Funded by the National Science and Engineering Research Council (NSERC) to JFXD, Faculty of Health, York University and author SO has PhD funding by The Ontario Problem Gambling Research Centre (OPGRC).

1. Before Entering the MRI Room
<ol>
	<li>Participants complete a consent form explaining all the experimental risks (e.g. pacemaker, claustrophobia, metallic implants, chance of pregnancy, etc), and benefits of their participation.</li>
	<li>All participants are required to fill out the MRI safety and screening questionnaire (brief medical history, previous surgical procedures etc.) Participants with contraindications must be excluded.</li>
</ol>
2. Task Overview and Training
<ol>
	<li>Provide training on task performance on anti-saccade.
	<ol>
		<li>Green fixation indicates a pro-saccade trial. Instruct participants to look to the target appearing in the periphery of the screen, at a visual angle of 8-10&deg;.</li>
		<li>Red fixation indicates an anti-saccade trial. Instruct participant to look to the mirror opposite of target appearing in the periphery of the screen, at a visual angle of 8-10&deg; (e.g. for right target, look to the left).</li>
	</ol>
	</li>
	<li>Provide training on task performance for the emotional Stroop outside the scanner.
	<ol>
		<li>Include 15 practice trials with different combinations of face-word expressions on a computer outside the scanner. The purpose of the practice is for the participants to learn the task and what is expected of them in pressing the appropriate button in the MRI scanner. Instruct participants as to which buttons are pressed for reporting a happy expression/word, neutral expression/word and sad expression/word. Additionally, when inside the scanner, remind participants of which emotion each button represents.</li>
		<li>Descriptive words indicating the expressions (Happy, Neutral, Sad) are superimposed over the pictures of individual faces. These words are either congruent or incongruent with the emotion depicted by the face in the picture (Figure 1). Begin each scan with a written instruction on the screen reminding the participants to either report the "FACE EXPRESSION (happy, neutral, sad)" or the "WRITTEN WORD (happy, neutral, sad)" by pressing the corresponding button as quickly as possible.</li>
		<li>The instruction is displayed for 1 second, followed by a fixation cross, which the participants fixated on for another 1 sec. The fixation cross is followed by the face stimuli presented for 250 milliseconds and then followed by the response image for 2 seconds. The response image is used to give the participants time to report their responses by pressing the appropriate button. The next visual presentation of the fixation cross begins after the end of this response image. Each participant repeats the experimental scan in one of the two instruction groups (i.e. face expression OR written word). All stimuli were created and presented using Presentation 12.1 (<a href="http://www.neurobs.com" target="_blank">www.neurobs.com</a>).</li>
	</ol>
	</li>
</ol>
3. Scanner and Eye Tracking Setup
<ol>
	<li>To begin the set up of the experiment, start by projecting the computer stimulus as a focused image onto the screen in the MRI with a digital projector.</li>
	<li>Participants are asked to get up from their chair in the control room and walk into the scanner room. Earplugs and/or head phones are provided and subjects place them in their ears canals.
	<ol>
		<li>The subject lies supine with their head position to the centre of the head coil. We stabilize participant's body and head position with pillows or foam inserts to make them as comfortable as possible but also to aide in restricting their movement of the head since head motion during scanning causes loss of data. Especially if the head motion is greater than 1 mm in any direction.</li>
		<li>Slide/place the headcoil over the participant's head and have them tilt their head as comfortably as possible while looking straight ahead to view the mirror that reflects the projector's screen. The eyes must be as close to primary position as possible8 in order to maintain the participant's comfort over the scanning session which can last up to two hours.</li>
	</ol>
	</li>
	<li>Ask the participant how the focus of the projected image is once they are in the scanner. If it is not sharp, re-adjust the lens to improve the image on the screen.</li>
	<li>The eyetracker is now tested by means of a calibration to ensure that the IRED camera is in the correct location. If the reflection on the cornea is not ideal or working properly, the IRED source must be adjusted, or the mirror reflecting the IRED source near the participant's head position must be realigned/adjusted. If the participant's head was adjusted, ask the subject if more padding/foam or pillows are needed to maintain this head/body position. During scanning monitor and record participants' horizontal and vertical eye positions using an infrared eye tracker (i.e. Sensomotoric Instruments, Needham/Boston, MA) and correlate these with the behavioral paradigm when analyzing brain activity.5,7</li>
</ol>
4. Scanning Procedures
<ol>
	<li>Place the emergency contact squeeze ball on the participant's abdomen in their left hand and the joystick/button box in the right hand. Place a vitamin E capsule on the right side near the head. This will be viewable on the anatomical scans which will make certain an error will not flip the images from left to right. Raise and slide the bed into the centre of the MRI.</li>
	<li>Ensure all of the experimenters leave the MRI room and close the door to the MRI.</li>
	<li>Communicate with the participant through the intercom in the control room and confirm that they are prepared to begin scanning and are as comfortable as possible. If not, readjust as needed.</li>
	<li>Remind the participant that the noises in the scanner will be loud and this is normal.
	<ol>
		<li>The first scan collects a few brain images along the sagittal region to be able to localize/prescribe the exact orientation of the slices for the full anatomical and functional data. The participants are told this scan will take a few minutes.</li>
