We thank Dr. S. Stella and Helen Vuong for contributions to the calcium imaging protocol. We thank Dr. K. Sheets for filming the interview scenes. We thank Dr. Arlene Hirano for her comments on the manuscript. This research and development project was conducted by the authors at the David Geffen School of Medicine at UCLA and is made possible by a contract agreement that was awarded and administered by the U.S. Army Medical Research &#38; Materiel Command (USAMRMC) and the Telemedicine &#38; Advanced Technology Research Center (TATRC), at Fort Detrick, MD under Contract Number: W81XWH-10-2-0077. Support for these studies also came from NIH EY04067 and a VA Merit Review (NB). NCB is a VA Career Research Scientist.

All experiments were carried out in accordance with the guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the University of California-Los Angeles (UCLA) Animal Research Committee. Male and female adult Sprague-Dawley rats (Charles River Lab, Wilmington, MA) between the age of 3-5 weeks were used.
1. Animal and Tissue Preparation for Wholemount Retina and Immunohistochemistry Protocol
<ol>
	<li>Prepare the following materials and tools: A dissecting microscope, 2 forceps with very fine tips, scissors, cellulose filter paper, plastic pipette and a microscope slide.</li>
	<li>Euthanize the rat in a closed chamber by deeply anesthetizing the animal with 1-3% isoflurane followed by decapitation with a guillotine. Remove the eyes with a pair of iris scissors and place in a Petri dish containing the extracellular solution. Hibernate A, Ames Medium or physiological solutions are recommended.</li>
	<li>[Optional step if backfilling of RGCs with Fluo-4 is desired]. Perform the Fluo-4 labeling as described in the steps 2.3-2.4.</li>
	<li>Using the dissecting microscope (light intensity: 1.6 x 108 photons/mm2&#8901;sec), remove the cornea by cutting off the front of the eye ball. Remove the lens and vitreous from the inner retinal surface with forceps. To remove the vitreous, stabilize the eyeball by holding the sclera firmly with one forcep. Gently peel the vitreous base toward the center of the retina with the other forcep. The preparation at this stage is called the eyecup, which is used for cryosections. More details about cryosections can be found in references 14,15.</li>
	<li>Remove the retina from the eyecup. Then, make four cuts to allow the retina to lay flat. Gently mount the retina on a microscope slide with the photoreceptor layer up. Add one drop of solution to the retina and put cellulose filter paper on the retina. Once the retina attaches to the filter paper, place the retina back in solution. If needed, flatten the retina with a brush or forceps.</li>
	<li>Place the wholemounted retinas into 4% PFA for 10-15 min for fixation.</li>
	<li>Wash the wholemounted retinas three times for 30 min in 0.1 M phosphate buffer (PB), pH 7.4. Block the retinas with 5% normal goat serum or donkey serum in 0.1 PB containing 0.3% Triton-X 100 and 0.1% NaN3 at 4 &#176;C overnight. We recommend a final volume of 500 &#956;l for all the steps to save blocking solutions and antibodies.</li>
	<li>Next day, incubate the retinae wholemounts with the primary antibody 5-7 days at 4 &#176;C. Optimal dilutions must be determined by the user.</li>
	<li>Wash the retinas 3 x 30 min in PB 0.1 M and incubate overnight at 4 &#176;C in the corresponding fluorophore-conjugated secondary antibody that is directed against the species of the primary antibody (concentration 1:1,000).</li>
	<li>After three final washes of 30 min each with PB, mount the retinas in mounting medium. Seal the coverslips with nail polish for prolonged storage. Store samples at 4 &#176;C and protect from light.</li>
</ol>
2. Labeling of Ganglion Cells and Axons in Rat Wholemount Retinas with a Calcium Indicator Dye
<ol>
