저자는 전문 기술 지원을 다 Roderigo을 감사드립니다. 우리는 비디오 제작에 도움을 S. 쾰러 (고급 이미징 센터, 하인리히 하이네 대학 뒤셀도르프를) 감사합니다. (MC에) R01NS032123를 부여합니다 (롬 2327 / 8-1 CRR에 DFG), (NH까지) 하인리히 하이네 대학 뒤셀도르프와 건강의 국립 연구소에 의해 저자의 실험실에서 연구는 독일 연구 협회에 의해 투자되고있다.

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The authors declare no competing interests.

액체 캐리어 기반이, 이온 선택성 전극이 성공적으로 수십 년 동안 많은 이온을 위해 사용되어왔다, 매우 구체적인 센서는 22 ~ 26 사용할 수 있습니다. ECS의 폭은 단지 약 20 내지 50 nm 인, 선택적 이온의 직경 인 동안 : 척추 뇌 제제의 세포 외 공간 (ECS)에서 사용되는 경우, 하나의 문제는 상당히 침습적 방법이 있다는 것이 유념해야 미소 전극은 약 1 ㎛ (더블 질주 전극) 이상 (동심 전극)입니다. 이온 선택성 미소 전극의 끝은 이렇게뿐만 아니라 조직의 자신의 꿰 뚫기 동안 조직을 손상시킬뿐만 아니라, 이온 과도의 과소 평가를 선호, ECS을 확대 할 것이다. 이 함정에도 불구하고, 신경 세포의 활동에 응답 세포 외 이온 과도은이 방법의 신뢰성을 입증 다른 실험실 7, 8, 사이 현저하게 일치한다. 이온 선택성 전극의 성능 및 적합성사용되는 센서 칵테일 ( &#39;액체 막 이온 운반체&#39;)에 의해 정의되는 자신의 감도와 선택도에 따라 달라집니다. 센서 칵테일 칼륨 (27)에 대한 높은 선택성을 나타내는 K + -selective 미소 전극을 위해 발리 노마 이신 (Valinomycin) 예를 들어, 특별한 캐리어 분자가 포함되어 있습니다. 에도 불구하고, 다른 이온과 교차 반응이 발생할 수 있으며, 테스트해야합니다. 발리 노마 이신 (Valinomycin)는 결과를 해석 할 때 고려되어야 암모늄 대한 상당한 교차 반응 (예, 11, 12)를 나타낸다. 이오 노 포어의 전압 응답 Nernstian 동작 (참조, 수학 식 1)을 따르기 때문에 또한, 신호 대 잡음비와 검출 임계 값이 측정되는 이온의 농도에 의존한다. 따라서, 동안 작은 [K +] 오 과도 과도 훨씬 더 어려운 인사에 대해 감지 할 수있는 O를 낮은 기준 [K +] 오, 작은 [나 +]에 대한 큰 전압 변화를 보여주고GH 기준 [나 +] O (참조 그림 5, 6). 이온 선택성 전극의 성능은 대부분의 전기 시정이 적용됩니다 시간적 해상도에 의해 결정된다. 후자는 주로 센서의 축 방향의 저항에 의해 결정되고, 내부 용액 및 외부 사이의 유체 피펫의 길이를 따라 분산 커패시턴스에 의해된다. 이중 목적 구성에서, 저항은 백필 이온 센서의 컬럼 길이로 인해 높다. (이 경우 붕규산 유리) 지정된 절연 유전체의 경우, 용량은 유전체 두께의 적용을받습니다. 이중 목적의 전극에서, 유전체층의 폭은 피펫의 유리 벽에 달한다. 유리 선단 부근 얇아 같이, 유전체 폭이 떨어지는 및 용량이 증가한다. 이러한 요인은 수백 밀리 초에서 몇 범위 응답 시간 전극을 생성하기 위해 결합초, 이러한 요인은 다양하다. 동심 설계의 주요 장점은 축 및 저항 욕 용량 모두 크게 감소된다는 것이다. 팁 전에 마지막 몇 마이크로 미터 만 남은 떠나는 채워서 이온 교환기의 저항의 대부분 동심 피펫 션트. 또한, 동심 피펫 내의 도금액 물리적 용량을 크게 줄일 수 개의 유리 벽의 두께에 의해 분리 조로부터 이격된다. (10) 이전에 도시 된 바와 같이, 감소 된 저항 및 커패시턴스의 결합 된 효과는, 2 차의 크기의 시간적 해상도의 향상이다. 