This work was supported in part by grants from the NIH R01EY012949, R21EY018885, T32 EY0707133. We thank Darwin Hang for his technical assistance.

Animal Use Statement: Animals were cared for in accordance with guidelines described in Guide for the Care and Use of Laboratory Animals, using protocols approved by the University of Minnesota Institutional Animal Care and Use Committee.
1) Preparation of solutions
<ol>
 <li> Before performing electrophysiological recording, intracellular, extracellular, and enzyme solutions need to be prepared.</li>
 <li> Extracellular solution: mix one bottle (8.8g) of powdered Ames' medium (Sigma) with 1 l of H2O and 1.9 g of sodium bicarbonate (23 mM). At all points the extracellular solution is saturated with 95% O2/5% CO2 gas mixture. Transfer 200 mL to a beaker for retina storage. Place the remaining solution in the perfusion system container for gravity perfusion once retina is mounted.</li>
 <li> Intracellular solution: Intracellular solution should have been made previously and then aliquoted into 500-1000 &mu;L aliquots and stored at -20 &deg;C. Intracellular solution varies depending on the experiment needed, for current clamp recordings shown here we use (in mM): 125 K-gluconate, 2 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, 2 Na2-ATP, 0.5 mM NaGTP pH to 7.2 with KOH 5. A fluorescent tracer such as 10 &mu;M Alexafluor-594 hydrazide (Invitrogen) can be added to visualize dendrites during the experiment, and neurobiotin (0.3%) can also be included for analysis of cell anatomy following recording. When ready to record, thaw intracellular solution.</li>
 <li> Enzyme solution: Combine 10000 Units Collagenase (Worthington Chemicals) with 83 mg Hyaluronidase (Worthington Chemicals) into 4.150 mL of extracellular solution. Store in 50 &mu;L aliquots at -20&deg;C. Aliquots can be used for several weeks. When ready to treat the retina, dilute aliquot into 500 &mu;L of bubbled extracellular solution.</li>
 <li> Phosphate Buffered Solution (PBS): In 800 mL of H2O add 8 g of NaCl, 0.2 g of KCl, 1.4 g of Na2HPO4 and 0.24 g of KH2PO4. Bring the pH to 7.4 with NaOH. Adjust the volume to 1 L.</li>
 <li> 0.2 M Phosphate Buffer: In 800 ml of H2O add 21.8 g of Na2HPO4 and 6.4 g of NaH2PO4. Bring volume to 1 L.</li>
 <li> 4 % Paraformaldehyde Solution: Heat 50ml H2O to 60&deg;C. Add 4g paraformaldehyde, stir for several minutes. Add few drops of 1M NaOH (about 5 drops), keep stirring until solution is clear. Add 50 ml 0.2 M phosphate buffer into beaker. Check pH using pH strips (~7.4). Filter and cool. </li>
 <li> Blocking solution (10% goat serum, 0.5% TritonX100): In 1.8 ml of PBS add 0.2 ml of goat serum and 10 &mu;L Triton X-100. TritonX-100 serves to permeabilise the tissue.</li>
 <li> Streptavidin solution (5% goat serum, 0.5% TritonX100, 1:500 Streptavidin 594): In 1.9 ml of PBS add 0.1 ml of goat serum, 10 &mu;L Triton X-100 and 4 &mu;L of Streptavidin 594.</li>
</ol>
2) Isolation of retina from mouse
Dissection tools needed: 1 #2 Forceps, 2 #5 Forceps, ophthalmologic scissors
<ol>
 <li> Euthanize mouse according to approved protocols, in this case CO2 asphyxiation and enucleate eyeball by gently inserting an angled #2 forceps behind the eye and lifting out the eyeball.</li>
 <li> Place the eyeball into a 35 mm Petri dish with extracellular solution. Cut the optic nerve and any attached extraocular muscles or connective tissue using ophthalmologic scissors. Cut away the cornea with an ophthalmologic scissor and pull out the lens using #5 forceps. Tear the sclera using a pair of #5 forceps and use the forceps to sever the optic nerve where the retina and sclera meet at the optic disk. Gently peel the retina away from the eyecup. Tip: Do each step on both retinas before moving to next step to minimize time.</li>
 <li> Use forceps to remove vitreous attached to the retina. This can be performed by "grabbing" the transparent vitreous above the retina with forceps. The vitreous, if successfully removed, should be visible as a clear, gelatinous substance on the forceps. Tip: Slicing retinas in half following removal of vitreous will maximize the amount of usable tissue and make mounting in the chamber easier.</li>
 <li> Keep the retinas in oxygenated extracellular solution at room temperature in a dark environment with minimal ambient light until they are ready to be digested. Retinas can be stored in this way for ~6 hours.</li>
</ol>
3) Treatment of retina with enzyme mixture and mounting in recording chamber
<ol>
 <li> Dilute an aliquot of collagenase/hyaluronidase in 500 &mu;L extracellular solution, place solution in a 35 mm Petri dish and transfer the retina.</li>
 <li> Keep the retina-containing Petri dish, covered and in darkness, in a 95% O2/5% CO2 environment for 15 min with gentle agitation. This step aids in digesting any remaining vitreous and allows for easier access to the ganglion cell layer. Tip: for retinas from younger animals with less vitreous or if adverse effects are observed, time can be shortened to ~5 minutes.</li>
 <li> Remove retina from enzyme-containing medium and wash extensively in extracellular solution</li>
 <li> Transfer the retina into recording chamber 6 with the photoreceptor layer facing down. Wick away remaining fluid with either a pipette or tissue and roll out sides of retina to flatten tissue being careful to touch only the edges of the retina, particularly avoiding contact with the ganglion cell layer. Place a platinum ring with nylon mesh over retina to anchor the tissue and immediately fill the chamber with extracellular solution.</li>
