이 연구는 NIH 보조금 NS047073 및 NSF 보조금 DMS1010434에 의해 지원되었다.

<ol>
	<li>Neustadter, D. M., Herman, R. L., Drushel, R. F., Chestek, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinematics+of+multifunctionality:+comparisons+of+biting+and+swallowing+in+Aplysia+californica.">The kinematics of multifunctionality: comparisons of biting and swallowing in Aplysia californica.</a> J. Exp. Biol. 210, 238-260 (2007).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+buccal+nerve+activity+that+distinguishes+ingestion+from+rejection+can+be+used+to+predict+behavioral+transitions+in+Aplysia.">In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia.</a> J. Comp. Physiol. A. 172, 17-32 (1993).</li><li>Hurwitz, I., Neustadter, D., Morton, D. W., Chiel, H. J., Susswein, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+patterns+of+the+B31/B32+pattern+initiators+innervating+the+I2+muscle+of+the+buccal+mass+during+normal+feeding+movements+in+Aplysia+californica.">Activity patterns of the B31/B32 pattern initiators innervating the I2 muscle of the buccal mass during normal feeding movements in Aplysia californica.</a> J. Neurophys. 75, 1309-1326 (1996).</li><li>Cullins, M. J., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrode+fabrication+and+implantation+in+Aplysia+californica+for+multi-channel+neural+and+muscular+recordings+in+intact,+freely+behaving+animals.">Electrode fabrication and implantation in Aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals.</a> J. Vis. Exp. (40), e1791 (2010).</li><li>Warman, E. N., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+chronic+single+extracellular+recording+in+freely+behaving+animals+using+pipette+electrodes.">A new technique for chronic single extracellular recording in freely behaving animals using pipette electrodes.</a> J. Neurosci. Methods. 57, 161-169 (1995).</li><li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extracellular+stimulation+of+individual+neurons+in+ganglia.">Selective extracellular stimulation of individual neurons in ganglia.</a> J. Neural Eng. 5, 287-309 (2008).</li><li>Church, P. J., Lloyd, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+diverse+neuropeptide+cotransmitters+by+identified+motor+neurons+in+Aplysia.">Expression of diverse neuropeptide cotransmitters by identified motor neurons in Aplysia.</a> J. Neurosci. 11, 618-625 (1991).</li><li>Church, P. J., Lloyd, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+multiple+identified+motor+neurons+recorded+intracellularly+during+evoked+feedinglike+motor+programs+in+Aplysia.">Activity of multiple identified motor neurons recorded intracellularly during evoked feedinglike motor programs in Aplysia.</a> J. Neurophys. 72, 1794-1809 (1994).</li><li>Nargeot, R. N., Baxter, D. A., Byrne, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contingent-dependent+enhancement+of+rhythmic+motor+patterns:+an+in+vitro+analog+of+operant+conditioning.">Contingent-dependent enhancement of rhythmic motor patterns: an in vitro analog of operant conditioning.</a> J. Neurosci. 17, 8093-8105 (1997).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Behavioral biology of Aplysia. , (1979).</li><li>Scott, M. L., Govind, C. K., Kirk, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+organization+of+the+buccal+system+in+Aplysia+californica.">Neuromuscular organization of the buccal system in Aplysia californica.</a> J. Comp. Neurol. 312, 207-222 (1991).</li><li>Susswein, A. J., Rosen, S. C., Gapon, S., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+buccal+motor+programs+elicited+by+a+cholinergic+agonist+applied+to+the+cerebral+ganglion+of+Aplysia+californica.">Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica.</a> J. Comp. Physiol. A. 179, 509-524 (1996).</li><li>Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+behavior+in+Aplysia:+A+simple+system+for+the+study+of+motivation.">Feeding behavior in Aplysia: A simple system for the study of motivation.</a> Behav. Biol. 10, 1-26 (1974).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+timing+of+activity+in+motor+neurons+that+produce+radula+movements+distinguishes+ingestion+from+rejection+in+Aplysia.">The timing of activity in motor neurons that produce radula movements distinguishes ingestion from rejection in Aplysia.</a> J. Comp. Physiol. A. 173, 519-536 (1993).</li><li>Weiss, K. R., Chiel, H. J., Koch, U., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+an+identified+histaminergic+neuron,+and+its+possible+role+in+arousal+of+feeding+behavior+in+semi-intact+Aplysia.">Activity of an identified histaminergic neuron, and its possible role in arousal of feeding behavior in semi-intact Aplysia.</a> J. Neurosci. 6, 2403-2415 (1986).</li><li>Jing, J., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+variants+of+a+motor+act+in+a+modular+and+hierarchical+motor+network.">Generation of variants of a motor act in a modular and hierarchical motor network.</a> Curr. Biol. 15, 1712-1721 (2005).</li><li>Jing, J., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+motor+program+switching+in+Aplysia.">Neural mechanisms of motor program switching in Aplysia.</a> J. Neurosci. 21, 7349-7362 (2001).</li><li>Morgan, P. T., Jing, J., Vilim, F. S., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneuronal+and+peptidergic+control+of+motor+pattern+switching+in+Aplysia.">Interneuronal and peptidergic control of motor pattern switching in Aplysia.</a> J. Neurophysiol. 87, 49-61 (2002).</li><li>Jing, J., Cropper, E. C., Hurwitz, I., Weiss, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+construction+of+movement+with+behavior-specific+and+behavior-independent+modules.">The construction of movement with behavior-specific and behavior-independent modules.</a> J. Neurosci. 24, 6315-6325 (2004).</li></ol>

