Name: Video: Video-oculography in Mice
Uploaded: 2012-07-19T12:00:00.000+00:00
Duration: 9 min 43 s

We kindly thank the Netherlands Organisation for Health Research and Development (M.D.J, C.D.Z), The Netherlands Organisation for Scientific Research (C.D.Z), NeuroBasic (C.D.Z), Prinses Beatrix Fonds (C.D.Z), The SENSOPAC (C.D.Z), C7 (C.D.Z) and the CEREBNET (C.D.Z) program of the European Community for their financial support.

1. Preparation
The following experiments were conducted in accordance with The Duch Ethical Committee for Animal Experiments.
<ol>
	<li>Preparing mice for video-oculography. In order to measure eye movements of a mouse, the head of the mouse needs to be immobilized. Therefore, a pedestal construction is made on the skull of the mouse (Figure 1).
	<ol>
		<li>Anesthetize the mouse by a mixture of isoflurane (isofluran 1-1.5%; Rhodia Organique Fine LtD, France) and oxygen in a gas chamber. The excessive gas is scavenged. Maintain anesthesia via nose cone. Confirm depth of anesthesia via a toe pinch.</li>
		<li>Maintain the body temperature at 37 &deg;C with the use of an anal thermosensor and a heating pad (FHC, Bowdoinham, ME).</li>
		<li>Protect the eyes by covering them with an eye ointment (duratears, Alcon, Belgium). Shave the dorsal cranial fur, and clean the surgical area with a rotation of scrub and betadine or chlorhexidine solution.</li>
		<li>Make a middle line incision to expose the dorsal cranial surface of the skull. Make the surface clean and dry.</li>
		<li>Apply a drop of phosphoric acid (phosphoric acid gel etchant 37.5%; Kerr, CA) on the dorsal cranial surface of the skull from bregma to lambda. Remove the etchant after 15 seconds and make the cranial surface clean with saline and dry again.</li>
		<li>Apply on top of this etched cranial surface a drop of OptiBond prime (Kerr, CA) and air-dry it for 30 seconds.</li>
		<li>Place a drop of OptiBond adhesive (Kerr, CA) on top of the OptiBond prime and cure with light for 1 minute (Maxima 480 visible light curing unit; Henry Schein, USA).</li>
		<li>Cover the adhesive layer with a thin layer of Charisma composite (Heraeus Kulzer, Germany). Embed two connected nuts (diameter: 3 mm) in the composite. Cure the composite afterwards with light. When necessary, apply additional layers of composite and cure them with light.</li>
		<li>Administer buprenorphine (0.015 mg/kg, s.c) for post-operative analgesia. The animal should be back on its feet within approximately 5 min. Allow the mouse to recover in the home cage at room temperature for at least 3 days after the surgery. </li>
	</ol>
	</li>
	<li>Video-oculography setup for mice (Figure 2).
	<ol>
		<li>Place the mouse in the restrainer and fix his head to the restrainer by two screws (Figure 1). The mouse does not need to be anesthetized for this procedure. Restraining time should not exceed 1 hr/day.</li>
		<li>Mount the mouse head-and-body restrainer on an X-Y platform, which in turn is mounted upon the turntable (diameter: 60 cm). Using the X-Y platform the mouse head can be placed above the center of the turntable. The mouse can be moved over the pitch, yaw and roll axes. The head of the mouse is placed in the correct pitch, yaw and roll angle by aligning the eye using the visual image of the eye generated by the ISCAN system. Alternatively, the pedestal construction can be placed on the head of mouse in a stereotactic frame11.</li>
		<li>The turntable is attached to an AC servo-controlled motor (Harmonic drive AG, the Netherlands) and the position of the turntable is monitored by a potentiometer (Bourns inc., CA) attached to the turntable axis.</li>
		<li>A cylindrical surrounding screen (diameter: 63 cm; height: 35 cm) with a random dotted pattern (each element 2&deg;) covers the turntable; this drum is also equipped with an AC servo-controlled motor (Harmonic drive AG, the Netherlands). The position of the cylindrical screen is monitored by a potentiometer (Bourns inc., CA) attached to its axis and the screen can be lit by a halogen light (20 Watt). Both the surrounding screen and the turntable are driven independently.</li>
		<li>The movement of the turntable and surrounding screen is controlled by a computer that is connected to an I/O interface (CED limited, Cambridge, United Kingdom). Table and surrounding screen position signals are filtered (cut-off frequency: 20 Hz), digitized by the I/O interface and stored on this computer.</li>
		<li>The eye of the mouse is illuminated by three infrared emitters (600 mW, dispersion angle: 7&deg;, peak wavelength: 880 nm, RS components, the Netherlands). Two infrared emitters are fixed to the turntable and the third emitter is attached to the camera. This third emitter produces a reference corneal reflection (CR), which is used during the calibration procedure and during the eye movement recordings.</li>
		<li>An infrared CCD camera equipped with a zoom lens (Zoom 6000, Navitar inc., NY) is attached to the turntable and is focused on the mouse head in the center of the turntable. The camera can be unlocked and can be yawed about the turntable axis over exactly 20&deg; during the calibration procedure.</li>
		<li>The video signal is processed by an eye tracking system (ETL-200, ISCAN, Burlington, MA). The ISCAN system uses an algorithm to track the centers of the pupil and the reference CR. The system can track the pupil and reference CR in horizontal and vertical direction at a sample rate of 120 Hz.</li>
