Name: horizontal slice preparation of the retina
Uploaded: 2006-11-20T00:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Horizontal Slice Preparation of the Retina at JoVE.com

אנו מודים בני הזוג. Richard H. Masland ו Akimichi Kaneko שנתן לנו עצות ערך והצעות להקים את הליך הכנת הפרוסה האופקי של הרשתית.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן לחסום אגר (30 מ&quot;ג / מ&quot;ל, 3%, כלומר במים מזוקקים). ממיסים במיקרוגל ועל זה קודם ושופכים אותו לתוך צלחת זכוכית. שמור את המנה במקרר ב 4 o C. טיפ: הבלוק אגר אמור להיות מוכן מראש. </li><li style=";text-align:right;direction:rtl"> הכן בטמפרטורה נמוכה ההיתוך agarose ג&#39;ל (25 מ&quot;ג / מ&quot;ל, 2.5% כלומר, בינוני). ממיסים במיקרוגל ועל זה. שמור את זה באמבט מים חמים על 35 ° C. טיפ: שמרו על ג&#39;ל agarose לא חם מדי. אם זה חם מדי, זה לוקח זמן רב להיות הקרושה, ג&#39;ל עשוי להרוג את הרשתית . </li><li style=";text-align:right;direction:rtl"> להרדים את enucleate, חיה גלגל העין ופתח אותו כמו עיינית. כדי לנזל הומור זגוגי, עיינית נפתחה ספוגה במשך 10 דקות ב hyaluronidase במדיום (0.07 מ&quot;ג / מ&quot;ל), אם בהכרח. </li><li style=";text-align:right;direction:rtl"> חותכים את אגר לגוש (למשל, 1 ס&quot;מ X 1.5 ס&quot;מ הרשתית העכבר) ו מחובר היטב על ידי דבק מיידית על הבמה vibratome. שמור את בלוק אגר רטוב. </li><li style=";text-align:right;direction:rtl"> מניחים את להב ב 5 מעלות על מבצעה. בעדינות לחתוך את המשטח העליון של לחסום את אגר במהירות של כ. 50 מיקרומטר / sec, freq. 50 הרץ. טיפ: ודאו כי המשטח העליון של לחסום את אגר חלקה לגמרי שטוח. כמה חתכים (לפחות 3 פעמים) עשוי להיות נחוץ כדי לקבל את משטח חלק ושטוח של גוש אגר. </li><li style=";text-align:right;direction:rtl"> שוטפים את עיינית עם המדיום ללא hyaluronidase. לבודד את הרשתית של אפיתל הפיגמנט ולמקם אותו photoreceptor בצד על נייר הסינון. כדי בתוקף לצרף את הרשתית לנייר סינון, ואקום הרשתית על הנייר מסנן מספר פעמים. טיפ: ודא הרשתית מחובר נייר הסינון שטוח לחלוטין. לאחר לשים הרשתית על הנייר לסנן, לחתוך את נייר הסינון עודף סביב הרשתית (Fig.2A). העיתון לסנן עודף לעתים קרובות מונע להב מללכת לתוך שכבת המתאים של הרשתית. </li><li style=";text-align:right;direction:rtl"> נגב בעדינות את עודף בינוני ברחוב אגר ובזהירות המקום הרשתית על הנייר סינון לחסום את אגר. כדי בתוקף במקום נייר סינון לחסום את אגר, דבק לצרף מיידית על קצה נייר הסינון. עצה: כדי להימנע סדק unexpectable, המקום הרשתית כחזית ככל האפשר על פני השטח העליון של לחסום את אגר. </li><li style=";text-align:right;direction:rtl"> יוצקים בעדינות את הג&#39;ל בטמפרטורה נמוכה ההיתוך agarose על הרשתית. חכו דקות 1 בעדינות לשפוך את המדיום בו כדי לצנן אותה. הג&#39;ל הופך agarose הקרושה במהירות. טיפ: חם מדי agarose ג&#39;ל הורג את הרשתית. </li><li style=";text-align:right;direction:rtl"> הרם את עמדת להב מיקרומטר כמה מאות מעל המשטח העליון של גוש אגר בעדינות לחתוך את החלק העליון של הקרושה agarose ג&#39;ל. </li><li style=";text-align:right;direction:rtl"> תחתון למטה את המיקום להב. חותכים את הרשתית ברמה של הפרוקסימלית שליש שכבת הגרעין הפנימית (בערך בין 200 לבין 250 מיקרומטר מעל המשטח העליון של גוש אגר). טיפ: מהירות רבה מדי של הלהב יפגע קשות ברשתית. כ. 50 מיקרומטר / sec (50 הרץ) המתאים. </li><li style=";text-align:right;direction:rtl"> שימו לב כי תאים amacrine עכשיו עם הפנים כלפי מטה. בזהירות להרים את הפרוסה של הרשתית יחד עם הקרושה agarose ג&#39;ל ולמקם אותו עם הפנים כלפי מעלה בתא או צלחת. טיפ: מכיוון הרשתית העכבר הזעיר וגלי, קשה לקבל פרוסת אופקי מושלמת שכבת המתאים. פרוסת &quot;עקיפה&quot; עשוי להיות שיטה חלופית כדי לקבל את פרוסת שטוח יחסית של הרשתית. כדי לקבל את פרוסת אלכסונית, לשים עוד נייר מסנן בין הרשתית על הנייר לסנן את הבלוק אגר כפי שמוצג Fig.2B. למרות שלא ניתן לצפות פרוסת אופקי מושלם, תמיד יש כמה תחומים שבהם סומה תא amacrine הוא על פני השטח של הפרוסה. </li></ol>

