This work is supported by the National Natural Science Foundation of China (32271060). Y.-t.L. designed the research, performed the experiment, analyzed the data, and wrote the manuscript. Z.L. and R.W. performed the experiment.

All experimental procedures were performed in accordance with the animal welfare guidelines and approved by the IACUC at the Chinese Institute for Brain Research, Beijing.
NOTE: The timeline for this protocol is as follows: 1) make the suction cup; 2)&#160;inject the virus; 3) implant the headplate; 4)&#160;after 3 weeks, implant the plug; 5)&#160;after a ~3 day recovery and habituation on the treadmill, perform two-photon/wide-field imaging.
1. Preparation of a suction cup (Figure 1A)
<ol>
	<li>Deposit a drop of phosphate-buffered saline (PBS, 1x) in an acrylic dish and touch it with a 21 G flat needle to fill the tip by capillarity.</li>
	<li>Cover the tip of the needle with a translucent silicone adhesive and set for about ~10 min.</li>
	<li>Cut the silicone adhesive with scissors to a disk with a ~2 mm diameter.</li>
</ol>
2. Preparation of plugs (Figure 1B)
<ol>
	<li>Prepare a 0.75 mm thick plastic shim stock and cut out a 1&#160;cm x 1&#160;cm&#160;square in the center. Prepare two acrylic blocks. Clean the shim stock and acrylic blocks with alcohol and wipe them with lens paper.</li>
	<li>Put the shim stock on one acrylic block, deposit the silicone adhesive into the center to fill ~90% of the opening (avoid bubbles), add another acrylic block and ~1 kg force, and wait 20 min.</li>
	<li>Under a surgical microscope, cut the silicone adhesive with a scalpel to 1 mm high by 1.5 mm wide triangular prisms above a piece of paper with triangles printed on it.</li>
	<li>Remove any dust from the triangular prisms with plastic sticky tape and put it on a glass coverslip of 5 mm diameter and 0.15 mm thickness.</li>
	<li>Put the coverslip with the triangular prisms under a corona treater, turn on the corona treater, and bring it closer to the coverslip until lightening appears. Hold it at that position for a few minutes until they are attached to each other. 
	NOTE: This step might be different for other corona treaters.</li>
	<li>Put the plugs in a Petri dish and place it in an incubator at 70 &#176;C overnight.</li>
</ol>
3. Preparation of animals and injection of viral construct
<ol>
	<li>Use C57BL/6J (wild-type) and Emx1-Cre:Pals1flox/wt (partial-cortex)20 mice of both sexes at 6 weeks of age (9 weeks at the time of imaging).</li>
	<li>Sterilize all materials and instruments used for the surgical procedure.</li>
	<li>Anesthetize the mouse with 5% isoflurane and then maintain the isoflurane at 1%-2% with a flow rate of 1 L/min during the surgery. Confirm the level of anesthesia by the lack of toe pinch reflex.</li>
	<li>Head-fix the mouse on a stereotaxic frame with ear bars. Throughout the surgery, put ophthalmic ointment on the mouse eyes to prevent drying, and maintain its body temperature at 37 &#176;C with a thermostatic heating pad.</li>
	<li>Shave the fur and sterilize the skin surface with iodophor and 75% ethanol. Inject lidocaine (5 mg/kg, subcutaneous injection [SQ]), tilidine (2 mg/kg, SQ), and meloxicam (2 mg/kg, SQ). 
	NOTE: The surgical procedure for sterilization, analgesia, and local anesthesia may differ across institutions.</li>
	<li>Remove the skin over the SC with surgical scissors to expose the skull. Clean the skull with a cotton swab.</li>
	<li>Adjust the stereotaxic frame until the skull surface is flat. Drill a hole 0.5 mm lateral and 0.42 mm anterior to the lambda until the dura is exposed.</li>
	<li>Inject adeno-associated virus (AAV) expressing GCaMP6m (rAAV2/9-hsyn-GCaMP6m; 1 x 1013 GC/mL dissolved in 1x PBS) at depths of 1 mm and 1.6 mm from the lambda with a beveled glass micropipette (tip is beveled to 25&#176; with a diameter of 40-50 &#181;m).</li>
	<li>At each depth, inject 100 nL at a speed of 50 nL/min and wait 5 min after each injection. After the injection, slowly withdraw the injector.</li>
</ol>
4. Implantation of the headplate
<ol>
	<li>Clean the muscles and tissues over the skull and scratch the skull with a blade. If bleeding occurs, stop it with gel foam and clean the blood. Attach the headplate (Figure 1C) to the skull with a self-curing dental adhesive resin cement. Specifically, mix a 3/4 spoon of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture on the headplate and the skull. Attach them together and wait 5 min for the adhesive to set.</li>
	<li>Inject antibiotics (ceftiofur sodium, 5 mg/kg, SQ) to prevent infections. Remove the mouse from the stereotaxic frame and place it on a heating pad for recovery. Return it to the home cage after recovering from anesthesia. For pain management, provide ibuprofen-containing drinking water&#160;(0.5 mL/50 mL) to the mouse for 3 days after the surgery.</li>
</ol>
5. Implantation of the plug (Figure 2)
<ol>
	<li>Implant the plug 3 weeks after the viral injection. Anesthetize and fix the mouse on a stereotaxic frame, as described in steps 3.2-3.5. Prepare gel foam soaked in saline to stop potential bleeding.</li>
