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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	<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. 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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 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 ...

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Research

JoVE Journal

Neuroscience

5.4K Views.  OIST. 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.

Video: In vivo Calcium Imaging in Mouse Inferior Olive

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 connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

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

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, ...

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 ...

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 stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

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 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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging 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 ...

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 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 ...