Funding for this work was provided in part by the Commonwealth of Pennsylvania # 4100037709 subcontract #240468 and Drexel University subcontract #280773.

Maze-Suite consists of three main applications; an editing program to construct maze environments (MazeMaker), a visualization/rendering module (MazeWalker), and finally an analysis and path visualization tool (MazeAnalyzer). The following sections describe the use of MazeSuite and more details are available in the three supplementary videos, one for each MazeSuite module. 
1. Designing mazes 
The MazeMaker application within Maze-Suite, allows creating three-dimensional (3D) environments by simply drawing them on a two-dimensional (2D) canvas from a birds' eye view. Walls and floors can be drawn by mouse clicks to indicate coordinates, and users can import 3D object files or edit properties of items within the maze such as position, orientation, color, texture and lights.
Maze start and end regions can be assigned in addition to other exit criteria such as a timeout period. Separate text messages can also be displayed to inform a participant for each exit condition. During the design phase, mazes can be tested by using the &#x2018;quick run' function within MazeMaker. 
For functional imaging studies, participants are usually asked to perform a series of tasks with repeated trials. To facilitate this testing of repeated trials, MazeMaker can create maze list files, which are sorted lists of mazes and text messages for an experimental session. It is critical that the maze list file for the experiment is prepared and thoroughly pilot-tested before the day of the experiment.
A cursory tutorial for the use and development of environments with MazeMaker is provided in 'Supplementary video II &#x2013; MazeMaker'.
2. fNIR Setup and placing the fNIR sensor-pad
Preparing the fNIR Box
<ol>
	<li>There are two cable connectors behind the fNIR box. One of the slots is for USB connection and the other connection is for the power cord.</li>
	<li>Connect the fNIR device box through the USB cable to a PC or Laptop that will be used for data acquisition. </li>
	<li>Connect the power adapter to the device and turn on the switch.</li>
	<li>The ribbon cable is used to connect the sensor pad with the fNIR box.
		<ol class="lalpha">
			<li>The sensor pad houses light source (LEDs) and photo detectors. </li>
			<li>The LEDs emit infrared light at 730nm and 850nm wavelengths that are absorbed mainly by deoxygenated- and oxygenated- hemoglobin, respectively and thus can penetrate through biological tissue. </li>
		</ol>	
	</li>
</ol>
Placing the Sensor-pad
<ol class="lalpha">
	<li>Ask the participant to lift their hair off the forehead before sensor placement. Place the sensor strip just above the eyebrows. Match the center of the sensor with the vertical axis of symmetry that passes also through the nose</li>
	<li>Press the sensor pad firmly against the forehead and use a clip to hold the cables together at the back of the head. While not strictly necessary, head-wrap or tennis bandana is recommended to secure the sensor pad.</li>
	<li>Upon placing the sensor pad on the forehead, attach the two ends of the ribbon cable to the fNIR box. The ribbon cables should be attached by matching the 'I' and 'II' sides of the ribbon cable with the corresponding 'I' and 'II' connectors on the device.</li>
</ol>
Starting the COBI Studio Software for data collection
<ol>
	<li>Click on the Cognitive Optical Brain Imaging (COBI) Studio13 icon on the desktop to open the main window.</li>
	<li>The program will come with certain preset settings for data collection and visualization.</li>
	<li>It is important to check and confirm data acquisition parameters and triggering (marker synchronization) at device settings dialog if needed.</li>
	<li>COBI Studio can automatically name all related data files if "Experiment mode" is activated by using the "New Experiment" wizard.</li>
	<li>In the data folder, 3 types of files will be created: (*.nir) for fNIR data, (*.mrk) for marker data and (*.txt) for a log of the experiment session. </li>
</ol>
3. Running the experiment: Rendering the mazes
Device Setting and starting fNIR data acquisition
<ol>
	<li>LED Drive Current defines how bright each LED shines. The default value for LED Drive Current is 20mA. This value may have to change based on the skin pigmentation and other characteristics of the participant. Suggested range for the LED current is between 5mA to 20mA</li>
	<li>The default value for initial gain for all channels is 20. Suggested values for gain are 1,5,10,15,20.</li>
	<li>Click on the 'Start current device' link, and examine the signal quality. If the raw intensity values shown in COBI approach 4000 or are below 1000, click 'Stop Device', and adjust the LED Drive Current and Device Gain until suitable values are obtained. In low signal circumstances, increase LED Drive Current before increasing Device Gain. Under saturated signal circumstances reduce the Device Gain before reducing the LED Drive Current. Once signal quality is acceptable, continue to Step 4.</li>
	<li>Start Baseline. This will collect 10 seconds of data and use it as baseline in a modified Beer-Lambert equation set to calculate concentration changes for oxy- and deoxy-hemoglobin8.</li>
	<li>Let the Baseline complete (may take 10-20 seconds)</li>
