Name: optical coherence tomography: imaging mouse retinal ganglion cells in vivo
Uploaded: 2017-09-22T13:00:00
Description: Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

This work was supported by Inserm, Universit&#233; Montpellier, Retina France, Union National des Aveugles et D&#233;ficients Visuels (UNADEV), Association Syndrome de Wolfram, Fondation pour la Recherche M&#233;dicale, Fondation de France, and the Laboratory of Excellence EpiGenMed program.

The experimental protocol was approved by the Institut national de la sant&#233; et de la recherche m&#233;dicale (Inserm; Montpellier, France), is consistent with the European directives, and complies with the ARVO Statement for the Use of Animals in Ophthalmic Research. It was carried out under the agreement of the Languedoc Roussillon Comity of Ethics in Animal Experimentation (CEEALR; nuCEEA-LR-12123).
1. Equipment Setup and Pre-imaging Preparation
NOTE: Here, OCT...

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	<li>Drexler, W., Fujimoto, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-of-the-art+retinal+optical+coherence+tomography.">State-of-the-art retinal optical coherence tomography.</a> Prog Retin Eye Res. 27 (1), 45-88 (2008).</li><li>Grenier, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WFS1+in+Optic+Neuropathies:+Mutation+Findings+in+Nonsyndromic+Optic+Atrophy+and+Assessment+of+Clinical+Severity.">WFS1 in Optic Neuropathies: Mutation Findings in Nonsyndromic Optic Atrophy and Assessment of Clinical Severity.</a> Ophthalmology. 123 (9), 1989-1998 (2016).</li><li>Zmyslowska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+thinning+as+a+marker+of+disease+progression+in+patients+with+Wolfram+syndrome.">Retinal thinning as a marker of disease progression in patients with Wolfram syndrome.</a> Diabetes Care. 38 (3), e36-e37 (2015).</li><li>Fischer, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive,+in+vivo+assessment+of+mouse+retinal+structure+using+optical+coherence+tomography.">Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.</a> PLoS One. 4 (10), e7507 (2009).</li><li>Grieve, K., Thouvenin, O., Sengupta, A., Borderie, V. M., Paques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+the+Retina+With+Full-Field+Optical+Coherence+Tomography.">Appearance of the Retina With Full-Field Optical Coherence Tomography.</a> Invest Ophthalmol Vis Sci. 57 (9), OCT96-OCT104 (2016).</li><li>Chang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42 (4), 517-525 (2002).</li><li>Mustafa, S., Pandit, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+diagnosis+and+management+of+optic+neuropathy.">Approach to diagnosis and management of optic neuropathy.</a> Neurol India. 62 (6), 599-605 (2014).</li><li>Sarzi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+OPA1delTTAG+mutation+induces+premature+age-related+systemic+neurodegeneration+in+mouse.">The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.</a> Brain. 135 (Pt 12), 3599-3613 (2012).</li><li>Sarzi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+steroidogenesis+promotes+early-onset+and+severe+vision+loss+in+females+with+OPA1+dominant+optic+atrophy.">Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.</a> Hum Mol Genet. 25 (12), 2539-2551 (2016).</li><li>Delettre-Cribaillet, C., Hamel, C. P., Lenaers, G., Pagon, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Atrophy+Type+1.">Optic Atrophy Type 1.</a> Gene Reviews. 7 (2007), (2007).</li><li>Lenaers, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+optic+atrophy.">Dominant optic atrophy.</a> Orphanet J Rare Dis. 7 (46), (2012).</li><li>Delettre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+gene+OPA1,+encoding+a+mitochondrial+dynamin-related+protein,+is+mutated+in+dominant+optic+atrophy.">Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy.</a> Nat Genet. 26 (2), 207-210 (2000).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+retinal+morphologic+and+functional+changes+in+mice+following+optic+nerve+crush.">Monitoring retinal morphologic and functional changes in mice following optic nerve crush.</a> Invest Ophthalmol Vis Sci. 55 (6), 3766-3774 (2014).</li></ol>

The OCT system, a non-invasive in vivo imaging method, provides high-resolution retinal cross-section-like scans. Thus, its main advantage is its potential for detailed analysis, with the wonderful opportunity to transpose protocols routinely applied to humans to mouse models.
In the example of Opa1delTTAG mutant mice, SD-OCT results showed an increase of RNFL and GC complex layer thickness, which allowed for further exploration of DOA pathophysiology<sup class="xr...

