This work has been supported by the National Institutes of Health (R01-AA016624). We are grateful to Dr. Sanja Kovacevic for her important contributions.

This experimental protocol has been approved by the Human Subjects Protection Committee at the University of California, San Diego.
1. Human Subjects
<ol>
	<li>Recruit healthy right-handed adult volunteers, obtain their consent, and screen them on the inclusion/exclusion criteria. 
	NOTE: In this study, twenty young, healthy individuals (mean &#177; standard deviation [SD] age = 25.3 &#177; 4.4 years) including 8 women were recruited who drink in moderation, who have never been in treatment or arrested for drug or alcohol related offenses, who report no alcoholism-related symptoms on the Short Michigan Alcoholism Screening Test28, who do not smoke nor use illegal substances, who do not have a history of neuropsychiatric disorders or any current health problems, and who are medication free and have no internal ferromagnetic objects or implants.</li>
</ol>
2. Experimental Design
<ol>
	<li>Scan each participant four times, including three MEG sessions (a no-beverage introductory session and two experimental beverage sessions in which alcohol and placebo are administered in a counterbalanced manner), and one structural MRI scan. 
	NOTE: In this within-subject design, participants serve as their own controls by participating in both alcohol and placebo sessions. This design reduces error variance and increases statistical power by minimizing influence of individual variability in brain anatomy, activity patterns, and alcohol metabolism.</li>
</ol>
3. Collecting MEG Scans
<ol>
	<li>Perform familiarization session.
	<ol>
		<li>During the initial introductory session, administer questionnaires to obtain more information about the participants&#39; medical history, their drinking patterns and severity of alcoholism-related symptoms28,29, family history of alcoholism30, and personality traits including impulsivity31,32.</li>
		<li>Carry out an initial recording in the MEG scanner following the protocol described below in steps 3.2, 3.3, and 3.5. Do not provide any beverage. Explain the task and run the practice version allowing participants to get familiarized with it beforehand. 
		NOTE: The acclimation to the experimental situation serves the purpose of minimizing potential effects of situation-induced arousal33, thereby equating subsequent alcohol and placebo sessions on that dimension.</li>
	</ol>
	</li>
	<li>Perform the alcohol/placebo experimental sessions. 
	NOTE: Follow the same experimental procedures during both alcohol and placebo sessions with the exception of the administered beverage. Counterbalance beverage order by administering alcohol beverage first to one half of participants and placebo to the other half in a random order.
	<ol>
		<li>Upon their arrival to the MEG lab, run a brief test scan by putting the participant in the scanner and checking the channels for possible magnetization. Measure their weight. Screen them with an electronic breathalyzer. Query them about compliance with the requirements to abstain from alcohol for 48 h and from food for 3 h prior to the experiment.</li>
		<li>Collect urine samples for a multi-drug test panel from all participants and exclude those who test positive for any drug. In addition, check female participants for pregnancy with a urine test and exclude those who test positive or if they suspect that they might be pregnant.</li>
		<li>Assess dynamic changes in the subjective effects of alcohol by asking participants to rate their momentary feelings and states on a standardized scale34 prior to drinking and on two additional occasions during the experiment - on the ascending limb (~15 min after consuming beverage) and descending limb of the breath alcohol concentration curve (BrAC), after the MEG recording.</li>
		<li>Administer a practice run of the Stroop task on a laptop with stimulus presentation software to ensure that the participants understand the task before recording. 