	<li>Drill a 3 mm x 2 mm oval with a microdrill centered at 0.5 mm posterior to the lambda. Thin the boundary of the oval until it cracks. Thin the skull in front of the oval recording window to make the coverslip close to the SC.</li>
	<li>Take off bone flaps using fine forceps. Make a cut on the dura posterior to the transverse sinus and remove the piece of dura. If necessary, add artificial cerebrospinal fluid (ACSF) to the brain with a 3 mL syringe to prevent drying. Dry the skull and recording window with gel foam and cotton swabs.</li>
	<li>Deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure. Use a motorized micromanipulator to move the suction cup and lower the plug into the silicone adhesive until the coverslip touches the skull. Then, move the plug forward ~1 mm to push away the transverse sinus and expose the SC. This step should be done in 1-2 min before the silicone adhesive becomes sticky.</li>
	<li>Clean the headplate and apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the headplate. 
	NOTE: Provide postoperative care, as described in step 4.2.</li>
</ol>
6. Two-photon imaging (Figure 3)
<ol>
	<li>Head-fix the animal on a rotating treadmill with its headplate. Place the treadmill under a two-photon microscope and adjust the height to an appropriate position. Put a black aluminum cone between the objective and the headplate to prevent light contamination from the monitor for visual stimulation. Habituate the mouse for ~15 min.</li>
	<li>Perform two-photon imaging on the microscope with a 16x magnification, 0.8 numerical aperture (NA), and 3 mm working distance (WD) objective. Use a Ti:sapphire laser with mode-locking technique controlled by two galvo scanners to excite GCaMP6m at 920 nm. Adjust the laser power at the sample plane between 20-80 mW.</li>
	<li>Scan a 600 &#181;m x 600 &#181;m field of view at 4.8 Hz with a spatial resolution of 2.4 &#181; m/pixel, and image neural activity across the depth up to 350 &#181; m. 
	NOTE: The sampling rate and spatial resolution are for galvo scanners, and should be adjusted for resonant scanners.</li>
	<li>Collect emitted fluorescence with a dichroic mirror and detect it using a photomultiplier tube (PMT)&#160;after passing it through a bandpass filter.</li>
	<li>Record the mouse&#39;s locomotion on the treadmill with a rotary encoder. Use a camera with a 50 mm lens to record its pupil size and position. Synchronize these recordings and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer.</li>
</ol>
7. Analysis of calcium responses measured by two-photon imaging
<ol>
	<li>Correct the brain motion during imaging using SIMA21 or NoRMCorre22.</li>
	<li>Manually draw a region of interest (ROI) using the Cell Magic Wand tool (ImageJ) and fit it with an ellipse in MATLAB. Extract fluorescence traces of each ROI after removing neuropil contamination: 
	<img alt="Equation 1" src="/files/ftp_upload/65181/65181eq01.jpg" style="margin: auto;" /> 
	Ftrue&#160;and Fraw are the corrected and raw fluorescence amplitudes of the ROI, respectively, Fneuropil is the fluorescence amplitude of the surrounding neuropil, and r is the out-of-focus neuropil contamination factor (0.7 for our setup). The r is estimated by measuring the signals on a blood vessel for which Ftrue = 0, as described previously23,24.</li>
	<li>Remove slow baseline fluctuations by subtracting the eighth percentile value from a 15 s window centered on each frame25.</li>
	<li>Define visual responses of a neuron as the relative change of fluorescence amplitude in its ROI during the stimulus period: 
	<img alt="Equation 2" src="/files/ftp_upload/65181/65181eq02.jpg" style="margin: auto;" /> 
	Fpeak&#160;is the peak amplitude of the fluorescence trace during the visual stimulation, and Fbaseline is the mean amplitude during a 0.5 s period prior to stimulation.</li>
</ol>
8. Wide-field imaging and data analysis (Figure 4)
<ol>
	<li>Build a wide-field macroscope with a tandem lens4 (Figure 4A). The tandem-lens macroscope is two camera lenses (50 mm and 105 mm) connected via an adapter, and the magnification is ~2x.</li>
	<li>Prepare partial-cortex mutant mice as described in steps 3.2-3.5. Inject 100 nL of AAV expressing GCaMP6m at a depth of 200 &#181;m at the center of each side of the SC.</li>
	<li>After ~3 weeks, implant a 5 mm diameter coverslip over the SC. Perform an oval craniotomy of 4 mm x 3 mm centered at the SC and thin the bone around it. Then, press the coverslip directly onto the SC and fix it with resin cement.</li>
	<li>Excite GCaMP6m with a blue light emitting diode (LED) with an excitation filter (469 nm &#177; 35 nm). Acquire images using a complementary metal oxide semiconductor (CMOS) camera after passing an emission filter (525 nm &#177; 39 nm) at 10 Hz. The camera covers an area of 5.36 mm x 2.85 mm at a spatial resolution of 2.63 &#181;m/pixel.</li>
	<li>Convolve each acquired image with a normalized box filter and downsample it to 1/4 of the original size. For each pixel in the image, define the response, as described in step 7.4. 
	NOTE: Provide postoperative care, as described in step 4.2.</li>
</ol>