		<li>Once the experimenters view the results of the localizer scan, we prescribe a series of anatomical slices that cover the whole brain. In our cases we typically scan axial oblique slices that encompass the whole brain (170 to 256 slices). Tell the participant that this scan will take around 6 to 10 minutes depending on the prescribed number of slices. In some cases the anatomical scan can be done after the functional scans. There are some advantages of the latter; usually in long experiments subjects will experience fatigue. Thus, the anatomical scan needs no attention by the subjects so they may close their eyes. It might be useful to do these scans at the end of the imaging session.</li>
		<li>Once anatomical scans are completed the participant is reminded of the specific instructions of the upcoming scan through communication via the microphone/speaker system.</li>
	</ol>
	</li>
	<li>In this example, a pseudo event-related design2 is used to identify the brain regions activated by the emotional stroop task but any sensory, internal perception9 or motor stimulus10 could be directed for use when needed. After these are scanned then we will instruct the subject that the anti-saccade paradigm will be scanned next. Depending on the imaging parameters chosen the scan will be close to 6 minutes long. We find that scans longer than this induce subjects to fall asleep.</li>
	<li>The total imaging session takes approximately 60 to 120 of minutes, depending on the total scans needed for the analysis.</li>
</ol>
5. fMRI Analysis
<ol>
	<li>Analyze the data using BrainVoyager QX software (or any analysis package such as AFNI or SPM).</li>
	<li>Begin by superimposing functional data statistical maps onto anatomical brain images. Functionally define the brain regions of interest (ROIs) using the general linear model (GLM), with separate predictors (i.e. congruent and incongruent, face instruction and word instruction, anti-saccade, pro-saccade) for each of the conditions in the task during the two types of scans2.</li>
	<li>Examine the signal intensity in all the activated frontal regions from the GLM contrasts (i.e. all incongruent versus all congruent to produce a map of areas), compute the standardized BOLD signal across all participants and compare the incongruent word/face expression with the congruent word/faces expressions for both conditions2.</li>
	<li>Correlate the reaction times collected on the trials that were used for the GLMs, then correlate brain activity across each individual with their own reaction times for the specific trials2 as in Figure 4.</li>
</ol>
6. Representative Results
After the analysis we show brain regions that correlate with the emotional stroop and anti-saccade tasks recorded during scanning. The results from the emotional stroop paradigm showed an interaction effect between all three factors of expression, instruction, and brain region but there was no main effect of expression and no main effect of instruction2. We found that when the expression of the face was incongruent to the superimposed emotional word, this incongruency produced from reporting the written word showed higher BOLD signal intensity in the left IFG2 (Figure 2). The larger signal intensity on the incongruent expressions compared to the congruent expressions was statistically significant, with happy congruent showing the largest difference2.
Most importantly the RTs for the three incongruent conditions tested (sad, happy and neutral) predicted an increased BOLD signal within left IFG compared to all the congruent conditions (Figure 3). For this analysis we specifically examined the reaction times and conducted a regression analysis to test whether RT for the incongruent and congruent conditions were predictive of the BOLD signal activity within this brain region (Figure 3). We found that RT accounts for 81% of the variation in left IFG activity when reporting the word expressions of Happy, Neutral, and Sad during the incongruent and congruent conditions2. Higher RT is predictive of larger left IFG activation, with the incongruent sad condition yielding the greatest RT/signal intensity ratio compared to all other expression conditions. We analyzed the anti-saccade paradigms using similar methods as above to be able to compare the two networks of activity. In this example, we found that there was no increased signal in left IFG for the anti-saccade compared to the pro-saccade task. For more details, we refer the readers to Ford et al. (2007).
<img alt="Figure 1" src="/files/ftp_upload/3237/3237fig1.jpg" /> 
Figure 1. An example of an incongruent trial (face with a happy expression superimposed by the word SAD). The experiment will begin with the fixation dot (1 second), proceeding by the face stimulus (250 ms) and the masked image (2 seconds) which requires the participant's button response.
<img alt="Figure 2" src="/files/ftp_upload/3237/3237fig2.jpg" /> 
Figure 2. All fixation volumes were used as the baseline. Error bars signify the standard error of mean (SEM). Incongruent expressions (Happy, Neutral, Sad) showed significantly larger BOLD signal change compared to congruent expressions2. The inset image shows left inferior frontal gyrus (IFG) that was functionally localized using the contrast describe in section 5.2 for the incongruent emotional stroop versus the congruent condition during the attend to word instruction set.
<img alt="Figure 3" src="/files/ftp_upload/3237/3237fig3.jpg" /> 
Figure 3. During the "Attend to Word" instruction, incongruent-congruent contrast showed a positive correlation between the RTs and BOLD signal intensity. This graph is an average of all 10 subjects' RTs and BOLD signal during each of the six conditions. Error bars signify the standard error of mean (SEM)2 .
<img alt="Figure 4" src="/files/ftp_upload/3237/3237fig4.jpg" /> 
Figure 4. Two repetitions of each expression were displayed to the subjects. Top row is an schematic illustration of a trial sequence from one block of trials. Bottom section is a depiction of the Two-Gamma hemodynamic response function (HRF) used to discover brain regions involved in the emotional face expressions.

