이 작품은 건강의 국립 연구소에서 교부금에 의해 투자되었다 : EY015748, EY017964 (MEZ), 및 EY019517 (ASV). 이 작품은 또한 안과 부서 및 중앙 뉴욕의 사자 실명 무제한 부여를 방지하기 위해 연구에 의해 지원되었다. 우리는 또한 동물의 자신의 우수한 치료를 위해이 비디오에서 주연하는 스테핑에 대한 우리의 동물 기술자, 마 멜리을, 감사의 말씀을 전합니다.

동물 관리 Xenopus의이 연구에 사용 된 올챙이는 실험 동물의 관리 및 사용에 대한 북부 의과 대학 IACUC 및 가이드에 의해 승인 된 절차에 따라, 성장 제기 및 처리 하였다 laevis의. . 1 전 동작 설정 : 올챙이 <ol><li> 시판 소스에서 수정 된 Xenopus의 배아를 얻거나 이전에 6 바와 같이 체외에서 난자를 비옥. </li><li> 20 mM의 KCl을,, 20 mM의 염화칼슘, 50 mM의 HEPES 0.1X MMR [10 X 마크의 수정 링거 (MMR) 솔루션 주식 (10 mM의 MgCl2를 2를 포함 60mm 배양 접시에 18 ˚ C에서 신경 판 단계의 배아에 blastula를 놓고; 1 M의 NaCl, 50 ㎍ / ml의 젠타 마이신 항생제), pH를 7.5로 조정하고 오토 클레이브 RT에서 보관. 매일 0.1X MMR을 변경하고 죽은 태아를 제거합니다. </li><li> 배아 ~ 27 Nieuwkoop와 페이버 단계에 도달하면 - 30 7 번째 전환항생제없이 0.1X MMR에 전자 배아 장내 박테리아의 성장을 허용합니다. </li><li> 그들은 먹이 단계​​ (단계 45)에 도달 할 때까지 올챙이 성장과 0.1X MMR 가득 100mm 배양 접시로 이동합니다. 그 사이 날에 0.1X MMR을 변경, 일주일 동안 매일 그들에게 쐐기 분말 상층 액을 넣습니다. </li><li> 100mm 페트리 접시에 한 주 후에, 개구리 물이 가득 반 갤런 탱크에 동물 이동 [0.5 g / L 즉시 바다와 2 ㎜ 2 인산 나트륨 (나 2 HPO 4 : 141.96 MW)의 pH 6.8]. 단계 2.4로 피드,하지만 물이 흐린 경우 물에게 일주일에 2-3 시간을 변경합니다. 빛이 오전 6시에 점등과 12 시간의 light/12 어두운 빛의주기에서 동물을 유지합니다. </li><li> 실험실 벤치있는 underpad, 적어도 12 시간의 O / N, 시험 전에 같은 흰색 표면에 시험 과목으로 하프 갤런 탱크를 배치합니다. </li><li> 단계 45-50에서 테스트 올챙이은 아래 3 단계에서 설명. </li></ol> . 2 전 동작 설정 : 기술 장비티 <ol><li> 동작 분석을위한 실험실의 영역을 따로 설정합니다. 그것은 표준 형광 조명 조용하고, 트래픽이 낮은 지역에 있어야합니다. </li><li> 동물 시험 탱크를 준비합니다. 중첩, 하프 갤런 테스트 탱크는 두 부분으로 구성되어있다 : 물과 올챙이를 개최한다 내부 탱크; 그리고 시각적 인 자극과 외부 탱크. <ol><li> 내부 탱크, 빈틈없는, 바닥에서 5cm와 외부 코너에 작은 표시를합니다; 이 물 라인 (그림 1A, 녹색 점선)입니다. </li><li> 또한, 내조에 대해, 실 가드 엘라스토머와 같은 불활성 화합물로 (모서리와 중앙) 탱크 디벳 채우기. 참고 : 그들은 채워하지 않는 경우 올챙이는이 지역에 머무르게됩니다 </li><li> 외부 탱크, 흰색 조직 종이 그림 1B 검은 전기 테이프, 다른 절반 하나의 탱크의 외부의 정확히 절반을 커버합니다. </li></ol></li><li> 에 탱크를 배치턴테이블을 접종. </li><li> 설치 웹캠 / 삼각대는 테스트 탱크 위에 있도록. 도 1C에 도시 된 바와 같이, 테스트 영역의 시각화를 허용하도록 카메라를 조정. </li><li> 5를 설치 퀵타임 플레이어 소프트웨어를 컴퓨터에 웹캠을 연결합니다. 카메라의 전원을 켭니다. QuickTime 플레이어에서 파일에서, &#39;새 동영상 저장&#39;을 선택합니다. 행동 분석 설정은 바로 컴퓨터에 표시됩니다. </li><li> 올챙이 동작에 영향을 미칠 수있는 외부 신호를 줄일뿐만 아니라, 물의 표면에서의 반사광을 감소시키는 전체 셋업 위에 경량 면천 드레이프. </li><li> 확인이 35-50 CD / m 2 사이 피복 대책 하에서 휘도. </li></ol> 3. 