This research 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 (to FD). The authors wish to thank Trisha Chakrabarty and Peter Seres for assistance with fMRI data collection and Kristina Suen for assistance with data analysis.

1. Task Design, Stimuli, and Experimental Protocol
The overarching protocol involves assessments of emotional ratings of pictures (immediate impact), and of the memory for 
these pictures (long-term impact), as a result of inducing the goal to regulate emotional responses. The goal to regulate is induced explicitly or implicitly, and the immediate and long-term impact is assessed relative to ratings of and memory for pictures presented during baseline blocks/runs that precede the ER manipulations (pre-ER induction baseline); see Figure 1 for a diagram of the overall experimental design. Below we provide details regarding all these aspects.
<img alt="Figure 1" src="/files/ftp_upload/2430/2430fig1.jpg" /> 
Figure 1. Diagram of the protocol. The ER goal is induced in each participant both consciously/explicitly and non-consciously/implicitly, but the order of induction is different and counterbalanced across participants - i.e., those assigned to explicit ER in the first part complete the implicit manipulation in the second part, and vice versa; each manipulation is preceded by its own baseline runs. See below for detailed descriptions of the Rating and Memory tasks.
Emotion Regulation Manipulation
<ol>
	<li>Explicit ER manipulation. In the explicit ER condition, the emotion rating task is preceded by instructions to suppress emotional experience and expression of emotional responses. Previous evidence suggests that explicit ER strategies can reliably influence emotional responding2.</li>
	<li>Implicit ER manipulation. For this, an adaptation of the Scrambled Sentence Task (SST)3 is used to non-consciously induce the goal to regulate emotions. In this task, participants have to construct 20 four-word grammatically correct sentences from five-word jumbles that have embedded words that convey the idea of emotion control (e.g., "restrains", "stable", "covered"), and thus prime the participants to suppress their emotional response4. The emotion control words were taken from previous studies4, and selected by asking undergraduate students to "list the 20 words that come to your mind when you think of the concept 'emotion control'."</li>
	<li>Of note, to maintain the same structure of the task in both ER conditions, a SST is also performed in the explicit ER condition, but in this case participants are presented with 20 sentences containing only neutral terms regarding the goal to control emotions.</li>
</ol>
Assessments of the Immediate Impact of ER manipulation: The Emotional Rating Task
<ol start="4">
	<li>Selection of Stimuli. Stimuli consist of emotional and neutral pictures selected from the International Affective Picture System (IAPS)5, based on their normative scores for emotional valence and arousal. Careful attention should be paid to balancing the emotional content of picture across all study runs, and hence avoid confounding effects due to possible differences in these basic properties.</li>
	<li>Timing of the task. Each picture is presented on the screen for 4 seconds, and then is removed to minimize confounding effects of eye movements associated with prolonged scanning of images6; a fixation cross follows each picture for 12 seconds. Participants' task is to watch the picture and rate their subjective emotional experience triggered by the pictures on an 8-point scale (1 = neutral, 8 = extremely negative). To avoid mood induction, pictures are pseudo-randomized so that no more than three pictures of the same valence are consecutively presented.</li>
</ol>
Assessment of the Long-Term Impact of ER manipulation: The Memory Retrieval Task
<ol start="6">
	<li>One week later, participants view an equal number of Old (seen in the scanner) and New pictures (never seen before), which are presented in black and white to prevent ceiling performance. Following an Old/New decision based on whether they remember seeing the pictures while in the scanner or not, participants also rate the confidence of their responses on a 3-point scale (1 = low; 2 = medium; 3 = high confidence).</li>
</ol>
2. Preparing the Subject for the Scan
All participants provide written informed consent prior to running the experimental protocol, which is approved by an Ethics Board. They are also warned that many of the pictures depict distressing events, such as acts of violence or trauma, and are provided with printed examples of representative pictures.
Before Scanning
<ol>
	<li>On the day of scanning, participants' current state of mind is assessed7, to control for the effect of mood on the emotional experience and ability to control emotions. In conjunction with post-scanning assessments, these initial evaluations can be also used to screen for changes in mood as a result of study participation. Assessments of personality traits, such those indexing habitual engagement of emotion regulation strategies8 can also be made to investigate their possible influences on the success of the ER manipulation and the associated neural correlates.</li>
	<li>Prior to the scan, the participants are informed in detail of the scan procedures, and are given specific instructions for the behavioural task. To avoid discomfort and to create increased familiarity with the task, participants are also given abbreviated practice runs for both the emotion rating task and the SST.</li>
</ol>
In the Scanning Room
<ol start="3">
	<li>Participants are instructed to lie supine on the scanning bed, with additional cushioning for the head to ensure comfort during the scan and minimize movement. The non-adhesive side of a length of tape may be wrapped lightly around the participants' forehead to further minimize head movement. Participants are given ear protection as well as isolation headphones to communicate with the experimenter during the MRI scan.</li>
	<li>Subjects' right hand is positioned comfortably on the response box, thus allowing the left hand to be used for support or for other measurements (e.g., skin conductance responses). An emergency stop button is placed nearby so that the subject may indicate any urgent need to stop the scanner. Before starting data collection, it is critical to make sure that the subject can see the screen projection clearly for stimulus presentation and that the response buttons work properly.</li>
	<li>The study is divided into runs/blocks of trials, to allow participants time to rest, and to avoid massive data loss in case of equipment malfunction. Run order is counterbalanced between participants, and each run begins with six seconds of fixation, to allow for MR signal stabilisation.</li>
