This research was supported by a Young Investigator Award from the US National Alliance for Research on Schizophrenia and Depression and a CPRF Award from the Canadian Psychiatric Research Foundation (to FD). ED was supported by a Wyeth-CIHR Post-Doctoral Fellowship. The authors wish to thank Peter Seres for assistance with fMRI data collection and Kristina Suen for assistance with data analysis.

1. Collection and Selection of Memories, Imaged Task, and Experimental Protocol
Collection of the Emotional AMs
<ol>
 <li>Personal memories are elicited from each participant during an interview performed prior to the fMRI session, similar to the procedure employed for non-emotional AMs (e.g., 11, 12). Unlike most previous techniques, our AMQ is specifically constructed to target the assessment of emotional personal episodes and their recollective properties. A commonly used method in behavioural research and adapted for use in neuroimaging studies involves the Crovitz and Schiffman technique 13-15, where subjects remember specific, personally experienced events in response to cue words. A photo-paradigm has been also used to investigate AMs in more controlled settings in fMRI studies 16, 17. However, these techniques do not typically target examination of the emotional component of AMs, that are matched in terms of recollective qualities.</li>
 <li>Our AMQ comprises a list of 115 verbal cues for distinct life events (e.g., the birth of a family member, the death of a relative), which is a combination and extension of lists used by others 18, 19. For each cue, participants are asked to remember a unique episode from their life, that occurred in a specific place and time (e.g., one instance when s/he played in a specific basketball game), rather than remembering general or repeated events (e.g., playing high school basketball). Importantly, the memories must be accompanied by the recollection of being personally involved, rather than hearing about them from others.</li>
 <li>Upon recollection, participants are asked to provide a brief description of the memory (see Figure 1), which will then be used as a personalized memory cue during fMRI scanning (participants are na&iuml;ve to the specific purpose of the pre-scanning interview).</li>
 <li>Each memory is also dated and rated on six Likert scales 20, 21, to assess its phenomenological properties (see Figure 1). Our scales included emotional valence (using a 7-point scale: -3 = very negative, 0 = neutral, +3 = very positive), emotional intensity, personal significance, the amount of contextual details, the amount of visuo-perceptual details (i.e., vividness), and the frequency of retrieval (all of the latter using a 7-point scale: 1 = not at all, 7 = extremely).</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/2396/2396fig1.jpg" /> 
Figure 1. Illustration of AMQ Administration. For each cue, participants remember and briefly describe a specific event, and then date and rate it on 6 scales.
Selection of Highly Emotional AMs
<ol start="5">
 <li>Next, the 40 most emotional memories (20 positive and 20 negative) are selected for each participant, based on the ratings provided on the AMQ (i.e., rated 2 or 3 and -2 or -3, respectively). The positive and negative AMs are then matched in terms of age and phenomenological properties, to ensure that any differences in brain activity during recollection are not confounded by differences in these basic properties.</li>
 <li>If necessary, the descriptions of the selected memory cues are slightly adapted to be matched as closely as possible for length and grammatical complexity; it is also recommended to select a few additional memories for practice purposes.</li>
</ol>
The fMRI Task
<ol start="7">
 <li>The fMRI task is designed to allow comparison of the AM task with a semantic memory (SM) control task; the AM and SM retrieval trials have a similar general structure (see Figure 2A). We used CIGAL (<a href="http://www.nitrc.org/projects/cigal/">http://www.nitrc.org/projects/cigal/</a>) for stimulus presentation in the MR scanner, but other stimulus presentation software may also be used.</li>
 <li>The AM task is based on the personalised cues collected prior to scanning. Each trial begins with a cue that triggers AM recollection, which is indicated by the participant with a button press. Then, participants continue remembering details of the event until cued again to rate the recollected memory (Figure 2B).</li>
 <li>The SM task involves generation of exemplars from different semantic categories (e.g., musical instruments, sports), which like AM retrieval involves search in memory and extended retrieval time 22 (Figure 2B). After a semantic category is cued, participants press a button as soon as they start recalling exemplars from that category and then continue recalling until cued again for memory ratings.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/2396/2396fig2a.jpg" /> 
<img alt="Figure 2" src="/files/ftp_upload/2396/2396fig2b.jpg" /> 
Figure 2. Structure of the fMRI Trials. A. General Structure of the trials. B. Specific structure of the AM and SM trials.
<ol start="10">
 <li>In addition to the basic comparison of AM and SM trials, other manipulations can also be involved. For instance, the focus of the AM retrieval can be manipulated by directing the participants to pay attention to emotional or non-emotional aspects of the remembered events (Figure 2B). This manipulation allows investigation of how retrieval focus may influence the experiencing of AMs and any associated changes in the associated neural correlates.</li>
 <li>Each AM or SM trial is followed by rating screens presented in a counterbalanced order, using 5-point Likert scales (see Figure 2B).</li>