		NOTE: This version of the Stroop task combines reading and color naming (Figure 1). The congruent condition consists of color words (i.e., red, green, blue, yellow) that are printed in the matching font color (i.e., the word &#34;green&#34; is printed in green). In the incongruous condition, color words are printed in color that does not match their meaning (i.e., the word &#34;green&#34; is printed in yellow). Ask the participants to press one of four buttons corresponding to the font color whenever a word is written in color, or, when a word is written in gray, to press a button corresponding to the meaning of the word18,23.</li>
	</ol>
	</li>
	<li>Prepare the MEG/EEG recording. 
	NOTE: Details of MEG data acquisition have been described in previous publications35,36,37.
	<ol>
		<li>Position the EEG cap or individual EEG electrodes on the head of the participant and check that all impedances are below 5 k&#937;.</li>
		<li>Attach the head position indicator (HPI) coils on either side of the forehead and behind each ear. 
		NOTE: This step is specific to Neuromag systems.</li>
		<li>Digitize positions of the fiducial points including the nasion and two preauricular points, positions of HPI coils, EEG electrodes, and obtain a large number of additional points (~200) delineating the head shape. Use this information for the co-registration with anatomical MRI images (Figure 2).</li>
	</ol>
	</li>
	<li>Administer beverage.
	<ol>
		<li>Prepare alcohol beverage by mixing premium quality vodka with chilled orange juice (25% v/v), based on each participant&#39;s gender and weight (0.60 g/kg alcohol for men, 0.55 g/kg alcohol for women), targeting a BrAC of 0.06%38. Serve the same volume of orange juice in glasses with rims swabbed with vodka as a placebo beverage. Ask the participant to consume the beverage in approximately 10 min.</li>
		<li>Check the participants&#39; BrAC with the breathalyzer starting at ~15 min after drinking and then every 5 min until they enter the recording chamber. Since electronic devices cannot be used in the shielded room, use a saliva alcohol test, which consists of a cotton swab that is saturated in saliva and is inserted into a receptacle that provides a readout.</li>
	</ol>
	</li>
	<li>Acquire MEG/EEG data.
	<ol>
		<li>Position the participant comfortably in the scanner. Since the prefrontal activity is of particular interest, ensure that the participant is positioned so that his/her head is touching the top of the helmet and is aligned along the front. 
		NOTE: Head position can affect activity estimates in significant ways because the magnetic field gradients decrease with the cube of the distance between the sensors and the brain sources39.</li>
		<li>Connect HPI coils and all of the electrodes to their respective inputs on the scanner. Position response pads so that the buttons can be pressed comfortably. Ascertain that the font is clearly legible on the projection screen in front of the participant.</li>
		<li>Back in the console room, check that the intercom is functioning properly. Remind the participant to minimize blinking and to avoid movements including head motion caused by talking. Instruct the participant to reply to questions by pressing response buttons instead.</li>
		<li>Check that all response and stimulus triggers are recorded correctly. Examine all channels for artifacts and measure the head position in the scanner.</li>
		<li>Start data acquisition and begin the task. Give breaks every ~2.5 min to rest the eyes. Save the data upon task completion and escort the participant out of the recording chamber.</li>
		<li>When the participant has exited the scanner, acquire approximately two minutes of data from the empty room as a measure of instrumental noise.</li>
		<li>Ask the participant to rate perceived task difficulty, content of the imbibed beverage, how intoxicated they felt, as well as their momentary moods and feelings34.</li>
	</ol>
	</li>
</ol>
4. Image Acquisition and Cortical Reconstruction of Structural MRI
<ol>
	<li>Obtain a high-resolution anatomical MRI scan for each participant, and reconstruct each participant&#39;s cortical surface with FreeSurfer software40,41,42.</li>
