(; U54GM104942 MNR), NIA (MNR, R15AG045812), 알츠하이머 협회 (MNR, NIRG-12-242187), WVU 교수 연구 상원 그랜트 (MNR) 및 WVU PSCOR 보조금이 작품은 국립 일반 의학 연구소에 의해 지원되었다 (MNR).

<ol>
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GG는이 연구에서 글루탐산 측정에 사용되는 FAST-16 기록 시스템을 만드는 Quanteon, LLC의 유일한 소유자입니다. JEQ는 Quanteon에 대한 유료 컨설턴트이다.

MEA를 기술은 시험 관내 및 생체 내에서 신경 전달 물질 방출 및 흡수 빠른 반응 속도의 측정을 허용한다. 따라서, 기술 토닉 신경 전달 물질의 농도를 포함하는 데이터 출력 유발 신경 전달 물질 방출, 및 신경 전달 물질 간극 다양한 생산한다. MEA의 사용이 상대적으로 복잡한 과정이기 때문에, 성공적인 사용을 위해 최적화 될 필요가있다 수많은 요인이있다. 예를 들어, 교정 ?...

뇌의 신경 전달 물질의 변화를 측정하는 것은 종종 신경 전달 물질 조절 곤란을 특징으로 중추 신경계 (CNS) 질환을 연구 신경 과학자을위한 필수적인 도구입니다. 고압 액체 크로마토 그래피 (HPLC / EC)와 함께 미세 투석은 세포의 신경 전달 물질의 레벨 1, 2의 변화를 측정하기 위해 가장 널리 사용되는 방법 하였지만 3, 4, 미세 투석 프로브의 공간 및 시간 해상도는 신경 전달에 적합하지 않을 수도 이러한 단단히 외 공간 (5, 6)에 규정되는 글루타메이트 등. 때문에 유전 촬상 최근의 진보, 생체 내에서의 글루타메이트를 매핑하는 데 사용할 수있는 추가의 방법이있다. 유 전적으로 인코딩 된 글루타메이트 형광성 리포터를 사용하여 (iGluSnFR)를D 이광자 촬상 연구자들은 시험 관내 및 생체 7, 8, 9 모두 뉴런 및 성상으로의 글루타메이트 방출을 시각화 할 수있다. 특히,이보다 넓은 시야에서 기록이 가능하며 뇌의 고유 연결을 방해하지 않습니다. 이러한 새로운 광학 기술의 글루타메이트 반응 속도와 감각 유발 반응과 신경 활동의 측정 시각화 할 수 있지만, 그들은 분리 된 뇌 영역의 세포 외 공간에서의 글루타메이트의 양을 정량화 할 수있는 능력이 부족하다. 다른 방법은 선택적으로 자기 - 참조 기록 방식의 사용을 통해 이러한 세포 외 글루타메이트 신경 전달 물질의 수준을 측정 할 수있는 효소 - 결합 된 미세 배열 (MEA)이다. MEA를 기술은 외상성 뇌 다음 세포의 글루타메이트의 변화를 연구하는 데 사용되었습니다노화 부상 10, 11, 12, 13, 14, 스트레스 (15, 16), 간질 (17), (18), 알츠하이머 병 (19), (20), 및 바이러스 성 미믹 (21)의 주입은 미세 투석에 내재하는 공간 및 시간 제한에 비해 개선을 나타낸다. 