Author response: Connectivity map of bipolar cells and photoreceptors in the mouse retina
2016-10-28

Article Figures and data Abstract Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract In the mouse retina, three different types of photoreceptors provide input to 14 bipolar cell (BC) types. Classically, most BC types are thought to contact all cones within their dendritic field; ON-BCs would contact cones exclusively via so-called invaginating synapses, while OFF-BCs would form basal synapses. By mining publically available electron microscopy data, we discovered interesting violations of these rules of outer retinal connectivity: ON-BC type X contacted only ~20% of the cones in its dendritic field and made mostly atypical non-invaginating contacts. Types 5T, 5O and 8 also contacted fewer cones than expected. In addition, we found that rod BCs received input from cones, providing anatomical evidence that rod and cone pathways are interconnected in both directions. This suggests that the organization of the outer plexiform layer is more complex than classically thought. https://doi.org/10.7554/eLife.20041.001 Introduction Parallel visual processing already starts at the very first synapse of the visual system, where photoreceptors distribute the signal onto multiple types of bipolar cells. In the mouse retina, two types of cone photoreceptors differing in their spectral properties – short (S-) and medium wavelength-sensitive (M-) cones – and rod photoreceptors provide input to 14 types of bipolar cell (reviewed in Euler et al., 2014). The precise connectivity rules between photoreceptors and bipolar cell (BC) types determine which signals are available to downstream circuits. Therefore, the connectome of the outer retina is essential for a complete picture of visual processing in the retina. For some mouse BC types, specific connectivity patterns have already been described: For example, based on electrical recordings and immunohistochemistry cone bipolar cell type 1 (CBC1) have been suggested to selectively contact M-cones, whereas CBC9 exclusively contacts S-cones (Haverkamp et al., 2005; Breuninger et al., 2011). The other BC types are thought to contact all M-cones within their dendritic field, but their connectivity to S-cones is unclear (Wässle et al., 2009). In addition, two fundamental cone-BC contact shapes have been described: invaginating contacts with the dendritic tips extending into the cone pedicle and flat (basal) contacts that touch the cone pedicle base, commonly associated with ON- and OFF-BCs, respectively (Dowling and Boycott, 1966; Kolb, 1970; Hopkins and Boycott, 1995). Rod bipolar cells (RBCs) are thought to exclusively receive rod input and to feed this signal into the cone pathway via AII amacrine cells (reviewed by Bloomfield and Dacheux, 2001). However, physiological data indicate that RBCs may receive cone photoreceptor input as well (Pang et al., 2010). Also, types CBC3A, CBC3B and CBC4 have been reported to receive direct rod input (Mataruga et al., 2007; Haverkamp et al., 2008; Tsukamoto and Omi, 2014), suggesting that rod and cone pathways are much more interconnected than their names implicate. Here, we analyzed an existing electron microscopy dataset (Helmstaedter et al., 2013) to quantify the connectivity between photoreceptors and bipolar cells in the mouse. We did not find evidence for additional M- or S-cone selective CBC types in addition to the reported CBC1 and 9. However, we found interesting violations of established rules of outer retinal connectivity: The newly discovered CBCX (Helmstaedter et al., 2013), likely an ON-CBC (Ichinose et al., 2014), had unexpectedly few and mostly atypical basal contacts to cones. CBC5T, CBC5O and CBC8 also contacted fewer cones than expected from their dendritic field. In addition, we provide anatomical evidence that rod and cone pathways are connected in both directions: Not only OFF-types CBC3A, CBC3B and CBC4 get direct input from rods but also RBCs from cones. Results Identification of S- and M-cones We used the serial block-face electron microscopy (SBEM) dataset e2006 published by Helmstaedter et al. (2013) to analyze the connectivity between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the mouse retina (Figure 1A). To this end, we reconstructed the volume of all cone axon terminals (cone