	<li>Prepare 1,000 ml of mammalian Ringer&#39;s containing (in mM) 125 NaCl, 3 KCl, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 25 NaHCO3, and 10 glucose bubbled with 95% O2/ 5% CO&#173;2.</li>
	<li>Perform the dissection as described in the steps 1.2-1.4.</li>
	<li>To load retinal ganglion cells (RGCs) and their axons with a calcium indicator dye, inject 0.5 &#956;l of Fluo-4 pentapotassium salt (40 mM stock in H&#173;2O) into the optic nerve stump approximately 1 mm posterior to the eyeball using a syringe. To measure dynamic increases in ganglion cell [Ca2+]i an indicator with high affinity, such as Fluo-4 with its Kd of 345 nM, is optimal. Fluo-4 offers the additional benefit of having a very high quantum efficiency resulting in an emission increase of &#62;100 fold when bound to calcium.</li>
	<li>Place the eyecup in mammalian Ringer&#39;s bubbled with 95% O2/ 5% CO&#173;2 for 1 hr at room temperature in the dark.</li>
</ol>
3. Calcium Imaging Protocol for Retinal Ganglion Cells and Axons.
<ol>
	<li>Remove the eye, lens and vitreous as described in steps 1.1-1.4. Isolate the retina from the eyecup and divide into quadrants. Mount one quadrant ganglion cell side up on a glass slide and use a harp slice grid to stabilize it. Keep the other pieces dark-adapted in mammalian Ringer&#39;s bubbled with 95% O2/ 5% CO&#173;2 on ice for later use.</li>
	<li>Superfuse the recording chamber with mammalian Ringer&#39;s solution and keep the solution bubbling continuously with 95% O2/5% CO2.</li>
	<li>Image the tissue under the microscope. Conduct experiments at room temperature (~23 &#176;C) or achieve physiological temperatures by warming the stage, heating the water bath under the specimen or applying warm air to the surface of the specimen. 
	Note: It is important to note that many cellular functions are regulated by temperature, which plays a key role in maintaining intracellular homeostasis16. However, introduction of warming measures may increase the rate of photobleaching, evaporation of the medium and focus drift caused by thermal expansion17. Use of room temperature decreases intracellular compartmentalization of Fluo-418.</li>
	<li>Perform a control of two paired high K+ pulses in the absence of drugs. Depolarize RGCs and RGC axons by raising the [K+]o from 3 mM to 60 mM for 33 sec during each pulse to activate VGCCs with an eight channel gravity driven superfusion system. Reduce Na+ by 57 mM to maintain isosmolarity in the elevated K+ solution.</li>
	<li>Assess the contribution of different VGCCs to the calcium signal with the use of general or specific Ca channel blockers. For example, the reduction in calcium signal following the administration of a non-specific calcium channel blocker, cobalt, provided a semi-quantitative measure of the contribution of the VGCCs to the high K+-evoked calcium signal.</li>
	<li>Acquire fluorescence intensity values by placing a region of interest (ROI) around the RGCs of interest (Figure 2).</li>
	<li>Normalize the change in fluorescence intensity produced by the second high K+ peak to the change produced by the first high K+ peak. Then, for testing of specific calcium channel blockers, apply drugs only during the second high K+ pulse of a pair and normalize this peak against the first peak value. To measure the affect of the drug on the high K+ peak, divide the amplitude of the second K+ peak by that of the first. Analysis should be performed on these values in respect to their matching controls derived from paired high K+ peaks measured in the absence of drug.</li>
	<li>Acquire images at 5 sec intervals. To avoid photo-bleaching, keep the laser excitation as low as possible. Provide excitation by the 488 nm line of the argon laser, while the photomultiplier tube collects the fluorescent emission through a 505 nm LP filter.</li>
</ol>

