August 2007
Volume 48, Issue 8
Free
Retina  |   August 2007
Synaptic Contact between Melanopsin-Containing Retinal Ganglion Cells and Rod Bipolar Cells
Author Affiliations
  • Jens Østergaard
    From the Departments of Ophthalmology and
    Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark.
  • Jens Hannibal
    Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; and the
    Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark.
  • Jan Fahrenkrug
    Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark.
Investigative Ophthalmology & Visual Science August 2007, Vol.48, 3812-3820. doi:10.1167/iovs.06-1322
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      Jens Østergaard, Jens Hannibal, Jan Fahrenkrug; Synaptic Contact between Melanopsin-Containing Retinal Ganglion Cells and Rod Bipolar Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(8):3812-3820. doi: 10.1167/iovs.06-1322.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Evidence indicates that the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) receive input from rods and cones, which are thought to modulate the irradiance detecting system driving entrainment of the circadian system and pupillomotor control. This study was performed to identify retinal cells that have synaptic contact with ipRGCs.

methods. Immunohistochemistry and high-power confocal microscopy were used to generate stacks of digital images of sections stained with antibodies against melanopsin, protein kinase C (PKCα), tyrosine hydroxylase (TH), presynaptic terminal markers (C-terminal binding protein 2 [CtBP2], vesicular monoamine transporter 2 [VMAT2] and postsynaptic marker (glutamate receptor subunit 4 [GluR4]). Results were analyzed in a computer-based three-dimensional reconstruction program for cellular contacts.

results. Markers and melanopsin rod bipolar processes were found to have axosomatic and axodendritic contact with melanopsin-containing RGCs. Typically, three to four contacts were found on the soma of the melanopsin-containing RGCs, together with contacts on proximal dendrites. Contacts visualized by only CtBP2 immunoreactivity could also be demonstrated on melanopsin cell bodies and processes representing contacts with other types of bipolar cells. At the border of the inner plexiform layer (IPL) and inner nuclear layer (INL), where melanopsin processes stratify, contacts between melanopsin and TH or VMAT2 immunoreactivity processes were observed.

conclusions. Through confocal microscopy and computer-based three-dimensional analyses, this study demonstrates that melanopsin-containing RGCs have synaptic contact with PKC/CtBP2-containing rod bipolar cells and TH/VMAT2-immunoreactive amacrine cells through axodendritic and axosomatic contact, supporting electrophysiological observations that rods and cones signal to the melanopsin-containing intrinsically photosensitive RGCs.

