purchased from The Jackson Laboratory (Bar Harbor, Maine). Mice were euthanized by CO2 inhalation followed by cervical dislocation. All experiments on animals were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic and were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

11.2.2 Immunofluorescent Staining of Retinal Sections

Mouse eyes were prepared as previously described (Xi et al. 2007). Briefly, eyes were fixed in 4% paraformaldehyde, immersed through a graded series of sucrose solutions, embedded in OCT and flash frozen. The tissue was sectioned at 10 μm thickness using a cryostat. Retinal sections were blocked before incubation with primary antibodies overnight at 4ºC. Primary antibodies and dilutions were as follows: Tulp1: rabbit polyclonal M-tulp1N 1:250 (Hagstrom et al. 2001); Piccolo: rabbit polyclonal 1:500 (ab20664: Abcam Inc); Bassoon: mouse monoclonal 1:500 (SAP7F407: Assay Designs Inc); Ribeye/CtBP2: mouse monoclonal 1:500 (612044: BD Biosciences); Protein Kinase C-α (PKC): rabbit polyclonal 1:1,000 (SC208: Santa Cruz Biotechnology Inc); Rhodopsin: mouse monoclonal 1:100 (B630N: P. Hargrave, Univ. of Florida). Sections were washed and incubated in fluorescent secondary antibodies at room temperature for 1 h. Secondary antibodies were: Alexa Fluor R 488 goat anti-rabbit IgG and goat anti-mouse IgG; Alexa Fluor R 594 goat anti-rabbit IgG and goat anti-mouse IgG (Invitrogen). The sections were then coverslipped in mounting media with DAPI (Vector Laboratories). Sections were imaged using an Olympus BX-60 fluorescent microscope equipped with a CCD monochrome camera (Hamamatsu Photonics, Bridgewater, NJ). For the imaging of ribbon-associated synaptic proteins (Bassoon, Piccolo and Ribeye), 2 μm Z-stacks were acquired using nearest neighbor deconvolution, followed by a maximum intensity Z-axis projection (SlideBook, Intelligent Imaging Innovations). For the co-localization of Bassoon and Piccolo, three-dimensional surface plots were generated from the Z-stacks.

11.3 Results

To describe the synaptic terminal architecture of tulp1–/– mice, we examined the distribution of synaptic proteins at ages prior to photoreceptor degeneration. Photoreceptor ribbon synapses are thought to be critical for the transport, release and recycling of synaptic vesicles (Morgans 2000; tom Dieck and Brandstätter 2006). Piccolo and Bassoon are proteins that have been associated with the organization of the photoreceptor synapse as well as the functioning of the ribbon (Dick et al. 2001; 2003). These proteins normally localize together at the presynaptic membrane with a crescent-like arrangement. We recently confirmed that Tulp1 is present in the photoreceptor synapse by its co-localization with Bassoon in the wt retina.

Fig. 11.2 Immunofluorescent staining of Ribeye in the OPL of mouse retinal sections. In the wt OPL at P16 (a), rd10 at P16 (c) and P21 (d), the synaptic ribbons decorated with Ribeye immunoreactivity display the classic crescent-like appearance (arrows highlight examples in magnified insets). In contrast, few intact ribbons are detectable in the tulp1–/– OPL at P16 (b). Scale bar, 10 μm

We further characterized the synaptic architecture using antibodies against Ribeye, a protein thought to constitute the core of the ribbon (Schmitz et al. 2000). Ribeye co-localizes and binds with Piccolo and Bassoon (tom Dieck et al. 2005; Heidelberger et al. 2005). Figure 11.2a shows that in the P16 wt OPL, numerous distinct crescent-shaped Ribeye-positive ribbons (see arrows) can be observed. This is also the case in rd10 retinas at P16 (Fig. 11.2c) and P21 (Fig. 11.2d), prior to and after the commencement of retinal degeneration. In contrast, the P16 tulp1–/– retina contains few normal ribbons (Fig. 11.2b), providing further evidence of a malformation of the ribbon synapse.

Next, we investigated the downstream consequences of the photoreceptor synaptic malformation on postsynaptic targets in tulp1–/– retinas using antibodies against Protein Kinase C-α (PKC), which labels rod DBCs and their dendrites (Greferath et al. 1990). At P16, wt DBC dendrites have elongated dendrites that penetrate the OPL, and each termination has a high degree of branching (Fig. 11.3a; vertical lines track a process of an individual DBC – indicated by an arrow). In contrast, DBC dendrites of P16 tulp1–/– mice are notably shorter and present far less branching (Fig. 11.3b). Interestingly, DBC terminals of the

Fig. 11.3 Immunofluorescent staining of DBC bodies (arrows) and dendrites (vertical lines) in the OPL of mouse retinal sections. In the wt OPL at P16 (a) and rd10 at P16 (c), long dendrites display ornate branching. In contrast, both the tulp1–/– OPL at P16 (b) and rd10 at P21 (d) have severely shortened dendrites. However, branching is still observed in the rd10 OPL at P21. Scale bar, 10 μm

P16 rd10 retina resemble those of wt (Fig. 11.3c). DBC dendritic retraction was, however, noted in the P21 rd10 retina (Fig. 11.3d), which resembled the P16 tulp1–/– phenotype.

11.4 Discussion

In young tulp1–/– mice, two key ribbon-associated proteins, Piccolo and Bassoon, are rarely united into the horseshoe shape characteristic of the wt photoreceptor ribbon synaptic complex. These proteins are, however, situated in close proximity to one another and are both confined to the OPL. These observations indicate that in the absence of Tulp1, Piccolo and Bassoon are able to arrive at their correct destination, but are unable to coordinate into the normal synaptic architecture. In addition, immunostaining for Ribeye showed that in the tulp1–/– retina, few intact ribbons are present. We note that prior to and during the height of photoreceptor degeneration, normal synaptic architecture is readily seen in the rd10 retina. This does not necessarily indicate that the observed synaptic defects are unique to the tulp1–/– retina, but it does make clear that generalized photoreceptor degeneration is not sufficient to induce synaptic alterations. However, it may be the case that