켄터키 대학, 생물 학부, 학부 연구과 예술 &amp; 과학 대학 사무실에서 지원됩니다.

관심 없음 충돌 선언하지 않습니다.

막 잠재 마찬가지로 초기 1902와 같은, 번스타인은 오징어의 축삭에서 휴식 가능성의 문제를 처리했습니다. 그것은 번스타인 (1902)와 Nernst (1888)의 초기 아이디어와 관찰 나중에 막 생리 연구 영향을 어떻게 고려하는 흥미로운 것입니다. (Malmivuo 및 Plonsey 1995의 리뷰를 참조하고, WWW에서도 가능 <a href="http://www.bem.fi/book/">http://www.bem.fi/book/</a> ). 이온 채널 기능과 조직, 장기 및 시스템의 기능에 관한 세포 생리를 이해하는 매우 중요한 아르 생물 세포막의 속성에 대해 만들어지고 획기이 오늘날까지 여전히있다. 외부의 실험과 이론적으로 파생 효과의 비교 [K +] 쉬고 막 잠재력에 막 잠재력에 대한 이온의 영향을 나타냅니다. 이 같은 준비를 사용하여 추가 실험은 근본적인 생리 질문을 해결하기 위해 수행할 수 남아 있습니다. 일부는 앳우드와 Parnas에 의해 1968 년 다시 강조되었고 아직 완전히 달려드는해야합니다. 이 운동에서 얻은 기술로, 하나는 다른 실험 준비뿐만 아니라 의학 및 건강에 관한 생리 응용 프로그램에 남아있는 많은 질문에 답변을 진행할 수 있습니다. 우리는 동물 관련 근본적인 문제를 해결하기 위해 모델 무척추 준비의 유용성을 증명하고있다. 지식이 위 운동 이온의 전기 그라디언트에서 얻은으로, 당신은 지금 왕새우에 신경근육학 준비에 신경 전송을 검토하여 세포막의 흥분을 진행하실 수 있습니다. 시냅틱 응답 측정 이 실험의 첫번째 부분과 관련된 영화가 제공하는 내용은, 멤브레인 잠재력을 기록하고 근육 구조를 조사하는 주요 단계를 제공합니다. 이 연구소의 두 번째 부분에서는, phasic 및 토닉 모터 단위의 NMJs에서 절개 및 녹음 시냅스 전달의 데모는 생리학 기본 개념에 노출을 제공했습니다. 부분에 자신의 실험실 운동 내에서 다양한 기한이 질문을 조사하기 위해 학생뿐만 아니라의 연결 회로에 대한 미래의 연구뿐만 아니라 잠재적인 우물에있는 그대로 동​​물에 관련된 행위를 설명하는 데 사용할 수있는 수있는 신경 회로에 노출 설립 무척추 준비 (케네디 외, 1969;. Antonsen와 에드워즈, 2003) 이러한 준비는 시냅스 촉진, 우울증 및 장기 소성 (이 실험실 연구에서 조사되지 않음) 조사하는 데 사용할 수 있습니다. 뿐만 아니라 자연 환경, 심지어 왕새우 종 (种) 이내의 연결을 소성의 실험 자극 조건 (. 쿠퍼 외, 1998 머시 및 앳우드, 1989)에 따라 달라집니다. 어느 정도하기 위해 시냅스 효능과 근육 역학을 변경하는 능력은 동물이 조사에 남아 역할을합니다. 크레이 피쉬는 계절 변화 및 ​​털갈이 사이클과 관련하여 자신의 행동을 변경합니까 때문에, 자신의 신경근육학 시스템에 비교적 장기간의 활동 차이가 있습니다. 이것은 클로 가까이 근육의 phasic 모터 신경 터미널은 겨울 동안 고전 phasic 형태를 전시하지만, 여름 동안 터미널의 길이를 따라 더 정맥류의 (; Lnenicka 및 조 1991 Lnenicka 1993) 팽창하고 될 것으로 나타났습니다. 복부 신경 코드 내에서 왕새우 측면 대형 (LG) interneurons 실시 몇몇 초기 연구 갑 분기점 (; 와타나베와 Grundfest, 1961 존슨 1924 년)의 존재를 보여주었다. 이곳은 CO 2 갑 분기점을 (아레 야노 외, 1990) uncoupling하여 전기 통신에 효과를내는 것으로 알려져 있습니다. 이것은 최근에 본 보고서에 설명되어있는 감각 - CNS - 모터 근육 회로, 내의 신경 코드와 통신 간격 분기점 (Bierbower 2010의 존재를 나타내는 2 노출 CO 또한 민감한 것을 보여주었다 Bierbower와 쿠퍼, 2010) 1960이 동일하거나 인접한 세그먼트 내의 정적 근육 수용체 기관 (MRO)을 해고 토닉은 자연 모터 드라이브에 대한 계정이있다면 에커트 (1961)는 검사 때부터 3 차 모터 뿌리의 자발적인 활동 주제를했습니다. 