	<li>Use the inner skull surface derived from the segmented structural MRI images to generate a boundary element model of the volume conductor, which is used to provide a model for the forward solution that is consistent with each individual&#39;s brain anatomy43,44.</li>
</ol>
5. MEG Data Analysis
NOTE: Analyze the data with the anatomically-constrained MEG approach which uses each participant&#39;s reconstructed cortical surface to constrain source estimates to the cortical ribbon40,45,46. The analysis stream relies on custom functions with dependencies on publicly available packages including FieldTrip47, EEGLab48, and MNE49.
<ol>
	<li>During data preprocessing, use a permissive band-pass filter (e.g., 0.1 - 100 Hz) and epoch data with respect to stimulus onset into segments that include padding intervals on each end (e.g., -600 to 1100 ms for an interval of interest spanning -300 to 800 ms after the removal of padding).</li>
	<li>Remove noisy and flat channels, as well as trials containing artifacts by visual inspection and using threshold-based rejection. Use independent component analysis48 to remove eyeblink and heartbeat artifacts. Eliminate trials with incorrect responses.</li>
	<li>Apply Morlet wavelets (Figure 3)47 to calculate complex power spectrum for each trial in 1 Hz increments for theta frequency band (4 - 7 Hz). Remove any additional artifacts. Compute the noise covariance from empty room data.</li>
	<li>Co-register the MEG data with MRI images using the three-dimensional (3D) head digitization information (Figure 2).
	<ol>
		<li>Open the MRIlab module.</li>
		<li>Select File| Open| Select subject&#39;s structural MRI.</li>
		<li>Select File| Import| Isotrak data| select raw data.fif file| Make Points.</li>
		<li>Select Windows| Landmarks| Adjust fiducial landmarks until co-registration of MEG data and MRI are acceptable.</li>
		<li>Select File| Save.</li>
	</ol>
	</li>
	<li>Calculate noise-sensitivity normalized estimates of theta source power and phase with a spectral dynamic statistical mapping approach18,50. Express event-related theta source power as percent signal change relative to baseline.</li>
	<li>Create group averages of event-related theta source power by morphing each participant&#39;s estimates onto an average cortical representation51.</li>
	<li>Visualize the source estimates on an inflated average surface to enhance visibility of sulcal estimates (Figure 4).
	<ol>
		<li>Open the MNE software.</li>
		<li>Select File| Load Surface| Load inflated group-average FreeSurfer cortical surface.</li>
		<li>Select File| Manage overlays| Load stc| Load group-averaged data| Select loaded file from available overlays.</li>
		<li>Select overlay type as Other.</li>
		<li>Adjust Color Scale thresholding| Show.</li>
		<li>View brain movies and examine spatio-temporal stages of processing by identifying areas and time windows characterized by highest activation.</li>
	</ol>
	</li>
	<li>Create unbiased regions of interest (ROIs) based on overall group-averaged estimates to incorporate cortical locations with most notable source power. Calculate time courses for each subject, condition, and ROI (Figure 5).</li>
	<li>Submit the obtained theta source power estimates to the statistical analysis.
	<ol>
		<li>Extract time windows of interest from each ROI time course and perform analysis of variance (ANOVA) with beverage (alcohol, placebo) and trial type (congruous, incongruous) as within subject factors. Use a nonparametric cluster-based permutation test52 to examine beverage and condition comparisons of event-related theta power as well as phase-locking values (PLV).</li>
	</ol>
	</li>
	<li>Estimate task-related changes in the long-range synchronization between the main activation foci in the ACC and the latPFC by computing the PLV12. Express PLV as percent change relative to baseline. 
	NOTE: The PLV is an indicator of consistency of the phase angle between the two ROIs across trials as it measures the extent to which they co-oscillate at a particular frequency and in real time (Movie 1).</li>
	<li>Calculate correlations between ROI MEG activity estimates, indices of behavioral performance, and questionnaire scores to inform interpretation of the observed results.</li>
</ol>

