저자는 승진의 과학 KAKENHI 그랜트 JP16K11200 및 17 H 02223, 일본 사회와 가나자와 의료 대학 S2016-8 및 C2017-3에서 연구 보조금에 의해 지원 되었다. 우리는 사진 촬영에 그의 지원에 대 한 Yuhichi 것을 감사 합니다.

모든 절차는 동물 관리 위원회의 가나자와 의과대학의 승인와 일본의 생리 사회의 원칙에 따라 수행 됩니다.
1입니다. 건설 유리 Optrode 홀더
참고: 유리 optrode 홀더를 구축 하는 상업 전극 홀더 사용 됩니다 (그림 1A).
<ol><li>부드럽게 소유자의 배럴에서 압력 제어를 위해 스틸 튜브 밖으로 당겨.</li>
	<li>드릴링에 의해 소유자의 배럴에서 핀 좌석 쪽의 구멍을 확대. 깊이 3 m m 직경에서 그리고 12mm 구멍을 확인 합니다.</li>
	<li>스테인리스 강 관 (직경에서 3 m m, 두께, 0.5 m m 그리고 길이; 8 m m를 넣어 그림 1A 2) 구멍에.</li>
	<li>구멍에 ⌀2.5 m m 깃 봉 (그림 1A1)에 대 한 짝짓기 소매를 분할 하는 세라믹을 삽입 합니다.</li>
	<li>에폭시 접착제는 L 자 모양의 사 개에 전극 홀더의 배럴을 수정 합니다.</li>
	<li>사 개에 구멍 (직경에서 3 m m)를 확인 하 고 나사와 조작에 첨부.</li>
</ol>2. 머리 포스트 설치
<ol><li>회로 보드 스페이서의 만든 맞춤식 머리 게시물을 준비. 열 회로 보드 스페이서 밀링 커터 cuboidal 모양으로 만들고 (그림 1B) 톱으로 2 머리 게시물에 그것을 잘라. 밀링 커터 헤드 게시물의 하단에 평평 하 게.</li>
	<li>빛에 민감한 opsin 연구에 대 한 특정 뉴런 형식에 표현 하는 유전자 변형 동물을 준비 합니다. 이 작품에는 억제 신경 channelrhodopsin-2 (ChR2)11 을 표현 하는 유전자 변형 마우스 (VGAT-ChR2 마우스) 사용 됩니다.</li>
	<li>복 관리에 의해 medetomidine의 0.3 mg/kg, 4.0 mg/kg, midazolam의 및 butorphanol의 5.0 mg/kg의 혼합물으로 동물을 anesthetize. 동물 마 취 완벽 하 게 확인, 곤란 꼬리의 반응을 테스트 합니다.</li>
	<li>생리대에 stereotaxic 프레임에 마우스를 놓습니다. 청소 stereotaxic 프레임 및 난방 패드 70% 알코올로 미리. 치아를 물린 바의 구멍에 놓고 가볍게 코 클램프를 조여. 귀 막대를 사용 하 여 머리의 양쪽 모두를 수정 합니다. 머리 귀 막대로 고정 올바르게 때 코 클램프를 조입니다. 눈에 눈 연 고를 적용 합니다.</li>
	<li>작은 위로 머리 피부를 면도 하 고 액체 글 루 콘 산의 면봉으로 두 피를 청소. 수술 하기 전에 수술 도구와 머리 압력솥 (121 ° C, 15 분)와 게시 모든 소독 합니다.</li>
	<li>두 피 중간 선 따라가 위로 잘라 고 옆으로 밀어. 눈 (약 20 m m)를 머리 뒤쪽에서 절 개를 확인 합니다. 70% 알코올에 담근 면봉으로 여과 제거 하 고 건조 한 두개골.</li>
	<li>조작자를 머리 게시물을 연결 합니다. 머리 게시물 두개골 bregma 조작자를 사용 하 여 주위에 배치 합니다.</li>
	<li>단위체 (4 정), 촉매 (1 방울), 및 냉장고에 미리 냉장은 작은 접시에 치과 시멘트의 폴리머 (1 작은 스푼) 섞는다. 정면에 해골 머리 게시물 수정 두개골의 정수 리 뼈 (1 g) 아래 치과 시멘트를 넣어.</li>
	<li>치과 시멘트 하드 후, stereotaxic 프레임에서 마우스를 제거 합니다. 노출 된 피부에 항생제 연 고를 넣어. Medetomidine 길 항 제 (0.3 mg/kg)과 항생제 (oxytetracycline; 20 mg/kg) 주사. 깨우 고, 주택 케이지를 반환 때까지 장소와 모니터에 마우스에에서 패드.</li>
	<li>하우스 몇 가지 농축 장치 개별 주택 장에 동물. 케이지를 청결 한 유지 하 고 감염을 방지 하기 위해 매일 침구 동물 종이 변경 합니다. 신중 하 게 모니터링 동물 표시 불편의 어떤 표시 든 지. 상처에 적용 lidocaine 통증이 의심 되는 경우. 관리 meloxicam 또는 carprofen 두 머리 포스트 설치 및 craniotomy 수술 후 48 h 한 번 모든 24 h. 로컬 lidocaine 주어질 수 있다 또한 경우에 NSAID 적합 하지 않습니다.