이러한 초기 연구에서는이 활동을 가능성이 높은 센터 (; 케네디와 다케다, 1965a, B;. 스트론 외, 2000 에커트, 1961)에서 복부 신경 코드 (VNC) 내에서 구동 것을 분명했다. CO 2의 존재가 자발적 활동을 중단 이후, 한 갑 분기점 또는 glutamatergic 흥분성의 드라이브가있을 어딘가 드라이브에 모터 뉴런을 추측할 수있다. NMJs가 차단 또는 전시 CO 2의 존재에 조미료로 감도를 감소, 그들은 블록 수 아르(; Bierbower와 쿠퍼, 2010; Badre 외 또보고, 2005. Bierbower 2010) CNS 내에서뿐만 아니라 ked. 다양한 neuromodulators의 동작도 쉽게 NMJs의 다양한 유형 (; 그리피스 외, 2000;. 서더드 외, 2000;.. 스트론 외, 2000, 쿠퍼와 쿠퍼, 2009)에서 공부합니다. 또한, 다양한 영향은 CNS 회로에 neuromodulators로 끼쳤다 있습니다. . 슈나이더 외, 1996,,. 호르너 외, 1997;. 이것은 5 - HT 및 octopaminergic 뉴런이의 연결 회로의 출력 (MA 외, 1992를 변경에서 &#39;이득 세터스&#39;로 기능을 수있는 제안되었습니다 에드워즈 외., 2002). 우리가 완전히 개별 타겟 세포에 neuromodulators의 영향을 이해하기 전에 많은 작업이 남았 는데요. 다른 neuromodulators가 서로 콘서트에서 일할 수 있습니다 감안할 때, 그들의 혼합 행동의 분석 미래 연구 (Djokaj 외., 2001)을위한 영역입니다. 또한, 몇 가지 연구, 특히 척추 동물에서 특정 동작을 조절할 수있는 전체 경로에 neuromodulators의 효과를 주소를 입력합니다. 이 감각 - CNS - 모터 유닛 준비 하나는 모터 뉴런 (케네디 외., 1969)의 활동에 감각 입력 및 neuromodulators 모두의 영향을 검토할 수 있습니다. 그것이 5 - HT는 왕새우의 행동 상태, 바다 가재 규제의 역할을하고, 게는 (.. 리빙스턴 외, 1980; 스네돈 외, 2000)을 postulated되어 있기 때문에, 여러 시도에 그 농도를 결정되었습니다 VNC, hemolymph, 그리고 바다 가재 (리빙스톤 외, 1980., 해리스 - 워릭과 크래빗 1984;. Fadool 외, 1988)의 절연 신경 인치 그러나, 행동 행동에 대한 계정을 수있는 구체적인 선량 - 반응 관계를 precluding, 기록된 측정에 상당한 변화가 발생했습니다. 제기 위치하고 복부 아래에 자리잡고 꼬리 개최 발톱과 왕새우가 (리빙스턴 외., 1980) 지배 자세를 전시하는 것으로 생각되었습니다. 크레이 피쉬의 복부 굴곡의 상태는 지배 계층 상태 (Listerman 외., 2000) 유지하면서 지배 왕새우는 한 쌍의 내에서 사회적 상호 작용을하는 동안 전시 또는 자세로 나타나지 않습니다. 그들은 상대의 휴양지로 복종 왕새우도 스스로하에 자신의 abdomens 넣어 것입니다. 이러한 꼬리 집어넣고 또한 방위 태세 (Listerman 외., 2000)으로 여겨진다. 이러한 행동은 쉽게 (; Bruski 및 던햄, 1987; 리 외, 2000;.. Listerman 외, 2000 Bovbjerg, 1953, 1956) 현장 및 실험실 설정에서 관찰되었습니다. 흥미롭게도, 행동 자세 랍스타 (리빙스턴 외., 1980)에 명시된 바와는 5 - HT 및 호주 왕새우, Cherax의 소멸자 (맥레이, 1996)에 octopamine 주사에 대해 반대하고 있습니다. 아마, 완전히 다른 응답은 호주 왕새우의 표면 flexor 준비에서 관찰됩니다. 지배력은 일반적으로 왕새우 중에서 관련 크기이기 때문에 또한, 하나는 신속하게 변경 사회적 조건 (스트론 외., 2000)에 대한 매우 플라스틱 응답 시스템을 기대합니다. 무척추 동물에서 neuromodulators에 대응에서 소성 조사의 빈 공간입니다. Wyttenbach, 존슨, 그리고 호이 (1999)는 디지털 미디어 및 기타 왕새우 준비 외에이 보고서에 표시되는 동일한 근육을 포함한 다양한 크레이 피쉬 experimentations위한 실험실 매뉴얼을 제작했습니다. 이것은 학생 운동에 대한 훌륭한 리소스입니다.

