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Evidence for synergistic and complementary roles of Bassoon and darkness in organizing the ribbon synapse
Neuroscience 236 (2013) 149–159
EVIDENCE FOR SYNERGISTIC AND COMPLEMENTARY ROLES OF BASSOON AND DARKNESS IN ORGANIZING THE RIBBON SYNAPSE I. SPIWOKS-BECKER, a* R. LAMBERTI, b S. TOM DIECK c,d AND R. SPESSERT b
INTRODUCTION Sensory neurons of the retina and inner ear face the challenge of transmitting sensory signals over a broad range of intensity magnitude. For this reason, they need dynamic and adjustable synaptic machinery, which is provided by a unique type of chemical synapse, the ribbon synapse (tom Dieck and Brandsta¨tter, 2006). Their most prominent organelle is the so-called synaptic ribbon (SR), which is thought to tune the synaptic vesicle cycle in a dynamic manner for speed, precision, and endurance (Zanazzi and Matthews, 2009). SRs constitute electrondense structures of various forms and considerable size (Vollrath and Spiwoks-Becker, 1996). They are capable of tethering hundreds of synaptic vesicles (Rao-Mirotznik et al., 1995), which are positioned by the ribbon in close proximity to the presynaptic neurotransmitter release site, namely the active zone. Most of the ribbon synapses in the outer plexiform layer of the mouse retina form part of the rod terminals, whereas the cone terminals account for less than 2% of the photoreceptor terminals. In the rod photoreceptor ribbon synapse, the prototype of a ribbon synapse, SRs mostly appear as rod-like structures in ultrathin sections, whereas three-dimensionally, they are thin plates with a horseshoelike shape that is easily detected by immunofluorescence light microscopy. They are 30–50 nm in thickness, protrude upward with an average of 300 nm into the presynaptic cytoplasm, and can extend up to 2 lm in length parallel to the presynaptic plasma membrane. The concave inner base of the ribbon is anchored to the presynaptic membrane by means of the arciform density, a second electron-dense structure belonging to the ribbon complex. The morphology of photoreceptor SRs is dynamic (Vollrath et al., 2001; Regus-Leidig et al., 2010a) and undergoes daily changes in BALB/c mice (Adly et al., 1999; Spiwoks-Becker et al., 2004). Although they are large and smooth during the dark phase, after light exposure in the morning, they form distal swellings that bud off, thus forming spherical synaptic bodies (synaptic spheres, SSs), whereas at the onset of darkness, the reverse process occurs (Spiwoks-Becker et al., 2004). Accordingly, SSs can be considered as being the disintegrated building block pool of ribbon material. A major component of the SRs is the protein RIBEYE (Schmitz et al., 2000; Magupalli et al., 2008; Venkatesan et al., 2010). Other molecular components found to be enriched at the photoreceptor SRs are a variety of proteins of the cytomatrix at the active zone (CAZ) as found in conventional active zones, including the
a
Institute of Microanatomy and Neurobiology, University Medical Center of the Johannes Gutenberg University, 55099 Mainz, Germany
b
Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University, 55099 Mainz, Germany
c Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/M, Germany d
Department of Synaptic Plasticity, Max Planck Institute for Brain Research, D-60528 Frankfurt/M, Germany
Abstract—Ribbon synapses are tonically active highthroughput synapses. The performance of the ribbon synapse is accomplished by a specialization of the cytomatrix at the active zone (CAZ) referred to as the synaptic ribbon (SR). Progress in our understanding of the structure–function relationship at the ribbon synapse has come from observations that, in photoreceptors lacking a full-size scaffolding protein Bassoon (BsnDEx4=5 ), dissociation of SRs coincides with perturbed signal transfer. The aim of the present study has been to elaborate the role of Bassoon as a structural organizer of the ribbon synapse and to differentiate it with regard to the ambient lighting conditions. The ultrastructure of retinal ribbon synapses has been compared between wild-type (Wt) and BsnDEx4=5 mice adapted to light (low activity) and darkness (high activity). The results obtained suggest that Bassoon and environmental illumination synergistically and complementarily act as organizers of the ribbon synapse. Thus, light-dependent and Bassoon-independent regulation involves initial SR tethering to the membrane and a basic shape transition of ribbon material from spherical to rod-like, since darkness induces these features in BsnDEx4=5 rod spherules. However, the tight anchorage of the SR via an arciform density and the proper assembly of SRs to the full-sized horseshoe-shaped complex depend on Bassoon, as these steps fail in BsnDEx4=5 rod spherules. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: bassoon, retina, ribbon synapse, synaptic ribbon, light/dark-cycle.
*Corresponding author. Tel: +49-6131-3922365; fax: +49-61313922167. E-mail address: [email protected] (I. Spiwoks-Becker). Abbreviations: Bc, bipolar cells; CAZ, cytomatrix at the active zone; hc, horizontal cells; PBS, phosphate-buffered saline; SS, spherical synaptic bodies; SR, synaptic ribbon; wt, wild-type.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.031 149
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scaffolding protein Bassoon (tom Dieck et al., 2005). These findings have led to the concept that SRs are a specialization of the CAZ present at conventional synapses (Zhai and Bellen, 2004). The ribbon complex of photoreceptor cells can be segregated into a ribbonassociated compartment and an active zone compartment. Bassoon is enriched at the proximal/ concave site of the ribbon and appears to contribute essentially to the link between the two compartments (tom Dieck et al., 2005). At the ribbon site, it physically interacts with RIBEYE (tom Dieck et al., 2005), whereas the interacting protein at the active zone compartment has not yet been identified. In BsnDEx4=5 mutant mice, the region of Bassoon interacting with RIBEYE is deleted (Altrock et al., 2003; Dick et al., 2003; tom Dieck et al., 2005; Frank et al., 2010). These mice produce a 180-kDa Bassoon mutant protein, which is not tightly anchored to the CAZ and is easier to extract (Altrock et al., 2003; Dick et al., 2003). Consistent with the loss of the Bassoon-RIBEYE interaction, SRs in light-adapted BsnDEx4=5 rod spherules are not anchored to the presynaptic active zone but float freely in the cytoplasm (Dick et al., 2003). Since the function of SRs is to tether synaptic vesicles close to the release sites, this seems to explain why retina signal transfer from photoreceptors to the second-order neuron is disturbed in the BsnDEx4=5 mutant (Dick et al., 2003). The observation that, in BsnDEx4=5 rod spherules, the shape of anti-RIBEYE-stained SRs changes from horseshoe-like to punctate (tom Dieck et al., 2005) hints at the possibility that Bassoon also influences the morphology of SRs, and that perturbed signal transmission in BsnDEx4=5 mutant photoreceptor terminals might therefore be attributable to several structural deficiencies (tom Dieck et al., 2005; Specht et al., 2009). The aim of the present study was to dissect the role of Bassoon as a structural organizer of the ribbon synapse in dependence of the ambient lighting conditions. To this end, the ultrastructure of ribbon synapses was systematically compared between BsnDEx4=5 and wild-type (wt) mice during the light phase when synaptic transmission is low and the dark phase when synaptic transmission is high.
EXPERIMENTAL PROCEDURES Animals All animal experiments were performed in accordance with the guidelines issued by the Max Planck Society. In total, 17 wt and 11 Bassoon mutant mice (BsnDEx4=5 ; Altrock et al., 2003; Dick et al., 2003) were examined under light- (4 h after light onset) or dark-adapted (4 h after dark onset) conditions from two independent experiments with an age of 8–15 weeks. Homozygous BsnDEx4=5 mice and wt controls (mixed background: 129/Sv 129/SvJ from R1 ES-cells, backcrossed to C57Bl/6) were both obtained as littermate offspring from intercrosses of heterozygous BsnDEx4=5 mice. The mice were kept under constant laboratory conditions (12-h light, 12-h dark; lights on at 6 a.m. and off at 6 p.m.; less than 100–200 lux at the bottom of the cages; food and water ad libitum) for a minimum of one week prior to the experiments. They were anesthetized with isoflurane and decapitated prior to removal of the eyes during darkness under dim red light. Usually, both eyes/retinae per animal were removed by using the Winkler method (Winkler, 1972).
Electron microscopy and morphometry For the electron-microscopical evaluation, the retinae were dissected from rapidly removed eye balls and fixed in freshly prepared fixative, viz., 2% paraformaldehyde, 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 h. Subsequently, tissue was rinsed in PBS containing 6.8% (w/v) sucrose, postfixed in osmium tetroxide (2% (w/v) in PBS) for 90 min, washed three times in PBS, and dehydrated in a graded series of acetone. Tissue was flat-embedded in Epon (Serva, Heidelberg, Germany). Transverse sections (50–60 nm thick) were mounted onto one-hole Formvar-coated copper grids (Serva), stained with 8% (w/v) uranyl acetate (10 min), and contrasted with lead citrate for 5 min. Sections were viewed in an LEO 906 transmission electron microscope (Zeiss, Oberkochen, Germany), and all synapses were photographed. Morphometry was carried out by using Analysis 3.2. Software (Soft Imaging Systems, Mu¨nster, Germany).
