우리는 성령 연구소의 회원을 감사드립니다. 이 작품은 청각 및 통신 장애에 대한 국립 연구소 (R01 DC005964로)와 NSF IGERT 0548890 (하)에 의해 지원되었다. 동물 실험은 미국 동물 복지 행위 및 건강 지침 국립 연구소에 따라 수행되었으며 워싱턴 대학 동물 케어 및 사용위원회에 의해 승인되었습니다.

1 부. 전기 녹음 계측 <ol><li> 우리는 실리콘 질화물과 절연 10 μm의 평면 티타늄 질화물 전극과 평면 multielectrode 배열을 (에이러 과학 악기)을 사용합니다. 60 전극 30 μm의 중심 - 투 - 중심 간격 5x6 격자로 이루어진 두 분야로 정렬됩니다. (다른 구성도 가능합니다, 직경 녹음 표면에 10 μm의와 전극은 단일 뉴런의 활동을 격리의 관점에서 큰 전극보다 수 있습니다.) </li><li> 전기 신호는 MEA 1,060 앰프 (에이러 과학 악기)를 사용 (1000x 증폭) 증폭됩니다. 신호는 데이터 수집 카드 (내쇼날 인 스트 루먼트) 및 사용자 정의 데이터 수집 소프트웨어 (하나도 상용 소프트웨어를 구입할 수 있습니다)를 사용하여 10 kHz에서에서 찾았습니다. </li><li> 조직은 MEA의 문화 관 반지 안쪽 snugly 맞는 사용자 정의 삽입에 연결된 나일론 메쉬 (53 μm의)에 의해 MEA 아래로 개최됩니다. </li></ol> 2 부. 액체 배달 계측 <ol><li> 조직은 우리가 링어의 솔루션 (115 MM 나트륨 염화물, 5 MM 염화칼륨, 2 MM의 염화칼슘, 2 MM 마그네슘 염화물, 25 MM 나트륨 중탄산염, 10 MM HEPES, 10 MM D - (+를 제공하여 보증 지속 superfusion을 필요로 HPLC 펌프에서) - 포도당). 자극은 HPLC 밸브에 주입을 수행하고 -를 통해 흐름과 루프 사이의 전환에 의해 제공됩니다. HPLC 펌프 및 사출 로봇 주사기 펌프 모두 링어의 솔루션을 제공하고 있습니다. </li><li> 링어의 솔루션은 실험을하는 동안 42 온수 물을 욕조 (ISOTEMP 105) ° C에 보관하고 지속적으로 95% O 2 / 5% CO 2와 함께 부풀어 오른 모양이다. 포도당없이 링어의 별도의 플라스크는 주사기 펌프를 백업을위한 준비가되어 있습니다. 링어의 솔루션은 3 ML / 분 속도로 307 HPLC 펌프 (길슨)를 사용하여 조직에 배달됩니다. 전형적인 5~6시간 실험에서 각 링어의 솔루션 1 L은 충분합니다. </li><li> 실험을하는 동안, 조직은 지속적으로 37 ° C 링어의 솔루션을 superfused입니다. 히터 프로브 직접 조직 위에 위치합니다. </li><li> 자극은 길슨 215 액체 처리기 로봇 팔을 사용하여 전달됩니다. 자극은 고무 덮인 유리 튜브에 저장하고, 일정한 유속과 압력이 유지되는 동안 길슨 소프트웨어의 통제, 로봇 팔 재관류 라인에 자극을 제공하고 있습니다. </li><li> HPLC 밸브 (루프 약혼 / 약혼하지 않음)의 상태의 연결 활동으로 동기화를 보장하기 위해 별도의 채널로 수집 컴퓨터에 의해 기록됩니다 전기적 전압으로 표시됩니다. </li></ol> 부 3. MEA와 자극 전달 장치를 준비 <ol><li> 전에 절개를 수행하기 위해 상온에서 최소 5 분 dH2O의 BSA와 잘 MEA를 입력합니다. </li><li> 따뜻한 - 부풀어 오른 링어의 솔루션을 사용하여 MEA를 씻어 얼음에 MEA를 놓으십시오. </li><li> 프라임 액체 처리기와 HPLC는 제조 업체의 지시에 따라 펌프. </li></ol> 부 4. VNO의 해부 <ol><li> 절개를 수행하기 전에, 얼음에 부풀어 오른 링어의 솔루션 및 장소 2 60x15 mm 배양 접시를 입력하십시오. 또한 얼음 링어의 솔루션과 장소에서 동일한 크기의 2 Sylgard 코팅된 배양 접시를 입력하십시오. 해부하는 동안 사용하기 위해 따뜻한 부풀어 오른 링어의 추가 50mL를 풀어. </li><li> 마우스는 자궁 전위 다음 CO 2 질식을 통해 희생이다. </li><li> 머리의 뒤쪽에, 턱 라인에 평행하게 실행 입의 측면에서 양국 incisions하십시오. 