Name: juxtacellular monitoring and localization of single neurons within sub-cortical brain structures of alert, head-restrained rats
Uploaded: 2015-04-27T13:00:00
Description: Watch this Scientific Journal Video about Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats at JoVE.com

We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.

연방 정부가 규정 된 동물 관리 및 사용 지침에 따라 - (350 g을 250)와 캘리포니아 샌디에고 대학의 기관 동물 케어 및 사용위원회에 의해 승인되었습니다 실험 프로토콜은 여성 롱 에반스 쥐에서 수행되었다. 1. 신체 구속에 쥐 익숙해 질 참고 : 제한된 다이어트에 쥐를 놓습니다. 구속 (아래에서 설명)에 쥐를 적응 즉시 각 매일 처리 세션 후 하루에 한 번 ?...

<ol>
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실험 지그의 건설 이 다양한 방법으로 구성 될 수있는 지그 실험 (도 4)를 구축하는 데 사용되는 기계 부품의 설명은 생략 프로토콜. 이 데모 표준에서 광학 기계 부품 및 지원 클램프 헤드 구속 바, 신체 구속 튜브 (자료 섹션 참조) 마운트하는 데 사용됩니다. 유사 광학 기계식 전동 부를 미세 조작기에 전극 홀더를 장착하는데...

적극적으로 행동 작업에 종사하는 경고 동물에서 신경 세포의 활동을 모니터링 신경계의 기능과 조직을 이해하는 것이 중요합니다. 하나의 신경 세포 단위에서 전기 활동의 세포 외 녹음이 긴 시스템 신경 과학의 주식 도구 왔으며 현재 광범위하게 사용되고 여전히있다. 전극의 종류와 구성의 다양 한 특정 실험의 과학적, 기술적 요구에 따라 사용할 수 있습니다. 만성 또는 마이크로 드라이브를 주입 전극 배열은 종종 조류, 설치류 및 영장류를 포함 1-4 자유롭게 움직이는 동물에서 사용된다. 또한, 외부 미세 조작기를 통해 금속 또는 유리 미세 전극 급성 관통 자주 마취 또는 머리를 억제 동물에서 기록하는 데 사용됩니다. 유리 마이크로 피펫 전극들이 사용될 juxtacellular 또는 &quot;세포 부착&quot;될 수있는 이점이 명백하게 구성을 분리5 정렬 사후 스파이크의 합병증없이 단일 뉴런의 활동. 이들이 염료 또는 신경 해부학 트레이서 작은 예금을 주입하는, 또는 심지어는 개개의 셀을 기록 채우기 위해 사용될 수있다 이들 전극은 또한, - 식별 된 해부학 세포 또는 위치에서의 기록을 허용한다. 이 구성은 성공적으로 쥐, 마우스, 조류 6-10에 적용되었습니다. 현재 기술 기술은 juxtacellular 모니터링 및 경고에 ​​세포 외 색소 침착, 머리 억제 쥐에 초점을 맞추고있다. 이러한 염료 보증금은 세포 형태 또는 축삭 돌기 (11)에 대한 정보를 제공하지 않는 채 웁니다 juxtacellular는 달리 하나의 셀을 참고하지만 그들은 경고 동물에서 상당히 높은 수율을 가지고 비판적으로, 약 50 μm의 정확한 해부학 적 위치 파악을 가능하게합니다. 그럼에도 불구하고 해부학 라벨링을위한 대안 전략으로 제공됩니다 채워 juxtacellular 단일 셀에 대한 정보. 요컨대,프로토콜은 세 가지 주요 단계로 구성됩니다. 첫 번째 단계에서, 래트 6 일의 기간에 걸쳐 천 깔창 (도 1) 신체 구속에 순응된다. 두 번째 단계에서, 헤드 레스트 장치 (도 2) 기록 챔버 수술 래트에 여러 후속 기록 회의 (도 3) 중에 정위 평면에서 유지 될 수 있도록 주입; 이 절차는 표준 참조 (12) 좌표를 기반으로 전기 생리학 연구를 위해 뇌의 특정 하위 대뇌 피질의 영역을 대상으로 실험을 할 수 있습니다. 세 번째 단계는 명백하게 하나의 단위로 6-9을 분리 juxtacellular 신경 녹음을, (그림 5) 석영 모세관 튜브에서 전극을 구성, 행동 적, 전기 생리 실험 (그림 4)를 수행하기에 적합한 지그에 쥐를 배치 포함하고, 해부학 locati 마킹시카고 스카이 블루 염료 (도 6 및도 7)와 기록 사이트에서. 녹음 동시 행동 모니터링을 수행; 그러나, 행동의 기술적 인 세부 사항은 각 실험의 과학적 목표에 따라 달라집니다 단일 프로토콜의 범위를 벗어 포함된다. 여러 일에 반복 될 수 실험 절차의 종료 후, 동물은 안락사된다. 뇌는 시야 또는 형광 현미경을 사용하여 표준 신경 해부학 적 기술에 따라 추출 및 처리됩니다.

