알코올 남용과 알코올 중독에 국립 연구소 (; AA - 016967 - 01S1과 AA016967, NIAAA TAM)에서 제공하는 기금, 웨인 주립 대학은 기금을 시작하고, 웨인 주립 대학 연​​구 보조금 프로그램. 내용은 전적으로 저자의 책임이며, NIAAA 또는 국립 보건원의 공식 견해를 대변하지 않습니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

여기에 제시 프로토콜 FSCV 실험을위한 마우스 코로나 뇌 조각을 준비하고 사용하는 방법을 보여줍니다. 11 -이 방법은 도파민 역학을 취득하고 측정하기 위해 특정이지만, 같은 다른 신경 전달 물질은 아데노신, 과산화수소, norepinephrine, 그리고 세로토닌은 FSCV 3 8 생체내 또는 체외에서 모니터링하고 있습니다. FSCV은 작업 전극 세, 11에 적용된 파형의 간단한 변경을 통해 이러한 다른 neurochemicals의 일부를 모니터하는 데 사용할 수 있습니다. 이러한 neurochemical 종의 많은 비슷한 산화 잠재력을 가지고 있기 때문에, 생성된 순환 voltammograms 화학 식별을 허용 각 oxidizable 종족에 대한 아주 독특한 화학 지문을 제공합니다. 15 - 또한, FSCV 이러한 모델 생물 12 neurotransmission의 더 나은 이해를 얻기 위해, 과일 파리에서 비 인간 영장류에 다양한 수종에서 사용되었습니다. FSCV가 B를 가지고있는 주된 이유 중 하나종 등 다양한에서 사용 een 직경은 일반적으로 탄소 섬유 microelectrodes, 이하 7 μm의의 작은 직경에 의한 것입니다. 결과적으로 이러한 microelectrodes은 가능한 과일 파리 두뇌 (NL)의 경우와 같이, 매우 작은 환경에서 조직 샘플, 또는 큰 종 12 쉘 비해 NAC 코어와 같은 이산 하위 해부학 지역에서 차별 있도록 -14. 결론적으로, 결과는 슬라이스 voltammetry는 마우스 striatum에서 presynaptic 도파민 역학을 탐사하는 소중한 전기 도구입니다 보여주는 여기서 제시. 대표 데이터는 &#39;정상 또는 건강한&#39;컨트롤에서 뇌 슬라이스와 도파민 릴리스와 이해의 매개 변수를 특징하는 능력을 통해 약리 요원을 perfusing에 초점을 맞추고 있습니다. 또한, FSCV은 자신이나 pharmac 이후 유전자 변형 또는 치료 동물에 전기 자극 릴리스의 차이와 이해 매개 변수를 평가하는 데 사용할 수 있습니다ological 치료 15-16. 슬라이스 FSCV는 밀리초의 timescale에서 발생하는 이산 해부학 지역 내의 도파민 neurotransmission의 역학을 조사하기 위해 독특한 기회를 제공합니다. 전체 FSCV의 전기 기술은 다른 neurochemical 기술에 비해 모두 향상된 공간과 시간적 해상도를 제공합니다.

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> CAS 번호 </td></tr><tr><td width="274"> 시약 </td><td></td><td></td></tr><tr><td width="274"> 염화칼륨 </td><td> 어부 </td><td width="142" valign="top"> 7,447,407 </td></tr><tr><td width="274"> 나트륨 염화물 </td><td> EMD 화학 </td><td width="142" valign="top"> 7,647,145 </td></tr><tr><td width="274"> 마그네슘 염화물 </td><td width="194" valign="top"> 어부 </td><td width="142" valign="top"> 7,791,186 </td></tr><tr><td width="274"> 염화칼슘 </td><td width="194" valign="top"> 어부 </td><td width="142" valign="top"> 10035048 </td></tr><tr><td width="274"> 나트륨 중탄산염 </td><td width="194" valign="top"> EMD 화학 </td><td width="142" valign="top"> 144,558 </td></tr><tr><td width="274"> 인산 나트륨, 이염 </td><td width="194" valign="top"> EMD 화학의 </td><td width="142" valign="top"> 7,558,794 </td></tr><tr><td width="274"> D - 글루코오스 </td><td width="194" valign="top"> 어부 </td><td width="142" valign="top"> 50,997 </td></tr><tr><td width="274"> 아스코르비 산 </td><td width="194" valign="top"> 어부 </td><td width="142" valign="top"> 50,817 </td></tr><tr><td width="274"> 자당 </td><td width="194" valign="top"> 어부 </td><td> 57,501 </td></tr><tr><td width="274">&nbsp;</td><td width="194" valign="top">&nbsp;</td><td>&nbsp;</td></tr><tr><td width="274"> 자료 </td><td width="194" valign="top"> 공급 업체 </td><td> 카탈로그 번호 </td></tr><tr><td width="274"> 탄소 섬유 </td><td> Goodfellow Oakdale, PA </td><td></td></tr><tr><td width="274"> 유리 모세관 </td><td> AM 시스템 Carlsborg WA. </td><td> 602,000 </td></tr><tr><td width="274"> 실버 와이어 </td><td> A -M 시스템 Carlsborg WA. </td><td> 787,000 </td></tr><tr><td width="274"> 텅스텐 전극 자극 </td><td> 플라스틱 하나, 로어 노크, VA </td><td></td></tr><tr><td width="274"> 백금 와이어 </td><td></td><td></td></tr><tr><td width="274"> 와이어 리드 </td><td> 종복인 전자, 고넬료, 또는 </td><td></td></tr><tr><td width="274"> Loctite 404 인스턴트 접착제 </td><td> Hankal 주식 회사 록키 힐 중부 표준시. </td><td></td></tr><tr><td width="274"> 면도날 </td><td> 세계 정밀 계측기 주식 회사 FL. </td><td></td></tr><tr><td width="274"> BD 척수 바늘 </td><td> BD 의료 시스템, Franlin 호수, NJ </td><td> REF 405,234 </td></tr><tr><td width="274"> 외과 블레이드 </td><td> 깃털 안전 면도기 (주) LTD. 일본 </td><td></td></tr><tr><td width="274"></td><td></td><td></td></tr><tr><td width="274"> 소프트웨어 </td><td width="194" valiGN = &quot;위로&quot;&gt; 공급 업체 </td><td> 카탈로그 번호 </td></tr><tr><td width="274"> TH 소프트웨어 </td><td> ESA 주식 회사, Chelmsford, MA </td><td></td></tr><tr><td width="274"> 기계 </td><td width="194" valign="top"> 공급 업체 </td><td> 카탈로그 번호 </td></tr><tr><td width="274"> 잠수 기록 챔버 </td><td> 맞춤 과학, 덴버, CO </td><td></td></tr><tr><td width="274"> Neorolog의 자극 아이 솔 레이터 </td><td> Digitmeter, 하트 퍼 드셔, 영국 </td><td></td></tr><tr><td width="274"> 자동 온도 컨트롤러 </td><td> 워너 악기 주식 회사 </td><td></td></tr><tr><td width="274"> 현미경 (SZX7) </td><td> 올림포스 산 </td><td></td></tr><tr><td width="274"> 현미경 </td><td> 어부 </td><td></td></tr><tr><td width="274"> Vibratome 3000 sectioning 시스템 </td><td> 세인트 루, MO입니다. </td><td></td></tr><tr><td width="274"> 재관류 펌프 </td><td> 왓슨 Marlow 제한, Falmouth 콘월 - 영국 </td><td> H110708 </td></tr><tr><td width="274"> Micropipette 풀러 </td><td> Narishige, 도쿄, 일본 </td><td></td></tr><tr><td width="274"> ChemClamp potentiostat </td><td> Dagan 기업, 미네 아 폴리스, 미네소타. </td><td></td></tr></tbody></table>

