우리는 꿀벌에 촉각 학습 패러다임에 우리를 소개를 위해 요아킴 Erber 감사드립니다. 이 작품은 독일의 우수 프로젝트의 프레임 워크에 재정 지원 우수 277 CITEC의 클러스터에 의해 지원되었다.

1. 꿀벌 준비 <ol><li> foraging 여행에서 돌아하면서 꿀이나 꽃가루 일개미는 자당 피더이나 직접 벌집 입구에서 어느 분야에서 잡은 수 있습니다. 각 단일 벌은 폼 플러그와 폐쇄 및 추가 처리를위한 실험실로 바로 이동되는 유리관에 캡처됩니다. </li><li> 사람들이 부동의 첫 징후를 보여 ° C까지 실험실에서 캡처 꿀벌은 잠시 4에 냉장고에 냉각되어 있습니다. </li><li> 각 단일 고정 벌은 머리와 가슴 사이와 복부 이상 접착 테이프로 작은 금속 튜브에 장착되어 있습니다. 케어는 코와 안테나 자유롭게 움직일 수 있다는주의해야한다. </li><li> 비전을 막다하기 위해 흰색 페인트 (예 : 용매 무료 Tipp-예)로 고정 벌의 화합물 눈과 ocelli 그림을 그리기도 하죠. </li><li> 녹음하는 동안 머리의 움직임을 방지하기 위해 머리와 가슴 사이의 테이프에 문제를 해결하기 위해 꿀벌의 머리 뒤에 녹은 왁스의 작은 방울을 추가. </li><li> 더 나은 식별 테이프에 번호를 각각 하나의 벌을 표시하고 탈수를 방지하기 위해 습윤 분위기에 고정 벌과 튜브를 놓습니다. </li><li> 주사기가 표시 30 % 자당 솔루션의 방울을 5 초 각 하나의 벌을 먹이고 촉각 시설 프로토콜로 시작하기 전에 모든 꿀벌이 30 분에 복구 보자. </li></ol> 2. 촉각 컨디셔닝 <ol><li> 에어컨하기 전에, 각 단일 벌은 안테나에 적용 30 % 자당의 자극에 코 확장 응답 (PER)에 대한 검사를해야합니다. 따라서 코의 끝이 열려있는 개미 사이에 가상의 선을 넘어있다. 자당의 자극에 PER으로 응답하지 않는 모든 꿀벌을 폐기하십시오. </li><li> 촉각 에어컨이 설치된 자극 (CS)와 같은 부드러운 또는 조각 패턴, 예를 들어, 수평 또는 150 μm의 파장과 격자를 형성하는 수직 홈이있는 황동 큐브를 (예를 들어 3 × 5 ㎜), 사용하십시오. 에프또는 무조건 자극 (미국)는 30 %의 자당 솔루션을 (물에 희석 가정 설탕,)을 사용합니다. </li><li> 황동 큐브 (CS)는 컨디셔닝 절차를 수행하는 동안 정확한 위치를 보장하기 위해 (예를 들어, Märzhäuser MM33) micromanipulator에 홀더에 배치됩니다. 미국은 30 % 자당 솔루션들로 가득 찬 주사기로 벌에게 수여됩니다. </li><li> 에어컨 절차는 5 분 간 시험 간격 (ITI)와 촉각 자극 (CS)과 자당 솔루션 (미국) 다섯 pairings로 구성되어 있습니다. 마운트 된 촉각 자극 (CS)과 micromanipulator 앞에 한 벌을 배치합니다. 촉각 자극의 표면이 꿀벌의 머리 (그림 1 A와 B)에 평행되도록 천천히 CS를 배치합니다. 동물과 촉각 객체 사이의 거리가 테스트 꿀벌의 antennal 작업 반경의 범위에 있어야, 비 편안한 자세 w에 촉각 자극을 스캔 할 수 있어야한다 즉i 번째 안테나 모두. 그림 1에 표시된 예제에서, 거리가 3mm했다. 꿀벌이 5 초에 대한 촉각 자극 (CS)를 스캔 보자. 처음 3 초 후, 30 % 자당 (미국) 코 아래에 주사기와 솔루션의 비말을 제시한다. 부드럽게 코를 높이 주사기의 끝을 사용합니다. 코 아래에 자당의 자극은 15 당 무조건을 이끌어내는 것입니다. 꿀벌은 자당 보상을 치유 할 수 있습니다. 컨디셔닝 동안 정확한 시간 간격을 유지하기 위해 경보 신호 스톱워치를 사용하십시오. </li><li> 에어컨, PER은 벌의 학습 성공을위한 조치로 사용됩니다. 첫 번째 보상 pairings 후, 꿀벌들은 향후 보상을 기대하는 의미, 자신의 코의 확장에 의한 CS 프리젠 테이션에 대응하기 위해 시작합니다. 촉각 자극 프리젠 테이션의 3 초 시간 창 동안과 자당 프리젠 테이션하기 전에 언제든지 관찰 완전히 확장 코는 긍정적 인 반응으로 채점됩니다. 