동심 칼슘하여 pH 미소 전극의 경우 90 %의 응답 시간은 낮은 밀리 10-20 : 10이었다. 동심 디자인 관련 장점은 낮은 노이즈 레벨 (참조 그림 8)입니다. 어떤 수면제에서 크게 감소 저항에 전압 과도 때문에T 소음은 최소화된다. 또한, 이러한 과도로부터 회복하기 때문에 빠른 시간 상수, 빠르다. 이러한 유물 따라서 작고 빠른, 그리고 생리 학적 기록 (참조 그림 8)에 덜 파괴적인 영향을 미친다. 동심 기술의 단점도 있습니다. 우선, 이들 어셈블리는 더 복잡하고, 시간 소모적이다. 두 번째 단점은 별도의 micromanipulator 또는 특수한 이중 매니퓰레이터 하나의 사용을 수반하는 그 팁 별도 기준 미세 전극을 배치 할 필요가있다. 마지막으로, 이중 목적 미소 전극 동심 전극 용 불가능 동시에 28에서 두 개의 서로 다른 이온 종의 검출을 가능하게 트리플 질주 설계로 확장 될 수있다. 가장 일반적인 함정 비효율적 인 실란 화. 모든 액체 SENS의 제조에있어서 가장 중요한 공정 및 주요 장애물또는 기반 이온 선택적 미세 전극은 실란 화의 절차입니다. 전극이 특정 이온 농도의 변화에 반응, 또는 서브 Nernstian 응답 (10 배 농도의 차이에 따라, 즉, 58도 미만 MV)에 대응하지 않을 때, 실란 화의 불량한 효능은 일반적 원인이다. 대기 습도가 여름 또는 겨울의 높이에서 조건의 전형적인, 너무 높거나 너무 낮은 경우 우리의 경험에 의하면,이 각각 발생할 수 있습니다. 그것은 실내 습도 일부 제어 발휘 가능한 경우, 이러한 문제는 극복 할 수있다. 전극 저항이 너무 높다. 필요한 경우, 이온 성 배럴의 저항은 베벨함으로써 감소 될 수있다. 이를 위해 몇 초 동안 물에 현탁 연마제 강한 제트 선단을 노출. 이것은 그것의 최상의 팁 깨고 원하는 저항 값을 저하시킬 것이다. 소금 다리. 소금이온 및 참조 배럴 사이의 다리는 잘못 또는 없음 - 응답 전극을 초래할 따라서도 크게 교정에서의 성능을 혼동 할 수 있습니다. (포인트 1.6 참조.) 상술 한 바와 같이, 이는 이중 목적 세타 유리가 선택 문제는 주로하지만 여기에 설명 오프셋 꼬인 배럴 기법을 사용할 때 드문하다. 염두에두고 제작의 용이성과 함께, 럭스 (29)의 원래 더블 질주 디자인은 종종 유익하게 사용할 수 있습니다. 이 방법은 또한 팁을 통해, 이온 교환기의 도입에 의해 다음, 소금 솔루션, 끝에서의 흡입 및 퇴학으로 실란 솔루션을 빠르고 노출 이온 및 참조 배럴의 사전 충전을 활용 (30, 31 참조) . 이 전극은 약 10 분으로 제작하지만, 자신의 팁 크기는 일반적으로 4 ㎛ 이상을 그리고 그들은 실험 기간 동안 실패 할 경향이있다 할 수있다. 대조적으로, 그 방법은 실란 화 실란 증기 및 열에 대한 노출을 포함ING는 작은 일 마지막 팁, 때로는 주와 전극을 생성 할 수 있습니다. 함께 찍은 여러 프로토콜이있다 이온 선택성 미소 전극을 준비하는 방법에 접근한다. 여기, 우리는 가까운 100 %의 성공률로, 우리의 실험실에서 잘 안정적으로 작동 트위스트 질주 더블뿐만 아니라 동심 미소 전극의 제조를 위해 두 가지 방법을 설명했다. 중요한 것은, 이들 기술은 산도 또는 칼슘 이온을 포함하는 다른 종의 측정에 양도 할 것이며, 또한 일반적으로 유체로 채워진 캐비티 또는 체액을 포함한 뇌 이외의 제제에 적용 할 수있을 것이다. 마지막으로,하지만 적어도, 이온 선택성 미소 전극은 세포 내부의 이온 농도의 결정을 할 수 있습니다. 상대적으로 큰 팁 크기 (~ 1 ㎛)의 때문에,이 뜻은, 그러나, 무척추 준비 28, 32에서 발견 된 같은 단지 예를 들어 큰 세포체와 세포에서 가능하다.

