splitting of murine retina for immunofluorescence analysis

Étude des cellules neuronales bipolaires et horizontales dans la rétine divisée murine

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.

Pleins feux sur l’auteur : Utilisation de la technique de la rétine fendue pour un meilleur accès et des expériences accélérées 

Split Retina en tant que préparation améliorée à montage plat pour l’étude des neurones de la couche nucléaire interne dans la rétine des vertébrés

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  

Scission de la rétine murine pour l’analyse d’immunofluorescence

Commencez par remplacer le milieu d’Ames de la boîte de Pétri qui contenait la rétine de la souris par un nouvel approvisionnement d’Ames carbogé. À l’aide d’une pipette de transfert à large ouverture, placez un morceau de rétine de souris disséquée sur une lame de verre, les cellules ganglionnaires vers le haut. Retirez l’excès de liquide à l’aide d’une lingette délicate pour l’aplatir.Si nécessaire, utilisez un pinceau à pointe fine pour tirer doucement les bords de la rétine sous un microscope à dissection. Utilisez une pince pour placer un morceau sec de membrane nitrocellulosique sur la rétine, en le faisant adhérer au côté des cellules ganglionnaires. Retournez la rétine pour déposer la nitrocellulose sur la lame de verre et placez un autre morceau de membrane sèche du côté photorécepteur de la rétine.Maintenant, mouillez la pointe du pinceau avec le média Ames. Appliquez une légère pression vers le bas sur la membrane supérieure pour favoriser une adhérence uniforme. Ensuite, épinglez la membrane inférieure au verre avec une paire de pinces.Ensuite, utilisez soigneusement une autre paire pour décoller la membrane supérieure dans un mouvement lent et régulier. Éliminez la membrane supérieure qui contient les photorécepteurs. Enfin, remettez la membrane inférieure contenant la rétine interne dans le milieu d’Ames carbogé.

splitting-of-murine-retina-for-immunofluorescence-analysis

Research

JoVE Journal

Neuroscience

Splitting of Murine Retina for Immunofluorescence Analysis

568 vues.  Oregon Health and Science University. Commencez par remplacer le milieu d’Ames de la boîte de Pétri qui contenait la rétine de la souris par un nouvel approvisionnement d’Ames carbogé. À l’aide d’une pipette de transfert à large ouverture, placez un morceau de rétine de souris disséquée sur une lame de verre, les cellules ganglionnaires vers le haut. Retirez l’excès de liquide à l’aide d’une lingette délicate pour l’aplatir.Si nécessaire, utilisez un pinceau à pointe fine pour tirer doucement ...

Vidéo: Splitting of Murine Retina for Immunofluorescence Analysis  

Split Retina as an Improved Flatmount Preparation for Studying Inner Nuclear Layer Neurons in Vertebrate Retina

Bipolar cells and horizontal cells of the vertebrate retina are the first neurons to process visual information after photons are detected by photoreceptors. They perform fundamental operations such as light adaptation, contrast sensitivity, and spatial and color opponency. A complete understanding of the precise circuitry and biochemical mechanisms that govern their behavior will advance visual neuroscience research and ophthalmological medicine. However, current preparations for examining bipolar and horizontal cells (retinal whole mounts and vertical slices) are limited in their capacity to capture the anatomy and physiology of these cells. In this work, we present a method for removing photoreceptor cell bodies from live, flatmount mouse retinas, providing enhanced access to bipolar and horizontal cells for efficient patch clamping and rapid immunolabeling. Split retinas are prepared by sandwiching an isolated mouse retina between two pieces of nitrocellulose, then gently peeling them apart. The separation splits the retina just above the outer plexiform layer to yield two pieces of nitrocellulose, one containing the photoreceptor cell bodies and another containing the remaining inner retina. Unlike vertical retina slices, the split retina preparation does not sever the dendritic processes of inner retinal neurons, allowing for recordings from bipolar and horizontal cells that integrate the contributions of gap junction-coupled networks and wide-field amacrine cells. This work demonstrates the versatility of this preparation for the study of horizontal and bipolar cells in electrophysiology, immunohistochemistry, and in situ hybridization experiments.

This work presents an alternative flatmount retina preparation in which the removal of photoreceptor cell bodies enables faster antibody diffusion and improved patch pipette access to inner retinal neurons for immunohistochemistry, in situ hybridization, and electrophysiology experiments.