Quantitative analysis From one randomly selected retinal section, synaptic body profiles in approximately 50 neighboring photoreceptor terminals were systematically examined according to the following criteria: type of SR profile (e.g., rodlike, spherical, or polymorphic) and size and location of the SR within the terminal. The SRs were classified as ‘‘tethered’’ when they bordered on the presynaptic membrane, in wt photoreceptors, typically via the arciform density and, in bipolar cells (BC), via small dense plaques. SR profiles not bordering on the presynaptic membrane were referred to as being ‘‘free’’. Because rod-like SRs of rod photoreceptor are sections through horseshoe-shaped structures, and because the structure might be cut at various angles, transverse cuts yield shorter profiles with a smaller variability than horizontal cuts, which represent the SR length, and therefore sections were taken through the distal SR area (see also Fig. 3A). However, transversely cut profiles can be easily recognized, since, unlike the horizontally cut profiles, the underlying arciform density is seen together with the ribbon. In general, within a given synapse, the number of SRs and SR fields were also estimated. Synapses without SR profiles were classified as ‘‘empty’’ endings. Furthermore, measurements of the area of the postsynaptic horizontal and bipolar processes were performed. Tissue and data analyses were performed blind, i.e., without knowledge of the genotype and by using coded specimens.
Serial section analysis To draw conclusions on the three-dimensional shape, the location of the SRs, and their arrangement at the synaptic complex, serial sections of randomly chosen profiles of light- and dark-adapted mutant photoreceptor terminals were photographed. The synaptic areas of adjacent sections were superimposed by using the Adobe Photoshop (Adobe Systems Inc.).
Statistical analysis The data obtained were expressed as means ± standard error of the mean. For statistical analysis, Student’s t-test or the Wilcoxon-Mann–Whitney U-test was used. A p-value of smaller than or equal to 0.05 was regarded as significant. The values are based on pooled data from two independent experiments.
RESULTS Light-dependent ultrastructural changes in rod ribbon synapses lacking functional Bassoon In adult wt mice (8–15 weeks), presynaptic electron-dense SRs typically anchored to the so-called arciform density
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facing four postsynaptic elements: two horizontal cell processes and two rod bipolar cell dendrites (Fig. 1A–C). The SR showed a rod-like appearance in vertically sectioned (Fig. 1A, C) and a horseshoe-shaped appearance in sagitally sectioned (Fig. 1B) rod spherules. No obvious differences were seen in single sections between light- and dark-adapted wt animals (Fig. 1A, C). In contrast, light-adapted rod spherules in BsnDEx4=5 retinas (2 sections from each retina; in total 243 terminals were examined) often either did not contain SR profiles (stars in Fig. 1D, E) or had multiple and often very small SSs (considered as the disintegrated building block pool of ribbon material) floating freely in the cytoplasm (arrowheads in Fig. 1D, E). SSs tended to aggregate to ‘‘ribbon fields’’ (Fig. 1D–F). Furthermore, ‘‘anchorage’’ places of SRs were typically ‘‘empty’’ (arrow in Fig. 1D) and did not show a typical arciform density. In sagitally sectioned rod spherules, SRs never appeared horseshoe-shaped as in wt mice (Fig. 1B) but rather as a rectangular-like plate of smaller dimensions than wt SRs (Fig. 1E). In addition, the area of invaginating postsynaptic elements was smaller when compared with wt (Fig. 1A versus D).
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In BsnDEx4=5 retina, empty spherules and those with only spherical material occurred less often in mice adapted to the dark than in those adapted to the light. In dark-adapted BsnDEx4=5 mice, retina spherules containing SRs were tethered to the membrane, often (16.4%) two in parallel (Fig. 1F). The membrane tethering mostly occurred in locations lacking the typical features of wt anchorage spots. Thus, the typical arciform densities and proper tetradic postsynaptic arrangements were absent. These observations suggest that light/dark regulation might still take place in BsnDEx4=5 rod spherules and can be used to unravel the characteristics of normal light-dependent structural changes. Dissecting steps in ribbon anchorage: membrane tethering of SR but not tight anchorage in BsnDEx4=5 rod spherules With respect to the membrane tethering, we found that, in wt retinas, >90% of ribbons showed tight membrane anchorage, regardless of the ambient lighting conditions (Fig. 2A). Although bona fide ribbon anchorage as defined by the appearance of an arciform density was never observed in the mutant (Fig. 1D–F), membrane
Fig. 1. Transmission–electron micrographs of rod photoreceptor synapses in wild-type and homozygous Bassoon mutant retina in mice adapted to the light or dark. The rod spherules are sectioned vertically (A, C, D, F) and sagittally (B, E). In wild-type (wt) mice (A–C), presynaptic electron-dense synaptic ribbons (SRs, arrowheads) anchor to the so-called arciform density (arrows) facing four postsynaptic elements: two horizontal cell processes (hc) and two rod bipolar cell dendrites (bc). The SR shows a rod-like appearance in vertically sectioned spherules (arrowheads in A, C) and a horseshoe-like appearance in sagittally sectioned spherules (arrowhead in B). In Bassoon mutant (BsnDEx4=5 ) rod endings (D–F), spherules without SRs are seen (stars in D, E), anchorages of SRs are typically ‘‘empty’’ (e.g. arrow in D), and in the cytoplasm, ‘‘free floating’’ multiple synaptic spherical bodies (SSs) are abundant (arrowheads in D, E). In horizontally sectioned spherules of mutant mice, the shape of SRs changes from horseshoe-like (B) to plate/rectangular-like (E). Note also that, in mutants, the area of the postsynaptic elements is decreased (e.g., C versus F). As exemplified by SR anchoring, the characteristics of the mutant ribbon synapses are more pronounced during the light phase (D, E) than during darkness (F). Scale bars, 0.2 lm (A–C, F); 0.5 lm (D, E).
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Fig. 2. Membrane-tethering of synaptic ribbons in wild-type and homozygous Bassoon mutant retina in mice adapted to the light or dark. (A) Quantification of membrane-tethered synaptic ribbons (SRs). Note the decrease in the number of membrane-tethered SRs in Bassoon mutants (BsnDEx4=5 ) versus wild-type (wt) mice, which is more pronounced under light conditions. L = light, D = Dark, n = SRs examined (wt: L n = 464, D n = 387; BsnDEx4=5 : L n = 391, D n = 414. n = rod spherules examined wt: L n = 428, D n = 323; BsnDEx4=5 : L n = 243, D n = 291. Data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 dark-adapted wt mice, and 6 dark-adapted mutants (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001. (B–I) Ultrastructural appearance of SR profiles in serial sections of photoreceptor synapses (numbered 1–5) from BsnDEx4=5 in the light (B–E) and dark (F–I) phases. The figure exemplifies that, in a follow-up series of rod spherules, only 15% of the SRs (from 21 section series analyzed) contact the membrane in light-adapted mice, but all SRs (from 30 section series analyzed) contact the membrane at least at one point throughout the series in dark-adapted mice. The arrows in D, E, and F mark ‘‘empty’’ anchorages of SRs. Note also that, in light-adapted mutant rod spherules, multiple small and membrane-detached spherical ribbon profiles (SSs, arrowhead) are present, often appearing in only one section. Dark-adapted mutant rod spherules show a decrease in the number of SSs and often contain two parallel ribbon profiles (e.g. F2–I2). Cone pedicles (CP) do not show SSs, but sometimes SRs with a direct attachment to the presynaptic membrane are seen (⁄in D, E). Scale bars, 1 lm.