	<li>Click 'Start recording'. This will start saving all the data. Experiment protocol should start after this.</li>
	<li>Experimenter may choose to add manual markers throughout the experiment session to denote certain events by pressing the &#x2018;add manual marker' buttons using the manual marker menu located in the lower left corner of the screen.</li>
	<li>Start the Maze Suite to present visual stimuli.</li>
</ol>
Using MazeWalker to render mazes
<ol>
	<li>Run MazeWalker from menu at Start&gt;Maze Suite&gt;MazeWalker</li>
	<li>Turn on marker synchronization by selecting 'Enable Serial Port' from the in the menu under Advanced>Serial Port Options. Ensure that the correct COM port address is selected.</li>
	<li>Select the maze list file (that was created by MazeMaker), and also select name for the new log file for this session. Name or participant number can be recorded within walker field. Optionally the autolog may be used to automatically record to a time-stamped log file instead of manually specifying the log file.</li>
	<li>Click 'Start' to initiate the process. Depending on the video settings, protocol can run full screen or in windowed mode.</li>
</ol>
4. Visualizing the subject's path
Using MazeAnalyzer, the researcher can visualize the maze and the participant's path from the maze and log files. Additionally, a summary report including total path length and time to completion of the each maze are produced as cursory behavioral measures. Log files contain information in millisecond time resolution about the path that a subject traveled as well as subject's view vector and interactions with objects.
A tutorial video is provided in 'Supplementary video I &#x2013; MazeAnalyzer' and describes the use of MazeAnalyzer's basic functionality along with methods to produce the behavioral metrics described in the results section.
5. Processing the fNIR data and analysis
Noise removal is the first step for processing the data. Sources of noise include 1) Head Movement 2) Physiological signals such as heart rate and respirations and 3) Instrument and environment-related noise.
Head movement can cause the fNIR detectors to shift and lose contact with the skin, exposing them to: 1) ambient light; 2) light emitted directly from the fNIR sources; or 3) light reflected from the skin, rather than being reflected from tissue in the cortex. This type of motion artifact is readily recognizable because it causes sudden, large spikes in the fNIR data. A more subtle artifact of head movement is due to the effects of gravity on the cerebral blood. Rapid head movement can cause the blood to move toward (or away from) the area that is being monitored, rapidly increasing (or decreasing) blood volume with a concomitant skewing of the data. Since the dynamics of this type of motion artifact are slower than LED "pop" they can be confused with the actual hemodynamic response due to brain activation. Hence, removing motion artifact from fNIR data is an important and necessary step if fNIR is to be deployed as a brain monitoring technology in natural environments16.
Physiological signals such as heart rate (over 0.5 Hz) and respiration (over 0.2 Hz) are at higher frequency ranges than hemodynamic responses, thus, they can be eliminated using a linear phase low-pass FIR filter with cut-off frequency between 0.1 to 0.15 Hz9. 
Instrument and environmental noise can originate due to ambient light such as daylight (DC) and room light (60Hz) or light from a computer monitor (60-75Hz). It is suggested that the best way to eliminate this type of noise is to prepare the experimental environment and data acquisition accordingly. An analog low pass filter (anti-aliasing filter) has been implemented in the fNIR box to eliminate folding of high frequency noise onto sampling frequency range. 
There are many advanced noise reduction algorithms available that utilize different characteristics of the signal17-20. However if a subject or session can be excluded if data is irrecoverable (i.e. saturated).
<img src="/files/ftp_upload/3443/3443fig1.jpg" /> 
Figure 1. fNIR signal components
The fNIR raw signals are light intensity measurements (See Figure 1). By measuring optical density (OD) changes at two wavelengths, the relative change of oxy-Hb and deoxy-Hb versus time can be obtained using the modified Beer&#x2013;Lambert Law21-23. OD at a specific input wavelength (&lambda;) is the logarithmic ratio of input light intensity (Iin) and output (detected) light intensity (Iout). OD is also related to the concentration (c) and extinction coefficient (e) of chromophores, the corrected distance (d) between the light source and detector, plus a constant attenuation factor (G). 
<img src="/files/ftp_upload/3443/3443eq1.jpg" alt="Equation 1" />
Having the same Iin at two different time instances and detected light intensity during baseline (Irest) and during performance of the task (Itest), the difference in OD for wavelength &lambda; is 
<img src="/files/ftp_upload/3443/3443eq2.jpg" alt="Equation 2" />
Measuring the OD at two different wavelengths gives 
<img src="/files/ftp_upload/3443/3443eq3.jpg" alt="Equation 3" />