<ol><li> 크레이 피쉬 (Procambarus clarkii). 아차 팔 라야 생물학 공급 주식 레이스 랜드, LA. 미국. </li><li> 표준 크레이 피쉬 호수 : 반 Harreveld의 ​​솔루션 (1936)에서 수정. (MM)를 205 NaCl, 5.3 KCl, CaCl 2 13.5 2 시간 2 O, 2.45 MgCl 2 6H 2 O, 5 HEPES 및 산도 7.4로 조정. 세로토닌, 글루 탐 산염과 도파민은 크레이 피쉬 호수에서 만들어집니다. 부왕의 고정력있는 솔루션은 직접 사용되었고 메틸렌 블루는 크레이 피쉬 호수에서 이루어집니다. 모든 화학 물질은 시그마 화학 회사 (세인트 루이스, MO)에서 얻을 수 있습니다. </li><li> 해부 도구 : 좋아요 # 5 핀셋, 고급 가위, 나이프 블레이드 홀더, # 26002-20 곤충 핀 (모든 파인 과학 도구 (미국), 주식 회사, 373 - G 빈티지 파크 드라이브, 포스터 시티, CA 94404-1139에서 얻은) . </li><li> Sylgard 바닥 유리 접시 </li><li> 비커 (화학 솔루션을 개최) </li><li> 전기 신호는 컴퓨터 (ADInstruments, 콜로라도 스프링스, CO, 미국)에 PowerLab 26T 인터페이스 라인에 기록됩니다. 우리는 차트 또는 범위 이름을 ADInstruments에서 표준 소프트웨어를 사용합니다. </li><li> 세포내뿐만 아니라 세포 레코딩을위한 모델 3000 AC / DC 앰프를 사용할 수 있습니다. </li><li> 세포내 레코딩을위한 줌 기능과 함께 현미경을 해부. 시각 터미널에 초점 녹음 똑바로 목표 (4 x와 20X)와 복합 현미경이 사용됩니다. 하나는 HG 광원이 필요합니다. </li><li> 세포내 녹음을 위해 우리는 유리 모세관 튜브 (FHC, 브런즈윅, ME, 04011, 미국의 카탈로그 # 30-31-0)를 사용합니다. 세포내 전극은 20-30 mOhm의 저항이 있어야합니다. 세포내 전극은 3 M KCl로 가득 차 있었다. </li><li> 흡입 전극은 세포 신호를 기록하는 데 사용됩니다. </li><li> 패러데이 케이지. </li><li> 고강도 조명기 (광원). </li><li> Micromanipulator </li></ol>

membrane potentials, synaptic responses, neuronal circuitry, neuromodulation and muscle histology using the crayfish: student laboratory exercises