관심 없음 충돌이 선언 없습니다.

이전 작업은 그대로 동물과 같은 고립 된 신경절 등의 감소 준비에서 Aplysia 모터 프로그램을 특징으로하고 있습니다. 개별 뉴런의 녹음은 5를 획득 한하지만 그대로 동물에서 같은 실험은 매우 어려운이며, 전극은 먹이를하는 동안 신경 세포로 신경 세포에서 이동할 수 없습니다. 절연 신경절에서 신경 활동에 의해 유도 먹이 움직임이 관찰 할 수 없습니다. 정지 구강 대량 준비는이 두 극단 사이의 간격을 다리. 다른 반 그대로 준비가 이전 연구에서 사용하지만, 정지 구강 대량 준비 상당한 장점이있다. 한 반 그대로 준비에서 입술과 구강 질량은 여전히 첨부되었지만, 준비도 14 관찰 할 전체 먹이의 움직임에 감소했다. 체외의 움직임을 먹이의 먹이 헤드 준비 15,16 허용 관찰. 그러나, 일머리 준비를 공급하는 것은 중지 구강 대량 여기서 설명한 것보다 설치가 더 복잡했고, 만 구강 신경절에서, 대뇌 신경의 개별 뉴런에 액세스 할 수 없습니다 제공됩니다. 먹이 헤드 준비에 장점이있다, 그의 입술과 앞 촉수의 존재는 무는 패턴의 생성은 김의 응용, 천연 자극을 통해 허용됩니다. 이 실험을 더 어렵게 만들지 것이지만, 개별 뉴런에 대한 액세스 구강 신경절을 달아 동안 구강 질량에 부착 입술을 남겨두고 준비를 결합 할 수도 있습니다. Aplysia의 먹이 패턴이 모터 프로그램의 길게 끌기과 후퇴 단계에 비해 음식 움켜 잡 기사를 종료 radular 신경의 활동의 타이밍에 따라 ingestive 또는 egestive으로 분류 할 수 있습니다. radular 신경 활동이 후퇴 단계와 동시에 발생하는 경우, 패턴은 ingesti 아르VE, 그것은 구강 캐비티에 음식을 끌어하므로 움켜 잡 기사는 폐쇄하고 retracting 때문입니다. radular 신경 활동이 길게 끌기 단계에서 발생하는 경우 움켜 잡 기사는 폐쇄하고 구강 캐비티 (2)의 자료를 밀어 protracting 때문에, 패턴, egestive 있습니다. , 무는 음식을 파악하려고하고, 삼키거나, 이미 파악 식품 1,2의 교통 : Ingestive 패턴은 적어도 두 개의 서브 타입 구분으로 나눌 수 있습니다. 그러나, 체외 작업 (예 : 14,17,18)의 대부분은 삼키는에서 무는 구별하려고 시도하지 만 섭취 / egestion 이분법에 초점을 맞추고있다. 두 연구 16,19가 무는 같은과 삼키는 같은 그룹에서 체외 ingestive 패턴에 분류했습니다. 이러한 연구의 첫 번째에서 패턴은 절연 신경절에서 관찰 된, 구강 질량의 관련 먹이 움직임을 관찰 할 수 있다면,이 인수를 강화 겠어요SE 패턴 물고 및 생체에서 볼 삼키는를 나타냅니다. 두 번째 연구에서는 패턴이 먹이 헤드 준비에서 관찰되었다, 그래서 패턴이 관찰 움직임에 따라 분류되었지만, 구강 신경절의 뉴런의 더 녹음 얻을 수 없습니다. , 물고 삼키는 및 거부 - - 정지 구강 대량 준비 행동의 세 가지 유형을 관찰 할 수있는 방법을 제공 동시에 주요 구강 신경과 근육의 활동을 기록, 개인 뉴런에서 자극 및 녹음을 모두 동안. elicited 강한 먹이와 같은 움직임이 거의 확실 세포 전극를 제거하기 때문에 세포 뉴런 기술 6이 준비 다른 키 사전입니다. 이 단독으로 신경 녹음과 어려운 것 때 여러 단위가 F 때문에 개별 뉴런의 세포 녹음으로, 모터 프로그램 동안 이러한 뉴런의 활동의 타이밍은 ascertained 수 있습니다동시에 iring. 우리는 정지 구강 대량의 먹이 움직임이 생체에서 본 것과 질적으로 유사하게 나타납니다 것이 관찰되었습니다. 중요한 자극은 (carbachol 각각 구강 신경이 지점 자극)보다 감소 준비에 사용, 우리의 자극은 생리 움직임을 생성하는 찾는 일은 다른 설정에서의 사용에 신뢰성을 대여 한 무는 및 거부 패턴을 생성하기 위해 사용됩니다. 대조적으로, 삼키는 패턴을 생성하기 위해, 우리는 격리 된 신경절에 적용 할 수없는 자연 식품 자극 (김 스트립)와 인공 자극 (carbachol)을 결합. 우리는 또한 중지 구강 대량의 신경 패턴이 감각 피드백 CPG에서 패턴을 형성에 중요 할 수 있습니다 제안, 더 감소 준비에서 얻은 패턴보다 생체 패턴에 더 유사한 것으로 나타 관찰했다. 절연 갱의 예를 들어, 모터는 프로그램정지 구강 대량의 모터 프로그램은 일반적으로 손상 동물 (게시되지 않은 데이터)의 모터 프로그램에 기간이 더 가까워지는 반면 리아는 그대로 동​​물의 모터 프로그램보다 기간에 여러 번 더 일반적입니다. 중지 된 구강 대량의 또 다른 장점은 동시 측 있으며, 먹이 장치의 전경을 얻을 수 있습니다. 턱 근육 수축을 더 잘 관찰을 허용하면서 정면은 생체에서 볼 수있는 것입니다 거기에 입과 비슷한 전망을 제공합니다. 측면 뷰는 쉽게 다른 행동을하는 동안 biomechanical 변경 사항을 이해하고, 구강 근육의 수축이 관찰하고 근육 아래에 움켜 잡 기사의 앞으로 및 뒤로 이동을 할 수 있습니다. 이 준비에서, 우리는 동시에 다섯 신경 (radular 신경과 양국 구강 신경 2, 3)과 I2 근육에에서 최대 기록했으며, 확인 두번 세포 전극까지 배치신경절의 신경 세포. 이 실험 원하는 경우, 추가 신경 및 / 또는 근육에서 기록하는 것이 가능해 질 것입니다. 또한 뇌성 구강 interneurons의 자극, 일반적으로 17,19가 생체 행동과 비슷한 모터 프로그램을 유도하는지에 대한 통찰력을 줄 수 대뇌 신경의 뉴런에서 자극하고 기록하는 것이 가능해 질 것입니다. 이렇게하면 준비의 기술적 어려움을 증가했지만, 구강 동맥 재관류 15는 준비가 강하고 오래 지속되는 행위 (게시되지 않은 관찰)을 생성하기 위해 허용 할 수 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S671</td>
			<td>Biological, Certified</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher Scientific</td>
			<td>P217</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Acros Organics</td>
