Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses.

Central retina tissue from C57BL/6 mice was used.

Laser scanning confocal microscopy

(LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms.

Electron microscopy

(EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca 2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted.

Methods Materials All chemicals were purchased as analytical or molecular biology grade from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise noted. The pH of all solutions was 7.40 at indicated temperatures.

Experimental animals

All experimental and animal care procedures complied with the principles of the American Physiological Society, the NIH Guide for the Care and Use of Laboratory Animals and Maintenance (NIH publication No. 85-123, 1985) and were approved by the Institutional Animal Care and Use Committee of the University of Houston. Wild-type C57BL/6J mice (Harlan Sprague Dawley, Indianapolis, IN), from litters bred at our facility, were maintained on a 12:12 light:dark cycle (10-20 lux cage luminance) with food and water available ad libitum. For most studies, 21 and 60 day old female mice were decapitated between one and two hours after light-onset and their eyes were rapidly removed and immersed in ice-cold phosphate buffered saline (PBS). For a few studies, 60 day old female mice were dark-adapted overnight and decapitated two hours after scheduled light-onset under dim red light (λ >650 nm). The corneas were gently punctured at the limbus. Then the eyes were either immersion-fixed in room temperature 4% paraformaldehyde in 0.1 M cacodylate buffer for 30 min for LSCM studies or in ice-cold 3% glutaraldehyde, 2% paraformaldehyde and 0.1% CaCl 2 in 0.1 M cacodylate buffer (Karnovsky's fixative) for 12 h at 4 °C for conventional EM or ET studies as described [ 40 , 55 , 56 , 64 ]. Three to seven retinas from different mice were used for each independent analysis. There were no age-dependent differences on any analysis.