미세 투석은 시냅스 (22,23) 근처에 측정 할 수있는 능력을 제한하는 반면, MEA의 세포 외 글루타메이트의 선택적 유출 대책 근처 24, 25 시냅스 허용하는 높은 공간 해상도를 가지고있다. 둘째, 미세 투석 낮은 시간 해상도 (1 - 20 분)을 조사 할 수있는 능력을 제한범위 제 26 밀리 초에서 발생 글루타메이트 방출 및 클리어런스의 신속한 역학. 글루타메이트의 방출 또는 간극의 차이 글루타메이트 농도 쉬고 토닉의 측정에서 분명하지 않을 수도 있기 때문에 글루타메이트 방출 및 클리어런스를 직접 측정하는 것이 필수적 일 수있다. MEA를 인해 높은 시간 분해능 (Hz의 2)의 검출 하한 (&lt;1 μM)에 조치 허용한다. 셋째, MEA를는 래트 나 마우스 해마 같은 특정 뇌 영역 내에서 신경 전달 물질의 소 변형 검사 허용한다. 예를 들어, 세포 외 글루타메이트의 소 차이를 조사하기 위해, 우리는 별도로 치아 이랑 (DG)와, trisynaptic 회로 (27)를 통해 연결되어 해마의 cornu의 ammonis 3 (CA3)와 cornu ammonis 1 (CA1)를 타겟팅 할 수있는 MEA를을 사용. 주입 28 &lt;기인 - (4 mm 길이 1) 및 손상으로 인해 미세 투석 프로브의 크기/ SUP&gt;, 29, 소 지역적 차이는 해결하기 어렵다. 또한, 광학 시스템은 예컨대 자극 소 7을 허용하지 않는 수염 자극 또는 광 플리커 등의 외부 자극을 통해 자극을 허용한다. 다른 방법을 통해 다자간 환경의 최종 이익은 외적인 연결을 방해하지 않고 생체 내에서 이러한 하위 영역을 연구 할 수있는 기능입니다. 여기서는 세라믹 기반 다중 미세 이루어지는 MEA를 함께 결합하는 기록 시스템 (예 FAST16mkIII)는, 차분 검출 및 분석 신호로부터 제거 될 에이전트의 간섭을 허용하는 기록 위치에 도포 할 수있는 방법을 설명한다. 우리는 이러한 배열을 기초 amperometry-DG, CA3 내의 생체 글루타메이트 규제 연구 및 마취 RTG의 해마 CA1의 하위 영역에 사용할 수있는 증명 (TauP301L) 4510 마우스, 일반적으로 사용되는 m알츠하이머 병의 여러개 모델. 또, 릴루 졸과 쥐를 치료함으로써 글루타메이트 방출 및 클리어런스의 빠른 동역학에 MEA 시스템의 감도의 확인을 제공 시험관에 나타내는 약물 글루타메이트 방출을 감소시키고 글루타메이트 흡수 30, 31, 32, 33을 증가시키기 그리고 TauP301L 마우스 모델에서 생체 내에서 이러한 각각의 변화를 보여주는.

