Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Neuroscience
뇌의 세포 외 공간의 부피비와 비틀림을 정량화하기위한 테트라 메틸 암모늄을 이용한 실시간 이온 도입
John Odackal1*, Robert Colbourn2,3*, Namrita Jain Odackal4, Lian Tao5, Charles Nicholson5, Sabina Hrabetova2
1Department of Medicine, University of Virginia, 2Department of Cell Biology, SUNY Downstate Medical Center, 3Neural and Behavioral Science Graduate Program, SUNY Downstate Medical Center, 4Division of Neonatology, University of Virginia, 5Department of Neuroscience and Physiology, New York University School of Medicine
* These authors contributed equally이 프로토콜은 살아있는 두뇌의 세포 외 공간 (ECS)의 물리적 매개 변수를 측정하는 방법 인 실시간 이온 삼투압을 설명합니다. ECS로 방출 된 불활성 분자의 확산은 ECS 부피 분율 및 비틀림을 계산하는 데 사용됩니다. 그것은 뇌의 ECS에 대한 급격한 가역적 인 변화를 연구하는데 이상적입니다.
이 리뷰에서는 살아있는 두뇌의 세포 외 공간 (ECS)을 탐구하고 정량화하기위한 금 이온 표준 인 실시간 이온 삼투압 (RTI) 방법을 수행하기위한 기본 개념과 프로토콜에 대해 설명합니다. ECS는 모든 뇌 세포를 둘러싸고 있으며 간질 액과 세포 외 기질 모두를 포함합니다. 신경 전달 물질, 호르몬 및 영양소를 포함한 뇌 활동에 필요한 많은 물질의 운반은 ECS를 통한 확산에 의해 발생합니다. 이 공간의 부피와 기하 구조의 변화는 수면과 허혈과 같은 병리학 적 상태와 같은 정상적인 뇌 과정에서 발생합니다. 그러나, 특히 병이있는 상태에서 뇌 ECS의 구조와 조절은 아직 미개척이다. RTI 방법은 살아있는 두뇌의 두 가지 물리적 매개 변수, 즉 체적 분율과 비틀림을 측정합니다. 부피 분율은 ECS가 차지하는 조직 부피의 비율입니다. 구불 구불 한 (tortuosity)은 뇌를 통해 확산 될 때 물질이 마주 치는 상대적인 방해의 척도이다장애물이없는 매체에 비해 RTI에서 불활성 분자는 근원 미세 전극에서 뇌 ECS로 펄싱된다. 분자가이 소스에서 멀리 확산함에 따라 이온의 변화하는 농도는 약 100 μm 떨어진 이온 선택 미세 전극을 사용하여 시간에 따라 측정됩니다. 결과적인 확산 곡선으로부터 부피 분율 및 비틀림을 계산할 수 있습니다. 이 기술은 여러 종 (인간 포함)의 뇌 조각과 생체 내 에서 ECS의 급성 및 만성 변화를 연구하는 데 사용되었습니다. 다른 방법과 달리 RTI는 실시간으로 두뇌 ECS의 가역적 및 비가 역적 변화를 검사하는 데 사용할 수 있습니다.
세포 외 공간 (ECS)은 모든 뇌 세포 외부의 상호 연결된 채널의 네트워크이며 간질 액과 세포 외 기질 모두를 포함합니다 ( 그림 1a 및 그림 1b ). 영양소, 호르몬 및 신경 전달 물질을 포함한 뇌 세포 기능에 필요한 많은 물질의 분포는 ECS를 통한 확산에 의해 발생합니다. 체적, 기하학 및 세포 외 기질을 포함한이 공간의 물리적 매개 변수의 변화는 뇌 세포 기능에 중대한 영향을 미치는 뇌 세포를 목욕시키는 ECS 및 국소 이온 농도를 통한 확산에 크게 영향을 미칠 수 있습니다 1 , 2 .
실시간 이온 삼투압 (RTI)은 뇌 영역의 두 가지 구조적 특성을 결정하는 데 사용됩니다 : 체적 분율과 비틀림 3 , 4 ,"xref"> 5. 부피 분율 ( α )은 대표적인 기본 부피의 총 조직 부피 ( V 조직 )에 대한....
조직 샘플을 얻는 데 사용 된 모든 동물 절차는 SUNY Downstate Medical Center의 동물 윤리위원회의 승인을 받았습니다.
1. 솔루션 및 장비 준비
- ISM의 기준 배럴에 150mM NaCl 백필 용액을 준비하십시오. 0.22 μm 필터 (박테리아 또는 입자를 제거하기 위해)에 부착 된 10 mL 주사기에 보관하십시오.