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			<td height="22" style="height:22px;width:191px;">Name of the Reagent</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Ketaset (Ketamine HCl)</td>
			<td style="width:179px;">Fort Dodge&#160;</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
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			<td height="21" style="height:21px;width:191px;">Anased (Xylazine solution)</td>
			<td style="width:179px;">Lloyd Laboratories</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Betadyne (Povidone-Iodine)</td>
			<td style="width:179px;">CVS Pharmacy</td>
			<td align="right" style="width:175px;">269281</td>
			<td style="width:315px;"></td>
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			<td height="21" style="height:21px;width:191px;">Loctite 495</td>
			<td style="width:179px;">Grainger Industrial Supply</td>
			<td style="width:175px;">4KL86</td>
			<td style="width:315px;">20-40 cp cyanoacrylate</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Vetbond</td>
			<td style="width:179px;">3M</td>
			<td style="width:175px;">1469SB</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement powder</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675571</td>
			<td style="width:315px;">For the base of the recording chamber</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement liquid</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675572</td>
			<td style="width:315px;">For the base of the recording chamber</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Silicone Gel</td>
			<td style="width:179px;">Dow Corning</td>
			<td align="right" style="width:175px;">Mar-80</td>
			<td style="width:315px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Jet denture repair acrylic powder</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Ortho-Jet Fast curing orthodontic acrylic resin liquid</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Chicago sky blue</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C8679</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma</td>
			<td align="right" style="width:175px;">158127</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Phosphate-buffered saline</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">P3813</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cytochrome C</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C2506</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Diaminobenzidine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">D5905</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Material Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Rat sock</td>
			<td style="width:179px;">Sew Elegant (San Diego, CA)</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 1, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">PVC tube 2 &#189;&#8221;</td>
			<td style="width:179px;">U.S. Plastic Co.</td>
			<td align="right" style="width:175px;">34108</td>
			<td style="width:315px;">Figure 4</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Subminiature D pins &#38; sockets</td>
			<td style="width:179px;">TE Connectivity</td>
			<td style="width:175px;">205089-1</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel music wire 0.010&#8221; diameter</td>
			<td style="width:179px;">Precision Brand Products, Inc.</td>
			<td align="right" style="width:175px;">21010</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic holding frame</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 900</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic ear bars</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 957</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic manipulator</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 960</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">&#189; mm drill burr</td>
			<td style="width:179px;">Henry Schein</td>
			<td style="width:175px;">100-3995</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quiet-Air dental drill&#160;</td>
			<td style="width:179px;">Midwest Dental</td>
			<td style="width:175px;">393-1600</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel 0-80 1/8&#8221; screw</td>
			<td style="width:179px;">Fastener superstore</td>
			<td align="right" style="width:175px;">247438</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">0.2mL centrifuge tube</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:175px;">05-407-8A</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Custom head-holding bar</td>
			<td style="width:179px;">UCSD SIO Machine Shop</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 2, 3, 4</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:191px;">Custom head-holding plate</td>
			<td rowspan="2" style="width:179px;">UCSD SIO Machine Shop</td>
			<td rowspan="2" style="width:175px;">N/A</td>
			<td rowspan="2" style="width:315px;">Custom made, Figure 2, 3, 4</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Right angle post-clamp</td>
			<td style="width:179px;">Newport</td>
			<td style="width:175px;">MCA-1</td>
			<td style="width:315px;">Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">8-32 3/4&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">240181</td>
			<td style="width:315px;">For head-restraint, Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">4-40 &#188;&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">239958</td>
			<td style="width:315px;">For head restraint, Figures 3, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quartz capillary tubing</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">QF-100-60-10</td>
			<td style="width:315px;">Figure 5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Carbon dioxide laser puller</td>
			<td style="width:179px;">Sutter instruments</td>
			<td style="width:175px;">P-2000</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Motorized micromanipulator</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">MP-285</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">Multiclamp 700B</td>
			<td style="width:315px;">Alternate part: Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier head stage</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">CV-7B</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Isolated pulse stimulator</td>
			<td style="width:179px;">A-M Systems</td>
			<td style="width:175px;">Model 2100</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Audio monitor</td>
			<td style="width:179px;">Radio Shack</td>
			<td style="width:175px;">32-2040</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:191px;">Pipette holder</td>
			<td rowspan="2" style="width:179px;">Warner Instruments</td>
			<td rowspan="2" style="width:175px;">#MEW-F10T</td>
			<td style="width:315px;">Alternate parts: see Discussion</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Figure 6A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Electrode lead wire</td>
			<td style="width:179px;">Cooner wire</td>
			<td style="width:175px;">NEF34-1646</td>
			<td style="width:315px;">(optional), Figure 6D</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Relay for amplifier head-stage</td>
			<td style="width:179px;">COTO Technology</td>
			<td style="width:175px;">#2342-05-000</td>
			<td style="width:315px;">(optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Digital video camera</td>
			<td style="width:179px;">Basler</td>
			<td style="width:175px;">A602fm</td>
			<td style="width:315px;">(optional) For behavioral monitoring, Figure 7</td>
		</tr>
	</tbody>
</table>