OCT is a diagnostic tool that facilitates the examination of retinal structures1, including the optic nerve head (ONH). Over the years it has become a dependable indicator of disease progression in humans2,3, as well as in rodents4,5. It uses interferometry to create cross-sectional images of retinal layers at a 2-&#181;m axial resolution. The innermost layer is the retinal nerve fiber layer (RNFL), containing RGC axons, which is followed by the ganglion cell layer (GCL), containing mostly RGC bodies. Next is the inner plexiform layer (IPL), where RGC dendrites meet bipolar, horizontal, and amacrine cell axons. These, together with horizontal cells, form the inner nuclear layer (INL), and their protrusions connect with photoreceptor axons in the outer plexiform layer (OPL). This is followed by the outer nuclear layer (ONL), with photoreceptor cell bodies, and is separated from the photoreceptor layer by the outer limiting membrane (OLM), also called the inner segment/outer segment (IS/OS) layer. Finally, the last observable layers in the mouse retina are the retinal pigment epithelium (RPE) and the choroid (C). The RNFL alone is normally too thin to be measured in mice; thus, analyzing the RNFL/GCL instead is preferable4,5. Another possibility is the GC complex layer, which contains the latter in addition to the IPL, making it thicker and thus even easier to measure on OCT scans4. Consequently, OCT can provide insight into the pathological status of the retina, such as in OPNs. 
Alternatively, the thickness of the mouse retina is often analyzed with post mortem histology. However, this technique faces limitations relating to tissue collection, fixation, cutting, staining, mounting, etc. Hence, some defects, such as subtle thickness changes, cannot be detected. Finally, because the same mouse cannot be tested at several time points, the number of animals per study greatly increases, unlike for OCT. All in all, the non-invasiveness, high-resolution, possibility for repetition, time monitoring in time, and ease of use of the OCT technology make it the method of choice in retinal disease studies.
Mouse models are used to identify gene defects and to elucidate molecular mechanisms underlying retinopathies6. OPN is a form of retinopathy with substantial damage to the optic nerve (ON), which is made up of approximately 1.2 million RGC axons. OPN can be focused on the ON or can be secondary to other disorders, inborn or not7, leading to visual field loss and later, blindness. Characteristic traits of OPN are RGC loss and ON damage, which can be observed in human OCT as RNFL and GCL thinning2,3. Meanwhile, the pathophysiology of OPN is still poorly understood, and hence the need to test mouse retinas remains. 
This manuscript describes the imaging and quantification of retinal layer thickness, using the example of the Opa1delTTAG mouse line8,9, a model of dominant optic atrophy (DOA)10. To assess RGC pathophysiology, radial, rectangular, and annular scans were quantified. This was done either with standard calipers provided by the OCT software or with a homemade macro developed for an open-source image processing program. The standard calipers are difficult to manipulate and often thicker than the RNFL/GCL, while the homemade calipers are easy to use, reproducible, and more precise. The macro performs a measurement for an automatically detected layer, in 5 points and at fixed positions, on both sides of the ONH in the peripapillary region. The goal of the presented protocol is to describe OCT scan acquisition to specify retinal positioning, with a focus on RGCs.

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			<td style="width:324px;">Institute for Neurosciences in Montpellier, INSERM UMR 1051, France</td>
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			<td>Opa1 knock-in mice carrying&#160; OPA1 c.2708_2711delTTAG mutation on C57Bl6/J background</td>
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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.

The SD-OCT technology enables retinal imagining and thickness analysis that is comparable to histology, but is faster and more detailed (Figure 3). As presented with wildtype C57Bl/6 mice, even though the quality of an SD-OCT scan (Figure 3A, right) is not as good as that of an image of a retinal cross-section (Figure 3A, left), it visualizes more layers (e.g., OLM). Moreover, it takes only ...

Watch this Scientific Journal Video about Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo at JoVE.com

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.

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 ...

