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Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

Wenzhou Medical University

α-Synuclein (α-Syn) is enriched in presynaptic terminals of the central nervous system including the retina and plays a role in the synaptic vesicle cycle and synaptic transmission. Abnormal aggregation of α-Syn is considered to be the main component of the Lewy bodies that are the pathological hallmarks of Parkinson's disease. Although expression pattern of α-Syn has been described in the retinas, its precise cellular and subcellular locations are poorly understood. We investigated the precise expression of α-Syn using light microscopy (LM) and electron microscopy (EM) with antibodies against α-Syn in the mouse retina. We found that the majority of α-Syn immunoreactivity (IR) is located in GABAergic, glycinergic, and dopaminergic amacrine cells, and their processes often make a direct synapse to other labeled or unlabeled amacrine profiles, bipolar cell terminals, or ganglion cell dendrites. Further, our LM and immuno-EM results confirm the absence of α-Syn in excitatory photoreceptors, bipolar cell bodies, and their ribbon synapses, providing evidence, for the first time, that ribbon synapses do not express α-Syn. Additionally, α-Syn IR is located in the ganglion cells, some of which are intrinsically photosensitive retinal ganglion cells. These results reveal a previously unappreciated inhibitory synapse-specific expression pattern of α-Syn in the retina, suggesting that α-Syn may play a distinct role in the modulation and integration of inhibitory synaptic transmission in the retina.

Expression of α-Synuclein in the mouse retina is confined to inhibitory presynaptic elements.

The light pathways are segregated into rod and cone pathways in which rods synapse with rod bipolar cells (RBCs), while cones contact cone bipolar cells (CBCs). However, previous studies found that cones can make synapse with RBCs (cone-RBC synapses) and rods can contact OFF CBC in primate and rabbit retinas. Recently, such cone-RBC synapses have been reported physiologically and morphologically in the mouse retina. Nevertheless, the precise subcellular evidence to determine whether it is the invaginating synapse or the flat contact remains absent. This is due to a lack of immunochemically verified ultrastructural data. Here, we investigated the precise expression of protein kinase C alpha (PKCα) using pre-embedding immunoelectron microscopy (immuno-EM) with a monoclonal antibody against PKCα, a biomarker for the RBCs. We determined the nanoscale localization of PKCα in the outer plexiform layer of the mouse and guinea pig retinas. Our results demonstrate the existence of both the direct invaginating synapse and the basal/flat contact of the cone-RBCs, providing for the first time immunochemically verified ultrastructural evidence for the cone-RBC synapse in the mouse and guinea pig retinas. These results suggest that the cross talk between cone and rod pathways is much more extensive than previously assumed.

Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.

Substance P (SP), a neuroprotective peptidergic neurotransmitter, is known to have immunoreactivity (IR) localized to amacrine and/or ganglion cells in a variety of species' retinas, but it has not yet been studied in the mouse retina. Thus, we investigated the distribution and synaptic organization of SP-IR by confocal and electron microscopy immunocytochemistry in the mouse retina. SP-IR was distributed in the inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Most of the SP-IR somas belonged to amacrine cells (2.5% of all) in the INL and their processes stratified into the S1, S3, and S5 layers of the IPL, with the most intense band in the S5 layer. Some SP-IR somas can also be observed in the GCL, which were identified as displaced amacrine cells (82%, 1269/1550) and ganglion cells (18%, 281/1550) by antibodies against AP2α and RBPMS, respectively. Such SP-IR ganglion cells (1.2% of all RGCs) can be further divided into 3 subgroups expressing SP/α-Synuclein (α-Syn), SP/GAD67, and/or SP/GAD67/α-Syn. Possible physiological and pathological roles of these ganglion cells are discussed. Further, electron microscopy evidence demonstrates that SP-IR amacrine cells receive major inputs from other SP-IR amacrine cell processes (146/242 inputs) and output mostly to SP-negative amacrine cell processes (291/673 outputs), suggesting series inhibition among amacrine cells. These results reveal for the first time an explicit distribution, novel ganglion cell features, and synaptic organization of SP-IR in the mouse retina, which is important for the future use of mouse models to study the roles of SP in healthy and diseased (including Parkinson's disease) retinal states.

Distribution and synaptic organization of substance P-like immunoreactive neurons in the mouse retina.

β-synuclein, a member of the synuclein family, is frequently co-expressed with α-synuclein in the neural system, where it serves to inhibit abnormal aggregation of α-synuclein in neurodegenerative diseases. Beyond its role in pathological conditions, β-synuclein plays various functions independently of α-synuclein. In our investigation, we discovered a broader expression of β-synuclein in the mouse retina compared to α-synuclein. This widespread pattern implies its potential significance in the retina. Through detailed examination via light- and electron-microscopic immunocytochemistry, we identified β-synuclein expression from the inner segment (IS) and outer segment (OS) of photoreceptor cells to the ganglion cell layer (GCL). Our findings unveiled unique features, including β-synuclein immunoreactive IS and OS of cones, higher expression in cone pedicles than in rod spherules, absence in horizontal cells, limited expression in cone bipolar dendrites and somas, higher expression in cone bipolar terminals, presence in most amacrine cells, and expression in almost majority of somas in GCL with an absence in intrinsically photosensitive retinal ganglion cell (ipRGCs) processes. Notably, all cholinergic amacrine cells express high β- but not α-synuclein, while dopaminergic amacrine cells express α-synuclein exclusively. These distinctive expression patterns offer valuable insights for further exploration into the functions of β-synuclein and its potential role in synuclein pathology within the retina.

Bilin Rao

Wenzhou Medical University

Preparing Retinal Organoid Samples for Transmission Electron Microscopy