The project was supported by Award AAES 461Hatch/Multistate Grants ALA08-045 and ALA015-1-10026 to N.L.

악기, 자극 솔루션, 그리고 침대 버그 1. 준비 <ol><li> 20 ㎖ 병 (V / W) 50 % KNO 2 솔루션을 준비합니다. </li><li> 반복과 솔루션에서 텅스텐 전극을 담그고 5 V에서 KNO 2 용액에 두 개의 텅스텐 미소 전극을 선명하게. <ol><li> 대략 텅스텐 와이어의 선단부를 소비 크게 할 수는 약 5 분 동안 2 딥 / 초의 속도 및 KNO 2 용액 중에서 텅스텐 와이어의 약 10mm 침지하여 텅스텐 와이어를 선명. </li><li> 전극의 미세하고 날카로운 포인트가되도록 섬세 적어도 1 분 동안 2 딥 / 초의 속도 및 용액 중 와이어 팁의 약 1mm 침지하여 전극을 선명. 이 침대 버그 후각 sensillum의 표피에 구멍을 충분히 괜찮을 0.2 ~ 0.5 μm의에 도달 할 때까지 자주 현미경으로 전극의 끝 부분의 직경을 확인합니다. 참고 : 수동으로 날카로운 동안전극 위할, KNO 2 용액에 텅스텐 와이어의 침지 속도는 항상 일정하지 않다. 더 연습, 그것은 전극 선명에서 비교적 일정한 속도를 유지하는 것이 훨씬 쉽다. 선명 화 시간은 전극이어야 방법에 따라 미세도 불확실하다. 여기서, ~ 0.2 ㎛의 직경을 가진 전극 팁은 후각 sensillum 뚫 정도로 충분하다. </li></ol></li><li> 원액 1:10의 V / V의 초기 농도로 단정 화합물에서 디메틸 설폭 시드 (DMSO)에 화학적 자극의 각각의 희석. 각 화학 물질에 대한 재고 솔루션의 각에서, DMSO 다시, 실험에 필요한 얼마나 많은 용량에 따라 십진 희석 시리즈를 만듭니다. β - 피넨과 유칼 립톨 - 여기, 10 % (+)를 사용합니다. </li><li> unfed 또는 칠일 배치 후 공급 포트에서 (남성 또는 여성 중) 성인 침대 버그. 딕스 식민지 (켄터키 대학에서 박사 헤인즈의 선물)은 T에 사용되는그는 배양 접시에서 실험. 참고 : 페트리 접시에 배치 침대 버그에 대한 정확한 수는 없습니다. 이 적거나 많은 수있다. </li></ol> 2. 침대 버그 더듬이 준비 <ol><li> 얼음 (2-3 분)에 침대 버그를 마취. </li><li> 양면 테이프로 현미경 커버 슬립에 안테나와 곤충의 몸을 모두 수정하고 미세 가위 다리를 제거합니다. </li><li> 꾸준히 테이프를 부착 할 수 있도록 부드럽게 안테나를 만져 작은 핀을 사용합니다. </li><li> 조작을 용이하게하고, 기록 전극 (그림 1)에 대한 적절한 각도 (~ 90 °)로 조정하는 치과 왁스의 작은 공 (~ 1cm 직경)에 대한 커버 슬립을 놓습니다. </li><li> 일단, 스테레오 현미경 침대 버그를 배치 차가운 광원을 켜고 안테나가 명확하게 제시 될 때까지 조명의 강도를 조정하고, 높은 배율에서 침대 버그 안테나의 두 번째 편모에 현미경을 집중 확보 (720X) . 노트: 실험에 사용 된 조명의 강도는 정말 실험자의 눈이 조명의 강도를 어떻게 생각하는지에 의존하는, 정량화되지 않습니다. </li></ol> 3. 단일 Sensillum 기록 <ol><li> 신호 기록 및 시각화를위한 컴퓨터와 연결되어 신호 포착 컨트롤러와 프리 앰프 (10X)를 연결한다. 컴퓨터를 켜고 소프트웨어, 예를 들어, AutoSpike32를 시작하고 메뉴 바에서 &quot;녹음&quot;모드를 클릭합니다. 이어서 웨이브 신호 기록을 시작하도록 &quot;웨이브&quot;선택. 참고 : 반복적으로 모니터의 오른쪽 왼쪽에서 실행하는 평면 라인은 이제 볼 수 있어야합니다. 