The authors have nothing to disclose.

The multimodal imaging method used in this study comprises distributed source modeling of the temporally precise MEG signal along with spatial constraints of inverse estimates derived from each participant&#39;s structural MRI. The aMEG approach combines the strengths of these techniques to provide insight into the spatio-temporal stages of oscillatory dynamics and the long-range integration subserving cognitive control. This method provides greater temporal precision than other neuroimaging techniques such as fMRI-BOLD whose temporal resolution is on the magnitude of seconds due to its indirect sensitivity to neural changes via neurovascular coupling9. In comparison, the millisecond precision of MEG signal allows for the investigation of neural processing stages, as demonstrated by the present study. The aMEG model assumes distributed sources of the MEG signal along the cortical surface which, when reconstructed from structural MRI images, provides spatial constraints for activity estimates45,53. These spatial estimates can be used to investigate not only local activation but long-range communication at an interactive network level in the form of phase-locking16,20. Moreover, the aMEG approach is well suited for investigating the effects of pharmacological manipulation on neural functions, given that the fMRI-BOLD signal is confounded by the vasoactive effects of pharmacological manipulations such as alcohol and may not accurately reflect the magnitude of neural changes10.
The high sensitivity of this method to minute neural changes means that it is also sensitive to non-neural noise including muscle movements or eye blinks, so the various artifacts need to be detected and carefully removed from the raw signal. Moreover, head position can have significant effects on activity estimates due to sensor sensitivity to magnetic field gradients39. Given the assumptions of the aMEG model, source estimates are constrained to the cortical surface45,46, so the activity elicited from subcortical structures cannot be estimated.
Based on previously published results18, the present study has illustrated changes in event-related theta (4 - 7 Hz) power during Stroop-induced conflict as a function of acute alcohol intoxication in healthy social drinkers. As shown in Figure 5, theta power is differentially sensitive to cognitive demands imposed by the Stroop task conditions. Incongruity is especially effective in engaging cognitive control as reflected in greater theta power in the prefrontal cortex compared to prestimulus baseline. The principal estimated generator of theta oscillations is the ACC that is sensitive to response conflict during both early and late processing stages18. These findings support the role of the ACC in monitoring for conflict in concordance with prominent accounts7,8. Thus, the aMEG method has provided a temporally-sensitive insight into the sustained engagement of the ACC during trials imposing higher load on cognitive control. Together with extensive anatomical connections between the ACC and distributed brain regions5,6, this evidence corroborates its multifaceted role in self-regulation. On that view, the ACC is a key hub in the neurofunctional system that subserves cognitive control by aligning goals and intentions with contextual and motivational constraints54,55. Inferolateral prefrontal cortex, especially on the right, is another important area within that system which has been associated with inhibition of prepotent responses, attentional control, and working memory in the service of updating task representations56,57,58.
It has been established that theta oscillations mediate neural integration necessary for cognitive and affective processing13,16,59,60. Neural communication may thus rely on synchronized excitability of distant neuronal ensembles in theta band with nested fast rhythms mediating local processing61,62. PLVs reflect phase consistency between cortical areas and are commonly used to estimate their oscillatory synchrony as it is assumed that two areas interact when they co-oscillate63. Indeed, transient increases in PLV are observed in those intervals of neural activity that would be expected to necessitate synchronous interactions12,20. The present study confirms previous evidence and adds spatio-temporal refinement to the functional synchronization between the sources estimated to the ACC and the latPFC. Consistent with previous reports64, the present results indicate that PLVs are increased and sustained on incongruous trials in the Stroop task. By quantifying phase synchronization between these two areas with high temporal precision, these findings extend the conflict monitoring account and indicate that their interaction is particularly prominent after ~350 ms on incongruous trials. During this cognitive integration stage, the medial and lateral prefrontal cortices are likely to interact to support behavioral performance during more difficult task conditions imposing demands on attention, response inhibition, and working memory. Extensive evidence from fMRI-based functional connectivity studies indicates that these cortical areas form a dynamic, interactive cingulo-opercular network that supports top-down cognitive control65,66,67. More broadly, the brain optimizes responding to environmental demands in an adaptive and coherent manner via flexible and dynamic synchronization of distributed neurofunctional systems68,69.
The anatomically-constrained MEG approach used in the present study relies on a combination of complementary imaging methods. It can characterize the spatio-temporal sequence of neural activity and can provide insight into the dynamics of long-range interactions important for integrating top-down influences during engagement of cognitive control. The MEG signal reflects synaptic currents directly, which allows for testing hypotheses about co-oscillatory interactions within and across neurofunctional systems with high temporal precision. Furthermore, this method is suitable for pharmacological manipulations because it is not susceptible to vasoactive confounds. Research from this lab and others indicates that prefrontally-mediated cognitive control functions are particularly vulnerable to alcohol intoxication17,18,19,20,23,24,70,71,72,73,74. The present study shows that acute alcohol intoxication decreases activity in the prefrontal areas subserving response conflict. Furthermore, alcohol disrupts synchronized co-oscillations20,75 that may underlie impaired or maladaptive response suppression. As a result, intoxicated individuals exhibit deficient self-control resulting in disinhibition which may contribute to compulsive drinking and the development of alcohol dependence25,26,76. In sum, estimates of synchronous co-oscillations can illuminate real-time interactions of the neural systems engaged by a particular cognitive demand and can inform a realistic brain-based model. They can characterize the selective sensitivity to alcohol challenge across networks and serve as biomarkers of individual vulnerability to pharmacological effects.

