FD was supported by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression and a CPRF Award from the Canadian Psychiatric Research Foundation.

1. Experimental Design, Methodological Issues, &amp; Stimuli
The SMP consist of two tasks, an encoding task and a retrieval task, and recording of brain activity may occur during the encoding task or during 
both encoding and retrieval tasks (see Figure 1 for a diagram illustrating the design of the SMP).
<img src="/files/ftp_upload/2433/2433fig1.jpg" alt="Figure 1" /> 
Figure 1. General Diagram of the SMP design. Brain regions whose activity during encoding is greater for subsequently remembered than for subsequently 
forgotten items (R &gt; F) are associated with encoding success (ES). Similarly, if this difference in memory (Dm) 3 is measured based on comparing brain activity 
recorded during retrieval (Hits &gt; Misses) it identifies regions associated with retrieval success (RS). 
Several important aspects must be considered in studies using the SMP with emotional stimuli:
<ol>
 <li>It is important to control for the emotional properties of stimuli, such as emotional valence and arousal/intensity, as they have different effects on 
 emotional perception and thus on emotional memory10, 26, 27. Commonly used emotional stimuli are pictures from the International Affective Picture 
 System (IAPS)23 and words from the Affective Norms for English Words (ANEW)24. IAPS pictures and ANEW words are controlled on two major affective 
 dimensions (i.e., valence - the degree of pleasantness or unpleasantness, and arousal - the emotional intensity) that define the emotional 
 properties of a stimulus (see 25). Another category of stimuli frequently used in emotion research are human faces with emotional expressions, such us 
 those from the Pictures of Facial Affect (<a href="http://www.paulekman.com">http://www.paulekman.com</a>) and the 
 NimStim (<a href="http://www.macbrain.org/">http://www.macbrain.org/</a>) sets.</li>
 <li>Other factors whose manipulations may also affect memory performance, and hence contribute to the versatility of this protocol include: a. variation in the 
 type of encoding task used (i.e. incidental11, 13, 19 or intentional 8, 20), b. the encoding focus with respect to the emotional content of the stimuli (i.e. with 
 emotional 8, 11 or non-emotional aspects as the primary focus 13, 19), and c. the type of retrieval task used (i.e. recall 8, 11 
 or recognition 12, 20). In addition, stimulus duration also affects memory performance 21, and hence it should be considered as well, along with 
 the factors mentioned above.</li>
 <li>Other possible confounding variables, such as the human presence in and visual complexity of emotional and neutral pictures 11 should also be controlled for.</li>
 <li>It is critical that memory performance for the conditions of interest falls in reasonable ranges that allow fair statistical comparisons of both behavioral and brain 
 imaging data. For example, in our cued-recall tasks, the proportion of remembered and forgotten trials was around 50% 8, 11. However, because in two-choice 
 recognition tasks this performance level would be close to chance, higher overall performance level is expected. In this case, fair comparison between remembered and 
 forgotten conditions can be accomplished by randomly selecting a smaller number of remembered trials to equate the number of forgotten trials. In addition, an 
 enhancing effect of emotion on memory should also be present in the behavioral data, reflected in greater proportion of remembered trials for emotional than 
 for neutral items.</li>
 <li>It is also advised that mood induction be avoided as this may also affect performance 22. This can be done by pseudo-randomizing the emotional 
 stimuli to ensure that no more than 2-3 stimuli of the same valence are consecutively presented 11. </li>
</ol>
2. Preparing the Subject for the Scan
All Subjects provide written informed consent prior to running the experimental protocol, which has been approved by an Ethics Board.
Prior to Entering the Scanning Room
<ol>
 <li>Approximately 45 minutes prior to the scan each subject completes questionnaires to assess overall emotional state and anxiety level 28, 29. These initial 
 assessments are used in combination with post-session assessments to screen for changes in mood/anxiety as a result of participating in the study.</li>
 <li>After the completion of the questionnaires, the subject is informed of the scanning procedures and given specific instructions for the behavioral task. The 
 subject also completes a short practice session to become familiar with the task.</li>
</ol>
Entering the Scanning Room
<ol start="3">
 <li>The subject is instructed to lie supine on the scanning bed, with additional cushioning for the head, and is given ear protection. Also, the non-adhesive 
 side of a length of tape may be wrapped lightly around the subject&#x2019;s forehead to help minimize movement.</li>
 <li>Prior to scanning, the subject&#x2019;s right hand is positioned on a response box and the response box is tested. Before starting data collection, it should also be ensured that the subject can see the screen projection clearly. An emergency stop button is placed nearby the subject&#x2019;s free hand so that s/he may indicate any urgent need to stop the scanner.</li>
 <li>In our studies, stimuli were presented using CIGAL, an in-house data presentation software program 30, but other software are also available, 
 including E-prime, SuperLab, Presentation, and the Matlab-based Psychtoolbox.</li>
 <li>Based on our investigations, we recommend that the duration of scanned tasks involving emotion stimulation should be no longer than 1h, and the overall 
 scanning time should not exceed 2 hrs; also, if possible the latter should be reduced in clinical groups 17.</li>
</ol>
3. Data Recording and Processing
Scanning Parameters
In the original study by Dolcos and colleagues 11, MRI data were collected on a 1.5 Tesla GE scanner. Full-brain coverage was 
acquired via 34 contiguous gradient-echo echoplanar images (EPIs) sensitive to blood oxygen level dependent (BOLD) contrast, defined parallel to the AC-PC plane in 
