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;

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			<td>Invitrogen</td>
			<td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=A1247501">A12475-01</a></td>
			<td>Media</td>
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			<td height="21" style="height:21px;width:216px;">Vectashield</td>
			<td>Vector</td>
			<td>H-1000&#160;</td>
			<td><a href="https://www.google.com/url?sa=t&amp;rct=j&amp;q=&amp;esrc=s&amp;source=web&amp;cd=1&amp;cad=rja&amp;sqi=2&amp;ved=0CC0QFjAA&amp;url=http%3A%2F%2Fwww.vectorlabs.com%2Fcatalog.aspx%3FcatID%3D279&amp;ei=VAHbUb_fBceeiQLK2IHIDw&amp;usg=AFQjCNHijFTTB8p19FMPQ5hg_qTawXU9mA&amp;sig2=FJCfB9fzKR_TmjxoGvhLcg&amp;bvm=bv.48705608,d.cGE">Mounting Media for Fluorescence</a></td>
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			<td height="21" style="height:21px;width:216px;">Fluo-4 pentapotassium</td>
			<td>Invitrogen</td>
			<td>F-14200</td>
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			<td>Abbott Laboratories</td>
			<td>05260-05</td>
			<td>anesthesia</td>
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			<td height="21" style="height:21px;width:216px;">Microscope slide</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">22-178-277</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss LSM 5 Pascal microscope</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Axioplan 40x (NA 0.8) objective lens</td>
			<td style="width:112px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:112px;">Molecular Probes</td>
			<td style="width:119px;">A-11034</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RBPMS</td>
			<td style="width:112px;">ProSci, Poway, CA</td>
			<td style="width:119px;"></td>
			<td>A rabbit polyclonal antibody&#160;was generated against the N-terminus of the RBPMS&#160;polypeptide (RBPMS4-24), GGKAEKENTPSEANLQEEEVR, by a commercial vendor (ProSci, Poway, CA).</td>
		</tr>
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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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...