Name: an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation
Uploaded: 2018-06-02T14:45:00
Description: Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

(하이 타오 우), 중국과 그랜트 Z161100000216154에서의 기초 연구에 대 한 국가 주요 개발 프로그램에서 973 (2014CB542203) 프로그램 31522029, 31770929 및 31371149 (하이 타오 우) 중국의 국가 자연과학 기초에 의해 지원 되는 베이징 시 과학 및 기술 위원회 (하이 타오 우). 저자는 그들의 격려와 토론에 대 한이 타오 우 연구소의 모든 구성원을 감사합니다. 저자는 그의 도움 장치 디버깅에 대 한 Zhenwei 류를 매우 감사.

동물 실험 절차는 베이징의 기본적인 의료 과학 연구소 (베이징, 중국) 관리 및 실험 동물의 사용에 대 한 중국 정부 규정의 기관 지침에 따 랐 다. 쥐 (성인 남성, 26 ~ 30 g) 지 내게 되었고 12 h 빛/12-h 어두운 주기 (오전 7 시에 조명) 아래 물과 음식 광고 libitum, 23 ° C의 일정 한 온도에서 보관. 모든 실험 절차는 가벼운 주기 동안 수행 했다.
1. 수술 준비
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HF DBS 기술 널리 사용 되었습니다 강력한 도구로 1990 년대 이후 많은 신경 성 질환의 치료에 대 한. 지금까지, HF DBS의 랜드마크 일 많은 관심과 모두 클리닉 및 사회 과학에 관심을 끌고있다 진전, 파 킨 슨 병의 치료입니다. 특정 신경 및 정신 장애32,33에서 HF-DBS의 치료 응용 프로그램에 대 한 많은 그룹에 의해 지속적인 HF DBS 연구의 다양 한 유형이 있다. ...

HF DBS 18701이후 개발 된 두뇌에 전기 자극에 대 한 신경외과 기술 이다. 1980 년대 후반에, HFS 처음 파 킨 슨 병에 대 한 잠재적인 치료 적 개입으로 사용 하 고 다른 운동 장애2. 지난 몇 십년에서 HF-DBS 점점 더 널리 사용 되었습니다 현재 치료 되지 않은 뇌 질환의 치료에는 전통적인 치료 전략에 의해. 특히, 때문에 HFS 전극, 매우 효과적인 결과, 최소한의 부작용의 정확도 향상, 뇌 장애 HF DBS에 의해 처리 수 크게 증가 지난 수십 년간3,4, 5. 예를 들어 HF-DBS 승인 되었습니다 미국 식품의 약 청 (FDA)에 의해 파 킨 슨 병 (PD), Alzheimer 유형 치 매, 진전, 운동 장애2,6, 의 다른 종류의 치료에 대 한 7. PD 환자에서 dopaminergic 약물 감소 HF DBS8동안 최대 50%. 또한 운동 장애의 성공적인 치료, HF-DBS 또한 보여주었다 정신 질환 클리닉, 그리고 잘2,9, 로 인식 확대의 치료에는 강력한 효과 10 , 11. 환자12많은 약속을 제공 하는 다양 한 단계에서 다른 정신 장애의 치료에 대 한 HFS의 연구는 주목 해야 한다.
많은 학문은 초점 HFS 뇌13를 통해 로컬 및 원격 효과 설명 했다, 비록 효과의 신경 및 분자 메커니즘 애매2,14남아 있습니다. 일반적으로 클리닉, 치료 HF-DBS는 파 킨 슨 병 및 만성 통증, 등 많은 의견 설명 어떤 하나의 가능성 중 HF DBS 치료에 의해 생성 된 개선 발생의 치료에 대 한 장기적인 방식으로 적용 HFS 전류 조절 한다 신경 네트워크 활동을 아마 이식된 HFS 전극 주변 축 삭의 반복적인 도발은 여. 또는, HF-DBS 출력 뉴런 및 예상된 대상의 방전 율 변경 될 수 있습니다. 또한, HF-DBS 이어질 수 있습니다 장기 potentiation (LTP) 장기 불경기 (주식 회사), 등 장기 시 냅 스 변화는 증상 개선에 기여할 수 있습니다. 지금까지, 그것은 아직 불분명 HFS inﬂuences 휴대 규제 키 분자 이벤트 등을 처리 하는 여부 성체 비보로. 여러 줄 연구의 HFS 설치류에 임상 적용된 DBS15,16의 유사한 신경 응답을 모방 수 설명 했다. 이 연구에서 HF-DBS의 기본 셀룰러 메커니즘을 이해 하기 우리 먼저 설정는 vivo에서 HFS 방법론 쥐에서에 급성 (1 일) 또는 만성 (5 일) 방식으로. 둘째, 우리는 HF DBS 납품 후 신경 활동과 성체의 변경 확인 하려면 활성화 분석 방법론을 설정 합니다.
신경 줄기 세포에서 신경 생산 배아 개발 하는 동안 풍부한 이지만 성인 생활 내내 계속, hippocampal subgranular 영역은 신생 발생 하는 주요 지역 중 하나입니다. 성체의 과정은 많은 생리 적 및 병 적인 요인에 의해 영향을 받습니다. 경우에 따라 간 질 hippocampal 신생 극적으로 감소17,18이다. 또한, 단일 electroconvulsive 치료 크게가 이랑19신경 생산을 증가할 수 있었다. 이러한 관측 제안 electrophysiological 활동 성체와 hippocampal 신경에 시 냅 스가 소성의 규칙에 있는 중요 한 역할을 한다. 따라서, 더 신경 활동 및 신생에 HF DBS의 효과 보여, 우리가 처음 실시는 즉시 이른 유전자 (IEG) c-fos 단기 신경 활동에서 결과의 잘 알려진 마커는의 immunostaining 분석 결과 경험20. HFS 배달21,22후 신호 활성화를 모니터링 하 Notch1 신호 감지 됩니다. 또한, 우리 또한 감지 BrdU 다양 한 매너에 HF DBS 유도 후 분석을 라벨에 의해 신경 생산 BrdU 얼룩은 또한 gliogenesis에 대 한 마커 수 있지만.
현재 연구에서 두 HFS 방법론 직접 및 간접적으로 hippocampal DG의 활성화를 대상에 맞게 있습니다. 전극은 DG에 직접 이식 또는 DG 뉴런을 활성화 하는 계획을 보내는 중간 perforant 경로 (PP)로 이식. HF DBS 유도 대 한 프로그래밍 가능한 자극 기는 마우스 머리에 고정된 전극에 지속적인 자극을 통해 에 대 한 제공 됩니다. 신경 활성화와 신경 신생에 HFS의 효과 확인 하려면 immunofluorescent 얼룩이 지 고 hippocampal DG 지역에서 긍정적인 뉴런 BrdU 통합의 수에 의해 c-fos 와 Notch1의 식 감지 후 각각, HFS 치료입니다. 특히,는 DG에서 성체에 HF-DBS의 효과 비교는 급성 및 만성 자극 방식으로, 또는 직접 및 간접적인 자극 방법, 각각.

