이 작품과 같은 2,10를 인용, 자금은 노화 연구, 에드워드 P. 스타일즈 신탁 기금 및 노스 웨스트 루이지애나의 바이오 메디컬 연구 재단에 대한 미국 연방에서 MF 살바토레에 대한 연구 기금 수상에 의해, 부분적으로 제공된 그리고 아이크 Muslow Predoctoral 원정대에서 학사 Pruett까지 LSU 보건 과학 센터 - 위조.

<ol>
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관심의 어떠한 충돌 선언 없습니다.

그림 1에 명시된 바와 같이 위에 설명된 방법은 도파민의 여러 설치 했어요 그리고 쥐 드나 마우스 중 하나에서 얻은 SN 또는 VTA 중 하나를 샘플로부터 조절 단백질의 TH, DAT, 그리고 VMAT2를 얻을 수 있습니다. 다시 말하지만,이 프로토콜을 수행의 장점은 조사가 도파민은 거의 모든 실험적인 패러다임 하에서 생체내에서 규제하고, 그동안, 어떤에서 필요한 동물의 수를 줄임으로써 상당한 실?...

HPLC 시스템 : 기본 시스템은 Shimadzu LC10-ADvp HPLC 펌프, 워터스시키는 717 자동 시료 주입기, 250 x 4.4 mm 5 미크론 Spherosorb ODS-1 C18 리버스 상 열 (워터스), Bioanalytical 시스템 (BAS) TL12 이중 유리로 구성되어 있습니다 탄소 전극, 두 BAS LC4B 전기 화학 감지기 및 워터스는 2 데이터 수집 및 통합 시스템의 능력을 주소서. 칼럼은 30-45에서 관리하고 있습니다 ° C (BAS LC22A 열 히터). 모바일 단계는 0.1 M의 인산 나트륨 (산도 3.0), 0.1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산), 1-옥탄 설폰산 (이스트만 - 코닥) 0.2-0.4 밀리미터, 그리고 0.45 미크론 필터를 통해 여과 0.35 %의 acetonitrile (V / V)입니다. 유속은 1.2 ML / 분입니다. 모바일 단계의 네 리터 배치는 산도, 옥탄가 Sulfonic의 산성과 열 온도를 조정하여 분판을 위해 최적화되었습니다. 모바일 단계는 재활용하고 있으며, 지속적으로 용존산소를 제거하는 헬륨 가스를 정화한다. 모바일 위상의 재활용시간의 합리적인 기간에 좋은 해상도를 유지하는 데 거의 필수적입니다. 모바일 위상 보관 수명은 각 실행의 첫 2-7 분 시간을 낭비 돌린다 플로우 스위치 (통합에 의해 제어)를 사용하여 유지됩니다. 전극은 Ag/AgC1 기준 전극에 대하여 약 0.78와 0.95V의 가능성에 유지 관리됩니다. 높은 잠재력의 전극은 트립토판 (및 NMDA 내부 표준)의 결정에 대해 독점적으로 사용됩니다. 0.78 V의 잠재력은 트립토판 이외 monoamines과 화합물의 검출을위한 잡음 비율로 우수한 신호를 제공합니다. chromatograms는 주소서 워크 스테이션의 하드 드라이브에 저장하고, 이후 처리 및 데이터 신진 대사 금액 및 그룹 데이터를 컴파일 계산을위한 Excel 스프레드 시트로 직접 전송됩니다. 펌프 : Shimadzu LC-10AD 셀 : BAS 크로스 흐름. 유리 탄소가 0.780과 0.9에서 전극을 조사50 V의 잠재력. 감지기 : BAS LC-4B는 듀얼 채널 모드에서 운영. 데이터 Acq. 시스템 : 워터스는 프로이 능력을 주소서. 분사기 : 워터시키는 717 칼럼 : 워터스 Spherosorb ODS-1, 5 μm의 입자, 4.4 mm X 250mm. <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 나트륨 Dodecyl 황산 (SDS) </td><td> - JT 베이커 </td><td> 4095-02 </td></tr><tr><td> Trizma 자료 </td><td> 시그마 </td><td> T1503-1KG </td></tr><tr><td> Trizma HCL </td><td> 시그마 </td><td> T3253-1KG </td></tr><tr><td> 글리세린 </td><td> 시그마 </td><td> G8773-500 ML </td></tr><tr><td> PVP-40 </td><td> 시그마 </td><td> PVP40-1KG </td></tr><tr><td> dPBS </td><td> Gibco </td><td> 21600-069 </td></tr><tr><td> Tween20 </td><td> 시그마 </td><td> P1379-500 ML </td></tr><tr><td> 글리신 </td><td> 시그마 </td><td> G8898-1KG</td></tr><tr><td> 개양귀비빛의 S </td><td> Fluka </td><td> 81,460 </td></tr><tr><td> Bromophenol 블루 </td><td> 시그마 </td><td> B8026-5G </td></tr><tr><td> Dithiothreitol </td><td> 시그마 </td><td> D-9163 </td></tr><tr><td> 단백질 표준이 MG BSA </td><td> 시그마 </td><td> P5619-25VL </td></tr><tr><td> 피어스 BCA 단백질 분석 시약 </td><td> 열 피셔 과학 </td><td> 23,223 </td></tr><tr><td> 정밀 플러스 단백질 표준 </td><td> 바이오는 힘이에요 </td><td> 161-0373 </td></tr><tr><td> [125I]-단백질, 특정 활동 </td><td> Perkin-엘머 </td><td></td></tr></tbody></table> 표 2. 특정 시약. <table border="1"><tbody><tr><td> 시약 </td><td> 수식 </td></tr><tr><td> 10% SDS </td><td> 10g SDS, 100 ML DI H 2 0 </td></tr><tr><td> 1퍼센트 SDS (산도 8.2) </td><td><ol><li> 10 ML10% SDS </li><li> 60.5 MG Trizma 자료 </li><li> 37.22 MG EDTA (에틸렌 다이아 민 테트라 초산) </li><li> 90 ML DI H 2 0 </li></ol></td></tr><tr><td> 구리 II 황산 솔루션 </td><td><ol><li> 1g 구리 II 황산 </li><li> 25 ML DI H 2 0 </li></ol></td></tr><tr><td> 배 샘플 버퍼 </td><td><ol><li> Trizma 자료 2.27 g </li><li> SDS 6g </li><li> Dithiothreitol 0.463 g </li><li> 글리세롤 30g </li><li> Bromophenol 블루 10 밀리그램 </li><li> DIH 2 0 (처음 H의 40 ML에 시약 위에 눈금 실린더에 2 0을 추가, 부피가 100 ML에 도달할 때까지 H 2 0 추가) </li><li> HCL (추가 등 6.85의 산도에 도달하는 데 필요한) </li><li> 50 2.0 ML 튜브의 솔루션을 고정. </li></ol> (100 ML하게) : 3 배 샘플 버퍼의 볼륨이 필요 = 샘플에서 사용되는 SDS의 ½ 볼륨. </td></tr><tr><td> 1X 샘플 버퍼 </td><td> DI H 2 0을 사용하는 1X 샘플 버퍼에 3 배 샘플 버퍼를 희석 </td></tr><tr><td> 10X<html> 버퍼를 실행하면 (4 패를 만듭니다) </td><td><ol><li> Trizma베이스 121.1 g </li><li> 글리신 577g </li><li> SDS 40g </li></ol></td></tr><tr><td> 10X 전송 버퍼 : (4 패를 만듭니다) </td><td><ol><li> 글리신 360g </li><li> Trizma베이스 96g </li></ol></td></tr><tr><td> 개양귀비빛의 </td><td><ol><li> 개양귀비빛의 S. 0.5 g </li><li> 아세트산의 산성 5 ML </li><li> DI H 2 0 95 ML </li></ol></td></tr><tr><td> 0.2 % HCL 솔루션 </td><td> DI H 2 0 500mL에서 5.2 ML HCL </td></tr><tr><td> PVP-T20은 Soln 차단. (4 패를 만듭니다) </td><td><ol><li> PVP-40 40g </li><li> dPBS 38.2 g </li><li> Tween20 2g </li><li> Thimerisol 0.4 g </li><li> 1M 트리스 산도 7.6 (60.6 g 트리스 HCL + 500 ML DI H 2 0에서 13.9 g 트리스 자료) - 50 ML </li></ol></td></tr><tr><td> 10X 얼룩 버퍼 (4 패를 만듭니다) </td><td><ol><li> 십대 초반 20 20g </li><li> 트리스베이스 14g </li><li> 트리스 HCL 61g </li></ol></td></tr></tbody></table>ontent &quot;&gt; 표 3. 단백질 분석과 서양의 모래 바닥 수식. 티로신 hydroxylase 표준은 :이 실험실에서 사용하는 보정 TH 단백질 및 인산화 표준은 궁극적으로 닥터 존 Haycock 11 실험실에서 유래 이전에 보정 TH 표준에 대한 TH 단백질 함량과 인산화의 stoichiometries에 대해 분석했다 PC12 세포 추출물에서 파생됩니다 .

comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area

도파민은 CNS에 적극적으로 공부 신경 전달 물질이다. 사실, 전위의 활동 및 보상 관련 행동의 개입은 도파민 조절과 관련된 분자 결함으로 문의 50 여년간을 육성하고있다. nigrostriatal 및 mesoaccumbens 경로의 터미널 필드 지역의 규제에 대한 분자 기초시 뇌 초점에서 도파민 규제 이러한 질문의 대부분은, striatum과 핵의 accumbens. 또한, 이러한 연구는 서부 유럽 표준시 조직 무게로 정상화와 도파민 조직 내용의 분석을 집중하고 있습니다. 같은 티로신 hydroxylase (TH) 단백질 번째 인산화, 도파민 수송 (DAT) 및 기공을 갖는 모노 아민 전송기 2 (VMAT2) 단백질로, 도파민 조절 단백질의 조사는 종종 동일한 예제에서 도파민 조직 내용의 분석에 포함되지 않습니다. 도파민 조직 내용과 그 조절 단백질 (POST-tra를 포함하여 모두를 분석할 수있는 능력nslational 수정)은 단백질 수준과 TH, DAT, 또는 VMAT2의 기능과 도파민의 관계를 해석에 내재된 능력을 제공, 또한 샘플 경제를 확장뿐만 아니라. 아직은 적은 비용,로이 번역은 수사관 &#39;선택의 거의 모든 패러다임에서 도파민의 분자 조절에 대한 식견을 생산하고 있습니다. 우리는 midbrain에 분석을 집중. SN 및 VTA는 일반적으로 도파민 규제 대부분의 연구에서 소홀히하고 있지만, 이러한 핵 쉽게 실천으로 해부한다. 종합 도파민 조직 컨텐츠와 TH의 판독, DAT, 또는 VMAT2가 실시됩니다. 이 행위에 대한 SN 및 VTA의 도파민 기능의 영향에 문학 급성장, 그리고 그 안에 1-5 외인성 물질 또는 질병 프로세스의 impingements있다. 또한, 이러한 성장 요인과 같은 화합물은 SN 또는 VTA에서 비교적 큰 범위까지, 도파민과 도파민 조절 단백질에 지대한 영향을 미칠 <sup&gt; 6-8. 따라서이 방법은 특정 치료 행태 및 도파민 조절을 조절하는 방법에 자신의 질문을 연장하려는 실험실에 대한 참조용으로 제공됩니다. 자, 다단계 방식은 도파민 조직 내용의 분석, TH의 단백질 수준, DAT, 또는 VMAT2, 그리고 쥐 midbrain의 substantia nigra 및 VTA의 TH의 인산화 위해 제공됩니다. 회 인산화의 분석뿐만 아니라 번째 활동 규제하는 방법뿐만 아니라, 주어진 패러다임의 somatodendritic 핵에 영향 신호 폭포에 상당한 통찰력을 얻을 수 있습니다. 우리는이 두 핵 각각의 핵 (그림 1) 특정 생체내에서 도파민 규정의 분자 메커니즘을 공개 프로필을 생산하고 해부 조직의 샘플 처리를 분리하는 해부의 기술을 설명합니다.

Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

도파민은 분명히 세포 기관 및 도파민 뉴런의 dendrites을 포함 midbrain의 핵에서 규제하고 있습니다. 여기 substantia nigra (SN)과 설치류의 복부 tegmental 지역 (VTA)의 midbrain의 핵에 도파민 규정에 따라서 절개 및 샘플 처리 결과를 극대화하는 접근 방식과 결론 및 통찰력을 설명합니다.

Substantia Nigra 및 복부 Tegmental 지역의 도파민 규정의 종합적인 프로파일

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

comprehensive-profiling-dopamine-regulation-substantia-nigra-ventral

Research

JoVE Journal

Neuroscience

25.6K 조회수.  Louisiana State University Health Sciences Center. 도파민은 분명히 세포 기관 및 도파민 뉴런의 dendrites을 포함 midbrain의 핵에서 규제하고 있습니다. 여기 substantia nigra (SN)과 설치류의 복부 tegmental 지역 (VTA)의 midbrain의 핵에 도파민 규정에 따라서 절개 및 샘플 처리 결과를 극대화하는 접근 방식과 결론 및 통찰력을 설명합니다.

비디오: Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

동시 전압 클램프 및 Amperometry과 도파민 릴리스의 단일 세포 측정

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

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In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

복부 병아리 중뇌에있는 마이크로 RNA의 오보, 식

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

성인 마우스의 중간 뇌 도파민 뉴런의 수의 환경 변조

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

레이저 캡처 이외에 Microdissection - 개별 도파민 신경 세포의 분리 및 전체 복부 피개 영역의 데모

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

고성능 액체 크로마토그래피 분석 및 Synaptosomal 도파민 통풍 관의 사용에 의해 쥐에서 Dopaminergic 항상성의 평가

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

광학 Fractionator 표준 현미경 장비를 사용 하 여 마우스 Substantia Nigra에서 Dopaminergic 신경 수 stereological 추정

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

탐지 기술 현장에서 혁신적인 RNA에 의해 설치류 핵 Accumbens에서 도파민 D1-알파 수용 체의 식별

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

급성 척수 조각 Substantia Gelatinosa 신경에 있는 전체 셀 패치 클램프 기록에 대 한 준비

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

다발성 경화증을 가진 개별을 위한 포괄적인 부검 프로그램

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

빠른 스캔 순환 볼탐법 (CIS-FSCV)과 결합 된 주입 및 자극을 결합하여 Phasic 도파민의 복부 테자멘트 영역 수용체 조절을 평가합니다.

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...