목 뒤에 따라 2 상처에 참여하고 마우스에 부착된 아래턱을두고, 머리 등의 부분을 제거합니다. </li><li> 좋은 가위를 사용하여 조직과 뼈가에서 조직을 분리 위턱 뼈의 양쪽 사이 했네요. </li><li> 비강을 폭로하는 미각의 조직에서 껍질, # 3 포셉을 사용합니다. </li><li> 작은 가위 또는 뼈를 vomeronasal 캡슐을 무료로 사이의 상단이 앞니의 양쪽에 상처 메스를 사용합니다. </li><li> # 3 포셉을 사용하여 얼음에 페트리 접시에있는 평면 골과 장소로 vomeronasal 캡슐을 선택합니다. 최대 집중을 통해 단계는 3보다 분 이내에 수행되어야합니다. </li><li> 반사광과 조명 stereomicroscope 아래 접시를 놓으십시오. # 3 포셉를 사용하여 한 번에 한 VNO에서 뼈다귀 캡슐을 제거합니다. , 멀리 VNO에서 뼈 껍질에 꼬리 끝 및 다른 손에서 재밌 VNO을 손상하지 않도록주의를 취하여 뼈에 캡슐을 안정화시키기 위해 한 손으로를 사용합니다. </li><li> 뼈가 제거되면 Sylgard 코팅 페트리 접시에 VNO을 전송하기만하면됩니다. 다른 VNO에 대해 남아있는 요리를 사용합니다. 전송 빛을 조명을 전환합니다. </li><li> 마이크로 가위 또는 # 5 포셉를 사용 VNO의 가장자리 절단하여 혈관에서 VNO를 구분한다. 이 시점에서 당신 것입니다완전히 혈관에서 분리 신경 시트 수 있습니다. </li><li> 내부 (돌기) 측면까지 함께 VNO를 놓습니다. # 3 포셉 사용하여 조직의 모서리에있는 Sylgard에 VNO를 앵커. 클램프와 함께 개최되는 테플론 코팅 면도날의 작은 조각을 사용하여 기초 라미나에서 신경 상피 껍질. 얕은 각도 (&lt;30 ° 그릇 표면에서)에서 칼날을 개최하는 것이 가장 좋습니다. </li><li> 유리 피펫을 사용하여 MEA로 요리에서 신경 상피의 조각을 전송합니다. 최대 돌기 면을 전극 필드 위에 VNO를 놓습니다. 피펫과 초과 링어의 솔루션을 제거합니다. 장소에 VNO를 개최하고, 냉장 링어의 솔루션을 작성하기 위해 배열의 잘에 메쉬 조직 홀더를 놓습니다. </li></ol> 제 5 부. 녹음 <ol><li> MEA 1,060 증폭기의 MEA를 삽입하여 직접 조직 위의 히터 프로브를 위치. </li><li> 적절한 히터 프로브 위치를 확인하려면, 링어의 솔루션은 2 초 50 MM의 칼륨을 (몰 나트륨에 대한 대체) 포함과 자극. 칼륨 용액에 신경 응답의 지연 시간을 최소화하기 위해 위치를 조정합니다. </li><li> 이전에 조직이 적응 수 있도록 녹화 45 분 1 시간 온수 링어의 솔루션으로 조직을 Superfuse. </li><li> 전기 신호는 사용자 정의 레코딩 소프트웨어를 사용하여 디스크에 저장됩니다. </li></ol> 6 부. 데이터 분석 <ol><li> 오프라인 데이터 분석은 원시 전극 기록을 사용하여 수행할 수 있습니다. 예를 들어, 특정 리간드에 대한 응답으로 기록된 세포의 발사 속도 변화를 측정합니다. 그 결과 데이터 세트는 대형이며, 다양한 질문을 해결하는 데 사용할 수있는, 프로그래밍이나 분석 언어 (C, Matlab, R, 파이썬, 또는 이와 유사한 것)과 친숙이 필요합니다. </li></ol> 7 부. 대표 결과 이 준비는 일반적으로 데이터 (60 전극에서 기록 6 시간)의 대량 항복, 6 시간 동안의 기록 정도로 안정됩니다. <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="그림 1" border="0" /> 그림 1. 배열의 절반에 걸쳐 동시 활동의 스냅샷이 표시됩니다. 시간 창 400 MS에 걸쳐. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="그림 2" border="0" /> 그림 2. 전형적인 실험에서는, 우리는 10 초 동안 관심의 특정 화합물과 자극. 이 추적은 100 배 희석 여성 마우스 소변에 대한 응답의 예입니다. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">여기를 클릭</a> 그림 2의 더 큰 버전을 볼 수 있습니다.

