Name: Splitting of Murine Retina for Immunofluorescence Analysis
Uploaded: 2024-01-16T11:00:00
Duration: 1 min 52 s
Description: Watch this Scientific Journal Video about Splitting of Murine Retina for Immunofluorescence Analysis at JoVE.com

splitting of murine retina for immunofluorescence analysis

研究小鼠分裂视网膜中的双极和水平神经元细胞

The retina is a thin neural tissue located in the posterior eye where light is intercepted and processed into an electrochemical signal that can be interpreted by the brain. At the back of the retina, rod and cone photoreceptors are stimulated by light, which reduces the tonic release rate of the neurotransmitter, glutamate1. The first neurons to experience and respond to this light-induced change in glutamate concentration are the bipolar cells (BCs) and horizontal cells (HCs), whose somas reside in the outermost region of the inner nuclear layer (INL). These second-order neurons perform the first stage of signal processing in the retina and shape critical features of vision such as light adaptation, contrast sensitivity, and spatial/color opponency2. While these functions have been ascribed to BCs and HCs, the circuitry and biochemical mechanisms underlying these processes are not fully understood3. Therefore, the advancement of tools and methods for exploring BC and HC physiology is of paramount importance.
Vertical (transverse) retina sections have long&#160;proven the most practical model for studying BCs and HCs; however, certain aspects of BC and HC physiology are inaccessible to the experimenter under this model. Direct recordings from HCs or indirect measurements of their effects on BCs do not reflect the retina&#39;s endogenous connectivity since the lateral processes of these cells are severed during slicing. Whole mount retina preparations circumvent this issue by preserving these lateral processes, but the surrounding retinal layers pose a challenge for accessing these cells4. While there are abundant examples of immunostaining5,6,7,8 and patch clamp recordings9 from INL neurons in whole mount retinas, there is an opportunity to expedite and simplify the collection of these data. The inherent limitations of transverse sections and the challenges of the whole mount model thus inspired the development of this alternative flatmount retina preparation.
The following work describes a protocol for easily removing the photoreceptor layer from live, flatmount retinas to enhance access to BCs and HCs for simplified patch clamping and faster, more efficient immunolabeling. Peeling apart two pieces of nitrocellulose membrane attached to either side of an isolated retina tears the tissue through the photoreceptor axons, leaving a split retina that retains the outer plexiform layer (OPL) and all inner retinal layers. While others have described protocols for mechanically separating layers of the retina, these methods are either poorly suited to patch clamping and microscopy applications or require tedious manipulation of the tissue. Several of these methods require frozen or lyophilized tissue for layer separation, making them incompatible with electrophysiology experiments10,11,12. Others are designed for live tissue, but necessitate 5-15 sequential peels with filter paper4,11 or treatment with trypsin13 to remove the photoreceptors. The technique described here improves upon its predecessors by simplifying the photoreceptor removal procedure and expanding the repertoire of downstream applications.

作者聚焦：使用 Split Retina 技术增强访问和加速实验 

分裂视网膜作为研究脊椎动物视网膜内核层神经元的改进平装制剂

Our group is interested in the neuronal circuitry and synaptic mechanisms underlying visual processing in the retina. The Morgans Lab is investigating molecular mechanisms of light adaptation in bipolar cells, and the Sivyer Lab is interested in how inner retinal neurons contribute to ganglion cell function. Accessing internuclear layer neurons in whole mount retina is a challenge for both anatomical and physiological studies.Internuclear layer neurons are accessible in vertical sections, but they're very few neurons in the field of view. Furthermore, slicing severs lateral processes and connections which can impact physiological studies. The removal of the photoreceptors in the split retina technique dramatically improves antibody diffusion into the inner retina, which makes immunolabeling of inner retinal targets over 20 times faster when compared to traditional whole mount retina.The split retina also greatly improves access to internuclear layer neurons during patch clamp electrophysiology. The split retina technique will open doors to new approaches and accelerate the pace of our experiments. For example, we plan to use this technique to study bipolar cell inputs to melanopsin ganglion cells by expressing channelrhodopsin in the bipolar cells.

Watch this Scientific Journal Video about Splitting of Murine Retina for Immunofluorescence Analysis at JoVE.com

Splitting of Murine Retina for Immunofluorescence Analysis  

小鼠视网膜分裂用于免疫荧光分析

首先用新鲜的碳化Ames替换含有小鼠视网膜的培养皿中的Ames培养基。使用广口移液管将一块解剖的小鼠视网膜放在载玻片上，神经节细胞面朝上。用精细的任务擦拭将其抹平，去除多余的液体。如有必要，使用细尖画笔在解剖显微镜下轻轻拉动视网膜边缘。用镊子将一块干燥的硝酸纤维素膜放在视网膜上，将其粘附在神经节细胞侧。将视网膜翻转过来，将硝酸纤维素放在载玻片上，然后在视网膜的感光侧放置另一片干燥的膜片。现在，用艾姆斯介质润湿画笔的尖端。对上膜施加轻微的向下压力，以帮助均匀粘附。接下来，用一对镊子将下膜固定在玻璃上。然后小心地使用另一对以缓慢、稳定的动作剥离上膜。处理含有感光器的上膜。最后，将含有内视网膜的下膜放回碳化的Ames培养基中。