この研究は、NIHの助成金NS047073とNSFの助成DMS1010434によってサポートされていました。

<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>

特別な利害関係は宣言されません。

前の仕事は無傷動物で、そのような隔離された神経節などの還元剤、でアメフラシモータープログラムを特徴付けている。個々のニューロンのレコーディングは5を取得してきたが無傷の動物では、このような実験は非常に困難であり、電極が授乳中のニューロンへのニューロンから移動することはできません。隔離された神経は、神経活動によって誘発される摂食の?...

<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

多機能、複数の、そして、異なった行動を生成するには1つの周辺構造の能力は、動物が急速に変化する環境に自分の行動を適応することができます。海洋軟体動物アメフラシカリフォルニは多面的機能の研究のための扱いやすいシステムを提供します。授乳中は、 アメフラシは同じ送り装置、頬質量を用いて行動のいくつかの異なったタイプを生成します。これらの行動を制御する神経節の電気生理学的研究からアクセス可能な、大規模で識別されたニューロンの数を含んでいます。これらのニューロンの活性がprotracts縮みグラスパー2その神経活動に対する相対食品グラスパーを閉じ神経活動のタイミングに基づいて2種類、ingestiveとegestiveプログラム、に分けることができ、モータのプログラムに記載されている。しかし、単離された神経節で、これらの行動を生み出すであろう筋肉の動きは、それを作って、欠席している観測されたモータのプログラムは、実際の行動の相関があるかどうか。 生体 、神経と筋肉のレコーディングでは、特定することが難しいが2,3,4給餌プログラムに対応して得られますが、それは直接個々のニューロン5から録音することは非常に困難であるされています。さらに、in vivoで 、ingestiveプログラムは、さらに刺され、ツバメの1,2、最も以前にin vitro標本で説明で行うことが困難である区別に分けることができます。 中断頬大量調製（ 図1）は、孤立した核と無傷の動物の間のギャップを埋めます。この準備として、ingestive行動-かむと嚥下の両方を含む-とegestive行動（拒否）は、個々の神経細胞は、細胞外から記録電極6を使用して刺激することができると同時に、誘発することができます。これらの異なる動作に関連付けられている餌の動きがrecorすることができますDEDに、定量化、およびモータのプログラムに直接関係する。中断頬大量調製におけるモータープログラムは孤立核において誘発運動プログラムよりもin vivoで観察されたものにもっと似ているように見える。したがって、この準備のモータのプログラムは、より直接的に生体内挙動にに関連することができます。同時に、個々の神経細胞は無傷の動物よりも記録と刺激によりアクセス可能です。また、孤立した核と無傷の動物との間に中間のステップとして、中断頬塊から得られた知見は、より減少し、より完全な両方の設定で得られたデータの解釈を助けることができます。中断頬マスの準備はアメフラシにおける多面的機能の神経制御を特徴づけるための便利なツールです。

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...

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

我々は細胞外記録し、神経、筋肉、および個々の識別されたニューロンから刺激するテクニックを説明<em&gt; in vitroで</em&gt;の送り装置では摂食行動の異なったタイプを引き出すと観察しながら、<em&gt;アメフラシ</em&gt;。

アン<em&gt; in vitroで</em生理的動きと送りモータプログラムを引き出すと録音用&gt;準備<em&gt;アメフラシカリフォルニ</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

Research

JoVE Journal

Neuroscience

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

10.5K ビュー.  Case Western Reserve University. 我々は細胞外記録し、神経、筋肉、および個々の識別されたニューロンから刺激するテクニックを説明 in vitroでの送り装置では摂食行動の異なったタイプを引き出すと観察しながら、アメフラシ。

ビデオ: 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.

の電極の作製と注入アメフラシカリフォルニ無傷、自由に振る舞う動物における多チャンネル神経と筋肉の録音のための

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

神経科学

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.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

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

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.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</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.

げっ歯類におけるランニングホイール活動の概日リズムの記録および分析

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.

体外における筋モータープールのために運動ニューロンの識別<em&gt;アメフラシカリフォルニ</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.

の感覚ニューロンの分離<em&gt;アメフラシカリフォルニ</em&gt;グルタミン酸作動電流のパッチクランプ記録のために

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.

幼虫給電回路の機能解析ショウジョウバエ</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.

の記録細胞内の電圧応答のための電気生理学的手法<em&gt;ショウジョウバエ</em&gt;光刺激に感光体と介在ニューロン<em&gt;インビボ</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.

大人のホールマウントの準備のためのシンプルなワンステップ解剖プロトコル<em&gt;ショウジョウバエ</em&gt;ブレインズ

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

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.

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

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