The overall goal of this experiment is to use optical coherence tomography or OCT to quickly obtain an accurate assessment of retinal thickness. OCT can help answer key questions in the field of ophthalmology such as, optic neuropathy. The main advantage of this technique is that, it is a non-invasive method for detecting structural changes in the mouse retinal ganglion cells.This technique is really interesting because it is non-invasive, safe and reputable method to test the effect of treatment in the optic neuropathy. To begin, first adjust the reference arm settings appropriate for the lens. Next, turn on the power supply and the computer.When the computer is ready, launch the imaging program. Create a new clinical study and add desired protocols. Go to patient exam section and set exam defaults.Click, add patient in the patient exam section to add a new patient and enter the relevant patient information. Then click, add exam, start exam and add preset scans from the list to select the desired protocol starting with the eye that will be measured first. At least 15 minutes before the data acquisition, instill drops of room temperature 10%phenylephrine into each eye of all the mice that will be tested.Then remove the excess reagent and instill drops of room temperature 0.5%tropicamide. Approximately five minutes before the acquisition, induce general anesthesia. Immediately after, administer 0.4%oxybuprocaine hydrochloride to each eye, leaving the solution in place for three seconds to anesthetize and immobilize the eyes.Then, remove the drops and repeat the 10%phenylephrine and 0.5%tropicamide installation. When the eyes are sufficiently dilated for the imaging, lubricate the eyes with viscous glycol based eye drops to provide corneal hydration and wrap the mouse in a sheet of surgical gauze to keep it warm. Using a sponge or cotton wick, apply a thin layer of ophthalmic gel supplemented with 0.3%hypromellose onto each eye.While doing so, set the eyelashes and whiskers aside. Position the mouse in the cassette with the head straight and pointing forward and carefully place the bite bar in its mouth. Place a cotton roll under the side of the animal for the eye that is being examined.Then, keeping the clip on aiming tip on the mouse specific lens, rotate the z translator screw counter clockwise to bring the lens towards the eye. Preset the position of the mouse by rotating and swiveling the cassette and turning the bite bar and the x-translator screws until the experimental eye is positioned to look directly into the lens. When the optical axis of the eyes and the lens are aligned, select the first scan in the exam.Click start aiming and use the z-translator screw to move the retina vertically in the left panel and horizontally in the right panel. By rotating the cassette, move the optic nerve head up or down to bring it into the middle of the right panel. Use the bite bar screw to straighten the retina within the right panel and swivel the cassette to position the optic nerve head in the middle of the left panel.Then use the x-translator screw to level the retina within the left panel and keeping in mind the major function of each modulator, further adjust the position of the retina for the perfect centralization on the optic nerve head. When the retina is in position, click start snapshot to begin the spectral domain optical coherence tomography scanning. Do not forget to save the scan and the report.Adjust the centralization if necessary. When the scanning is complete, retract the lens and position the second eye as just demonstrated. After the second eye has been scanned, remove the mouse from the cassette, apply ophthalmic gel with 0.3%hypromellose to each eye and place the mouse on a heating plate with monitoring until full recovery.To focus on retinal ganglion cells, use the MRI retina tool macro created for image J.Open the MRI retina tool and modify polygon selection tool and load the image of interest. Click M to begin the measurement, then click E to adjust the horizontal position. Select the first cassette in the newly opened region of interest manager window.Click on the blue polygon and click on the borders of the measured layer in the picture to adjust the position of the first cassette. Select the second cassette in the region of interest manager window and adjust the second cassette. Then click R, to re-measure the retina and view the results in the measurements window.Even though the quality of a spectral domain OCT scan is not as good as that of an image of a retinal cross section, the OCT allows more layers to be visualized. The retina layers are easily measured in an OCT scan and encompass the retinal nerve fiber in ganglion cell layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner and outer segment layers retina pigment epithelium and the choroid, allowing a complex study of the entire retina. OCT measurement of retinal thickness is just demonstrated, indicates that visually impaired genetically mutant OPA1 female mice exhibit a progressive thickening of the ganglion cell complex and the peripapillary retina nerve fiber layers.A comparison of the retina nerve fiber in ganglion cell layer thickness using standard and homemade calipers, demonstrates that standard calipers measure a significantly lower thickness than do homemade calipers, as the standard calipers are much thicker and more difficult to place on a thin layer border. Once mastered, this technique can be done in 15 minutes if performed correctly. After its development, OCT paved the way for the researchers in the field of ophthalmology to analyze structural changes in the mouse retina.After watching this video, you should have a good understanding of how to perform and analyze OCT imaging for retina ganglion cells in mice.

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Research

JoVE Journal

Neuroscience

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.

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 ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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 ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.