The overall goal of this study is to examine the effects of acute alcohol intoxication on spatio-temporal changes in the brain oscillatory dynamics and long-range functional integration during cognitive control. The employed multimodal imaging approach combines magnetoencephalography (MEG) and structural magnetic resonance imaging (MRI) to provide insight into the neural basis of decision making with high temporal precision and at the level of an interactive system.
Flexible behavior makes it possible to adapt to changing contextual demands and to switch strategically between different tasks and requirements in agreement with one&#39;s intents and goals. The capacity to suppress automatic responses in favor of goal-relevant but non-habitual actions is an essential aspect of cognitive control. Extensive evidence suggests that it is subserved by a predominantly frontal cortical network, with the anterior cingulate cortex (ACC) as a central node in this interactive network1,2,3,4. While the abundant anatomical connectivity between the ACC and lateral frontal cortices is well-described5,6, the functional characteristics of communication between these regions during cognitive control, response selection and execution, are poorly understood.
The highly influential conflict monitoring theory7,8 proposes that cognitive control arises from a dynamic interaction between the medial and lateral prefrontal cortices. This account purports that the ACC monitors conflict between competing representations and engages the lateral prefrontal cortex (latPFC) to implement response control and optimize performance. However, this account is primarily based on the functional MRI (fMRI) studies using the blood oxygenation level dependent (BOLD) signal. The fMRI-BOLD signal is an excellent spatial mapping tool, but its temporal resolution is limited because it reflects regional hemodynamic changes mediated by neurovascular coupling. As a result, the BOLD signal changes unfold on a much slower time scale (in seconds) than the underlying neural events (in milliseconds)9. Moreover, the BOLD signal is sensitive to alcohol&#39;s vasoactive effects10 and may not accurately represent the magnitude of neural changes, which makes it less suitable for studies of acute alcohol intoxication. Therefore, the presumed interplay between the medial and lateral prefrontal cortices and its sensitivity to alcohol intoxication need to be examined by methods that record neural events in a temporally precise manner. MEG has an excellent temporal resolution since it directly reflects postsynaptic currents. The anatomically-constrained MEG (aMEG) methodology employed here is a multimodal approach that combines distributed source modeling of the MEG signal with structural MRI. It allows for the estimation of where the conflict- and beverage-related brain oscillatory changes are occurring and to understand the temporal sequence (&#34;when&#34;) of the involved neural components.
Decision making relies on the interactions of distributed brain regions that are dynamically engaged to deal with increased demands on cognitive control. One way to estimate event-related changes in long-range synchrony between two cortical regions is to calculate their phase coupling as an index of their co-oscillations11,12. The present study applied a phase-locking analysis to test the basic tenet of the conflict monitoring theory by examining the co-oscillatory interactions between the ACC and latPFC. Neural oscillations in theta range (4 - 7 Hz) are associated with cognitive control and have been proposed as a fundamental mechanism supporting the long-range synchronization needed for top-down cognitive processing13,14,15,16. They are generated in prefrontal areas as a function of task difficulty and are significantly attenuated by acute alcohol intoxication17,18,19,20.
Long-term excessive alcohol intake is associated with a range of cognitive deficits with prefrontal circuitry being especially affected21,22. Acute alcohol intoxication is detrimental to cognitive control under conditions of increased difficulty, ambiguity, or those that induce response incompatibility17,23,24. By affecting decision making, alcohol may interfere with goal-directed behavior, may result in poor self-control and increased drinking, and may also contribute to traffic- or work-related hazards25,26,27. The present study uses an aMEG approach to measure the oscillatory activity in theta band and synchrony between the principal executive areas with excellent temporal resolution. The effects of alcohol on theta activity and co-oscillations between the ACC and the latPFC are examined as a function of conflict elicited by the Stroop interference task. We hypothesize that increased cognitive demands are associated with greater functional synchrony and that alcohol-induced dysregulation of synchronous activity of the medial and lateral prefrontal cortices underlies impairments in cognitive control.

