The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3

Abstract

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (Dnm1-3), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

Keywords: dynamin; endocytosis; retina; ribbon; rod photoreceptor; synapse.

Figure 1.

Working model for synaptic vesicle cycling at the rod ribbon synapse. Cytoplasmic vesicles attach and replenish vacant sites on the ribbon (1). As tethered vesicles move down the ribbon (2), they are primed for release (3). At the base, the opening of L-type calcium channels allows an influx of Ca²⁺ and triggers the fusion of vesicles with the plasma membrane. Vesicle exocytosis releases glutamate into the synaptic cleft (4). Vesicular proteins and lipids released into the membrane are removed via vesicle endocytosis. Membrane invagination occurs (5) followed by scission of the formed vesicle from the plasma membrane by dynamin (6). Reformed vesicles are refilled with glutamate (7) and returned to the large cytoplasmic pool of vesicles.

Figure 2.

Conditional deletion of dynamins 1 and 3 in rod photoreceptors. Retinal cross sections were obtained at P28 and stained with antibodies against dynamin 1 (magenta; A–D) and dynamin 3 (magenta; E–H). Rod and cone synapses were labeled with an antibody against PMCA (green). In control mice, dynamin 1 (A) and dynamin 3 (E) are highly abundant in synaptic terminals of rod photoreceptors. Staining for dynamin 1 was absent in ^rodDnm1^−/− (B) and ^rodDnm1^−/−/3^−/− (D) mice but present in ^rodDnm3^−/− mice (C). Staining for dynamin 3 was absent in ^rodDnm3^−/− (G) and ^rodDnm1^−/−/3^−/− (H) mice but present in ^rodDnm1^−/− mice (F). Cone synapses, indicated with an asterisk, have normal levels of dynamin 1 and dynamin 3 staining.

Figure 3.

Scotopic ERG responses are reduced in Dnm double cKO mice. Average scotopic ERG responses (n = 3–7 animals per genotype) from control (black) and ^rodDnm1^−/−/3^−/− (red) mice were recorded at P28, P90, and P180 at different single-flash intensities (A). The a-wave (B) and b-wave amplitudes (C) are plotted as a function of increasing flash intensity (mean ± SD). Scotopic ERG responses from single cKO mice (^rodDnm1^−/−, blue; ^rodDnm3^−/−, green) were comparable to their littermate controls (black). In ^rodDnm1^−/−/3^−/− mice (red), a significant decrease in the scotopic b-wave at P28 (C) is followed by a reduction in the scotopic a-wave at P90 and P180 (B), indicating impaired synaptic transmission to rod bipolar cells, followed by impaired photoreceptor function. D, Average photopic ERG responses (n = 3–9 animals per genotype) from control (black) and dynamin double cKO mice (red) are shown at P28, P90, and P180 at 2.0 log cd·s/m². Amplitudes of photopic b-waves (E) were plotted over increasing intensity of single-flash stimuli. Cone-driven ERG b-waves recorded from single and double cKO mice are normal at all tested ages. Comparisons reflect average response over two flash intensities within the gray bars; *p < 0.005; **p < 0.001.

Figure 4.

Scotopic ERG responses from Dnm double cKO mice lacking cone function (GNAT2^−/−). Scotopic ERG responses (n = 4–9 mice per genotype) from GNAT2^−/− (black) and ^rodDnm1^−/−/3^−/−;GNAT2^−/− (red) mice were recorded at P28/P40 and P90 at different single-flash intensities (A). The a-wave (B) and b-wave amplitudes (C) are plotted as a function of increasing flash intensity (mean ± SD). In ^rodDnm1^−/−/3^−/−;GNAT2^−/− mice (red) at P28/P40, the scotopic a-wave is modestly reduced, whereas the scotopic b-wave is more severely decreased. At P90, the a-wave shows a trend toward impairment (p = 0.024), while the b-wave is significantly diminished in Dnm double cKO mice. Photopic ERG responses are eliminated in GNAT2^−/− mice (D). Dashed lines in (B) through (D) show comparable data with intact GNAT2 expression (from Fig. 3). For scotopic responses, comparisons reflect average response over two flash intensities within the gray bars; *p < 0.01; **p < 0.005; ***p < 0.0001.

Figure 5.