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an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation

전기-고주파 자극 (HFS), 이식된 전극 다양 한 두뇌 지역 타겟팅을 사용 하 여 다양 한 신경 및 정신 질환에 대 한 효과적인 치료로 입증 되었습니다. 또한 라는 깊은 뇌 자극 (DBS), 뇌의 깊은 지역에서 HFS 임상 시험에서 점점 더 중요 해지고 있다. 높은 주파수 (HF-DBS) DBS 수술의 분야에서 최근 진행 주요 우울증 장애 (MDD), 강 박 장애 (강 박 증)에 대 한 치료와 같은 다른 상황에이 침략 적인 기술 활용의 가능성을 확산 하기 시작 했다 그래서 .
이러한 확대 징후에도 불구 하 고 HF-DBS의 유익한 효과의 기본 메커니즘 수수께끼 남아 있습니다. 이 문제를 해결 하려면 한 가지 방법은 사용 하는 이식된 전극 띄엄띄엄 HFS에 의해 뉴런의 분산된 모집단을 활성화 하는. 그것은 알려졌다 시상의 앞쪽 핵에서 HFS 내 화 간 질 클리닉에서의 치료를 위해 사용 될 수 있습니다. 기본 메커니즘 증가 신생 관련이 있을 수도 있습니다 하 고 신경 활동을 변경. 따라서, 우리는 HFS 치료 전후 생리 변경 마우스가 이랑 (DG)에서 성체 신경 활동의 탐지에 의해 탐험에 관심이 있습니다.
이 원고에서 우리가 직접 또는 간접적으로, 급성 또는 만성 방식 마우스에 DG의 활성화를 목표로 HFS에 대 한 방법론을 설명 합니다. 또한, c fos 와 얼룩 신경 활동을 모니터링 하 고 활성화 신호 immunofluorescent Notch1 및 bromodeoxyuridine (BrdU) 결정을 라벨에 대 한 뇌 조각의 준비를 위한 상세한 프로토콜 설명 합니다 HF DBS 유도 후의 신경 신생 HF DBS 치료 후 신경 활동과 성체의 활성화 직접 neurobiological 증거 및 잠재적인 치료 혜택을 제공합니다. 특히,이 방법론 수정 하 고 기초 중추 등 다른 관심된 뇌 영역 및 시상 지역 병원에서 특정 뇌 질환에 대 한 대상에 적용 될 수 있습니다.