<table border="0" cellpadding="0" cellspacing="0" style="width:871px;" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:222px;">Elekta Neuromag</td>
			<td style="width:125px;">Elekta</td>
			<td style="width:354px;"></td>
			<td style="width:171px;">Magnetoencephalography system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">1.5 T GE EXCITE HG</td>
			<td>General Electric</td>
			<td></td>
			<td>Magnetic Resonance Imaging scanner</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gold Cup Electrodes</td>
			<td>OpenBCI</td>
			<td></td>
			<td>Electroencephalography electrodes for optional simultaneous EEG recording</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Prep Check Impedance Meter</td>
			<td>General Devices</td>
			<td></td>
			<td>Check electrode impedances</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HPI Coils</td>
			<td>Elekta</td>
			<td></td>
			<td>Head position indicator coils for co-registration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alcotest</td>
			<td>Draeger</td>
			<td></td>
			<td>Breathalyzer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fiber Optic Response Pad</td>
			<td>Current Designs, Inc</td>
			<td></td>
			<td>MEG-compatible response pad</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Grey Goose Vodka</td>
			<td>Bacardi</td>
			<td></td>
			<td>Vodka is used during the alcohol session</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Orange Juice</td>
			<td>Naked</td>
			<td></td>
			<td>Orange juice is used as the beverage during the placebo session as well as mixed with vodka during the alcohol session</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Discover Drug Test Card</td>
			<td colspan="2">American Screening Corp</td>
			<td>Multi-screen drug test</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">QED Saliva Alcohol Test</td>
			<td colspan="2">OraSure Technologies</td>
			<td>Saliva alcohol test</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Urine Hcg Test Strips</td>
			<td>Joylive</td>
			<td></td>
			<td>Pregnancy test</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Short Michigan Alcohol Screening Test</td>
			<td>Selzer et al., 1975</td>
			<td></td>
			<td>Alcoholism screening questionnaire</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Zuckerman Sensation Seeking Scale</td>
			<td>Zuckerman, 1971</td>
			<td></td>
			<td>Questionnaire: disinhibitory, novelty-seeking, and socialization traits</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eysenck Impulsivity Inventory</td>
			<td colspan="2">Eysenck &#38; Eysenck, 1978</td>
			<td>Questionnaire: impulsivity traits</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eysenck Personality Questionnaire</td>
			<td colspan="2">Eysenck &#38; Eysenck, 1975</td>
			<td>Questionnaire: personality traits</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Biphasic Alcohol Effects Scale&#160;</td>
			<td>Martin et al., 1993</td>
			<td></td>
			<td>Questionnaire: subjective experience of the effects of alcohol</td>
		</tr>
	</tbody>
</table>

disruption of frontal lobe neural synchrony during cognitive control by alcohol intoxication

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance imaging (fMRI) studies indicates that the anterior cingulate (ACC) and the lateral prefrontal cortices (latPFC) are essential nodes subserving cognitive control. However, because of its limited temporal resolution, fMRI cannot accurately reflect the timing and nature of their presumed interplay. The present study combines distributed source modeling of the temporally precise magnetoencephalography (MEG) signal with structural MRI in the form of &#34;brain movies&#34; to: (1) estimate the cortical areas involved in cognitive control (&#34;where&#34;), (2) characterize their temporal sequence (&#34;when&#34;), and (3) quantify the oscillatory dynamics of their neural interactions in real time. Stroop interference was associated with greater event-related theta (4 - 7 Hz) power in the ACC during conflict detection followed by sustained sensitivity to cognitive demands in the ACC and latPFC during integration and response preparation. A phase-locking analysis revealed co-oscillatory interactions between these areas indicating their increased neural synchrony in theta band during conflict-inducing incongruous trials. These results confirm that theta oscillations are fundamental to long-range synchronization needed for integrating top-down influences during cognitive control. MEG reflects neural activity directly, which makes it suitable for pharmacological manipulations in contrast to fMRI that is sensitive to vasoactive confounds. In the present study, healthy social drinkers were given a moderate alcohol dose and placebo in a within-subject design. Acute intoxication attenuated theta power to Stroop conflict and dysregulated co-oscillations between the ACC and latPFC, confirming that alcohol is detrimental to neural synchrony subserving cognitive control. It interferes with goal-directed behavior that may result in deficient self-control, contributing to compulsive drinking. In sum, this method can provide insight into real-time interactions during cognitive processing and can characterize the selective sensitivity to pharmacological challenge across relevant neural networks.

