저자는 재정 지원에 대한 기술 지원, CIMO 재단과 FGSN에 대한 Neurotar (주)에게 감사의 말씀을 전합니다.

동물 과목을 포함하는 절차는 국립 동물 실험위원회, 핀란드에 의해 승인되었습니다. 이미징을위한 1 동물 준비 <ol><li> intraperitional 케타민 (IP) 주입 (체중 당 0.08 mg)의과 자일 라진 (체중 당 0.01 ㎎)을하여 마우스를 마취. 후면 발가락 핀치 반사와 마취를 확인합니다. </li><li> 눈 탈수으로부터 눈을 보호하기 위해 (Viscotears를) 삭제에 동물의 눈을 담근다. </li><li> 저체온증을 방지하기 위해 37 ° C에서 가열 패드 (SUPERTECH)에 마우스를 넣습니다. </li><li> 70 % 에탄올로 영상에 대해 지정된 발 패드를 청소합니다. </li><li> 피부와 커버 글라스 사이에 침지 커플 링 풋 패드의 피부에 물 방울을 추가한다. </li><li> 커버 클래스와 금속 링 아래 위치되는 뒷발 하에서 플라스틱 포장재를 넣어. </li><li> 피부를 평탄화 플라스틱 포장 재료의 두께를 조정한다. </li><li> 커버 GLAS에 물 방울을 넣어커버 유리와 대물 사이 침지들. </li></ol> 2 금속 고정 장치 반지 준비 <ol><li> 피부의 안정화 금속 고정 장치를 이용한다. 우리는 금속 링 (Neurotar (주), 핀란드의 제공) 커뮤니티 디자인 보호 두 날개 고정 장치를 사용합니다. , superglue과 주사기를 채우기 링에 바늘을 통해 superglue의 작은 방울을 넣어 조심스럽게 링 표면의 균일 한 커버리지 접착제를 확산. 접착제의 양이 과대 시야를 감소시키고 후속 이미징을 방해 할 수 있기 때문에, 단지 균일 따르면에 필요한 접착제의 최소량을 넣어 중요하다. NOTE : (아래에서)과 금속 바아 (커버)을 표준 현미경 유리 슬라이드의 선택적 조합은 피부를 평평하게하고 적절한 어셈블리 (14)의 광 결합을 보장하기 위해 사용될 수있다. 두 종이 클립 유리 및 금속 바아 표면 사이의 발을 고정하는 데 사용될 수있다 (도 4 </strong&gt;). </li><li> 현미경 커버 슬라이드 (5mm 직경, 전자 현미경 과학)와 함께 반지를 커버. </li><li> 전동 현미경 무대에 montaged되는 단단한 금속 막대에 링 고정 장치에 나사. </li></ol> 3 이미징 절차 <ol><li> thy1-YFP-H 마우스를 이용하는 경우, 신경 섬유를 시각화하는 형광 램프 스펙트럼의 청색 부분을 선택한다. 표면 형광 모드에 대한 관심의 신경을 찾아 초점을 맞 춥니 다. </li><li> 길이 이미징을위한 장소의 좌표를 기록합니다. 기준점으로 손목 패드를 사용한다. 다음 촬상 세션 중에 제 손목 패드를 감지하고 동일한 상류 큰 신경 섬유를 찾아 저장된 좌표를 사용 돌아가서. 그것은 참조 동일한 시스템을 유지하는 금속 바아에 직교하는 동일 방향으로 다리 패드를 배치하는 것이 필수적이다. </li><li> 두 광자 모드를 켭니다. thy1-YFP-H 마우스의 경우, 레이저 방사의 950 nm 파장을 사용하는 것이 적합신경 섬유를 시각화합니다. </li><li> (450부터 520에서 YFP 표지 된 신경의 촬상 550 ㎚에 대한 580에서 YFP 형광 및 모발의 자기 형광의 검출을 위해 630 ㎚에 제 고조파 발생 검출을위한 480 nm의, 행) 선택 레이저 파장 및 배출 채널, 시작 표백 광 손상을 방지하기 위해 낮은 레이저 파워 (~ 5 %). 