</li>
</ol>3입니다. 새 환경 순응
<ol><li>2 일 (최소), 마 취에서 회복 후 녹음 실에서 머리 고정 상태로 동물 적응. 적어도 5 일 동안 새 환경 순응 세션을 수행 합니다.</li>
	<li>새 환경 순응 세션 동안 사용자 클램프에 고정 머리 게시물 사용 녹음 실에서 동물을 놓습니다. 움직임을 제한 하기 위하여 세션 동안 커버 맞춤 수 지 반 튜브 (그림 1C), 시체 3 차원 프린터를 사용 하 여 만든. 참고: 1 일, 새 환경 순응 세션 5 분 긴를 이어야 한다. 그것은 다음 증가 매일 10, 20, 40, 및 60 분 동안 2, 3, 4, 및 5, 각각.</li>
	<li>각 세션의 끝에, 동물을 음식 보상을 제공 합니다. 동물에 새 환경 순응 기간 동안 지속적으로 투쟁을 하는 경우는 세션을 중지 합니다.</li>
</ol>4입니다. craniotomy
참고: 새 환경 순응, 후 한 craniotomy가 이루어집니다 녹음에 대 한 뇌 영역을 통해. craniotomy 마 취, 머리로 설치 게시물 stereotaxic 프레임에서 수행 됩니다. 사후 작업 절차는 머리 게시물 설치는 동일 합니다.
<ol><li>Craniotomy, 하기 전에 액체 글 루 콘 산 및 70% 알코올 면봉으로 두개골을 청소.</li>
	<li>조심 스럽게 치과 함께 녹음 사이트를 통해 두개골을 다쳤어요. 뼈를 충분히 얇은 되 면 메스의 팁으로 넘어가지 고 그것을 제거 합니다.</li>
	<li>두개골을 강화 노출 지역의 치과 시멘트를 넣어.</li>
	<li>치과 시멘트 하드 후 실리콘 접착제로 노출 된 뇌 영역을 커버. 수술 후 절차는 2.8 단계에서 설명한 대로.</li>
</ol>5. electrophysiological 기록
<ol><li>동물 electrophysiological 녹화를 수행 하기 전에 craniotomy 후 최소 1 일에 대 한 복구를 허용 합니다. 같은 방법으로 그것을 녹음 시작 될 때 새 환경 순응 세션 동안 한 녹음 실에서 동물을 놓습니다.</li>
	<li>붕 규 산 유리 모 세관에서 유리 펫 만들기 (OD = 1.5 m m, ID = 0.9 m m, 길이 90.0 m m) 전극 끌어당기는 사람에. 그들의 팁 직경은 2-3 µ m 이다, 그리고 그들의 저항에서 4-6 m ω가 때 그들은 10mm PBS로 가득. 전극의 길이 40-50 m m입니다. 일부 녹음에 2% neurobiotin 10 m m PBS에 추가 됩니다.</li>
	<li>주문 품 optrode 홀더를 채워진된 유리 피 펫을 연결 합니다.</li>
	<li>유리 피 펫에 Ag Cl 코팅 실버 와이어 (직경에서 0.2 m m)를 넣어. 실버 와이어를 핀을 통해 얇은 코팅된 전기 와이어를 연결 합니다. 녹음 앰프의 headstage에 핀을 연결 합니다.</li>
	<li>광섬유 패치 코드를 연결 (코어 = 960 µ m, 클 래 딩 1000 µ m, NA = = 0.63) 지 르 코니 아 깃 봉을 가진 (OD = 2.5 m m) optrode 홀더에 소매 짝짓기 분할. 아무의 팁 연락처 유리 피 펫의 뒷면까지 소매에 패치 코드를 밀어. 패치 코드 제공 빛 (파장 = 465 nm) LED에서. LED 빛은 단일 채널 LED 드라이버에 의해 제어 됩니다.</li>
	<li>craniotomy 통해 실리콘 접착제를 제거 합니다. 넣어 예 열된 염 분 (38 ° C) 기록 세션 동안 건조에서 뇌 표면 방지 하기 위해 주기적으로 뇌 표면에. 필요한 경우 날카로운 핀셋으로 경질을 제거 합니다.</li>
	<li>유리 전극 조작자와 뇌 조직에 삽입 합니다. 이 삽입 될 때 전극 저항을 모니터링 합니다. 저항 되는 전극의 팁 손상 될 수 있습니다 때문에 예상 보다 낮은 전극을 교체 합니다. 관심의 깊이에서 검색 하 고 2 ~ 5 µ m 단계에 깊이 조정 하 여 단일 단위 스파이크를 분리 합니다.</li>
	<li>단일 단위 격리 된 후에, LED 빛을 전달 하 고 빛 자극에 응답을 관찰.</li>
	<li>세포 유형의 식별 후 신경 활동을 기록 합니다. 2-3 h에 대 한 녹음을 수행 합니다. 녹음에서 만든 연속 일 (최대 2 일의), 경우 실리콘 접착제 craniotomy 커버. 녹음을 완료 하 고 동물 희생, 머리 게시물을 검색 하 고 세척 하 고 그것을 다시 아세톤에 넣어.</li>
</ol>