		<li>Reference CR position, pupil position and pupil size signals are digitized by the I/O interface and are stored in the same file as the table and surrounding screen position signals. The video pupil-tracking system induces a delay of the eye movement signals of approximately 27 ms.</li>
	</ol>
	</li>
</ol>
2. Calibrating and Measuring Eye Movements Using Video Pupil-tracking
The eye tracking system captures the movement of the pupil as a translational motion. The translational motion of the tracked pupil contains a translational component due to axial difference between the rotational center of the eye and the anatomical center of the eye (i.e. center of corneal curvature), and a rotational component due to the angular rotation of the eyeball. By subtracting the reference CR from the pupil movement/position, the undesired translational component is eliminated from the signal, resulting in a translational motion that is only due to the rotation of the eyeball. Although they are often very small, this subtraction also eliminates the translations between the head and the camera. The residual isolated translational motion is converted into the angular rotation of the eyeball by the following calibration method8,12. This calibration was performed prior to any eye movement experiment.
<ol>
	<li>Adjust the mouse head position to the camera in such a way that the video image of the pupil is situated at the middle of the monitor and that the representation of the reference CR is located on the vertical midline of the eye preferably direct above the pupil. Minimize the movements of the reference CR due to angular camera rotations, which can be accomplished by placing the center of the corneal curvature over the camera/table axis.</li>
	<li>Rotate the camera several times by +/- 10&deg; (i.e. 20 degrees peak to peak) around the vertical axis of the turntable. Use the positions of the tracked pupil (P) and the reference CR recorded in the extreme positions of the camera rotation to calculate the radius of rotation of the pupil (Rp; Rp = &Delta;/sin(20&deg;); where &Delta;=(CR-P), see Figure 3A).</li>
	<li>Due to the fact that the Rp value depends on the pupil size, a pupil size correction needs to be implemented12 (Figure 3B). Repeat step 2.2 many times under various illumination conditions (i.e. manipulating the pupil size; Figure 3C) in order to determine the pupil size - Rp relationship and compose an Rp correction curve (Figure 3D). The Rp value also depends on the vertical eye position. When the experiment will cause vertical eye movements then a correction of the calibration for vertical eye positions is highly recommendable13.</li>
	<li>Determine the angular position of the eye (E) by measuring the reference CR position, P position and the pupil size. The reference CR position is subtracted from the pupil position generating a translational free pupil position. By measuring the pupil size the Rp value can be extracted from the Rp correction curve and E can be calculated by using the following formula E= arcsin {(&Delta;1)/Rp} (Figure 4A; where &Delta;1=(P2-P1) and P1 and P2 are corrected by subtraction of the reference CR).</li>
	<li>A large repertoire of turntable and/or surrounding screen rotations can now be used to stimulate the oculomotor system. In order to perform video oculography in the dark, the mouse eye needs to be pretreated with a miotic drug to limit the pupil dilatation and allow pupil tracking under these circumstances. In our experiments, we use pilocarpine (4%, Laboratories Chauvin, France) to limit pupil dilatation in the dark.</li>
</ol>
3. Data Analysis
<ol>
	<li>Eye positions, table positions and surrounding screen positions are all converted into angular positions (see Figure 4B and formula in 2.4). Eye signals are corrected for their delay of 27 ms induced by the imaging processing of the pupil-tracking system.</li>
	<li>Angular positions of eye, table and surrounding screen are differentiated and filtered with a Butterworth low-pass filter using a cut off frequency of 20 Hz.</li>
	<li>Saccades are removed from the eye velocity signal using a detection threshold of 40&deg;/s. Data is removed starting from 20 ms before and up to 80 ms after crossing the detection threshold.</li>
	<li>Table, surrounding screen and eye velocity signals are averaged using each individual cycle in the trail (Figure 4C).</li>
	<li>Averaged signals are fitted with an appropriate function. In general, a sinusoidal velocity stimulation is used and the averaged cycles are fitted with sinus or cosinus function (Figure 4C). Then, the gain can be computed as the ratio of eye velocity to stimulus velocity, whereas the phase can be computed as the difference (in degrees) between the eye velocity and stimulus velocity.</li>
</ol>
4. Representative Results