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tethering of 42.1 ± 6.1% of dark-adapted and 19 ± 4.1% of light-adapted BsnDEx4=5 ribbons (Fig. 2A) was seen. Although quantification from single sections is a valid measure of the distance between SRs and cell membrane in a given plane, it does not inherently provide the precise number of SRs tethered to the cell membrane. Accordingly, quantification from single sections might miss short stretches of membrane contact and therefore will usually lead to underestimates of attachment numbers. Serial section analysis (Fig. 2B–I) revealed that, in BsnDEx4=5 mice, SRs seemingly detached from the membrane in a given section were often found to be tethered to the membrane at least at one point. Consistent with this observation, Bassoon promoted long stretches of membrane contact, whereas short stretches of membrane contact occurred in the absence of a functional Bassoon. In BsnDEx4=5 rod spherules, the tethering of SRs was found to be increased in darkness (Fig. 2A) and therefore seemed to depend on the lighting conditions. We conclude from these observations that the anchorage of the SR to the presynaptic membrane involves two distinct steps: a light-dependent step ensuring that tethering takes place, and a Bassoon-dependent step responsible for tight anchorage via the arciform density. Basic transition of SSs to SRs but not the assembly of mature ribbon units in BsnDEx4=5 rod spherules To confirm and specify the influences of Bassoon and environmental lighting conditions on the ribbon material itself, its structural integrity (in terms of the frequency of SSs), its size (in terms of the diameter of SSs, the height of SRs, and the length of SRs), and its tendency to aggregate with other ribbon material (in terms of the frequency of so-called synaptic fields) were compared in wt and BsnDEx4=5 rod spherules during the light and dark phases. In wt rod spherules, SSs were rarely seen during the light phase (2.2 ± 0.7%) and were absent during the dark phase (Fig. 3B). In BsnDEx4=5 rod spherules, almost 50% of light phase ribbons were SSs, whereas only 16% of dark phase ribbons appeared in the SS form (Fig. 3B). Therefore, not only the number of SS structures increased in the mutant when compared with the respective condition in wt, but also an approximately threefold difference was seen between the light and dark state in BsnDEx4=5 rod spherules. Based on the assumption that spherical ribbon material represents spare or ‘‘storage’’ ribbon material (Adly et al., 1999; Spiwoks-Becker et al., 2004; Regus-Leidig et al., 2010a), we conclude that Bassoon and lighting conditions not only synergistically mediate anchorage, but also enhance the structural integrity of SRs. A change in structural integrity might also influence the size of ribbon profiles. Consistent with this hypothesis, the diameter of SSs (Fig. 3C) and the length of SRs (Fig. 3D) were found to be smaller in BsnDEx4=5 mice than in wt animals. In contrast, the height of SRs was unaltered in BsnDEx4=5 rod spherules (Fig. 3E) suggesting that the decrease in SR size is
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directional and primarily attributable to a shortening of the horseshoe’s arms. In BsnDEx4=5 rod spherules, darkness affected neither the diameter of SSs (Fig. 3C) nor the size of SRs (Fig. 3D, E). Therefore, the size of ribbon profiles appears to be influenced by Bassoon and not by ambient lighting conditions. The tendency of ribbon profiles to aggregate with each other in ribbon fields might correlate with the frequency of membrane-detached synaptic profiles. Consistent with this assumption, ribbon fields were not observed in wt retinas but in 30.4 ± 5.0% of light-adapted and 23.2 ± 3.2% of dark-adapted BsnDEx4=5 rod spherules (Fig. 3F). Taken together, these findings suggest that lighting conditions and Bassoon complementarily act as structural organizers in the proper assembly of ribbon units or cytomatrix material. Whereas darkness primarily ensures the basic transition of SSs to SRs, Bassoon appears to promote the proper assembly of ribbon units to their full size and the formation of the horseshoeshaped appearance.
Formation of the synaptic complex is altered in BsnDEx4=5 rod spherules and depends on lighting conditions During the maturation of ribbon synapses in wt mice, anchored presynaptic ribbons appear at sites at which postsynaptic elements contact photoreceptor terminals (Blanks et al., 1974), and in BsnDEx4=5 mice, the loss of SRs anchoring coincides with an incomplete interdigitation of postsynaptic elements (Dick et al., 2003; Regus-Leidig et al., 2010b). Therefore, either anchored presynaptic SRs can initiate the interdigitation of postsynaptic elements or vice versa. In accordance with a correlation of ribbon anchoring and interdigitation, the postsynaptic interdigitation of processes from horizontal (Fig. 3G) and bc (Fig. 3H) into the rod spherule was found to be decreased in the BsnDEx4=5 retina. Irrespective of the genotype, the interdigitation of processes from horizontal (Fig. 3G) and bc (Fig. 3H) into the rod spherules was more prominent during the dark phase. These findings suggest that interdigitation of the ribbon synapse complex is promoted by both Bassoon and darkness.
Proper anchorage of SRs is also disturbed in BsnDEx4=5 cones and bc To address the question of whether the role of Bassoon in ribbon synapse architecture is a general one, the localization of SRs was also investigated in cone terminals (Fig. 4) and rod bipolar cell terminals (Fig. 5) from BsnDEx4=5 and wt retinas. As seen for rods, the lack of full-size Bassoon increased the occurrence of SR fields in cones (Fig. 4E) and decreased the proportion of membrane-tethered or membrane-anchored SRs in cones (Fig. 4F) and BC (Fig. 5E). Unlike in rod spherules, the lighting conditions did not significantly influence the tethering of SRs in BsnDEx4=5 terminals of cones (Fig. 4F) and BC (Fig. 5E). This suggests that, in
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Fig. 3. The lack of a functional Bassoon affects the ultrastructure of synaptic ribbons and the area of postsynaptic elements in rod ribbon synapses. (A) Schematic three-dimensional drawing of a half-moon-shaped SR (black) typical for rod cells. Note also the so-called arciform density (ad) to which the SR is attached at the presynaptic membrane. The two planes of sectioning of the organelle are indicated (1, 2) resulting in profiles representing the length (1, horizontal cuts) or the height (2, vertical cuts). The SR is located at the apex of an invagination that comprises a tetrad of postsynaptic elements: two lateral processes of horizontal cells (hc) and two central bipolar cell dendrites (Bc, modified after Vollrath and SpiwoksBecker, 1996). (B)Frequency of spherical ribbon profiles (SSs), (C) diameter of SSs, (D) length of synaptic ribbons (SRs), (E) height of SRs, (F) frequency of SR fields, (G) area of invaginated processes from horizontal cells, and (H) area of invaginated processes from bipolar cells in rod synaptic complexes of wild-type (wt) and homozygous Bassoon mutant (BsnDEx4=5 ) mice under light and dark conditions. All parameters in mutants are different from those in wt mice, except for SR height. Note also that, in BsnDEx4=5 mice, darkness decreases the frequency of SSs (B). (B–F) L = light, D = Dark, n = SRs examined, wt: L n = 464, D n = 387; BsnDEx4=5 : L n = 391, D n = 414. (B) n = SSs observed, wt: L n = 19, D n = 0; BsnDEx4=5 : L n = 172, D n = 58. (B–F) n = rod spherules examined E: wt: L n = 428, D n = 323; BsnDEx4=5 : L n = 243, D n = 291. (G,H) n = postsynaptic elements. (G) wt: L n = 537, D n = 437; BsnDEx4=5 : L n = 122, D n = 148, (H) wt: L n = 207, D n = 127; BsnDEx4=5 : L n = 21, D n = 21). Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 dark-adapted wt mice, and 6 darkadapted mutants (pooled values from two independent experiments). Statistical differences: ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.
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Fig. 4. Cone photoreceptor synapses in wild-type and homozygous Bassoon mutant retinae in mice adapted to the light or dark The wild-type cone terminal (wt, A, B) features single synaptic ribbons (SRs) each of which faces processes of horizontal cells (hc) and bipolar cells (Bc). As in rod synapses, many cone endings of Bassoon mutants (BsnDEx4=5 , C, D) contain a synaptic complex without an attached SR (arrows in C) or SRs predominately ‘‘free floating’’ and arranged in aggregates, forming stacks of several ribbons (D, insets in C, D). Note also a group of postsynaptic ectopic SRs (arrowhead in C; see also Spiwoks-Becker et al., 2000). Scale bars, 0.5 lm (except D: 1 lm. (E) Irrespective of the lighting conditions, SRs aggregate in so-called synaptic fields in BsnDEx4=5 compared with wt mice. L = light, D = Dark, n = cone terminals examined, wt: L n = 60, D n = 40; BsnDEx4=5 : L n = 45, D n = 66. (F) The numbers of SRs not attached to the presynaptic membrane is increased in BsnDEx4=5 . n = SRs examined E: wt: L n = 73, D n = 53; BsnDEx4=5 : L n = 47, D n = 58; B: wt L n = 193, D n = 99; BsnDEx4=5 : L n = 115; D n = 109. Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted BsnDEx4=5 , 8 dark-adapted wt mice, and 6 dark-adapted BsnDEx4=5 (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001.
these types of ribbon synapses, Bassoon promotes the anchoring of SRs independent of the lighting conditions.