This equation set can be solved for concentrations if the 2x2 matrix is non-singular. Typically, the two wavelengths are chosen i) within 700-900nm where the absorption of oxy-Hb and deoxy-Hb are dominant as compared to other tissue chromophores, and ii) below and above the isosbestic point (~805nm where absorption spectrums of deoxy- and oxy-Hb cross each other) to focus the changes in absorption to either deoxy-Hb or oxy-Hb, respectively. The fNIR instrument used in this study employs 730nm and 850nm wavelengths.
Finally, using the markers (time synchronization signals), periods of baseline/rest and tasks are labeled and select features from oxygenation data are extracted such as mean, min and max values. COBI Studio saves both raw light intensity measurements and calculated oxygenation values (using modified beer lambert law) to text based files as well as time synchronization (marker) files. COBI Studio can also be used to apply noise removal methods such as finite impulse response low-pass or band-pass filters. Output files can be exported to common analysis software such as (Matlab, Excel, SPM and SPSS) or dedicated analysis software such as fnirSoft24 for further processing.
6. Representative Results:
fNIR Signal Examples
Figures 2-5 preset fNIR raw signals from a single measurement location with two wavelength components (730nm and 850nm) shown separately. Figure 2 represents a valid and acceptable signal epoch whereas Figure 3 and 4 are unacceptable and needs to be discarded. Figure 5 presents raw signal that was contaminated with motion artifact and needs to be cleaned or discarded. 
<img src="/files/ftp_upload/3443/3443fig2.jpg" alt="Figure 2" /> 
Figure 2. A good fNIR raw signal sample
<img src="/files/ftp_upload/3443/3443fig3.jpg" alt="Figure 3" /> 
Figure 3. A bad fNIR raw signal sample where 850nm channel is saturated.
<img src="/files/ftp_upload/3443/3443fig4.jpg" alt="Figure 4" /> 
Figure 4. A bad fNIR raw signal sample where there's hardware problem or cable connectivity issue.
<img src="/files/ftp_upload/3443/3443fig5.jpg" alt="Figure 5" /> 
Figure 5. A bad fNIR raw signal sample where there are motion artifacts.
Study Protocol
Assessments of learning are best exemplified by retention (i.e., memory) and transfer (i.e., generalizability) tests. In our study, three mazes (maze1, maze2 and maze3) were practiced during the acquisition phase for a total of 105 trials each across three days. The two conditions; random practice order (RND) and blocked practice order (BLK) are summarized in Figure 6. Two subjects volunteered for the four day study. On the 4th day, 10 retention trials were conducted for each maze and the mazes were presented in a random order. Two novel mazes (maze4 and maze5) were created that had additional corridors and different starting and ending points than the mazes originally practiced. Subjects completed ten trials for each of the two new mazes. These mazes constituted a transfer phase, and were presented in a random order to all. The transfer phase mazes were used to determine the extent to which each subject was able to generalize their learning and practice with acquisition mazes. 
<img src="/files/ftp_upload/3443/3443fig6.jpg" alt="Figure 6" /> 
Figure 6. Representative experiment protocol outline.
Study Behavioral Results
The following figures 7 through 9, display average feature values (path length, total time of completion, and velocity) of subjects across each day. First, results from maze1, maze2 and maze3 are listed for both RND and BLK practice. Next, maze4 and maze5 results are listed to compare random versus blocked practice results. All error bars are standard error of the mean (SEM).
<img src="/files/ftp_upload/3443/3443fig7.jpg" alt="Figure 7" /> 
Figure 7. RND practice behavioral performance summary for acquisition and retention tasks including total path length, total time of completion and average velocity.
<img src="/files/ftp_upload/3443/3443fig8.jpg" alt="Figure 8" /> 
Figure 8. BLK practice behavioral performance summary for acquisition and retention tasks including total path length, total time of completion and average velocity.
<img src="/files/ftp_upload/3443/3443fig9.jpg" alt="Figure 9" /> 
Figure 9. Behavioral performance comparison of BLK vs. RND practice for transfer tasks including total path length, total time of completion and average velocity.
Study fNIR Results
Oxygenated hemoglobin concentration changes from baseline were averaged over the time course of each individual maze using marker data received from MazeWalker (indicates start and end of each maze). Larger magnitude concentration changes are taken to represent higher levels of regional brain activation. A higher level of activation was observed during both retention and transfer tasks for BLK practice compared to RND practice. During transfer tasks, a higher level of activation was seen under BLK practice as compared with RND practice (See Figure 10).
For the BLK practice subject, transfer trials required higher activation compared to retention trials. For the RND practice subject, activation level DID NOT differ for transfer and retention tasks as opposed to BLK practice subject (See Figure 11).
<img src="/files/ftp_upload/3443/3443fig10.jpg" alt="Figure 10" /> 
Figure 10. Comparison of average oxy-Hb concentration changes for BLK vs. RND practice for both retention tasks (left) and transfer tasks (right).
<img src="/files/ftp_upload/3443/3443fig11.jpg" alt="Figure 11" /> 
Figure 11. Comparison of average oxy-Hb concentration changes for retention vs. transfer tasks for both BLK (left) and RND (right) practice.