Antibodies and lectins

The primary antibodies and lectin used in these studies were selected carefully for their tissue and cellular specificity, ability to recognize single or multiple protein isoforms, ability to recognize all splice variants of selected isoforms, and commercial availability ( Table 1 ). We conducted extensive preliminary experiments to ensure that every antibody utilized in these studies had the appropriate specificity and penetration. All antibodies utilized in this study were titrated through a broad range of dilutions (most over 3 orders of magnitude) to determine optimal working dilutions. In addition, the concentration of Triton X-100 in the blocking agent was titrated to ensure optimal penetration without a significant loss in epitope. Immunolabeling specificity was confirmed by processing retinal sections as described below in the absence of the primary antibodies, by substituting normal rabbit or goat serum for polyclonal antibodies, or by using immunizing peptides for neutralization experiments. These procedures eliminated all specific labeling and revealed no false-positive labeling. Table 1 Cell-specific primary antibodies and lectin. Primary antigen or lectin Structure labeled Host Source Dilution References Calreticulin ER Rabbit Chemicon 1:100 78,79 Middle wavelength-sensitive cone arrestin (M-CAr) Cones Rabbit Kind gift from Cheryl Craft 1:1000 109 Cytochrome oxidase subunit IV (COX IV) Mitochondrial inner membrane system Mouse Molecular Probes 1:500 40,73,94 Kinesin KIF3A Photoreceptor ribbon and synaptic vesicles Mouse BD Biosciences 1:100 71 Na+/Ca2+ exchanger isoform 1 (NCX1) Synaptic terminals Rabbit Swant 1:100 81,82 Middle wavelength-sensitive opsin (M-opsin) Cones Rabbit Kind gift from Cheryl Craft 1:1000 65 Short wavelength-sensitive opsin (S-opsin) Cones Rabbit Kind gift from Cheryl Craft 1:1000 65 pan-Plasma membrane Ca2+ ATPase (PMCA) Synaptic terminals Mouse Affinity Bioreagents 1:100 81 Mitochondrial DNA polymerase-γ (POLG) Mitochondrial matrix Rabbit Lab Vision 1:500 76 Protein kinase C-α (PKCα) Rod bipolar cells Rabbit Sigma 1:1000 68,69 Rhodopsin (1D4) Rods Mouse Chemicon 1:1000 72 Sarcoplasmic-endoplasmic reticulum Ca2+ ATPase isoform 3 (SERCA3) ER Rabbit Affinity Bioreagents 1:400 80 Synaptotagmin 1 Photoreceptor and bipolar synaptic vesicles Mouse Chemicon 1:100 70,113 pan-Voltage-dependent anion channel (VDAC) Mitochondrial outer membrane Rabbit Calbiochem 1:1000 74 Vesicular glutamate transporter 1 (VGluT1) Photoreceptor and bipolar cell terminals Guinea pig Chemicon 1:1000 69 Peanut agglutinin (PNA)-Alexa Fluor 647 Conjugate Cone outer segments and terminals Molecular Probes 1:50 58,69 The primary antigen or lectin, structure(s) they label, host, source, and reference are presented. An extensive panel of well-characterized primary antibodies directed against cell- and organelle-specific markers in the retina was used in double and triple labeling experiments. These were antibodies for rhodopsin, M-opsin, S-opsins, M-cone arrestin, vesicular glutamate transporter 1 (VGluT1), protein kinase C α (PKCα), kinesin KIF3A, synaptotagmin 1, and peanut agglutinin (PNA) [ 58 , 65 - 72 ]. The details about the other antibodies, previously not used for retinal immunocytochemistry studies, are immediately below. COX is the terminal electron transport complex of the mitochondrial respiratory chain and standard activity-dependent inner boundary membrane (IBM) and cristae marker [ 41 , 47 ]. The anti-COX subunit IV (COX IV) mouse monoclonal 20E8 antibody (Molecular Probes, Eugene, OR) is a molecular marker of the inner membrane system [ 73 ] and detects a single 16 kDa band on Western blots [ 40 , 105 ]. The anti-voltage-dependent anion channel (VDAC: mitochondrial porin) rabbit polyclonal antibody Ab-5 (Calbiochem, San Diego, CA) is an established marker of the outer mitochondrial membrane (OMM), was raised against amino acids 185-197 of the human VDAC, recognizes all three VDAC isoforms and detects a single 31 kDa band on Western blots [ 74 ]. We did not use the anti-VDAC mouse monoclonal antibody 31HL, used in retinal studies by Gincel et al. [ 104 ], since it only recognizes the VDAC1 isoform [ 75 ]. The anti-mitochondrial DNA polymerase-γ (POLG) rabbit polyclonal antibody Ab-1 (Lab Vision, Fremont, CA) is a nuclear-encoded protein responsible for mitochondrial DNA (mtDNA) repair and replication that is located in the mitochondrial matrix, was raised against amino acids 714-1061 of the human POLG, and detects a single 140 kDa band on Western blots [ 76 ]. POLG expression and message level are maintained regardless of the mtDNA status [ 77 ]. The anti-calreticulin rabbit polyclonal antibody AB3825 (Chemicon, Temecula, CA) is specific for the Ca 2+ -binding chaperone located in the lumen of all ER that actively modulates Ca 2+ transport across the ER membrane [ 78 ]. It was raised against amino acids 412-417 of the C-terminus of calreticulin, does not cross react with other ER proteins and detects a single 60 kDa band on Western blots [ 79 ]. The anti-pan-sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase isoform 3 (SERCA3) rabbit polyclonal antibody PA-1-910A (Affinity BioReagents, Golden, CO) was raised against amino acids 29-39 of the mouse and rat SERCA3 isoform, recognizes all splice variants of human and rodent SERCA3, does not cross react with other SERCA isoforms, and detects a single 97 kDa band on Western blots [ 80 ]. The anti-pan-PMCA mouse monoclonal antibody MA3-914 (Affinity BioReagents) was raised against amino acids 724-783 of the human erythrocyte Ca 2+ pump, recognizes all four isoforms of PMCA and detects a 140 kDa band on Western blots [ 81 ]. The anti-NCX1 rabbit polyclonal antibody p11-13 (Swant, Switzerland) was raised against the full length canine cardiac NCX1, recognizes all splice variants of NCX1, does not cross react with other NCX isoforms, and detects the 120 and 160 kDa bands on Western blots [ 81 , 82 ].