The mammalian eye contains the classical vision (image)-bearing system of rods and cones located in the outer retina and a non-image-forming photoperception (irradiance detecting) system in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) of the inner retina. 1 The ipRGCs project as the retinohypothalamic tract (RHT) to brain areas involved in the regulation of circadian timing, acute modulation of locomotor activity (masking), and regulation of the pupillary light reflex. 2 The ipRGCs constitute approximately 1% to 2% of the total number of RGCs and are widely distributed in the entire retina, forming a photosensitive network. 3 4 5 The ipRGCs are photosensitive because of the expression of melanopsin, 6 7 8 a photopigment found primarily in the membrane of the ipRGCs. 3 9 10 11 12 13 The ipRGCs in melanopsin knockout mice are photoinsensitive, 6 but these mice are still able to photoentrain, 14 15 and they have preserved negative masking behavior 16 and a pupillary light response. 6 Mice lacking melanopsin and rods and cones have, however, a complete loss of photoresponsiveness, 17 18 indicating that input from rods and cones is of functional importance for the ipRGCs. Electrophysiological studies in primates have provided evidence that the ipRGCs receive inhibitory signals from short wavelength-sensitive cones and excitatory input from rods and medium- and long-wavelength cones. 5 This is in accordance with an ultrastructural study in mice showing that melanopsin-expressing ganglion cells located in the ganglion cell layer (GCL) and displaced to the inner nuclear layer (INL) have close contact with amacrine and bipolar cells of unknown phenotype. 19 In the present study, we performed immunohistochemical double/triple staining for melanopsin and distinct retinal markers, combined with high-resolution confocal laser scanning microscopy and three-dimensional computer analysis to clarify which retinal cells have contact with the melanopsin-containing RGCs. 
Materials and Methods
Animals
Fifteen adult male Wistar rats, each weighing 150 to 200 g (M & B Breeding Centre Ltd., Ll., Skensved, Denmark), were given free access to food and water under regulated temperature conditions and were entrained to a 12-hour light/12-hour dark cycle. Eyes were obtained during the subjective day by decapitation and enucleation. One eye was removed, immersion fixed in Stefanini fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.2) for 12 to 24 hours at 4°C, cryoprotected in 30% sucrose, and frozen at −80°C until cutting in 12- to 25-μm-thick sections in a microtome. The anterior segment and the vitreous body of the other eye were removed, and the posterior segment, including the retina, was fixed in Stefanini fixative for 12 to 24 hours at 4°C. The retina was subsequently removed from the eyecup, transferred to cryoprotectant, and stored at −20°C until it was processed for immunohistochemistry. All experiments were performed in accordance with Danish law on animal experiments (publication no. 382; June 10, 1987) and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Antibodies
In the initial experiments, we immunostained the retina with a number of antibodies against markers for different retinal cell types. Two antibodies against protein kinase Cα (PKCα; code P-5704, clone MC5, diluted 1:500 [Sigma, St. Louis, MO], or goat anti-PKCα, code SC12356, diluted 1:500 [Santa Cruz Biotechnology, Santa Cruz, CA]) found in rod bipolar cells 20 21 and one anti-tyrosine hydroxylase antibody (mouse anti-TH; code mAb 318, diluted 1:500 [Chemicon, Temecula, CA]) staining dopaminergic amacrine cells 22 23 were selected for further study. These antisera were used for double immunohistochemistry, together with antibodies against melanopsin (an in-house rabbit anti-melanopsin antibody, code 41K9, characterized previously 9 and used in a dilution of 1:100,000) or a rabbit anti-melanopsin antibody (code PAI-781, diluted 1:500 [Affinity BioReagents, Inc., Golden, CO]). A mouse monoclonal antibody against C-terminal binding protein 2 (CtBP2), a RIBEYE homolog, was purchased from BD Biosciences (Heidelberg, Germany; no. 612044, used at a dilution of 1:10,000). The antibody was raised against mouse CtBP2 and recognizes synaptic ribbons in retinal bipolar cells of mammals. 24 25 As postsynaptic marker a rabbit anti-glutamate receptor 4 (GluR4) antibody was used 26 27 (code AB1508, diluted 1:10,000). A rabbit polyclonal anti-vesicular monoamine transporter 2 (VMAT2) antiserum was used to visualize areas of dopamine release (code PA1–4680, diluted 1:2000 [Affinity BioReagents]). 28  
Immunohistochemistry
Immunofluorescence procedures for double- or triple-antigen localization were performed as described previously 9 29 30 using a mixture of the primary antibodies and Texas Red-conjugated donkey anti-goat antibodies (code 705–075-147, diluted 1:100 [Jackson ImmunoResearch Laboratories, Inc., West Grove, PA]), Cy5-conjugated donkey anti-rabbit antibodies (code 711–175-152, diluted 1:100 [Jackson ImmunoResearch Laboratories]), and Cy2-conjugated donkey anti-mouse antibodies (code 715–225-151 diluted 1:200 [Jackson ImmunoResearch Laboratories]). Because the melanopsin, GluR4, and VMAT2 antisera were raised in rabbits, colocalization was visualized by the reported use of the tyramide amplification technique for primary antisera raised in the same species. 30 31 Sections were incubated overnight with the primary rabbit antibody (melanopsin 41K9 diluted 1:100,000 or GluR4 diluted 1:10,000) and were visualized by biotinylated tyramide (Tyramide System Amplification; DuPont NEN, Boston, MA) and streptavidin-conjugated red fluorescent dye (Texas Red; Amersham, Bioscience) or visualized using an immunohistochemical detection system (EnVision, code 4065; DAKO, Copenhagen, Denmark) and conjugate (Alexa 488 tyramide, diluted 1:200; Molecular Probes, Eugene, OR). After they were washed and blocked in 1% H2O2 for 10 minutes, the sections were incubated overnight with the second primary rabbit antibody (melanopsin, code PAI-781, diluted 1:500, or anti-VMAT2 antiserum, diluted 1:2000). On the third day, melanopsin/VMAT2 antibodies were visualized by Cy5/Cy2-conjugated donkey anti-rabbit antibodies. Controls were made by omission and/or adsorption of primary antibodies, which eliminated all staining. 
Analysis for Contact