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

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The authors have nothing to disclose.

Critical steps in the protocol 
The most critical step is the craniotomy in steps 5.2 and 5.3. First, the bone at 0.5 mm posterior to the lambda is thick and has blood vessels inside, which can cause bleeding during the drilling process. Adequate gel foam should be prepared to stop the bleeding. Second, there is a good chance of angiorrhexis when removing the bone just above the transverse sinus. For troubleshooting, one alternative approach is to thin the bone inside the oval and remove it piece by...

The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at&#160;single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-mm lens</td>
			<td>Computar</td>
			<td>M5018-MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mm coverslip</td>
			<td>Warner instruments</td>
			<td>CS-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma Technology</td>
			<td>HQ575/250 m-2p</td>
			<td></td>
		</tr>
		<tr>
			<td>butyl cyanoacrylate</td>
			<td>Vetbond, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for monitoring pupil</td>
			<td>FLIR</td>
			<td>BFS-U3-04S2M-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>camera for widefield imaging</td>
			<td>Basler</td>
			<td>acA2000-165&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic</td>
			<td>Chroma Technology</td>
			<td>T600/200dcrb&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bead sterilizer</td>
			<td>RWD</td>
			<td>RS1502</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>QUAD</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td></td>
		</tr>
		<tr>
			<td>rotory encoder</td>
			<td>USdigital</td>
			<td>MA3-A10-125-N</td>
			<td></td>
		</tr>
		<tr>
			<td>self-curing dental adhesive resin cement&#160;</td>
			<td>SuperBond C&#38;B, Sun Medical Co, Ltd. Moriyama, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic heating pad&#160;</td>
			<td>RWD</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>translucent silicone adhesive&#160;</td>
			<td>Kwik-Sil, World Precision Instruments</td>
			<td></td>
			<td></td>
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			<td>treadmill</td>
			<td>Xinglin Biology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV2/9-hsyn-Gcamp6m</td>
			<td>Vector Core at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild type</td>
			<td>Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emx1-Cre</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>5628</td>
			<td></td>
		</tr>
		<tr>
			<td>Pals1flox/wt</td>
			<td>Christopher A. Walsh Lab</td>
			<td></td>
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		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
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			<td>ImageJ</td>
			<td>NIH Image</td>
			<td></td>
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		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figures 1A,B show&#160;how to make the suction cup and the plugs, respectively. Figure 2 shows how to implant the plug successfully. After implanting the plug, the posterior-medial SC is exposed, as shown in Figure 2D. Figure 3 shows calcium responses of SC neurons from an example wild-type mouse imaged using two-photon microscopy. The triangular prism, which is easily captured under th...

Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

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.

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 ...

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

3.1K Views.  Chinese Institute for Brain Research. 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. 

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

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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, ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

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+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures1,2,3,4,5,6. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory7. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

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

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

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

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