</ol>
3. Data Recording and Processing
Scanning Parameters
We collected MRI data from 24 young healthy participants, using a 1.5 Tesla Siemens Sonata scanner for MRI recordings. Our anatomical images are 3D MPRAGE anatomical series (repetition time (TR) = 1600 ms, echo time (TE) = 3.82 ms; number of slices = 112; voxel size = 1x1x1mm). The functional images consist of series of 28 functional slices (voxel size = 4x4x4 mm), acquired axially using an echoplanar sequence (TR = 2000 ms; TE = 40 ms; field of view FOV = 256x256mm), thus allowing for full-brain coverage.
Data Analysis
We use Statistical Parametric Mapping (SPM: <a href="http://www.fil.ion.ucl.ac.uk/spm/">http://www.fil.ion.ucl.ac.uk/spm/</a>) in combination with in-house MATLAB-based tools. Pre-processing involves typical steps: quality assurance, TR alignment, motion correction, co-registration, normalization, and smoothing (8 mm3 kernel)9. Individual and group-level statistical analyses may involve comparisons of brain activity according to ER manipulation (ER vs. baseline runs; see Figure 2), emotional valence (negative vs. neutral), arousal (low vs. high), and memory performance (remembered vs. forgotten). Correlations of brain imaging data with behavioural data (e.g., picture ratings) and/or scores indexing personality measures (e.g., personality traits indexing habitual engagement of ER strategies) can also be performed, to investigate how brain activity co-varies with individual differences in behaviour and personality.
4. Representative Results:
<img alt="Figure 2" src="/files/ftp_upload/2430/2430fig2.jpg" /> 
Figure 2. Decreased amygdala activity and increased prefrontal cortex activity following ER manipulation. Inducement of the ER goal was associated with reduced activity in brain areas associated with emotion processing, including the amygdala (a), and increased activity in brain regions associated with cognitive control and emotion regulation10, including ventrolateral prefrontal cortex (PFC) (b), dorsolateral PFC (d), and medial PFC (c)11,12. As shown in the bar graph illustrating the vlPFC activity, explicit and implicit ER were associated with similar patterns of response, suggesting that these changes reflect joint contribution of explicit and implicit ER. The "activation maps" are superimposed on high resolution brain images displayed in coronal views; the color bars indicate the gradient of t values of the activation maps, based on group statistics (N = 24), reflecting brain activity in voxels that show decreased (left panel) or increased (right panel) activity as a result of ER manipulation. It should be noted that activation blobs in the PFC may cover more areas, as follows: vlPFC/Insula (b) and dorsal Anterior Cingulate Cortex/Premotor Cortex (c). The bar graphs illustrate percent signal changes in response to emotional (Emo) and neutral (Neu) pictures before (i.e., BaseRuns) and after the induction of the ER goals (i.e., Combined ER = Explicit and Implicit ER averaged together, Explicit ER, and Implicit ER), as extracted from voxels showing pre- to post-ER differences. L = Left; R = Right.
<img alt="Table 1" src="/files/ftp_upload/2430/2430table1.jpg" /> 
Table 1. Decreased emotional ratings following ER manipulation. Similar to the fMRI results, there was a reduction of emotional ratings following ER induction, and this effect resulted from decreases following both explicit and implicit ER. Importantly, emotional ratings did not change significantly in a control group that performed the same task following similar delays but in the absence of ER goal induction. Supporting these ideas, a 2 (Manipulation: Baseline vs. ER Induction) x 2 (Valence: Emotional vs. Neutral) repeated-measures ANOVA on data from the experimental group* yielded a significant main effect of Manipulation (F[1, 19] = 11.21, p &lt; 0.002) and a significant Manipulation x Valence Interaction (F[1, 19] = 3.02, p &lt; 0.05), and a similar 2-way ANOVA on data from the control group (N = 9) did not yield a significant main effect of Delay (Baseline vs. Delayed Runs) or a significant Delay x Valence interaction. These findings suggest that the decrease in ratings in the experimental group was due to the induction of the goal to control emotions rather than due to habituation following repeated exposure to emotional stimulation. *because of data attrition due to common causes (e.g., technical failure), behavioral analyses in the experimental group were based on data from 20 participants.
Taken together, these behavioral and brain imaging findings validate the present experimental design, which can be used to compare explicit and implicit inductions of the goal to regulate emotions. It should be noted that the present report emphasizes aspects that result from the joint contribution of the Explicit and Implicit ER manipulations, but this does not exclude the possibility that their effects may be dissociable at behavioral and/or brain level.

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No conflicts of interest declared.

We described a protocol that involves explicit and implicit manipulation of the goal to regulate emotion, and allows investigation of the associated neural correlates. This design has the potential to advance our knowledge of how the brain regulates emotions, being well suited for comparisons of explicit ER to implicit ER, which is unintentional and can happen without participant's insight and awareness13,14. Hence, the protocol can be particularly useful for investigations of habitual opposite affective biase...

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.

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Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

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 ...

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Video: Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

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 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.

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 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 ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

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

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

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

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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

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

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

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

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...