Analysis for contacts between melanopsin-containing RGCs and rod bipolar or RGCs and amacrine cells were performed using high-resolution confocal microscopy to generate stacks of images obtained from flat mounts or 12 to 25-μm-thick cross-sections of the retina using a confocal microscope (IX70; Olympus, Copenhagen, Denmark) or a laser scanning microscope (Zeiss LSM 510; Brock og Michelsen, Birkerød, Denmark) equipped with appropriate filter settings for detecting Cy2/Alexa 488, Texas Red, and Cy5. A typical stack consisted of 40 to 90 images, each 0.23- to 0.30-μm thick, equal to a scan depth of 9.2 to 20.7 μm (Z-axis). Intensity of the channels was carefully adjusted within the borders of the gray tone scale to preserve information about relative intensities in the digital image and to prevent overexposure. Image analysis (Volocity 3.6 software; Improvision, Coventry, UK) consisted of deconvolution of each stack of images (using the 3D Restoration Module [Improvision] and fast restoration based on a calculated point spread function) followed by colocalization analysis and three-dimensional-rendered images. Cellular extension was determined by the classification module of the software (Volocity 3.6; Improvision) according to a set of well-defined criteria in which the lower threshold level of fluorescence intensity defining cell borders was set as high as possible to maintain a natural three-dimensional image of the cytoarchitecture (see Figs. 2H 2I 6B 6C ). The mean lower threshold was 25% (range, 12%–35% greater than background level). To eliminate user bias, the threshold was set independently for each of the three channels. Given these conditions, colocalization (overlap) was calculated (using the classification module) and visualized with the three-dimensional rendering function (Visualization module; see http://www.improvision.com/pdfs/guides/ VolocityUserGuide.pdf). Control experiments in which one channel was flipped with respect to the other channel were performed routinely to ensure that the detected signal was identical independently of the laser channel used. Furthermore, random colocalization arising from two populations of dense puncta (illustrated by the number and density of CtBP2-IR puncta colocalized with PKC-IR bipolar axon terminals) were calculated according to the description of Jusuf et al. 26 We used Volocity to determine the number of colocalized puncta in focus where after one channel was flipped in the vertical axis and pseudorandom colocalization was determined (Fig. 1) . Image-editing software (Adobe Photoshop and Adobe Illustrator; Adobe Systems, San Jose, CA) was used to combine the obtained images into plates. 
Results
Melanopsin-Immunoreactive RGC Contact with PKC-Containing Bipolar Cells
We used two different antibodies against PKCα in combination with two well-characterized anti-melanopsin antibodies and found that the distribution and localization of PKC and melanopsin were identical independently of the antibodies used. In cross-section of the retina PKCα immunoreactivity (IR) was found in bipolar cell bodies and in bipolar axons reaching the border between the inner plexiform layer (IPL) and the ganglion cell layer (GCL). In all parts of the retina, melanopsin-containing RGCs were found in close contact with PKC-containing bipolar axon terminals (Fig. 2) . Often several axon terminals were found close to the melanopsin soma or in close contact with proximal dendrites making axodendritic contacts. In contrast, melanopsin-containing processes, which descend through the IPL to stratify in the border between the IPL and INL, showed no contact to PKC-containing processes; similarly, no contacts in the IPL/INL zone were found. Whole mount preparation confirms this distribution and shows that each melanopsin-containing RGC had approximately three to six contacts (Fig. 2) . A three-dimensional reconstruction of stacks of digital images was analyzed for contacts between PKC-IR bipolar axons and melanopsin-IR RGCs (see Materials and Methods). This analysis, which is illustrated in Figures 2G 2H 2I 2J 2K 2L , substantiates that PKC immunoreactive axon terminals have axosomatic and axodendritic contact with proximal dendrites of the melanopsin-containing RGCs. Because the synapses of bipolar cells, including rod bipolar cells, contain synaptic ribbons, 24 we verified the existence of contacts between melanopsin-immunoreactive RGCs and PKC-containing bipolar cell axons by triple immunohistochemistry for the ribbon marker CtBP2 (Figs. 3 and 4) . Strong CtBP2 immunostaining was found in ribbons located in the photoreceptor synapses (Figs. 3A 3B) . Furthermore, punctuate staining occurred in ribbons located in the IPL and in the border of the IPL/GCL (Figs. 3 4) . CtBP2 and PKC were colocalized in almost all PKC-containing axons (Fig. 3) , including those that had close contact with melanopsin-containing somata or proximal dendrites (Fig. 3) . Within a single PKC-containing terminal, as many as six to eight CtBP2 immunoreactive ribbons could be found (Fig. 3E) . To further substantiate the existence of contacts between melanopsin-IR RGCs and PKC-containing bipolar cells, stacks of high-power images that also stained for CtBP2 were analyzed. Three-dimensional reconstruction of an area representing contacts between melanopsin-IR RGCs and PKC/CtBP2-IR axon terminals is shown in Figure 4 . An extended view of 61 digital sections of the two cells is shown in Figure 4A , followed by the three-dimensional reconstruction model in Figure 4B . In Figure 4D , the calculated overlaps (contacts) between CtBP2 and melanopsin are shown at lower magnification. The results indicated contact between CtBP2-containing ribbons and the melanopsin-IR dendrite and cell soma. Detailed analysis of the contact with the melanopsin-IR soma is shown at high magnification in Figure 4F . The analysis shows that areas of the soma membrane have contact with PKC (Fig. 4C , yellow) and that these areas overlap with areas of contact between melanopsin and CtBP2, representing synaptic contacts (Figs. 4E 4F) . Contacts between CtBP2-containing ribbons and melanopsin-lacking PKC (non-PKC, ribbon contacts) were also demonstrated, located mainly on the proximal dendrites (Fig. 4D , double arrows). These contacts most likely represent cone bipolar cells. These ribbons seem to represent approximately half the ribbon-containing inputs to the melanopsin-containing RGCs. To support that the CtBP2-IR contacts with melanopsin-IR ganglion cells represent synaptic contact, triple-labeling analysis was performed on sections stained for melanopsin, CtBP2, and the postsynaptic glutamate receptor GluR4. Previous studies in primate retina have demonstrated a high degree of colocalization between these presynaptically and postsynaptically located markers in the IPL. 26 27 As shown in Figure 5 , CtBP2 was stored with many GluR4-IR punctuates that overlap with melanopsin-IR ganglion cells (Fig. 5A) . Three-dimensional reconstruction of a stack of digital images confirming these observations are shown in Figure 5B , and the calculated overlap between melanopsin and CtBP2 (Fig. 5C) , between melanopsin and GluR4 (Fig. 5D)and between melanopsin, CtBP2, and GluR4, are shown in Figure 5E
Contacts between Dopaminergic Amacrine Cells and Melanopsin-Immunoreactive Processes