<ol>
	<li>Azuma, T., Enoki, R., Iwamuro, K., Kaneko, A., Koizumi, A., Koizumi, A., TC, J. a. k. o. b. s., Masland, R. H., MA, M. a. c. N. e. i. l., RH, M. a. s. l. a. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+spatiotemporal+patterns+of+dendritic+Ca2++signals+in+goldfish+retinal+amacrine+cells.">Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells.</a> Brain Res. , 64-73 (2004).</li></ol>

כל הפרוטוקולים הניסוי נערכו על פי המכון הלאומי לבריאות הנחיות לשימוש בבעלי חיים.

ישנם מספר יתרונות חשובים של הכנת החתך האופקי של הרשתית. ראשית, מורפולוגיה של תאים דנדריטים להרחיב את אופקי, כגון תאים amacrine תאים אופקיים, נשמר. הוכח כי קיימים מעל 20 סוגים של תאים amacrine ברשתית (מקניל ו Masland, 1998), ולכן חשוב מאוד להשוות את המורפולוגיה של התאים עם הנכס פיזיולוגיים. שנית, ברשתית כל הר, את הסיקור הנרחב של endfeets תא של גליה מונע גישה של pipettes תיקון לתאי amacrine. ב פרוסות אופקיות, סומה של תאים amacrine חשופה אל פני השטח של הפרוסות. שלישית, ריאגנטים כימיים יכול בקלות להגיע אל התאים הממוקדים והדנדריטים שלהם, כי אלה נמצאים על פני השטח של הפרוסות. שטפי-in-ולשטוף מתוך כימיקלים מהירה וקלה. רביעית, הדמיה יון ניאון כגון הדמיה סידן המקובלת היא performable. ב פרוסות אנכי המסורתית או את הרשתית כל הר, אור עירור הדמיה עצמה מפעילה את קולטני האור. בהכנת פרוסה אופקי, photoreceptors ו photopigments יוסרו לחלוטין כך שאין זיהום של עירור אור עורר חפצים. בנוסף, יחס אות לרעש של הדמיה הוא הרבה יותר גבוה, כי פעילות רקע של photoreceptors מסולק. החיסרון הגדול ביותר של הכנת החתך האופקי הוא הקשר האנכי בין photoreceptors ותאי גנגליון הוא חתך כך שאין עיבוד מידע חזותי בתוך הפרוסה. טכניקה זו החלה על הרשתית של כל בעלי חיים סטנדרטיים כגון דגי זהב, העכבר ארנב (Fig.1B). השיטה של ​​פרוסת האופקי עשוי להציע דרך חלופית לחקור את תפקוד הנוירונים המעגלים העצביים ברשתית.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A-1296</td><td></td></tr><tr><td>Agarose L</td><td>Wako Pure Chemical Industries, Ltd.</td><td>317-01182</td><td>Low-temperature melting agarose </td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td></td><td>Type I-S, 300 U/mg</td></tr><tr><td>Medium</td><td></td><td></td><td>in mM: 102 NaCl, 3.1 KCl, 2.0 CaCl&lt;sub&gt;2&lt;/sub&gt;, 1.0 MgCl&lt;sub&gt;2&lt;/sub&gt;, 23 NaHCO&lt;sub&gt;3&lt;/sub&gt;, and 10 glucose, maintained at pH 7.8, or, Ames&amp;rsquo; Solution (Sigma)</td></tr><tr><td>Microwave</td><td></td><td></td><td></td></tr><tr><td>Hot water bath</td><td></td><td></td><td>35 &amp;deg;C</td></tr><tr><td>Vibratome slicer</td><td>Leica Microsystems</td><td>VT1000S</td><td></td></tr><tr><td>MF-Membrane filters</td><td>EMD Millipore</td><td>HAWP01300</td><td>Filter paper , pore size 0.45 &amp;#181;m</td></tr><tr><td>Dissection tools</td><td></td><td></td><td>forceps, blade, pinch, twizzer, &lt;em&gt;etc&lt;/em&gt;.</td></tr><tr><td>Instant glue</td><td></td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; Alonalpha, Krazy Glu</td></tr><tr><td>50 mL Syringe</td><td></td><td></td><td></td></tr><tr><td>Syringe filter</td><td>EMD Millipore</td><td>SX0001300</td><td></td></tr></tbody></table>