Dynamin cKO lines lack an outer segment phenotype. Retinal cross sections of control (A) and ^rodDnm1^−/−/3^−/− mice (B) obtained at P28, P90, and P180 were stained with an antibody against rhodopsin to label rod outer segments (magenta), and Hoechst staining was used to label photoreceptor nuclei (blue). In all mice, rhodopsin localizes normally to rod outer segments, indicating normal vesicular trafficking from ER to Golgi in the absence of both dynamin isoforms. C–E The measurements of the ONL thickness in mice of indicated ages and genotypes (n = 3–5 mice per condition; mean ± SD). After deletion of dynamins 1 and 3, the ONL thickness is normal at P28 (C; control, 60.6 ± 2.4 µm; ^rodDnm1^−/−/3^−/−, 60.0 ± 1.9 µm), but is slightly reduced at P90 (D; control, 59.1 ± 2.6 µm; ^rodDnm1^−/−/3^−/−, 55.2 ± 2.5 µm) and reduced at P180 by ∼20% (E; control, 59.3 ± 3.9 µm; ^rodDnm1^−/−/3^−/−, 47.4 ± 5.0 µm). ONL thickness in single cKOs is comparable to controls. ****p < 0.0001. The ultrastructure of rod outer segments (ROS) assessed by TEM at P90 appears normal in all dynamin cKO lines (F–I).

Figure 6.

Reduced synaptic ribbon length in Dnm1/3 double cKO mice. Rod synaptic ribbons and postsynaptic neurites were colabeled with antibodies against CtBP2/RIBEYE (magenta) and WGA (green), respectively. In retinal cross sections from control (A,D,G) and ^rodDnm1^−/−/3^−/− mice (B,E,H) at P28 (A–C), P90 (D–F), and P180 (G–I), rod synaptic ribbons in double cKO mice are significantly shorter at P28 (C; control, 2.1 ± 0.3 µm; ^rodDnm1^−/−, 2.0 ± 0.2 µm; ^rodDnm3^−/−, 1.9 ± 0.2 µm; ^rodDnm1^−/−/3^−/−, 1.6 ± 0.2 µm; mean ± SD) and progressively reduce their length at P90 (F; control, 2.1 ± 0.2 µm; ^rodDnm1^−/−, 1.9 ± 0.2 µm; ^rodDnm3^−/−, 2.0 ± 0.3 µm; ^rodDnm1^−/−/3^−/−, 1.1 ± 0.2 µm) and P180 (I; control, 2.1 ± 0.3 µm; ^rodDnm1^−/−, 2.0 ± 0.3 µm; ^rodDnm3^−/−, 2.1 ± 0.2 µm; ^rodDnm1^−/−/3^−/−, 1.1 ± 0.2 µm). This reduced length is accompanied by a reduction in the number of identified ribbons in the OPL. The shape and number of synaptic ribbons in rods from single Dnm cKOs (C, F, I) are comparable to control animals. ****p < 0.0001.

Figure 7.

Synaptic proteins are absent in rods after Dnm1/3 cKO. Immunostaining of retinal cross sections from control (A) and ^rodDnm1^−/−/3^−/− mice (B) with an antibody against Syt1 (magenta) shows severe reduction in the Syt1 staining in double cKO rods at P28, which is followed by severe degeneration of rod synapses at P90 and P180; cones, labeled with PNA (green), show normal intensity of Syt1 staining in double cKO retinas. In ^rodDnm1^−/− (C) and ^rodDnm3^−/− (D) retinas at P180, Syt1 staining of rods and cones resembles the staining in control retinas (A). At P90, retinal cross sections from control (E, I), ^rodDnm1^−/− (G, K), ^rodDnm3^−/− (H, L), and ^rodDnm1^−/−/3^−/− mice (F, J) were stained for synaptic vesicle proteins (E–H, vesicular glutamate transporter 1, VGluT1, magenta; I–L, complexin 3, magenta) and ribbon-associated proteins (E–H, bassoon, yellow; I–L, CtBP2, yellow). Only in double cKO retinas (F, J) the staining of these marker proteins is significantly reduced, indicating impaired synaptic vesicle machinery in rods. Immunostaining of single cKO retinas resembles that in control mice. Cone synapses are labeled with antibodies against cone arrestin (cArr, E–H) and PNA (I–L).

Figure 8.