An author's middle initial was omitted from the publication, <a href="http://www.jove.com/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

Erratum: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Abrasive&#160;&#160;</td>
			<td>MicroPolish&#160;</td>
			<td>Buehler GmbH</td>
			<td>Dissolved in A.dest</td>
		</tr>
		<tr>
			<td>Borosilicate-glass capillaries</td>
			<td>1405059</td>
			<td>Hilgenberg</td>
			<td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC 150 F-15&#160;</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the sensor of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC100-F-15</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the reference of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-200TF-15&#160;</td>
			<td>Science Products</td>
			<td>Concentric, outer channel. o.d. 2.0 mm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-120TF-10</td>
			<td>Science Products</td>
			<td>Concentric, inner channel. o.d. 1.2 mm</td>
		</tr>
		<tr>
			<td>Digidata</td>
			<td>1322A</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrometer amplifier with headstage</td>
			<td></td>
			<td>Custom-made</td>
			<td>Rin = 10T&#937; and Ibias=50fA-1pA (commercially available&#160; alternatives: e. g. Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td>
		</tr>
		<tr>
			<td>Experimental chamber</td>
			<td></td>
			<td>Custom-made</td>
			<td>Commercially available from e.g.&#160; Warner Instruments,USA;&#160; Scientifica, UK</td>
		</tr>
		<tr>
			<td>Furnace</td>
			<td></td>
			<td>Heraeus</td>
			<td>Must stay constant at 200&#176;C</td>
		</tr>
		<tr>
			<td>Hard sticky wax / dental wax&#160;</td>
			<td>Deiberit 502&#160;</td>
			<td>Siladent Dr. Boehme &#38; Schoeps GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td></td>
			<td>Custom-made</td>
			<td>Must stay constant at 40&#176;C</td>
		</tr>
		<tr>
			<td>Microelectrodes holder made of plexiglas</td>
			<td></td>
			<td>Custom-made</td>
			<td>Double-barreled: o.d.&#160; capillaries 1.5 mm, concentric: o.d.&#160; capillaries 2 mm</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td></td>
			<td>Leitz</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>MD4R</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>M205C</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Plan 0.8xLWD</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model PP-830</td>
			<td>Narishige</td>
			<td>Concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model P-97</td>
			<td>Sutter Instruments&#160;</td>
			<td>Sensor of concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic drug ejection system</td>
			<td>Picospritzter Type II</td>
			<td>General Valve TM Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>Travel dovetail stage</td>
			<td>DT 25/M</td>
			<td>Thorlabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-component glue</td>
			<td>&#160;Araldite</td>
			<td>Huntsman advanced materials GmbH</td>
			<td>One may also use a small stripe of aluminum foil to stick the capillaries together&#160;</td>
		</tr>
		<tr>
			<td>Silverwire</td>
			<td>99.9%&#160;</td>
			<td>Wieland Edelmetalle</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer / Vibratome</td>
			<td>Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>AxoScope 8.1&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical puller&#160;</td>
			<td>Type PE-2</td>
			<td>Narishige Scientific Instruments</td>
			<td>With a revolvable chuck for&#160; double-barreled microelectrodes</td>
		</tr>
		<tr>
			<td>x/y translational stage</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Compound</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>1(S),9(R)-(&#8722;)-Bicuculline methiodide</td>
			<td>Sigma aldrich</td>
			<td>14343</td>
			<td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma aldrich</td>
			<td>C-127</td>
			<td>AMPA/kainate receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma aldrich</td>
			<td>A9256</td>
			<td>Activates NMDA and non-NMDA and EAATs</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma aldrich</td>
			<td>G1626</td>
			<td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&#160; mGluRs and EAATs</td>
		</tr>
		<tr>
			<td>Potassium ionophore I - cocktail B</td>
			<td>Fluka&#160;</td>
			<td>60403</td>
			<td>Based on valinomycin; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Sodium ionophore II - cocktail A</td>
			<td>Fluka&#160;&#160;</td>
			<td>71178</td>
			<td>Based on ETH 157</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Water, ultra pure</td>
			<td>Sigma aldrich</td>
			<td>W3500&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