여기서, 기록 창 40 초 정도. 맥스 웨이브 녹음은 10 초입니다. 선택된 샘플링 속도가 96,000이고 디지털 샘플링 속도 오프셋은 0 % 및 필터링없이, 기록 신호의 부정은 정류가 240이다. 필요에 따라 소프트웨어의 이러한 모든 파라미터 설정이 수정 될 수있다. </li> &lt;리&gt; 더듬이 sensillum에서 신경 응답을 토닝 모드를 제공하는 데 사용되는 프리 앰프에 연결 스피커의 전원을 켭니다. </li><li> 안정화 된 침대 버그의 복부에 기준 전극을 삽입합니다. 주 : 기준 전극은 자기 에어 테이블 스탠드에 부착 된 금속에 의해 유지되었다. </li><li> 기준 전극은 침대 벌레의 복부에 접속 된 후, 침대 벌레의 안테나의 후방 단부를 향해, ​​미세 조작기에 의해 전치 증폭기에 접속되고 조작되는 기록 전극을 이동. </li><li> 기록 전극은 안테나의 오른쪽 끝과 접촉 할 때, 현미경 켜고 저배율에서 전극을 찾아. </li><li> 전극과 더듬이 sensillum 모두 동일 평면에 있고 현미경으로 명확하게 표시 될 때까지 점차적으로 배율을 증가시키면서 기록 전극을 조정한다. 참고 :이 시간까지, 현미경 보통이다가장 높은 배율 LY. </li><li> 미세 조작기를 사용 sensillum의 샤프트에 기록 전극을 삽입하고 배경 잡음이 활동 전위에 비해 높은 경우 조금 더 깊게. </li><li> β - 피넨 - 일단 명확한 활동 전위가 기록 된 sensillum에서 관찰된다, 10 % (+)와 마이크로 피펫을 입력합니다. 유리 파스퇴르 피펫 내부에 배치 필터 종이 스트립 (~ 3 × 15mm)에 β-피넨 - 10 % (+)의 10 μL 나누어지는을 입금 마이크로 피펫을 사용합니다. <ol><li> 자극 컨트롤러의 펄스 흐름 튜브의 콘센트에로드 된 피펫을 연결하고 안테나 지향 튜브에있는 작은 구멍에 피펫의 팁을 배치합니다. </li></ol></li><li> 모든 연결이 안정되면, 연속 가습 공기 흐름에 자극 (/ 분 0.5 L)의 0.5 초 퍼프를 제공하기 위해 자극 컨트롤러의 풋 스위치를 우울. 활동 전위의 녹음 할 때 footswitc 동시에 시작됩니다시간은 우울하다. 기록 처리는 자극 전에 1 초부터 10 초 지난 것이다. </li><li> 이 500 밀리 초 기간 전에 하나는 자극 후 한 오프 라인 활동 전위를 계산합니다. 선행하는 500 밀리 초 동안 기록 운동량로부터 500 밀리 초 후의 자극 동안 스파이크 속도의 변화를 빼고 2별로 곱하여 스파이크 / (S)의 크기로 종래의 수를 변환한다. </li></ol> SSR 4. 자극 교체 <ol><li> 침대 벌레 안테나 상으로 피펫에 β-피넨과 피펫이 제거 된 후 10 초이 특정 취제에 응답하여 기록 - 풋 스위치가 트리거되면, 10 % (+)를 제공한다. </li><li> 0.001 %의 유칼 립톨와 다른 새로운 피펫 레이블을 테스트 할 수 있습니다. 자극의 10 μL가 새로운 피펫으로, 적용된 필터에 종이의 작은 조각을 놓습니다. </li><li> 자극이 완전히 vapori 때까지 2-5 분 동안 기다립니다유리 피펫에 데이빗. 펄스 흐름 튜브의 출구에 피펫을 연결합니다. </li><li> 안테나 지향 튜브의 작은 구멍에 피펫 팁을 삽입합니다. 풋 스위치를 누르고 전송 10 초 녹화를 시작합니다. </li><li> 피펫을 분리하고 0.01 %의 유칼 립톨와 다른 피펫을 준비합니다. </li><li> 용량 의존적 반응을 관찰하는 더듬이 sensilla에 유칼 립톨의 용량 (0.001 %에서 10 %)의 모든 나머지를 테스트합니다. 대부분의 시험은 희석 최소 용량으로 희석. </li></ol>

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The authors have nothing to disclose.

단일 Sensillum 녹화 기술은 광범위하게 이러한 환경에서 다른 화학적 자극에 과일 파리, 모기와 빈대 같은 곤충의 신경 반응을 테스트에 사용되어왔다. 이러한 화학적 자극에 종종 용해 치료의 상이한 투여 량을 준비하기 위해 공통의 용매로 희석된다. 그러나, 다른 용매는 자극을위한 상당히 다른 방출 속도를 생산할 수있다. 이러한 초파리 melanogaster의,과 아노 펠 레스 gambiae, <...

임시 기생충으로, 생존, 개발 및 재생이 인간과 동물 1, 2 모두를 포함하는 호스트의 혈액 소스를 필요로 의미 의무가 피를 빨아 먹는 곤충이다 : 일반적인 침대 버그 CIMEX는 L (Cimicidae 매미목)를 lectularius. 바이러스 전달은 거의 C. 인해보고되었지만 lectularius은 감염에 의해 발생 무는 성가신 심각한 신체적으로나 심리적으로 3 호스트에 영향을 미친다. 소개 및 화학 살충제의 광범위한 사용, 특히 DDT, 침략의 위험을 감소하고, 1950 년대 침략의 말에 더 이상 심각한 공공 우려했다 이러한 낮은 수준이었다. 그러나, 가능한 요인은 살충제의 사용 감소로, 전 세계적으로 침대 버그 인구에 부활하는 주도, 대중의 인식의 감소, 활동을 여행 증가, 살충제 4-9 저항의 개발. &lt;/ P&gt; 환경 화학 단서가 감지 및 안테나와 상악 palps으로 후각 기관을 통해 곤충에 의해 인식됩니다. 곤충 안테나에 후각 sensilla이 화학 신호를 검출하는 데 중요한 역할을한다. 화학 분자는 표피 표면에 구멍을 통해 더듬이 표피를 입력합니다. 취제이 화학 분자 더듬이 림프 바인드에 결합 단백질과 후각 수용체 (10)에 그들을 수송. 후각 수용체 이러한 화학 분자되면 탈 분극 될 신경 막에 비 선택적 양이온 이온 채널에서 자신의 공동 수용체는 후각 수용체 (11)에 의해 인식됩니다. 단일 sensillum 기록 (SSR)은 화학적 또는 비 화학 자극 중 하나의 애플리케이션에 의한 활동 전위에서 세포의 변화를 검출하기 위해 개발되었다. sensillum 림프 및 기준 전극에 기록 전극을 삽입하여곤충의 몸 (보통 화합물 눈 또는 복부 중 하나)의 다른 부분으로, 자극에 반응하여 신경 세포의 발사 속도는 12을 기록 할 수 있습니다. 스파이크의 수의 변화는 특정 자극에 대한 곤충의 감도를 나타낸다. 다른 ID 및 농도의 화학적 자극이 다른 연소 속도와 시간적 구조와 다른 신경 반응을 유발하며, 따라서 특정 화학 물질에 대한 곤충의 부호화 처리를 조사하는데 사용될 수있다. 구 홈 페그 C의 sensilla, 29 머리와 같은 E (E1 및 E2) sensilla, 한 쌍 Dα, Dβ, Dγ 부드러운 말뚝을 각각 : 일반 침대 버그, 모두 성적 형태의 안테나에 후각 sensilla의 동일한 패턴을 공유 sensilla (13, 14). 여러 뉴런 sensillum 각 유형에서 식별 된 바와 같이, 그것은, 동일한 sensillum 수납 다른 뉴런에서 활동 전위를 구별하는 것은 쉽지 않다 실험자는 tota 이처럼활동 전위의 L 번호는 이전과 자극 후 500 밀리 초 동안 오프라인 계수 하였다. 자극 후 활동 전위의 수는 전 자극 활동 전위의 수로부터 감산하고 15 초 당 스파이크 각 sensillum에서 소성 율의 변화를 정량화하기 위해 두 곱 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="1064">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:299px;">Tungsten wire</td>
			<td style="width:139px;">A-M SYSTEMS</td>
			<td style="width:117px;">#716500</td>
			<td style="width:511px;">Used for preparing the electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KNO2</td>
			<td>Sigma</td>
			<td>#310484</td>
			<td>Used for sharpening the tungsten wire</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AC Power Supply</td>
			<td>BK Precision</td>
			<td>1653A</td>
			<td>Providing the voltage in sharpening the tungsten wire</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Z6 APO Microscope</td>
			<td>Leica</td>
			<td>10447424</td>
			<td>Used for observing the sensilla on antennae</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Simulus controller</td>
			<td>Syntech</td>
			<td>CS-55</td>
			<td>Used for controlling the stimulus application</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Channel USB Acquisition Controller</td>
			<td>Syntech</td>
			<td>IDAC-4</td>
			<td>Real-time on screen display of all signals before and during recording</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Source</td>
			<td>SCHOTT</td>
			<td>A20500</td>
			<td>Providing light sources for observation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanupulator</td>
			<td>Leica</td>
			<td>115378</td>
			<td>Used for minor movement of electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Speaker</td>
			<td>Juster</td>
			<td>95a</td>
			<td>Connected with Acquisition Controller IDAC-4 and providing sound for the signal</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td>Used to hold the reference electrode, stablized bed bug and stimulus delivery tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMC Vibration Isolation Table</td>
			<td>TMC</td>
			<td>63-500</td>
			<td>Used for isolating the vibration from the equipments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverslip</td>
			<td>Tedpella</td>
			<td>2225-1</td>
			<td>Used for holding the bed bug</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double-sided Tape</td>
			<td>3M</td>
			<td>XT6110</td>
			<td>Used for stablizing the bed bug on the coverclip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dental Wax</td>
			<td>Dentakit</td>
			<td>DK-R012</td>
			<td>Used for supporting the coverclip where bed bug is stablized&#160;</td>
		</tr>
	</tbody>
</table>

using single sensillum recording to detect olfactory neuron responses of bed bugs to semiochemicals

곤충 후각 시스템은 환경에 semiochemicals 검출에 중요한 역할을한다. 특히, 내부에 하나 또는 여러 개의 뉴런을 수용 더듬이 sensilla는 화학적 자극에 반응에서 중요한 기여를 할 것으로 간주됩니다. 직접 자극에 노출 된 후 후각 sensillum에서 활동 전위를 기록함으로써, 단일 sensillum 레코딩 (SSR) 기술은 화학적 자극에 곤충의 신경 반응을 조사하기위한 강력한 방법을 제공합니다. 악명 높은 인간의 기생충 인 베드 버그를 들어, 후각 sensillum의 여러 유형이 특징으로하고있다. 이 연구에서 우리는 두 가지 화학 자극과 SSR 방법을 사용하여 그들 중 하나에 용량 의존적 반응에 침대 버그 후각 sensilla의 신경 반응을 보여 주었다. 이 방법은 (STABLE)에 대한 중요한 정보를 제공 할 것이다 침대 버그 후각의 sensilla 개별 화학적 자극, 조기을 조회 연구자 수새 침대 버그 유인 또는 방충제 및 혜택 침대 버그 제어 노력의 opment.

단일 sensillum 녹음 곤충 화학 생태학 및 신경 생리학의 연구에 사용되는 강력한 수사 기법이다. 다른 휘발성 화합물, 생각, 특히에 곤충의 신경 반응을 조사하는 곤충의 생존과 발전에 생태 학적으로 관련 될뿐만 아니라, 우리에게 곤충의 후각 과정에 귀중한 통찰력을 제공 할뿐만 아니라 잠재적으로 이어질 수있는 유망한 새로운 길을 열어 해충 제어를위한 유용한 새로운...

Watch this Scientific Journal Video about Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals at JoVE.com

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

Semiochemicals에 침대 버그의 후각 신경 세포 응답을 감지하기 위해 단일 ​​Sensillum 기록을 사용하여

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

using-single-sensillum-recording-to-detect-olfactory-neuron-responses

Research

JoVE Journal

Neuroscience

9.8K 조회수.  Auburn University. Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

비디오: Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

곤충의 단일 Sensillum 레코딩 Drosophila melanogaster과 아노 펠 레스 gambiae</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

연구

신경과학

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

에어 - 상 Electroolfactogram 기록을 사용하여 마우스 강한 감각 뉴런의 분석 응답

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

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 ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

아버지의 응답의 신경 생물학을 맡은 이후로 비교 종의 접근법을 사용

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (<em&gt; Schistocerca 아메리카나</em&gt;) 강한 회로

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

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

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

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

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

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

인간의 후각 기능을 연구하기위한 자유 호흡 fMRI 방법

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Semiochemicals에 침대 버그의 후각 신경 세포 응답을 감지하기 위해 단일 ​​Sensillum 기록을 사용하여

Semiochemicals에 침대 버그의 후각 신경 세포 응답을 감지하기 위해 단일 Sensillum 기록을 사용하여