<ol>
	<li>Stroop, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+interference+in+serial+verbal+reactions.">Studies of interference in serial verbal reactions.</a> Journal of Experimental Psychology. 18, 643-662 (1935).</li><li>Ovaysikia, S., Tahir, K. A., Chan, J. L., DeSouza, J. F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Word+wins+over+face:+emotional+Stroop+effect+activates+the+frontal+cortical+network.">Word wins over face: emotional Stroop effect activates the frontal cortical network.</a> Front Hum. Neurosci. 4, 234 (2011).</li><li>Hallett, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+and+secondary+saccades+to+goals+defined+by+instructions.">Primary and secondary saccades to goals defined by instructions.</a> Vision Res. 18, 1279-1296 (1978).</li><li>Connolly, J. D., Goodale, M. A., DeSouza, J. F. X., Menon, R. S., Vilis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+frontoparietal+fMRI+activation+during+anti-saccades+and+anti-pointing.">A comparison of frontoparietal fMRI activation during anti-saccades and anti-pointing.</a> J. Neurophysiol. 84, 1645-1655 (2000).</li><li>DeSouza, J. F. X., Menon, R. S., Everling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparatory+set+associated+with+pro-saccades+and+anti-saccades+in+humans+investigated+with+event-related+FMRI.">Preparatory set associated with pro-saccades and anti-saccades in humans investigated with event-related FMRI.</a> J. Neurophysiol. 89, 1016-1023 (2003).</li><li>Everling, S., DeSouza, J. F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rule-dependent+activity+for+prosaccades+and+antisaccades+in+the+primate+prefrontal+cortex.">Rule-dependent activity for prosaccades and antisaccades in the primate prefrontal cortex.</a> J. Cogn. Neurosci. 17, 1483-1496 (2005).</li><li>Ford, K. A., Goltz, H. C., Brown, M. R. G., Everling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+processes+associated+with+antisaccade+task+performance+investigated+with+event-related+fMRI.">Neural processes associated with antisaccade task performance investigated with event-related fMRI.</a> J. Neurophysiol. 94, 429-440 (2005).</li><li>DeSouza, J. F. X., Nicolle, D. A., Vilis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Task-dependent+changes+in+the+shape+and+thickness+of+Listing's+plane.">Task-dependent changes in the shape and thickness of Listing's plane.</a> Vision Res. 37, 2271-2282 (1997).</li><li>Hadjikhani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+migraine+aura+revealed+by+functional+MRI+in+human+visual+cortex.">Mechanisms of migraine aura revealed by functional MRI in human visual cortex.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 4687-4692 (2001).</li><li>DeSouza, J. F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+position+signal+modulates+a+human+parietal+pointing+region+during+memory-guided+movements.">Eye position signal modulates a human parietal pointing region during memory-guided movements.</a> J. Neurosci. 20, 5835-5840 (2000).</li></ol>