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fNIR Devices, LLC manufactures the optical brain imaging instrument and licensed IP and know-how from Drexel University. H. Ayaz, M. Izzetoglu, K. Izzetoglu and B. Onaral were involved in the technology development and thus offered a minor share in the new startup firm fNIR Devices, LLC.

The prefrontal cortex (PFC) in the human brain facilitates cognitive control over the coordination of thoughts and actions in relation to internal goals. In particular, the anterior/dorsolateral PFC is known to mediate higher cognitive functions such as task management, planning and spatial navigation25. fNIR is a portable, safe and noninvasive brain monitoring tool that has been used in clinical, laboratory and natural settings to study brain activation. This exploratory study demonstrated the use of Maze Suite and fNIR to study neurobehavioral aspects of spatial navigation. In this exploratory study, the MazeSuite platform is used in conjunction with fNIR to study the neurobehavioral aspects of spatial navigation in the dorsolateral PFC and to demonstrate the combination of these two tools.
MazeSuite is an experimental design, presentation and analysis platform. It enables the creation and application of simple 3D environments with a user friendly graphical interface and automatically records behavioral measures for within subject or across subject comparisons. During the presentation of MazeSuite environments, simultaneous time-synchronized fNIR measurements were taken using a commercially available, portable, continuous wave fNIR system (Imager 1000, fNIR Devices, LLC) and COBI Studio software15. fNIR previously has been established as a safe and effective noninvasive brain monitoring tool in clinical, laboratory and natural settings for the study of brain activation7,11 and is used in this study to investigate cognitive responses associated with contextual interference during of spatial navigation tasks.
To examine the effects of practice order contextual interference, subjects were presented with either a low interference (BLK) or high interference (RND) practice order. These distinct practice schedules were used to test the effect of learning multiple virtual spatial navigation maze tasks across acquisition, retention and transfer tests. The behavioral results indicate that for both practice orders, there is a monotonic decreasing trend in the total time necessary to complete maze, suggesting that as subjects practice, they completed each maze in shorter periods of time. In addition, the average speed with which subjects navigated the maze (maze velocity) increased with practice. These improvements in behavioral measures across time are expected inferences of learning. Mean oxy-Hb concentration changes during practice trials indicate that the BLK practice required higher brain activation when compared with RND practice order. 
RND practice resulted in faster completion time and shorter path lengths when compared to the BLK practice order for both retention and transfer, respectively. A decreasing trend observed in the mean oxy-Hb for BLK and RND practice across retention trials indicating reduced activity in the PFC. This finding is expected as previous research has suggested that there is a reduction in the activity of the PFC during the later stages of learning12,26.
Moreover, the navigation in new mazes during the transfer phase required higher brain activation for BLK practice subject compared to RND practice subject. Given that the practice order was different for the tasks already learned (i.e., maze1, maze2, and maze3), this stratified random practice order for the subject that learned the tasks in a sequential (BLK practice) order may have been sufficiently novel to require additional effort and cognitive resources to perform the tasks12,26. However, for the RND practice, the transfer phase of neural activation was not higher than the retention phase. These findings corroborate the PET findings with spatial navigation of virtual mazes reported by Van Horn and colleagues12. 
In summary, we described the use of MazeSuite in combination with COBI Studio for a study on the effects of contextual interference related to practice order when learning spatial navigational tasks. The fNIR methods discussed here are not limited to spatial navigation tasks and can be used for a variety of tasks in other neuroimaging studies. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