Rod synapses severely degenerated in rods lacking Dnm1 and Dnm3. Ultrastructural analysis of rod (A) and cone synaptic terminals (B) from control (left panels) and ^rodDnm1^−/−/3^−/− (right panels) mice at P180. A, Rod synaptic terminals in control mice have one ribbon (yellow arrow) and form an invaginating synapse with HC axon terminals and RB dendrites; see enlargements for details. B, Cone synaptic terminals are generally larger, contain multiple ribbons (yellow arrows) and invagination sites, and are formed with HC and CB dendrites; see enlargements for details. In dynamin double cKO mice, the rod terminal (two examples shown) contains a detached ribbon, and the invagination site is degenerating (A), whereas the neighboring cone synapses remain intact and are structurally comparable to those in control mice (B). C, Relative sizes of the example terminals shown in (A,C).

Figure 9.

Lack of dynamin 1/3-dependent endocytosis in rod synaptic terminals causes formation of enlarged endosome-like vesicles and Omega profiles. The ultrastructure of rod (A, B, D) and cone (C) synaptic ribbons was assessed by TEM in control (A, C), Dnm1/3 cKO (B, C), and Dnm1 cKO and Dnm3 KO mice (D) at P180. The synaptic ribbon (yellow arrow) is intensively stained and attached to the membrane at its base (arciform density, dashed yellow line, Aii). Control synapses contain a homogeneous pool of tethered and cytoplasmic vesicles: the tethered pool (red ellipses, Aii) are within 100 nm of the ribbon (shaded yellow area surrounding the yellow line, Aii), and the cytoplasmic pool (red ellipses within yellow shaded area, Aiv) are free floating in the cytoplasm. B, In double cKO mice, vesicles are heterogenous in size and enlarged (evs); impaired vesicle endocytosis halts vesicle release from the plasma membrane resulting in the formation of Omega profiles (*; Bi–iii). Ultrastructure from cone ribbon synapses appears similar in double cKO mice and control mice (C). The cone pedicles remain intact, with multiple ribbons attached to invagination sites and a homogenous pool of normal-sized vesicles present in the cytoplasm, without signs of enlarged evs. Rod ribbon synapses from single cKO/KO mice (D) appear relatively normal with a homogeneous pool of vesicles. E–H, Quantification of the number (E, G) and size (F, H) of the tethered (Aii, E, F) and cytoplasmic vesicle pools (Aiv, G, H) in control, single cKO/KO, and double cKO mice at P180 (mean ± SD). E–H, Double cKO mice have a significant reduction in the vesicle number and form larger vesicles, whereas Dnm3 KO (green) are indistinguishable from controls (black); at this age, Dnm1 cKO (blue) show a moderate reduction in vesicle number for both tethered (18.1%) and cytoplasmic pools (15.2%), but no change in vesicle size. *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 10.

Dynamin-dependent endocytosis is impaired in rod synapses lacking dynamin 1/3 at P40. Ultrastructure from control (A) and ^rodDnm1^−/−/3^−/− retinas (B) was performed at P40, when the rod synaptic terminals start to degenerate in double cKO mice. Ultrastructure is shown from side (i-ii: upper panel) and top views (iii: lower panels). In the absence of dynamin-dependent endocytosis, enlarged evs and Omega profiles (*) develop along the plasma membrane of double cKO rod synapses (B); see enlargements for details. The processes from the invaginating horizontal axon terminals in control and double cKO rod synapses are indicated as HC. M, mitochondria. C–F, Quantification of tethered (C, D) and cytoplasmic (E, F) vesicle pools in control (black) and double cKO (red) mice (mean ± SD): in the absence of dynamin 1/3, the number of vesicles is significantly reduced in both pools (C, E), and the size of the tethered and cytoplasmic vesicles is significantly enlarged (D, F). *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 11.