<html> 모니터링하려면 [K +]는 O 및 변경은 내부에 흥분성 활동에 대한 응답으로, 이중 질주 칼륨 선택적 미세 전극은 CA1 영역의 지층 radiatum에 삽입되었다. 몇 분 꿰 뚫기 후에 전극의 이온 선택 배럴은 약 2.8 mm의 사용 ACSF의 [K +]에 가까운 값 (아 [K +]에 대응하는, 안정한 기준선 (도 7a)에 도달 2.5 mm)이다. 10 초 동안 0.5 mM의 글루타메이트의 목욕 응용 프로그램은 ~ 0.7 밀리미터 (그림 7A)에 달하는 기준선 아래 언더 다음, 약 4 mm 정도 O를 [K +]에서 가역적 인 증가를 유도. 0.4, 0.3 글루타메이트 농도를 낮추고, 0.2 mm의 일시적 증가 [K +] 오 (그림 7A)에 언더 모두의 진폭에 대응 감소를 일으키는 원인이되었다. OAD / 53058 / 53058fig7.jpg &quot;/&gt; 그림 흥분성 활동에 응답 7. 세포 외 칼륨 과도. (A) [K +] 오 과도, 지시 된 바와 같이 10 초 동안 글루타메이트의 서로 다른 농도의 목욕 응용 프로그램에 의해 유도 된 기준 아래 언더 다음의 증가, 구성. (B) 글루타메이트 수용체 차단제와 관류의 영향 (CNQX, 100 μM, APV, 100 μM)과 테트로도톡신 (TTX 0.5 μM) [K +] O 과도에 10 초 동안 0.5 mM의 글루타메이트의 목욕 응용 프로그램에 의해 유도. 0Mg 2+ / BIC의 존재 (C) 자발적인 [K +] 오 과도. AC에 표시된 실험은 두 번 질주 전극을 사용하여 수행 하였다. 그림을 위해, 흔적 Sawitzky - 골 레이 필터 (폭 20)와 함께 부드럽게했다. <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">카스티하세요</a>이 그림의 더 큰 버전을 보려면 여기를 ICK. 이전 실험은 글루타메이트에 의한 [K +] O 신호가 크게 TTX의 응용 프로그램 중 변경되지 것을 보여, 따라서 전압 문이 나트륨 채널과 신경 활동 전위 세대 (15, 16)의 개방에 거의 독립적있다. 글루타메이트에 반응하여 관찰 된 [K +] O 변화를 기본 메커니즘을 연구하기 위해, 우리는 글루타메이트 수용체 차단제, 즉 CNQX (100 μM; AMPA 수용체 차단제) 적용 및 APV를 (100 μM; NMDA 수용체 차단제)의 존재하에 TTX (0.5 μM). (도 7B 예를 들어, 17)이 차단제 목욕 관류시, 글루타메이트에 의한 [K +] 변화 o를하기 전에보고 된 이온 성 글루타메이트 수용체 채널의 개방에 대한 의존도를 확인, 사실상 폐지했다. 더 T를 보여주기 위해세포의 생성을위한 글루타메이트 여기의 그는 관련성 [K +] 신호, 조각은 명목상의 Mg 2+ - 무료 식염수 10 μm의 bicuculline의 메티 (0Mg 2+ / BIC)를 포함하여 관류 하였다. 이는 NMDA 수용체의 전압 의존성의 Mg 2+ - 블록을 완화하고 (예 : 18,19) 네트워크에서 자발적인 간질 재발 활동을 일으키는, GABA 수용체를 차단함으로써 억제를 감쇠시킨다. 예상대로 20, 자연, 재발 [K +] 오 과도, 약 1.5 밀리미터에 달하는이 0Mg 2+ / BIC 식염수 (그림 7C)에서 검출되었다. 약 0.2의 평균 진폭이 응답 사이의 작은 [K +] 오 과도 전월 (그림 7C)를 발생했습니다. 제 2 세트의 실험에서는, 도포에 의해 유발 [나 +] O 변화를 결정하기 위해 [나 +] 민감한 미소 전극을 사용글루타민산 작용제. 여기, 우리는 두 개의 서로 다른 응용 프로그램의 패러다임을 사용하는 더블 질주과 동심 미소 전극의 응답 특성을 비교 하​​였다. [나 +]는 민감한 미소 전극은 약 50 μm의 깊이의 지층 radiatum에 위치했다. 안정한 기준선을 달성 한 후, 글루타메이트 작용제 L 아스파라긴산은 (2.5 ㎖ / 분으로 10 mm의 120 초, 목욕 관류) 목욕 관류 당 도포 하였다. 이전 21 관찰, 아스 파르 테이트의 응용 프로그램은 약 5 분을 지속 한 후베이스 라인에 다시 (그림 8A)를 복구하기 시작 약 15 밀리미터에 의해 O를 [나 +]에 느린 감소를 일으키는 원인이되었다. 