The rod pathway of the microbat retina has bistratified rod bipolar cells and tristratified AII amacrine cells

Abstract

We studied the retinal rod pathway of Carollia perspicillata and Glossophaga soricina, frugivorous microbats of the phyllostomid family. Protein kinase Cα (PKCα) immunolabeling revealed abundant rod bipolar cells (RBCs) with axon terminals in the innermost sublamina of the inner plexiform layer (IPL), which is typical for mammals. Extraordinarily, the RBC axons showed additional synaptic contacts in a second sublamina further out in the IPL. Dye injections of PKCα-prelabeled RBCs of C. perspicillata confirmed the bistratified axon morphology. The functional partition of the IPL into ON and OFF sublayers was shown by using antibodies against vesicular glutamate transporter 1 [labeling all ON and OFF bipolar cell (BC) axon terminals] and G-protein γ13 (labeling all ON BCs). The ON sublayer occupied 75% of the IPL thickness, including both strata of the RBC axons. RBC output onto putative AII amacrine cells (ACs), the crucial interneurons of the rod pathway, was identified by calretinin, PKCα, and CtBP2 triple immunolabeling. Dye injections of calretinin-prelabeled ACs revealed tristratification of the AII ACs corresponding to the bistratified RBCs. Triple immunolabeling for PKCα, nitric oxide synthetase (NOS), and either GABA(C) or CtBP2 indicated GABAergic feedback onto RBCs via NOS-immunoreactive ACs. AII output analysis showed glycineric synapses with glycine receptor α1 expression between AII cells and OFF cone BCs and connexin 36-labeled gap junctions between AII cells and ON cone BCs. We conclude that microbats have a well developed rod pathway with great similarities to that of other mammals, but with an unusual IPL stratification pattern of RBCs and AIIs.

Figure 1.

A, C, Vertical cryostat sections of C. perspicillata (A) and G. soricina (C) retina, immunolabeled for PKCα. The cell bodies are located in the outer half of the INL, the dendritic branches in the OPL extend to the ONL border, and the axons terminate deep in the IPL close to the GCL. In the outer third of the IPL, additional PKCα-ir varicosities form a second stratum in S2. B, D, Retinal layers of sections A and C, respectively, by DIC optics. Retinal thickness differs between the two species. E, H, J, Individual DiI-injected RBCs in vertical vibratome slices of C. perspicillata retina. F, Retinal slice in DIC optics with a digitally superimposed contour of a DiI-injected RBC. The IPL is divided into five strata, S1–S5. G, Slices in H and J were prelabeled for PKCα. I, K, Merge of DiI-injected RBCs and PKCα staining, where varicose presumed synaptic sites are marked by arrows. Asterisk in G and I indicates the cell body of the PKCα-ir RBC that was injected with DiI. The filled round soma below the RBC soma in E is a cell without processes injected en passant. The filled process to the left of the cell in H and to the right of the soma in J belongs to another cell. GCL, Ganglion cell layer. Scale bars: A–D (in A), E, F (in E), 20 μm; G–K (in G), 10 μm.

Figure 2.

Vertical cryostat sections of C. perspicillata retina, double labeled for vGluT1 and PKCα (A–C) or Gγ13 and PKCα (E–K). D, H, Retinal layers of sections C and G with DIC optics (asterisks in C and D mark the same RBC). A, vGluT1 is expressed in all BC axon terminals throughout the IPL and in all photoreceptor terminals in the OPL. B, PKCα labels all RBCs with axon varicosities in S2 and in S4 plus S5. C, Colocalization of both labels in S2 and S4 plus S5 is visible as yellow labeling. E, Gγ13-ir cell bodies in the outer INL and axon terminals in the inner IPL (ON sublayer). G, Colocalization of Gγ13 and PKCα in S2 and S4 plus S5. I–K, Double immunolabeling for Gγ13 and PKCα in the IPL at higher magnification. Colocalization is visible in S2 (arrows) and S4 plus S5. Scale bars: A–H (in E), A–K (in I), 20 μm. GCL, Ganglion cell layer.

Figure 3.

A–C, DiI-injected AII amacrine cell in a vertical vibratome slice of C. perspicillata retina prelabeled for calretinin; a collapsed stack of 10 confocal optical slices of 1 μm each. The IPL is divided into five strata, S1–S5. Presumed lobular appendages of the AII AC in S1 are marked by arrows in B. Dendrites of the AII AC are obvious at two additional levels, in S2 and S5. D–I, Single confocal images of vertical cryostat sections of C. perspicillata retina labeled for PKCα, calretinin, and CtBP2. D–F, Double immunolabeling for PKCα and calretinin. The calretinin label is present in horizontal cells and cells in the INL and ganglion cell layer (GCL). Throughout the IPL, numerous calretinin-ir AII dendrites are labeled. The merge reveals a close association between PKCα- and calretinin-ir processes. Lobular appendages of AII ACs are mostly out of focus. G–I, Triple immunolabeling for PKCα, CtBP2, and calretinin. CtBP2-ir puncta in the OPL and IPL correspond to synaptic ribbons. Almost all somata in the INL are CtBP2 immunoreactive as well. RBC axon terminals in S2, which contain ribbons (magenta; G, I), are in close association with calretinin-ir AC dendrites (H, I). Typical lobular appendages in S1 are visible too but slightly out of focus. Scale bars: A–C (in C), 20 μm; D–F (in F), G–I (in I), 10 μm.

Figure 4.

