This work was supported by grants from the National Natural Science Foundation of China (31771219, 31871170), the Science and Technology Division of Guangdong (2013KJCX0054), and the Natural Science Foundation of Guangdong Province (2014A030313418, 2014A030313440).

All animals were paired-housed under standard conditions (12 h light/dark, constant temperature environment, free access to food and water) according to the Chinese Ministry of Science and Technology Laboratory Animals Guidelines and experiments were approved by the local ethical committee of Guangzhou University. This is a non-survival procedure.&#160;
NOTE: For data shown in the representative results, APP/PS1 (B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J) double-transgenic mice and littermate wild-type (WT) controls at 3-5 months of age, were used for recordings (n = 10, per group).
1. Animal anesthesia and surgery
<ol>
	<li>Weigh and anesthetize the mouse by your approved anesthesia regimen from your local animal care committee.&#160;</li>
	<li>Perform a tail or toe pinch with forceps to confirm deep anesthesia prior to surgery.</li>
	<li>Position the mouse in a stereotaxic apparatus and fix its head.</li>
	<li>Apply eye ointment on both eyes to keep moist. Follow your local animal care guidelines regarding pre- and postoperative analgesia.&#160;</li>
	<li>Shave the hair using surgical clippers. Make a small incision (12-15 mm) in the middle of the exposed surgical area with scissors. Using forceps, gently pull the scalp away from the midline.</li>
	<li>Separate the skin gently and remove residual tissue. Clean the skull using hydrogen peroxide-coated cotton buds.</li>
	<li>Drill two small holes of radii 1.0-1.5 mm on both left and right sides of the skull to allow insertion of the recording microelectrodes into the M2 regions under a stereomicroscope (Figure 1A). 
	NOTE: Stereotaxic locations of bilateral M2: 1.94 mm anterior to the bregma, 1.0 mm lateral to the midline, and 0.8-1.1 mm ventral to the dura.</li>
	<li>Remove the dura mater carefully with a tungsten needle.</li>
	<li>Pull glass borosilicate micropipettes (outer diameter: 1.0 mm) as recording microelectrodes with resistance of 1-2 M&#937;.</li>
	<li>Insert two separate recording microelectrodes filled with 0.5 M NaCl into the holes using mechanical micromanipulators (at 60&#176;,&#160;Figure 1B).</li>
</ol>
2.&#160;LFP recordings in bilateral M2 of mice
<ol>
	<li>Lower the left and right glass electrodes slowly into appropriate coordinates of bilateral M2&#160;(Figure 1C).</li>
	<li>For quality control, test the resistance of each electrode using the differential amplifier before capturing LFPs.</li>
	<li>Set the recording process at 0.1 Hz high-pass and 1,000 Hz low-pass with 1,000x amplification.</li>
	<li>Collect digitized&#160;raw LFP data of at least 60 s spontaneous activities in stable state, with mice breathing evenly at a respiratory rate of 2 breaths per second&#160;under anesthesia.</li>
	<li>After recording, slowly raise the electrodes out of the brain, then euthanize the mice by fast cervical dislocation.</li>
	<li>Save the data and analyze offline.</li>
</ol>
3. Cross-correlation analysis
<ol>
	<li>Click Analysis - Waveform correlation in the analysis software and import the data.</li>
	<li>Parameter settings
	<ol>
		<li>Define one waveform channel signal as the first channel and the other as the reference. Set width as 2 and offset as 1 (Figure 2A).</li>
		<li>Set the duration of both LFPs for 100 s by selecting the start time and end time. Press the Process button to perform cross-correlation analysis (Figure 2B). 
		NOTE: Simultaneous bilateral signals with such durations would be long enough to show neuronal spontaneous activities, thereby revealing the basic properties of synchronization.</li>
	</ol>
	</li>
	<li>Click File - Export As, then save the cross-correlation results corresponding to the resulting pop-up chart in .txt format.</li>
	<li>Open the .txt file (Figure 2C), remove the correlation values at time lags ranged 0 &#177; 0.01 s (since two continuous gamma waves have at least 0.01 s interval), then average the rest of the cross-correlation data in the negative time lag part or average the rest of the cross-correlation data in the positive time lag part.</li>
</ol>
4. Coherence analysis
<ol>
	<li>Import and run the data in the analysis software.</li>
	<li>Assign the two LFP signals to be the first and second waveform channels separately. Then set the block size value (Figure 3A). 
	NOTE: Block size means the number of data points used in the FFT. The larger the block size, the better the frequency resolution. Here we recommend setting it as 4096.</li>
	<li>Move the dotted lines manually to ensure the time accuracy for signals in both channels are being set as the same period (Figure 3B). Press the Add Area button to load the area and perform coherence analysis.</li>
	<li>Click File - Save As to save the coherence results corresponding to the resulting pop-up chart in .txt format (Figure 3B).</li>
</ol>

The authors have nothing to disclose.