DISCUSSION The ribbon synapse represents an ideal model for studying the relationship between structure and function in synapses (Sterling and Matthews, 2005; Prescott and
Zenisek, 2005; Regus-Leidig and Brandsta¨tter, 2012). Progress in our understanding of this matter has come from the finding that, in BsnDEx4=5 rod spherules, the detachment of the SRs from the active zone compartment coincides with perturbed synaptic transmission (Dick et al., 2003). By using this experimental system in the present study, a more comprehensive concept is provided on
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Fig. 5. Comparison of light- and dark-adapted rod bipolar synapses in wild-type mice and homozygous Bassoon mutant retinae. In the wt mice (wt, A, B), synaptic ribbons (SRs) are predominately attached to small plaques differing from the typical arciform density of rods. In mutants (BsnDEx4=5 , C, D), ‘‘empty’’ rod bipolar terminals are typical, without anchorages of SRs at the presynaptic active zone (arrows in D). ‘‘Free floating’’ SRs and SSs are also present (D, insets). Note the long and free SR detached from its presynaptic density compartment (D, left inset). Abbreviation: ac, amacrine cell. Scale bars, 0.25 lm (including insets), except D (1 lm). (E) Quantification of SRs tethered to the presynaptic membrane. BsnDEx4=5 show lower numbers of SRs attached to the membrane than wt and this effect tends to be more pronounced during the light phase. L = light, D = Dark, n = SRs examined (wt: L n = 73, D n = 53; BsnDEx4=5 : L n = 47, D n = 58). n = rod bipolar terminals examined, wt: L n = 147, D n = 78; BsnDEx4=5 : L n = 76, D n = 79. Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 darkadapted wt mice, and 6 dark-adapted mutants (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001.
the complementary roles of ambient lighting conditions and Bassoon in organizing a functionally proper CAZ. Whereas darkness appears to ensure a basic structuring of the CAZ, including the membrane tethering of SRs and the transition of SSs to SRs, Bassoon appears to promote full maturation, including the tight anchorage of SRs and the assembly of fullsized and horseshoe-shaped SRs. Since, in darkness,
synaptic activity is higher than in light, darkness might influence the CAZ in terms of synaptic activity. However, on considering that the retina and photoreceptor cells are under circadian regulation (for review, see Tosini et al., 2008), the effect of the lighting conditions on the CAZ might also be mediated through the entrainment of a circadian clock. Although these findings are of interest in the interpretation of our data,
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we should bear in mind that the phenotype of BsnDEx4=5 photoreceptor synapses observed in this study might reflect not only a direct action of Bassoon, but also secondary effects downstream of Bassoon (Specht et al., 2009). The molecular basis for the detachment of ribbons from the active zone in BsnDEx4=5 mice (Dick et al., 2003) has been explained by the finding that Bassoon physically interacts with the B-domain of the ribbon protein RIBEYE, i.e., within a domain that is lacking in the BsnDEx4=5 mutant (tom Dieck et al., 2005). In this study, we show that Bassoon is not essential for the initial attachment of SRs, but rather is required for the tight association of SRs to the cell membrane. This is evident from the observation that, in dark-adapted BsnDEx4=5 retina, SRs remain in contact with the cell membrane at one point (Fig. 2F–I) but generally loosen their attachment to the cell membrane (Fig. 2A). Thus, a mechanism independent of Bassoon contributes to the tethering of ribbon material at the plasma membrane, a concept consistent with the observation that the initial tethering of ribbon material at the rod spherule base seen during development is also observed in BsnDEx4=5 animals (Regus-Leidig et al., 2010b). Whereas the role of Bassoon in ribbon anchoring has been well described in rods and cones (Dick et al., 2003), a similar role in bipolar cell terminals is controversial, since Bassoon immunoreactivity in bc has been observed in some studies (Deguchi-Tawarada et al., 2006) but not in others (Brandsta¨tter et al., 1999; Dick et al., 2001). Furthermore, the lack of a functional Bassoon results in unstructured bipolar cell ribbons in aged mice (Specht et al., 2009) but has not been found to affect the anchoring of bipolar cell ribbons (Dick et al., 2003). However, the similar behavior of bipolar cell, cone, and rod ribbons seen in this study also indicates the dependence of bipolar cell ribbons on Bassoon and suggests that Bassoon, although present only at low concentrations, is nevertheless essential for the tight anchoring of SRs in BC. The results presented here indicate, in addition, a role for Bassoon in the organization of the structure of the ribbon itself. BsnDEx4=5 rod spherules exhibit an increased fraction of SSs. This finding is of particular interest, because a modular assembly of SRs has been proposed in which SRs are assembled from individual SS-like modules, each composed of RIBEYE (Magupalli et al., 2008; Schmitz, 2009). Therefore, the enhanced appearance of SSs in BsnDEx4=5 rod spherules can be interpreted as an increased segregation of RIBEYE modules from the SR. If this is valid, then Bassoon would appear to ensure the structural integrity of the ribbon by tethering RIBEYE modules to the SR. Consistent with this suggestion, segregation of SSs occurs from the distal part of the SR (Adly et al., 1999; Spiwoks-Becker et al., 2004) at which point its Bassoon content is low (tom Dieck et al., 2005). The lack of a functional Bassoon also decreases the size of SSs, i.e., the size of the segregations of the ribbon. This suggests that the segregations of the ribbon also consist of multiple RIBEYE modules.
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Consistent with the concept that the segregation of SSs occurs primarily at the apical bow of the horseshoe-shaped SR (Adly et al., 1999; SpiwoksBecker et al., 2004), the occurrence of SSs in BsnDEx4=5 rod spherules correlates with a decrease in SR length. Since, in BsnDEx4=5 rod spherules, the appearance of SRs changes from horseshoe-like to punctate by light microscopy (tom Dieck et al., 2005) and to rectangular plates by electron microscopy (Fig. 1B, E), the reduction in the SR length appears to occur primarily at the expense of the arms of the horseshoe. In the present study, we show that Bassoon is also required for the normal interdigitation of the postsynaptic elements to the rod terminal. During the maturation of the ribbon synapse, SRs bound at the presynaptic membrane mark the sites of invagination of the postsynaptic elements (Blanks et al., 1974). This raises the possibility that either the anchoring of SRs at the presynaptic membrane is essential for inducing the interdigitation of postsynaptic elements to the rod terminal or that, vice versa, intact interdigitation is a prerequisite for SR attachment. Depending on which interpretation is valid, the effect of Bassoon on SR anchoring might be the reason or, alternatively, the consequence of the defect in interdigitation. SRs are considered to tether synaptic vesicles close to the release sites (Lenzi and von Gersdorff, 2001) suggesting that a functioning photoreceptor terminal requires SRs attached to the active zone compartment. Therefore, the loss of anchoring might be a reason for disturbances in the synaptic transmission in BsnDEx4=5 ribbon synapses (Dick et al., 2003). However, the finding that sustained transmitter release is mostly unchanged in BsnDEx4=5 hair cells, even though SRs are undocked (Khimich et al., 2005), suggests that the loss of a functional Bassoon affects synaptic transmission not only through the detachment of SRs from the active zone compartment. Additional explanations for the perturbed signal transmission in the BsnDEx4=5 retina are provided by the present observation that the loss of a functional Bassoon decreases the stability and the size of SRs and might subsequently result in the reduced capability of the SR to tether and immobilize synaptic vesicles close to the release sites (Morgans, 2000). The latter is consistent with the finding that SRs devoid of a functional Bassoon in rod spherules tether fewer synaptic vesicles (Frank et al., 2010). A reduced capability of the SR to tether synaptic vesicles might also contribute to the decrease in the reloading of rapidly recruitable vesicles in Bassoon mutant ribbon synapses (Frank et al., 2010; Joselevitch and Zenisek, 2010). The results of the present study indicate that the regulation of the ribbon synapse not only depends on Bassoon, but also involves Bassoon-independent and light-dependent steps. This is evident from the observation that, in BsnDEx4=5 rod spherules, darkness increases the tethering of SRs to the cell membrane/ active zone (Fig. 2A), the transition of ribbon material from spherical to rod-like (Fig. 3B), and the invagination of postsynaptic elements (Fig. 3G, H). Thus, darkness
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and Bassoon function synergistically to ensure the proper morphology and high synaptic transmission in the active ribbon synapse during darkness. Whereas darkness promotes punctuate SR tethering to the cell membrane and the shape transition of ribbon material from spherical to rod-like, Bassoon is responsible for the tight anchorage of SR to the arciform density and the proper assembly of the ribbon to the full-sized horseshoe-like complex.
CONCLUSION In this study, the regulatory role of Bassoon in the ribbon synapse has been refined and confined to that of ambient illumination. Although SRs appear to represent a specialization of the CAZ of conventional synapses, and although darkness might influence the CAZ in terms of synaptic activity, we need to investigate whether Bassoon and its activity also play synergistic and complementary roles in conventional synapses (Hallermann et al., 2010), an assumption that is in agreement with recent evidence (Mukherjee et al., 2010). A more general aspect arising from our study is that structural and hence also functional changes occurring between light and dark states in photoreceptor mutants might be more substantial than previously thought. This deserves particular attention, because morphological studies are usually carried out in lightadapted animals, whereas physiological studies often use dark-adapted animals, so that the correlation of morphological and physiological data might be misleading. Acknowledgements—The authors thank Ilse von Graevenitz for excellent technical assistance and Debra Bickes-Kelleher for linguistic assistance. Data in this study form part of the thesis of R.L. as a partial fulfillment of his medical doctorate degree at the University Medical Center of the Johannes Gutenberg University, Mainz. S.t.D. was supported by DFG grant BR 1643/4-1.