Laser scanning confocal microscopy studies

Fixed eyes were rinsed in ice-cold PBS for 10 min. Fixed and washed eyes were cryoprotected in 30% sucrose/PBS solutions. The anterior segments were removed, eyecups were embedded in Tissue-Tek® OCT mounting media (Electron Microscopy Sciences, Fort Washington, PA) for 30 min and then frozen by immersion in liquid nitrogen. Retinas were sectioned along the vertical meridian on a cryostat at a thickness of 10-15 μm, collected onto Superfrost®/Plus microscope slides (Fisher Scientific) and stored at -20 °C until used. For all LSCM experiments, sections were fixed and immunolabeled in parallel to insure identical processing. All analyzed sections were obtained 200-400 μm from the optic nerve head. Immunofluorescent labeling of frozen sections was essentially as described [ 79 ]. Briefly, sections were thawed for 60 min before use and postfixed by immersion in 4% paraformaldehyde for 15 min to improve tissue adherence to the slides. Sections were rinsed in nanopure water (npH 2 O), treated with 1% sodium borohydride to reduce nonspecific tissue autofluorescence, and immediately rinsed in npH 2 O. Sections were rinsed in PBS and treated for two hours at RT with 5% bovine serum albumin, 1% fish gelatin, 10% normal goat serum and 0.1-0.3% Triton X-100 in ice-cold PBS to block non-specific immunolabeling. Primary antibodies were applied for two days at 4 °C. For double and triple labeling experiments, primary antibodies from different host animals were applied simultaneously. After incubation in primary antibody, the sections were rinsed three times in PBS and blocked for 30 min. Dilutions (1:500) of Cy3- or Cy5- (Jackson ImmunoResearch Laboratories, West Grove, PA) or Alexa Fluor 488 (Molecular Probes) -conjugated secondary antibodies were applied and incubated for 60 min in the dark at RT. For double and triple labeling experiments, secondary antibodies directed against primary antibodies from different species were applied simultaneously. After incubation with secondary antibody, the sections were rinsed in PBS and npH 2 O. The immunolabeled slides were dried and cover-slipped with Vectashield® anti-fade mounting medium (Vector Laboratories, Burlingame, CA) and stored at 4 °C until visualized. For double and triple labeling experiments using PNA, PNA-Alexa Fluor 647 (1:50 dilution: Molecular Probes) was applied simultaneously with the secondary antibody/antibodies. LSCM images were acquired using a Leica TCS SP2 LSCM (Leica Microsystems, Exton, PA). Stacks of images from different Z-planes were obtained using a step size of 0.3-0.5 μm. "Bleedthrough" of fluorescent signals from different channels was eliminated by adjusting laser power, detector sensitivity and by sequentially imaging each fluorescent channel. Confocal images were identically and minimally processed by importing them into Adobe Photoshop CS software (Adobe Systems, Inc., Mountain View, CA). The results shown are representative of three to six separate immunolabeling experiments from three to five different mouse retinas. In all double and triple labeling experiments, the voxel dimensions in the X-Y dimensions were smaller than in the Z-dimension. Epitopes were designated as "colabeled" when the fluorescent pixels overlapped in the images. For all figures, the designation colabeled implies that the epitopes were within 290-400 nm of each other. Semi-quantitative assessment of immunolabeling intensity The lamination-specific intensity of COX IV, VDAC, POLG, calreticulin, PMCA, and NCX immunolabeling was assessed by three independent viewers. Each viewer examined a minimum of five confocal immunofluorescent sections per retina from three to five mice and ranked the immunolabeling intensity on a relative five-point scale. The fluorescent labeling scale was intense (++++), strong (+++), moderate (++), weak (+) or absent (0). The combined results had a 90-95% concordance between viewers and are presented in Table 2 . Table 2 Retinal lamination and corresponding staining intensity of mitochondria, calcium transporters and ER. Mitochondrial antibody Calcium transporters Retinal Area or Structure COX IV VDAC POLG PMCA NCX Calreticulin ROS and COS 0 0 0 0 0 0 RIS and CIS ++++ ++/+++ ++++ + ++++ ++++ Cone juxtanuclear mitochondria +++/++++ +++/++++ +++ na na na Rod juxtanuclear mitochondria ++/+++ ++ +++ na na na Overall ONL +/++ ++/+++ +/++ + +++ ++ OPL ++++ ++++ ++++ ++++ ++/+++ ++/+++ Distal INL somas ++ + ++++ ++ ++ ++/+++ Middle INL somas +/++ + + ++ ++ ++ Proximal INL somas +/++ + ++++ ++ ++ ++/+++ IPL sublamina-α +++/++++ ++/+++ +/++ +++ ++/+++ 0/+ IPL sublamina-β +++/++++ ++/+++ ++ +++ ++ 0/+ RGC +++ ++ +++ ++ ++ +++ Müller glial end-feet ++++ 0/+ + ++ 0/+ 0/+ COX IV represents cytochrome oxidase subunit IV; VDAC represents voltage-dependent anion channel; POLG represents mitochondrial DNA polymerase-γ; PMCA represents plasma membrane Ca 2+ ATPase; NCX represents Na + /Ca 2+ exchanger isoform 1; ER represents endoplasmic reticulum ROS and COS represents rod and cone outer segments; RIS and CIS represents rod and cone inner segments; ONL and INL represents outer and inner nuclear layer; OPL and IPL represents outer and inner plexiform layer; GCL represents ganglion cell layer intensity staining key: ++++ represents intense; +++ represents strong; ++ represents moderate; + represents weak; 0 represents absent; na represents not applicable.