We have nothing to disclose.

Identifying brain regions relies on creating an accurate contrast between the tasks scanned (i.e. in either the Stroop, incongruent versus congruent emotion and facial expression; or anti-saccade versus pro-saccade) in order to produce a map of activation related to the task. These functional maps can be more refined when behavior is collected in the scanner to remove trials where the subject made errors. These errors can be removed and if there was enough numbers of errors than functional maps could be made of these<sup...

A correction was made to <a href="http://https://www-jove-com.remotexs.ntu.edu.sg/video/3237">Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis</a>. Joseph DeSouza and Laura Pynn middle initials were omitted at publication.
These have been corrected to:
Joseph F.X. DeSouza Laura K. Pynn

A correction was made to <a href="http://https://www-jove-com.remotexs.ntu.edu.sg/video/3237">Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis</a>. Some of the authors middle initials were omitted.

Erratum: Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>3-Tesla MRI machine</td>
 <td>Siemens Magnetom Trio (Erlangen, Germany)</td>
 </tr>
 <tr>
 <td>iViewX Eye Tracking </td>
 <td>SensoMotoric Instruments, Inc. </td>
 </tr>
 <tr>
 <td>BrainVoyager QX software </td>
 <td>Brain Innovation, Maastricht, The Netherlands</td>
 </tr>
 <tr>
 <td>Four-button Joystick</td>
 <td>Current Designs, Inc., Philadelphia, PA, USA </td>
 </tr>
 </tbody>
</table>
Table 1. Specific Reagents and Equipment.

correlating behavioral responses to fmri signals from human prefrontal cortex: examining cognitive processes using task analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

Watch this Scientific Journal Video about Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis at JoVE.com

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

correlating-behavioral-responses-to-fmri-signals-from-human

Research

JoVE Journal

Neuroscience

12.7K Views.  Centre for Vision Research, York University. The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

Video: Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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

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

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

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

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by inspecting the relationship between the neural and hemodynamic signals. EEG is an electrophysiological technology that can measure the rapid neuronal activity of the cortex, whereas fNIRS relies on the hemodynamic responses to infer brain activation. The combination of EEG and fNIRS neuroimaging techniques can identify more features and reveal more information associated with the functioning of the brain. In this protocol, fused EEG-fNIRS measurements were performed for concurrent recordings of evoked-electrical potentials and hemodynamic responses during a Flanker task. In addition, the critical steps for setting up the hardware and software system as well as the procedures for data acquisition and analysis were provided and discussed in detail. It is expected that the present protocol can pave a new avenue for improving the understanding of the neural mechanisms underlying various cognitive processes by using the EEG and fNIRS signals.

The present protocol describes how to perform concurrent EEG and fNIRS recordings and how to inspect the relationship between the EEG and fNIRS data.&#160;

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.