행동 분석 <ol><li> 개구리 물 단계 1.2.1 제 5 cm 물 표시로 내부 테스트 탱크를 채우십시오. </li><li> 작은 그물을 사용하여 조심스럽게 내부 테스트 탱크로 동물을 이동합니다. </li><li> 오픈 Q컴퓨터와 가이드로 외부 탱크를 사용하는 방법에 대한 uickTime 소프트웨어는 카메라 테스트 영역을 시각화하고 기록 할 수 있는지 확인합니다. </li><li> 카메라로 종이와 기록의 조각에 동물 이름, 날짜와 시간을 기록합니다. </li><li> 오른쪽 그림 2 탱크의 검은면이 외부 탱크에 시험 탱크를 배치합니다. </li><li> 탱크를 배치 한 후 바로 동영상 촬영을 시작하고 2 분 동안 타이머를 설정합니다. </li><li> 타이머 비프, 내부 시험 탱크를 제거하면, 외부 탱크를 180도 회전과 외부 탱크 내부의 내부 시험 탱크를 배치합니다. 타이머를 시작합니다. 참고 : 검은 색면 지금 다른 측면에 있어야합니다. </li><li> 열 시험의 총 단계 3.7, 여덟 번 이상 반복합니다. 각 시험을 확인, 가이드로 그림 2를 사용합니다. </li><li> 두 개의 분리 된 일에 행동 분석을 반복합니다. </li></ol> 4. 망막 Axotomy <ol><li> 그들은이 응답 할 때까지 0.02 %의 tricaine에 동물을 배치# 3 집게로 꼬리 핀치에. </li><li> 0.1X MMR 1 % 아가로 오스를 용융 한 후 60mm 페트리 접시에 추가합니다. 동물의 일부가 액체에 빠져들도록 냉각되면, 아가로 오스의 작은 직사각형의 잔디를 만들어 작은 0.02 %의 tricaine와 잔디에 올챙이를 추가합니다. </li><li> 피어스 25 G 바늘을 눈의 등쪽 영역 뒤에 45도 각도로 피부, 반대편 집게 대하여 동물을 보강하면서. </li><li> 시신경 옆에 자리 잡고 정맥을 자르지 않도록주의하면서 조심스럽게 # 5 집게로 구멍에 도달 시신경을 싹둑 꺼 그것을 뒤집어. 심한 출혈로 인해 실수로 동맥을 자르는에있는 경우, 다음 신속하게 안락사에 2 % tricaine에 동물을 배치합니다. 주의 : 2 % tricaine 솔루션은 인간의 마비를 일으킬 수 있습니다. 이 솔루션은 장갑으로 취급되어야한다. </li><li> 동물을 복구하기 위해 수술 후, 0.7X MMR과 100mm 배양 접시에 20 분 동안 50 ㎍ / ㎖의 젠타 마이신에 동물을 배치합니다. 네브라스카XT, 개구리 물 회수 탱크로 전송 올챙이. 동물 나머지 O / N.에게하자 </li><li> 다음날 아침, (3.1-3.9 단계) 전술 한 바와 같이 시각 기능을 테스트합니다. </li></ol> 5. 결과 분석 <ol><li> 시험이 종료 된 후, 올챙이는 탱크의 검은면에 남아있는 시간의 양을 측정합니다. <ol><li> 수동으로 시간을 표시하고 자주 일시 중지 할 수 있습니다 소프트웨어를 사용하여 비디오를 볼 수 있습니다. 비디오 디스플레이 시간을 본다. 내부 탱크가 성공적으로 외부 탱크 내부에 둥지와 카메라의 관점에서 제곱 때입니다 두 분 재판의 시작을 기록합니다. </li><li> 동물에 수영 탱크의 어느 쪽이 정의하는 올챙이의 눈의 위치를​​ 사용합니다. 두 눈은 검은 색 / 흰색 선을 넘은 경우에만 다른 탱크의 한쪽면에 교차 정의합니다. </li></ol></li><li> 동물 블랙 측에 소비 한 시간 (초 단위) 각 간격의 시작과 끝을 적는다. 일시 정지 및 재정확도를 보장하기 위해 비디오 감는다. 각 시험에서 초 총. </li><li> 연결된 동작 워크 시트에 입력이 숫자를. 워크 시트는 동물이 초 총 시간으로 총 120 초 시행 분열에서 탱크의 검은면에 소요되는 시간을 빼서 흰색면에 소요되는 시간의 비율을 계산합니다 [(120 - 전체 초에 지출 검은면) / 120 초]. 10 시험에 워크 시트의 평균은이 비율. </li><li> 일 사이의 실험 (0.05 ≤ P) 중요한 있는지 확인하기 위해 학생의 T-테스트, 쌍, 양측 분포를 사용합니다. </li></ol>

