We thank Andrew Scott from media center of OIST for his help with video recording and editing. Also, we thank Hugo Hoedemaker for his help with developing the surgery to expose the brainstem and Dr. Kevin Dorgans for his help with drawing diagrams for figures. In addition, great thanks to Salvatore Lacava for his voice-over narration, as well as all nRIM members and pets for continuing support for wellbeing in the tough times of COVID-19.

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GitHub - PeyracheLab/miniscoPy: A package to analyse calcium imaging data recorded with the Miniscope Available from: <a target="_blank" href="https://github.com/PeyracheLab/miniscoPy">https://github.com/PeyracheLab/miniscoPy</a> (2020)</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. , (1998).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+accurate+extraction+of+in+vivo+calcium+signals+from+microendoscopic+video+data.">Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data.</a> eLife. 7, 1-37 (2018).</li><li>. GitHub - zhoupc/CNMF_E: Constrained Nonnegative Matrix Factorization for microEndoscopic data Available from: <a target="_blank" href="https://github.com/zhoupc/CNMF_E">https://github.com/zhoupc/CNMF_E</a> (2020)</li><li>De Gruijl, J. R., Bosman, L. W. J., De Zeeuw, C. 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The authors have nothing to disclose.

As the surgical procedure involves operations performed in the throat region with numerous vitally critical structures (arteries, nerves), it is essential that it is conducted by a researcher with high-level surgical skills. In following, we highlight and comment on several key points of the procedure; however, it must be reminded that no amount of written advice can supplant the experience, skill, and intuition of the researcher.
The most critical step in the surgery is tracheotomy. It involves cutting the trachea, switching isoflurane from the nose cone to the intubation tube, securing the trachea to the chest skin and tying the trachea and the intubation tube together. All these operations must be completed in a smooth and fast manner to avoid accidents, such as inadequate anesthesia, inflow of fluid into trachea or intubation tube slip-off. One must keep the protocol clear in mind before cutting the trachea.
Hemorrhage is one of the major causes of animal death in this surgery. Since the neck area is dense with blood vessels, cut should only be executed when the line of sight is clear to avoid cutting unseen veins and arteries. Therefore, muscles and connective tissues obscuring vision must be removed, and blood from broken capillaries must be cleaned before advancing.
Animal can be kept alive for a long time (more than 8 hours) from the start of surgery. However, it is important to finish the surgical procedure quickly so there is more time to examine brainstem neurons when animal physiological condition is good. A skilled researcher can finish the whole procedure in 70 min.
While the method provides a clean view of ventral brain surfaces, it is unfortunately impossible to do so without performing a tracheotomy as well as removing significant amount of tissue in the throat region. Therefore, the animal cannot be allowed to wake from anesthesia. Furthermore, even though it is possible to keep the animal alive for many hours with careful adjustment of anesthetic delivery, maintaining body temperature and hydration, it is inevitable that prolonged experimentation will eventually lead to weakening of the animal condition. It is left to the expertise of the researcher to consider the maximal duration of stable recordings.
Another potential limitation of the method as described here is that as the GRIN lens is not inserted into the brain parenchyma, only relatively superficial neurons (~150-200 &#181;m) can be examined. While surgical implantation of GRIN lens is technically possible, acute surgery method does not allow sufficient time for neurons to recover from oxidative stress and presence of blood after implantation likely will degrade image quality beyond acceptable.
Despite the above concerns, we believe this is the first time a method for in vivo imaging of IO neurons is presented. It allows examination of spatiotemporal activity in the IO neurons in the context in vivo in the presence of intact afferent inputs from sensory systems as well as the signals from the cerebellar nuclei and the mesodiencephalic junction22, a feat that has not been possible hitherto. With this method, the function of the IO can now be investigated in greater depth with combination of sensory and optogenetic stimulation. Notably, with the evolution of voltage imaging (such as our recent method for voltage imaging in the IO 23), we hope the presented surgical method will inspire numerous researchers to take up the challenge of investigating how the IO contributes to the generation of cerebellar complex spikes.

The main goal of systems neuroscience is to understand how spatiotemporal activity patterns of neuronal networks contribute to generation of animal behavior. Thus, fluorescent imaging methodology utilizing calcium-sensitive probes has in the past decade become a main tool for examining neuronal network activity in living animals1,2, as it allows visualization of such dynamics across spatial scales ranging from single cells to mesoscale circuitry. In recent years, the common approach where neural circuits in superficial brain structures (such as cerebral or cerebellar cortices) are imaged through a transparent cranial window3 has been complemented with the use of gradient-refractive index (GRIN) lenses4 allowing examination of network dynamics in deep brain structures. Currently-available GRIN lenses allow reaching into structures several millimeters deep, such as the mouse amygdala, hippocampus and basal ganglia5. However, many regions of interest such as various nuclei in the ventral medulla lie significantly deeper, placing them at the extreme of the GRIN lens reach.
Here, we describe how to overcome this difficulty by taking advantage of the relatively easy accessibility of medulla through the ventral aspect of the brain. Using adult mice where the inferior olive (IO), a nucleus in the ventral medulla, has been virally transfected with a calcium sensor GCaMP6s, we describe the surgical steps (modified from the method described originally in Khosrovani et al. 20076) to place a GRIN lens on the ventral surface of the brain of an anesthetized mouse. Using a miniature microscope, we demonstrate the feasibility of recording neuronal activity in such extremely ventral brain regions. While the procedure is necessarily a non-survival surgery and no experimentation can be performed in awake animals, the method allows examination of intact network dynamics in the context of sensory or other afferent pathway stimulation, providing clear advantages over ex vivo-approaches such as using acute slice preparations.

<table><tbody><tr><td>AAV.CAG.GCaMP6s.WPRE.SV40</td><td>Addgene, USA</td><td>100844-AAV9</td><td></td></tr><tr><td>Absorbable suture with 6 mm half circle needle</td><td>Natume, Japan</td><td>L6-60N2</td><td>hook needle with thread</td></tr><tr><td>Absorption triangles</td><td>FST, Germany</td><td>18105-03</td><td>Surgical sponges</td></tr><tr><td>Stereo microscopes</td><td>Leica, Germany</td><td>M50</td><td></td></tr><tr><td>Castroviejo curved tip needle holder with lock</td><td>FST, Germany</td><td>12061-01</td><td>Surgery tool</td></tr><tr><td>cotton swabs</td><td>Sanyo, Japan</td><td>HUBY-340</td><td></td></tr><tr><td>Delicate suture tying forceps</td><td>FST, Germany</td><td>11063-07</td><td>Surgery tool</td></tr><tr><td>Delicate Suture Tying Forceps</td><td>FST, Germany</td><td>11063-07</td><td>Surgery tool</td></tr><tr><td>Dumont #5/45 forceps</td><td>FST, Germany</td><td>11251-35</td><td>Surgery tool</td></tr><tr><td>Fine Iris scissors</td><td>FST, Germany</td><td>14060-09</td><td>Surgery tool</td></tr><tr><td>Friedman-Pearson rongeur curved tip</td><td>FST, Germany</td><td>16221-14</td><td>Surgery tool</td></tr><tr><td>Gelfoam absorbable gelatin sponge</td><td>Pfizer, USA</td><td>0315-08</td><td>Hemostatic gelatin sponge</td></tr><tr><td>Glass-Capillary Nanoinjection</td><td>Neurostar, Germany</td><td>n/a</td><td>For virus vector injection</td></tr><tr><td>Graefe Forceps with serrated tip</td><td>FST, Germany</td><td>11052-10</td><td>Surgery tool</td></tr><tr><td>Implantation rod</td><td>Inscopix, USA</td><td>n/a</td><td>It is part of the nVoke2 system. It&#039;s designed to nVoke2 miniature microscpe and GRIN lens can be mounted on it</td></tr><tr><td>IsoFlo</td><td>Zoetis, UK</td><td>n/a</td><td>Isoflurane</td></tr><tr><td>KETALAR FOR INTRAMUSCULAR INJECTION</td><td>Daiichi Sankyo, Japan</td><td>n/a</td><td>Ketamine</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark, USA</td><td></td><td>Cleaning tissue</td></tr><tr><td>Laser-Based Micropipette Puller</td><td>Sutter Instrument, USA</td><td>P-2000</td><td></td></tr><tr><td>Micropipette Beveler</td><td>Sutter Instrument, USA</td><td>BV-10</td><td></td></tr><tr><td>Motorized Stereotaxic based on Kopf, Model 900</td><td>Neurostar, Germany</td><td>n/a</td><td>Stereotaxic frame</td></tr><tr><td>mouseOxPlus with rectal temperature sensor and thigh clamp pulse oximeter</td><td>Starr Life Sciences, PA, USA</td><td>MouseOxPlus</td><td>Measures animal heart rate, arterial oxygen saturation (SpO2), breath rate, and temperature</td></tr><tr><td>nVoke2 integrarted Calcium imaging micro camera system</td><td>Inscopix, USA</td><td>1000-003026</td><td>Miniature microscope</td></tr><tr><td>Ohaus Compact Scales</td><td>Ohaus, USA</td><td>CS 200</td><td>Scale used to weight animal</td></tr><tr><td>Otsuka Normal Saline</td><td>Otsuka Pharmaceutical Factory, Japan</td><td>n/a</td><td></td></tr><tr><td>Physiological-biological temperature controller system</td><td>SuperTech Instruments, Hungary</td><td>TMP-5b</td><td>Thermal pad for mouse</td></tr><tr><td>ProView Lens Probe 1.0 mm diameter, 9.0 mm length</td><td>Inscopix, USA</td><td>1050-002214</td><td>Gradient-refractive index (GRIN) lens</td></tr><tr><td>Q114-53-10NP glass capillaries</td><td>Sutter Instrument, USA</td><td>112017</td><td>Customized&amp;nbsp; quartz glass capillaries</td></tr><tr><td>Safety IV Catheter 20G</td><td>B. Braun, Germany</td><td>4251652-03</td><td>20 gauage catheter used to prepare intubation tube</td></tr><tr><td>Sand paper</td><td>ESCO, Japan</td><td>EA366MC</td><td>Used to polish the tip of 25G needle to prepare curved and blunt needle</td></tr><tr><td>Scalpel blade</td><td>Muromachi Kikai, Japan</td><td>10010-00</td><td>Used to cut the tip of quartz glass pipette</td></tr><tr><td>SomnoSuite low flow inhalation anesthesia system</td><td>Kent Scientific, USA</td><td>SOMNO</td><td>Provides precise control of isoflurane flow</td></tr><tr><td>Surgic XT Plus drill</td><td>NSK</td><td>Y1002774</td><td>For virus vector injection</td></tr><tr><td>Syringe 1 ml</td><td>Terumo, Japan</td><td>SS-01T</td><td></td></tr><tr><td>Syringe needle 25G</td><td>Top, Japan</td><td>00819</td><td>Used to make blunt and bended needle</td></tr><tr><td>Syringe needle 26G</td><td>Terumo, Japan</td><td>NN-2613S</td><td></td></tr><tr><td>Thrive 2100 Professional Trimmer</td><td>Thrive, Japan</td><td>n/a</td><td>Shaver</td></tr><tr><td>Vannas-T&amp;uuml;bingen spring scissors</td><td>FST, Germany</td><td>15004-08</td><td>Surgery tool</td></tr><tr><td>Vaseline</td><td>Hayashi Pure Chemical, Japan</td><td>22000255</td><td></td></tr><tr><td>Veet sensitive skin</td><td>Veet, Canada</td><td>n/a</td><td>Hair removal cream</td></tr><tr><td>Xylocaine Jelly 2 % 30ml</td><td>Aspen Japan,&amp;nbsp; Japan</td><td>871214</td><td></td></tr></tbody></table>

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.

Here we present a representative recording obtained with the method as described. Figure 6a shows the location of brightly labeled IO cells visualized during the experiment. The dark diagonal stripes are blood vessels. Note the variable brightness of individual cells, resulting from variable transfection efficacy. In panel Figure 6b we show the mean-normalized fluorescence intensity (deltaF/F) traces obtained from the somata indicated with colors and numbers in panel a. Upward deflections represent transient increases in intracellular calcium. Note how different level of GCaMP6s expression (reflected in cell brightness in panel a) lead to variable signal-to-noise-ratios (SNR).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62222/62222fig06.jpg" /> 
Figure 6: Example recording of activity of IO neurons in anesthetized mouse. (a) Representative example frame from a recording after spatial filtering. Bright spots are IO neuronal somata, several of which have been indicated as regions of interests (ROIs, colored numbers). Dark stripes are blood vessels. (b) Example deltaF/F traces obtained from the ROIs indicated in panel a. Upwards deflections reflect increases in calcium signal. Scale bar in a=10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62222/62222fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In vivo Calcium Imaging in Mouse Inferior Olive at JoVE.com

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.

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 ...

in-vivo-calcium-imaging-in-mouse-inferior-olive

Nanyang Technological University

Research

JoVE Journal

Neuroscience

5.5K Views.  OIST. Inferieure olijf is het meest ventrale deel van de medulla en is dus extreem moeilijk te bereiken bij een levend dier. Hier introduceren we een protocol om de volwassen muishersenen aan de ventrale kant bloot te leggen en gebruiken we GRIN-lenzen om neuronale activiteit in de inferieure olijfneuronen op te nemen die calciumsensoren uitdrukken. Deze methode voorkomt schade aan levenskritische hersenstam en maakt onderzoek mogelijk naar ruimtelijk-temporele activiteitspatronen en inputintegrati...

Video: In vivo Calcium Imaging in Mouse Inferior Olive

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

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 ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

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

Calcium Imaging in Mouse Superior Colliculus

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

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

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

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

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

In Vivo Calcium Imaging of Neuronal Ensembles in the Intact Dorsal Root Ganglia of a Mouse

Source: <a href='https://app-jove-com.remotexs.ntu.edu.sg/v/64826/in-vivo-calcium-imaging-of-neuronal-ensembles-in-networks-of-primary-sensory-neurons-in-intact-dorsal-root-ganglia'>Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. Vis. Exp. (2023).</a>The video demonstrates an in vivo calcium imaging technique to monitor neuronal activity in the dorsal root ganglia (DRG) of a transgenic mouse. DRG neurons express a calcium indicator that increases fluorescence intensity upon binding to calcium. Mechanical or thermal stimuli applied to a hind paw generate action potentials that propagate to the DRG, inducing calcium influx in the neurons and resulting in increased fluorescence emission.

Source: Shannonhouse, J., et al.In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia. J. ...

In vivo Calcium Imaging in Mouse Inferior Olive

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