Double staining for melanopsin and TH showed throughout the entire retina that the melanopsin-IR dendritic processes found in the OFF layer (stratum 1) of the IPL have close contact with the TH-IR processes (Fig. 6) . Axodendritic contacts between TH-IR processes and proximal dendrites of melanopsin-IR RGCs and few axosomatic contacts were also seen in the ON layer of the IPL and in the GCL. To confirm that overlap between melanopsin-IR RGCs and TH-IR amacrine cells represented contacts, analyses were performed on stacks of high-power digital images (see Material and Methods). Three-dimensional reconstruction of areas representing melanopsin and TH-IR contacts are shown in Figures 6Dto 6F . The three-dimensional reconstruction shows that the melanopsin-IR dendrite projects toward the border between the INL/IPL and forms distinct synaptic contacts. A typical melanopsin-IR ganglion cell sends one to three dendritic processes toward the outer retina, where the processes stratify within the same stratum as the TH-IR processes. Three-dimensional analysis, as illustrated in Figure 6 , shows that many contacts between TH-IR processes and melanopsin-IR dendritic processes exist (Figs. 6D 6E 6F , light blue). To substantiate that melanopsin-containing dendrites make synaptic contacts, the localization of a presynaptic marker for dopamine release, VMAT2, was analyzed. As shown in Figures 7A and 7B , VMAT2-IR processes were identical to those of TH-IR processes stratifying in the border between the IPL and INL. Three-dimensional reconstruction of areas representing melanopsin and VMAT2-IR contacts are shown in Figures 7C 7D 7E 7F . The three-dimensional reconstruction shows that the melanopsin-IR dendrite projects toward the border between the INL/IPL and forms distinct contacts with VMAT2-containing processes. 
Discussion
In the present study, we showed for the first time that melanopsin-containing ipRGCs have synaptic contact with rod bipolar cells identified by presynaptic and postsynaptic retinal markers. Furthermore, we extended previous findings 19 32 by showing that melanopsin-IR RGCs have contact with TH-IR and VMAT2-IR processes. 
High-resolution electron microscopy has for years been the method of choice for the demonstration of synapses in the retina. In the present study, we identified synaptic contacts using well-characterized markers for two subsets of retinal cells located in the inner retina in combination with CtBP2, a RIBEYE homolog found in presynaptic ribbons 24 25 and the postsynaptic located GluR4 receptor, 26 27 followed by high-power confocal microscopy, deconvolution, and three-dimensional reconstruction analysis. This approach gives an overview of the entire retina and highly detailed information at the same time, whereas electron microscopy is laborious and often limited to examination of smaller areas. A similar approach has been used to examine presynaptically and postsynaptically localized receptors, 26 27 and these studies support that the contacts identified between melanopsin-IR RGCs and bipolar/amacrine cells represent synaptic contacts. Bipolar cells mediate light information from the rods and cones to the inner retina through synaptic contacts in the IPL. The contacts, named dyads, are characterized by a presynaptic ribbon and two postsynaptic processes. 33 34 Ultrastructural ribbons have been used as anatomic markers for bipolar terminals. Bipolar axon terminals containing ribbons have previously been shown in close apposition to melanopsin-containing dendrites in the ON layer of the IPL. 19 In the present study, we demonstrated that PKCα/CtBP2-containing rod bipolar cells most likely make synaptic contact at GluR4 receptors on melanopsin-containing cell bodies and proximal dendrites. This finding seems to extend the previous findings of Belenky et al., 19 who found, at the electron microscopic level, bipolar terminals close to melanopsin-containing soma membranes in the IPL/GCL. These axon terminals had no ribbons but presynaptic vesicles suggesting that they represented classic chemical synapses. 19 One explanation for this discrepancy is that rod bipolar input could be missed at the EM level because the melanopsin RGCs accounted for only 1% of the total numbers of RGCs, 3 and the relatively few PKC-immunoreactive terminals on each ganglion cell. We also found non-PKC ribbon contacts on proximal dendrites of the melanopsin-containing RGCs. Although not proven, it is likely that these contacts represent input from cone bipolar cells. Because the classical input from rods is through AII amacrine cells and cone ON bipolar cells, it is possible that melanopsin-containing RGCs receive input from rods through these two different input pathways. That dendrites of melanopsin-IR RGCs make contact with dopaminergic amacrine processes was demonstrated with TH-IR as a marker for dopamine-expressing cells and was supported by the demonstration of similar contacts using a marker for dopamine release, VMAT2. 28 Similar findings were reported recently in normal and dystrophic rat retina 32 and in accordance with the ultrastructural study by Benlenky et al., 19 which shows that melanopsin-immunoreactive processes found in the OFF layer of the IPL and the border of the INL had synaptic contact with undefined amacrine cells, characterized by numerous presynaptic vesicles near the membrane specialization but no synaptic ribbons. 19 Amacrine cells made also synaptic contact with melanopsin-containing dendrites in the ON layer of the IPL and at the soma membrane of the melanopsin-containing RGCs, 19 as found in the present study. 
The functional implications of outer retinal input to the melanopsin-containing RGCs are unknown. Recent electrophysiological studies in primates have shown that the melanopsin-containing RGCs are strongly influenced by excitatory and inhibitory inputs from rods and cones. 5 It is likely that this modulation represents functional fine-tuning of the irradiance detecting system, driving entrainment of the circadian system and pupillomotor control during irradiance changes at twilight. Melanopsin expression is regulated by a circadian clock and by the light/dark cycles. 35 36 37 Input from the classical photoreceptors also seems to influence the expression level of melanopsin. In rats lacking the outer retina because of retinal degeneration (Royal College of Surgeons rats with a defect in the retinal dystrophy gene RCS/N-rdy) cyclic changes in melanopsin mRNA are eliminated. 37 indicating that rods, cones, or both are involved in the regulation of melanopsin mRNA expression. Recently, evidence was provided that dopaminergic amacrine cells are involved in melanopsin expression. 38 When cells of the INL, including TH-containing amacrine cells, were significantly reduced by intraocular injection of kainic acid, a significant reduction was observed in the diurnal changes of melanopsin expression. 38 Furthermore, injection of the specific dopamine D2 agonist quinpirole provoked a dose- and time-dependent increase in melanopsin mRNA level, most likely through the dopamine D2 receptor expressed in the melanopsin-containing RGCs. 38  
By confocal microscopy and computer-based three-dimensional analysis, we have demonstrated that melanopsin-containing RGCs make synaptic contact with rod bipolar cells along the borders of the IPL and the ganglion cell layer and with TH-IR amacrine cells through axodendritic and axosomatic contact along the borders of the INL/IPL, supporting electrophysiological observations that rods and cones signal to ipRGCs. 
 