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The authors have no disclosures.

In this article, we have described two different techniques: 1) Immunohistochemistry to show Fluo-4 localization to the ganglion cells in retinal wholemounts, and 2) Calcium imaging to analyze calcium dynamics in retinal ganglion cells and their axons.
Immunohistochemistry using wholemounts has been used to reveal the localization of proteins in the rodent retina20-23. However, there are a few limitations. The quality of the staining relies heavily on the ability of the antibody to ...

Retinal ganglion cells (RGCs) express L-, N-, P/Q- and T-type VGCC as determined through pharmacological blockade of these components of the whole cell Ca channel current1,2. VGCC are transmembrane multimeric proteins that are involved in transmitter release, gene transcription, cell regulation and synaptic plasticity3,4,5. Functional VGCCs are made up of at least three distinct classes of subunits: large, transmembrane &#945;&#173;1 pore forming subunits that establish the channel&#8217;s biophysical and pharmacological properties, primarily extracellular auxiliary &#945;&#173;2&#948; subunits and intracellular &#946; subunits. The latter two form heteromeric complexes with different &#945;&#173;1 subunits and alter the gating kinetics and trafficking of the channels to the plasma membrane6.
Over recent decades, many techniques have been employed to study protein expression, such as immunohistochemistry, enzyme-linked immunosorbent assay, western analysis and flow cytometry. These techniques require the use of specific antibodies for the detection of a given protein of interest and provide powerful tools for the localization and distribution of specific proteins in different tissues. Techniques used to detect and quantify mRNA expression levels of a particular protein such as northern blot analysis, RT-PCR, real-time quantitative RT-PCR, in situ hybridization, cDNA microarrays and ribonuclease protection assay provide an alternative approach when antibodies are not readily available or if expression levels of a particular protein are low7. However, one limitation to the use of such molecular techniques is the required identification of the gene sequence.
To localize proteins in the retina, immunohistochemistry can be performed on wholemount retinas. Due to the accessibility of the RGCs, the wholemount preparation provides an excellent platform to study the localization of specific proteins to the RGC somata and their axons.
In addition to their localization, some functional properties of the VGCCs in RGCs can be demonstrated through the use of calcium imaging techniques. We describe a calcium imaging protocol to selectively label RGCs with a calcium indicator dye to measure intracellular calcium dynamics. The contribution of different VGCCs to the calcium signal in different cellular compartments can be isolated with the use of subtype-specific Ca channel blockers.
Perhaps one of the most beneficial aspects of the calcium imaging technique described here is the ability to simultaneously and independently record from multiple RGCs and their axons. Although many physiological techniques, such as whole cell patch clamp recording, provide high temporal resolution recordings of membrane currents, the somatic or axonal source of these currents recorded cannot be discriminated and recordings can only be made from a single neuron at a time. Multielectrode arrays (MEAs) are capable of simultaneously recording spikes from many cells, but can neither detect nor discriminate the activation of, for example, different subtypes of calcium channels.&#160; MEAs preferentially record from cells that are located in close proximity to a given electrode8 and record from cells that generate large spikes9. Optical imaging methods provide an alternative strategy to enable the simultaneous and independent recordings of whole populations of cells that can be integrated with the information obtained from single cell microelectrode and patch clamp recording and MEA recording.&#160; Although the calcium imaging techniques described here were employed to study the calcium dynamics of RGCs, patch clamp and MEAs can also be used in parallel to further elucidate the ionic currents and spiking properties of RGCs.
Since displaced amacrine cells make up approximately 60% of the neuronal population in the ganglion cell layer in the mouse retina10, our goal was to use a loading technique that selectively labels RGCs with a synthetic calcium indicator dye in a wholemount preparation. Although synthetic calcium indicator dyes provide an excellent platform for the study of intracellular calcium dynamics, its widespread use has been hindered by the inability to effectively load specific populations of neurons within a given network.&#160; Many techniques such as bulk loading11 and electroporation8,12 have been performed to load entire populations of cells, however, such techniques do not discriminate between specific cell types. Genetically encoded calcium indicators provide the ability to selectively label specific populations of cells, however, such methods require the generation of transgenic animals13. Our technique describes a method to selectively label RGCs in the wholemount preparation via optic nerve stump injection of a calcium indicator dye.
Taken together, the structural and physiological techniques outlined in this article provide a platform to study the localization and contribution of the VGCCs to the calcium signal in RGCs and their axons.&#160;

<table><tbody><tr><td>Straight scissors</td><td>WPI</td><td>14124-G</td><td>dissecting tools</td></tr><tr><td>Straight Forceps</td><td>WPI</td><td>501985</td><td>dissecting tools</td></tr><tr><td>curved iris scissors</td><td>WPI</td><td>504487</td><td>dissecting tools</td></tr><tr><td>Cellulose filter paper</td><td>Millipore</td><td>HABP04700</td><td></td></tr><tr><td>Hibernate A</td><td>Invitrogen</td><td>&lt;a href=&quot;http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;amp;productID=A1247501&quot;&gt;A12475-01&lt;/a&gt;</td><td>Media</td></tr><tr><td>Vectashield</td><td>Vector</td><td>H-1000</td><td>&lt;a href=&quot;https://www.google.com/url?sa=t&amp;amp;rct=j&amp;amp;q=&amp;amp;esrc=s&amp;amp;source=web&amp;amp;cd=1&amp;amp;cad=rja&amp;amp;sqi=2&amp;amp;ved=0CC0QFjAA&amp;amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;amp;bvm=bv.48705608,d.cGE&quot;&gt;Mounting Media for Fluorescence&lt;/a&gt;</td></tr><tr><td>Fluo-4 pentapotassium</td><td>Invitrogen</td><td>F-14200</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott Laboratories</td><td>05260-05</td><td>anesthesia</td></tr><tr><td>Microscope slide</td><td>Fisher Scientific</td><td>22-178-277</td><td></td></tr><tr><td>Zeiss LSM 5 Pascal microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Axioplan 40X (NA 0.8) objective lens</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td><td>Molecular Probes</td><td>A-11034</td><td>Secondary antibody</td></tr><tr><td>RBPMS</td><td>ProSci, Poway, CA</td><td></td><td>A rabbit polyclonal antibody&amp;nbsp;was generated against the N-terminus of the RBPMS&amp;nbsp;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td></tr></tbody></table>

immunohistochemical and calcium imaging methods in wholemount rat retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunolabeling with specific antibodies provides a platform to study the localization of particular proteins of interest in the retina and the calcium imaging technique permits the study of the contribution of the VGCCs to the calcium dynamics in the retinal ganglion cells and their axons.
By using an antibody against the ganglion cell protein RBPMS (RNA binding protein with multiple splicing) that selectively labels RGCs19, we were able to show that the Fluo-4 labeling is limited t...

Watch this Scientific Journal Video about Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina at JoVE.com

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

immunohistochemical-calcium-imaging-methods-wholemount-rat

Research

JoVE Journal

Neuroscience

14.4K Views.  University of California, Los Angeles. Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Video: Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Bioengineering

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.

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

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Biology

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

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.

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 ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

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Rozdziały w tym wideo

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

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

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

In vivo Calcium Imaging in Mouse Inferior Olive

Calcium Imaging in Mouse Superior Colliculus

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes