Name: in vivo calcium imaging in mouse inferior olive
Uploaded: 2021-06-10T14:30:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about In vivo Calcium Imaging in Mouse Inferior Olive at JoVE.com

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.

<ol>
	<li>Russell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+signals+in+vivo:+A+powerful+tool+in+physiology+and+pharmacology.">Imaging calcium signals in vivo: A powerful tool in physiology and pharmacology.</a> British Journal of Pharmacology. , (2011).</li><li>Zhang, T., 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=Kilohertz+two-photon+brain+imaging+in+awake+mice.">Kilohertz two-photon brain imaging in awake mice.</a> Nature Methods. , (2019).</li><li>Lin, X., Zhao, T., Xiong, W., Wen, S., Jin, X., Xu, X. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+the+primary+somatosensory+cortex+using+Thy1-GCaMP6s+transgenic+mice.">Imaging neural activity in the primary somatosensory cortex using Thy1-GCaMP6s transgenic mice.</a> Journal of Visualized Experiments. 2019 (143), 1-8 (2019).</li><li>Lee, H. S., Han, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+In+vivo+Calcium+Imaging+with+a+Head-Mount+Miniaturized+Microscope+in+the+Amygdala+of+Freely+Behaving+Mouse.">Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse.</a> Journal of visualized experiments. (162), 1-19 (2020).</li><li>Aharoni, D., Khakh, B. S., Silva, A. J., Golshani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+the+light+that+we+can+see:+a+new+era+in+miniaturized+microscopy.">All the light that we can see: a new era in miniaturized microscopy.</a> Nature Methods. 16 (1), 11-13 (2019).</li><li>Khosrovani, S., Van Der Giessen, R. S., De Zeeuw, C. I., De Jeu, M. T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mouse+inferior+olive+neurons+exhibit+heterogeneous+subthreshold+oscillations+and+spiking+patterns.">In vivo mouse inferior olive neurons exhibit heterogeneous subthreshold oscillations and spiking patterns.</a> Proceedings of the National Academy of Sciences. 104 (40), 15911-15916 (2007).</li><li>Osten, P., Cetin, A., Komai, S., Eliava, M., Seeburg, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+gene+delivery+in+the+rodent+brain.">Stereotaxic gene delivery in the rodent brain.</a> Nature Protocols. 1 (6), 3166-3173 (2007).</li><li>Berg, L., Gerdey, J., Masseck, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Manipulation+of+Neuronal+Activity+to+Modulate+Behavior+in+Freely+Moving+Mice.">Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice.</a> Journal of visualized experiments : JoVE. , e61023 (2020).</li><li>Green, C. J., Knight, J., Precious, S., Simpkin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine+alone+and+combined+with+diazepam+or+xylazine+in+laboratory+animals:+A+10+year+experience.">Ketamine alone and combined with diazepam or xylazine in laboratory animals: A 10 year experience.</a> Laboratory Animals. 15 (2), 163-170 (1981).</li><li>Tsukamoto, A., Serizawa, K., Sato, R., Yamazaki, J., Inomata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+signs+monitoring+during+injectable+andn+inhalant+anesthesia+in+mice.">Vital signs monitoring during injectable andn inhalant anesthesia in mice.</a> Experimental Animals. 64 (1), 57-64 (2015).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harbor Protocols. 6 (2), 174-178 (2011).</li><li>Vrieler, N., 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=Variability+and+directionality+of+inferior+olive+neuron+dendrites+revealed+by+detailed+3D+characterization+of+an+extensive+morphological+library.">Variability and directionality of inferior olive neuron dendrites revealed by detailed 3D characterization of an extensive morphological library.</a> Brain Structure and Function. , 1677-1695 (2019).</li><li>Esposito, M. S., Capelli, P., Arber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brainstem+nucleus+MdV+mediates+skilled+forelimb+motor+tasks.">Brainstem nucleus MdV mediates skilled forelimb motor tasks.</a> Nature. , (2014).</li><li>Martin, E. M., Devidze, N., Shelley, D. N., Westberg, L., Fontaine, C., Pfaff, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+neuroanatomical+characterization+of+single+neurons+in+the+mouse+medullary+gigantocellular+reticular+nucleus.">Molecular and neuroanatomical characterization of single neurons in the mouse medullary gigantocellular reticular nucleus.</a> Journal of Comparative Neurology. , (2011).</li><li>Cold Spring Harbor. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine/Xylazine.">Ketamine/Xylazine.</a> Cold Spring Harbor Protocols. 2006 (1), (2006).</li><li>Giovannucci, A., 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=CaImAn+an+open+source+tool+for+scalable+calcium+imaging+data+analysis.">CaImAn an open source tool for scalable calcium imaging data analysis.</a> eLife. 8, 1-45 (2019).</li><li>Lu, J., 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=MIN1PIPE:+A+Miniscope+1-Photon-Based+Calcium+Imaging+Signal+Extraction+Pipeline.">MIN1PIPE: A Miniscope 1-Photon-Based Calcium Imaging Signal Extraction Pipeline.</a> Cell Reports. 23 (12), 3673-3684 (2018).</li><li>. 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. I., De Jeu, M. T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferior+olive:+All+ins+and+outs.">Inferior olive: All ins and outs.</a> Handbook of the Cerebellum and Cerebellar Disorders. , (2013).</li><li>Dorgans, K., Kuhn, B., Uusisaari, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Subthreshold+Voltage+Oscillation+With+Cellular+Resolution+in+the+Inferior+Olive+in+vitro.">Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro.</a> Frontiers in Cellular Neuroscience. , (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#160; 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&#252;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,&#160; 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/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 ...

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Neuronal Calcium Imaging in C. elegans

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, ...

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

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 ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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 ...

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 ...

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 ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

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 ...

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

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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 ...