the axial plane (TR = 3000ms; TE = 40ms; FOV = 642 image matrix; FA = 90&deg;; slice thickness = 3.75 mm). The scanning parameters used here have been well suited to 
identify activations in the medial-temporal lobe (MTL) structures, but other scanning parameters optimized for higher spatial resolution can also be used 33-35.
Post-Scanning Procedures
<ol>
 <li>The subject completes questionnaires to assess state mood and anxiety levels and, if not performed before the scanning session, assessments of personality 
 traits (e.g. neuroticism, emotional arousability) can also be collected.</li>
 <li>If retrieval activity is not recorded during the scanning session, the retrieval task may be performed following the scanning session, or at a later time. If 
 not already performed in the scanner, an emotional rating task may be used to assess the subject&#x2019;s emotional response to the stimuli. This way, the separation of 
 emotional and neutral categories can be done based on the participants&#x2019; ratings, rather than based on the normative scores. However, this task should be performed 
 following the retrieval task.</li>
 <li>To maximize the effect of emotion on memory, a minimum delay of ~20 minutes needs to occur between encoding and retrieval 18. However, the delay 
 can be much longer, from minutes to days, weeks, and months, as the impact of emotion on memory can be long-lasting and emotional recollection tends to 
 persist overtime compared to neutral recollection 12, 13.</li>
</ol>
The Memory Task
The main focus in the present protocol is on findings from a study using a cued-recall memory test 11, in which participants 
were provided with verbal cues for each picture and they had to provide in writing details about the pictures they could remember. However, other retrieval tasks 
can also be used. Of particular relevance is the so-called R-K paradigm 31, a memory recognition task in which participants categorize each retrieved 
item based on whether they recollect specific contextual details from encoding (Remember responses) or just have the feeling of familiarity that they 
encountered the items (Know responses). Used with fMRI and with emotional stimuli, this task allows not only dissociation of the neural correlates of 
recollection- vs. familiarity-based retrieval 32 but also identification of differential effects of emotion on these two types of retrieval, and the 
associated neural correlates 12.
fMRI Data Analysis
Statistical Parametric Mapping (SPM: (<a href="http://www.fil.ion.uc.uk/spm/">http://www.fil.ion.uc.uk/spm/</a>) in combination with in-house 
Matlab-based tools were used for data analysis. Pre-processing typically involves: quality assurance, image alignment, motion correction, co-registration, 
normalization, and smoothing (8 mm3 Kernel), but if analyses involve anatomically defined regions of interest (ROIs), increased spatial specificity is obtained 
if data are not smoothed and normalized (see 11). Individual and group-level statistical analyses involve comparisons of encoding/retrieval brain activity based 
on subsequent memory performance. The modulatory effect of emotion on memory-related activity can be seen in areas showing greater ES / Dm or RS 
activity for emotional than for neutral stimuli (see Figure 2). Investigations targeting MTL activity allow identification of functional 
dissociations, which are linked to the emotional content of stimuli (emotional vs. neutral memory; 11), the type of memory being studied 
(item vs. source memory; 19), or the type of retrieval task (recollection- vs. familiarity-based; 12, 36). Investigation of brain regions outside of the 
MTL particularly target areas in the prefrontal cortex (PFC), that are differentially involved in emotional vs. neutral memory 10, 20.
<img src="/files/ftp_upload/2433/2433fig2.jpg" alt="Figure 2" /> 
Figure 2. Identification of Memory-Related Brain Activity Susceptible to Emotion Modulation. In addition to allowing identification of memory-related 
brain activity that is sensitive to emotion modulation (EmoDm / RS &gt; NeuDm / RS; i.e., showing emotion x memory interactions), this design also allows 
identification of brain regions whose activity reflects emotion effects on general/perceptual processing [(Emotion R + Emotion F) &gt; (Neutral R + Neutral F ); 
i.e., showing main effects of emotion], as well as overall effects of memory [(Emotion R + Neutral R) &gt; (Emotion F + Neutral F); i.e., showing main 
effects of memory].
4. Representative Results:
The employment of the SMP in studies of emotional memory has proved successful in shedding light on the neural correlates of the 
memory-enhancing effect of emotion. Providing strong support for the modulation hypothesis 37, 38, studies of memory encoding have found evidence supporting 
the idea that emotion enhances memory through modulatory influences from the amygdala, a brain region involved in emotion processing, on activity in 
memory-related brain regions, such as the medial-temporal lobe (MTL 11, see Figure 3) and the prefrontal cortex (PFC10). Moreover, studies of memory 
retrieval provide further evidence that the modulation hypothesis also applies to retrieval, and that various MTL regions are differentially involved 
in recollection- vs. familiarity-based retrieval of emotional memories 12.
<img src="/files/ftp_upload/2433/2433fig3.jpg" alt="Figure 3" /> 
Figure 3. Evidence for the Modulation Hypothesis. Supporting the modulation hypothesis, the memory-enhancing effect of emotion was associated with 
increased ES/Dm activity (A) in and interactions (B) between the amygdala and the MTL memory system (illustrated in C &amp; D). The bar graphs compare the percent 
signal change for emotional and neutral ES / Dm, as extracted from the peak activation slice and averaged across hemispheres. The scattergrams show the co-variation 
between ES / Dm activity in the amygdala and the entorhinal cortex for emotional and neutral pictures. ES / Dm = Encoding Success / Difference due to Memory. 
From Dolcos et al. 11, with permission.