 <li> Mount the recording chamber onto the microscope stage, attach tube for gravity perfusion and vacuum suction and attach ground wire.</li>
</ol>
4) Localization of EGFP expressing ganglion cells and light stimulation
<ol>
 <li> Perfuse the retina by gravity with extracellular solution at 1 mL/min or faster rates. Tip: If perfusion rate is too slow, the tissue will not be sufficiently oxygenated and synaptic signaling may be disrupted.</li>
 <li> Fill a recording pipette with intracellular solution and insert it into the pipette holder. Tip: Pipettes with resistances in the range of 3 to 5M&Omega; give the best results.</li>
 <li> Visualize the retina under infrared differential interference contrast (DIC) optics at low magnification (10X objective, NA 0.3) to localize the recording pipette. Infrared light is used to minimize retina exposure to visible light and minimize light adaptation. Move to higher magnification (40X objective, NA 0.8) and bring electrode into view just above tissue.</li>
 <li> At high magnification (40X objective), illuminate the retina with epifluorescence (480 nm for EGFP) and select a fluorescent ganglion cell and mark on computer monitor using tape (Figure 1A).</li>
 <li> Under DIC and infrared illumination, position the recording electrode near the targeted ganglion cell (Figure 1A). Clean area immediately covering cell by applying gentle positive pressure to recording electrode via a 12cc plastic syringe connected to the pipette holder with polyethylene tubing. On the amplifier, zero any voltage offsets. Place electrode onto selected ganglion cell until a dimple appears on cell surface.</li>
 <li> With the amplifier in voltage clamp mode, apply a test pulse and monitor the current passing through the pipette. Apply negative pressure to create seal between electrode and cell. Release negative pressure when cell begins to seal and wait until holding current is indicative of a G&Omega; electrical seal between the cell's membrane and pipette. Apply gentle pulses of negative pressure until capacitive transients appear and then release any application of pressure. Tip: if access (series) resistance is high (>30M&Omega;), this can often be ameliorated by additional, very gentle, pulses of negative pressure.</li>
 <li> Once a stable seal has been reached apply a pulse of negative pressure. After whole cell mode is obtained, allow cell to dark adapt for 5 minutes. Record, in current clamp mode, response to full-field, white light stimulation (Figure 1B).</li>
 <li> Following recording, Alexafluor-594 will have diffused to the dendrites. The tracer can be visualized under epifluorescence at 594 nm and used to determine whether a ganglion cell is On-, Off-, or Bi-stratified by switching between epifluorescence and DIC infrared optics 5.</li>
 <li> If neurobiotin is included in pipette and retina is going to be processed for immunohistochemistry, then pipette must be gently retracted to provide minimal disruption of the membrane.</li>
</ol>
5) Analysis of anatomy of recorded ganglion cells
Filling with neurobiotin allows for more detailed morphological analysis of ganglion cells following recording. Tip: If specific correlations between a recorded ganglion cell and detailed morphology is desired, it may be best to record and fill only one RGC per preparation.
<ol>
 <li> If neurobiotin was included in the intracellular solution then place the retina in paraformaldehyde solution and fix overnight at 4&deg;C.</li>
 <li> Rinse in PBS, at least 2 hours, at least 6 changes. Transfer the retina to a plate containing blocking solution. Leave in blocking solution for 2 hours, room temperature on shaker.</li>
 <li> Replace the blocking solution with the Streptavidin solution. Incubate 2 days at 4&deg;C on shaker. Rinse the retina in PBS, 6 rinses, 20 min each.</li>
 <li> Mount the retina with Vectashield in a glass slide. The retina must be placed with the ganglion cells up. Perform confocal microscopy (Figure 1C).</li>
</ol>
Representative Results
If the retina is healthy and the dissection was successful, individual ganglion cells should be visible under DIC at high magnification (Figure 1A). Unhealthy retinas will have large, granulated nuclei and should be discarded. If the synaptic response of a given ganglion cell is intact, then the cell should respond at light onset or offset with action potentials and, in the case of an intrinsically-photosensitive ganglion cell (Figure 1B) , a sustained depolarization.
<img src="/files/ftp_upload/2367/2367fig1.jpg" alt="Figure 1" /> 
 Figure 1. Localization, recording, and staining of a fluorescently labeled retinal ganglion cell. (A) GFP positive RGC visualized under epifluorescent illumination at 40X magnification (left panel) and under DIC illumination (right panel). (B) Synaptic light response of an M2 intrinsically photosensitive retinal ganglion cell recorded in whole-cell current clamp mode from fluorescently labeled RGC. (C) M1 intrinsically photosensitive retinal ganglion cell that was identified under epifluorescence, targeted for whole-cell recording, filled with neurobiotin, and then processed for immunohistochemistry.