<table><tbody><tr><td>FAST-16mkIII-8 channel</td><td>Quanteon</td><td>16mkIII</td><td></td></tr><tr><td>Microelectrode arrays</td><td>CenMet</td><td>W4 or 8-TRK</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A-3059</td><td>10 g (expires after 1 month)</td></tr><tr><td>Glutaraldehyde</td><td>Sigma-Aldrich</td><td>G-6257</td><td>100 mL (expires after 6 months)</td></tr><tr><td>Glutamate Oxidase</td><td>US Biological or Sigma Aldrich</td><td>G4001-01 or 100646</td><td>50 UI (expires after 6 months)</td></tr><tr><td>Hamilton Syringes</td><td>Hamilton</td><td>#80383</td><td>2 syringes</td></tr><tr><td>Methanol</td><td>BDH</td><td>UN1230</td><td>4 L</td></tr><tr><td>m-Phenylenediamine dihydrochloride (mPD)</td><td>ACROS Organics</td><td>1330560250</td><td>25 g</td></tr><tr><td>Reference Electrodes (RE-5B)</td><td>BAS</td><td>MF-2079</td><td>3 electrodes</td></tr><tr><td>Battery-powered Magnetic stir plate</td><td>Cole-parmer</td><td>EW-04804-01</td><td>Can purchase from different supplier</td></tr><tr><td>Glutamate</td><td>Sigma-Aldrich</td><td>G-1626</td><td>100 g</td></tr><tr><td>Ascorbic Acid</td><td>TCI</td><td>50-81-7</td><td>500 g</td></tr><tr><td>Dopamine Hydrochloride</td><td>Alfa Aesar</td><td>62-31-7</td><td>5 g</td></tr><tr><td>Perchloric acid</td><td>VWR</td><td>UN2920</td><td>500 mL</td></tr><tr><td>Postassium chloride</td><td>VWR</td><td>7447-40-7</td><td>1 kg</td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>7647-40-7</td><td>1 kg</td></tr><tr><td>Calcium Chloride</td><td>MP</td><td>153502</td><td>100 g</td></tr><tr><td>Sodium Hydroxide</td><td>BDH</td><td>1310732</td><td>500 g</td></tr><tr><td>Glass pressure ejection pipettes</td><td>CenMet</td><td></td><td></td></tr><tr><td>Sticky wax</td><td>Kerrlab</td><td>625</td><td>Can purchase from different supplier</td></tr><tr><td>Microsyringe</td><td>World Precision Instruments</td><td>MF28G-5</td><td></td></tr><tr><td>Modeling clay</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Picospritzer III</td><td>Parker</td><td></td><td></td></tr><tr><td>Silver wire</td><td>AM systems</td><td>#786500</td><td></td></tr><tr><td>Hydrochloric acid</td><td>BDH</td><td>7647010</td><td>2.5 L</td></tr><tr><td>Platinum wire</td><td>AM Systems</td><td>778000</td><td></td></tr><tr><td>Solder gun</td><td>Lowes or Home Depot</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Multimeter</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>PhysioSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>SomnoSuite</td><td>Kent Scientific</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Stereotaxic device</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Digital Lab Standard</td><td>Stoelting</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Meiji EMZ microscope</td><td>Meiji</td><td>EMZ-5</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>Micro</td><td></td></tr><tr><td>Metricide</td><td>Metrex</td><td>102800</td><td></td></tr><tr><td>Scalpel</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Surgery scissors</td><td>VWR</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile cotton swabs</td><td>Puritan</td><td>25806</td><td>Can purchase from different supplier</td></tr><tr><td>Eye ointment</td><td>Puralube Vet Ointment</td><td></td><td>Obtain from the vet</td></tr><tr><td>Iodine swabs</td><td>VWR</td><td>S48050</td><td>Can purchase from different supplier</td></tr><tr><td>Alcohol swabs</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Sterile surgery drape</td><td>Dynarex</td><td>4410</td><td>Can purchase from different supplier</td></tr><tr><td>Sterile saline</td><td>Teknova</td><td>S5815</td><td>Can make own soltuion using filters</td></tr><tr><td>Hydrogen Peroxide (3%)</td><td>Local drug store</td><td></td><td>Can purchase from different supplier</td></tr><tr><td>Heating Pad</td><td>WalMart</td><td></td><td>Can purchase from different supplier</td></tr></tbody></table>