Figure 1.
 
Histogram showing the number of presumed ribbon synapses visualized by CtBP2-IR found in PKC-IR axon terminals (n = 48) of the rat retina. Gray bars: number of colocalized IR puncta in correctly superimposed terminals that were significantly higher than the population (P < 0.027, Wilcoxon paired test) of the pseudorandom flipped controls (black bars).
Figure 1.
 
Histogram showing the number of presumed ribbon synapses visualized by CtBP2-IR found in PKC-IR axon terminals (n = 48) of the rat retina. Gray bars: number of colocalized IR puncta in correctly superimposed terminals that were significantly higher than the population (P < 0.027, Wilcoxon paired test) of the pseudorandom flipped controls (black bars).
Figure 2.
 
Synaptic contact between PKC-IR rod bipolar cells and melanopsin-IR RGCs. (AF) Stacks of confocal digital images of cross-sections of rat retina showing melanopsin-IR RGCs located in the GCL with a dendritic process passing through the IPL toward the zone between the IPL and INL (A, C, D, F, red). PKC-IR (B, C, E, F, green) are found in bipolar cell bodies located in the INL and in bipolar axons, with terminal buttons found at the borders between the IPL and the GCL. (C, F, yellow) Potential synaptic contacts analyzed by computer. (GL) Extended view of 85 digital sections of melanopsin-IR RGCs (red) and PKC-IR processes (green) in a flat-mount preparation analyzed for synaptic contacts. (G) Extended view of all digital sections. Computer-based selection of (H) red (melanopsin) and (I) green (PKC-IR) used for the analyses. (J) Three-dimensional model of the two melanopsin-IR RGCs, together with the calculated synaptic contacts with PKC-IR (light blue). (K, L) High-power magnification of this synaptic contact. (K) PKC, melanopsin, and synaptic contact. (L) Melanopsin and the calculated synaptic contact. Scale bars: 20 μm (AF), 15 μm (GJ), 7.5 μm (KL).
Figure 2.
 
Synaptic contact between PKC-IR rod bipolar cells and melanopsin-IR RGCs. (AF) Stacks of confocal digital images of cross-sections of rat retina showing melanopsin-IR RGCs located in the GCL with a dendritic process passing through the IPL toward the zone between the IPL and INL (A, C, D, F, red). PKC-IR (B, C, E, F, green) are found in bipolar cell bodies located in the INL and in bipolar axons, with terminal buttons found at the borders between the IPL and the GCL. (C, F, yellow) Potential synaptic contacts analyzed by computer. (GL) Extended view of 85 digital sections of melanopsin-IR RGCs (red) and PKC-IR processes (green) in a flat-mount preparation analyzed for synaptic contacts. (G) Extended view of all digital sections. Computer-based selection of (H) red (melanopsin) and (I) green (PKC-IR) used for the analyses. (J) Three-dimensional model of the two melanopsin-IR RGCs, together with the calculated synaptic contacts with PKC-IR (light blue). (K, L) High-power magnification of this synaptic contact. (K) PKC, melanopsin, and synaptic contact. (L) Melanopsin and the calculated synaptic contact. Scale bars: 20 μm (AF), 15 μm (GJ), 7.5 μm (KL).
Figure 3.
 
(AE) Cross-sections of the rat retina showing PKC-IR terminals (red) costoring CtBP2, a synaptic ribbon marker (green) in close contact with melanopsin-IR RGCs and their proximal dendrites (blue). (A, B) Strong immunostaining of CtBP2 is also found in the zone of synaptic contacts between photoreceptors and bipolar cells (asterisks). PKC-IR terminals costoring CtBP2 with close contact to melanopsin-IR RGCs (arrows). Computer analyses of stacks of digital images obtained from these areas are illustrated in Fig. 4 . Scale bars: 20 μm (A, B), 10 μm (CE).
Figure 3.
 
(AE) Cross-sections of the rat retina showing PKC-IR terminals (red) costoring CtBP2, a synaptic ribbon marker (green) in close contact with melanopsin-IR RGCs and their proximal dendrites (blue). (A, B) Strong immunostaining of CtBP2 is also found in the zone of synaptic contacts between photoreceptors and bipolar cells (asterisks). PKC-IR terminals costoring CtBP2 with close contact to melanopsin-IR RGCs (arrows). Computer analyses of stacks of digital images obtained from these areas are illustrated in Fig. 4 . Scale bars: 20 μm (A, B), 10 μm (CE).
Figure 4.
 