juxtacellular monitoring and localization of single neurons within sub-cortical brain structures of alert, head-restrained rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

<html> 쥐가 적극적으로 쫓기대로 vibrissa 운동의 위상 자체 생성이 15, 16 쫓기 동안 인코딩 시상 복부 후방 내측 (VPM)의 신경 단위. 그림 7a는 VPM 시상 단위의 샘플 스파이크 활동을 보여줍니다. 그림 7b는 스파이크의 히스토그램을 보여줍니다 시간 vibrissa 운동 (17)의 순시 위상 정렬. 쫓기의 후퇴 단계에서 더 스파이크가 있습니다. 도 7c에 도시 된 ?...

Watch this Scientific Journal Video about Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats at JoVE.com

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats (Video) | JoVE

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

경고, 헤드 억제 쥐의 하위 대뇌 피질의 뇌 구조 내에서 Juxtacellular 모니터링 및 현지화 단일의 뉴런

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Research

JoVE Journal

Neuroscience

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

Single-cell Profiling of Developing and Mature Retinal Neurons

Single-cell Profiling of Developing and Mature Retinal Neurons (Video) | JoVE

개발하고 성숙한 망막 신경 세포의 단일 세포 프로 파일링

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

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신경과학

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury (Video) | JoVE

외상성 뇌 손상 후 뉴런 또는 유전자 발현 분석을위한 단일 뉴런의 강화 인구의 레이저 캡처 Microdissection

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method (Video) | JoVE

골지 - 콕스 방법을 사용하여 두꺼운 뇌 섹션 내에서 이미징 뉴런

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃŽÂ²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain (Video) | JoVE

아 밀 로이드-β42 독성 그리고 Zebrafish 두뇌에서 Neurodegeneration 모델링

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial Electrical Brain Stimulation in Alert Rodents (Video) | JoVE

경고 설치류에 Transcranial 전기 두뇌 자극

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains (Video) | JoVE

절연 및 애벌레 Zebrafish 두뇌에서 신경 세포, 대 식 세포 및 Microglia RNA 추출

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Localization of the Locus Coeruleus in the Mouse Brain

Localization of the Locus Coeruleus in the Mouse Brain (Video) | JoVE

쥐의 뇌에서 로커 스 Coeruleus의 지역화

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury (Video) | JoVE

머리 맡, 단일 버 구멍 접근 Multimodality 심각한 뇌 손상에서 모니터링

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Three-Dimensional Shape Modeling and Analysis of Brain Structures (Video) | JoVE

뇌 구조의 3차원 형상 모델링 및 분석

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips (Video) | JoVE

사전 조립된 플라스틱 미세유체 칩 내에서 인간 줄기 세포 유래 뉴런의 구획화

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...