 <li>The experiment is split into runs/blocks of trials both to allow participants time to rest and to avoid data loss in case of equipment malfunction. Run order is counterbalanced between participants. Each run begins with six seconds of fixation, to allow stabilization of the MR signal. The AM and SM conditions are presented in a random order separated by an inter-trial interval of variable duration (5-9 sec, average = 7sec.).</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. Typically, to avoid confounds in the lateralization of brain activations, scanned participants are right-handed.
Prior to Entering the Scanning Room
<ol>
 <li>On the day of scanning, participants' current affective state is assessed 23, to control for the effect of mood on recollection of emotional AMs. In conjunction with post-scanning assessments, these initial evaluations can also be used to screen for changes in mood as a result of the study participation, and as covariates in the fMRI analyses to investigate brain activations influenced by current states. Similarly, assessments of personality traits can also be made (e.g., neuroticism), to investigate their possible biases on the AM task 24 and the associated neural correlates.</li>
 <li>Prior to the scan, participants are informed in detail of the scan procedures, and are given specific instructions for the behavioral task. Participants also complete a short practice session, to familiarize themselves with the task.</li>
</ol>
Entering the Scanning Room
<ol start="3">
 <li>Participants are instructed to lie supine on the scanning bed, and are provided with additional head cushioning, to ensure comfort during the scan and to minimize movement. To further minimize head movement, the non-adhesive side of a length of tape may be wrapped lightly around the subjects' forehead. Subjects are given ear protection as well as isolation headphones to communicate with the experimenter during the MRI scan.</li>
 <li>Subject's 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.</li>
 <li>Before starting data collection, it is critical to make sure that subjects can see the screen projection clearly for stimulus presentation and that the response buttons work properly.</li>
</ol>
3. Data Recording and Processing
Scanning Parameters
We collected MRI data using a 1.5 Tesla Siemens Sonata scanner for MRI recordings. Anatomical images were 3D MPRAGE anatomical series (repetition time (TR) = 1600 ms, echo time (TE) = 3.82 ms; number of slices = 112; voxel size = 1x1x1 mm), and functional images were series of 28 functional slices, acquired axially using an echoplanar sequence (TR = 2000 ms; TE = 40 ms; field of view FOV = 256x256 mm; voxel size = 4x4x4 mm), thus allowing for full-brain coverage.
Data Analysis
We used 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 involved typical steps: quality assurance, TR alignment, motion correction, co-registration, normalization, and smoothing (8 mm3 Kernel). Individual and group-level statistical analyses may involve comparisons of brain activity according to memory type (AM vs. SM), emotional valence (positive vs. negative), and retrieval focus (emotional vs. non-emotional content).
4. Representative Results
<img alt="Figure 3" src="/files/ftp_upload/2396/2396fig3.jpg" /> 
Figure 3. Neural Correlates of AM Retrieval. Validating the present protocol, retrieval of AMs yielded increased activity in the AM retrieval network 4, 25, including hippocampal areas (a), involved in general memory retrieval, the medial prefrontal cortex (b), associated with personal engagement, the cuneus/precuneus regions and parieto-occipital junction (c, e, respectively), associated with processing visuo-spatial representation, and the frontal-temporal junction (d), involved in affective AM retrieval; similar effects to the latter were also found in the amygdala (not shown). The "activation maps" are superimposed on high resolution brain images displayed in coronal (left panel) and saggital (middle and right panels) views; the color bars indicate the gradient of t values of the activation maps (p &lt; 0.005, 10 contiguous voxels 26), reflecting brain activity time-locked to the onsets of the memory cues. The line graphs illustrate the time courses of the fMRI signal (% signal change), for each trial type and TR (1 TR = 2 seconds). L = Left; R = Right.

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No conflicts of interest declared.

The experimental design introduced here allows investigation of the neural correlates of remembering emotional autobiographical memories. This design has the potential to advance our knowledge of how the brain generates affective biases (positive or negative) in remembering personal memories, and how those biases may be modulated by the retrieval focus (on emotional or non-emotional aspects). This protocol has additional benefits in that it can also be used with clinical populations (e.g., in patients with depression and...

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.

Watch this Scientific Journal Video about Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection at JoVE.com

Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection

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 ...

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9.7K Views.  University of Alberta, Edmonton. Vi presentiamo un protocollo che permette di indagine dei correlati neurali della ricordando ricordi autobiografici emotivo, utilizzando la risonanza magnetica funzionale. Questo protocollo può essere utilizzato con i partecipanti sani e clinica.

Video: Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection

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 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 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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

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 ...