Computer-based analysis for synaptic contacts between melanopsin-IR RGCs (blue) and PKC-IR bipolar cells (red) containing the synaptic ribbon marker CtBP2 (green). (A) Extended view of 86 digital sections of two melanopsin-IR RGCs located in the GCL. Arrows: areas of potential synaptic contact. A three-dimensional model is constructed (B) and analyzed in (CF). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and PKC-containing bipolar processes (yellow). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with CtBP2-IR terminals (white). Note that not all white areas correspond to contacts between melanopsin and PKC seen in (C). Some represent contacts with other types of bipolar cells (D, double arrows). (E) Areas that represent synaptic contact between PKC-IR bipolar terminals containing CtBP2-IR and the melanopsin membrane (purple). (F) Higher magnification of (E). Scale bars: 7 μm (AE), 5 μm (F).
Figure 4.
 
Computer-based analysis for synaptic contacts between melanopsin-IR RGCs (blue) and PKC-IR bipolar cells (red) containing the synaptic ribbon marker CtBP2 (green). (A) Extended view of 86 digital sections of two melanopsin-IR RGCs located in the GCL. Arrows: areas of potential synaptic contact. A three-dimensional model is constructed (B) and analyzed in (CF). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and PKC-containing bipolar processes (yellow). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with CtBP2-IR terminals (white). Note that not all white areas correspond to contacts between melanopsin and PKC seen in (C). Some represent contacts with other types of bipolar cells (D, double arrows). (E) Areas that represent synaptic contact between PKC-IR bipolar terminals containing CtBP2-IR and the melanopsin membrane (purple). (F) Higher magnification of (E). Scale bars: 7 μm (AE), 5 μm (F).
Figure 5.
 
Computer-based analysis for synaptic contact between melanopsin-IR RGCs (blue) and a presynaptic marker of the synaptic ribbon marker CtBP2 (red) and the postsynaptically located glutamate receptor 4 (GluR4 in green). (A) Extended view of 53 digital sections of a melanopsin-IR RGC located in the GCL. A three-dimensional model is constructed (B) and analyzed (C-E). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and CtBP2-containing processes (light blue). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with GluR4-IR (pink). Note that almost all areas containing CtBP2 on the cell soma correspond to contacts between melanopsin and GluR4. (E) The computer-based overlap representing synapses showed as contacts among melanopsin, CtBP2, and GluR4. Scale bars, 5 μm.
Figure 5.
 
Computer-based analysis for synaptic contact between melanopsin-IR RGCs (blue) and a presynaptic marker of the synaptic ribbon marker CtBP2 (red) and the postsynaptically located glutamate receptor 4 (GluR4 in green). (A) Extended view of 53 digital sections of a melanopsin-IR RGC located in the GCL. A three-dimensional model is constructed (B) and analyzed (C-E). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and CtBP2-containing processes (light blue). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with GluR4-IR (pink). Note that almost all areas containing CtBP2 on the cell soma correspond to contacts between melanopsin and GluR4. (E) The computer-based overlap representing synapses showed as contacts among melanopsin, CtBP2, and GluR4. Scale bars, 5 μm.
Figure 6.
 
TH-IR (dopaminergic) amacrine cells have synaptic contact with melanopsin-IR processes in the rat retina. (A) Flat-mount preparation and an extended view of 100 digital sections of a melanopsin-IR RGC (red) located in the GCL and a dendritic process reaching the border between the IPL and the INL, where it bifurcates in a network of TH-positive processes (green). Yellow: potential contacts. (B) Computer-based selection of red (melanopsin) and (C) green (TH-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR RGCs, together with the calculated synaptic contacts with TH-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) TH (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 25 μm (AD), 12.5 μm (E, F).
Figure 6.
 
TH-IR (dopaminergic) amacrine cells have synaptic contact with melanopsin-IR processes in the rat retina. (A) Flat-mount preparation and an extended view of 100 digital sections of a melanopsin-IR RGC (red) located in the GCL and a dendritic process reaching the border between the IPL and the INL, where it bifurcates in a network of TH-positive processes (green). Yellow: potential contacts. (B) Computer-based selection of red (melanopsin) and (C) green (TH-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR RGCs, together with the calculated synaptic contacts with TH-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) TH (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 25 μm (AD), 12.5 μm (E, F).
Figure 7.
 
Contact between dopaminergic amacrine cells illustrated by staining of vesicular monoamine transporter 2 (VMAT2) and melanopsin-IR processes in the rat retina. (A) Cross-section of the rat retina shows two melanopsin-IR ganglion cells (red) with distal processes in the stratum 1 of the IPL in close contact with dopaminergic processes containing VMAT2-IR (green). (B) High-powered photomicrograph of stratum 1 used for three-dimensional analyses is shown in extended view (68 digital sections). Yellow: potential contacts. (C) Computer-based three-dimensional reconstruction of red (melanopsin) and green (VMAT2-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR processes, together with the calculated synaptic contacts with VMAT2-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) VMAT2 (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 20 μm (AD), 10 μm (E, F).
Figure 7.
 
Contact between dopaminergic amacrine cells illustrated by staining of vesicular monoamine transporter 2 (VMAT2) and melanopsin-IR processes in the rat retina. (A) Cross-section of the rat retina shows two melanopsin-IR ganglion cells (red) with distal processes in the stratum 1 of the IPL in close contact with dopaminergic processes containing VMAT2-IR (green). (B) High-powered photomicrograph of stratum 1 used for three-dimensional analyses is shown in extended view (68 digital sections). Yellow: potential contacts. (C) Computer-based three-dimensional reconstruction of red (melanopsin) and green (VMAT2-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR processes, together with the calculated synaptic contacts with VMAT2-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) VMAT2 (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 20 μm (AD), 10 μm (E, F).
The authors thank Anita Hansen for her skillful technical assistance. 
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Figure 1.
 