<ul>
	<li>Maze Suite www.mazesuite.com</li>
	<li>fNIR Imager 1000 www.fnirdevices.com</li>
</ul>

using mazesuite and functional near infrared spectroscopy to study learning in spatial navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

Watch this Scientific Journal Video about Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation at JoVE.com

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

using-mazesuite-functional-near-infrared-spectroscopy-to-study

Research

JoVE Journal

Neuroscience

30.4K Görüntüleme.  Drexel University. MazeSuite hazırlama, sunma ve seyir ve mekansal deneyleri analiz için tam bir araç setidir. Fonksiyonel yakın kızılötesi spektroskopi (fNIR) serebral kan oksijenlenme değişikliklerin noninvaziv ve taşınabilir izleme sağlayan bir optik beyin görüntüleme tekniğidir. Bu kağıt bir bilişsel işleme öğrenme paradigması içinde MazeSuite ve fNIR kolektif kullanımını özetlemektedir.

Video: Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

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

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

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

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

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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 ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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 ...

Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design

Neuroimaging studies play a pivotal role in the evaluation of pre- vs. post-interventional neurological conditions such as in rehabilitation and surgical treatment. Among the many neuroimaging technologies used to measure brain activity, functional near-infrared spectroscopy (fNIRS) enables the evaluation of dynamic cortical activities by measuring the local hemoglobin levels similar to functional magnetic resonance imaging (fMRI). Also, due to lesser physical restriction in fNIRS, multiple variants of sensorimotor tasks can be evaluated. Many laboratories have developed several methods for fNIRS data analysis; however, despite the fact that the general principles are the same, there is no universally standardized method. Here, we present the qualitative and comparative analytic methods of data obtained from a multi-channel fNIRS experiment using a block design. For qualitative analysis, we used a software for NIRS as a mass-univariate approach based on the generalized linear model. The NIRS-SPM analysis shows qualitative results for each session by visualizing the activated area during the task. In addition, the non-invasive three-dimensional digitizer can be used to estimate the fNIRS channel locations relative to the brain. To corroborate the NIRS-SPM findings, the amplitude of the changes in hemoglobin levels induced by the sensorimotor task can be statistically analyzed by comparing the data obtained from two different sessions (before and after intervention) of the same study subject using a multi-channel hierarchical mixed model. Our methods can be used to measure the pre- vs. post-intervention analysis in a variety of neurological disorders such as movement disorders, cerebrovascular diseases, and neuropsychiatric disorders.

We describe the analysis of continuous-wave functional near-infrared spectroscopy experiment using a block design with a sensorimotor task. To increase the reliability of the data analysis, we used the qualitative general linear model-based statistical parametric mapping and the comparative hierarchical mixed models for multi-channels.

Neuroimaging studies play a pivotal role in the evaluation of pre- vs. post-interventional neurological conditions such as in rehabilitation and surgical ...

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the concurrent brain activity of two or more interactive individuals with a novel index called inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is presented here. Further, a pipeline of analyzing IBS of oxygenated hemoglobin (Oxy-Hb) signal is explained. Specifically, the experimental design, the process of NIRS data recording, data analysis methods, and future directions are all discussed. Overall, implementing a standardized fNIRS hyperscanning pipeline is a fundamental part of second-person neuroscience. Also, this is in line with the call for open-science to aid the reproducibility of research.

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.