양 전극에 의해 결정 [나 +] O 신호의 피크 진폭과 동력학은 이들 조건 하에서 거의 동일했다하면서 특히, 동심 전극보다 안정한 기준선 및 낮은 잡음 레벨 (도 8a)를 나타냈다. R을 테스트하려면<html> 더 빠른 애플리케이션 패러다임에서 다른 전극의 특성을 저 응답, 우리는 이온 선택적 미세 전극의 끝에서 20 ~ 40 ㎛의 거리에서 지층 radiatum에 위치 미세 유리 피펫을 통해 글루타메이트를-인가 압력. (21) 예상대로, 200 MS에 대한 글루타메이트의 응용 프로그램 (10 mM)을 [나 +] O (그림 8B)의 저하를 일으키는 원인이되었다. [나 +] O 감소의 피크 진폭이 전극의 두 가지 유형에 대해 동일한 범위에 있었다 (:; : 19.1 밀리미터, N = 15 - - 2.0 동심 13.5 밀리미터, N = 14 4.5을 두 번 질주). 그러나, 느린 목욕 관류 (위 참조), 신호 대 잡음비, 또한 전극의 두 가지에 의해 검출 [나 +] O 신호의 시간 경로뿐만 얻어진 결과와 대조적 크게 달랐다. 피크 평균 시간은 동심 미소 전극의 질주 - 더블, 1.3 초 3.5 초였다. ve_content &quot;&gt; 따라서, 이러한 결과를 보여 주 및 대 동심을 두 번 질주 나 + -selective 미소 전극의 빠른 반응 속도론을 확인, (참조 그림 6C 및도 8b)으로는 CA의 유명했다 2+ pH를 선택적 대응 (10) . 짧은 버스트 시냅스 자극이 빠른 시냅스에 의한 이온 과도를 연상하기 위해 수행되었던 이전의 연구와는 달리, 단지 경향이 있지만, 우리의 응용 프로그램과 동심을 두 번 질주 전극 사이의 평균 피크 진폭에서 유의 한 차이가 있었다 패러다임. 이는 사실에 아마이었다 그 결과적으로 두 개 (20 ~ 40 μm의)의 인자에 의해 변경 이온 선택 미세 전극의 팁으로부터 애플리케이션 피펫의 거리, 즉 타깃 영역에서 최대 글루타메이트 농도 피크 진폭의 기존 차를 막고, 다른 실험에서 같은 아니었다. (가) 위쪽 추적 : 볼타의 변화를이중 질주 전극 (검은 색 점선 추적)와 욕조 나 + 농도의 변화에 따라 동심 전극 (회색 추적)의 기준 배럴 (V 심판)의 GE ([나 +는] b)에 나타낸 바와 같이. 양 전극의 전압 응답 사실상 동일하다. 바닥 흔적 : 이중 질주 전극 (블랙 추적)와 [나 +] B 변화에 대응 동심 전극 (회색 추적)의 나 + -potential (V 나 +)의 전압의 변화. 양 전극의 V 나 + 대 B (B) [나 +]의 하프 로그 플롯. 데이터의 선형 그래프는 양 전극에 대한 약 48 MV의 기울기를 알 수있다. 70 밀리미터에 152 밀리미터에서 B를 두 번 질주 (블랙 추적)의 V 나 +의 (C) 응답하고, [나 +]의 빠른 변화에 동심 전극 (회색 추적). 동심 전극의 응답 시간이 크게 FAS 유의터. 예시를 위해, 트레이스 Sawitzky - 골 레이 필터와 평활화 된 (폭 20). <img alt="그림 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> 그림 8 : 두 번 질주와 동심 전극에 의해 검출 된 세포 외 나트륨 과도. (A) 과도 V 심판의 변화와 [나 +] 막대로 나타낸 바와 같이 O 120 초 동안 10 mM의 아스 파르 테이트 목욕 관류에 의해 유도. 상단 트레이스는 이중 질주 전극 수행 기록, 낮은 흔적 동심 전극을 사용하여 기록 하였다 보여준다. (B)에 화살표로 표시된 바와 같이 0.2 초 동안 10 mM의 글루타메이트 현지 압력 응용 ​​프로그램에 의해 유도 O를 V 심판와 [나 +]에 과도 변경됩니다. 상단 트레이스는 이중 질주 전극 수행 기록, 낮은 흔적 동심 전극을 사용하여 기록 하였다 보여준다.빨간 점선 라인은 최대로 신호의 시작 사이의 시간 선형 발작을 나타낸다. 동심 전극의 응답 시간이이 조건에서 상당히 빠르다는 것을주의. 그림을 위해, 흔적 Sawitzky - 골 레이 필터 (폭 20)와 함께 부드럽게했다. <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