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(Accepted 9 December 2012) (Available online 4 January 2013)
EVIDENCE FOR SYNERGISTIC AND COMPLEMENTARY ROLES OF BASSOON AND DARKNESS IN ORGANIZING THE RIBBON SYNAPSE I. SPIWOKS-BECKER, a* R. LAMBERTI, b S. TOM DIECK c,d AND R. SPESSERT b
INTRODUCTION Sensory neurons of the retina and inner ear face the challenge of transmitting sensory signals over a broad range of intensity magnitude. For this reason, they need dynamic and adjustable synaptic machinery, which is provided by a unique type of chemical synapse, the ribbon synapse (tom Dieck and Brandsta¨tter, 2006). Their most prominent organelle is the so-called synaptic ribbon (SR), which is thought to tune the synaptic vesicle cycle in a dynamic manner for speed, precision, and endurance (Zanazzi and Matthews, 2009). SRs constitute electrondense structures of various forms and considerable size (Vollrath and Spiwoks-Becker, 1996). They are capable of tethering hundreds of synaptic vesicles (Rao-Mirotznik et al., 1995), which are positioned by the ribbon in close proximity to the presynaptic neurotransmitter release site, namely the active zone. Most of the ribbon synapses in the outer plexiform layer of the mouse retina form part of the rod terminals, whereas the cone terminals account for less than 2% of the photoreceptor terminals. In the rod photoreceptor ribbon synapse, the prototype of a ribbon synapse, SRs mostly appear as rod-like structures in ultrathin sections, whereas three-dimensionally, they are thin plates with a horseshoelike shape that is easily detected by immunofluorescence light microscopy. They are 30–50 nm in thickness, protrude upward with an average of 300 nm into the presynaptic cytoplasm, and can extend up to 2 lm in length parallel to the presynaptic plasma membrane. The concave inner base of the ribbon is anchored to the presynaptic membrane by means of the arciform density, a second electron-dense structure belonging to the ribbon complex. The morphology of photoreceptor SRs is dynamic (Vollrath et al., 2001; Regus-Leidig et al., 2010a) and undergoes daily changes in BALB/c mice (Adly et al., 1999; Spiwoks-Becker et al., 2004). Although they are large and smooth during the dark phase, after light exposure in the morning, they form distal swellings that bud off, thus forming spherical synaptic bodies (synaptic spheres, SSs), whereas at the onset of darkness, the reverse process occurs (Spiwoks-Becker et al., 2004). Accordingly, SSs can be considered as being the disintegrated building block pool of ribbon material. A major component of the SRs is the protein RIBEYE (Schmitz et al., 2000; Magupalli et al., 2008; Venkatesan et al., 2010). Other molecular components found to be enriched at the photoreceptor SRs are a variety of proteins of the cytomatrix at the active zone (CAZ) as found in conventional active zones, including the
a
Institute of Microanatomy and Neurobiology, University Medical Center of the Johannes Gutenberg University, 55099 Mainz, Germany
b
Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University, 55099 Mainz, Germany
c Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/M, Germany d
Department of Synaptic Plasticity, Max Planck Institute for Brain Research, D-60528 Frankfurt/M, Germany
Abstract—Ribbon synapses are tonically active highthroughput synapses. The performance of the ribbon synapse is accomplished by a specialization of the cytomatrix at the active zone (CAZ) referred to as the synaptic ribbon (SR). Progress in our understanding of the structure–function relationship at the ribbon synapse has come from observations that, in photoreceptors lacking a full-size scaffolding protein Bassoon (BsnDEx4=5 ), dissociation of SRs coincides with perturbed signal transfer. The aim of the present study has been to elaborate the role of Bassoon as a structural organizer of the ribbon synapse and to differentiate it with regard to the ambient lighting conditions. The ultrastructure of retinal ribbon synapses has been compared between wild-type (Wt) and BsnDEx4=5 mice adapted to light (low activity) and darkness (high activity). The results obtained suggest that Bassoon and environmental illumination synergistically and complementarily act as organizers of the ribbon synapse. Thus, light-dependent and Bassoon-independent regulation involves initial SR tethering to the membrane and a basic shape transition of ribbon material from spherical to rod-like, since darkness induces these features in BsnDEx4=5 rod spherules. However, the tight anchorage of the SR via an arciform density and the proper assembly of SRs to the full-sized horseshoe-shaped complex depend on Bassoon, as these steps fail in BsnDEx4=5 rod spherules. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: bassoon, retina, ribbon synapse, synaptic ribbon, light/dark-cycle.
*Corresponding author. Tel: +49-6131-3922365; fax: +49-61313922167. E-mail address: [email protected] (I. Spiwoks-Becker). Abbreviations: Bc, bipolar cells; CAZ, cytomatrix at the active zone; hc, horizontal cells; PBS, phosphate-buffered saline; SS, spherical synaptic bodies; SR, synaptic ribbon; wt, wild-type.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.12.031 149
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scaffolding protein Bassoon (tom Dieck et al., 2005). These findings have led to the concept that SRs are a specialization of the CAZ present at conventional synapses (Zhai and Bellen, 2004). The ribbon complex of photoreceptor cells can be segregated into a ribbonassociated compartment and an active zone compartment. Bassoon is enriched at the proximal/ concave site of the ribbon and appears to contribute essentially to the link between the two compartments (tom Dieck et al., 2005). At the ribbon site, it physically interacts with RIBEYE (tom Dieck et al., 2005), whereas the interacting protein at the active zone compartment has not yet been identified. In BsnDEx4=5 mutant mice, the region of Bassoon interacting with RIBEYE is deleted (Altrock et al., 2003; Dick et al., 2003; tom Dieck et al., 2005; Frank et al., 2010). These mice produce a 180-kDa Bassoon mutant protein, which is not tightly anchored to the CAZ and is easier to extract (Altrock et al., 2003; Dick et al., 2003). Consistent with the loss of the Bassoon-RIBEYE interaction, SRs in light-adapted BsnDEx4=5 rod spherules are not anchored to the presynaptic active zone but float freely in the cytoplasm (Dick et al., 2003). Since the function of SRs is to tether synaptic vesicles close to the release sites, this seems to explain why retina signal transfer from photoreceptors to the second-order neuron is disturbed in the BsnDEx4=5 mutant (Dick et al., 2003). The observation that, in BsnDEx4=5 rod spherules, the shape of anti-RIBEYE-stained SRs changes from horseshoe-like to punctate (tom Dieck et al., 2005) hints at the possibility that Bassoon also influences the morphology of SRs, and that perturbed signal transmission in BsnDEx4=5 mutant photoreceptor terminals might therefore be attributable to several structural deficiencies (tom Dieck et al., 2005; Specht et al., 2009). The aim of the present study was to dissect the role of Bassoon as a structural organizer of the ribbon synapse in dependence of the ambient lighting conditions. To this end, the ultrastructure of ribbon synapses was systematically compared between BsnDEx4=5 and wild-type (wt) mice during the light phase when synaptic transmission is low and the dark phase when synaptic transmission is high.
EXPERIMENTAL PROCEDURES Animals All animal experiments were performed in accordance with the guidelines issued by the Max Planck Society. In total, 17 wt and 11 Bassoon mutant mice (BsnDEx4=5 ; Altrock et al., 2003; Dick et al., 2003) were examined under light- (4 h after light onset) or dark-adapted (4 h after dark onset) conditions from two independent experiments with an age of 8–15 weeks. Homozygous BsnDEx4=5 mice and wt controls (mixed background: 129/Sv 129/SvJ from R1 ES-cells, backcrossed to C57Bl/6) were both obtained as littermate offspring from intercrosses of heterozygous BsnDEx4=5 mice. The mice were kept under constant laboratory conditions (12-h light, 12-h dark; lights on at 6 a.m. and off at 6 p.m.; less than 100–200 lux at the bottom of the cages; food and water ad libitum) for a minimum of one week prior to the experiments. They were anesthetized with isoflurane and decapitated prior to removal of the eyes during darkness under dim red light. Usually, both eyes/retinae per animal were removed by using the Winkler method (Winkler, 1972).
Electron microscopy and morphometry For the electron-microscopical evaluation, the retinae were dissected from rapidly removed eye balls and fixed in freshly prepared fixative, viz., 2% paraformaldehyde, 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 h. Subsequently, tissue was rinsed in PBS containing 6.8% (w/v) sucrose, postfixed in osmium tetroxide (2% (w/v) in PBS) for 90 min, washed three times in PBS, and dehydrated in a graded series of acetone. Tissue was flat-embedded in Epon (Serva, Heidelberg, Germany). Transverse sections (50–60 nm thick) were mounted onto one-hole Formvar-coated copper grids (Serva), stained with 8% (w/v) uranyl acetate (10 min), and contrasted with lead citrate for 5 min. Sections were viewed in an LEO 906 transmission electron microscope (Zeiss, Oberkochen, Germany), and all synapses were photographed. Morphometry was carried out by using Analysis 3.2. Software (Soft Imaging Systems, Mu¨nster, Germany).
