Name: quantifying infra-slow dynamics of spectral power and heart rate in sleeping mice
Uploaded: 2017-08-02T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice at JoVE.com

We thank all lab members for their contribution to the writing and careful reading of this manuscript. We are grateful to Paul Franken for stimulating discussions, Dr. Gis&#232;le Ferrand for helpful comments on the surgical protocol, and Dr. Jean-Yves Chatton for providing the original Labview executable files for the noise exposure. Funding was provided by the Swiss National Science Foundation (Grants 31003A_146244 and 31003A_166318) and the Etat de Vaud.

All experimental procedures were performed in accordance with the University of Lausanne Animal Care Committee and the Service de la Consommation et des Affaires V&#233;t&#233;rinaires of the Canton de Vaud.
1. Surgery for EEG/EMG-ECG Recordings
<ol>
	<li>Animal housing and selection.
	<ol>
		<li>Keep animals (C57Bl/6J, 7 - 9 weeks, 25 - 30 g) in a 12:12-h dark/light cycle, singly housed, and under standard conditions (40% humidity, 22 &#176;C), with food and water available...

<ol>
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Here, we show how to establish a continuous temporal profile of non-REM sleep that integrates EEG, EMG, and ECG variables. This is a first step towards developing an integrative description of mouse sleep, which can help to identify the previously unrecognized timescale over which high and low resilience to noise are organized during non-REM sleep1. A similar temporal structure was also described in human non-REM sleep through an analogous analysis1.
<p class="jove_cont...

