 9C). Each DC had one member possessing a middle wavelength (M) visual pigment with mean λmax at 478 nm (Fig. 9E), whereas the other member housed a long wavelength (L) visual pigment with mean λmax at 566 nm (Fig. 9G). Rods had a visual pigment with mean λmax at 500 nm (Fig. 9I). These λmax values denote visual pigments based primarily on vitamin A1 chromophore (Hárosi, 1994). 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 (
Variability in mitochondria of zebrafish photoreceptor ellipsoids
R. TARBOUSH,1,* I. NOVALES FLAMARIQUE,2 G.B. CHAPMAN,3 and V.P. CONNAUGHTON1 1Department
of Biology, American University, Washington DC of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada 3Department of Biology, Georgetown University, Washington DC *Present address: Department of Neurotrauma, Navy Medical Research Center, Silver Spring, Maryland 20910 2Department
(Received June 25, 2013; Accepted November 25, 2013)
Abstract Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter. Keywords: Development, Retina, Inner segment
are another type of atypical mitochondria that is present in the photoreceptor ellipsoids of many fish and mammalian species, including zebrafish (Collins et al., 1996; Knabe et al., 1997; Lluch et al., 2003; Kim et al., 2005). Megamitochondria are large, electron dense, and located in the distal portion of inner segments (Collins et al., 1996; Lluch et al., 2003; Kim et al., 2005). They contain irregularly shaped and/or reduced numbers of cristae (Lluch et al., 2003) and are significantly larger in size (Tarboush et al., 2012) than the surrounding “regular” mitochondria. Branchek and Bremiller (1984) and Kljavin (1987) 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. (2005) 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., 2012). In that work, we reported the presence of megamitochondria in photoreceptor inner segments and measured their longest dimension (Tarboush et al., 2012). 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
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, 1953), but later work showed that they can exhibit great morphological diversity (Ishikawa & Yamada, 1969). 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, 1969; Kljavin, 1987; Lluch et al., 2003; Kim et al., 2005). 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., 2002), similar to the function of oil droplets in the retinas of birds, reptiles, and some fishes (Ishikawa & Yamada, 1969; Mariani, 1987; Hárosi & Novales Flamarique, 2012). One example of modified photoreceptor mitochondria is the ellipsosome in the retina of some teleost fishes (MacNichol et al., 1978; Novales Flamarique & Hárosi, 2000). Giant mitochondria, also referred to as megamitochondria,
Address correspondence to: V.P. Connaughton, Department of Biology, Hurst Hall, Rm. 101, American University, 4400 Massachusetts Ave, NW, Washington DC 20016. E-mail: [email protected]
11
12 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 glutaraldehydebased primary fixatives (Chapman et al., 2009): Millonig’s fixative (5% glutaraldehyde in double strength phosphate buffer with CaCl2, Millonig, 1961; Sabatini et al., 1963), cacodylate-containing fixative (2.5% glutaraldehyde in 0.18 M sodium cacodylate, Osinchak, 1964), and 2.5% glutaraldehyde in 0.135 M sodium cacodylate and 1% tannic acid (Hayat, 2000). 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, 1961). 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
Tarboush et al. 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., 1960). 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, 1960) followed by 0.4% lead citrate for 10 min (Venable & Coggeshall, 1965). These were viewed and micrographs acquired with a JEOL-1010 transmission electron microscope (JEOL Ltd., Akashima, Japan). 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, 1987; Novales Flamarique & Hárosi, 2000, 2002). 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, 1987).
Results Adults Adult zebrafish rods had long uniform outer segments of similar width to the inner segments (Fig. 1) and contained fewer mitochondria
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Mitochondria of zebrafish cone ellipsoids
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 electronlucent (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, 2014), 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 electrondense 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, 2004; Suliman & Novales Flamarique, 2013), had ellipsoids with large highly electrondense mitochondria immediately beneath the outer segment (SC, Figs. 2A and 4), and these were surrounded by less electrondense 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
Fig. 1. Ellipsoid region of adult rod photoreceptors. Rod ellipsoid is packed with megamitochondria, as previously reported by Kim et al. (2005), 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 electronlucent 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.)
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. 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, 1969; Fishelson et al., 2004). Additionally, some of the large mitochondria were so electron-dense that cristae were visible (E-DM, Fig. 3).
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, 1987). 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., 2010).
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Fig. 2. 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.)
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.
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 electrondense 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
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Mitochondria of zebrafish cone ellipsoids
Fig. 3. 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.
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.
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).
15 days postfertilization
Photoreceptor photopigments in adult zebrafish
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
In accordance with previous microspectroscopy studies (Robinson et al., 1993; Cameron, 2002), we found four cone visual pigments and one rod visual pigment in the retina of adult zebrafish (Fig. 9).
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Fig. 4. 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.
The short SCs had an UV visual pigment with maximum wavelength of absorbance (λmax) mean at 361 nm (Fig. 9A), whereas the long SCs had a short wavelength (S) visual pigment with mean λmax at 417 nm (Fig. 9C). Each DC had one member possessing a middle wavelength (M) visual pigment with mean λmax at 478 nm (Fig. 9E), whereas the other member housed a long wavelength (L) visual pigment with mean λmax at 566 nm (Fig. 9G). Rods had a visual pigment with mean λmax at 500 nm (Fig. 9I). These λmax values denote visual pigments based primarily on vitamin A1 chromophore (Hárosi, 1994). 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 (