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저자는 공개 없다.

Optogenetics 신경 과학에 강력한 도구가 되고있다. 그것은 특정 신경 통로의 활동을 조작 뿐만 아니라 특정 신경 형식에서을 식별 비보에 이용 되었습니다. 신경 종류의 신경 활동의 명확한 신경 회로의 메커니즘의 이해를 촉진합니다. 여기, 우리는 깨어 VGAT ChR2 쥐의 IC에 유리 전극을 통해 녹음 사이트에 빛을 전달 하는 방법을 설명 했다.
설명 하는 방법을 몇 가지 중요 ?...

다양 한 종류의 다른 기능을 하는 신경 중추에 의하여 이루어져 있다. 이러한 서로 다른 유형의 신경 신경 회로 내에서 작동 하는 어떻게 신경 과학에서 중요 한 관심사 중 하나입니다. 그러나, 많은 두뇌 지구에서 그것은 되었습니다 하지 몇 가지 예외와 함께 전기 스파이크 신호 자체에 분명 차이가 있기 때문에 전기 활동의 비보에 녹음에 신경 종류를 구별 하는 것. Optogenetics의 최근 발전 돌파구1,2를 만들었습니다. 빛에 민감한 opsin (예를 들어, channelrhodopsin-2) 특정 신경 종류에 표현 하는 유전자 변형 동물을 사용 하 여, 그것은 되었다 vivo에서 녹음3, 에서 효율적으로 신경 종류를 구별할 수 4,,56. 이 동물에 빛에 민감한 opsin와 뉴런 전기 녹음 하는 동안 가벼운 자극을 제공 하 여 흥분 하지만 다른 신경 있습니다. Opsin 양성 신경, 따라서 쉽게 구별 된다 다른 신경 종류에서 빛에 대 한 답변으로.
Optogenetics 실험, 그것이 기록 사이트에 빛을 전달 하기 위해 중요 합니다. 비-침략 적 방법으로 빛은 종종 뇌의 표면에서 감독. 그러나, 빛의 강도 감소 뇌 조직 속으로, 때문에 두뇌의 표면에서 깊은 두뇌 지구를 자극 하기 어렵다. 특히, 그것은 종종 깨어 있는 동물에서 녹음의 경우 뇌 표면의 광 투명성은 낮은, 깊은 두뇌 지역 도달 자극 빛 어렵습니다. 마 취 동물에 electrophysiological 실험 자주 수행 되었습니다 몸 움직임 녹음에 잡음을 발생 하기 때문에. 그러나 잘 설명 된 대로, 마 취 신경 응답7,8,,910변경으로 알려져 있다. 따라서, 그것은 깨어 있는 동물을 사용 하 여 마 취의 인공 효과 없이 신경 응답을 공부 하기 위하여 필요가 있다. 마 취 동물 실험, 달리 electrophysiological 녹음 깨어 동물 실험에서 수술에서 회복 후 수행 됩니다. 수술과 기록 사이 간격 동안 조직 exudate 자주 뇌 표면에 축적 하 고 낮은 뇌 표면의 광 투명성을 만든다.
여기, 우리는 주문 품 유리 optrode를 사용 하 여 깨어 마우스에서 단일 단위 기록 기록 하는 방법을 설명 합니다. 이 방법에서는, 빛은 안정적으로 깊은 두뇌 지구에서 빛으로 기록 된 신경 자극 수 있도록 녹음 유리 전극을 통해 배달 됩니다. 이 주문 품 optrode 시스템 접근 가능 하 고 저렴 한 재료를 이루어져 있고 조립 하기 쉽습니다.

