Name: calcium imaging in freely behaving caenorhabditis elegans with well-controlled, nonlocalized vibration
Uploaded: 2021-04-29T15:00:00
Description: Watch this Scientific Journal Video about Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration at JoVE.com

We thank the Caenorhabditis Genetics Center for providing the strains used in this study. This publication was supported by JSPS KAKENHI Grant-in-Aid for Scientific research (B) (Grant no. JP18H02483), on Innovative areas &#34;Science of Soft Robot&#34; project (Grant no. JP18H05474), the PRIME from Japan Agency for Medical Research and Development (grant number 19gm6110022h001), and the Shimadzu foundation.

1. Cultivation of worms until calcium imaging
<ol>
	<li>Four days before a calcium imaging experiment, transfer two adult ST12 worms to a new nematode growth medium (NGM) plate (Table of Materials) on which Escherichia coli OP50 are streaked in a square pattern (approximately 4 mm x 4 mm) using a cell spreader so that the worm spends most of the time in the bacteria during calcium imaging12.</li>
	<li>Incubate this NGM plate for 4 days at 20 &#176;C in an incubator (<st...

<ol>
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Generally, the quantification of neural activity requires introduction of a probe and/or restraints on animal body movement. However, for studies of mechanosensory behavior, the invasive introduction of a probe and restraints themselves constitute mechanical stimuli. C. elegans provides a system to circumvent these problems, because its features are transparent and because it has a simple, compact neural circuit comprising only 302 neurons. Combining these advantages with the previously developed method of evoki...

