Name: dynamic inter-subject functional connectivity reveals moment-to-moment brain network configurations driven by continuous or communication paradigms
Uploaded: 2019-03-21T13:30:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about Dynamic Inter-subject Functional Connectivity Reveals Moment-to-Moment Brain Network Configurations Driven by Continuous or Communication Paradigms at JoVE.co

This work was supported in part by each of the following: the Swiss National Science Foundation (grant number 205321_163376 to DVDV), the Bertarelli Foundation (to TB and DVDV), the Center for Biomedical Imaging (CIBM), and the National Agency for Research (tempofront grant number 04701 to ALG).&#160;The authors would like to thank Roberto Martuzzi and Giulia Preti for their contribution to the video content of this work as, respectively, the MRI operator and scanned volunteer.

The authors have nothing to disclose.

On a dataset of healthy subjects, we demonstrated how synchronous cross-subject increases and decreases in FC, the ISFC transients, would match temporally localized movie cues, providing information that goes beyond a static description. Although the use of cross-subject correlation measures enables to focus the analysis on stimulus-driven functional reconfigurations, one must also be aware that it limits the findings to the effects that are shared across the studied population: hence, low-level sensory processing is expected to be over-represented compared to frontal processing40. To bypass this limitation, new methods that also have the ability to extract the regions that most strongly vary across subjects are being developed41.
Another limitation from the introduced methodology arises from the sliding-window aspect, as temporal resolution of ISFC transient time courses is lowered compared to frame-wise approaches15. As we showed, a trade-off is needed between a sufficiently low window length to properly resolve dynamic ISFC reconfigurations, and a large enough size to obtain robust estimates. Two critical steps in our framework ensure that the extracted ISFC transients reflect truly occurring changes in connectivity: first, the high-pass filtering of regional time courses with the inverse of the window length32; second, the use of resting-state ISFC data for the generation of a relevant null distribution, with identical acquisition parameters as compared to the stimulus-related data. Of course, the latter also requires a lengthier global acquisition time, so that resting-state data can be gathered on top of stimulus-related sessions. As an alternative approach to avoid the additional resting-state recordings, we also offer the possibility to generate phase randomized data directly from the stimulus-related time courses, an approach often used in dynamic functional connectivity analyses23,24. Further evaluation on a subset of sessions revealed that although the resting-state null method is more conservative, and thus less prone to false positives, the global patterns of ISFC excursion detection were similar across both schemes (see Supplementary Figure 5).
<img alt="Supplementary Figure 5" src="/files/ftp_upload/59083/59083sfig5.jpg" /> 
Supplementary Figure 5: Detection of ISFC excursions across null data generation methods. For resting-state (left column, blue plots) or phase randomization (right column, red plots) null data generation methods, percentage of ISFC excursions extracted across connections. The bottom plots are an inset on the connections emanating from the first three considered brain regions. Error represents standard deviation across subjects. <a href="https://www.jove.com/files/ftp_upload/59083/59083sfig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The duration of the resting-state acquisitions actually relates to a critical parameter of the analyses: the &#945;-value. As exemplified above, a too lenient choice will lead to a large amount of false positives in the detected ISFC transients. The larger the amount of available resting-state data, the more stringent the achievable false positive rate, because thresholding can be based on more extreme values from the null distribution. As an indication, for n = 299 atlas regions as here and given our tally of 5,762 resting-state data points, we could at best achieve an &#945;-value close to 0.01% (see step 5.2.6 for mathematical details).
Another key point pertaining to any fMRI analysis lies in the rigorous removal of possible motion-related artifacts from the analyzed data30,42. In particular, if one wishes to apply the introduced pipeline to diseased populations exhibiting marked motion in the scanner, we recommend that on top of including motion variables as covariates in the performed statistical analyses, additional preprocessing steps be run, such as wavelet denoising43 or ICA-AROMA44. Group comparison, for instance to compare ISFC transients between a healthy and a diseased group, can readily be performed by running the described approach in parallel on both groups of interest (see Bolton&#160;et al.25 for an example on a population diagnosed with autism spectrum disorders). However, a difference between the groups can then arise in two distinct settings: (1) an absent ISFC change in one group, or (2) a more heterogeneous evolution in that group. To disentangle those two factors, the pipeline should be run once more for the diseased group, using the healthy subject set as the reference group at the bootstrapping step. The former case would still result in an absent response, whereas the latter would not.
On top of what we described here, the introduced methodology also opens up promising future avenues: from an analytical side, ISFC transient maps could be viewed as brain graphs from which metrics quantifying brain connectivity could be derived45, or dynamic ISFC states could be extracted through clustering approaches and assessed in terms of their spatial and temporal characteristics17,46. In addition, one could also envisage the use of more sophisticated connectivity measurement tools than Pearson&#39;s correlation coefficient to reveal subtler sides of FC47,48.
From the experimental side, the application of our pipeline to a more extended set of paradigms is a promising perspective: for example, instead of a movie as studied here, one could envisage to use a piece of music49 or a narrative story13,50 as a time-locked stimulus. Alternatively, it could even be envisioned, through hyperscanning51, to probe naturalistic social communication52,53.