<ol>
	<li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li><li>Schmidt, T. M., Taniguchi, K., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+extrinsic+light+responses+in+melanopsin-expressing+ganglion+cells+during+mouse+development.">Intrinsic and extrinsic light responses in melanopsin-expressing ganglion cells during mouse development.</a> J Neurophysiol. 100, 371-384 (2008).</li><li>Huberman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+identification+of+an+On-Off+direction-selective+retinal+ganglion+cell+subtype+reveals+a+layer-specific+subcortical+map+of+posterior+motion.">Genetic identification of an On-Off direction-selective retinal ganglion cell subtype reveals a layer-specific subcortical map of posterior motion.</a> Neuron. 62, 327-334 (2009).</li><li>Siegert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+address+book+for+retinal+cell+types.">Genetic address book for retinal cell types.</a> Nat Neurosci. 12, 1197-1204 (2009).</li><li>Schmidt, T. M., Kofuji, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+differences+among+intrinsically+photosensitive+retinal+ganglion+cells.">Functional and morphological differences among intrinsically photosensitive retinal ganglion cells.</a> J Neurosci. 29, 476-482 (2009).</li><li>Newman, E. A., Bartosch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+eyecup+preparation+for+the+rat+and+mouse.">An eyecup preparation for the rat and mouse.</a> J Neurosci Methods. 93, 169-175 (1999).</li><li>Haverkamp, S., Inta, D., Monyer, H., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+analysis+of+green+fluorescent+protein+in+retinal+neurons+of+four+transgenic+mouse+lines.">Expression analysis of green fluorescent protein in retinal neurons of four transgenic mouse lines.</a> Neuroscience. 160, 126-139 (2009).</li><li>Zhang, D. Q., Zhou, T. R., McMahon, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+retinal+dopaminergic+neurons+underlying+their+multiple+roles+in+vision.">Functional heterogeneity of retinal dopaminergic neurons underlying their multiple roles in vision.</a> J Neurosci. 27, 692-699 (2007).</li><li>Hu, E. H., Dacheux, R. F., Bloomfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flattened+retina-eyecup+preparation+suitable+for+electrophysiological+studies+of+neurons+visualized+with+trans-scleral+infrared+illumination.">A flattened retina-eyecup preparation suitable for electrophysiological studies of neurons visualized with trans-scleral infrared illumination.</a> J Neurosci Methods. 103, 209-216 (2000).</li><li>Wu, S. M., Gao, F., Pang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+circuitry+mediating+light-evoked+signals+in+dark-adapted+mouse+retina.">Synaptic circuitry mediating light-evoked signals in dark-adapted mouse retina.</a> Vision Res. 44, 3277-3288 (2004).</li></ol>

This technique is applicable to any retinal ganglion cell recordings from an isolated retina. Though in the mouse line used above intrinsically photosensitive, melanopsin-expressing RGCs are labeled with EGFP2, 5, this protocol is readily transferable to other fluorescently labeled RGC lines or can be generalized to record from randomly selected RGCs as well. This technique is particularly valuable because the dendritic arbor of each RGC and its respective input neurons are left entirely intact. Recordings ca...

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<td>Ames&#x2019; Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase</td>
<td>&nbsp;</td>
<td>Whorthington Biochemical</td>
<td>LS005273</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Whorthington Biochemical</td>
<td>LS002592</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5761</td>
<td>&nbsp;</td>
<tr>
<td>AlexaFluor 594 Hydrazyde</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A10442</td>
<td>&nbsp;</td>
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<td>Neurobiotin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>SP1120</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>&nbsp;</td>
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<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>&nbsp;</td>
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<td>Ophthalmologic Scissor</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
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<td>Streptavidin 594</td>
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<td>Invitrogen</td>
<td>S32356</td>
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<td>Vectashield</td>
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<td>005-000-001</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>T8787</td>
<td>&nbsp;</td>
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<td>Paraformaldehyde</td>
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<td>Electron Microscopy Sciences</td>
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an isolated retinal preparation to record light response from genetically labeled retinal ganglion cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

Watch this Scientific Journal Video about An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells at JoVE.com

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

an-isolated-retinal-preparation-to-record-light-response-from

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Neuroscience

16.7K ビュー.  University of Minnesota. この記事では、細かく分析し、マウスで分離された網膜の準備から記録する方法の説明を提供します。特に、我々は、蛍光標識神経節細胞集団からの光の応答を記録し、続いてその形態を特定し分析する方法について説明します。

ビデオ: An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

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.

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

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.

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

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...