Hippocampal DG 하위 직접 또는 PP 하위는 DG를 직접 활성화 하기 위해 HF DBS 자극 통해 삽입 전극을 사용 하 여 정위 적 조정, 설치류 pentobarbital로 취 했다 고 3 h 샘플링 후 c-fos 와 Notch1 immunostaining에 대 한 마지막 HF DBS 자극. BrdU 얼룩, 1 일 또는 HF DBS 자극의 5 일 후 마지막 BrdU 주입 후 36 h에 대 한 설치류 뇌 섹션의 준비에 대 한 pentobarbital로 취 했다. ?...

Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

이 프로토콜에는 생쥐에서 신뢰할 수 있는 HFS 메서드를 설정 하는 방법을 보여 줍니다. Hippocampal가 뇌 전체 신경 전기 HFS 직접 및 간접적으로 비보에 의해 자극 된다. 신경 활동 및 분자 신호 검사 c-fos 와 Notch1 immunofluorescent 얼룩, 각각; 성체는 bromodeoxyuridine 분석 결과 라벨에 의해 정량 이다.

고주파 자극에 의해 마우스가 뇌의 활성화에 대 한 침략 적 방법

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

The overall goal of this experiment is to observe the effect of high-frequency stimulation on neuronal activity in neurogenesis. This method can help answer key questions in an aligned cellular and molecular mechanisms of high-frequency deep brain stimulation, such as the induction of neuron activity and the neurogenesis in hippocampus. The advantage of this technique is that it demonstrates the powerful effect of high-frequency TBS on neuron activity and the neurogenesis in the adult brain.Though this method can provide insight into the effects of high-frequency TBS on the mouse dentate gyrus, it can also be applied to other systems such as the basal ganglia and the subthalamic regions for the brain disorders in the clinic. To begin this procedure, place an anesthetized mouse onto the stereotactic frame. Align the ear bands with its head, and gently tighten them to support the centered head.Grasp the skin anterior to its ears and remove about one square centimeter of it over the skull. Then remove the muscle and other attachment overlying the skull with a cotton swab to expose the skull surface. Next, apply hydrogen peroxide solution to dry the skull in order to identify the sagittal and lambda suture lines.Then, identify the bregma and lambda. The bregma point will be used as the original point to set the nucleus position. After that, level the skull on the horizontal plane using these two points.Make sure that the heights of these two points are almost the same in the dorsal ventral plane. In addition, make sure that the skull is leveled in the medial lateral plane. Next, mount a homemade two channel copper micro wire electrode into the rotating electrode holder on the stereotactic surgical frame.Place the electrode directly above the bregma and set it as original site. Then, move the electrode gently to the correct position above the skull, without touching it. Use the digital display on the stereotactic surgical frame to indicate the anterior posterior, medial lateral, and dorsoventral positions.When the desired site is identified for the electrode insertion mark the position with a black marker. Afterward, use the dorsoventral stereotactic adjustments to raise the electrode holder. Then, sterilize the drill bit with 70%ethanol.Use a handheld micromanipulator assisted drill, make a burr hole precisely at the marked position. Use a sterile cotton swab to stop any bleeding during the operation. Keep drilling until dura is exposed.Use a bent needle to remove any bone scraps that are generated during the drilling, and make a pinhole on the dura without damaging the underlying soft brain tissue. To confirm that the burr hole is made properly at the desired position, lower the electrode to make sure that it inserts through the burr hole smoothly without touching any obstacle. If no resistance is detected, slowly insert the electrode to the target depth from the surface of the skull.Then, apply dental cement using a small spatula to hold the electrode in place. As the cement thickens, mold it around the inserted electrode and other bone screws to form a nice, smooth cap. Next, disengage the electrode from the electrode holder.Remove the mouse from the stereotactic frame, and return it to a warm cage. Let the mouse move freely and allow it to recover for two days in the cage. To deliver stimulation, connect the implanted electrode to a programmable stimulator via leads.Set up the software interface. Then, set the HFS parameters as six series of six trains of six pulses at 400 Hertz, and the stimulation intensity of 200 milliamps. Deliver HFS for the desired period of time as set up.For the control group, implant electrodes in the mice without delivering any stimulation. For CFAS staining, stimulate the experimental mice two times for one day. For BrdU labeling, apply appropriate stimulations to both the acute stimulation group and the chronic stimulation group.During the course of the stimulation, make sure the mouse is awake. Remember to insure the leads are not twisted. When the stimulation is done, carefully disconnect the electrode pin from the electrode holder in the connector of the recording system.For the CFAS and notch one staining, about three hours after the last HFDBS, anesthetize the mice by injecting pentobarbital to achieve a surgical plane of anesthesia. For BrdU labeling, about 12 hours after the last HFDBS stimulation, give six injects of BrdU to the control and the stimulated mice at two hour intervals. 