Mammalian sleep is a behavioral state of rest and of resilience to environmental stimuli. In spite of this apparent uniformity, polysomnographic and autonomic parameters indicate that sleep moves between qualitatively and quantitatively different neural and somatic states on various temporal and spatial scales1. Over minutes to tens of minutes, the switch between non-REM and REM sleep occurs. Non-REM sleep is accompanied by large-amplitude, low-frequency activity in the EEG, with a spectral peak around ~0.5 - 4 Hz, whereas REM sleep shows regular EEG activity in the theta band (6 - 10 Hz), together with muscle atonia2. Within non-REM sleep, humans cycle through light (S2) and deep slow-wave sleep (SWS). As their naming indicates, these two stages show lower and higher arousal thresholds3,4, respectively, and they differ primarily in the density of low-frequency cortical EEG power, referred to as slow-wave activity (SWA; 0.75 - 4 Hz). Non-uniformity persists throughout individual bouts of S2 and SWS on the minute- to sub-second-timescale, as extensively documented by the variable presence of SWA over the course of a bout5,6, but also by EEG and field potential rhythms at higher frequencies, including spindle waves in the sigma band (10-15 Hz) and gamma rhythms (80 - 120 Hz) (for a review, see7,8,9,10).
Rather than being subtle, these variations do shift the sleeping cortical state in humans to the extremes of the spectrum. For non-REM sleep, these range from a predominance of SWA to states that approximate wake-like activity because they contain a substantial proportion of high-frequency components11,12. In rodents and cats, although non-REM sleep is not subdivided into stages, a brief period called intermediate sleep (IS) emerges prior to REM sleep onset13. During IS, REM sleep features such as hippocampal theta activity and ponto-geniculo-occipital waves initiate, while non-REM sleep signatures, such as spindle waves and SWA, are still present, indicating a mix between the two sleep states14,15. Nevertheless, IS could be functionally distinct because it is modulated by antidepressants16 and through novel object presentation during prior waking17, and it contributes to setting the arousal threshold18. Furthermore, state space plots of EEG and EMG parameters of freely moving rats show a cluster of points14 that is continuous between non-REM sleep, REM sleep, and wakefulness. There are also sporadic declines in SWA, without entering wakefulness or REM sleep, which leads to substantial fluctuations in the relative presence of the low- and high-frequency components during a consolidated non-REM sleep bout14,19,20. Finally, variable ratios of SWA and higher frequency rhythms during non-REM sleep occur not only in time, but also show regional differences in amplitude and synchronization between cortical areas19.
Mammalian non-REM sleep is far from uniform. However, whether such non-uniformity leads to states that differ in function and behavioral attributes is not clear. In several types of sleep disorders, continuous sleep is disrupted by spontaneous awakenings and inappropriate motor behavior. Moreover, spectral analyses show alterations in the relative presence of higher frequencies in the EEG21 and in autonomic parameters, such as rates of breathing and cardiac beating22. The orderly sequence of stable sleep states is thus perturbed, and elements of cortical and/or autonomic arousal intrude in an uncontrolled manner. Therefore, understanding the continuum of sleep states is of possible relevance for disease. Additionally, the perturbance of sleep by environmental noise in urban environments is associated with general health risks, rendering it crucial to identify moments of heightened vulnerability during sleep23.
Behavioral arousal experiments in sleeping humans indicate that it is hardest to wake up from SWA-dominated non-REM sleep (stage S3), whereas light non-REM sleep (stage S2) and REM sleep show comparable and lower arousal thresholds4. The cortical processing of short sound stimuli varies substantially between REM sleep, S2, and S324,25, indicating that state-specific cortical activity patterns modulate the first stages of sensory processing. For non-REM sleep in humans, the propensity to wake up in response to noise varies with the presence of spindle waves and alpha rhythms in the EEG26,27,28. Thalamocortical rhythmicity during spindles is accompanied by enhanced synaptic inhibition at both the thalamic and cortical levels, which is thought to contribute to the attenuation of sensory processing7.
How are noise-resistant and vulnerable periods of sleep organized in time, and what are their determinants? In both mice and human, we recently identified an infra-slow, 0.02-Hz oscillation in neural rhythms. Depending upon the phase of this 0.02-Hz oscillation, mice showed variable reactivity to external stimuli, either waking up or sleeping through the noise. Interestingly, this oscillation was correlated with the rate of the heartbeat, indicating that the autonomic nervous system participates in the modulation of sleep&#39;s vulnerability to external stimuli1. Memory-related hippocampal rhythms were also organized within this rhythm, and, most strikingly, its strength correlated with the quality of memory consolidation in humans. The 0.02-Hz oscillation thus appears to be an organizing principle of rodent and human non-REM sleep that modulates both sensitivity to the environment and internal memory processing. This again highlights the need for multiparametric and continuous assessments of sleep states to recognize their functionality and to identify sites of potential vulnerability.
Here, we present a procedure to extract the waveform of these dynamics, including the surgical implantation of mice for combined EEG/ECoG and EMG-ECG measurements, exposure to sensory stimuli, and analysis routines. This procedure provides a basis for viewing sleep as a continuously varying yet highly organized vigilance state during which different fundamental sleep functions are sequentially executed. More generally, the procedure is applicable to approaches aiming to extract the spectral and autonomic features that precede a behavioral outcome during sleep in both health and disease states.