광전자 증 배관 튜브의 전형적인 전압은 이미징 이런 종류의 600-700 V이다. </li><li> (1-10 원하는 해상도 (512 X 512 또는 빨리 이벤트 측정 640 × 640, × 800 또는 형태학 이미징 1024 1024 X 800), 축 방향의 공정 (1-3 μM), 시료의 두께와 시간 간격을 설정할 분). 노출 시간은 하나의 화소 당 1 밀리 초 정도이다. </li><li> 먼저 소프트웨어 촬상 공간의 상부 및 하부 경계를 표시한다. </li><li> 병변 전에 참조 용 참조 화상 스택을 획득. 필요하면, 수 분 동안 기준을 기록 할 수있다. </li><li필요할 때&gt; 반복 (적어도 매일 10 ~ 15 분) 동물의 호흡 속도와 반사 신경을 확인 마취제의 추가 금액을 적용합니다. </li><li> 티슈로 패드를 깨끗한 영상과 완전히 깨어 때까지 36 ° C에서 복구 상자에 마우스를 넣어 후. 참고 : 일반적으로, 레이저 유도 병변과 함께 한 영상 세션의 지속 시간 (아래 참조) 1 시간을 초과하지 않는다. 우리는 동물이 깊이마다 10 ~ 15 분 마취되어 있는지 확인하는 것이 좋습니다; 이 자체 이미징을 방해하지 않아야하지만, 실험 절차가 계획되는 동안 고려되어야한다. 단일 케타민​​ / xylazyne 투여의 효과의 지속 시간은 따라서 우리가 25 ~ 30 분 후 복용량을 1/2 ¼ 추가로 적용하는 것이 좋습니다, 40 분에 30이다. NOTE : 실험은 각각의 동물에 대한 촬상 세션 주파수 재사용의 악영향을 방지하기 위해 하나의 세션 당 2 일을 초과하지 않도록 계획 될 때뿐만 아니라 고려되어야petitive 마취. </li></ol> (4) 레이저 병변 <ol><li> 참조 화상 스택을 획득 한 후, 마이크로 병변을 만드는 표백 프로토콜을 사용한다. </li><li> 100 %로 레이저 파워를 높입니다. </li><li> 이자 (100-1,000x 표준 이미징 가지 경우에 비해 이상)의 지역 당 100 ~ 1000 밀리 초에 노출 시간을 늘립니다. </li><li> 병변의 영역을 설명합니다. </li><li> 표백에 전환하여 병변을 확인합니다. </li><li> 으로 보통의 영상 모드로 전환하고 시간 경과 녹화를 계속합니다. </li></ol> (5) 데이터 처리 및 분석 <ol><li> ImageJ에 8 스펙트럼 Unmixing 플러그인을 사용하여 unmixing와 (요아킴 월터 진행 <a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html을</a> ). </li><li> 이미지 스택을 열고 채널을 분할합니다. </li><li> 배경 측정을위한 신경 또는 머리카락이없는 지역을 찾아보십시오. 지역 O를 넣어그 지역에서 F 관심. </li><li> 신중하게 신경 분명히 구별 될 모발의 부분 윤곽. </li><li> 이 머리 별개 이미지 부분에서 신경 개요. </li><li> 행렬을 unmixing 저장합니다. </li><li> 전체 스택에 대한 측정 unmixing 행렬을 적용합니다. </li><li> *의이 .tif 형식의 새로운 이미지 처리 스택을 저장합니다. </li></ol>