이 보고서의 목적은 생물 학적 막에 걸쳐 이온 기울기에 의한 영향의 이해를 개발하는 것입니다. 이 실험에서 세포의 막 잠재력에 영향을 우리가 주소를 두 가지 측면은 다음과 같습니다 막의 외부에 K +를 (1) 이온 농도, 그리고 특정 이온에 멤브레인 (2) 투자율. 크레이 피쉬 복부 신근 근육 일부중인 토닉 (느린) 및 기타 phasic (빠른) 자신의 생화 학적 및 생리 학적 phenotypes에서뿐만 아니라 그들의 구조와 마찬가지로 그룹에, 이러한 근육 신경을 분포시키다 모터 뉴런은 기능 특성 correspondingly 다릅니다. 우리는 시냅스 전달의 속성을 증명하기 위해뿐만 아니라 표면, 토닉 복부 flexor 근육 이러한 근육을 사용합니다. 또한, 우리는 회로의 특정 측면에 cuticular 감각 자극의 효과뿐만 아니라 neuromodulators의 영향을 설명하기 위해 감각 - CNS - 모터 신경 - 근육 회로를 소개합니다. 이 운동에서 얻은 기술로, 하나는 다른 실험 준비뿐만 아니라 의학 및 건강에 관한 생리 응용 프로그램에 남아있는 많은 질문에 답변을하실 수 있습니다. 우리는 동물 관련 근본적인 문제를 해결하기 위해 모델 무척추 준비의 유용성을 증명하고있다.

Watch this Scientific Journal Video about Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises at JoVE.com

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

실험 근육 구조, 시냅스 반응, 이온 그라디언트 및 막 잠재력에 대한 투자율의 효과를 조사 경험을 얻기 위해 학생들을위한 쉬운 접근 방법을 보여줍니다. 또한, 감각 - CNS - 모터 근육 회로의 연결 회로에서 화합물의 효과를 테스트하는 방법을 보여 제공됩니다.

멤브레인 후보, 시냅틱 응답,의 연결 회로, Neuromodulation과 크레이를 사용하여 근육 조직학 : 학생 실험실 연습

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

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58.4K 조회수.  University of Kentucky. 실험 근육 구조, 시냅스 반응, 이온 그라디언트 및 막 잠재력에 대한 투자율의 효과를 조사 경험을 얻기 위해 학생들을위한 쉬운 접근 방법을 보여줍니다. 또한, 감각 - CNS - 모터 근육 회로의 연결 회로에서 화합물의 효과를 테스트하는 방법을 보여 제공됩니다.

비디오: Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

크레이 피쉬에 리플렉션하는 행동 응답 녹음

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

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신경과학

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

크레이 레그 신근 근육에 높은 및 낮은 출력 NMJs의 생리 레코딩

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

크레이 피쉬 복부 근육 수용체 기관 : Proprioception에서 학생 실험실 연습

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

크레이 피쉬 Hindgut와 생리 실험 : 학생 실험실 연습

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

교양 뉴런의 막 단백질의 측면 보급과 Exocytosis은 형광 복구 및 형광 손실 Photobleaching를 사용하여 평가

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

게 사지에서 고유 감각과 긴장 수용체 : 학생 실험실 연습

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

심각한<em&gt; In Vivo</em&gt; 마취 된 래트에서의 Hyperdirect 경로로부터의 국소 장 전위 및 다중 단위 활성의 전기 생리 학적 기록

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivo에서경작-Murine 이다 빠르고, 간단 하 고 저렴 한 AWESAM 프로토콜을 사용 하 여 같은

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...