			<td>19753</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientifc</td>
			<td>C79</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Glucose (dextrose)</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td>BioXtra</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Acros Organics</td>
			<td>17263</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Carbachol</td>
			<td>Acros Organics</td>
			<td>10824</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>SS255</td>
			<td>Certified</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>SA49</td>
			<td>Certified</td>
		</tr>
		<tr>
			<td>Single-barreled capillary glass</td>
			<td>A-M Systems</td>
			<td>6150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flaming-Brown micropipette puller model P-80/PC</td>
			<td>Sutter Instruments</td>
			<td>&nbsp;</td>
			<td>Filament used: FT345B</td>
		</tr>
		<tr>
			<td>Enamel coated stainless steel wire</td>
			<td>California Fine Wire</td>
			<td>&nbsp;</td>
			<td>0.001D, coating h</td>
		</tr>
		<tr>
			<td>Household Silicone II Glue</td>
			<td>GE</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Duro Quick-Gel superglue</td>
			<td>Henkel corp.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A-M Systems model 1700 amplifier</td>
			<td>A-M Systems</td>
			<td>&nbsp;</td>
			<td>Filter settings: 300-500 Hz nerves,10-500 Hz I2 muscle</td>
		</tr>
		<tr>
			<td>Pulsemaster Multi-Channel Stimulator</td>
			<td>World Precision Instruments</td>
			<td>A300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulus Isolator</td>
			<td>World Precision Instruments</td>
			<td>A360</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AxoGraph X</td>
			<td>AxoGraph Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Veeder-Root Totalizing Counter</td>
			<td>Danaher</td>
			<td>C342-0562</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gold Connector Pins</td>
			<td>Bulgin</td>
			<td>SA3148/1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gold Connector Sockets</td>
			<td>Bulgin</td>
			<td>SA3149/1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100 x 50 mm Crystalizing Dish</td>
			<td>Pyrex</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>21-375D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Modeling Clay</td>
			<td>Sargent Art</td>
			<td>22-4400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silk Sutures</td>
			<td>Ethicon</td>
			<td>K89OH</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Whisper Air Pump</td>
			<td>Tetra</td>
			<td>77849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aquarium Tubing</td>
			<td>Eheim</td>
			<td>7783</td>
			<td>12/16 mm</td>
		</tr>
		<tr>
			<td>Elite Airstone</td>
			<td>Hagen</td>
			<td>A962</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Yaki Sushi Nori Seaweed</td>
			<td>Rhee Bros</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro preparation for eliciting and recording feeding motor programs with physiological movements in aplysia californica

Multifunctionality, 여러, 독특한 행동 하나를 생성 한 주변 구조의 기능은 동물이 빠르게 환경을 변화하는 자신의 행동을 적응 할 수 있습니다. 해양 연체 동물 Aplysia californica는 multifunctionality 연구를위한 다루기 쉬운 시스템을 제공합니다. 수유하는 동안, Aplysia 같은 먹이 장치, 구강 대량를 사용하여 행동의 여러 독특한 종류를 생성합니다. 이러한 행동을 제어하는​​ 신경절은 electrophysiological 연구 액세스 할 수 있습니다 대형 식별 뉴런의 번호가 포함되어 있습니다. 이러한 뉴런의 활동은 신경 활동에 음식 움켜 잡 기사의 친척을 종료하는 신경 활동의 타이밍에 따라 두 가지 종류, ingestive 및 egestive 프로그램으로 나눌 수 있습니다 모터 프로그램에 설명 된 해당 protracts 또는 retracts 움켜 잡 기사 2. 그러나, 절연 신경절에서 이러한 행동을 생산 것입니다 근육 운동은하는, 결석 아르더 생체, 신경과 근육 레코딩의 프로그램에게 2,3,4를 공급하는 해당 획득했습니다. 관찰 모터 프로그램은 실제 행동의 상관 관계 여부를 확신 할 수 있지만, 직접 5 개의 개별 뉴런에서 기록하는 것은 매우 어렵습니다 수 있습니다. 또한, 생체에 ingestive 프로그램은 더 물린 상처와 제비의 1,2, 대부분의 이전에 체외 준비에 설명에 할 어려운 차이로 나눌 수 있습니다. 정지 구강 대량 준비 (그림 1) 절연 신경절과 그대로 동물 사이의 간격을 다리. 이 준비에는 ingestive 행동 - 물고과 삼키는 모두 포함 - 개별 뉴런이에서 녹화 및 세포 전극에게 6을 사용하여 자극 할 수 있으므로 및 egestive 행동 (거부)가 동시에 elicited 수 있습니다. 서로 다른 행동과 관련된 먹이 움직임이 recor 할 수 있습니다ded, 정량화 및 모터 프로그램에 직접 관련된. 중지 된 구강 대량 준비의 모터 프로그램은 모터 프로그램이 분리 된 신경절에 elicited하는 것보다 생체에서 관찰 것과 더 유사하게 나타납니다. 따라서이 준비 모터 프로그램은보다 직접적으로 생체 행동에 관한 할 수 있습니다, 같은 시간에, 개별 뉴런은 그대로 동물에 비해 녹음 및 자극에 더 액세스 할 수 있습니다. 또한,로 분리 신경절과 그대로 동​​물 사이의 중간 단계, 정지 구강 대량의 연구 결과는 더 감소 등 그대로 두 설정에서 얻은 데이터의 해석에 도움이 수 있습니다. 정지 구강 대량 준비 Aplysia의 multifunctionality의 신경 제어를 특징위한 유용한 도구입니다.

When an extracellular electrode is positioned above a neuron's soma and used to stimulate the neuron, a one-for-one correspondence between spikes on the soma channel and on the nerve(s) the neuron projects to can be observed (Figure 6, left panel, stimulation of identified neuron B9). The soma channel (top channel) is set to stimulating mode when the current is applied (time 1 in the figure), and is then quickly switched to recording mode (time 2). By maintaining the position of the electrode, the activity of the neuron can be recorded during motor programs (Figure 6, right panel), along with activity from the I2 muscle, radular nerve, and bilateral buccal nerves 2 and 3.
Figure 7 shows video images taken from a series of swallowing patterns induced by application of carbachol to the cerebral ganglion and the subsequent placement of a seaweed strip into the grasper during carbachol-induced biting. In 7A, the radula is protracted to grasp the seaweed, and in 7B, the radula has retracted, pulling seaweed into the mouth. Videos and electrophysiological recordings can be directly synchronized to each other; the still images in Figure 7 correspond to times A and B indicated in Figure 6's right panel.
<img src="/files/ftp_upload/4320/4320fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/4320/4320fig1highres.jpg"/> 
Figure 1. Schematic of overall setup for experiment. The main image shows an overhead view. The cerebral ganglion and buccal ganglia are pinned to Sylgard. The cerebral ganglion is in an isolated chamber so that carbachol can be applied to it. The cerebral-buccal connectives pass through a notch in the Sylgard, which is sealed with vacuum grease. The buccal mass, in the front chamber, is suspended by the buccal nerves from its posterior and by a silk suture at its anterior edge. An inset shows a side view of the setup, including an extracellular electrode positioned above the buccal ganglion. The entire structure within the dish is constructed from Sylgard. Dimensions indicated within the inset are: a. Length of notch connecting cerebral and buccal chambers, 0.5 cm. b. Width of buccal ganglia platform, 0.5 cm. c. Height of buccal mass chamber from bottom to buccal ganglia platform, 1.7 cm. Note that the height of the buccal mass chamber can be adjusted, depending on the size of the buccal mass, by placing a small platform made of Sylgard underneath the buccal mass.
<img src="/files/ftp_upload/4320/4320fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/4320/4320fig2highres.jpg"/> 
Figure 2. Schematic of the Aplysia feeding apparatus, the buccal mass, showing the locations of key structures, including buccal nerves (BN) 1, 2, and 3, the radular nerve (RN), and the I2 muscle, and the attachment points for hook electrodes. The radular nerve projects ventrally from the rostral surface of the buccal ganglia and goes beneath the surface fibers of the I2 muscle. Buccal nerve 2 trifurcates into branches a, b, and c before going beneath the I1 muscle at the lateral groove. Branch a is the first branch to separate from the main trunk, and is adjacent to buccal nerve 3. The nomenclature of branches a, b, and c was used by Warman and Chiel5. Branches a, b, and c correspond to branches 3, 2, and 1, respectively, in the nomenclature of Nargeot et al.9 Furthermore, the RN, BN1, BN2, and BN3 correspond to nerves 1, 6, 5, and 4, respectively, in the nomenclature of Kandel10 and Scott et al.11
<img src="/files/ftp_upload/4320/4320fig3.jpg" alt="Figure 3"/> 
Figure 3. Attachment of hook electrode to nerve. The nerve is placed within the hook and lifted, and superglue is applied to the nerve and hook. The glue must completely cover the hook, and must not cover the end of the reference wire.
<img src="/files/ftp_upload/4320/4320fig4.jpg" alt="Figure 4"/> 
Figure 4. Setup of buccal ganglia, showing the arrangement of pins to position and secure the ganglia. Pins must be placed in the sheath surrounding the nerves and ganglia, being careful not to damage nerves or neurons. The left ganglion is shown as rotated forward to provide access to neurons that are proximate to the back chamber. To accomplish this rotation, excess sheath from the CBC is pulled forward and pinned between buccal nerves 2 and 3. Note that the caudal surface of the buccal ganglion (which is rounded, rather than flat) is schematically shown in the figure.
<img src="/files/ftp_upload/4320/4320fig5.jpg" alt="Figure 5"/> 
Figure 5. Positioning of extracellular electrodes over neuron somata in the buccal ganglion. Glass electrode tips should be gently pressed, using manipulators, into the thinned sheath above target cell bodies. Two electrodes can be simultaneously positioned above different cells, as pictured.
<img src="/files/ftp_upload/4320/4320fig6.jpg" alt="Figure 6" fo:content-width="5.5in" fo:src="/files/ftp_upload/4320/4320fig6highres.jpg"/> 
Figure 6. Example AxoGraph recordings. Channels are shown for one extracellular soma electrode (top channel) and (in sequence from the top) for hook electrodes on the I2 muscle, radular nerve, left buccal nerve 2, left buccal nerve 3, right buccal nerve 2, and right buccal nerve 3. At left, identification of a neuron by extracellular stimulation. The soma channel is used to apply a stimulating current (time 1, note stimulation artifact in all channels other than the soma channel, which is not recording), and is then switched to recording mode (time 2). Note the one-for-one correspondence between spikes on the soma channel and the BN2-L, BN3-L, and BN3-R channels. At right, recording of two cycles of feeding programs as the buccal mass swallows a strip of seaweed. Note that the activity of the neuron previously stimulated can be visualized on the soma channel (these spikes are highlighted by red bars above the soma channel). This is identified neuron B9 in the right buccal ganglion. Times A and B correspond to video frames shown in Figure 7. <a href="https://www.jove.com/files/ftp_upload/4320/4320fig6large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/4320/4320fig7.jpg" alt="Figure 7"/> 
Figure 7. Example video images corresponding to the recording shown in Figure 6. The suspended buccal mass is shown in a front view (left) and side view (right, a mirror positioned adjacent to the dish). A strip of seaweed has been placed in the mouth and the radula is in the process of swallowing the seaweed. A. The radula is protracted to grasp the seaweed. B. The radula has retracted, pulling seaweed into the mouth. Note that A and B correspond to the same times labeled A and B in Figure 6.

Watch this Scientific Journal Video about An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica at JoVE.com

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

우리는 extracellularly 기록하고 신경, 근육, 및 개인 식별 뉴런에서 자극 할 수있는 기술을 설명<em&gt; 체외에서</em&gt; 도출하도록 의도 및 공급 장치에서 동작을 공급 다른 종류의 관찰<em&gt; Aplysia</em&gt;.

<em&gt; 체외에서</em의 생리적 운동과 수유 모터 프로그램을 도출하도록 의도하고 녹음&gt; 준비<em&gt; Aplysia californica</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

an-vitro-preparation-for-eliciting-recording-feeding-motor-programs

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

10.5K 조회수.  Case Western Reserve University. 우리는 extracellularly 기록하고 신경, 근육, 및 개인 식별 뉴런에서 자극 할 수있는 기술을 설명 체외에서 도출하도록 의도 및 공급 장치에서 동작을 공급 다른 종류의 관찰 Aplysia.

비디오: An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

의 전극 제작 및 주입 Aplysia californica 손상, 자유롭게 행동 동물의 다중 채널 및 신경 근육질 레코딩을위한

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

연구

신경과학

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

신경 응답을 녹음하고 추적하는 주사에 대한 깨어 마우스의 작성

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

쥐에 활동 륜 실행에 녹음 및 Circadian 리듬 분석

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracellularly의 근육 모터 풀에 모터 뉴런을 확인<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

의 애벌레 먹이 회로의 기능적 분석 초파리</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

간단한 한 단계 성인의 전체 마운트 준비에 대한 해부 프로토콜<em&gt; 초파리</em&gt; 두뇌

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...