Animals are often exposed to nonlocalized mechanical stimuli such as vibrations or acoustic waves1,2. Because these stimuli influence homeostasis, development, and reproduction, animals must modify their behaviors to cope with them3,4,5. However, the neural circuits and mechanisms underlying such behavioral modification are poorly understood.
Mechanosensory behavior in the nematode, Caenorhabditis elegans, is a simple behavioral paradigm, in which worms usually change behavior from forward movement to a backward escape response when they encounter nonlocalized vibration6. The neural circuit underlying this behavior is composed primarily of five sensory neurons, four pairs of interneurons, and several types of motor neurons7,8. Additionally, worms habituate to such mechanical stimuli after spaced training involving repeated stimulation9,10,11. Therefore, this simple behavioral response constitutes an ideal system to investigate neural mechanisms underlying both nonlocalized vibration-evoked behavior and memory. A protocol for calcium imaging in freely behaving worms under the influence of nonlocalized vibrations is illustrated. Compared with previously reported systems, this system is simple in that it does not require an additional camera for tracking; however, it allows us to change the frequency, displacement, and duration of nonlocalized vibration. Because activation of the AVA interneurons induces the backward escape response, worms co-expressing GCaMP, a calcium indicator, and TagRFP, a calcium-insensitive fluorescent protein, under the control of an AVA-specific promoter were used as an example (see Table of Materials for details). The protocol demonstrates the activation of AVA neurons as a worm switches from forward to backward movement. This protocol facilitates understanding the neural circuit mechanism underlying mechanosensory behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Data anaylsis software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DualViewImaging.nb</td>
			<td>author</td>
			<td></td>
			<td>For analysis of acquired data</td>
		</tr>
		<tr>
			<td>Mathematica12</td>
			<td>Wolfram</td>
			<td></td>
			<td>For running data anaysis software DualViewImaging</td>
		</tr>
		<tr>
			<td>Escherichia coli and C. elegans strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td>Food for C. elegans. Uracil auxotroph. E. coli B.</td>
		</tr>
		<tr>
			<td>lite-1(ce314) strain</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>KG1180</td>
			<td>Light-insensitive mutant</td>
		</tr>
		<tr>
			<td>lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter</td>
			<td>author</td>
			<td>ST12</td>
			<td>lite-1(ce314) mutant carrying the genes expressing NLS-GCaMP5G-NLS (NLS; nuclear localization signal) and TagRFP under the control of the flp-18 promoter as an extrachoromosomal arrays</td>
		</tr>
		<tr>
			<td>Laser Doppler vibrometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lase Doppler vibrometer</td>
			<td>Polytec Japan</td>
			<td>IVS-500</td>
			<td>For quantifying&#160; frequency and displacement generated by the accoustic transducer</td>
		</tr>
		<tr>
			<td>Mouse macro system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assay.txt</td>
			<td>Author</td>
			<td></td>
			<td>Script for temporally and specially controlling mouse cursol in Windows</td>
		</tr>
		<tr>
			<td>HiMacroEx</td>
			<td>Vector</td>
			<td>https://www.vector.co.jp/download/file/winnt/util/fh667310.html</td>
			<td>Free download software for controling mouse cursor based on a script</td>
		</tr>
		<tr>
			<td>Nematode growth media plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar purified, powder</td>
			<td>Nakarai tesque</td>
			<td>01162-15</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Bacto pepton</td>
			<td>Becton Dickinson</td>
			<td>211677</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Wako</td>
			<td>036-00485</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Wako</td>
			<td>034-03002</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>di-Photassium hydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28727-95</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>LB broth, Lennox</td>
			<td>Nakarai tesque</td>
			<td>20066-95</td>
			<td>For culture of E. coli OP50</td>
		</tr>
		<tr>
			<td>Magnesium sulfate anhydrous</td>
			<td>TGI</td>
			<td>M1890</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogenphosphate</td>
			<td>Nakarai tesque</td>
			<td>28720-65</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Nakarai tesque</td>
			<td>31320-05</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Petri dishes (60 mm)</td>
			<td>Nunc</td>
			<td>150270</td>
			<td>For preparation of NGM plates</td>
		</tr>
		<tr>
			<td>Nonlocalized vibration device</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>LEPY</td>
			<td>LP-A7USB</td>
			<td>For stimulation with controllable vibration</td>
		</tr>
		<tr>
			<td>Acoustic transducer</td>
			<td>MinebeaMitsumi</td>
			<td>LVC25</td>
			<td>For stimulation with controllable vibration</td>
		</tr>
		<tr>
			<td>WaveGene Ver. 1.5</td>
			<td>Thrive</td>
			<td>http://efu.jp.net/soft/wg/down_wg.html</td>
			<td>Free download software for controling vibration property</td>
		</tr>
		<tr>
			<td>Noninvasive calcium imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Channel benchtop 3-phase brushless DC servo controller</td>
			<td>Thorlabs</td>
			<td>BBD202</td>
			<td>Compatible controller for MLS203-1 stages</td>
		</tr>
		<tr>
			<td>479/585 nm BrightLine dual-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-479/585-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitter</td>
			<td>Semrock</td>
			<td>FF505/606-Di01-25x36</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>512/25 nm BrightLine single-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-512/25-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>630/92 nm BrightLine single-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-630/92-25</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP)</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Precision T7600</td>
			<td>Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM</td>
		</tr>
		<tr>
			<td>High-speed x-y motorized stage</td>
			<td>Thorlabs</td>
			<td>MLS203-1</td>
			<td>Fast XY scannning stage</td>
		</tr>
		<tr>
			<td>Image splitting optics</td>
			<td>Hamamatsu photonics</td>
			<td>A12801-01</td>
			<td>For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics</td>
		</tr>
		<tr>
			<td>LED light source</td>
			<td>CoolLED</td>
			<td>pE-4000</td>
			<td>For generating 470 nm and 560 nm excitation light</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>MVX10</td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Zyla</td>
			<td></td>
		</tr>
		<tr>
			<td>x 2 Objective lens</td>
			<td>Olympus</td>
			<td>MVPLAPO2XC</td>
			<td>Working distance 20 mm and numerical aperture 0.5</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pKDK66 plasmid</td>
			<td>author</td>
			<td>pKDK66</td>
			<td>Co-injection marker</td>
		</tr>
		<tr>
			<td>pTAK83 plasmid</td>
			<td>author</td>
			<td>pTAK83</td>
			<td>Plasmid for expression of TagRFP under the control of&#160; the flp-18 promoter</td>
		</tr>
		<tr>
			<td>pTAK144 plasmid</td>
			<td>author</td>
			<td>pTAK144</td>
			<td>Plasmid for expression of NLS-GCaMP5G-NLS under the control of&#160; the flp-18 promoter</td>
		</tr>
		<tr>
			<td>Tracking software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>homingback.vi</td>
			<td>author</td>
			<td></td>
			<td>SubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National instruments</td>
			<td></td>
			<td>For running tracking software</td>
		</tr>
		<tr>
			<td>Zyla Control ver.2.6CI.vi</td>
			<td>author</td>
			<td></td>
			<td>For tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage</td>
		</tr>
	</tbody>
</table>