Video-oculography can be used to investigate various forms of oculomotor performances (i.e. optokinetic reflex: OKR; vestibulo-ocular reflex: VOR; visually enhanced vestibulo-ocular reflex: VVOR) as well as motor learning (VOR adaptation; OKR adaptation). The OKR compensates for low-frequency disturbances using visual feedback. The OKR can be induced by rotating the well-illuminated surrounding screen (Movie 1). Rotating the surrounding screen over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg; shows how the optokinetic system is a more efficient compensatory mechanism in the low-frequency range than in the high-frequency range (Figure 5A). The VOR compensates for high-frequency head movements using signals from the vestibular organs. The VOR can be induced by rotating the animal (i.e. turntable) in the dark (Movie 2). Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg; demonstrates how the vestibulo-ocular system is more efficient in generating compensating eye movements in the high-frequency range than in the low-frequency range (Figure 5A). When the optokinetic and vestibulo-ocular system act in concert, images can be stabilized on the retina over a broad range of head movements. Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6&deg;, while the surrounding screen is well-illuminated (Movie 3) shows how the eye generates "high gain" compensating movements over the entire frequency range (Figure 5A). All these gain and phase values are typical for mice, although gender14 and strain15,16,17 differences were reported.
The independent control over the turntable and the surrounding screen enables us to confront the mice with a mismatch between visual and vestibular information. After a long-term and uniform exposure of mismatched visual and vestibular information, the VOR of the mouse will change to compensate for the altered visual input (VOR adaptation; Movie 4). Rotating the turntable out of phase (i.e. 180&deg;) with the surrounding screen (1 Hz, 1.6&deg;) increases the VOR gain (Figure 5B). The maximal change in VOR gain, when using a one trial learning paradigm, is often reached after 30 minutes.
<img alt="Figure 1" src="/files/ftp_upload/3971/3971fig1.jpg" /> 
Figure 1. Schematic drawing of the mouse head-and-body restrainer. The body of the mouse is restrained using a plastic cylindrical tube with a diameter of 35 mm. The head of the mouse is immobilized by connecting the pedestal of the mouse to the iron bar with two screws. The iron bar makes an angle of 30 degree in order to position the head of the mouse in the normal pitch during ambulation. *, top view of the pedestal containing two nuts.
<img alt="Figure 2" src="/files/ftp_upload/3971/3971fig2.jpg" /> 
Figure 2. Schematic drawing of the mouse video-oculography setup.
<img alt="Figure 3" src="/files/ftp_upload/3971/3971fig3.jpg" /> 
Figure 3. Calibration of the video pupil-tracking system. A) The camera is rotated several times by +/- 10&deg; (i.e. 20 degrees peak to peak) around the vertical axis of the turntable. The tracked pupil (P) and the reference corneal reflection (CR) recorded in the extreme positions of the camera rotation are used to calculate the radius of rotation of the pupil (Rp). B) The radius of the pupil diameter is dependent on the size of the pupil. C) Example showing the effect of pupil size on pupil position during the calibration procedure (both measured in pixels (px)). D) Relationship between Rp and pupil diameter measured in a single mouse. The thirteen different pupil diameters were accomplished by altering the intensity of the surrounding light.
<img alt="Figure 4" src="/files/ftp_upload/3971/3971fig4.jpg" /> 
Figure 4. Measuring and analyzing eye movements using video pupil-tracking. A) The angular pupil position is calculated from radius of the pupil (Rp) and the position of the Pupil (P; corrected for CR position). B) Example of compensatory eye movement induced by stimulating the vestibular and visual system (visual enhanced VOR). The turntable was rotated sinusoidally at 0.6 Hz with an amplitude of 1.6&deg;, while the surrounding screen was well-illuminated. C) Analyses of the recording shown in B). Graph shows the averaged velocity trace of the turntable (blue) and pupil (red). These averaged traces were fitted with a sinusoidal function (black).
<img alt="Figure 5" src="/files/ftp_upload/3971/3971fig5.jpg" /> 
Figure 5. Performance and learning of the oculomotor system measured in one C57Bl6 mouse. A) Eye movements are generated by rotations of the surrounding screen (optokinetic reflex: OKR, top panels), by rotating the mouse in the dark (vestibulo-ocular reflex: VOR, middle panels) and by rotating the mouse in the light (visually-enhanced vestibulo-ocular reflex: VVOR, bottom panel) with frequencies ranging from 0.2 to 1.0 Hz at an amplitude of 1.6&deg;. The gain of the reflex was computed as the ratio of eye velocity to stimulus velocity (left panels) and phase of the reflex was computed from the phase difference between the eye velocity and stimulus velocity (right panels). B) Motor learning was accomplished by adaptively increasing the VOR using an out of phase training paradigm. The mouse was subject to a visuovestibular training paradigm in which the rotation of the mouse was out of phase (180&deg;) with the rotation of the surrounding screen (both rotating at 1.0 Hz, 1.6&deg;) for forty minutes. Every 10 minutes the VOR was tested (1.0 Hz, 1.6&deg;). In this mouse the out of phase training increased the VOR gain.
Movie 1. Animation showing the paradigm that induces OKR in mice <a href="https://www.jove.com/files/ftp_upload/3971/3971movie1.mov" target="_blank">Click here to view movie</a>.
Movie 2. Animation showing the paradigm that induces VOR in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie2.mov" target="_blank">Click here to view movie</a>.
Movie 3. Animation showing the paradigm that induces VVOR in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie3.mov" target="_blank">Click here to view movie</a>.
Movie 4. Animation showing the visuovestibular out of phase training paradigm that induces VOR adaptation (increase) in mice. <a href="https://www.jove.com/files/ftp_upload/3971/3971movie4.mov" target="_blank">Click here to view movie</a>.