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		<tr height="21">
			<td height="21" style="height:21px;width:316px;">2-components epoxy glue</td>
			<td style="width:128px;">Henkel</td>
			<td style="width:177px;">Loctite EA 3450</td>
			<td style="width:605px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Absorbable Suturing Fiber (Prolene)</td>
			<td>Ethicon</td>
			<td>5-0 FS-3</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td>FST</td>
			<td>11006-12</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Antiseptic swab</td>
			<td>VWR</td>
			<td>149-0332</td>
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			<td height="21" style="height:21px;">Attane Isoflurane</td>
			<td>Piramal</td>
			<td>Isoflurane 250mL</td>
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			<td height="21" style="height:21px;">Connectors 3x2-channels</td>
			<td>ENA AG</td>
			<td>2.316</td>
			<td>Raster 2.00 x 2.00 mm; size 5x8x9 mm; pin size 5mm; http://www.ena.ch/</td>
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			<td height="21" style="height:21px;">Dragonfly commutator</td>
			<td>Dragonfly</td>
			<td>Model #SL-10</td>
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			<td height="21" style="height:21px;">EMBLA amplifier</td>
			<td>EMBLA</td>
			<td>A10 amplifier</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>FST</td>
			<td>14108-09</td>
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			<td height="21" style="height:21px;">Flat Head Gold-plated steel screw</td>
			<td>J.I. Morris</td>
			<td>FF00CE125</td>
			<td>https://jimorrisco.com/</td>
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			<td height="21" style="height:21px;">Gold wire</td>
			<td>CMSA</td>
			<td>T.69 5gr</td>
			<td>http://www.cmsa.ch/en/</td>
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			<td height="21" style="height:21px;">Hemostatic sponge</td>
			<td>Pfizer</td>
			<td>Gelfoam</td>
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		<tr height="21">
			<td height="21" style="height:21px;">iodine-based disinfectant (Betadine)</td>
			<td>Mundipharma</td>
			<td>standart solution 60mL</td>
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			<td height="21" style="height:21px;">Komet drill steel 1/005PM104</td>
			<td>UNOR AG</td>
			<td>22310</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Matlab Analysis Software</td>
			<td>MathWorks</td>
			<td>R2016b</td>
			<td>https://ch.mathworks.com/products/matlab.html</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microdrill</td>
			<td>Fine Science Tools</td>
			<td>96758</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse Gas Anesthesia Head Holder</td>
			<td>Kopf Instruments</td>
			<td>Model 923-B</td>
			<td>http://kopfinstruments.com/product/model-923-b-mouse-gas-anesthesia-head-holder/</td>
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			<td height="21" style="height:21px;">Ophtalmic ointment</td>
			<td>Pharmamedica</td>
			<td>VITA-POS</td>
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			<td height="21" style="height:21px;">Paladur (liquid)</td>
			<td>UNOR AG</td>
			<td>2260215</td>
			<td>for dental cement</td>
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			<td height="21" style="height:21px;">Palavit (powder)</td>
			<td>UNOR AG</td>
			<td>5410929</td>
			<td>for dental cement</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Frame</td>
			<td>Kopf Instruments</td>
			<td>Model 930</td>
			<td>http://kopfinstruments.com/product/model-930-small-animal-stereotaxic-frame-assembly/</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Soldering wire</td>
			<td>Stannol</td>
			<td>593072</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller - Mini rectal probe</td>
			<td>Phymep</td>
			<td>4090502</td>
			<td>http://www.phymep.com/produit/dc-temperature-controller/</td>
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			<td height="21" style="height:21px;">Temperature controller- heating pad</td>
			<td>Phymep</td>
			<td>4090205</td>
			<td>http://www.phymep.com/produit/dc-temperature-controller/</td>
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	</tbody>
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quantifying infra-slow dynamics of spectral power and heart rate in sleeping mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Figure 2A (top panel) shows 100-min stretches of spontaneous sleep-wake behavior, recorded through polysomnographic electrodes implanted as described (see Figure 1). The increases and decreases of the EEG and EMG amplitude at non-REM sleep onset are clearly visible. Intermittent REM sleep is marked by a decrease in EEG amplitude and a further decrease in EMG tone that is not visible on this compressed time scale....

Watch this Scientific Journal Video about Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice at JoVE.com