We report here the procedure for in vivo bilateral extracellular recording, along with analyzing the synchronization of dual-region LFP signals, which is both flexible and easy to conduct for estimating brain hemisphere lateralization, as well as the connectivity, directionality or coupling between neural activities of two brain areas. This can be widely used to reveal not only group-neuronal activities, but also some basic properties of interregional electrophysiology, especially for labs which are interested in screening oscillatory activities or labs which do not have systems for multi-channel recording in behaving animals23.
In general, a series of techniques are available to monitor brain activities, including electroencephalography (EEG), magnetoencephalography (MEG), and functional magnetic resonance imaging (fMRI). Such methods have relatively lower temporal and spatial resolution in comparison with our presented recordings. For example, EEG is one of the oldest and most commercially available instruments for investigating extracellular activity of the brain. Although there are studies using &#34;high density&#34;&#160;EEG in freely moving rodents to improve the insufficient spatial resolution24,25,26, the skull always generates more noise and thus reduces the signal-to-noise ratio of cortical gamma oscillation, especially for small-sized mice. Our method with glass microelectrodes would be a good choice to prevent researchers from that &#34;distorting noise&#34;&#160;since microelectrodes could be inserted into the brain structure directly. Moreover, the recording glass pipettes used here are inexpensive, highly maneuverable, and can be applied to explore deeper brain areas not limited to cortical areas.
Close attention should be paid to the following. First, it is mandatory to carry out anesthesia strictly based on the body weight, and to test the depth of anesthesia hourly. This is because the physiological state of the mouse plays an important role in the quality of the LFP recorded, and any movement of the referencing sites caused by, e.g., sudden awakening of the animal, could generate background electrophysiological noise that would depreciate the availability. Second, because microelectrode resistance varies with the shape and diameter of the glass pipette tip, the heating must be carefully adjusted within the range for appropriate impedance when pulling microelectrodes. As described earlier in the protocol section, we found that the electrodes with impedance ranging from 1 to 2 M&#937; captured high qualitied cortical oscillatory activities.
Gamma oscillations reflect the neuronal synchronization of different brain regions when animals are engaged in learning or stimulation-cued tasks27,28,29. The synchronization of gamma-band modulates excitation rapidly to activate postsynaptic neurons effectively30. It is worth noting that although the gamma oscillation was defined in the present study as oscillatory activity with frequency in the range 25-80 Hz as shown by several groups28,31,32, there are studies that describe 30-70 Hz as low gamma and 70-100 Hz as high gamma33,34,35. Regardless of the definition, the principles for data analysis remain similar. In signal processing, cross-correlation is used for determining the time delay between electrical signals of two brain regions36. For signals under stimulation conditions, the durations selected for cross-correlation analysis could be shorter37.
Though there are limitations in the use of LFP recording for the evaluation of neural activities; for instance, it can neither distinguish between pre- and post-synaptic activities nor detect resting membrane potentials of the neurons recorded23, the approach introduced here serves as a useful tool for the measurement of activities of a group of neurons from different brain areas of mice, allowing the investigation of brain-area functional connectivity and the coupling of electrical signals before and after drug infusion.
Several explanations have been proposed for the emergence of hemispheric asymmetry, e.g., asymmetry enhances an individual&#39;s ability to perform two different tasks at the same time38; or asymmetry increases neural capacity, avoiding unnecessary duplication of neural networks39; or two different cognitive processes may be more readily performed simultaneously if they are lateralized to different hemispheres40. Hemisphere lateralization is assumed to provide cognitive advantages, but it changes with age12,41. Neuroimaging studies have shown consistently that prefrontal activation tends to be less lateralized in older adults than in younger individuals42,43. AD patients with early unilateral or bilateral pathological changes develop brain abnormalities, including lateralization associating with forgetfulness, slow responses to sound stimulation and cognitive decline11,44. We observed, in the present study, a disrupted level of hemisphere lateralization between left and right M2 of APP/PS1 mice at 3-5 months, which is the period when such mice do not aggregate apparent deposition of beta amyloid plaques45,46, implying that toxicity induced by soluble beta amyloid oligomers may contribute, at least in part, to aberrant cortical hemisphere lateralization, which could accelerate brain deterioration in AD pathogenesis16,47.