Histogram showing the number of presumed ribbon synapses visualized by CtBP2-IR found in PKC-IR axon terminals (n = 48) of the rat retina. Gray bars: number of colocalized IR puncta in correctly superimposed terminals that were significantly higher than the population (P < 0.027, Wilcoxon paired test) of the pseudorandom flipped controls (black bars).
Figure 1.
 
Histogram showing the number of presumed ribbon synapses visualized by CtBP2-IR found in PKC-IR axon terminals (n = 48) of the rat retina. Gray bars: number of colocalized IR puncta in correctly superimposed terminals that were significantly higher than the population (P < 0.027, Wilcoxon paired test) of the pseudorandom flipped controls (black bars).
Figure 2.
 
Synaptic contact between PKC-IR rod bipolar cells and melanopsin-IR RGCs. (AF) Stacks of confocal digital images of cross-sections of rat retina showing melanopsin-IR RGCs located in the GCL with a dendritic process passing through the IPL toward the zone between the IPL and INL (A, C, D, F, red). PKC-IR (B, C, E, F, green) are found in bipolar cell bodies located in the INL and in bipolar axons, with terminal buttons found at the borders between the IPL and the GCL. (C, F, yellow) Potential synaptic contacts analyzed by computer. (GL) Extended view of 85 digital sections of melanopsin-IR RGCs (red) and PKC-IR processes (green) in a flat-mount preparation analyzed for synaptic contacts. (G) Extended view of all digital sections. Computer-based selection of (H) red (melanopsin) and (I) green (PKC-IR) used for the analyses. (J) Three-dimensional model of the two melanopsin-IR RGCs, together with the calculated synaptic contacts with PKC-IR (light blue). (K, L) High-power magnification of this synaptic contact. (K) PKC, melanopsin, and synaptic contact. (L) Melanopsin and the calculated synaptic contact. Scale bars: 20 μm (AF), 15 μm (GJ), 7.5 μm (KL).
Figure 2.
 
Synaptic contact between PKC-IR rod bipolar cells and melanopsin-IR RGCs. (AF) Stacks of confocal digital images of cross-sections of rat retina showing melanopsin-IR RGCs located in the GCL with a dendritic process passing through the IPL toward the zone between the IPL and INL (A, C, D, F, red). PKC-IR (B, C, E, F, green) are found in bipolar cell bodies located in the INL and in bipolar axons, with terminal buttons found at the borders between the IPL and the GCL. (C, F, yellow) Potential synaptic contacts analyzed by computer. (GL) Extended view of 85 digital sections of melanopsin-IR RGCs (red) and PKC-IR processes (green) in a flat-mount preparation analyzed for synaptic contacts. (G) Extended view of all digital sections. Computer-based selection of (H) red (melanopsin) and (I) green (PKC-IR) used for the analyses. (J) Three-dimensional model of the two melanopsin-IR RGCs, together with the calculated synaptic contacts with PKC-IR (light blue). (K, L) High-power magnification of this synaptic contact. (K) PKC, melanopsin, and synaptic contact. (L) Melanopsin and the calculated synaptic contact. Scale bars: 20 μm (AF), 15 μm (GJ), 7.5 μm (KL).
Figure 3.
 
(AE) Cross-sections of the rat retina showing PKC-IR terminals (red) costoring CtBP2, a synaptic ribbon marker (green) in close contact with melanopsin-IR RGCs and their proximal dendrites (blue). (A, B) Strong immunostaining of CtBP2 is also found in the zone of synaptic contacts between photoreceptors and bipolar cells (asterisks). PKC-IR terminals costoring CtBP2 with close contact to melanopsin-IR RGCs (arrows). Computer analyses of stacks of digital images obtained from these areas are illustrated in Fig. 4 . Scale bars: 20 μm (A, B), 10 μm (CE).
Figure 3.
 
(AE) Cross-sections of the rat retina showing PKC-IR terminals (red) costoring CtBP2, a synaptic ribbon marker (green) in close contact with melanopsin-IR RGCs and their proximal dendrites (blue). (A, B) Strong immunostaining of CtBP2 is also found in the zone of synaptic contacts between photoreceptors and bipolar cells (asterisks). PKC-IR terminals costoring CtBP2 with close contact to melanopsin-IR RGCs (arrows). Computer analyses of stacks of digital images obtained from these areas are illustrated in Fig. 4 . Scale bars: 20 μm (A, B), 10 μm (CE).
Figure 4.
 
Computer-based analysis for synaptic contacts between melanopsin-IR RGCs (blue) and PKC-IR bipolar cells (red) containing the synaptic ribbon marker CtBP2 (green). (A) Extended view of 86 digital sections of two melanopsin-IR RGCs located in the GCL. Arrows: areas of potential synaptic contact. A three-dimensional model is constructed (B) and analyzed in (CF). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and PKC-containing bipolar processes (yellow). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with CtBP2-IR terminals (white). Note that not all white areas correspond to contacts between melanopsin and PKC seen in (C). Some represent contacts with other types of bipolar cells (D, double arrows). (E) Areas that represent synaptic contact between PKC-IR bipolar terminals containing CtBP2-IR and the melanopsin membrane (purple). (F) Higher magnification of (E). Scale bars: 7 μm (AE), 5 μm (F).
Figure 4.
 