응답이 부정적인 인정되지 않습니다. occPER의 urrence은 실험에 의해 주목해야합니다. CS 프리젠 테이션 동안 PER을 보여 꿀벌의 비율은 각 시험에 대한 도시된다. </li></ol> 3. 동적 레코딩 <ol><li> 하나 밝혀 벌의 Antennal 움직임이 적합한 매크로 렌즈 (예 : TechSpec VZM 200 장착 Basler A602f-2, 화재 와이어 연결을 통해 50 프레임에서 작동)와 디지털 비디오 카메라로 기록됩니다. 카메라는 하향식 (top-down)보기에서 동물 (그림 1 A) 위에 위치합니다. 그림 1에서 녹음의 공간 해상도는 0.02 mm / 픽셀입니다. </li><li> 카메라 목적에 따라 서로 다른 방향에서 1 mm의 가장자리 길이 10 × 10mm의 체커의 사진을 하나 기록하여 카메라를 보정합니다. 보정은 카메라 보정 도구 상자 MATLAB 16으로 수행 할 수 있습니다. </li><li> 카메라 렌즈 아래에 하나의 고정 벌을 배치합니다. micromanipulator에 고정 촉각 자극을 제시, 동물합니다. 촉각 시설에 대한 설명과 같은 방식으로 진행하고, 비 개체를 스캔하는 동안 antennal 움직임을 기록합니다. 그것은 전체 antennal 작동 범위와 촉각 자극이 볼 수 있습니다하는 것이 중요합니다. 자극 패턴의 선택 테스트 및 시험 번호는 실험 설계에 따라 달라집니다. </li></ol> 4. 데이터 분석 <ol><li> 이미지 배경의 계산은 MATLAB에서 이루어집니다. 첫째, 시간이 지남에 따라 중간 그레이 스케일 값은 각 픽셀에 대해 계산해야합니다. 정적 객체는, 꿀벌과 촉각 자극의 고정 머리처럼 이미지 배경을 구성합니다. 움직이는 물체는 두 개의 안테나 같은 배경의 일부가 될 수 없습니다. </li><li> 기록 된 비디오의 각 프레임이 순차적으로로드 할 수 있으며, 현재 프레임과 이미지 배경의 차이는 계산해야합니다. 이 뺄셈의 결과는 프레임에서 프레임으로 이동하는 이미지의 부분을 강조하고 있습니다. 신분증eally, 꿀벌의 안테나는 비 제로 값 (그림 1C)의 유일한 지역입니다. </li><li> 자세한 처리를 위해, 비 - 제로 픽셀 값과 함께 두 큰 영역은 안테나로 간주됩니다. 두 안테나에 대한 바이너리 마스크는 픽셀의 안테나와 달리 값을 0으로 속하는 경우, 각 픽셀에 대해 값 1을 포함하는 생성하는 것입니다. 이 마스크는 나중에 antennal 팁을 현지화의 기반으로 제공하고 있습니다. 전체 이미지에 대한 예비 마스크를 얻으려면 다른 이미지의 각 픽셀의 회색 값은 미리 정의 된 임계 값과 비교됩니다. 우리가 노이즈 처리하기 때문에 나중에이 임계 값이 매우 낮을 것으로 선택됩니다. 첫째, 이미지 노이즈 작은 지역으로 인해 아직도 쓰고의 일부입니다 : 예비 마스크는 여전히 오류 두 가지를 수행합니다. 둘째, 안테나에 속하는 지역은 반드시 완전히 연결되지 않을 수 있습니다. 배경은 약 안테나와 같은 발광가있는 경우 후자는 주로 발생합니다. 이러한 유물을 제거하려면, 표준영상 처리 형태학의 작업이 적용됩니다, 표준 MATLAB 함수는 imerode과 (자세한 설명은 17 pp. 158-205 참조) 각각 imdilate에 의존, 이미지 침식과 팽창의 조합을 말한다. denoising 절차 후 안테나의 가설은 여전히​​ 같은 마스크에 포함되어 있습니다. 따라서, 다음 단계로 바이너리 마스크는 표준 MATLAB 함수를 bwlabel을 (이미지 세분화 알고리즘에 대한 자세한 내용은 17 pp. 40-48를 참조)를 사용하여 연결이 끊긴 지역에 밀집되어 있습니다. </li><li> 클러스터 당 픽셀의 수는 계산되어 두 개의 큰 클러스터가 선택되었습니다. 무게 중심은 왼쪽과 오른쪽 안테나 (그림 1D)를 구별하는 계산됩니다. </li><li> 각 안테나에 대한 antennal 팁은 근위 - 투 - 말초 방향 (그림 1D 참조)에서 가장 높은 값을 가진 클러스터에서 픽셀로 정의 할 수 있습니다. </li></ol>