Alzheimer&#39;s disease (AD) is the most common form of dementia1,2. Extracellular beta amyloid protein (&#946;-amyloid protein, A&#946;) deposition and intracellular neurofibrillary tangles (NFTs) are the main pathological features of AD3,4,5, but the mechanisms underlying AD pathogenesis remain largely unclear. Cerebral cortex, a key structure in cognition and memory, is impaired in AD6, and motor deficits such as slow walking, difficulty navigating the environment and gait disturbances occur with advancing age7. A&#946; deposition and neurofibrillary tangles have also been observed in the premotor cortex (PMC) and supplementary motor area (SMA) in AD patients8 and cognitively impacted older adults9, indicating the involvement of an impaired motor system in AD pathogenesis.
The brain is formed by two distinct cerebral hemispheres that are divided by a longitudinal fissure. A healthy brain exhibits both structural and functional asymmetries10, which is called &#34;lateralization&#34;, allowing the brain to efficiently deal with multiple tasks and activities. Aging results in a deterioration in cognition and locomotion, together with a reduction in brain laterality11,12. The motor abilities of the left hemisphere are readily apparent in the healthy brain13, but in the AD brain aberrant laterality occurs as a consequence of the failure of left hemisphere dominance associated with left cortical atrophy14,15,16. Therefore, an understanding of a possible alteration of brain lateralization in AD pathogenesis and the underlying mechanisms may provide new insights into AD pathogenesis and lead to identification of potential biomarkers for treatment.
Electrophysiological measurement is a sensitive and effective method of evaluating changes in the neuronal activities of animals. The reduction of hemispheric asymmetry in elders (HAROLD)17 has been documented by electrophysiological research with synchronized interhemispheric transfer time, which shows weakening or absence of hemispheric asymmetry to monaurally presented speech stimuli in the elderly18. Utilizing APP/PS1, one of the most commonly used AD mouse models19,20,21,22, in combination with in vivo bilateral extracellular recording of LFPs in both left and right M2, we evaluated possible laterality deficits in AD. In addition, with simple parameter settings, the built-in function of data analysis software (see the Table of Materials) provides a faster and more straightforward way to analyze the synchronization of electrical signals than mathematically complex programming language, which is friendly to beginners with in vivo electrophysiology.

<table border="0" cellpadding="0" cellspacing="0" style="width:892px;" width="891">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">AC/DC Differential Amplifier</td>
			<td style="width:261px;">A-M Systems</td>
			<td style="width:137px;">Model 3000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Analog Digital converter</td>
			<td style="width:261px;">Cambridge Electronic Design Ltd.</td>
			<td style="width:137px;">Micro1401</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Glass borosilicate micropipettes</td>
			<td style="width:261px;">Nanjing spring teaching experimental equipment company</td>
			<td style="width:137px;">161230</td>
			<td>Outer diameter: 1.0mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Microelectrode puller</td>
			<td style="width:261px;">Narishige</td>
			<td style="width:137px;">PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NaCl</td>
			<td style="width:261px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:137px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Pin microelectrode holder</td>
			<td style="width:261px;">World Precision Instruments, INC.</td>
			<td style="width:137px;">MEH3SW10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Spike2&#160;</td>
			<td style="width:261px;">Cambridge Electronic Design Ltd.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Stereomicroscope</td>
			<td style="width:261px;">Zeiss</td>
			<td style="width:137px;">435064-9020-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Stereotaxic apparatus&#160;</td>
			<td style="width:261px;">RWD Life Science</td>
			<td style="width:137px;">68045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Urethane</td>
			<td style="width:261px;">Sigma-Aldrich</td>
			<td style="width:137px;">94300</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of hemisphere lateralization with bilateral local field potential recording in secondary motor cortex of mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