Conventional electron microscopy

The ultrastructure of mouse and rat photoreceptors has been described in several classic papers [ 28 , 53 , 83 , 84 ]. The fixation procedures used in these studies preserved the ultrastructure of the outer retina, although they were not optimal for maintaining the photoreceptor mitochondria ultrastructure and substructure as these were not the major goals of these studies. In contrast, one of our primary goals was to analyze and compare the ultrastructural and substructural features and characteristics of rod and cone photoreceptor mitochondria. Therefore, we used our well-validated fixation and embedding procedures for these endeavors, essentially as described [ 40 , 55 , 65 , 85 ]. Briefly, each eye was fixed overnight and a piece of the superior temporal retina 200-250 μm from the optic nerve was obtained. We chose this retinal area for two reasons. First, we used the same area of mouse retina for our previous ultrastructural and ET work on mouse cone inner segment mitochondria [ 55 ]. This allowed us to compare directly our results from rod and cone inner segment mitochondria to the current study on rod and cone synaptic terminal mitochondria. Second, this region contains mostly middle wavelength-sensitive (M) cones [ 169 ] and M cones in the mouse are similar to those in other mammals [ 86 , 87 ], which enables cross-species comparisons. Sections were dehydrated and embedded in Spurr's or Araldite resin as described [ 40 , 55 , 65 , 88 ]. Ultra-thin vertical sections of the retina were stained with uranyl acetate and lead citrate before being examined in a JEOL 100-C or 1200EX transmission EM (Tokyo, Japan). The number of mitochondria per rod spherule and cone pedicle was calculated from three to five different grids from each of five different mice. The mean number from each mouse was determined and the overall mean±SEM was calculated.