Quantitative analysis From one randomly selected retinal section, synaptic body profiles in approximately 50 neighboring photoreceptor terminals were systematically examined according to the following criteria: type of SR profile (e.g., rodlike, spherical, or polymorphic) and size and location of the SR within the terminal. The SRs were classified as ‘‘tethered’’ when they bordered on the presynaptic membrane, in wt photoreceptors, typically via the arciform density and, in bipolar cells (BC), via small dense plaques. SR profiles not bordering on the presynaptic membrane were referred to as being ‘‘free’’. Because rod-like SRs of rod photoreceptor are sections through horseshoe-shaped structures, and because the structure might be cut at various angles, transverse cuts yield shorter profiles with a smaller variability than horizontal cuts, which represent the SR length, and therefore sections were taken through the distal SR area (see also Fig. 3A). However, transversely cut profiles can be easily recognized, since, unlike the horizontally cut profiles, the underlying arciform density is seen together with the ribbon. In general, within a given synapse, the number of SRs and SR fields were also estimated. Synapses without SR profiles were classified as ‘‘empty’’ endings. Furthermore, measurements of the area of the postsynaptic horizontal and bipolar processes were performed. Tissue and data analyses were performed blind, i.e., without knowledge of the genotype and by using coded specimens.
Serial section analysis To draw conclusions on the three-dimensional shape, the location of the SRs, and their arrangement at the synaptic complex, serial sections of randomly chosen profiles of light- and dark-adapted mutant photoreceptor terminals were photographed. The synaptic areas of adjacent sections were superimposed by using the Adobe Photoshop (Adobe Systems Inc.).
Statistical analysis The data obtained were expressed as means ± standard error of the mean. For statistical analysis, Student’s t-test or the Wilcoxon-Mann–Whitney U-test was used. A p-value of smaller than or equal to 0.05 was regarded as significant. The values are based on pooled data from two independent experiments.
RESULTS Light-dependent ultrastructural changes in rod ribbon synapses lacking functional Bassoon In adult wt mice (8–15 weeks), presynaptic electron-dense SRs typically anchored to the so-called arciform density
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facing four postsynaptic elements: two horizontal cell processes and two rod bipolar cell dendrites (Fig. 1A–C). The SR showed a rod-like appearance in vertically sectioned (Fig. 1A, C) and a horseshoe-shaped appearance in sagitally sectioned (Fig. 1B) rod spherules. No obvious differences were seen in single sections between light- and dark-adapted wt animals (Fig. 1A, C). In contrast, light-adapted rod spherules in BsnDEx4=5 retinas (2 sections from each retina; in total 243 terminals were examined) often either did not contain SR profiles (stars in Fig. 1D, E) or had multiple and often very small SSs (considered as the disintegrated building block pool of ribbon material) floating freely in the cytoplasm (arrowheads in Fig. 1D, E). SSs tended to aggregate to ‘‘ribbon fields’’ (Fig. 1D–F). Furthermore, ‘‘anchorage’’ places of SRs were typically ‘‘empty’’ (arrow in Fig. 1D) and did not show a typical arciform density. In sagitally sectioned rod spherules, SRs never appeared horseshoe-shaped as in wt mice (Fig. 1B) but rather as a rectangular-like plate of smaller dimensions than wt SRs (Fig. 1E). In addition, the area of invaginating postsynaptic elements was smaller when compared with wt (Fig. 1A versus D).
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In BsnDEx4=5 retina, empty spherules and those with only spherical material occurred less often in mice adapted to the dark than in those adapted to the light. In dark-adapted BsnDEx4=5 mice, retina spherules containing SRs were tethered to the membrane, often (16.4%) two in parallel (Fig. 1F). The membrane tethering mostly occurred in locations lacking the typical features of wt anchorage spots. Thus, the typical arciform densities and proper tetradic postsynaptic arrangements were absent. These observations suggest that light/dark regulation might still take place in BsnDEx4=5 rod spherules and can be used to unravel the characteristics of normal light-dependent structural changes. Dissecting steps in ribbon anchorage: membrane tethering of SR but not tight anchorage in BsnDEx4=5 rod spherules With respect to the membrane tethering, we found that, in wt retinas, >90% of ribbons showed tight membrane anchorage, regardless of the ambient lighting conditions (Fig. 2A). Although bona fide ribbon anchorage as defined by the appearance of an arciform density was never observed in the mutant (Fig. 1D–F), membrane
Fig. 1. Transmission–electron micrographs of rod photoreceptor synapses in wild-type and homozygous Bassoon mutant retina in mice adapted to the light or dark. The rod spherules are sectioned vertically (A, C, D, F) and sagittally (B, E). In wild-type (wt) mice (A–C), presynaptic electron-dense synaptic ribbons (SRs, arrowheads) anchor to the so-called arciform density (arrows) facing four postsynaptic elements: two horizontal cell processes (hc) and two rod bipolar cell dendrites (bc). The SR shows a rod-like appearance in vertically sectioned spherules (arrowheads in A, C) and a horseshoe-like appearance in sagittally sectioned spherules (arrowhead in B). In Bassoon mutant (BsnDEx4=5 ) rod endings (D–F), spherules without SRs are seen (stars in D, E), anchorages of SRs are typically ‘‘empty’’ (e.g. arrow in D), and in the cytoplasm, ‘‘free floating’’ multiple synaptic spherical bodies (SSs) are abundant (arrowheads in D, E). In horizontally sectioned spherules of mutant mice, the shape of SRs changes from horseshoe-like (B) to plate/rectangular-like (E). Note also that, in mutants, the area of the postsynaptic elements is decreased (e.g., C versus F). As exemplified by SR anchoring, the characteristics of the mutant ribbon synapses are more pronounced during the light phase (D, E) than during darkness (F). Scale bars, 0.2 lm (A–C, F); 0.5 lm (D, E).
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Fig. 2. Membrane-tethering of synaptic ribbons in wild-type and homozygous Bassoon mutant retina in mice adapted to the light or dark. (A) Quantification of membrane-tethered synaptic ribbons (SRs). Note the decrease in the number of membrane-tethered SRs in Bassoon mutants (BsnDEx4=5 ) versus wild-type (wt) mice, which is more pronounced under light conditions. L = light, D = Dark, n = SRs examined (wt: L n = 464, D n = 387; BsnDEx4=5 : L n = 391, D n = 414. n = rod spherules examined wt: L n = 428, D n = 323; BsnDEx4=5 : L n = 243, D n = 291. Data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 dark-adapted wt mice, and 6 dark-adapted mutants (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001. (B–I) Ultrastructural appearance of SR profiles in serial sections of photoreceptor synapses (numbered 1–5) from BsnDEx4=5 in the light (B–E) and dark (F–I) phases. The figure exemplifies that, in a follow-up series of rod spherules, only 15% of the SRs (from 21 section series analyzed) contact the membrane in light-adapted mice, but all SRs (from 30 section series analyzed) contact the membrane at least at one point throughout the series in dark-adapted mice. The arrows in D, E, and F mark ‘‘empty’’ anchorages of SRs. Note also that, in light-adapted mutant rod spherules, multiple small and membrane-detached spherical ribbon profiles (SSs, arrowhead) are present, often appearing in only one section. Dark-adapted mutant rod spherules show a decrease in the number of SSs and often contain two parallel ribbon profiles (e.g. F2–I2). Cone pedicles (CP) do not show SSs, but sometimes SRs with a direct attachment to the presynaptic membrane are seen (⁄in D, E). Scale bars, 1 lm.