pedicles; n = 163) in the dataset as well as the dendritic trees of all BCs (n = 451; Figure 1B, see Materials and methods). Figure 1 with 1 supplement see all Download asset Open asset Identification of S- and M-cones. (A) Scheme showing vertical section through the mouse retina. (B) Volume-reconstructed cones and all CBC4 cells. (C) Cone pedicles (grey) with CBC9s. BC soma localization is indicated by colored dots. Dashed outlines indicate incomplete cones. (D) Same as in C, but with putative S-cones (blue) and M-cones (green) highlighted. Unidentified cones are shown in grey. Insets indicate the location of the examples shown below of cone pedicles contacted by CBC9 dendrites. (E) Contact parameters used for S-cone identification. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. https://doi.org/10.7554/eLife.20041.002 To identify S- and M-cones, we used the fact that type nine cone bipolar cells selectively target S-cones (Figure 1C,D) (Mariani, 1984; Kouyama and Marshak, 1992; Haverkamp et al., 2005; Breuninger et al., 2011). We found 48 contacts of CBC9s and cones, involving 43 cones (Figure 1—figure supplement 1A). We visually assessed all contacts and found that 29 of these were in the periphery of the cone pedicle, where no synapses are expected (Figure 1—figure supplement 1B) (Dowling and Boycott, 1966; Chun et al., 1996). This left 14 potential S-cones with invaginating contacts from at least one CBC9. It has been shown that S-cones are contacted by the dendrites of all neighboring CBC9s and that these contacts occur mostly at the tip of dendritic branches (Haverkamp et al., 2005). Out of the 14 candidate cones, eight cones had only one CBC9 contact. Some of these cones were contacted by a CBC9 dendritic branch that continued past the contact site. Other cones – although contact by one CBC9 – were not contacted by passing dendrites from other CBC9s. The other six cones had at least two invaginating contacts from CBC9s. These originated from two different CBC9s or – in case they originated only from a single CBC9 – at least one of them was formed by a dendritic branch ending at the cone (Figure 1E). We labeled the eight cones that featured only a single CBC9 contact as M-cones (Figure 1—figure supplement 1C), and defined the remaining six candidate cones as S-cones (Figure 1D and Figure 1—figure supplement 1C and D, see Materials and methods). This corresponds to a fraction of 4.8% S-cones (6/124 cones within the dendritic field of at least one CBC9), matching the 3–5% reported in previous studies (Röhlich et al., 1994; Haverkamp et al., 2005). An alternative scheme for identifying S-cones would have been to classify all cones with at least one invaginating contact from CBC9 as S-cones. This would have resulted in a total of 14 S-cones out of 124 cones (Figure 3—figure supplement 2A) or a fraction of 11.3%. Because this S-cone percentage is much larger than the 3–5% reported earlier (Haverkamp et al., 2005), we consider this scenario as very unlikely (p=0.0037, binomial test, null hypothesis: 5% S-cones, n = 124). Classification of photoreceptor-BC contacts We next developed an automatic method to distinguish contacts likely corresponding to synaptic connections from false contacts. As the tissue in the dataset was stained to enhance cell-surface contrast to enable automatic reconstruction, it is not possible to distinguish between synaptic contacts based on explicit ultrastructural synaptic markers, such as vesicles, synaptic ribbons or postsynaptic densities (see also discussion in Helmstaedter et al., 2013). In contrast to the synaptic contacts in the inner plexiform layer studied by Helmstaedter et al. (Helmstaedter et al., 2013), the highly stereotypical morphology of synapses at photoreceptor axon terminals allowed us to classify the contacts (Haverkamp et al., 2000): The ribbon synapses of the cones are placed exclusively in the presynaptic area at the bottom of the cone pedicles. Here, ON-cone bipolar cells (ON-CBCs) make invaginating contacts, where the dendritic tips reach a few hundred nanometers into the presynaptic area of cone pedicles (Figure 2A) (Dowling and Boycott, 1966). In contrast, OFF-cone BCs (OFF-CBCs) make basal contacts in the same area (Figure 2B). These 'true' contacts have to be distinguished from contacts in the periphery or at the (out)sides of the cone pedicle as well as contacts between dendrites and cone telodendria, which can happen, for instance as dendrites pass by (Figure 2C). Figure 2 with 2 supplements see all Download asset Open asset Classification of cone contacts. (A) Invaginating ON-CBC contact. Schematic drawing (left), EM side view (center) and top view (right). Red and dark grey, BC dendrites; light grey, horizontal cell dendrites; cyan, cone pedicles. (B) Basal/flat OFF-CBC contact as in A. (C) Peripheral (non-synaptic) BC contact as in A. (D–F) Contact area (D) eccentricity (E) contact height (F) of invaginating/basal and non-synaptic contacts for OFF-/ON-CBCs and rod bipolar cells (RBCs). (G–I) Contact area versus eccentricity for OFF-CBC (G), ON-CBC (H) and RBC (I) contacts indicating correctly and incorrectly classified contacts. https://doi.org/10.7554/eLife.20041.004 In total, we found n = 20,944 contacts in n = 2620 pairs of cones and BCs. We trained a support vector machine (SVM) classifier to distinguish whether or not an individual BC obtains input from a cone (as opposed to classifying each individual contact site, see Materials and methods). To this end, we defined a set of seven features, including contact area, eccentricity and contact height, which allowed distinguishing between potential synaptic contacts and 'false' contacts (Figure 2D–F, Figure 2—figure supplement 1). For training of the classifier, we manually labeled a randomly selected set of contacts (n = 50 for OFF-CBCs, n = 108 for ON-CBCs and n = 67 for RBCs). Given the highly stereotypical anatomy of the photoreceptor-BC synapse, labeling performed by an experienced human observer is expected to be very accurate – we here consider therefore the human labels as 'ground-truth'. We trained separate classifiers for ON-CBCs, OFF-CBCs and RBCs and found that they could reliably distinguish between true and false contacts, with a success rate of ~90% (leave-one-out cross-validation accuracy, Figure 2G–I). Deviations between the labels of the automatic classifier and the human labels did not vary systematically with BC type (see Materials and methods). Such deviations typically occurred when human labels were assigned based on more global structural features of a contact; such more contextual features were not included in the features used for automatic classification (examples of misclassified contacts are shown in Figure 2—figure supplement 2 and Videos 1–3). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animated 3D stack of an ON contact (human: contact, algorithm: no contact). https://doi.org/10.7554/eLife.20041.007 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animated 3D stack of an ON contact (human: no contact, algorithm: contact). https://doi.org/10.7554/eLife.20041.008 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Animated 3D stack of a RBC contact (human: no contact, algorithm: contact). https://doi.org/10.7554/eLife.20041.009 Contacts between cones and CBCs We analyzed contacts between CBCs and S- and M-cones in the center of the EM stack where cones were covered by a complete set of all BC types. There was no difference in the number of CBCs contacted by S- and M-cones with 12.2 ± 1.5 CBCs (n = 5 cones, mean ± SEM) for S-cones and 12.2 ± 0.4 CBCs (n = 71 cones) for M-cones, respectively. Similarly, the total number of contact points per cone was almost identical for S- and M-cones with an average of 108 ± 24 per S- and 105 ± 5 per M-cone. We first studied the convergent connectivity onto the different CBC types and studied how many cones provide input to a single BC of each type (Figure 3A and B). To this end, we classified type 5 CBCs, which had not been further subdivided by Helmstaedter et al. (Helmstaedter et al., 2013), into three types (Figure 3—figure supplement 1, see Materials and methods) in agreement with recent reports (Greene et al., 2016). Figure 3 with 3 supplements see all Download asset Open asset Quantification of cone-to-CBC contacts. (A) Volume-reconstructed single BC dendrite (red) contacting numerous cone pedicles (cyan). (B) Number of S- and M-cones contacted by different CBC types. (C) Volume-reconstructed single cone (cyan) contacted by multiple BCs (orange/red). (D) Number of CBCs per type contacted by individual S- and M-cones. (E) Example cone array with CBC6 and CBC8 contacting cones. Grey, non-contacted cones; blue, contacted cones. (F) Number of contacted cones and cones within dendritic field for different CBC types. (G) Fraction of contacted cones/cones within the dendritic field. (H) Kernel density estimate of the distribution of contacted cones as function of distance from BC somata. (I) Same as H. but distance normalized by dendritic field size. Bars in B,D,F indicate 95% CI. https://doi.org/10.7554/eLife.20041.010 Most CBC types were contacted predominantly by M-cones, with an average of 2–6 cones contacting individual CBCs. One exception was the CBC9 that – by our definition of S-cones – received considerable S-cone input. We also detected a few contacts between CBC9s and M-cones; these are a consequence of our restrictive definition of S-cone and originate from those cones for which we found only single CBC9 contacts, such that they were classified as M-cone (see above, Figure 1; see also Figure 3—figure supplement 2). We next evaluated the divergent connectivity from S- and M-cones to CBCs and studied how many individual BCs of each type were contacted by a single cone (Figure 3C). We found that each M-cone contacted on average a little less than one CBC1, while S-cones contacted almost no CBC1, consistent with previous reports (Breuninger et al., 2011). Conversely, we found that M-cones almost never contacted CBC9s (see above), but S-cones contacted on average two. Both cone types contacted all other CBC types (Figure 3D), with each cone making contact with at least one CBC2, 3B, 4, 5I, 6 and 7. In contrast, some cones did not contact ON-CBC types 5T, 5O, X and 8, such that they were contacted by considerably less than one cone on average. In addition, we tested the hypothesis that CBCs other than type 1 and 9 unselectively contact all cones within their dendritic field (Wässle et al., 2009). To this end, we compared the number of contacted cones and the number of cones that are in reach of the BC dendrites (Figure 3E–G). OFF-CBCs (types 1–4) contacted on average 65–75% of the cones in their dendritic field, with very similar numbers across types (Figure 3G). In contrast, ON-CBCs showed greater diversity: The connectivity pattern of types 5I, 6 and 7 was similar to that observed in the OFF types (Figure 3G); these cells sampled from the majority of cones within their dendritic field (60–80%). CBC5T, 5O, X and 8, however, contacted less than half of the cones within their dendritic field (Figure 3G), with the lowest fraction contacted by CBCX (~20%). This result is not due to a systematic error in our contact classification: We manually checked volume-reconstructed dendritic trees of the respective types for completeness and frequently found dendrites passing underneath a cone with a distance of 1–3 μm without contacting it (Figure 3—figure supplement 3). Finally, we studied the contact density along CBC dendrites (Figure 3H and I). To check for systematic variation independent of the absolute size of the CBC dendritic tree, we normalized the cone contact density for the dendritic field size of each CBC type (Figure 3I). Almost all CBC types received input at a very similar location relative to their soma, except for CBCX, which received the majority of inputs closer to the soma than all other types relative to its dendritic field size. As a control, we also ran the connectivity analysis with the set of S-cones from our alternative, more liberal classification (Figure 3—figure supplement 2B,C). In this analysis, CBC9 was the only color specific BC type whereas all other BC types, including CBC1, contacted both S- and M-cones without preferences (Figure 3—figure supplement 2C). This contradicts the result of a previous analysis based on physiology, which implies that CBC1 does not receive S-cone input (Breuninger et al., 2011). The CBCX has few and atypical cone contacts CBCX had an atypical connectivity pattern compared to other CBC types, so we decided to study its connections in more detail. This BC type has only recently been identified by (Helmstaedter et al., 2013; Shekhar et al., 2016). It has a compact dendritic tree but a relatively wide axonal terminal system that stratifies narrowly at approximately the same depth as CBC5O and 5I do. Interestingly, CBCX seems to sample the cone input very sparsely, with input from only two cones on average, and contacting only about 20% of the cones available in its dendritic field (Figure 3C,D and G). In fact, dendrites of CBCX oftentimes passed underneath cones or even stopped shortly before cone pedicles without making contacts at all (Figure 4A and B). It is unlikely that this resulted from incomplete skeletons for these BCs, as all skeletons were independently verified for this study and corrected where necessary (see Materials and methods). Figure 4 Download asset Open asset CBCX makes few and atypical cone contacts. (A) Volume-reconstructed CBCX dendritic arbor (red) contacting few cone pedicles (cyan, invaginating contact; grey, tip contact). (B) Same exemplary cone array as in A. with CBCX dendritic arbor contacting cones. Light grey, non-contacted cones; cyan, invaginating contacts; dark grey, tip contacts. (C) EM image showing tip contact between CBCX (red) and cone pedicles (cyan), top view (left) and side view (right). (D) Invaginating and tip contacts in CBCXs and other ON-CBCs. Bars in D. indicate 95% CI. https://doi.org/10.7554/eLife.20041.014 We re-examined all detected contacts between CBCXs and cones and found that very few of those were 'classical' invaginating ON-CBC contacts (3 out of 19 contacts, n = 7 cells, Figure 4B–D). The vast majority were 'tip' contacts (16 out of 19 contacts, n = 7 cells), which were similar to basal contacts made by OFF-CBC dendrites (Figure 4B–D). The available data was not conclusive with regards to the question whether these tip contacts of CBCX are smaller than those of OFF-CBCs (median area: 0.05 μm2 for n = 22 CBCX contacts; 0.10 μm2 for n = 23 OFF-CBC contacts, but p=0.17, Wilcoxon ranksum test). In contrast to the CBCX, the other ON-CBC types made mostly invaginating contacts (71 out of 81 contacts, n = 12 cells, two cells per BC type, Figure 4D), indicating a significant effect of cell type on contact type (GLM with Poisson output distribution, n = 38, interaction: p=3.6x10−7, see Materials and methods). We checked if CBCX receive rod input instead but did not observe any rod contacts (see below). Thus, the CBCX appears to be an ON-CBC with both very sparse and atypical cone contacts similar to those made by based on the axonal depth (Helmstaedter et al., 2013) and recent and recordings (Ichinose et al., et al., this BC type is most likely an by its et al., 2016). RBCs make contacts with cones We next analyzed the connectivity between photoreceptors and rod bipolar cells (RBCs) to the hypothesis that RBCs may contact cones (Pang et al., 2010). cells labeled as RBC in Helmstaedter et al. (Helmstaedter et al., 2013) were from this analysis, as they did not contact any rods (Figure supplement 1). We also found some rods not contacted by any which is likely due to incomplete of the dendritic tips of some In fact, RBCs did not only contact rod but also cone pedicles (Figure These contacts were ON-CBC contacts with invaginating dendritic tips into the cone pedicles (Figure To quantify the connectivity in more we the number of cones contacted by an individual the vast majority contacted at least one only of all RBCs (n = did not contact any (Figure However, we did not find a of RBCs to S- or M-cones (Figure Conversely, of cones contacted a single their signal to two to and only 20% of the cones did not make any contact with an RBC (Figure an anatomical to the direct cone input into a of RBCs (Pang et al., 2010). we evaluated whether RBCs contacting only rods or both and rods two types of as by et al. However, the two of RBCs did not the depth of their axonal arbor (Figure supplement number of rod contacts (Figure supplement or potential connectivity to AII amacrine cells (Figure supplement and did not form independent (Figure supplement In addition, the dendritic field size n = and the number of dendritic tips n = 12 manually did not between the two Therefore, the available anatomical data two types of Figure 5 with 2 supplements see all Download asset Open asset contact rod bipolar cells. (A) Volume-reconstructed RBC (red) contacting both rods and cone pedicles (cyan). (B) EM showing invaginating contact between cone (cyan) and RBC side view and top view (C) Number of RBCs contacted by cones. Number of RBCs contacted by S- and cones. (E) Number of cones contacted by Bars in D. indicate 95% CI. Quantification of rod to OFF-CBC contacts to the