Behavioral results indicate that the Stroop task successfully manipulated response interference because the accuracy was the lowest and the response times the longest on incongruous trials (Figure 6). Alcohol intoxication lowered accuracy but did not affect reaction times18.
The spatio-temporal sequence of activity in theta frequency band revealed with the aMEG approach is overall in agreement with generally accepted models of cognitive functions in this type of task. As illustrated in the brain movies (Movie 2), the visual cortex is activated at around 100 ms after stimulus onset, followed by a posterior-to-anterior activation pattern that engages primarily frontal cortices during cognitive integration stages after ~300 ms. The ACC is particularly sensitive to incongruous (INC), high-conflict trials, indicating its engagement during conflict monitoring. The ACC is the principal generator of theta oscillations during tasks probing cognitive control but the latPFC is also active during the integration stage at around 350-600 ms. Activation of the motor cortex is visible after ~600 ms during the response preparation stage (Movie 2B). Event-related theta power is greatest on INC&#160;trials, which is consistent with its sensitivity to conflict demands (Figure 5), especially in the prefrontal cortex13,17,19,20. Theta power is decreased by acute alcohol intoxication overall. However, when compared to congruous (CONG) trials, alcohol decreases theta power on INC (high conflict) trials selectively in the ACC and latPFC18.
The present study extends the results from Kovacevic et al.18 by focusing on dynamic interactions between these areas during the processing of Stroop interference in light of a prevailing account of the cognitive control network7,8. To better understand the timing, degree, and nature of the interactions between these two principally engaged cortical areas, the PLVs were calculated for each beverage and task condition, and for each participant. As shown in a group average in Figure 7, co-oscillations between the ACC and latPFC vary across time with an overall early increase in co-oscillations during a stimulus processing stage. Under placebo, this is followed by a sustained increase after ~400 ms on incongruous trials during the integration and response preparation stage. Thus, synchronized co-oscillations between the medial and lateral prefrontal cortices are observed only on the more difficult, INC trials evoking response conflict F(1,19) = 5.5, p &#60; 0.05. This evidence supports the proposal that the ACC and the latPFC functionally interact in real time to subserve cognitive control. In contrast, acute alcohol intoxication significantly dysregulates the co-oscillations, yielding a Condition x Beverage interaction, F(1,19) = 5.1, p &#60; 0.05, in which incongruous trials specifically were affected by alcohol F(1,19) = 8.8, p &#60; 0.01 (Figure 7). This may underlie alcohol-induced impairments of inhibitory control and indicates the vulnerability of top-down regulative functions of the prefrontal cortex to acute intoxication.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58839/58839fig1.jpg" /> 
Figure 1: Stroop task combines color naming and reading. Trial examples for each of the three conditions along with the correct response color are presented. In the congruous&#160;condition (CONG), font color is consistent with the word meaning, while incongruous trials (INC) trails elicit response conflict due to interference from word meaning. Participants are instructed to press a button corresponding to the font color when words are written in color (CONG, INC) and to respond to the word meaning (Read) when they are written in gray. Trials are presented for 300 ms and then replaced by a fixation screen for 1700 ms. Trial types are presented in a randomized order. In this particular version, the CONG and INC conditions were equiprobable and were presented on 16.7% trials each out of 576 trials total. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig1large.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/58839/58839fig2.jpg" /> 
Figure 2: Co-registration of MEG and MRI. Digitized points across the head collected during the MEG recording are used for co-registration with anatomical MRI images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig2large.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/58839/58839fig3.jpg" /> 
Figure 3: Morlet wavelet. Morlet wavelets are used to calculate complex power spectrum for each trial in 1 Hz frequency increments for the theta band frequency (4 - 7 Hz). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig3large.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/58839/58839fig4.jpg" /> 