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tethering of 42.1 ± 6.1% of dark-adapted and 19 ± 4.1% of light-adapted BsnDEx4=5 ribbons (Fig. 2A) was seen. Although quantification from single sections is a valid measure of the distance between SRs and cell membrane in a given plane, it does not inherently provide the precise number of SRs tethered to the cell membrane. Accordingly, quantification from single sections might miss short stretches of membrane contact and therefore will usually lead to underestimates of attachment numbers. Serial section analysis (Fig. 2B–I) revealed that, in BsnDEx4=5 mice, SRs seemingly detached from the membrane in a given section were often found to be tethered to the membrane at least at one point. Consistent with this observation, Bassoon promoted long stretches of membrane contact, whereas short stretches of membrane contact occurred in the absence of a functional Bassoon. In BsnDEx4=5 rod spherules, the tethering of SRs was found to be increased in darkness (Fig. 2A) and therefore seemed to depend on the lighting conditions. We conclude from these observations that the anchorage of the SR to the presynaptic membrane involves two distinct steps: a light-dependent step ensuring that tethering takes place, and a Bassoon-dependent step responsible for tight anchorage via the arciform density. Basic transition of SSs to SRs but not the assembly of mature ribbon units in BsnDEx4=5 rod spherules To confirm and specify the influences of Bassoon and environmental lighting conditions on the ribbon material itself, its structural integrity (in terms of the frequency of SSs), its size (in terms of the diameter of SSs, the height of SRs, and the length of SRs), and its tendency to aggregate with other ribbon material (in terms of the frequency of so-called synaptic fields) were compared in wt and BsnDEx4=5 rod spherules during the light and dark phases. In wt rod spherules, SSs were rarely seen during the light phase (2.2 ± 0.7%) and were absent during the dark phase (Fig. 3B). In BsnDEx4=5 rod spherules, almost 50% of light phase ribbons were SSs, whereas only 16% of dark phase ribbons appeared in the SS form (Fig. 3B). Therefore, not only the number of SS structures increased in the mutant when compared with the respective condition in wt, but also an approximately threefold difference was seen between the light and dark state in BsnDEx4=5 rod spherules. Based on the assumption that spherical ribbon material represents spare or ‘‘storage’’ ribbon material (Adly et al., 1999; Spiwoks-Becker et al., 2004; Regus-Leidig et al., 2010a), we conclude that Bassoon and lighting conditions not only synergistically mediate anchorage, but also enhance the structural integrity of SRs. A change in structural integrity might also influence the size of ribbon profiles. Consistent with this hypothesis, the diameter of SSs (Fig. 3C) and the length of SRs (Fig. 3D) were found to be smaller in BsnDEx4=5 mice than in wt animals. In contrast, the height of SRs was unaltered in BsnDEx4=5 rod spherules (Fig. 3E) suggesting that the decrease in SR size is
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directional and primarily attributable to a shortening of the horseshoe’s arms. In BsnDEx4=5 rod spherules, darkness affected neither the diameter of SSs (Fig. 3C) nor the size of SRs (Fig. 3D, E). Therefore, the size of ribbon profiles appears to be influenced by Bassoon and not by ambient lighting conditions. The tendency of ribbon profiles to aggregate with each other in ribbon fields might correlate with the frequency of membrane-detached synaptic profiles. Consistent with this assumption, ribbon fields were not observed in wt retinas but in 30.4 ± 5.0% of light-adapted and 23.2 ± 3.2% of dark-adapted BsnDEx4=5 rod spherules (Fig. 3F). Taken together, these findings suggest that lighting conditions and Bassoon complementarily act as structural organizers in the proper assembly of ribbon units or cytomatrix material. Whereas darkness primarily ensures the basic transition of SSs to SRs, Bassoon appears to promote the proper assembly of ribbon units to their full size and the formation of the horseshoeshaped appearance.
Formation of the synaptic complex is altered in BsnDEx4=5 rod spherules and depends on lighting conditions During the maturation of ribbon synapses in wt mice, anchored presynaptic ribbons appear at sites at which postsynaptic elements contact photoreceptor terminals (Blanks et al., 1974), and in BsnDEx4=5 mice, the loss of SRs anchoring coincides with an incomplete interdigitation of postsynaptic elements (Dick et al., 2003; Regus-Leidig et al., 2010b). Therefore, either anchored presynaptic SRs can initiate the interdigitation of postsynaptic elements or vice versa. In accordance with a correlation of ribbon anchoring and interdigitation, the postsynaptic interdigitation of processes from horizontal (Fig. 3G) and bc (Fig. 3H) into the rod spherule was found to be decreased in the BsnDEx4=5 retina. Irrespective of the genotype, the interdigitation of processes from horizontal (Fig. 3G) and bc (Fig. 3H) into the rod spherules was more prominent during the dark phase. These findings suggest that interdigitation of the ribbon synapse complex is promoted by both Bassoon and darkness.
Proper anchorage of SRs is also disturbed in BsnDEx4=5 cones and bc To address the question of whether the role of Bassoon in ribbon synapse architecture is a general one, the localization of SRs was also investigated in cone terminals (Fig. 4) and rod bipolar cell terminals (Fig. 5) from BsnDEx4=5 and wt retinas. As seen for rods, the lack of full-size Bassoon increased the occurrence of SR fields in cones (Fig. 4E) and decreased the proportion of membrane-tethered or membrane-anchored SRs in cones (Fig. 4F) and BC (Fig. 5E). Unlike in rod spherules, the lighting conditions did not significantly influence the tethering of SRs in BsnDEx4=5 terminals of cones (Fig. 4F) and BC (Fig. 5E). This suggests that, in
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Fig. 3. The lack of a functional Bassoon affects the ultrastructure of synaptic ribbons and the area of postsynaptic elements in rod ribbon synapses. (A) Schematic three-dimensional drawing of a half-moon-shaped SR (black) typical for rod cells. Note also the so-called arciform density (ad) to which the SR is attached at the presynaptic membrane. The two planes of sectioning of the organelle are indicated (1, 2) resulting in profiles representing the length (1, horizontal cuts) or the height (2, vertical cuts). The SR is located at the apex of an invagination that comprises a tetrad of postsynaptic elements: two lateral processes of horizontal cells (hc) and two central bipolar cell dendrites (Bc, modified after Vollrath and SpiwoksBecker, 1996). (B)Frequency of spherical ribbon profiles (SSs), (C) diameter of SSs, (D) length of synaptic ribbons (SRs), (E) height of SRs, (F) frequency of SR fields, (G) area of invaginated processes from horizontal cells, and (H) area of invaginated processes from bipolar cells in rod synaptic complexes of wild-type (wt) and homozygous Bassoon mutant (BsnDEx4=5 ) mice under light and dark conditions. All parameters in mutants are different from those in wt mice, except for SR height. Note also that, in BsnDEx4=5 mice, darkness decreases the frequency of SSs (B). (B–F) L = light, D = Dark, n = SRs examined, wt: L n = 464, D n = 387; BsnDEx4=5 : L n = 391, D n = 414. (B) n = SSs observed, wt: L n = 19, D n = 0; BsnDEx4=5 : L n = 172, D n = 58. (B–F) n = rod spherules examined E: wt: L n = 428, D n = 323; BsnDEx4=5 : L n = 243, D n = 291. (G,H) n = postsynaptic elements. (G) wt: L n = 537, D n = 437; BsnDEx4=5 : L n = 122, D n = 148, (H) wt: L n = 207, D n = 127; BsnDEx4=5 : L n = 21, D n = 21). Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 dark-adapted wt mice, and 6 darkadapted mutants (pooled values from two independent experiments). Statistical differences: ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.
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Fig. 4. Cone photoreceptor synapses in wild-type and homozygous Bassoon mutant retinae in mice adapted to the light or dark The wild-type cone terminal (wt, A, B) features single synaptic ribbons (SRs) each of which faces processes of horizontal cells (hc) and bipolar cells (Bc). As in rod synapses, many cone endings of Bassoon mutants (BsnDEx4=5 , C, D) contain a synaptic complex without an attached SR (arrows in C) or SRs predominately ‘‘free floating’’ and arranged in aggregates, forming stacks of several ribbons (D, insets in C, D). Note also a group of postsynaptic ectopic SRs (arrowhead in C; see also Spiwoks-Becker et al., 2000). Scale bars, 0.5 lm (except D: 1 lm. (E) Irrespective of the lighting conditions, SRs aggregate in so-called synaptic fields in BsnDEx4=5 compared with wt mice. L = light, D = Dark, n = cone terminals examined, wt: L n = 60, D n = 40; BsnDEx4=5 : L n = 45, D n = 66. (F) The numbers of SRs not attached to the presynaptic membrane is increased in BsnDEx4=5 . n = SRs examined E: wt: L n = 73, D n = 53; BsnDEx4=5 : L n = 47, D n = 58; B: wt L n = 193, D n = 99; BsnDEx4=5 : L n = 115; D n = 109. Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted BsnDEx4=5 , 8 dark-adapted wt mice, and 6 dark-adapted BsnDEx4=5 (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001.
these types of ribbon synapses, Bassoon promotes the anchoring of SRs independent of the lighting conditions.