<ol>
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Neurosci. 4, 46 (2010).</li><li>Scheiner, R., Barnert, M., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+water+and+sucrose+responsiveness+during+the+foraging+season+affects+proboscis+extension+learning+in+honey+bees.">Variation in water and sucrose responsiveness during the foraging season affects proboscis extension learning in honey bees.</a> Apidologie. 34, 67-72 (2003).</li><li>Mujagic, S., Erber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sucrose+acceptance,+discrimination+and+proboscis+responses+of+honey+bees+(Apis+mellifera+L.)+in+the+field+and+the+laboratory.">Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.</a> J. Comp. Physiol. A. 195, 325-339 (2009).</li><li>Friedrich, A., Thomas, U., M&#252;ller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+at+different+satiation+levels+reveals+parallel+functions+for+the+cAMP-protein+kinase+A+cascade+in+formation+of+long-term+memory.">Learning at different satiation levels reveals parallel functions for the cAMP-protein kinase A cascade in formation of long-term memory.</a> J. Neurosci. 24, 4460-4468 (2004).</li><li>Erber, J., Pribbenow, B., Grandy, K., Kierzek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile+motor+learning+in+the+antennal+system+of+the+honeybee+(Apis+mellifera+L.).">Tactile motor learning in the antennal system of the honeybee (Apis mellifera L.).</a> J. Comp. Physiol. A. 181, 355-365 (1997).</li><li>Lambin, M., D&#233;glise, P., Gauthier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+movements+as+indicators+of+odor+detection+by+worker+honeybees.">Antennal movements as indicators of odor detection by worker honeybees.</a> Apidologie. 36, 119-126 (2005).</li></ol>

관심 없음 충돌이 선언 없습니다.

꿀벌의 준비 : 수집 및 꿀벌이 동물의 낮은의 스트레스 수준을 유지하기 위해 신속하게 수행해야 수정하는 것입니다. 스트레스는 당 응답에 영향을 가지고 있으며, 따라서 꿀벌 19, 20의 학습 성과에 간접적 인 영향을 미칠 수 있습니다. 응력 수준은 신속하게 고정하기 위해 수집 한 후 즉시 직접 얼음에 꿀벌과 유리 병을 배치하여 감소 될 수있다. 그것은 꿀벌이 마취 기간 전?...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 꽃가루의 일개미 API를 mellifera L. </td><td></td><td></td><td> 개인 두드러기 </td></tr><tr><td> 유리 병 (22 X 45mm) </td><td> 칼 로스 GmbH의 및 공동 </td><td> X655.1 </td><td></td></tr><tr><td> 황동 관, 10 X 30mm </td><td></td><td></td><td> 자체 제작 </td></tr><tr><td> 황동 큐브, 3 X 5mm </td><td></td><td></td><td> 자체 제작 </td></tr><tr><td> Tipp-전 (솔벤트 무료) </td><td> Tipp - 예 GmbH의 및 공동 KG </td><td></td><td></td></tr><tr><td> 자당 솔루션 (30 %) </td><td></td><td></td><td> 가정 suger은 물에 희석 </td></tr><tr><td> 에탄올 (70 %) </td><td> 칼 로스 GmbH의 및 공동 </td><td> 9065.3 &lt;/ TD&gt; <td></td></tr><tr><td> 주사기 (1 ML)와 바늘 (0.6 X 25) </td><td> 칼 로스 GmbH의 및 공동 </td><td> 59.1 </td><td></td></tr><tr><td> 보기 중지 </td><td> 칼 로스 GmbH의 및 공동 </td><td> L423.1 </td><td></td></tr><tr><td> Micromanipulator </td><td> Märzhäuser MM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td> 디지털 비디오 카메라 </td><td> Basler A602f-2 </td><td></td><td></td></tr><tr><td> 카메라의 매크로 렌즈 </td><td> TechSpec VZM 200 </td><td></td><td></td></tr><tr><td> MATLAB R2009b </td><td> MathWorks </td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