Figure 4: Cortical reconstruction and inflation. Individual cortical surfaces are reconstructed and are used to constrain estimated source power. Here shown is an average cortical surface which is inflated to enhance visibility of the sources estimated to cortical sulci. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig4large.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/58839/58839fig5.jpg" /> 
Figure 5: Group-average time courses of event-related theta source power estimates in selected regions of interest. Incongruous (INC) stimuli elicited increased event-related theta power compared to congruous (CONG) stimuli in the anterior cingulate cortex (ACC; F(1,19) = 34.1, p &#60; 0.0001) as well as the lateral prefrontal cortex (latPFC; F(1,19) = 11.0, p &#60; 0.01), during 480 - 670 ms. Conflict processing is particularly sensitive to alcohol intoxication as theta power to INC was attenuated by alcohol intoxication (F(1,19) = 9.9, p &#60; 0.01). The y-axis depicts baseline-corrected noise-normalized event-related theta source power. This figure has been modified from Kovacevic et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58839/58839fig6.jpg" /> 
Figure 6: Behavioral results on the Stroop task. Stroop interference was reflected in decreased accuracy and longer response times to incongruous (INC) trials. Alcohol intoxication (Alc) impaired accuracy compared to placebo (Plac) but did not affect reaction times. Error bars signify standard error of the mean. This figure has been modified from Kovacevic et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58839/58839fig7.jpg" /> 
Figure 7: Group-average time courses of phase-locking values (PLVs) in the theta band. Co-oscillatory synchrony between the anterior cingulate cortex (ACC) and lateral prefrontal cortex (latPFC) in theta band expressed as percent change from baseline for placebo (left) and alcohol (right) conditions. Following an early increase in PLVs during a stimulus processing stage (400 - 600 ms), a sustained increase in co-oscillations is observed on incongruous (INC) trails in response to increased cognitive control compared to congruous&#160;(CONG) trails under placebo, F(1,19) = 5.5, p &#60; 0.05. Acute alcohol intoxication selectively dysregulated the co-oscillations on INC trials, F(1,19) = 8.8, p &#60; 0.01. Activation maps (inset) show the incongruity effect (INC-CONG), which is prominent in the ACC and latPFC. The color scale denotes baseline-corrected source power estimates at 480 ms after stimulus onset, with red (activity &#62; 0.2) to yellow (activity &#62; 0.3) indicating stronger theta power to INC trials compared to CONG trials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/58839fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/58839/58839mov1.jpg" /> 
Movie 1: Co-oscillations. Phase-locking values were calculated in the theta frequency range (4 - 7 Hz) between the anterior cingulate cortex (ACC) and lateral prefrontal cortex (latPFC) as a measure of synchronization that is sensitive to the consistency of the phase difference between these two ROIs regardless of their theta power amplitude. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/Movie_1_Co-oscillations.mov" target="_blank">Please click here to download this movie.</a>
<img alt="Movie 2" src="/files/ftp_upload/58839/58370mov2v2.jpg" /> 
Movie 2: Brain movies. Distributed source modeling of the MEG signal combined with structural MRI allows for the estimation of principal cortical areas generating theta power and the temporal sequence of their activation in response to the Stroop interference. (A) Following early sensory processing, the anterior cingulate cortex (ACC) is selectively activated by incongruous, high-conflict trials after ~350 ms. (B) While the ACC is the principal generator of theta oscillations during tasks probing cognitive control, the lateral prefrontal cortex (latPFC) is also engaged during the integration stage around 350 - 600 ms. Activation of the motor cortex is observed after ~600 ms during response preparation. The color scale denotes differential baseline-corrected source power estimates, with red color indicating activation greater than 0.79 medially (0.57 laterally) and yellow indicates activation greater than 0.9 medially (0.8 laterally). Please note that these two movies should be shown together with unfolding time courses pertaining to the ACC and latPFC, respectively.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58839/Movie_2.zip" target="_blank">Please click here to download these movies.</a>

Watch this Scientific Journal Video about Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication at JoVE.com

Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance ...

disruption-frontal-lobe-neural-synchrony-during-cognitive-control

Nanyang Technological University

Research

JoVE Journal

Neuroscience

18.6K Views.  San Diego State University. This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

Video: Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...