Fluorescence micrographs of vertical cryostat sections of C. perspicillata retina. A, Immunolabeling for the α1 subunit of the glycine receptor (GlyRα1). GlyRα1-ir puncta are most numerous in the outer IPL (OFF sublayer), and smaller scattered puncta are found in the inner IPL (ON sublayer). In the OPL, sparsely distributed puncta are visible. GCL, Ganglion cell layer. B, DIC optics of the section in A showing the retinal layers. C, D, Double labeling for calretinin and GlyRα1. In the merge (D), yellow profiles indicate GlyRα1 puncta located at large calretinin-ir profiles in the outer part of the IPL (AII lobular appendages; arrows in C and D). E, F, Double labeling for PKCα and recoverin. E, Recoverin is strongly expressed in photoreceptors in the ONL. In the INL a few recoverin-ir bipolar cell somata are visible. Their axons form a distinct plexus in S1 of the IPL and a loose one in S4/S5. F, No colocalization of recoverin- and PKCα-ir axon terminals is visible in the merge. G, H, Triple immunolabeling for calretinin, GlyRα1, and recoverin. G, Recoverin-ir axon terminals in S1 are in close association with GlyRα1-ir puncta. H, In the merge of all three labels, calretinin-ir lobular appendages are in close association with recoverin-ir axon terminals indicating glycinergic input from the AII AC to the OFF cone BC. I, Immunolabeling for Cx36, a gap junction protein. Cx36-ir puncta are most numerous in the inner IPL. J, Double labeling for Cx36 and calretinin. Almost all Cx36-ir puncta associate with calretinin-ir AII dendrites. K, Double immunolabeling for Cx36 and recoverin. Cx36-ir puncta decorate the axon terminals of recoverin-ir ON cone BCs. L, M, Higher magnification of another recoverin-ir cone BC axon terminal in S4/S5, decorated with Cx36-ir puncta. Scale bars: A, B (in A), 20 μm; C–K (in C, E, J, K), 10 μm; L, M (in (M), 5 μm.

Figure 5.

Fluorescence micrographs of vertical cryostat sections of C. perspicillata retina. A, Immunolabeling for GABA_C receptors. There is strong punctate labeling throughout the IPL. B, DIC optics of the section in A showing the retinal layers. C, D, Double labeling for GABA_C and PKCα. In the merge (D), yellow profiles indicate GABA_C puncta located at the PKCα-ir RBC axon terminals (arrows). E, F, Double immunolabeling for NOS and ChAT. E, In the innermost INL, a NOS-ir AC soma is visible. NOS-ir processes stratify at three levels in the IPL, and the two prominent strata are located in S2 and S4 plus S5, the finer one in S1. F, The merge shows the typical appearance of ChAT-ir ACs with their processes stratifying at two levels in the IPL, the outer one in the OFF-lamina (S1) and the inner one in the ON-lamina (S3). There is hardly any overlap between the NOS and the ChAT AC dendrites in the IPL and hence no colocalization. G, Double labeling for PKCα and ChAT. Since the dendrites of the ChAT-ir ACs stratify in S1 and S3, no overlap with the PKCα-ir RBC axon terminals is visible. Arrows in F and G mark ChAT-ir somata. H, Double labeling for PKCα and NOS. Since dendrites of the NOS-ir ACs stratify in S2 and S4 plus S5, several NOS-ir profiles can be detected in close association with PKCα-ir RBC axon terminals, which makes GABAergic input to RBCs very likely. Scale bars: A, B (in A), E, G, H, 20 μm; C, D (in D), 10 μm.

Figure 6.

Synaptic interaction between NOS ACs and RBCs. A–F″, Single confocal images of triple immunolabeling for PKCα (blue), GABA_C (red), and NOS (green) (A–C″) and for PKCα (blue), CtBP2 (red), and NOS (green) (D–F″). A, D, Several PKCα-ir RBC axon terminals (blue) can be detected in close association with NOS-ir profiles in S2 (enlarged in A′, D′) and S4 plus S5 (enlarged in A″, D″). Double immunolabeling with GABA_C and NOS (B–B″) reveals GABA_C-ir puncta in close association with NOS-ir profiles in S2 and S4 plus S5 (arrows). C–C″, In the merge, aggregates of green, blue, and magenta label are obvious, making GABAergic input from the NOS-ir ACs to RBCs very likely. Double immunolabeling with CtBP2 and NOS (E–E″) reveals CtBP2-ir puncta in close association with NOS-ir profiles in S2 and S4 plus S5 (arrows). F–F″, In the merge, numerous ribbons in RBC axon terminals in S2 and S4 plus S5 of the IPL (arrows) are closely associated with NOS-ir profiles, indicating synaptic input. Scale bars: A–C (in C), D–F (in F), 20 μm; A′- F″ (in C″), 5 μm.

Figure 7.

Scheme of the rod pathway in C. perspicillata. Line drawings of DiI-injected cells prelabeled for PKCα (RBC), calretinin (AII amacrine cell), and recoverin (OFF and ON CB, cone bipolar cells) are shown. They were reconstructed from vertical retinal slices after intracellular injections. In the IPL (S1–S5), the immunoreactive plexus of ChAT and NOS ACs are marked as bars in different shades of gray. The RB makes glutamatergic synapses onto the AII AC in S4 plus S5 and S2 of the IPL, transmitting a light-ON signal. In S1, lobular appendages of the AII AC make glycinergic (sign-inverting) output onto the OFF CB. In S4/S5, the light-ON signal is transmitted from the AII AC onto the ON cone BC via (sign-conserving) gap junctions, labeled by connexin36 (Cx36). Dendrites of NOS-ir putative GABAergic ACs make feedback onto the RB.