Functional magnetic resonance imaging (fMRI) has become the tool of choice to non-invasively monitor the changes in brain activity resulting from external stimulation. More specifically, vivid interest has emerged about the understanding of statistical interdependence between regional activation time courses, known as functional connectivity (FC)1 and typically computed as Pearson&#39;s correlation coefficient. Functional interplays across the brain have been extensively shown to reconfigure as a function of the underlying task2,3,4.
Two analytical directions have separately been followed to go beyond this introductory characterization: on the one hand, the response induced in a given brain region by a time-locked stimulus was observed to strongly correlate across distinct subjects5. Quantifying this inter-subject correlation (ISC) showed potential to refine our understanding of cognition6,7,8,9 and brain disorders10,11. Further, this cross-subject correlational approach was also extended to the assessment of cross-regional synchronicity12, in what became known as the inter-subject functional correlation (ISFC) approach13.
On the other hand, the dynamic flavor of FC reconfigurations started to receive increased attention (see Hutchison&#160;et al.14, Preti, Bolton and Van De Ville15, Gonzales-Castillo and Bandettini16 for recent reviews on the resting-state and task-based sides of this question). In particular, whole-brain FC changes over time can be tracked through consecutive correlation measurements over a gradually shifted temporal sub-window17,18, revealing additional insight in the context of behavioral tasks19,20.
Here, we present a methodological framework that combines those two avenues. Indeed, we compute ISFC in sliding-window fashion to track the evolution of cross-regional synchronicity between the subjects exposed to a time-locked, naturalistic paradigm. Through the cross-subject aspect of the method, analyses are focused on stimulus-driven effects, while spontaneous fMRI changes (which are uncorrelated across subjects) are strongly damped. This is important because resting-state and task-evoked activity patterns are increasingly understood to be characterized by distinct properties21,22.
As for the dynamic component of the method, it enables a more complete and accurate characterization of task stimuli, particularly when probing a naturalistic paradigm in which a diverse set of cues (auditory, visual, social, etc.) are combined over time. Further, as the sound statistical evaluation of significant dynamic fluctuations has been hotly debated23,24, our approach takes particular care of this aspect of the analyses by isolating significant ISFC changes through comparison to appropriate null data.
We illustrate the method on a set of healthy subjects exposed to an audio-visual movie stimulus, for whom we show that the temporal and spatial ISFC change profiles arising from localized movie sub-intervals can be accurately extracted. In doing so, we also describe the influence of the main analytical parameters to be selected by the user. The presented findings are based on part of formerly published data25,26.

<table>
	<tbody>
		<tr>
			<td>Freesurfer version 6.0</td>
			<td>Laboratory for Computational Neuroimaging, Martinos Center for Biomedical Imaging, Boston (MA), USA</td>
			<td>https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall</td>
			<td>A MATLAB-compatible toolbox enabling to carry out various processing, visualisation and analytical steps on functional magnetic resonance imaging data</td>
		</tr>
		<tr>
			<td>MATLAB_R2017a</td>
			<td>MathWorks</td>
			<td>https://ch.mathworks.com/downloads/</td>
			<td>Working version of the MATLAB computational software (version 2014a or more recent should be used)</td>
		</tr>
		<tr>
			<td>Statistical Parametric Mapping version 12.0 (SPM12)</td>
			<td>Wellcome Trust Center for Neuroimaging, University College London, London, UK</td>
			<td>https://www.fil.ion.ucl.ac.uk/spm/software/spm12/</td>
			<td>A MATLAB-compatible toolbox enabling to perform statistical analyses on functional magnetic resonance imaging data</td>
		</tr>
		<tr>
			<td>Tim-Trio 3 T MRI scanner</td>
			<td>Siemens</td>
			<td>https://www.healthcare.siemens.ch/magnetic-resonance-imaging/for-installed-base-business-only-do-not-publish/magnetom-trio-tim</td>
			<td>Magnetic resonance imaging scanner in which subjects have their functional brain activity recorded (at 3 T)</td>
		</tr>
	</tbody>
</table>

dynamic inter-subject functional connectivity reveals moment-to-moment brain network configurations driven by continuous or communication paradigms

Task-based functional magnetic resonance imaging bears great potential to understand how our brain reacts to various types of stimulation; however, this is often achieved without considering the dynamic facet of functional processing, and analytical outputs typically account for merged influences of task-driven effects and underlying spontaneous fluctuations of brain activity. Here, we introduce a novel methodological pipeline that can go beyond these limitations: the use of a sliding-window analytical scheme permits tracking of functional changes over time, and through cross-subject correlational measurements, the approach can isolate purely stimulus-related effects. Thanks to a rigorous thresholding process, significant changes in inter-subject functional correlation can be extracted and analyzed.
On a set of healthy subjects who underwent naturalistic audio-visual stimulation, we demonstrate the usefulness of the approach by tying the unraveled functional reconfigurations to particular cues of the movie. We show how, through our method, one can capture either a temporal profile of brain activity (the evolution of a given connection), or focus on a spatial snapshot at a key time point. We provide a publicly available version of the whole pipeline, and describe its use and the influence of its key parameters step by step.