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

The overall goal of this procedure, is to describe how neural and cardiac activities are coordinated during mouse non REM sleep. Through monitoring and analyzing electroencephalographic, electromyographic, and electrocardiographic signals. This method can help answer key questions in the sleep field.Such as how sleep reconciles two essential and evolutionarily preserved needs. Which are to be both continuous and fragile. The main advantage of this technique is that it uses standard polysomnography to score non REM sleep in a novel way that is based on the variable behavioral responses to external stimuli.We first had the idea for this method when we observed that sleeping mice showed variable responses to noise such as waking up and sleeping through. Generally individuals new to this method will struggle because it combines surgical skill, bare minimal experimentation, and advancing analysis. Visual demonstration of this method is critical as several essential steps are new to the field.In particular concerning the behavioral design and data analysis. The implications of this technique extend towards diagnosis of sleep disorders in humans. Because it identifies the moments of sleep that are highly fragile.To begin the procedure, prepare to EEG ecogeolectrodes using 0.5 centimeter long pieces of gold wire, each soldered on top of a gold plated steel screw. Then clean them in 70%ethanol. Next prepare two EMG-ECG electrodes with three to four centimeter long gold wires.Bend the wires at a 90 degree angle one centimeter from one end and prepare a coil at the other end. Between the two ends bend the wire to create a small curvature that corresponds to the surface profile of the bone between the cerebellum and lambda. After that prepare a six channel female to male head connector by covering the connector of both female and male pins at the base with tape.Then add to the pins at the corners some tin to facilitate the soldering process. In this step, place an anesthetized animal on the stereotaxic apparatus by positioning the mouth. This will fix the mouse head.Then fix the head by positioning the ear bars at the skull with their blunt reverse ends. Subsequently lift the skin at the center of the skull and gently cut the lifted portion of the skin along the mid line from the top of the neck to the level of the eyes. Then remove the scalp.Ensure that the window is large enough to clearly see the bregma and the lambda fissures of the skull. Fix the skin on both sides with bulldog serafines to ensure access to the bone. Next carefully remove the conjunctive tissue with a scalpel.Clean the area with iodine based disinfectant and dry the skull with an antiseptic swab. Then using a sharp scalpel blade, scratch the skull to obtain a cleaned and mattified bone surface. Using only the scalpel tip, scratch a grid like meshwork of groves with a distance of one to two millimeters between the grooves.Afterward, perform four craniotomies in the skull at specific locations with a microdrill. While drilling, blow away the bone dust using a pasteur pipette and clean any bleeding with antiseptic swabs. If bleeding occurs, ensure that it is stopped completely before resuming the process.Use a hemostatic sponge to accelerate hemostasis. Fix the screw in a hemostatic clamp. On the left hemisphere, screw two gold plated screws through the craniotomies.Hold the clamp vertically above the craniotomy and rotate the screw while not deviating from the vertical position. Gently open the hemostatic clamp to release the screw. On the right hemisphere screw the previously prepared electrodes through the craniotomies.Add a drop of glue on the middle part of the skull to ensure tight adhesion of the left EMG-ECG. Carefully lift the border of the skin from the neck muscles with forceps and insert the EMG-ECG wires with the coiled ends in the muscles. To detect the heart ECG signals during sleep, ensure that the EMG-ECG wire is inserted into the muscle to a depth of about 0.8 to one centimeter.Position the left EMG-ECG next to the posterior left anchoring screw. Repeat the procedure to insert the right EMG-ECG. Position the right EMG-ECG next to the anterior left anchoring screw.Ensure that the loop ends are placed as far as possible from each other. It is important that the EMG-ECG wires are inserted deeply enough into the muscle in order for the heart rate to be detected reliably. Using a spatula apply the two component epoxy glue to the skull between and around the screws.Let it dry in the light but protect the eyes of the animal from excessive lighting. Try to place the connector pins as closely as possible to minimize the height of the implant using a small crocodile lamp. Solder the four pins at the corner of the connector to the four wires.Minimize the time in contact with the soldering tip as this rapidly heats the screws. Next, fill the space between the glue and the connector with dental cement and cover the soldered parts. Create smooth surface and avoid sharp edges that could hurt the animal.In addition, avoid touching the skin as this leads to itching. Now remove the bulldog serafines. Disinfect the wound with iodine based disinfectant.If necessary, close the wound with suture points in front and behind the connector. Afterwards, monitor the animal every day for a week and look for weight loss, reduced or abnormal activity, and signs of infection. Five to six days after surgery, connect the recording cable to the head connector on the animal, leaving it in its home cage.Wait an additional four to five days before the start of recording so that the animal is habituated to the condition and sleeps naturally. With a commercial polysomnographic software, record EEG and EMG-ECG data over 48 hours using typical settings such as 2000 x gain, a 2000 hertz sampling rate at acquisition, down sampled to 200 hertz after acquisition. Apply a 0.7 hertz high pass filter for all the EEG traces and a 10 hertz high pass filter for all the EMG-ECG traces.In this demonstration, the mouse has been sleeping for more than 40 seconds. Here are the characteristic low frequency, high amplitude signals in the EEG and the low muscle tone in the EMG traces. It is important that the mouse sleep stably for at least 40 seconds before the noise is played to ensure a long base line for the analysis of sleep, prior to wake up or sleep through.Now an acoustic stimulus, here a white noise, will be delivered. In this case, the mouse wakes up within 10 seconds. Note that the EEG desynchronizes when the mouse wakes up.In another case, the mouse does not wake up, even though the noise has been delivered for more than 20 seconds. Note that the EEG does not desynchronize. Shown here are the raw traces of the EEG in black and the corresponding EMG-ECG signals in gray, for 40 seconds before the noise onset and during the 20 seconds of noise.This is a sleep through event. And this is an example of the wake up event. The corresponding analyzed data for sigma power dynamics in the 40 seconds window before noise onset are shown here for sleep through and wake up.The key result of this analysis is that the sigma power is at its trough before noise onset for a sleep through event and at its peak for a wake up event. In contrast to the wake up and sleep through events presented before, we discarded the events with an early transition into REM sleep. And we also discarded the events with an early wake up before noise onset.While attempting this procedure, it's important to ensure that EMG signal detects the heart rate throughout non REM sleep. Moreover remember that the analysis procedure needs to be understood and implemented in a software script to extract the infra-slow oscillation. Following this procedure, other methods, such as local electrophysiological recordings, or imaging approaches can be performed to follow activity patterns in defined brain areas.After redevelopment, this technique paves the way for researchers in the field of human sleep to explore how neural and cardiac activities are organized in the different sleep stages in healthy sleepers and in sleep disorder patients. After watching this video, you should have a good understanding of how to score and analyze non REM sleep retrospectively based on the behavioral reactivity of the sleeping mouse to an acoustic stimulus.