horizontal slice preparation of the retina

באופן מסורתי פרוסה האנכי והכנת כל הר של הרשתית שימשו ללימוד פונקציה של מעגלים הרשתית. עם זאת, רבים של נוירונים ברשתית, כגון תאים amacrine, להרחיב הדנדריטים שלהם בצורה אופקית, כך מורפולוגיה של תאים אמור להינזק קשות פרוסות אנכי. לקראת כל הר, במיוחד עבור תיקון-clamp הקלטות, הנוירונים ברשתית בשכבת הביניים אינם נגישים בקלות בשל הסיקור הנרחב של גליה (תאים מולר) endfeets של התא. כאן, אנו מתארים את הרומן חיתוך שיטה לשמר את המורפולוגיה הדנדריטים של נוירונים ברשתית ללא פגע. פרוסה נעשתה אופקית על השכבה הפנימית של הרשתית באמצעות מבצעה vibratome אחרי הרשתית היה מוטבע נמוכה טמפרטורת ההתכה agarose ג&#39;ל. בהכנה זו פרוסה האופקי של הרשתית, למדנו את תפקוד הנוירונים ברשתית בהשוואה המורפולוגיה שלהם, באמצעות תיקון-clamp הקלטה, טכניקת הדמיה סידן, immunocytochemistry, ואת תא בודד RT-PCR.

Watch this Scientific Journal Video about Horizontal Slice Preparation of the Retina at JoVE.com

Horizontal Slice Preparation of the Retina

באופן מסורתי פרוסה האנכי והכנת כל הר של הרשתית שימשו ללימוד פונקציה של מעגלים הרשתית. כאן, אנו מתארים את הרומן חיתוך שיטה לשמר את המורפולוגיה הדנדריטים של נוירונים ברשתית ללא פגע.

פורסים הכנה אופקי של הרשתית

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

So the legendary is here, and this is a picture paper.

horizontal-slice-preparation-of-the-retina

Universidad San Sebastian

Research

JoVE Journal

Biology

15.7K Views.  Dalhousie University. So the legendary is here, and this is a picture paper.

Video: Horizontal Slice Preparation of the Retina

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

אור / חושך מעבר מבחן עכברים

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

הצמד תיקון הקלטות מתא עצב עכבר רשתית בהכנת Dark המותאמות פרוסה

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Neuroscience

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp מדידות קיבול ו Ca 2 + הדמיה על מסופי עצב יחיד ב Slices רשתית

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

השער המוח: בניתוח עיניים הפרימאטים

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

כל תא הקלטות בו זמנית מקולטני אור ועל נוירונים מסדר שני בהכנה פורסים רשתית אמפיבי

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

הקלטת אור עורר תגובות Postsynaptic בנוירונים מותאמים-Dark, תכשירי עכבר רשתית Slice באמצעות טכניקות הצמד התיקון

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

ההדמיה Ca<sup&gt; 2 +</sup&gt; Dynamics בסופי קון קולטי האור אקסון של רשתית העכבר

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

הכנת פרוסות אופקיות של רשתית למבוגרים עכבר עבור אלקטרו מחקרים

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

הכנת פרוסות דג זברה למבוגרים עבור לשעבר Vivo הדמיה ניסויים ברשתית טריים

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

חקר תאים עצביים דו-קוטביים ואופקיים ברשתית המפוצלת של מורין

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.

מחבר זרקור: שימוש בטכניקת הרשתית המפוצלת לגישה משופרת וניסויים מואצים 

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.

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