using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models

신경 전달 물질 중단은 알츠하이머 병, 파킨슨 병, 우울증, 불안의 기초가되는 병리의 역할을 주로 중추 신경계 (CNS) 질환의 주요 구성 요소입니다. 전통적으로, 미세 투석은이 질환에서 발생하는 신경 전달 물질의 변화를 검사하는 가장 일반적인 (찬양) 기술하고있다. 미세 투석은 조직의 넓은 영역에 걸쳐 천천히 1-20분 변화를 측정 할 수있는 능력을 가지고 있기 때문에, 그것은 잠재적으로 뇌와 느린 샘플링 기능 내에서 고유 연결을 파괴, 침입의 단점이있다. 비교적 새로운 기술은 미세 전극 배열 (MEA)가 그들이 발생하는 공간적 및 시간적 정확한 접근하게 이산 뇌 영역 내의 특정한 신경 전달 물질의 변화를 측정하기위한 많은 장점을 갖는다. 또한, MEA의를 사용하여 생체 내에서 신경 전달 물질의 변화의 측정을 허용 최소 침습적이다. 저희 연구실에서는, 우리는 헥타르알츠하이머 병의 병리와 관련된 신경 전달 물질 인 글루타메이트의 변화에 ​​특별히 관심을했습니다. 따라서, 여기에 설명 된 방법은 알츠하이머 병의 유전자 변형 마우스 모델에서의 글루타메이트의 잠재적 인 해마 중단을 평가하기 위해 사용되었다. 간단히, 방법은 관심과 배경 잡음 및 간섭 물질을 뺄 자체 참조 사이트를 사용하는 신경 전달 물질에 매우 선택적 효소와 다중 사이트 미세 전극을 코팅 포함 사용. 도금 및 교정 한 후, MEA는 마이크로 피펫으로 구성 될 수 있고, 정위 장치를 이용하여 관심있는 뇌 영역으로 낮췄다. 여기서, 상기 방법은 RTG (TauP301L) 4510 마우스를 마취하고 정확하게 해마의 서브 영역 (DG, CA1 및 CA3)를 대상으로하는 정위 장치를 사용하는 것 설명.

이 기술은 같은 외상성 뇌 손상, 노화, 스트레스, 그리고 간질 동물 모델의 많은 종류의 신호 글루타메이트의 변화를 측정 할 수 있지만, 여기에 우리는 MEA 기술은 유전자 변형 마우스 모델에서 글루타메이트의 변화를 검사하는 데 사용할 수있는 방법을 보여줍니다 인간 타우 병증 (19), (20). RTG를 (TauP301L)는 4510 마우스 염색체 17?...

Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

여기서는 토닉과 효소 결합 된 미세 배열 (MEA)를 사용하여 생체 내 세포 외 글루타메이트 phasic 변화를 측정하는 공간적 및 시간적 정확한 방법에 대한 설치 소프트웨어 탐색 및 데이터 분석을 기술한다.

효소 기반의 바이오 센서를 사용하면 알츠하이머 병 마우스 모델에서 토닉과 Phasic이 품목을 측정하는

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

using-enzyme-based-biosensors-to-measure-tonic-phasic-glutamate

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.2K 조회수.  West Virginia University.  여기서는 토닉과 효소 결합 된 미세 배열 (MEA)를 사용하여 생체 내 세포 외 글루타메이트 phasic 변화를 측정하는 공간적 및 시간적 정확한 방법에 대한 설치 소프트웨어 탐색 및 데이터 분석을 기술한다. 

비디오: Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

알츠하이머 질병 마우스 모델에서 Neuritic Plaques의 감지

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

연구

신경과학

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Cisterna 마그나 마우스에서 뇌척수의 직렬 그룹에 대한 기술

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

생물학

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

자연스럽 게 여성 시리아 햄스터에 동작을 보람 있는 하는 동안 Extracellular 신경 전달 물질의 생체 내에서 변화를 측정

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

아밀로이드 -β 및 타우 신경 병증의 유전자 변형 마우스 모델에서 자발적 교대, 새로운 대상 인식 및 사지 걸림의 평가

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

의학

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

마 취 유도 Neurotoxicity 그대로 돼지 뇌에서의 연구에 대 한 Microelectrode 배열 기술의 적응

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

글리신/D-떠들고와 조미료에 감도와 NMDA 수용 체를 공부 하는 높은 처리량 칼슘-플럭스 분석 결과

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

자유롭게 이동 하는 쥐에서 뇌 파 기록 하는 동안 흥분 성의 아미노산의 Microdialysis

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

정상 및 헌팅턴 마우스의 급성 뇌 슬라이스에서 글루타메이트 방출 및 섭취량의 단일 시냅스 지표

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

기본 마우스 중뇌 뉴런에서 자발적인 Ca2+ 플럭스와 다운스트림 효과를 정량화

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

근위축성 측삭 경화증 모델에서 시냅스 기능의 실시간 형광 측정

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...