Three-dimensional electron microscope tomography

Mouse retinas were prepared for ET essentially as described [ 40 , 55 ]. Briefly, the superior temporal retina 200-250 μm from the optic nerve was trimmed (vide supra) and the retinal sections were dehydrated, embedded in Durcupan resin, sectioned (500 nm thick) and imaged using the single- and double-tilt series techniques described by Perkins and co-workers [ 55 , 89 , 90 ]. Fiducial cues, consisting of 20 nm colloidal gold particles, were deposited on both sides of the section. For each reconstruction, a series of images at regular tilt increments was collected with a JEOL 4000EX intermediate-voltage EM operated at 400 kV. To limit anisotropic specimen thinning during image collection, the specimens were irradiated before each tilt series. Tilt series were recorded at 20,000X magnification with an angular increment of 2 ° from -60 ° to +60 ° about an axis perpendicular to the optical axis of the microscope. A computer-controlled goniometer accurately incremented the angular steps. A slow-scan CCD camera with pixel dimensions of 1960x2560 was used to collect images. The pixel resolution was 1.1 nm. Illumination was held to near parallel beam conditions and constant optical density was maintained constant by varying the exposure time. The IMOD package [ 91 ] was used for rough alignment with the fine alignment and reconstruction performed using the TxBR package [ 92 ]. Volume segmentation was performed by manual tracing in the planes of highest resolution with the program Xvoxtrace [ 90 ]. The mitochondrial reconstructions were visualized using Analyze (Mayo Foundation, Rochester, MN) or the surface-rendering graphics of Synu (National Center for Microscopy and Imaging Research, San Diego, CA) as previously described [ 55 , 93 ]. These programs allow one to step through slices of the reconstruction in any orientation and to track or model features of interest in three dimensions. Measurements of structural features were made within segmented volumes by the programs Synuarea and Synuvolume (National Center for Microscopy and Imaging Research, San Diego, CA). Overall, measurements from tomographic reconstructions were made from seven distinct mitochondria (four from rods and three from cones) using retinas obtained from three different mice. Ca 2+ imaging and correlative electron microscopy of rod and cone synaptic terminals in dark-adapted and light-adapted whole retinas Our fluo-3 Ca 2+ imaging and LSCM procedures [ 40 , 94 ], with modifications as described, were used to localize the distribution and to determine the relative concentrations of free Ca 2+ in dark- and light-adapted rod and cone synaptic terminals. All dark-adapted procedures were conducted under dim red light (λ >650 nm). Whole neural retinas were isolated from dark-adapted mice (n=3 mice), incubated in a Ca 2+ -free HEPES buffer (30 mM HEPES, 125 mM NaCl, 5 mM KCl, 3 mM MgCl 2 , 10 mM D-glucose: pH 7.4, 310±3 mOsm) containing rhodamine-labeled PNA (1:10 dilution; Vector Laboratories, Burlingame, CA) and bovine serum albumin (1 mg/ml) for 10 min at RT followed by three gentle aspiration/rinses with Ca 2+ -free HEPES buffer. Then the retinas were incubated in the Ca 2+ -free HEPES buffer containing 3 mM fluo-3 AM and 0.025% pluronic acid (Molecular Probes) for 30 min at RT followed by a gentle aspiration/rinse in Ca 2+ -free HEPES buffer. Preliminary experiments determined that the organic anion transport inhibitor probenecid (2.5 mM) did not significantly affect the fluo-3 fluorescence results in the rod or cone synaptic terminals, so it was not used in the present experiments. We did not use verapamil, an inhibitor of the multidrug resistance pump, because it also blocks L-type Ca 2+ channels and would have confounded our results [ 40 , 94 ]. The retinas were mounted retinal ganglion cell side down on nitrocellulose filter paper and several 100-150 μm thick slices were made in the central retinal area essentially as described [ 95 ]. The retinal slices and filter paper were placed on small volume glass bottom dishes coated with Matrigel (Collaborative Research, Palo Alto, CA) and incubated for 15 min at 27 °C in HEPES buffer containing 1.5 mM CaCl 2 in order to restore the normal extracellular Ca 2+ concentration and increase the esterase activity [ 96 ]. The HEPES buffer with CaCl 2 was aspirated and replaced with fresh buffer prior to the onset of Ca 2+ imaging. To confirm that the observed fluorescence signals reflected changes in internal Ca 2+ levels, we conducted three different experiments. First, we added 1 mM Pb 2+ , a potent fluo-3 fluorescence quencher [Kd for fluo-3 is 6 pM: 94,97], to the Ca 2+ -containing HEPES buffer. Similar to our previous results [ 40 , 94 ], this significantly quenched the Ca 2+ -enhanced fluo-3 fluorescence in dark-adapted photoreceptor synaptic terminals (data not shown). Second, we incubated retinal slices in Ca 2+ -free HEPES buffer with 5 mM BAPTA-AM [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] for 15 min at 27 °C. The fluo-3 fluorescence measured in dark-adapted photoreceptor synaptic terminals, from these retinal slices, was not above background (data not shown). Third, we attempted an in vivo calibration of fluo-3 fluorescence in photoreceptor synaptic terminals using the standard ionomycin, Mn 2+ and digitonin procedure for NIH 3T3 cells [ 98 , 99 ] and isolated cerebellar granule cells [ 97 ] as well as the Ca 2+ ionophore