뇌 조직에서 측정 세포 외 이온의 신호에 대한 두 번 질주과 동심 미소 전극

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

double-barreled-concentric-microelectrodes-for-measurement

Research

JoVE Journal

Neuroscience

13.4K 조회수.  Heinrich Heine University Düsseldorf. We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations. 

비디오: Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Immunoelectron 현미경을위한 마우스 뇌 조직의 준비

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

연구

신경과학

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

초점을 맞춘 이온 빔은 밀링 및 뇌 조직의 전자 현미경을 스캐닝

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

작은 동물의 만성 신경 레코딩을위한 초경량 동력 Microdrive의 설계 및 조립

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

만들기, 테스트 및 성인 뇌의 조직 조각에 칼륨 이온 선택적 Microelectrodes를 사용 하 여

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

로컬 Ca2 + 신호 막 대상 Ca2 + 지표에 의해 경작된 한 세포에의 해 부

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

용매 클리어 뇌 조직에서 광견병 바이러스 감염의 고해상도 3D 이미징

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

천연 폴리페놀 커큐민을 사용하여 뇌 조직에서 아밀로이드 플라크의 라벨링 및 이미징

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

마우스 뇌의 수평 해마 조각

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

쥐 뇌 조직을 위한 소수성 조직 청산 방법

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

급성 뇌 슬라이스에서 내인성 모아민 방출 측정을 위한 판 기반 분석

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...