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No conflicts of interest declared.

This experimental protocol allows investigation of the neural correlates of the memory-enhancing effect of emotion, by specifically separating 
the memory-related brain activity that is susceptible to modulation by emotion from activity in brain regions susceptible to emotion modulation during 
general/perceptual processing. This design has been instrumental in advancing our understanding of the neural mechanisms underlying the impact of emotion on 
memory, and given its versatility it is expected that SMP will remain cr...

brain imaging investigation of the memory-enhancing effect of emotion

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the neural mechanisms underlying this enhancing effect of emotion on memory. A method that has proven particularly influential in the 
investigation of the memory-enhancing effect of emotion is the so-called subsequent memory paradigm (SMP). This method was originally used to investigate the 
neural correlates of non-emotional memories3, and more recently we and others also applied it successfully to studies of emotional memory (reviewed in4, 5-7). 
Here, we describe a protocol that allows investigation of the neural correlates of the memory-enhancing effect of emotion using the SMP in conjunction with 
event-related functional magnetic resonance imaging (fMRI). An important feature of the SMP is that it allows separation of brain activity specifically 
associated with memory from more general activity associated with perception. Moreover, in the context of investigating the impact of emotional stimuli, 
SMP allows identification of brain regions whose activity is susceptible to emotional modulation of both general/perceptual and memory-specific processing. 
This protocol can be used in healthy subjects8-15, as well as in clinical patients where there are alterations in the neural correlates of emotion perception 
and biases in remembering emotional events, such as those suffering from depression and post-traumatic stress disorder (PTSD)16, 17.

Watch this Scientific Journal Video about Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion at JoVE.com

Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.

Emotional events tend to be better remembered than non-emotional events1,2. One goal of cognitive and affective neuroscientists is 
to understand the ...

brain-imaging-investigation-of-the-memory-enhancing-effect-of-emotion

Research

JoVE Journal

Neuroscience

16.2K Views.  University of Alberta. Vi presentiamo un protocollo che utilizza la risonanza magnetica funzionale per indagare i correlati neurali della memoria effetto di intensificazione delle emozioni. Questo protocollo permette l'identificazione di attività cerebrale specificamente legate al relativo alla memoria di elaborazione, contrariamente a quanto più generale di elaborazione percettiva, e può essere utilizzato con una popolazione sana e clinica.

Video: Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection

Recollection of emotional autobiographical memories (AMs) is important to healthy cognitive and affective functioning 1 - remembering positive AMs is associated with increased personal well-being and self-esteem 2, whereas remembering and ruminating on negative AMs may lead to affective disorders 3. Although significant progress has been made in understanding the brain mechanisms underlying AM retrieval in general (reviewed in 4, 5), less is known about the effect of emotion on the subjective re-experience of AMs and the associated neural correlates. This is in part due to the fact that, unlike the investigations of the emotion effect on memory for laboratory-based microevents (reviewed in 6, 7-9), often times AM studies do not have a clear focus on the emotional aspects of remembering personal events (but see 10). Here, we present a protocol that allows investigation of the neural correlates of recollecting emotional AMs using functional magnetic resonance imaging (fMRI). Cues for these memories are collected prior to scanning by means of an autobiographical memory questionnaire (AMQ), therefore allowing for proper selection of emotional AMs based on their phenomenological properties (i.e., intensity, vividness, personal significance). This protocol can be used in healthy and clinical populations alike.

We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Recollection of emotional autobiographical memories (AMs) is important to healthy cognitive and affective functioning 1 - remembering positive AMs is ...

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...