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No conflicts of interest declared.

In order to obtain high-quality video eye movements recordings in mice several requirements are necessary. The calibration procedure needs to be performed in the above mentioned standardized matter. For example off-center calibration, when the pupil is not positioned on the vertical midline with the reference CR during the calibration procedure, will result in an underestimation of RP and consequently an overestimation of the eye movement. Furthermore, we recommend integrating the pupil size correction method in the calibration procedure12, because trials that show a very stable pupil size are very rare. Even a small stressor during the trial can already alter the pupil diameter substantially.
When designing an eye movement experiment, the following factors need to be taken into account or controlled for because they are known to affect the eye movement response: age13,18, gender14 and strain15,16,19. Furthermore, the experimental animal should have pigmented irises since pupil detection and tracking is impossible when the contrast between pupil and iris is too low, like in the BALB/c mouse. Extremely nervous or anxious animals need to be trained, prior to the experiment, to get used to the experimental set up and the restrained condition. This animal handling procedure results in less closure or semi-closure of the eyes and prevents the generation of eye fluids during the experiment, and consequently a better pupil tracking is accomplished.
Finally, acquiring and analyzing the data requires two to three hours per animal. Therefore, eye movement recordings will likely remain a specific procedure applied to selected mice and is not suitable as a high throughput screening test.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Isofluran</td>
 <td>Rhodia Organique Fine LTD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>FHC</td>
 <td>40-90-8</td>
 </tr>
 <tr>
 <td>Duratears</td>
 <td>Alcon</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphoric acid gel</td>
 <td>Kerr</td>
 <td>31297</td>
 </tr>
 <tr>
 <td>Optibond prime</td>
 <td>Kerr</td>
 <td>35369</td>
 </tr>
 <tr>
 <td>Optibond adhesive</td>
 <td>Kerr</td>
 <td>35369</td>
 </tr>
 <tr>
 <td>Charisma composite</td>
 <td>Heraeus Kulzer</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Maxima 480 light curing unit</td>
 <td>Henry Schein</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AC servo-controlled motor</td>
 <td>Harmonic drive AG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cylindric screen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Halogen light (20 W)</td>
 <td>RS components</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potentiometers(precision)</td>
 <td>Bourns inc.</td>
 <td>6574</td>
 </tr>
 <tr>
 <td>Power 1401 (I/O interface)</td>
 <td>CED limited</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Computers</td>
 <td>Dell</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Infrared emmitters</td>
 <td>RS components</td>
 <td>195-451</td>
 </tr>
 <tr>
 <td>ETL-200</td>
 <td>ISCAN</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Zoom lens (zoom 6000)</td>
 <td>Navitar inc.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pilocarpinenitrate (minims)</td>
 <td>Laboratoire Chauvin</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

video-oculography in mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Watch this Scientific Journal Video about Video-oculography in Mice at JoVE.com

Video-oculography in Mice

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Research

JoVE Journal

Neuroscience

23.6K Views.  Erasmus MC, Rotterdam, The Netherlands. Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Video: Video-oculography in Mice

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

Performing Intracerebroventricular Injection in Mice Using a Free-Hand Approach

Free-Hand Intracerebroventricular Injections in Mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

Author Spotlight: Insights, Innovations, and Future Avenues in Reproductive Neurocontrol 

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...