RB dendrites disassemble in dynamin double cKO mice. RB dendrites in retinal cross sections obtained at P28 (A), P90 (B), and P180 (C) were costained with antibodies against PKCα (magenta) and mGluR6 (green). In control mice (A–C; left panels), RB dendrites sprout normally into the outer plexiform layer expressing mGluR6 at their postsynaptic terminals. After Dnm1/3 cKO in rods, RB dendrites sprout with normal mGluR6 expression at P28 (A; right panels), indistinguishable from control mice, but later undergo progressive degeneration at P90 and P180 with some dendrites extending into the ONL (indicated with white arrowheads; B, C; right panels). This degeneration of RB dendrites is accompanied by reduced staining of mGluR6 at their postsynaptic terminals. Enlarged images are shown to the right of each panel. Progressive degeneration of RB dendrites is also shown in retinal flat mounts, in which the dendrites are stained with anti-PKCα antibody (magenta). PNA marking cone terminals (green) was used to identify normal Z-projections of RB dendrites between the ONL and the outer plexiform layer (D, E). Enlarged images are shown to the right of each panel. Staining of retinal cross sections for the presynaptic protein bassoon (yellow) and the RB marker, PKCα (magenta), shows progressive loss of RB dendrites in ^rodDnm1^−/−/3^−/− retinas and the disintegration of their synaptic connections with rods (F, G). Staining intensity for bassoon becomes progressively reduced at P90 and P180 in the double cKO retina (G) compared with the control (F), indicating dynamin is required for the maintenance of the rod synaptic terminal.

Figure 12.

Horizontal cell axon terminals degenerate in dynamin double cKO retinas. The structure of HCs in control and ^rodDnm1^−/−/3^−/− retina was evaluated in retinal cross sections (A–F) and flat mounts (G–L) at P28, P90, and P180. HCs were stained for their marker calbindin-1 (green), and their axon terminals were labeled with an antibody against Syt2/ZNP-1 (magenta). In cross sections, HC bodies are visible at all ages; however, their axon terminals progressively degenerate: reduced staining for Syt2/ZNP-1 indicates that the degeneration starts at P28 (D) and progresses at P90 (E) and P180 (F); at P180, most axon terminals are gone. The remaining staining in (F) most likely originates from synaptic connections between cones and HC dendrites. Retinal flat mounts, stained for calbindin-1 (green; i: left panels) and Syt1/ZNP-1 (magenta; ii: right panels), confirmed that HC axon terminals start to degenerate at P28 (G, J) and continue degenerating at P90 (H, K) and P180 (I, L). PNA colabeling (green) in the right panels (ii) identified the layer connecting cone photoreceptor synapses with HCs. Enlargements are shown for details.

Figure 13.

HC somas and dendrites remain intact in dynamin double cKO retinas. Intravitreal injection of AAV2/1-CAG-ChR2-GFP was used to sparsely label HCs (green), which were costained for their marker calbindin-1 (magenta; A–F). Representative images from control (A, B, E) and double cKO mice (C, D, F) are shown at P180: somas with dendrites appear normal in both cases (A, C), whereas the axon in double cKO severely degenerated and is barely visible. Whole AAV-transfected HC from control and double cKO mice are shown in E and F, respectively. Staining of retinal cross sections from control (G) and double cKO (H) mice with an antibody against a neurofilament (NF-H) shows the progressive degeneration of the axon terminal (H). The axon colocalizes with calbindin-1 staining at P28 (H, upper panel), but the staining intensity of both markers progressively decreases with age; only calbindin-1-labeled somas are intensively stained in double cKO mice at P180 (H, lower panel). bv, blood vessel.

Figure 14.

Müller glia cells become stressed in dynamin double cKO mice. Retinal cross sections from control and dynamin double cKO mice were stained for GFAP, a marker identifying astrocytes and Müller glia cells (magenta). In control retina, GFAP staining was confined to astrocytes on the retinal surface. Staining of this area was more extensive in the double cKO retina at P28 (A) and spread toward the ONL at P90 (B) and P180 (C), indicating activation of Müller glia.

Figure 15.

Dnm1/Dnm3 deletion in rods impairs synaptic vesicle endocytosis and causes progressive degeneration of ribbon synapses. A normal rod synapse is filled with synaptic vesicles homogeneous in size; it has a single synaptic ribbon tethered with vesicles attached; and it forms one invagination site for the synaptic vesicle release of the neurotransmitter glutamate to HC axon terminals and RB dendrites (left illustration). In the absence of dynamins 1 and 3 at P40, enlarged evs in the cytoplasm and Omega profiles along the plasma membrane start forming; processes from HCs and rod bipolar cells retract from the invagination site; and the ribbon attached to the slowly disappearing vesicle release site becomes shorter (middle illustration). A more severe phenotype is observed by P180: enlarged evs accumulate in the cytoplasm, the synaptic ribbon detaches from the active site, HC axon terminals degenerate, and RB dendrites either retract or extend into the ONL (right illustration).