Here, we considered n = 15 typically developing (TD) subjects for whom we obtained written, informed consent. All were right-handed males (23.42 &#177; 7.8 years old). The chosen paradigm was an audio-visual scientific documentary for youngsters about the dangers of sun exposure. It contains a large array of visual, auditory and social stimuli, and can be watched at https://miplab.epfl.ch/index.php/miplife/research/supplement-asd-study.
We acquired two sessions per subject (RUN1 and RUN2) in which the assessed movie was displayed from 5 to 353 s (5.8 min duration). A resting-state segment also followed from 386 to 678 s (4.9 min duration). In addition, one solely resting-state session (RUN3) was acquired for each subject (excluding one who suffered from claustrophobia), lasting for 310 s (5.2 min). Example movie scenes and the timing of acquired data are summarized in Figure 1A. Importantly, the acquisition protocol was not optimal in the sense that resting-state recordings acquired just after movie exposure may be partly corrupted by spillover effects27; we make use of this data in the present findings to have a satisfying amount of samples for statistical thresholding, but this should be avoided whenever possible.
We excluded all sessions for which more than 10% of frames were scrubbed, at a threshold of 0.5 mm, and considered the parcellation from Craddock&#160;et al.34 (two-level temporal correlation algorithm) to generate regional time courses, for a total of 299 different brain regions.
ISFC was computed separately on (1) the movie-watching subparts of RUN1 and RUN2, (2) the resting-state subparts of RUN1 and RUN2, and (3) the resting-state RUN3 recordings. We used a window length W = 10 TR for the main presented results, and compare them to a lower value of W = 5 TR. Step size always remained equal to 1 TR. Bootstrapping was performed over 250 folds, including 6 session segments in each reference group.
Figure 1B&#160;displays ISFC time courses generated at W = 10 TR and W = 5 TR for three different representative connections: connection 1 involved a left inferior parietal region related to the expectation of moving objects (MNI coordinates: 41,9,32)35, and a right frontal opercular area linked to response inhibition (-34,-52,45)36. This latter region was also implicated in connections 2 and 3, respectively with an area implicated in sensory coordination (54,6,34)37, and one tied to the processing of the meaning of words (6,62,9)38.
A comparison across window lengths reveals that in the W = 5 TR setting, temporal variance in the subjects is overall larger in both the movie-watching and resting-state segment cases as compared to W = 10 TR, a known phenomenon in sliding-window analyses39. For connection 1, regardless of the window length, a localized subpart of the movie-watching recording (at around 55 s) shows a strong, synchronized ISFC increase across subjects, which largely exceeds the range of values taken in the resting-state case. Thus, we expect to capture this temporal subpart as a significant ISFC transient with our thresholding method.
For connection 2, we observe similar temporal dynamics, but for W = 5 TR, the increase becomes less easy to disentangle as compared to the resting-state time courses, due to the larger sliding-window methodology-related noise. As for connection 3, it reflects a case in which there is no clear response to the movie, and thus, the fluctuations from movie-watching and resting-state time courses are similar. The expected outcome at this analytical stage is a mix between connections that show clear stimulus-induced reconfigurations, and connections that do not respond.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59083/59083fig1.jpg" /> 
Figure 1: Acquisition timing and example ISFC time courses. (A) The movie watched by the subjects involved a wide array of social situations (example images 1 and 4), scientific explanations with colorful panels (example images 2 and 5), and landscape sceneries (example image 3). Three sessions were acquired per subject: two (RUN1 and RUN2) included the movie stimulation (from 5 to 353 s, highlighted in green) followed by a resting-state period (from 386 to 678 s, shown in yellow), while one (RUN3) solely consisted in a resting-state recording (310 s duration, displayed in orange). (B) For three indicative connections (C1, C2 and C3, respectively dark green/red, light green/orange and turquoise/yellow traces), evolution of ISFC over time during movie-watching (cold colors) or resting-state (hot colors). For W = 10 TR (left panel), movie-watching ISFC changes more largely stand out as compared to W = 5 TR (right panel). Each trace reflects the ISFC time course of one session. This figure has partly been modified from Bolton&#160;et al.25. <a href="https://www.jove.com/files/ftp_upload/59083/59083fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2A&#160;displays the results following statistical thresholding of ISFC time courses, for the same three connections as above. A time course value of 1 means that all subjects underwent the same ISFC increase at the same time point; a value of 0 means that no subject underwent a significant ISFC change; a value of -1 represents a synchronous ISFC decrease across all subjects. As before, we contrast W = 5 TR and W = 10 TR, and we also highlight two &#945;-value cases: &#945; = 0.01%, and &#945; = 5%.
Fitting with the above observations, a lower window length reduces the amount of extracted significant ISFC changes. For connection 1, both W = 5 TR and W = 10 TR, however, extract the same particular moment (t = 55 s) as showing a strong ISFC increase. Taking a hemodynamic delay of roughly 5 s into account, this corresponds to a subpart of the movie when colored lines were extending towards a doll, and abruptly stopped just in front of it (46-49 s), fitting with the role of the involved regions in moving object expectation and response inhibition35,36.
When increasing &#945; from 0.01% to 5%, one can observe a much lower specificity of the detected ISFC transients, likely including many false positives and expectedly showing much less temporal synchrony.
As another perspective that can be set on the data, Figure 2B shows the whole-brain spatial maps of significant ISFC changes at t = 55 s. It can be seen that the response to the movie scene extends far beyond the example connections described here.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59083/59083fig2.jpg" /> 
Figure 2: Temporal and spatial snapshots of ISFC patterns. (A) ISFC transient time courses, averaged across subjects, for three indicative connections (C1, C2 and C3, respectively dark green, light green and turquoise traces). The movie scene that drove the ISFC changes is highlighted in light grey, and depicted by example images. For W = 10 TR (left column of plots), ISFC changes are more strongly detected than for W = 5 TR (right column of plots). For &#945; = 0.01% (top row of plots), specificity to localized movie cues is larger than for &#945; = 5% (bottom row of plots). Each trace reflects the ISFC transient time course of one session, and the two-tailed 95% confidence intervals are displayed as error measure. (B) For W = 10 TR and &#945; = 0.01%, there is a neat, restricted spatial pattern of ISFC transients at t = 55 s (the peak ISFC transient value for C1); for W = 5 TR and &#945; = 5%, connections undergoing a significant ISFC change at this time are much more numerous. Note that we assume a hemodynamic delay of around 5 s in the described temporality (i.e.,&#160;a value of 55 s here relates to the movie stimulus at 50 s). This figure has partly been modified from Bolton&#160;et al.25. <a href="https://www.jove.com/files/ftp_upload/59083/59083fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dynamic Inter-subject Functional Connectivity Reveals Moment-to-Moment Brain Network Configurations Driven by Continuous or Communication Paradigms at JoVE.co