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The authors have nothing to disclose.

우리는 여기에 쉽게 최소한의 교육을받은 실험실 요원에 의해 주에서 수행 할 수있는 간단한 비전 가이드 행동 분석을보고합니다. 다른 분석은 동물의 행동에 전문 장비와 전문 지식을 필요로하지만,이 분석은 빠른 테스트가 시각 기능을 확인할 수 있습니다. 또 다른 행동 분석, 비주얼 회피 분석은, tectum는 Xenopus의 10에 시각적 인식에 기여하는 방법을 결정하기 위해 개발되었습니...

는 Xenopus laevis의 눈 형성을 연구하는 모델 생물로 사용되었습니다. 눈은 시각 발달과 기능에 영향을 테스트하는 유전자 또는 경로에 대한보다 주에 만기 성장하고, 신속하게 개발할 수 있습니다. 시각 기능을 테스트하기 위해 optokinetic 및 optomotor 응답은 각각 1, 2, 제브라 피쉬와는 Xenopus 올챙이에서 사용되었다. Xenopus의 올챙이의 눈이 제브라 피쉬에 비해 상대적으로 작은이기 때문에,이 분석은 Xenopus의 미묘한 꼬리 플립과 눈 운동 동작을 감지하는 특수 장비의 사용과 훈련 된 인력이 필요합니다. Xenopus의의보다 강력한 행동은 여기에 3 설명 흰색 배경에 탱크에서 수영에 대한 기본 설정입니다. 반 블랙 / 반 흰색 탱크에서 올챙이를 배치 할 때 미리 변성 올챙이 신속하게 탱크의 백색 옆에 수영. 우리는 이전에 다 능성 세포 유래 여부를 확인하기 위해이 분석을 사용눈은 4 기능이었다. 여기, 우리는 premetamorphic Xenopus의 올챙이의 시각 기능을 테스트하는 데 사용할 수있는이 분석의 세부 버전을보고합니다. 그것은 단지 대부분의 Xenopus의 실험실에서 발견 탑재 디지털 캠코더, 디지털 카메라 소프트웨어 및 표준 장비를 필요로하기 때문에이 분석은 optomotor 응답 분석보다 간단합니다. 또한, 기록 된 반응은 집계 결과에 특별한 훈련이 필요하지 않습니다. 우리의 대표적인 결과는 두 번 망막 axotomy을 시행 한 올챙이 같은 그룹, 탱크 주위에 무작위로 수영을 보여줍니다. 우리는 또한 하나의 눈은 시각적 인 반응을 테스트 할 수있는 방법을 보여주는 대표적인 올챙이의 행동 분석 결과를 포함했다. 분석 기간 동안 획득 한 숫자를 삽입하고 분석 할 수 있도록 워크 시트가 포함되어 있습니다. 이 워크 시트 테스트 올챙이가 시각적 응답을 가지고 있는지 여부를 결정하는데 사용될 수있다.