- microelectrodes에 대한 150 MM TMA 염화물 (TMA - 클럭) backfill 솔루션을 준비합니다. 0.......
도표 10 : 일반적인 기술적 인 문제점을 설명하는 비 이상적인 자료. ( a ) iontophoresis microelectrodes와 함께 일반적인 기술적 인 문제의 다이어그램 : 기능적인 iontophoresis microelectrode에서 TMA의 정상적인 릴리스와 기술적 인 문제를 보여주는 세 가지 소스의 비교. [고배율, a1] 이상적인 이온 삼투압 소스의 전류?.......
| Name | Company | Catalog Number | Comments |
| A/D and D/A converter | National Instruments Corporation | NI USB-6221 DAQ | The NI USB-6221 is still sold as a 'Legacy' device by NI. They recommend using NI USB-6341 X Series DAQs for new installations, however we have not tested the newer units. We describe the use of the NI USB-6221 with MATLAB and Windows 7 (32-bit). Alternatives: the much older PCI-MIO-16E-4 A/D converter (Used under Windows XP or older OS only) with BNC-2090 BNC connector panel and SH68-68-EP cable. As noted in the Wanda Manual, an experimental MATLAB program to use Axon Binary Files is available. |
| agarose | Lonza | NuSieve GTG Agarose #50081 | to prepare dilute agarose gel for RTI measurements |
| amplifier for ISM | Dagan | Model IX2-700 Dual Intracellular Preamplifier | ion and reference voltage amplifier with N=0.1 (for reference barrel) and N=0.001 (for ion barrel) headstages |
| biological compound miscroscope (with 4x and 10x objective) | for chipping the microelectrode tips and inspecting microelectrodes; various suppliers, e.g. AmScope | ||
| borosilicate theta capillary glass tubing | Harvard Apparatus | Warner Instruments model TG200-4; order #64-0811 | double-barreled glass tubing for ion-selective microelectrodes and iontophoretic microelectrodes; O.D. 2.0 mm, I.D. 1.4 mm, septum 0.2 mm, length 10 cm |
| brush | Winsor & Newton | University Series 233, size 0 | round shoft handle brush, available from Amazon |
| bunsen burner | Fisher | ||
| camera for visualizing micropipettes | Olympus | OLY-150 | requires monitor, IR filter on substage illuminator is optional |
| chart recorder | to record continuously voltages on ion-selective microelectrode during calibration in tetramethylammonium standards and during RTI experiment; e.g. Kipp & Zonen type BD112 dual-cannel chart recorded, available refurbished | ||
| chlorotrimethylsilane, puriss., > 99% | Sigma-Aldrich | catalog # 92360 | for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, reacts violently with water, see Sigma-Aldrich Safety Information for full description |
| Commercial Software | The MathWorks | MATLAB, Data acquisition toolbox | for data acquisition and analysis using Wanda and Walter programs. Note that an academic license is available. |
| eye protective goggles | Fisher | ||
| fixed-stage compound microscope | Olympus | BX51WI | can use other compound microscopes with fixed stages |
| forceps | Fine Science Tools | #11251-10 | to chip glass capillary; Dumond #5, preferably used and no longer needed for fine work |
| fume hood | for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; various supliers, e.g. Captair with approriate filter sold by Erlab | ||
| glass microscope slide | Fisher | #12-550A | to chip microelectrode tips |
| heater/stirrer | Fisher | Corning PC-420D | to prepare dilute agarose gel and stir solutions |
| iontophoretic unit | Dagan | ION-100 and PS-100 | ION-100 is a single channel iontophoresis unit +/- 130 V compliance; PS-100 is an external power supply; alternatives: e.g. Axoprobe-1A made by Axon Instruments (now Molecular Devices), out of production, check for availability of refurbished units (eBay and other sites) |
| liquid ion exchanger (LIX) for tetramethylammonium | World Precision Instruments | IE190 Potassium Ion Exchanger | Note: this is equivalent to the original Corning potassium exchanger 477317 based on tetraphenlyborate - do not confuse with neutral carrier potassium exchanger originating from the laboartory of Dr. Simon, ETH, Zurich, which does not sense tetramethylammonium, and is sold by Fluka. You can also make liquid ion exchanger for tetramethylammonium yourself: 3% by weight potassium tetrakis = (p-chlorophenyl) borate dissolved in 2,3-dimethylnitrobenzene. Buy chemicals from Fluka (now part of Sigma). See Oehme and Simon (1976) Anal. Chim. Acta 86: 21-25; CAUTION: The toxicological properties of this liquid ion exchanger have not been fully determined. Ingestion or contact with the human body may be harmful. Exercise due care! Liquid ion exchangers should be stored in a cool place out of direct sunlight. |