<ol>
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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

이 동영상 프로토콜에서는 단일 신경 말단의 비 침습적 종 이광자 촬상 방법을 보여준다. 피부 신경 밀도의 역학 건선 및 말초 신경 병증이, 등의 질환과 외상 구에 영향을받습니다. 두 광자 이미징은 콜라겐 매트릭스에있는 신경 섬유 구조의 상세한 분석을 할 수 있습니다. 리포터 유전자 변형 마우스의 사용은 신경 섬유의 염색에 관한 문제를 방지하는 데 ?...

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

피부 신경 말단 다른 병태 생리 학적 상태에 따라 동적 인 변화를 겪는다. 신경 섬유는 말초 신경 병증이 모톤 신경종 또는 3과 같은 질병의 과정에서 변성 및 재생 또는 재구성의 프로세스를 수행 할 수있다. 외상 후 피부의 신경 말단 역학의 중요한 부분은 손상된 영역의 reinnervation이다. 그러나, 신경 종말의 조사를위한 일반적인 방법은 진행중인 프로세스 4에 실시간 정보를 결여 생체의 조직 학적 절편이다. 유 전적으로 인코딩 된 형광 마커를 사용하여, 따라서 구조적 변화에 훨씬 더 풍부하고 관련 정보를 획득하는, 살아있는 동물의 피부에있는 신경 말단을 추적 할 수있다. 피부 신경 종말의 조사는 종래의 형광 현미경이 있지만, 피부 조직의 강한 광산란 강하게 품질을 저해하여 가능하다데이터 5를 인수했다. 다 광자 현미경에 의한 유일한 목적의 초점으로부터의 형광 발광을 초래 여기 광의 광자의 에너지의 비 - 선형 합에 강하게 산란 조직의 고해상도의 영상을 획득하는 것을 허용한다. 이 효과는 피부 조직 (6)에서의 측정 결과에 대한 강력한 침투 깊이의 증가와 신호 대 잡음 비의 향상을 이끈다. 이미징을위한 동일한 레이저를 사용하면, 신경 섬유 (7)의 선택적인 박리를 제조 할 수있다. 다음의 프로토콜에서는 선택적 레이저 병변 시판 다 광자 현미경 시스템을 사용하여 결합 된 리포터 유전자 변형 마우스에서 생체 내 피부 신경 종말의 길이의 촬상 방법을 보여준다.

<table border="0" cellpadding="0" cellspacing="0" width="1231">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

피부, 신경과 같은 신경 병증 성 통증은이 같은 일을 센싱뿐만 아니라 병리 생리적 과정의 프로세스에 관여된다. 그들의 근접에 표면의 위치는 그대로 동​​물을 생활에서 피부 신경 말단의 미세한 영상을 용이하게한다. 다 광자 현미경을 사용하면, 피부 조직의 강한 광 산란 문제를 극복 미세 영상을 얻을 수있다. (주변 감각 신경 포함) 뉴런 네-1 프로모터의 조절하에 EYFP을 표현 기자 형질 전환 마우스는 최대 몇 개월 또는 평생 오랜 기간 동안 개별 신경 종말의 종 연구에 적합하다. 또한, 촬상 용으로 동일한 펨토초 레이저를 이용하여, 이는 신경 섬유의 재구성 연구에 신경 섬유의 매우 선택적 병변을 제조 할 수있다. 여기서, 우리는 생체 내에서 촬상 종 다 광자위한 간단하고 신뢰성있는 프로토콜을 제시마우스 피부의 신경 말단에 레이저 기반 미세.

그것이 가능 기재된 기술을 사용하여 병변 후 동일한 광섬유를 추적하고 손상된 신경 종말 (도 1)의 분해를 연구. 120-150 μm의 두께의 적층 체의 취득은 시야에 전체의 섬유를 유지하기 위해 수 일 동안 반복 이미징 대개 적절하다. 플라스틱 포장재가 피부를 평평하게 조정되면 병변은 일반적으로 간결하게 제조 될 수 있으므로, 콜라겐 층에 균일 한 강도의 화상 <s...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

동적 길이 이미징 및 기자 형질 전환 생쥐의 신경 종말의 선택적 레이저 병변에 대한 프로토콜은 제공됩니다.

피부의 단일 신경 종말의 생체 두 광자 현미경에서

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

in-vivo-two-photon-microscopy-of-single-nerve-endings-in-skin

Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

10.6K 조회수.  University of Helsinki.  동적 길이 이미징 및 기자 형질 전환 생쥐의 신경 종말의 선택적 레이저 병변에 대한 프로토콜은 제공됩니다. 

비디오: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

두 광자 현미경을 사용하여 마우스 척수의 생체내 이미징에

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

연구

신경과학

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

동시에 두 광자<em&gt; 생체</em&gt; 마우스 Retrosplenial 코어 텍스에서 시냅틱 입력의 이미징 및 시냅스 대상

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

HIV 유도 된 신경 염증의 마우스 모델에서 이광자 현미경을 사용하여 뇌 혈관 구조의 정량

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

의 헤어 난포 피침 엔딩에서 생활 신경 터미널 라벨의 광학 모니터링<em&gt; 전의 VIVO</em&gt; 마우스 귀 피부

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

두뇌 조각에서 신경 모 수석에서 2 광자 칼슘 이미지

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo에서 신생아 쥐에 있는 대뇌 피 질의 뉴런의 두 광자 영상

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

유리 마이크로 파이펫을 사용하여 국소 ATP 배출에 의한 혈관 반응을 연구하기 위한 Vivo 3차원 2차원 광자 현미경 검사법

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

생체 내 2 광자 형광 평생 이미징 현미경 을 사용하여 머리 고정 행동 마우스에서 단백질 키나제 활동을 시각화

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...