我々は、データ収集の支援についてD.ジョンソンおよびK. Doellingに感謝します。我々はまた感謝M.-H.実験プロトコルに関する助言フィス、J.レドゥー、Y.ニヴとM.ミラド。この調査は健康（NIH）の助成金R21をMH072279（EAP）、NIHの助成金R37 MH038774、P50 MH058911、RO1 MH046516とK05 MH067048（JEL）、ポストドクトラルフェローシップNSERC、CIHRとAHFMRのジェームズS.マクダネル財団と国立研究所によって資金を供給された（M-HM）、およびフルブライトとBlavatnik賞（DS）。

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+neural+systems+mediating+extinction,+reversal+and+regulation+of+fear.">Overlapping neural systems mediating extinction, reversal and regulation of fear.</a> Trends in Cognitive Neuroscience. 14, 268-2627 (2010).</li><li>Esteves, F., Parra, C., Dimberg, U., Ohman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonconscious+associative+learning:+Pavlovian+conditioning+of+skin+conductance+responses+to+masked+fear-relevant+facial+stimuli.">Nonconscious associative learning: Pavlovian conditioning of skin conductance responses to masked fear-relevant facial stimuli.</a> Psychophysiology. 31, 375-385 (1994).</li><li>Knight, D. C., Cheng, D. T., Smith, C. N., Stein, E. A., Helmstetter, F. 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特別な利害関係は宣言されません。

我々はここで提示プロトコルは嫌悪結果と対中性刺激に対する単純な恐怖条件応​​答を作成できます。日後に我々は、その再固定をトリガーするために恐怖記憶を復活。この段階では、我々は絶滅の訓練の形で干渉を誘発した。絶滅が再固定に影響を与えていることを確認するために、我々は2つ​​の対照群を使用していました。一つのグループは再固定をトリガーリマインダー裁判なし...

extinction training during the reconsolidation window prevents recovery of fear

それは状況が安全になった後、長い持続するときの恐怖は不適応です。それが永続的に恐怖の戻りを防ぐアプローチを開発することが重要である。パブロフの恐怖コンディショニングパラダイムは、一般的に実験室で制御された、斬新な恐怖の関連付けを作成するために採用される。嫌悪結果（無条件刺激、米国）を用いて無害な刺激（条件刺激、CS）をペアリングした後、我々は一人で1,2だけ刺激を提示することによって、恐怖反応（条件反応、またはCR）を引き出すことができます。恐怖が取得されると恐怖は、もはや3を表明しなくなるまで、条件刺激が繰り返し嫌悪成果なしで提示される、それは、絶滅のトレーニングを使用して減らすことができる。この阻害学習は元の恐怖記憶の4を使用して式を競うCSのための新しい、安全な表現を作成します。絶滅が阻害恐怖で有効であるが、それは永久的ではありません。恐怖が自発的にすることができます時間の経過とともに回復します。初期学習の文脈へのストレスへの暴露や返品も浮上3,4に恐怖を引き起こす可能性があります。 我々のプロトコルは、より恒久的な方法で感情的なメモリを変更する再固定ウィンドウを標的とすることで、絶滅の過渡的な性質に対処しています。十分な証拠は、連結メモリを再度アクティブにすると、メモリが再び干渉5月9日の影響を受けやすくなり、その間に、不安定な状態に戻していることを示唆している。これはメモリ15の強さや年齢によって異なりますが、機会のこのウィンドウには、再活性化した直後に開いて、後で5,11,16約6hrs近いように見えます。それが10,11 reconsolidates、新しい情報が元のメモリ·トレースに組み込むようにすることで、このメモリが更新される場合があります。非ヒト動物を含む研究は、正常に再内薬理操作を導入することによって、恐怖記憶の発現をブロックしているコンソリデーション·ウィンドウは、しかし、使用されるほとんどの薬剤は人間に有毒であるのいずれか、またはヒトの研究12月14日で使用した場合あいまいな効果を示している。我々のプロトコルは、人間にとって安全で効果的な、まだ、非侵襲的、行動的な操作を提供することにより、これらの課題を解決します。 絶滅する前に恐怖記憶の想起を促すことで、私たちは本質的に再固定プロセスをトリガし、恐怖記憶がまだ干渉の影響を受けている間に新たな安全性情報（絶滅）を組み込むことができます。ラットではこの行動操作を採用した最近の研究では、正常にこれらの時間パラメータ11を用いて恐怖記憶をブロックしています。ヒトにおけるその他の研究では、従来から連結モータ16、エピソード17、または宣言18思い出の取得後に新たな情報を導入すると、元のメモリ·トレース14との干渉につながることが実証されている。我々は、新規プロ下概説トコールは人間に恐怖の回復を阻止するために使用されます。