quantifying-infra-slow-dynamics-spectral-power-heart-rate-sleeping

Research

JoVE Journal

Neuroscience

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain signal. Typically, researchers represent the brain's response as the mean across repeated experimental trials and disregard signal fluctuations over time as "noise". However, it is becoming clear that brain signal variability conveys meaningful functional information about neural network dynamics. This article describes the novel method of multiscale entropy (MSE) for quantifying brain signal variability. MSE may be particularly informative of neural network dynamics because it shows timescale dependence and sensitivity to linear and nonlinear dynamics in the data.

Neuroimaging researchers typically consider the brain's response as the mean activity across repeated experimental trials and disregard signal variability over time as "noise". However, it is becoming clear that there is signal in that noise. This article describes the novel method of multiscale entropy for quantifying brain signal variability in the time domain.

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain ...

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 ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Electroencephalographic, Heart Rate, and Galvanic Skin Response Assessment for an Advertising Perception Study: Application to Antismoking Public Service Announcements

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these approaches often appear poorly accurate in scientific terms. Neuroscience is increasingly applied to the investigation of the neurophysiological bases of the perception of and reaction to commercial stimuli to support traditional marketing methods. In this context, a particular sector or marketing is represented by public service announcements (PSAs). The objective of this protocol is to apply electroencephalography (EEG) and autonomic signal analysis to study responses to selected antismoking PSAs. Two EEG indices were employed: the frontal alpha band EEG asymmetry (the Approach Withdrawal (AW) index) and the frontal theta (effort index). Furthermore, the autonomic Emotional Index (EI) was calculated, as derived from the Galvanic Skin Response (GSR) and Heart Rate (HR) signals. The present protocol describes a series of operational and computational steps required to properly estimate, through the aforementioned indices, the emotional and cerebral reaction of a group of subjects towards a selected number of antismoking PSAs. In particular, a campaign characterized by a symbolic communication style (classified as "awarded" on the basis of the prizes received by specialized committees) obtained the highest approach values, as estimated by the AW index. A spot and an image belonging to the same PSA campaign based on the "fear arousing appeal" and with a narrative/experiential communication style (classified as "effective" on the basis of the economical/health-related improvements promoted) reported the lowest and highest effort values, respectively. This is probably due to the complexity of the storytelling (spot) and to the immediateness of the image (a lady who underwent a tracheotomy). Finally, the same "effective" campaign showed the highest EI values, possibly because of the empathy induced by the testimonial and the explicitness of the message.

The following protocol describes a series of operational and computational steps required to properly estimate the emotional and cerebral reaction of a group of subjects towards a selected number of Public Service Announcements (PSAs) against smoking, aired in the USA and Europe during the period between 1998 and 2015.

The evaluation of advertising, products, and packaging is traditionally performed through methods based on self-reports and focus groups, but these ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...