| microelectrode holder | WPI | M3301EH | to hold ion-selective microeletrode prefabricate for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; WPI sells two versions of this holder, clear M3301EH and black M3301EH. In our experience, the clear M3301EH appears to be sturdier then the black M3301EH. |
| micromanipulator | Narishige | MM-3 | to position ion-selective microelectrode prefabricate during silanization and filling the tip of ion-selective barrel with liquid ion exchanger; can be substituted with any three-axis micromanipulator in good working condition |
| micropipette puller | Sutter Instruments | Model P-97 | to pull double-barreled glass tubing; other pullers can be used as long as they can accommodate large diameter double-barreled glass tubing |
| microprobe thermometer | Physiotemp | Model BAT-12R | fine probe of this thermometer is placed close to recording site |
| needle | BD | Syringes and Needles # 305122 (25 gauge) | for silanization; BD PrecisionGlide needles 25 G x 5/8 in (0.5mm x 16mm) |
| objective 5x dry | Olympus | MPlan N | |
| objective 10x water immersion | Olympus | UMPlan FL N | 10x objective is water immersion, numerical aperture is 0.3, working distance is 3.3 mm |
| plastic containers (with lids) | Fisher | #14-375-148 | to store tetramethylammonium standard solutions and microelectrodes |
| platform and x-y translation stage for fixed-stage microscope | EXFO | Gibraltar Burleigh | platform holds slice chamber, micromanipulators and accesorries, x-y translational stage moves microscope without compromising recording stability |
| porous minicup | for RTI measurements in a dilute agarose gel; homemade | ||
| reusable adhesive | Bostik | Blu-Tack | for securing microelectrodes to holding vessel and other uses; various suppliers, available from Amazon |
| robotic micromanipulator with precise x,y,z positioning | Sutter Instruments | MP-285 | two mircomanipulators are needed to hold separately ion-selective microelectrode and iontophoretic microelectrode. Also possible to glue micropipettes in a spaced array (see text). |
| signal conditioning unit with low-pass filter | Axon Instruments | CyberAmp 320 or 380 | no longer available from the manufacturer but may be available from E-Bay; alternatives: e.g. FLA-01 Filter/Amplifier from Cygnus Technology. This is a single channel instrument with a minimum cutoff at 10 Hz using a multipole Bessel filter but the company may be willing to modify it for a lower cutoff frequency (2 Hz) if needed. |
| silver wire | A-M Systems | #7830 | diameter 0.015", bare (no coating) |
| slice chamber | Harvard Apparatus | Warner Model RC-27L | this is submersion slice chamber; do not use interface slice chamber |
| stereomicroscope | for silanization and filling the tip of ion-selective barrel with liquid ion exchanger; horizontally mounted; various suppliers | ||
| syringe, 10 mL | BD | Syringes and Needles #309604 | to backfill microelectrodes and for silanization; BD Luer-Lok tip |
| syringe filter 0.22µm pore | Whatman | #6780-1302 | to filter backfill solutions; available from Fisher |
| syringe needle, 28 gauge, 97mm | World Precision Instruments | MicroFil MF28G-5 | to backfill microelectrodes |
| Teflon (=PTFE) tubing | Component Supply | STT-28 PTFE tube light wall (28 gauge) | for silanization of ion-selective barrel; fits on BD PrecisionGlide needles 25 G x 5/8 in. Note: Teflon is essential, PVC tubing would melt by hot wax. |
| temperature control system | Harvard Apparatus | Warner Models TC-344B and SH-27A | TC-344B is a dual automatic temperature controller, SH-27A is an in-line heater; controller and heater work with Warner slice chambers |
| tetramethyammonium (TMA) chloride | Sigma-Aldrich | T-3411 | 5 M solution; CAUTION: acute toxicity (oral, dermal, inhalation), carcinogenicity, hazardous to the aquatic environment, see Sigma-Aldrich Safety Information for full description |
| vibrating blade microtome | Leica | VT1000S | to cut brain slices |
| xylenes | Fisher | X5-1 | for silanization; CAUTION: flammable, acute toxicity (oral, dermal, inhalation), skin corrosion, eye damage, carcinogenicity, see Fisher Safety Information for full description |
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