Computer-based analysis for synaptic contacts between melanopsin-IR RGCs (blue) and PKC-IR bipolar cells (red) containing the synaptic ribbon marker CtBP2 (green). (A) Extended view of 86 digital sections of two melanopsin-IR RGCs located in the GCL. Arrows: areas of potential synaptic contact. A three-dimensional model is constructed (B) and analyzed in (CF). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and PKC-containing bipolar processes (yellow). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with CtBP2-IR terminals (white). Note that not all white areas correspond to contacts between melanopsin and PKC seen in (C). Some represent contacts with other types of bipolar cells (D, double arrows). (E) Areas that represent synaptic contact between PKC-IR bipolar terminals containing CtBP2-IR and the melanopsin membrane (purple). (F) Higher magnification of (E). Scale bars: 7 μm (AE), 5 μm (F).
Figure 5.
 
Computer-based analysis for synaptic contact between melanopsin-IR RGCs (blue) and a presynaptic marker of the synaptic ribbon marker CtBP2 (red) and the postsynaptically located glutamate receptor 4 (GluR4 in green). (A) Extended view of 53 digital sections of a melanopsin-IR RGC located in the GCL. A three-dimensional model is constructed (B) and analyzed (C-E). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and CtBP2-containing processes (light blue). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with GluR4-IR (pink). Note that almost all areas containing CtBP2 on the cell soma correspond to contacts between melanopsin and GluR4. (E) The computer-based overlap representing synapses showed as contacts among melanopsin, CtBP2, and GluR4. Scale bars, 5 μm.
Figure 5.
 
Computer-based analysis for synaptic contact between melanopsin-IR RGCs (blue) and a presynaptic marker of the synaptic ribbon marker CtBP2 (red) and the postsynaptically located glutamate receptor 4 (GluR4 in green). (A) Extended view of 53 digital sections of a melanopsin-IR RGC located in the GCL. A three-dimensional model is constructed (B) and analyzed (C-E). (C) Three-dimensional model shows the computer-based analysis of contact between melanopsin-IR RGCs (blue) and CtBP2-containing processes (light blue). (D) Areas of computer-based analysis of the melanopsin-IR membrane, which has contact with GluR4-IR (pink). Note that almost all areas containing CtBP2 on the cell soma correspond to contacts between melanopsin and GluR4. (E) The computer-based overlap representing synapses showed as contacts among melanopsin, CtBP2, and GluR4. Scale bars, 5 μm.
Figure 6.
 
TH-IR (dopaminergic) amacrine cells have synaptic contact with melanopsin-IR processes in the rat retina. (A) Flat-mount preparation and an extended view of 100 digital sections of a melanopsin-IR RGC (red) located in the GCL and a dendritic process reaching the border between the IPL and the INL, where it bifurcates in a network of TH-positive processes (green). Yellow: potential contacts. (B) Computer-based selection of red (melanopsin) and (C) green (TH-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR RGCs, together with the calculated synaptic contacts with TH-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) TH (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 25 μm (AD), 12.5 μm (E, F).
Figure 6.
 
TH-IR (dopaminergic) amacrine cells have synaptic contact with melanopsin-IR processes in the rat retina. (A) Flat-mount preparation and an extended view of 100 digital sections of a melanopsin-IR RGC (red) located in the GCL and a dendritic process reaching the border between the IPL and the INL, where it bifurcates in a network of TH-positive processes (green). Yellow: potential contacts. (B) Computer-based selection of red (melanopsin) and (C) green (TH-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR RGCs, together with the calculated synaptic contacts with TH-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) TH (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 25 μm (AD), 12.5 μm (E, F).
Figure 7.
 
Contact between dopaminergic amacrine cells illustrated by staining of vesicular monoamine transporter 2 (VMAT2) and melanopsin-IR processes in the rat retina. (A) Cross-section of the rat retina shows two melanopsin-IR ganglion cells (red) with distal processes in the stratum 1 of the IPL in close contact with dopaminergic processes containing VMAT2-IR (green). (B) High-powered photomicrograph of stratum 1 used for three-dimensional analyses is shown in extended view (68 digital sections). Yellow: potential contacts. (C) Computer-based three-dimensional reconstruction of red (melanopsin) and green (VMAT2-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR processes, together with the calculated synaptic contacts with VMAT2-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) VMAT2 (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 20 μm (AD), 10 μm (E, F).
Figure 7.
 
Contact between dopaminergic amacrine cells illustrated by staining of vesicular monoamine transporter 2 (VMAT2) and melanopsin-IR processes in the rat retina. (A) Cross-section of the rat retina shows two melanopsin-IR ganglion cells (red) with distal processes in the stratum 1 of the IPL in close contact with dopaminergic processes containing VMAT2-IR (green). (B) High-powered photomicrograph of stratum 1 used for three-dimensional analyses is shown in extended view (68 digital sections). Yellow: potential contacts. (C) Computer-based three-dimensional reconstruction of red (melanopsin) and green (VMAT2-IR) used for the analyses. (D) Three-dimensional model of the melanopsin-IR processes, together with the calculated synaptic contacts with VMAT2-IR (light blue). (E, F) High-powered magnification of the synaptic contacts. (E) VMAT2 (green), melanopsin (red), and synaptic contact (light blue). (F) Melanopsin (red) and calculated synaptic contact (light blue). Scale bars: 20 μm (AD), 10 μm (E, F).
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