36 hours after the last injection, deeply anesthetize the mice with pentobarbital and transcardially profuse it with 50 milliliters of cold PBS followed by 50 milliliters of cold 4%paraformaldehyde.Remove its head and make an incision on the skin to expose the skill. Using forceps, chip off the skull slightly and carefully remove the whole brain. Then, post fix the brain is 4%paraformaldehyde for an additional two days, and transfer it to 30%sucrose solution at four degrees Celsius overnight.Using a cryostat, slice the brain into 20 micron coronal sections. Transfer and mount the sections to the glass slide with a brush, and store them a minus 20 degrees Celsius. Afterward, transfer the slides into normal PBS containing no fixative.Wash the sections with PBS three to four times for five minutes each to remove any excess fixative. For the BrdU labeled group, incubate the sections in two molar HCL for 30 minutes at 37 degree Celsius, and rinse them in a 0.1 molar borate buffer. After 10 minutes, wash the sections two times in PBS for five minutes each time.Subsequently, incubate the sections in blocking solution for one hour at room temperature. Then, incubate the sections with anti-CFAS polyclonal antibody, anti-notch one antibody, or anti-BrdU antibody in a blocking solution at four degrees Celsius overnight. Was the sections three times in PBS for 10 minutes each time, then incubate them with Alexa Fluor 568 conjugated anti-rabid, Alexa Fluor 488 conjugated anti-goat, or Alexa Fluor 488 conjugated anti-rat secondary antibody for one hour at room temperature.Was the sections three times in PBS for 10 minutes each time. Subsequently, cover the immuno labeled brain sections using an antifade reagent mounting medium containing DAPI. Let the sections air dry, and store them protected from light at four degrees Celsius.Shown here is an experimental paradigm for the HFS treatment in the generation of BrdU labeled proliferating neurons in the DG.The animals were divided into an acute and a chronic group to receive different patterns of stimulations. Afterward, the animals in both groups were injected with BrdU on day six. On day eight, the animals were anesthetized and profused for the preparation of cryostat sections.Here are the images of the BrdU immunofluorescent staining post-HFDBS in the DG.A quantitative analysis of the neuronal production indicated that the acute stimulation in the DG could not enhance neurogenesis. While the chronic stimulation in the DG significantly up regulated the neurogenesis. Compared to the control group, the number of BrdU labeling cells increased significantly in the chronic group, both in the ipsilateral and contralateral hemisphere.These are the images of the BrdU immunofluorescent staining post-HFDBS in the PP sub-region. A quantitative analysis of the neuronal production indicated that either the acute or chronic stimulation in the PP could not enhance the neurogenesis significantly. The number of BrdU labeling cells was relatively stable with or without the HFS treatment.While attempting this procedure, it's important to remember to locate the electrode properly, and to deliver the stimulation successfully. After it's development, this technique paved the way for researchers in the field neuroscience high-frequency stimulation enrolled in the models. After watching this video, you should have a good understanding of how to deliver high-frequency stimulation to deep brain nucleus.

Research

JoVE Journal

Neuroscience

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

기본 모드 네트워크를 검사하기 위해 Transcranial 자석 자극과 fMRI를 결합

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

연구

신경과학

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

TMS : 연약한 X 증후군 및 자폐증과 개인의 소성의 Mechasnism를 탐험하는 세타 - 버스트 프로토콜을 사용하여

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

NeuroStar TMS 장치 : FDA를 실시는 우울증 치료 프로토콜을 승인

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

비 침습적 실질, 혈관 및 대사 고주파 초음파 및 광 음향 쥐 뇌 심부 이미징

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

쥐의 해마 영역의 치아 이랑 (dentate gyrus)에서 수지상 Arborization의 평가

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

파상풍의 자극에 의​​해 지역의 CA1 γ 진동의 발생

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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

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

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

실시간 체 외 Odorant 수용 체 활성화는 Odorant 수증기 단계에서의 모니터링

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

전기 자극에 의한 쥐에 급성 폰틴 경색 의 설립

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...