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			<td>Narishige</td>
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			<td>Monofiberoptic patchcord, OD, 2.5 mm, core = 960 mm, cladding = 1000 mm, NA = 0.63</td>
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			<td>PB-7</td>
			<td>Dual-stage glass micropipette puller</td>
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			<td height="40" style="height:40px;width:247px;">Borosilicate glass capillary</td>
			<td style="width:140px;">Narishige</td>
			<td style="width:393px;">GD-1.5</td>
			<td style="width:191px;">Bolosilicate glass capillary, OD, 1.5mm, ID, 0.9 mm, 90.0 mm long</td>
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			<td height="40" style="height:40px;">GENTACIN</td>
			<td>MSD CO., Ltd</td>
			<td>185711173</td>
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			<td height="40" style="height:40px;">Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J</td>
			<td>Jackson Labs</td>
			<td>#14548</td>
			<td>VGAT-ChR2 mice</td>
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			<td height="40" style="height:40px;">Multiclamp 700B</td>
			<td>Molecular Devices</td>
			<td>2500-0157</td>
			<td>Microelectrode amplifier</td>
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optogenetics identification of a neuronal type with a glass optrode in awake mice

그것은 어떻게 다른 신경 과학에서 중요 한 관심사 종류 뉴런의 신경 회로에서 작동. Optogenetics의 최근 발전은 광범위 한 뇌 영역에 vivo에서 electrophysiological 실험에서 신경 유형 식별을 활성화 했습니다. Optogenetics 실험, 그것이 기록 사이트에 빛을 전달 하기 위해 중요 합니다. 그러나, 그것은 종종 뇌의 표면에서 깊은 두뇌 영역을 자극 빛을 제공 어렵다입니다. 특히, 그것은 종종 깨어 있는 동물에서 녹음의 경우 뇌 표면의 광 투명성은 낮은, 깊은 두뇌 지역 도달 자극 빛 어렵습니다. 여기, 우리는 주문 품 유리 optrode를 사용 하 여 깨어 마우스에서 빛을 스파이크 응답 하는 방법을 설명 합니다. 이 방법에서는, 빛은 안정적으로 깊은 두뇌 지구에서 빛으로 기록 된 신경 자극 수 있도록 녹음 유리 전극을 통해 배달 됩니다. 이 주문 품 optrode 시스템 접근 가능 하 고 저렴 한 재료를 이루어져 있고 조립 하기 쉽습니다.

그림 2, 우리는 팁 크기의 효과 유리 펫 펫 (그림 2A-B)의 끝에 빛 파워에의 길이 검사합니다. 가벼운 힘 1 m m 팁에서 배치 하는 광 파워 미터에 의해 측정 되었다. 팁 크기 전체 길이 변화 때 2.5 µ m 팁 크기 설정 하는 동안, 변화 하는 때 전체 길이 50 ± 2 mm로 설정 되었습니다. 유리 피 펫의 샹 크 8 m m로 설정 되었습니다...

Watch this Scientific Journal Video about Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice at JoVE.com

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

깨어 있는 쥐에 유리 Optrode 신경 형식의 Optogenetics 식별

이 작품은 optogenetic 단일-단위 맞춤 유리 optrode를 사용 하 여 깨어 마우스에서 안정적으로 녹화를 수행 하는 방법을 소개 합니다.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

optogenetics-identification-neuronal-type-with-glass-optrode-awake

Research

JoVE Journal

Neuroscience

6.7K 조회수.  Kanazawa Medical University. 이 작품은 optogenetic 단일-단위 맞춤 유리 optrode를 사용 하 여 깨어 마우스에서 안정적으로 녹화를 수행 하는 방법을 소개 합니다.

비디오: Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

연구

신경과학

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

동물 행동하자, 깨어있는 단일 단위 레코딩을위한 Microwires의 조직 된 배열을 주입하기위한 절차

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

신경 밀도 코어 소체의 수퍼 - 해상도 이미징

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

신경 발사 환율의 조작에 의해 성상 세포 수용체의 가소성을 유도

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

깨어 마우스의 신경 활성 물질의 Microiontophoretic 응용 프로그램과 결합하여 신경 활동의 세포 외 기록

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 쥐 행동에 Hippocampal 세타 진동의 유입

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.