To see whether early AD pathology impairs the capacity of hemisphere lateralization, we conducted bilateral extracellular LFP recordings in the left and right M2 of APP/PS1 mice and WT controls (aged 3-5 months), and analyzed the cross-correlation of these left and right LFPs. In WT mice, the results demonstrated that the mean correlation between left and right LFPs at positive time lags differed significantly from that at negative time lags, implicating the existence of hemispheric asymmetries in M2 areas of WT controls (Figure 4C; WT-positive, 0.08161 &#177; 0.01246; WT-negative, 0.0206 &#177; 0.01218; p = 4.74531E-4 &#60; 0.001 by a two sample t-test). In comparison, the left and right LFPs of APP/PS1 mice showed higher synchronized in time domain, suggesting a reduction of asymmetry between the left and right M2 (Figure 4C; APP/PS1-positive, 0.13336 &#177; 0.0105 APP/PS1-negative, 0.12635 &#177; 0.01066; p = 0.64157 &#62; 0.05 by a two sample t-test).
We then filtered gamma oscillations from the LFPs (Figure 5A) and performed a coherence analysis as described in the protocol to measure the similarity of electrical signals in the gamma frequency range. The result showed that the gamma coherence between left and right M2 in APP/PS1 was significantly higher than that in WT mice (Figure 5B,C; WT, 0.13267 &#177; 0.00598; APP/PS1, 0.17078 &#177; 0.0072; p = 0.00550 &#60; 0.01 by two sample t-test), indicating a higher synchronization, and consequently reduced lateralization, between left and right M2 in APP/PS1 mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59310/59310fig1.jpg" /> 
Figure 1: Diagram of the simultaneous LFP recording procedure. (A) Stereotaxic mouse with skull exposed and dura mater removed for in vivo bilateral recording of LFPs in left and right M2. (B) Two glass microelectrodes in touch with the cortical surface in the hole drilled simultaneously. (C) Recording microelectrodes along with the Ag/AgCl wires as reference electrodes positioned at appropriate sites. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59310/59310fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59310/59310fig2.jpg" /> 
Figure 2: Illustration of cross-correlation analysis. (A) Settings for the waveform correlation dialog box. This provides options for choosing which waveform channel is the reference and for analyzing the correlation of two signals. (B) The process dialog box. This provides options for setting the time length of the reference waveform and the duration of another waveform will be appended. The analysis is only done for regions of data in which both waveform channels exist. (C) Example .txt file with values of cross-correlation at negative and positive time lag ranges separately for statistics. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59310/59310fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59310/59310fig3.jpg" /> 
Figure 3: Illustration of coherence analysis. (A) Parameter settings for the coherence dialog box. The block size determines the number of data points used in the analysis, and the frequency resolution. (B) The dotted lines are adjustable for operator to move manually in order to set the duration of signals for analyzing. (C) After the software has created a chart, click File - Save As to save the coherence results as a file with a .txt filename extension&#160;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59310/59310fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59310/59310fig4.jpg" /> 
Figure 4: Cross-correlation indicates the declined hemisphere lateralization between left and right M2 of APP/PS1 mice. (A) Representative raw traces of LFPs recorded simultaneously in bilateral M2 of WT and APP/PS1 mice using extracellular recording method (L: left M2; R: right M2). (B) The cross correlation curve shows correlation of bilateral LFP signals at different time lags. (C) Between left and right M2, WT controls showed significantly higher cross-correlation value at positive time lag ranges than negative ones. In contrast, the cross-correlation value of APP/PS1 mice has a similarity, indicating a decline of asymmetry (n = 10, per group). Value represents mean &#177; standard error of the mean. ***p &#60; 0.001; two sample t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59310/59310fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59310/59310fig5.jpg" /> 
Figure 5: Coherence of gamma oscillations between left and right M2 of WT and APP/PS1 mice. (A) Representative traces of gamma oscillations filtered from LFPs in left and right M2. (B) Coherence distribution between LFPs simultaneously recorded in bilateral M2. APP/PS1 mice differ largely from WT controls in gamma frequency range. (C) The coherence between gamma oscillations of bilateral M2 in 
APP/PS1 mice are significantly higher than WT controls (n = 10, per group). Value represents mean &#177; standard error of the mean. **, p &#60; 0.01; two sample t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59310/59310fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice at JoVE.com

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

evaluation-hemisphere-lateralization-with-bilateral-local-field

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.7K Views.  Guangzhou University. We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Video: Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of 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 ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...