Dynamic Inter-subject Functional Connectivity Reveals Moment-to-Moment Brain Network Configurations Driven by Continuous or Communication Paradigms

The goal of the described approach is to determine at what moments of the paradigm (temporal perspective), and between which regions (spatial perspective), significant reconfigurations in functional connectivity occur on functional magnetic resonance imaging recordings during which a time-locked stimulus is played.

Task-based functional magnetic resonance imaging bears great potential to understand how our brain reacts to various types of stimulation; however, this ...

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Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

The Dig Task: A Simple Scent Discrimination Reveals Deficits Following Frontal Brain Damage

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in rodent models of brain injury is lacking. Borrowing from the operant literature our laboratory utilized a basic scent discrimination paradigm1-4 in order to assess deficits in frontally-injured rats. Previously we have briefly described the Dig task and demonstrated that rats with frontal brain damage show severe deficits across multiple tests within the task5. Here we present a more detailed protocol for this task. Rats are placed into a chamber and allowed to discriminate between two scented sands, one of which contains a reinforcer. The trial ends after the rat either correctly discriminates (defined as digging in the correct scented sand), incorrectly discriminates, or 30 sec elapses. Rats that correctly discriminate are allowed to recover and consume the reinforcer. Rats that discriminate incorrectly are immediately removed from the chamber. This can continue through a variety of reversals and novel scents. The primary analysis is the accuracy for each scent pairing (cumulative proportion correct for each scent). The general findings from the Dig task suggest that it is a simple experimental preparation that can assess deficits in rats with bilateral frontal cortical damage compared to rats with unilateral parietal damage. The Dig task can also be easily incorporated into an existing cognitive test battery. The use of more tasks such as this one can lead to more accurate testing of frontal function following injury, which may lead to therapeutic options for treatment. All animal use was conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee.

In this protocol, a novel application of scent discrimination is described. The Dig task is a reasonably inexpensive task that can be used to assess frontally-mediated cognition following brain damage.

Cognitive impairment is the most frequent cause of disability in humans following brain damage, yet the behavioral tasks used to assess cognition in ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

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Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

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This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

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In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

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Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...