calcium imaging in freely behaving caenorhabditis elegans with well-controlled, nonlocalized vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Here, a worm expressing both GCaMP and TagRFP under control of the AVA interneuron-specific promoter is used as an example of calcium imaging in freely behaving C. elegans. GCaMP and TagRFP channel data were obtained as a series of images, some of which are shown in Figure 6 and as a movie (Supplemental Movie 1). The displacement of the Petri plate induced by our nonlocalized vibration system (Figure 7) was also quantified. The displace...

Watch this Scientific Journal Video about Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration at JoVE.com

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

Our system allows researchers to evoke non localized vibrations with well-controlled properties at non-scaled displacement and to quantify calcium current during responses of the irritants to the vibrations. As a main advantage of this technique, we can noninvasively investigate the neural mechanisms, underlying mechanisms of behavior. Prepare a new nematode growth medium or NGM plate on which Escherichia coli OP50 is streaked in a square pattern using a cell spreader so that the worm spends most of the time in the bacteria during calcium imaging.Four days before a calcium imaging experiment, transfer two adult ST12 worms onto the plate, and place the plate in a 20 degree Celsius incubator for four days. Perform calcium imaging using a microscope equipped with an acoustic transducer device for stimulation, a 2x objective lens, a high speed CMOS camera, image-splitting optics. Turn on the precision computer in the X Y motorized stage controller.Then turn on the amplifier and set the volume adjuster to 10 in the base and treble adjusters to eight. To excite GCaMP and tag RFP, turn on the 488 and 560 nanometer lights of the LED light source. Set the 470 and 550 nanometer gauges of the control pod in the light source to 5%so that the intensity of the LED light is suitable for imaging.Download and install the following software packages and windows. Mouse macro software, software for vibration control, software for running tracking, and software for data analysis. Double click on the tracking software file, then set the exposure time to 0.033 seconds, and binning to two by two to ensure smooth image acquisition.Click the run button and wait for five minutes to stabilize the software. Input the information for image acquisition. For example, to record 500 images without an interval, enter 500 in the images total box, 500 in the images box, and zero in the intervals box.After turning on the acquisition button, split the fluorescence image using image splitting optics, projecting the GCaMP channel and tag RFP channel onto two halves of the CMOS camera. Calibrate the coordinates of the GCaMP and tag RFP channels and save the settings. Then set the directory to output the data file and turn off the acquisition.Read the assay. text file with the mouse macro system so that the mouse cursor is controlled based on the coordinates and the schedule in that file and double click on the software file for vibration control. Set the coordinates of the mouse cursor in wait time until stimulation after starting the recording, then set the frequency and the amplitude values in the software for vibration control.Transfer a single worm expressing both GCaMP and tag RFP from the incubated plate to a fresh NGM plate where E coli OP50 has been streaked. Attach the NGM plate to the acoustic transducer using transparent adhesive tape. For calcium imaging, turn on the acquisition button and find the worm at 2.5x magnification.Set the field of view as 1.1 by 1.1 millimeter with a resolution of 2.6 microns per pixel. Turn off the bright light and click on the homing button to track the fluorescent spot of a worm, which initiates the movement of the X Y stage to keep the maximum intensity region of the worm in the middle of the field of view in the tag RFP image. Ensure that the values of intensity are approximately 1000.If not, fine tune the 470 and 550 nanometer gauges of the control pod in the light source. Press the run button in the mouse macro system to allow mouse cursor control, and verify that the output BMP file is appropriately created. For data analysis, create a folder on the desktop and name it Calcium Imaging"then create a folder inside the Calcium Imaging folder and name it CI result for the output result files.Double click on the dual view imaging. MB file written in the data analysis software, and insert the file paths to the CI result folder in the folder with the BMP file. Push the shift and return keys simultaneously to start an automatic analysis, which uses the value of a maximum intensity region in the tag RFP image.Finally, check whether the output files are appropriately created in the CI result folder. In the representative analysis, GCaMP and tag RFP channel data was obtained as a series of images. Displacement of the Petri dish induced by non-localized vibration system was also quantified.The displacement can be controlled by setting the amplitude value in the software for vibration control, and the volume adjuster and the amplifier. Whereas the frequency can be regulated by setting the frequency value in the software. A transient calcium response of AVA neurons was observed upon stimulation with vibrations having a frequency of 630 Hertz and a displacement of approximately 4.5 micrometers per second, indicating that the Ava neuron was activated during a worm's backward response to the non localized stimulation.In our procedure, we can quantify only a single neuron. However, this system can also be combined with a biometric imaging method to noninvasively quantify multiple neurons or even the whole brain.