DISCUSSION The ribbon synapse represents an ideal model for studying the relationship between structure and function in synapses (Sterling and Matthews, 2005; Prescott and
Zenisek, 2005; Regus-Leidig and Brandsta¨tter, 2012). Progress in our understanding of this matter has come from the finding that, in BsnDEx4=5 rod spherules, the detachment of the SRs from the active zone compartment coincides with perturbed synaptic transmission (Dick et al., 2003). By using this experimental system in the present study, a more comprehensive concept is provided on
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Fig. 5. Comparison of light- and dark-adapted rod bipolar synapses in wild-type mice and homozygous Bassoon mutant retinae. In the wt mice (wt, A, B), synaptic ribbons (SRs) are predominately attached to small plaques differing from the typical arciform density of rods. In mutants (BsnDEx4=5 , C, D), ‘‘empty’’ rod bipolar terminals are typical, without anchorages of SRs at the presynaptic active zone (arrows in D). ‘‘Free floating’’ SRs and SSs are also present (D, insets). Note the long and free SR detached from its presynaptic density compartment (D, left inset). Abbreviation: ac, amacrine cell. Scale bars, 0.25 lm (including insets), except D (1 lm). (E) Quantification of SRs tethered to the presynaptic membrane. BsnDEx4=5 show lower numbers of SRs attached to the membrane than wt and this effect tends to be more pronounced during the light phase. L = light, D = Dark, n = SRs examined (wt: L n = 73, D n = 53; BsnDEx4=5 : L n = 47, D n = 58). n = rod bipolar terminals examined, wt: L n = 147, D n = 78; BsnDEx4=5 : L n = 76, D n = 79. Quantitative data are given as mean ± SEM from 9 light-adapted wt mice, 5 light-adapted mutants, 8 darkadapted wt mice, and 6 dark-adapted mutants (pooled values from two independent experiments). Statistical differences: ⁄⁄⁄P < 0.001.
the complementary roles of ambient lighting conditions and Bassoon in organizing a functionally proper CAZ. Whereas darkness appears to ensure a basic structuring of the CAZ, including the membrane tethering of SRs and the transition of SSs to SRs, Bassoon appears to promote full maturation, including the tight anchorage of SRs and the assembly of fullsized and horseshoe-shaped SRs. Since, in darkness,
synaptic activity is higher than in light, darkness might influence the CAZ in terms of synaptic activity. However, on considering that the retina and photoreceptor cells are under circadian regulation (for review, see Tosini et al., 2008), the effect of the lighting conditions on the CAZ might also be mediated through the entrainment of a circadian clock. Although these findings are of interest in the interpretation of our data,
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we should bear in mind that the phenotype of BsnDEx4=5 photoreceptor synapses observed in this study might reflect not only a direct action of Bassoon, but also secondary effects downstream of Bassoon (Specht et al., 2009). The molecular basis for the detachment of ribbons from the active zone in BsnDEx4=5 mice (Dick et al., 2003) has been explained by the finding that Bassoon physically interacts with the B-domain of the ribbon protein RIBEYE, i.e., within a domain that is lacking in the BsnDEx4=5 mutant (tom Dieck et al., 2005). In this study, we show that Bassoon is not essential for the initial attachment of SRs, but rather is required for the tight association of SRs to the cell membrane. This is evident from the observation that, in dark-adapted BsnDEx4=5 retina, SRs remain in contact with the cell membrane at one point (Fig. 2F–I) but generally loosen their attachment to the cell membrane (Fig. 2A). Thus, a mechanism independent of Bassoon contributes to the tethering of ribbon material at the plasma membrane, a concept consistent with the observation that the initial tethering of ribbon material at the rod spherule base seen during development is also observed in BsnDEx4=5 animals (Regus-Leidig et al., 2010b). Whereas the role of Bassoon in ribbon anchoring has been well described in rods and cones (Dick et al., 2003), a similar role in bipolar cell terminals is controversial, since Bassoon immunoreactivity in bc has been observed in some studies (Deguchi-Tawarada et al., 2006) but not in others (Brandsta¨tter et al., 1999; Dick et al., 2001). Furthermore, the lack of a functional Bassoon results in unstructured bipolar cell ribbons in aged mice (Specht et al., 2009) but has not been found to affect the anchoring of bipolar cell ribbons (Dick et al., 2003). However, the similar behavior of bipolar cell, cone, and rod ribbons seen in this study also indicates the dependence of bipolar cell ribbons on Bassoon and suggests that Bassoon, although present only at low concentrations, is nevertheless essential for the tight anchoring of SRs in BC. The results presented here indicate, in addition, a role for Bassoon in the organization of the structure of the ribbon itself. BsnDEx4=5 rod spherules exhibit an increased fraction of SSs. This finding is of particular interest, because a modular assembly of SRs has been proposed in which SRs are assembled from individual SS-like modules, each composed of RIBEYE (Magupalli et al., 2008; Schmitz, 2009). Therefore, the enhanced appearance of SSs in BsnDEx4=5 rod spherules can be interpreted as an increased segregation of RIBEYE modules from the SR. If this is valid, then Bassoon would appear to ensure the structural integrity of the ribbon by tethering RIBEYE modules to the SR. Consistent with this suggestion, segregation of SSs occurs from the distal part of the SR (Adly et al., 1999; Spiwoks-Becker et al., 2004) at which point its Bassoon content is low (tom Dieck et al., 2005). The lack of a functional Bassoon also decreases the size of SSs, i.e., the size of the segregations of the ribbon. This suggests that the segregations of the ribbon also consist of multiple RIBEYE modules.
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Consistent with the concept that the segregation of SSs occurs primarily at the apical bow of the horseshoe-shaped SR (Adly et al., 1999; SpiwoksBecker et al., 2004), the occurrence of SSs in BsnDEx4=5 rod spherules correlates with a decrease in SR length. Since, in BsnDEx4=5 rod spherules, the appearance of SRs changes from horseshoe-like to punctate by light microscopy (tom Dieck et al., 2005) and to rectangular plates by electron microscopy (Fig. 1B, E), the reduction in the SR length appears to occur primarily at the expense of the arms of the horseshoe. In the present study, we show that Bassoon is also required for the normal interdigitation of the postsynaptic elements to the rod terminal. During the maturation of the ribbon synapse, SRs bound at the presynaptic membrane mark the sites of invagination of the postsynaptic elements (Blanks et al., 1974). This raises the possibility that either the anchoring of SRs at the presynaptic membrane is essential for inducing the interdigitation of postsynaptic elements to the rod terminal or that, vice versa, intact interdigitation is a prerequisite for SR attachment. Depending on which interpretation is valid, the effect of Bassoon on SR anchoring might be the reason or, alternatively, the consequence of the defect in interdigitation. SRs are considered to tether synaptic vesicles close to the release sites (Lenzi and von Gersdorff, 2001) suggesting that a functioning photoreceptor terminal requires SRs attached to the active zone compartment. Therefore, the loss of anchoring might be a reason for disturbances in the synaptic transmission in BsnDEx4=5 ribbon synapses (Dick et al., 2003). However, the finding that sustained transmitter release is mostly unchanged in BsnDEx4=5 hair cells, even though SRs are undocked (Khimich et al., 2005), suggests that the loss of a functional Bassoon affects synaptic transmission not only through the detachment of SRs from the active zone compartment. Additional explanations for the perturbed signal transmission in the BsnDEx4=5 retina are provided by the present observation that the loss of a functional Bassoon decreases the stability and the size of SRs and might subsequently result in the reduced capability of the SR to tether and immobilize synaptic vesicles close to the release sites (Morgans, 2000). The latter is consistent with the finding that SRs devoid of a functional Bassoon in rod spherules tether fewer synaptic vesicles (Frank et al., 2010). A reduced capability of the SR to tether synaptic vesicles might also contribute to the decrease in the reloading of rapidly recruitable vesicles in Bassoon mutant ribbon synapses (Frank et al., 2010; Joselevitch and Zenisek, 2010). The results of the present study indicate that the regulation of the ribbon synapse not only depends on Bassoon, but also involves Bassoon-independent and light-dependent steps. This is evident from the observation that, in BsnDEx4=5 rod spherules, darkness increases the tethering of SRs to the cell membrane/ active zone (Fig. 2A), the transition of ribbon material from spherical to rod-like (Fig. 3B), and the invagination of postsynaptic elements (Fig. 3G, H). Thus, darkness
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and Bassoon function synergistically to ensure the proper morphology and high synaptic transmission in the active ribbon synapse during darkness. Whereas darkness promotes punctuate SR tethering to the cell membrane and the shape transition of ribbon material from spherical to rod-like, Bassoon is responsible for the tight anchorage of SR to the arciform density and the proper assembly of the ribbon to the full-sized horseshoe-like complex.
CONCLUSION In this study, the regulatory role of Bassoon in the ribbon synapse has been refined and confined to that of ambient illumination. Although SRs appear to represent a specialization of the CAZ of conventional synapses, and although darkness might influence the CAZ in terms of synaptic activity, we need to investigate whether Bassoon and its activity also play synergistic and complementary roles in conventional synapses (Hallermann et al., 2010), an assumption that is in agreement with recent evidence (Mukherjee et al., 2010). A more general aspect arising from our study is that structural and hence also functional changes occurring between light and dark states in photoreceptor mutants might be more substantial than previously thought. This deserves particular attention, because morphological studies are usually carried out in lightadapted animals, whereas physiological studies often use dark-adapted animals, so that the correlation of morphological and physiological data might be misleading. Acknowledgements—The authors thank Ilse von Graevenitz for excellent technical assistance and Debra Bickes-Kelleher for linguistic assistance. Data in this study form part of the thesis of R.L. as a partial fulfillment of his medical doctorate degree at the University Medical Center of the Johannes Gutenberg University, Mainz. S.t.D. was supported by DFG grant BR 1643/4-1.
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(Accepted 9 December 2012) (Available online 4 January 2013)