Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 current^1,2. VGCC are transmembrane multimeric proteins that are involved in transmitter release, gene transcription, cell regulation and synaptic plasticity^3,4,5. Functional VGCCs are made up of at least three distinct classes of subunits: large, transmembrane α­₁ pore forming subunits that establish the channel’s biophysical and pharmacological properties, primarily extracellular auxiliary α­₂δ subunits and intracellular β subunits. The latter two form heteromeric complexes with different α­₁ 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.  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.  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.  Many techniques such as bulk loading¹¹ and electroporation^8,12 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. 

Protokół

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

1. 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.
2. 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.
3. [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.
4. Using the dissecting microscope (light intensity: 1.6 x 10⁸ photons/mm²⋅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.
5. 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.
6. Place the wholemounted retinas into 4% PFA for 10-15 min for fixation.
7. 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 °C overnight. We recommend a final volume of 500 μl for all the steps to save blocking solutions and antibodies.
8. Next day, incubate the retinae wholemounts with the primary antibody 5-7 days at 4 °C. Optimal dilutions must be determined by the user.
9. Wash the retinas 3 x 30 min in PB 0.1 M and incubate overnight at 4 °C in the corresponding fluorophore-conjugated secondary antibody that is directed against the species of the primary antibody (concentration 1:1,000).
10. 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 °C and protect from light.

2. Labeling of Ganglion Cells and Axons in Rat Wholemount Retinas with a Calcium Indicator Dye

1. Prepare 1,000 ml of mammalian Ringer'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^­₂.
2. Perform the dissection as described in the steps 1.2-1.4.
3. To load retinal ganglion cells (RGCs) and their axons with a calcium indicator dye, inject 0.5 μl of Fluo-4 pentapotassium salt (40 mM stock in H­₂O) 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 K_d of 345 nM, is optimal. Fluo-4 offers the additional benefit of having a very high quantum efficiency resulting in an emission increase of >100 fold when bound to calcium.
4. Place the eyecup in mammalian Ringer's bubbled with 95% O₂/ 5% CO^­₂ for 1 hr at room temperature in the dark.

3. Calcium Imaging Protocol for Retinal Ganglion Cells and Axons.

1. 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's bubbled with 95% O₂/ 5% CO^­₂ on ice for later use.
2. Superfuse the recording chamber with mammalian Ringer's solution and keep the solution bubbling continuously with 95% O₂/5% CO₂.
3. Image the tissue under the microscope. Conduct experiments at room temperature (~23 °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.
4. 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.
5. 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.
6. Acquire fluorescence intensity values by placing a region of interest (ROI) around the RGCs of interest (Figure 2).
7. 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.
8. 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.

Wyniki

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...

Dyskusje

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 retina^20-23. However, there are a few limitations. The quality of the staining relies heavily on the ability of the antibody to ...

Materiały

Name	Company	Catalog Number	Comments
Straight scissors	WPI	14124-G	dissecting tools
Straight Forceps	WPI	501985	dissecting tools
curved iris scissors	WPI	504487	dissecting tools
Cellulose filter paper	Millipore	HABP04700
Hibernate A	Invitrogen	A12475-01	Media
Vectashield	Vector	H-1000	Mounting Media for Fluorescence
Fluo-4 pentapotassium	Invitrogen	F-14200
Isoflurane	Abbott Laboratories	05260-05	anesthesia
Microscope slide	Fisher Scientific	22-178-277
Zeiss LSM 5 Pascal microscope	Zeiss
Axioplan 40X (NA 0.8) objective lens	Zeiss
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody	Molecular Probes	A-11034	Secondary antibody
RBPMS	ProSci, Poway, CA		A rabbit polyclonal antibody was generated against the N-terminus of the RBPMS polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).