Figure 1.
Confocal microscopic evidence that calbindin ON cone bipolar cells make axonal synapses in the IPL.
A: An antibody to calbindin (green) labels a horizontal cell (arrow), three amacrine cells (arrowheads), and 3 bipolar cells (asterisks) in the INL. The axons of these calbindin labeled bipolar cells ramify in the border between strata 4 and 5 of sublamina b of the IPL, indicating that the calbindin labeled cells are ON-type cone bipolar cells. RIBEYE (red) labeled synaptic ribbons are seen in the OPL as horseshoes and in the IPL as puncta. The boxed area is magnified in B and C. B, C: Magnified images are taken from two 0.38-μm-thick single optical sections. A calbindin ON cone bipolar descending axon contains two synaptic ribbons in stratum 1. One is localized at the main axonal stem (arrow in B) and the other is localized on a small branch from the main descending axon (arrowhead in C). D, E: Both confocal micrographs were taken at the same location from a wholemount piece of the retina. The focal planes for D and E are in sublaminae a and b of the IPL, respectively. Three calbindin ON cone bipolar descending axons and an amacrine soma (asterisk) are seen in D. All of the labeled axons contain at least one RIBEYE labeled synaptic ribbon (arrows). Arrowheads indicate putative axonal synaptic sites formed by additional ON bipolar cell types. In E, numerous synaptic ribbons are found in calbindin ON cone bipolar axon terminals. In addition, there are many synaptic ribbons from other bipolar cells.
Figure 2.
A comparison of axonal synapses and axon terminal synapses of the calbindin ON cone bipolar cell.
A: The 3D reconstruction model for No. 3 calbindin ON cone bipolar cell axon (Fig. 3; Table 1). Magenta ellipses are the labeled profiles shown in B. B: Low magnification view of an electron micrograph used to make the 3D reconstruction model shown in A. Four immunolabeled axonal parts are seen. Among them, one (C) in the upper part of the figure is located in stratum 1 and the remaining three in the lower part are located in strata 4 and 5. The descending axonal part in stratum 1 and an axon terminal part in stratum 5 are depicted and magnified in C and D, respectively. C: The labeled axon descends obliquely to form an axonal ribbon synapse onto an amacrine process (A) represented as a monad. At this synapse, two synaptic ribbons (arrowheads) are engaged. Brackets indicate a putative ribbon in transport. D: A calbindin labeled bipolar axon terminal with a synaptic input from an amacrine cell (A) (arrow) forms a ribbon synapse (arrowhead) onto a postsynaptic dyad composed of a ganglion dendrite (G) and an amacrine process (A).
Figure 3.
The 3D reconstruction of calbindin ON cone bipolar cell axons in the IPL.
Ten reconstructed models of calbindin bipolar axons are demonstrated. Insets: Higher magnification views of calbindin ON cone bipolar axons at the border of the INL and the IPL. Insets show that all calbindin bipolar cells contain multiple synaptic ribbons in this IPL region.
Table 1.
Synapses made by calbindin ON cone bipolar axons in sublamina a of the IPL.a
Figure 4.
3D reconstruction of axonal and axon terminal synapses of a calbindin ON cone bipolar cell.
A–E: The 3D reconstruction of No. 6 axonal (A) synapse was derived from electron micrographs. A–C: Three electron micrographs of a calbindin ON cone bipolar axonal synapse show five synaptic ribbons (1–5, arrowheads) and a broad postsynaptic density on a postsynaptic amacrine process. D, E: Reconstructed synaptic ribbons (R, maroon) of a calbindin bipolar axon (gray) and the postsynaptic density (PSD, khaki) of an amacrine process (sea green) are displayed at two different angles and insets show magnified synaptic ribbons and the postsynaptic density. F, G: 3D reconstruction of No. 2 axon terminal (AT) synapse was derived from electron micrographs. F: An electron micrograph shows a calbindin ON cone bipolar axon terminal synapse containing a synaptic ribbon (arrowhead) and its postsynaptic dyad. G: A synaptic ribbon (R) of a calbinidn bipolar axon terminal (gray) and two postsynaptic densities (1, 2) of the postsynaptic elements composed of an amacrine (state blue) and a ganglion (sea green) are seen. H: Data from image analysis of surface area of the synaptic ribbon and the postsynaptic density from No. 6 axonal synapse (D, E) and No. 2 axon terminal synapse. The table shows that individual axonal ribbons are smaller but the post-synaptic density at axonal ribbons is much larger compared to the axon terminal synapses.
Table 2.
Comparison of axonal and axon terminal ribbon synapses of the calbindin ON cone bipolar cell in length and surface area of the synaptic ribbon and in postsynaptic density area.
Figure 5.
3D reconstruction of calbindin negative ON and OFF cone bipolar cell axons in the IPL.
A: Three different types of calbindin negative OFF cone bipolar cells (green) and seven calbindin negative ON cone bipolar cells (slate blue). The ON bipolar cells can be divided into at least 4 different types according to the branching level of the axon terminals. In these cells, only two different ON cone bipolar cells, which ramified in strata 4 and 5, made axonal synapses (boxes) in stratum 1 of the IPL. The second boxed area is shown in B and C at a higher magnification. B, C: These micrographs were taken from two consecutive ultrathin sections. The unlabeled ON cone bipolar axon (BC) makes axonal inputs onto a monadic amacrine process (A). At this axonal synapse, three ribbons (arrowheads) are engaged.
Figure 6.
Specificity of melanopsin antibodies.
A: Labeling of wholemount rabbit retina with the mouse monoclonal antibody MAB3101 (red). A sparsely branched set of ganglion cells was labeled with the morphological properties of ipRGCs. B: Same frame, wholemount rabbit retina, labeled with the rabbit antibody against the N-terminal of rabbit melanopsin (green). C: Double label image shows exact superposition of the two antibodies (yellow). We conclude both antibodies recognize ipRGCs in the rabbit retina.
Figure 7.
Identification of the synapse between an ipRGC and a putative ON cone bipolar cell in the IPL.
A: A low magnification electron micrograph shows the relationship between a labeled ipRGC dendrite (asterisk) and an unlabeled axon of a putative ON bipolar cell (BCAX), which is running vertically through the INL and sublamina a of the IPL. The boxed area is shown in B at a higher magnification. B: The unlabeled ON bipolar axon (BC) makes an en passant ribbon synapse onto a labeled ipRGC dendrite (asterisk). Note that two synaptic ribbons (arrowheads) are engaged in this monadic synapse. C: A low magnification electron micrograph illustrates a labeled ipRGC dendrite (asterisk) located just below the somata of amacrine cells (AC). The boxed area is shown in D at a higher magnification. D: A labeled dendrite (asterisk) makes a postsynaptic monad at the ribbon synapse (arrowhead) from the axon of a presumed ON bipolar cell (BC).
Figure 8.
Axonal synapse vs. axon terminal synapse of the ON cone bipolar cell. Diagram shows the differences in character between the axonal synapse and the axon terminal synapse of the ON cone bipolar cell in the mammalian retina. Post-synaptic elements are represented as green, for amacrine cells, and blue, for ganglion cell dendrites. The summary shows that axonal ribbon synapses are: 1) monadic, with a single post-synaptic process; 2) they have multiple synaptic ribbons; 3) they have shorter synaptic ribbons and 4) a much larger area of post-synaptic density, when compared to the dyads made by axon terminal synaptic ribbons.