presynaptic dopamine dynamics in striatal brain slices with fast-scan cyclic voltammetry

광범위한 연구 때문에 남용 약물 (예 : 코카인과 암페타민), 정신과 질환에가하는 역할 (예 : 정신 분열증 및주의 결여 과다 장애) 및 퇴행성 년에 ​​참여 행동의 메커니즘에서 그 중요성의 신경 전달 물질의 도파민에 주력했다 파킨슨병과 헌팅턴의 질환과 같은 질환. 정상적인 생리 조건에서 도파민은 전위의 활동,인지, 학습, 정서적 영향을, 그리고 neuroendocrine 호르몬 분비를 조절하는 것으로 알려져 있습니다. 도파민 뉴런의 가장 큰 밀도 중 하나는 핵의 accumbens와 꼬리가있는 - putamen으로 알려진 두 개의 neuroanatomical 지역 나눌 수 있습니다 striatum 이내입니다. 목표는 마우스 striatum 이내 슬라이스 빠른 스캔 순환 voltammetry (FSCV)에 대한 일반적인 프로토콜을 설명하는 것입니다. FSCV는 D에 실시간으로 도파민 자료 및 이해를 측정할 수있는 기회를 제공하는 잘 정의된 전기 기술이다iscrete 두뇌 영역. 탄소 섬유 microelectrodes는 (~ 7 μm의 직경) 도파민 산화를 감지 FSCV에 사용됩니다. 도파민을 감지 FSCV를 사용하는 분석 장점은 생체내의 microdialysis에로 보완 정보를 제공 미만보다 10 미크론의 100 밀리초와 공간 해상도, 자사의 향상된 시간적 해상도입니다.

Watch this Scientific Journal Video about Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry at JoVE.com

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

striatal 뇌 조각의 전기적 evoked presynaptic 도파민의 역학을 측정하는 빠른 스캔 순환 voltammetry를 사용하여.

빠른 스캔 순환 Voltammetry와 Striatal 뇌 조각의 Presynaptic 도파민 역학

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

presynaptic-dopamine-dynamics-striatal-brain-slices-with-fast-scan

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.8K 조회수.  Wayne State University. striatal 뇌 조각의 전기적 evoked presynaptic 도파민의 역학을 측정하는 빠른 스캔 순환 voltammetry를 사용하여.

비디오: Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

쥐 뇌 조각에서 뉴런의 Vibrodissociation는 시냅틱 전송 및 이미지 Presynaptic 터미널 공부

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

연구

신경과학

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

해마의 Organotypic 조각의 쌍으로 전체 셀 녹음

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

뇌 조각에서 전체 셀 패치 클램프 녹음

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

QNsim1.0을 이용한 전기 자극 도파민 신경 전달 데이터의 고속 스캔 순환 전압 측정 데이터 모델링

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

급성 Hippocampal 조각에 작은 볼륨 재활용-, 관류-침수 형 챔버 시스템 유지에 시 냅 스가 소성을 기록

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

Vivo에서 단일 분자 초파리 연 접 모터 신경 맨끝에 추적

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

신생아 마우스 두뇌의 Striatal 세포에서 유전자 조작에 대 한 stereotaxic 수술

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

성인 소뇌 슬라이스에 장기 우울증 유도의 평가

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...