A-23187. These calibration procedures saturated the fluo-3 fluorescent signal above the physiological range. However, they were unreliable because shortly after the fluo-3 fluorescent signal saturated the rod photoreceptors initiated apoptosis: as we reported [ 40 , 94 ]. Therefore, we established an in vitro relative fluorescence intensity (RFI) standard curve, as described [ 98 , 100 ], in order to estimate the free [Ca 2+ ] in the photoreceptor synaptic terminals. An intracellular buffer (25 mM HEPES, 130 mM KCl, 5 mM NaCl, 3 mM MgCl 2 , pH 7.2, 305±3 mOsm) that contained calibrated Ca 2+ buffers (0-40 μM Ca 2+ ; Molecular Probes) and 5 μM of the penta-ammonium salt of fluo-3 (Molecular Probes) was pipetted onto dual concave glass slides maintained in the dark at 27 °C. Fluo-3 fluorescence was measured with the Zeiss LSM-410 confocal microscope system (Zeiss, Thornwood, NY) as described below. Images were acquired on a Zeiss LSM-410 confocal microscope utilizing a Zeiss Axiovert 100 microscope equipped with an X63 oil immersion objective (1.4 numerical aperature). An argon laser excited fluo-3 at 488 nm and PNA-rhodamine at 568 nm, and bandpass filters of 530 and 590 nm collected the signals from fluo-3 and rhodamine, respectively. For each retina and all calibration procedures, the gain on the confocal system was kept constant for all recording conditions. To minimize light exposure to the retina and photobleaching, the OPL was identified rapidly and at low confocal gain by its PNA fluorescence and its retinal location. Once identified, the retinas were dark-adapted for an additional five minutes. Ten to 15 optical sections of the dark-adapted OPL were obtained using a Z-axis step size of 0.5 μm. Following these recordings, the retinas were dark-adapted for five minutes and then a rod saturating light illuminated the retina [ 49 , 97 ]. Ca 2+ image recordings began one minute after light onset as the light-adapted decrease in photoreceptor oxygen consumption stabilizes during the first minute of the light stimulation [ 49 , 50 , 101 ]. Z-axis reconstructions were made using Zeiss software. The image was a Z-stack maximum projection (single confocal section). For these experiments, the XY resolution was 200-250 nm and the Z resolution was 300 nm. The RFI of Ca 2+ -fluo-3 in dark- and light-adapted rod spherules and cone pedicles was determined using NIH Image, version 1.62. To directly compare the RFI, and thus the relative free [Ca 2+ ], in dark- and light-adapted rod and cone synaptic terminals, only experiments where relatively adjacent photoreceptor synaptic terminals were imaged are included in this data set. To normalize all the images a standard background subtraction was performed on each image. Then the overall RFI within each dark- and light-adapted rod spherule and cone pedicle was determined based on a grey scale with 256 levels. The light-adapted to dark-adapted fluorescence intensity ratio for each rod spherule and cone pedicle from each retina was calculated, means±SEMs were determined, and the data was statistically analyzed. The mean RFI of dark-adapted rod spherules and cone pedicles as well as light-adapted rod spherules and cone pedicles were determined and compared. To analyze and evaluate this data, the RFI values were normalized by the gain of the confocal system expressed on a linear scale as described [ 102 ]. The normalized intensity values were calculated, means±SEMs were determined, and the data was statistically analyzed. Similar imaging procedures were used to establish the Ca 2+ and fluo-3 fluorescence calibration curve. Confocal images for each retina were identically and minimally processed by importing them into Adobe Photoshop CS software (Adobe Systems, Inc., Mountain View, CA). For higher resolution and better visualization of the Ca 2+ microdomains, the pseudocolored images were transformed using the advanced "stained glass" imaging synthesis algorithm provided in Adobe Photoshop under the Filter and Texture pull-down menus (Adobe Systems, Inc.). Although the exact algorithm is proprietary, the values for cell size, border thickness and light intensity were 10, 1, and 2, respectively. The results shown are from a representative retina from three separate experiments from three different mouse retinas. Conventional EM identified the rod spherules and cone pedicles from where the Ca 2+ images were obtained. After the experiment, the small glass dish was placed on ice and the retinal slice was fixed for 30 min with ice-cold Karnovsky's fixative (described above). Then the dish and retina were placed in a larger volume of fresh Karnovsky's fixative for 12 h at 4 °C and the tissue was processed for conventional EM as described.

Statistical analysis

The electron tomography and fluo-3 intensity data were analyzed using a two-tailed Student's t-test (Kaleidagraph Synergy Software, Reading, PA). The differences were considered significant if p650 nm). The corneas were gently punctured at the limbus. Then the eyes were either immersion-fixed in room temperature 4% paraformaldehyde in 0.1 M cacodylate buffer for 30 min for LSCM studies or in ice-cold 3% glutaraldehyde, 2% paraformaldehyde and 0.1% CaCl 2 in 0.1 M cacodylate buffer (Karnovsky's fixative) for 12 h at 4 °C for conventional EM or ET studies as described [ 40 , 55 , 56 , 64 ]. Three to seven retinas from different mice were used for each independent analysis. There were no age-dependent differences on any analysis.