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			<td>Good for larger tadpoles but small ones are difficult to see</td>
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			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
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			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
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			<td>This can be used for connecting firewire to camera</td>
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			<td>Stored at room temperature</td>
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			<td>Stock solution = 10% stored at -20&#730;C</td>
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			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
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a simple behavioral assay for testing visual function in xenopus laevis

개구리의 올챙이의 시각 기능의 측정은,는 Xenopus laevis의, 살아있는 동물의 실명에 대한 검사를 할 수 있습니다. optokinetic 응답은 테스트 한 모든 척추 동물에서 관찰 된 비전 기반의 재귀 동작입니다. 그래서 꼬리 플립 응답이 미묘한 반응을 기록 할 수있는 숙련 된 기술자를 필요로 다른 측정으로 사용 된 올챙이의 눈은 작은 수 있습니다. 우리는 올챙이가 검은 색과 흰색 양면 모두 탱크에 주입 할 때 탱크의 백색 옆에 수영하는 것을 선호한다는 사실에 따라 다른 행동 분석을 개발했다. 여기에 제시된 분석은 쉽게 측정 된 응답을 만들어 저렴하고 간단한 대안입니다. 설치는 대부분의 Xenopus의 실험실에서 쉽게 사용할 수 삼각대, 웹캠과 중첩 된 시험 탱크로 구성되어 있습니다. 이 문서는 이전과 시신경을 단절 한 후, 올챙이의 동작을 보여주는 동영상이 포함되어 있습니다. 한쪽 눈의 기능을 테스트하기 위해, 우리는 또한 스와 템포 포함각각의 눈은 연속적인 일에 망막 axotomy을 시행하는 올챙이의 sentative 결과. 미래 연구는 한 번에 여러 올챙이의 비전을 테스트하기 위해이 분석의 자동화 된 버전을 개발할 수 있습니다.

이전 보고서는이 분석, 우리는 올챙이의 눈을 하나의 시각적 기능을 테스트하기 위해이 분석을 변경 한 배경 색상 기본 설정 분석 3 premetamorphic Xenopus의 올챙이가 검은 색 / 흰색 탱크의 백색 옆에 수영하는 것을 선호하는 것으로 나타와 호출 한 주 미만. 이러한 방식으로, 자신의 눈 조직 학적 검사를 위해 수집 될 수있다. 우리는 응답이 시각 신호에 의한 방법?...

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

제노는 올챙이가 검은 색 / 흰색 탱크의 흰색 측면에서 수영을 선호 ​​laevis의. 이 동작은 자신의 비전에 의해 유도된다. 이 동작을 바탕으로, 우리는 올챙이의 시각 기능을 테스트 할 수있는 간단한 분석을 제시한다.

테스트 시각 기능에 대한 간단한 행동 분석<em&gt;는 Xenopus laevis의</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

a-simple-behavioral-assay-for-testing-visual-function-xenopus

Research

JoVE Journal

Neuroscience

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

9.7K 조회수.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  제노는 올챙이가 검은 색 / 흰색 탱크의 흰색 측면에서 수영을 선호 ​​laevis의. 이 동작은 자신의 비전에 의해 유도된다. 이 동작을 바탕으로, 우리는 올챙이의 시각 기능을 테스트 할 수있는 간단한 분석을 제시한다. 

비디오: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

베르 미 첼리와 Capellini 처리 시험 : 쥐 및 생쥐의 손재주가 앞발 기능의 단순 양적 측정

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

연구

신경과학

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Hippocampal 인슐린 Microinjection 및<em&gt; 생체에</em공간 메모리 테스트 기간 동안&gt; Microdialysis

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

노래하는 새는에 청각 피드백을 조작하기위한 경량, 헤드폰 기반의 시스템

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

의 Mechanosensation에 대한 행동 분석 클론의 MARCM은 기반<em&gt; 초파리</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

의 후각 망울에서 칼슘의 변경 사항을 모니터링을 통해 온도에 의한 신경 세포의 활동을 기록<em&gt; Xenopus의의 laevis의</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

준비 및 Xenopus laevis Tectal 신경의 전체 셀 패치 클램프 기록에 대 한 프로토콜

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...