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><tbody><tr><td>AC/DC Differential Amplifier</td><td>A-M Systems</td><td>Model 3000</td><td></td></tr><tr><td>Analog Digital converter</td><td>Cambridge Electronic Design Ltd.</td><td>Micro1401</td><td></td></tr><tr><td>Glass borosilicate micropipettes</td><td>Nanjing spring teaching experimental equipment company</td><td>161230</td><td>Outer diameter: 1.0mm</td></tr><tr><td>Microelectrode puller</td><td>Narishige</td><td>PC-10</td><td></td></tr><tr><td>NaCl</td><td>Guangzhou Chemical Reagent Factory</td><td>7647-14-5</td><td></td></tr><tr><td>Pin microelectrode holder</td><td>World Precision Instruments, INC.</td><td>MEH3SW10</td><td></td></tr><tr><td>Spike2&amp;nbsp;</td><td>Cambridge Electronic Design Ltd.</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>435064-9020-000</td><td></td></tr><tr><td>Stereotaxic apparatus&amp;nbsp;</td><td>RWD Life Science</td><td>68045</td><td></td></tr><tr><td>Urethane</td><td>Sigma-Aldrich</td><td>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/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/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/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/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/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

6.7K Views.  Guangzhou University. Deze techniek kan worden gebruikt om een aantal basiseigenschappen van interregionale elektrofysiologie te beoordelen voor hemisfeer lateralisatie, evenals de connectiviteit, directionaliteit en koppeling. Elektrofysiologische meting is een gevoelige en effectieve methode om te evalueren bij de dieren, neuronale activiteiten. Dit protocol biedt een betere manier om prop in de synchronisatie van elektrische signalen.Het begrip van het onderliggende mechanisme van mogelijke veranderde he...

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

Combined Invasive Subcortical and Non-invasive Surface Neurophysiological Recordings for the Assessment of Cognitive and Emotional Functions in Humans

In spite of the success in applying non-invasive electroencephalography (EEG), magneto-encephalography (MEG) and functional magnetic resonance imaging (fMRI) for extracting crucial information about the mechanism of the human brain, such methods remain insufficient to provide information about physiological processes reflecting cognitive and emotional functions at the subcortical level. In this respect, modern invasive clinical approaches in humans, such as deep brain stimulation (DBS), offer a tremendous possibility to record subcortical brain activity, namely local field potentials (LFPs) representing coherent activity of neural assemblies from localized basal ganglia or thalamic regions. Notwithstanding the fact that invasive approaches in humans are applied only after medical indication and thus recorded data correspond to altered brain circuits, valuable insight can be gained regarding the presence of intact brain functions in relation to brain oscillatory activity and the pathophysiology of disorders in response to experimental cognitive paradigms. In this direction, a growing number of DBS studies in patients with Parkinson&#39;s disease (PD) target not only motor functions but also higher level processes such as emotions, decision-making, attention, memory and sensory perception. Recent clinical trials also emphasize the role of DBS as an alternative treatment in neuropsychiatric disorders ranging from obsessive compulsive disorder (OCD) to chronic disorders of consciousness (DOC). Consequently, we focus on the use of combined invasive (LFP) and non-invasive (EEG) human brain recordings in assessing the role of cortical-subcortical structures in cognitive and emotional processing trough experimental paradigms (e.g. speech stimuli with emotional connotation or paradigms of cognitive control such as the Flanker task), for patients undergoing DBS treatment.

The present protocol aims at assessing cognitive-emotional functions in the basal ganglia by simultaneous neurophysiological recording of local field potentials and non-invasive brain cortical activity (EEG). The procedure is exemplified by the use of paradigms involving speech stimuli with emotional connotation or the Flanker task involving cognitive control.

In spite of the success in applying non-invasive electroencephalography (EEG), magneto-encephalography (MEG) and functional magnetic resonance imaging ...

Behavior

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, typically 3 mm x 3 mm. This protocol introduces a method for creating a substantially larger 7 mm x 6 mm cranial window exposing most of a cerebral hemisphere over the mouse temporal and parietal cortices (e.g., bregma 2.5 - 4.5 mm, lateral 0 - 6 mm). To perform this surgery, the head must be tilted approximately 30&#176; and much of the temporal muscle must be retracted. Due to the large amount of bone removal, this procedure is intended only for acute experiments with the animal anesthetized throughout the surgery and experiment.
The main advantage of this innovative large lateral cranial window is to provide simultaneous access to both medial and lateral areas of the cortex. This large unilateral cranial window can be used to study the neural dynamics between cells, as well as between different cortical areas by combining multi-electrode electrophysiological recordings, imaging of neuronal activity (e.g., intrinsic or extrinsic imaging), and optogenetic stimulation. Additionally, this large craniotomy also exposes a large area of cortical blood vessels, allowing for direct manipulation of the lateral cortical vasculature.

This protocol presents a method for creating a large unilateral craniotomy over the temporal and parietal regions of the mouse cerebral cortex. This is especially useful for real time imaging over an expansive area of a cortical hemisphere.

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

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 ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Topographical EEG Recordings of Visual Evoked Potentials in Mice using Multichannel Thin-film Electrodes

Visual evoked potentials (VEP) allow the characterization of visual function in preclinical mouse models. Various methods exist to measure VEPs in mice, from non-invasive EEG, subcutaneous single-electrodes, and ECoG to fully invasive intracortical multichannel visual cortex recordings. It can be useful to acquire a global, topographical EEG-level characterization of visual responses previous to local intracortical microelectrode measurements in acute experimental settings. For example, one use case is&#160;to assess global cross-modal changes in VEP topography in deafness models before studying its effects on a local intracortical level. Multichannel epicranial EEG is a robust method to acquire such an overview measure of cortical visual activity.&#160;Multichannel epicranial EEG provides comparable results through a standardized, consistent approach to, for example, identify cross-modal, pathological, or age-related changes in cortical visual function. The current study presents a method to obtain the topographical distribution of flash-evoked VEPs with a 32-channel thin-film EEG electrode array in anesthetized mice. Combined with analysis in the time and frequency domain, this approach allows fast characterization and screening of the topography and basic visual properties of mouse cortical visual function, which can be combined with various acute experimental settings.

The present protocol describes a simple procedure to acquire and analyze the topography of epicranial visual evoked potentials with 32-multichannel thin-film electrodes in the mouse.

Visual evoked potentials (VEP) allow the characterization of visual function in preclinical mouse models. Various methods exist to measure VEPs in mice, ...

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 ...

LFP Recording in Mouse Brain for Cognitive Research and Drug Analysis

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

Author Spotlight: Studying Drug Impacts on Brain Signals Using Dual LFP Recording Protocol in Mice

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

Recording of Local Field Potential from Both Hemispheres of the Mouse Brain

Source: Chen, Y., et al. <a href="https://app.jove.com/v/59310">Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice.</a> J. Vis. Exp. (2019).
This video demonstrates in vivo recording of local field potential (LFP) from bilateral M2 areas of the mouse brain. An anesthetized mouse is secured and prepared for the procedure. To record LFPs, insert microelectrodes into the target coordinates of the bilateral M2 areas, which are responsible for complex motor movements.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

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

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