꿀벌 (API를 mellifera L.)은 eusocial 곤충이며, 잘 노동과 연관 학습 능력 1, 2, 자신의 복잡한 부문에 대한 알려져 있습니다. 그들은 애벌레를 받아야하거나 일반 육각 식품 빗 (예 : 꽃가루 나 꿀)과 알을 품다 3 구축 곳의 일벌은 어두운 벌집 내부의 인생의 첫 번째 절반을 보냅니다. 안테나는 특별한 multisensory feelers하고 하이브 건물 5 패턴 인식 6 등 다양한 촉각 인한 작업 4에서 중추적 인 역할을한다. 나중에 생활에서, 각 하나의 벌은 음식에 대한 사료에 하이브를 출발합니다. 그런 벌은 수익 음식 소스를 차별 위치를 기억, 7의 둥지 친구로 의사 소통을 할 수 있습니다. 꿀벌은 음식 소스의 multisensory 기억을 형성하기 위해 꽃잎 표면 9 색이나 냄새 7, 8, 9, 10와 같은 다른 꽃 신호뿐만 아니라, 촉각 신호를 사용합니다. 실험실 공동 이하nditions은 꿀벌이 같은 가장자리 또는 안테나 10, 11, 12, 13와 홈으로 촉각 객체 기능을 차별하는 appetitive 학습 패러다임에서 훈련을 할 수 있습니다. 이 학습 패러다임은 밀접하게 밝혀 꿀벌 14 코 확장 응답 (PER)의 클래식 후각 시설에 관련되어 있습니다. 실험실에서 촉각 학습 패러다임의 장점은 antennal 운동 패턴의 분석 등 다양한 생리 측정과 학습에 행동 실험을 결합의 가능성입니다.

다음과 같은 실험에서 우리는 촉각 학습 꿀벌의 antennal 샘플링 동작에 영향을 미치는 방법을 공부했습니다. 이를 위해, 우리는 이전에 순진하고 설치된 꿀벌의 antennal 팁과 촉각 자극을 제시하는 동안 움직임을 감시. 첫째, 꽃가루 일개미 집단의 자발적인 antennal 운동 (N = 42)이 1 분에 기록되었다. 꿀벌의 절반 (N = 21)가 다섯 번에게 30 %의 자당 보상과 촉각 자극을 페어링하여 ?...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

이 프로토콜에서 우리는 상태로 촉각 자극에 꿀벌을 밝혀와 고급 규모의 antennal 샘플링 패턴의 운동학을 분석하는 차원 모션 캡처 기술을 소개하는 방법을 보여줍니다.

촉각 시설과 꿀벌에 Antennal 샘플링 전략의 움직임 분석 (<em&gt; API를 mellifera</em&gt; L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

Research

JoVE Journal

Neuroscience

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

10.5K 조회수.  Bielefeld University. 이 프로토콜에서 우리는 상태로 촉각 자극에 꿀벌을 밝혀와 고급 규모의 antennal 샘플링 패턴의 운동학을 분석하는 차원 모션 캡처 기술을 소개하는 방법을 보여줍니다.

비디오: Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

꿀벌의 코 확장 응답 클래식 컨디셔닝에 Behavioural 약리학 ( 아피스 mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

연구

신경과학

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

여러 스케일에서 디지털의 연결 형태론의 자동 숄 분석

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

차 모터 및 Electrospun 폴리 - L - lactide Nanofiber 공사장 공중 발판을에 정의된 미디어 감각 뉴런의 문화

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

의 냄새-evoked 응답의 칼슘 이미징 Drosophila Antennal 엽

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Electrophysiological 측정과 인간 유아의 Nociception 분석

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

당뇨병 쥐의 열전달 Nociceptive 본부 신속한 결정

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

꿀벌의 RNAi를 매개로 두 유전자 넉다운과 미각 지각 측정 (<em&gt; 아피스 mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

절제된 자유 비행 꿀 꿀벌의 Neuropharmacological 조작,<em&gt; API를 mellifera이</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

태아 알코올 스펙트럼 장애의 쥐 모델에서 Hippocampal 기능 장애를 평가 하기 위해 추적 Eyeblink 클래식 컨디셔닝의 사용

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

학습과 기억의 척도 로서의 Drosophila Courtship 컨디셔닝

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...