Watch this Scientific Journal Video about Extinction Training During the Reconsolidation Window Prevents Recovery of Fear at JoVE.com

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

恐怖条件は絶滅と呼ばれるプロセスを介して抑制性の低下が、そのような時間やストレスへの暴露の通路のような条件の下で再浮上することができますすることができます。我々のプロトコルは再固定ウィンドウ（再活性メモリの再貯蔵期）に絶滅を導入することによって、恐怖の回復を防ぐ新しい方法を提示します。

再固定ウィンドウの間に絶滅の訓練は恐怖の回復を防ぎ

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

extinction-training-during-reconsolidation-window-prevents-recovery

Research

JoVE Journal

Neuroscience

24.9K ビュー.  Mt. Sinai School of Medicine. 恐怖条件は絶滅と呼ばれるプロセスを介して抑制性の低下が、そのような時間やストレスへの暴露の通路のような条件の下で再浮上することができますすることができます。我々のプロトコルは再固定ウィンドウ（再活性メモリの再貯蔵期）に絶滅を導入することによって、恐怖の回復を防ぐ新しい方法を提示します。

ビデオ: Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Human Fear Conditioning Conducted in Full Immersion 3-Dimensional Virtual Reality

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major challenge in conducting conditioning studies in humans is the ability to strongly manipulate or simulate the environmental contexts that are associated with conditioned emotional behaviors. In this regard, virtual reality (VR) technology is a promising tool. Yet, adapting this technology to meet experimental constraints requires special accommodations. Here we address the methodological issues involved when conducting fear conditioning in a fully immersive 6-sided VR environment and present fear conditioning data.
In the real world, traumatic events occur in complex environments that are made up of many cues, engaging all of our sensory modalities. For example, cues that form the environmental configuration include not only visual elements, but aural, olfactory, and even tactile. In rodent studies of fear conditioning animals are fully immersed in a context that is rich with novel visual, tactile and olfactory cues. However, standard laboratory tests of fear conditioning in humans are typically conducted in a nondescript room in front of a flat or 2D computer screen and do not replicate the complexity of real world experiences. On the other hand, a major limitation of clinical studies aimed at reducing (extinguishing) fear and preventing relapse in anxiety disorders is that treatment occurs after participants have acquired a fear in an uncontrolled and largely unknown context. Thus the experimenters are left without information about the duration of exposure, the true nature of the stimulus, and associated background cues in the environment1. In the absence of this information it can be difficult to truly extinguish a fear that is both cue and context-dependent. Virtual reality environments address these issues by providing the complexity of the real world, and at the same time allowing experimenters to constrain fear conditioning and extinction parameters to yield empirical data that can suggest better treatment options and/or analyze mechanistic hypotheses.
In order to test the hypothesis that fear conditioning may be richly encoded and context specific when conducted in a fully immersive environment, we developed distinct virtual reality 3-D contexts in which participants experienced fear conditioning to virtual snakes or spiders. Auditory cues co-occurred with the CS in order to further evoke orienting responses and a feeling of "presence" in subjects 2 . Skin conductance response served as the dependent measure of fear acquisition, memory retention and extinction.

Classical fear conditioning paradigm was adapted for human participants in a fully immersive virtual reality setting. Using a discrimination paradigm, conditioned fear, cue and context memory retention, and extinction was measured with skin conductance response to dynamic virtual snakes and spiders (the conditioned stimuli) in two distinct virtual contexts.

フルイマージョン3次元バーチャルリアリティのに対して、ヒト恐怖条件付け

Fear conditioning is a widely used paradigm in non-human animal research to investigate the neural mechanisms underlying fear and anxiety. A major ...

神経科学

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. 
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

fMRIの持つ認識と非認識恐怖記憶の神経機構を調査する

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

マウス脳の長期イメージングのためのポリッシュと強化間引き頭蓋骨ウィンドウ

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

円窓膜を介してマウスインナーイヤーにウイルスにより媒介遺伝子送達のための手術方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

脳スライスにおける恐怖回路のex vivo光遺伝学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

最適化されたNを使用して急性脳スライスの準備-メチル-D-glucamine 保護回復法

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

管理のリアルタイム fMRI ニューロフィード バック トレーニングのプロトコル

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

静脈瘤形成と中枢神経の軸索の回復を研究するための Microbiomechanical システム

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

マウスにおける線込みおよび脊髄窓移植

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...