analysis above, we and volume a complete set of neighboring rod of the EM field, Figure Figure supplement and identified cell In addition to the invaginating connections (Figure we also found basal contacts between OFF-CBCs and rods to the invaginating RBC dendrites (Figure as earlier et al., et al., 2007; Haverkamp et al., 2008; Tsukamoto and Omi, 2014). We did not find any contacts between ON-CBCs and rods agreement with Tsukamoto and Omi, but see Tsukamoto et al., Figure 6 with 1 supplement see all Download asset Open asset contact RBCs and (A) neighboring rod in one half of the field of the reconstructed cone pedicles (B) Rod with invaginating dendrites of two RBCs Schematic drawing (left), EM side view and top view (right). (C) Rod with basal contacts by OFF-CBCs Schematic (left), volume-reconstructed vertical view EM image with top view (right). The also an invaginating RBC dendrite Number of rods contacted by RBCs and OFF-CBC types Bars in D. indicate 95% CI. single RBC contacted about rods (Figure which is more than was recently reported Tsukamoto and Omi, 2013). single rod contacted one or two but very no RBC or more than two (Figure In all with two invaginating the dendrites to two different RBCs (n = The rods without RBC contacts were at the of the reconstructed where we could not all The number of rods contacting OFF-CBCs was much CBC1 and did not receive considerable rod CBC3A, CBC3B and CBC4 were contacted by with CBC3B the rod input (Figure Discussion We analyzed an existing electron microscopy dataset (Helmstaedter et al., 2013) to quantify the connectivity between photoreceptors and bipolar cells. We found interesting violations of established of outer retinal connectivity: The newly discovered CBCX (Helmstaedter et al., 2013), likely an ON-CBC (Ichinose et al., et al., had unexpectedly few and mostly atypical basal contacts to cones. CBC types 5T, 5O and 8 also contacted fewer cones than expected from their dendritic field, they invaginating synapses. In addition, we provide anatomical evidence that rod and cone pathways are showing contacts. The picture of BC types with their input are in Figure 7. Figure 7 Download asset Open asset between cone and rod photoreceptors and bipolar cells in the mouse retina. examples of bipolar cell types in the mouse retina are The number of cones in the dendritic field number and contacted photoreceptors are for each a a synaptic the dataset we used was not labeled for synaptic we used automatic classifiers based on structural to identify putative synaptic contacts between BCs and to the highly stereotypical anatomy of the photoreceptor-BC synapse, these of synaptic for trained (see also For example, we used the of the contact to the center of the cone pedicle as a where presynaptic ribbons have been reported at the ultrastructural (Dowling and Boycott, 1966; Chun et al., 1996). The of the classifiers evaluated with human labels was it is possible that a few contacts were control, however, no systematic it is unlikely that classification our between human and classifier labels occurred when the human used the global for the of a contact, that is not into an (Figure 2—figure supplement 2). human error cannot be For contact points labeled by as non-synaptic may a and are therefore than For all data including for classifying and contacts is available an effect of retinal the retinal location of the EM stack used here is (Helmstaedter et al., it may originate from the retina, where M-cones (Röhlich et al., 1994; et al., 2013) However, as 'true' S-cones were shown to be across the retina (Haverkamp et al., 2005), CBC9 connectivity can be used for of S-cones independent of it cannot be that in M-cones in the retina may the connectivity patterns between the M-cones and the remaining bipolar cell types. contacts between some ON CBC types and cones We found that ON-CBCs 5T, 5O, X and 8 contact fewer cones than expected from the size of their dendritic field. We observed that many of their dendrites passed by the cone pedicles with a distance of 1–3 μm or even a cone pedicle without contacting it (Figure 3—figure supplement 1). This is in agreement with a recent study that CBC8 does not contact all cones within its dendritic field and but in contrast to earlier studies that that different BC types receive input from all cones within their dendritic