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Research

JoVE Journal

Behavior

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about mechanosensory function that are relevant for clinical diagnosis as well as for basic research. Here, we describe two psychophysical tests designed to determine the vibration detection threshold (automated system) and tactile spatial acuity (handheld device). Both procedures implement a two-interval forced-choice and a transformed-rule up and down experimental paradigm. These tests have been used to obtain mechanosensory profiles for individuals from distinct human cohorts such as twins or people with sensorineural deafness.

Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man.

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Whole Body Vibration Methods with Survivors of Polio

The purpose of the original study was to examine the use of whole body vibration (WBV) on polio survivors with and without post-polio syndrome as a form of weight bearing exercise. The goal of this article is to highlight the strengths, limitations, and applications of the method used. Fifteen participants completed two intervention blocks with a wash-out period in between the blocks. Each block consisted of twice a week (four weeks) WBV interventions, progressing from 10 to 20 min per session. Low intensity (peak to peak displacement 4.53 mm, frequency 24 Hz, g force 2.21) and higher intensity (peak to peak displacement 8.82 mm, frequency 35 Hz, g force 2.76) WBV blocks were used. Pain severity significantly improved in both groups following higher intensity vibration. Walking speed significantly improved in the group who participated in higher intensity intervention first. No study-related adverse events occurred. Even though this population can be at risk of developing overuse-related muscle weakness, fatigue, or pain from excessive physical activity or exercise, the vibration intensity levels utilized did not cause significant muscle weakness, pain, fatigue, or sleep disturbances. Therefore, WBV appears to provide a safe method of weight bearing exercise for this population. Limitations included the lack of measurement of reflexes, muscular activity, or circulation, the difficulty in participant recruitment, and insufficient strength of some participants to stand in recommended position. Strengths included a standard, safe protocol with intentional monitoring of symptoms and the heterogeneity of the participants in their physical abilities. An application of the methods is the home use of WBV to reduce the barriers associated with going to a facility for weight bearing exercise for longer term interventions, and benefits for conditions such as osteoporosis, particularly for aging adults with mobility difficulties due to paralysis or weakness. Presented method may serve as a starting point in future studies.

The goal of this article is to highlight the strengths, limitations, and applications of the method used with whole body vibration on polio survivors with and without post-polio syndrome as a feasible and safe form of weight bearing exercise.

The purpose of the original study was to examine the use of whole body vibration (WBV) on polio survivors with and without post-polio syndrome as a form ...

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...