<ol>
	<li>Dulac, C., Torelo, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+pheromone+signals+in+mammals:+from+genes+to+behaviour.">Molecular detection of pheromone signals in mammals: from genes to behaviour.</a> Nat Rev Neurosci. 4, 551-562 (2003).</li><li>Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Vomeronasal+neurons+to+natural+stimuli.">Responses of Vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Nodari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+steroids+as+natural+ligands+of+mouse+pheromone-sensing+neurons.">Sulfated steroids as natural ligands of mouse pheromone-sensing neurons.</a> J Neurosci. 28, 6407-6418 (2008).</li></ol>

Multielectrode 배열 레코딩은 우리에게 뉴런의 큰 인구의 동시 활동을 모니터링하는 능력을 허용합니다. 이것은 특히, VNO를 감각 시스템의 속성을 탐색을위한 유용한 도구입니다. 메인 후각 시스템과는 달리, VNO는 액상의 자극을 감지합니다. 또한, VNO는 이질적인 조직이다. 아마도 다른 응답 프로필 마우스 1 수용체의 약 300 종류가 있습니다. 로봇 팔을와 결합된 MEA 레코딩은 우리가 안정적으...

multielectrode array recordings of the vomeronasal epithelium

신경 회로를 이해하는 것은 방법이 동시에 여러 뉴런에서 기록해야합니다. 에<em&gt; 체외에서</em&gt; 연구 하나의 현재 사용 가능한 기술은 평면 multielectrode 배열 (MEA) 기록입니다. 여기 수용체의 유형의 수백을 표현하는 감각 뉴런의 다양한 인구를 통해 페로몬과 사회적 단서의 검색에 필수적인 역할을 마우스 vomeronasal 기관 (VNO)을 공부하기 MEAs의 사용을 문서. 화학 자극을 제공하는 로봇 액체 처리기와 MEA의 녹음을 결합, 뉴런의 크고 다양한 인구의 감각 반응이 기록하실 수 있습니다. 준비는 우리가 마우스에서 VNO의 손상 neuroepithelium를 제거하고 몇 시간 동안 뉴런의 전기 활동을 모니터링하면서 화학 물질이나 잠재적인 리간드의 배터리와 자극 수 있습니다. 따라서이 기술은 리간드의 활동을 평가뿐만 아니라 수용체 뉴런의 속성을 탐험을위한 유용한 방법으로 제공하고 있습니다. 우리는 vomeronasal 상피, MEA 녹음, 화학 자극을 준비하는 데 필요한 기술을 제시한다.

Watch this Scientific Journal Video about Multielectrode Array Recordings of the Vomeronasal Epithelium at JoVE.com

Multielectrode Array Recordings of the Vomeronasal Epithelium

Multielectrode 어레이 (MEA) 녹음 뉴런의 대규모 인구의 전기적 활동을 연구하는 방법을 제공합니다. 여기, 우리는 동시에 조직을 자극하는 동안 마우스 vomeronasal 상피에서 기록 MEA 준비의 세부 사항을 제시한다.

Vomeronasal 상피의 Multielectrode 배열 레코딩

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

multielectrode-array-recordings-of-the-vomeronasal-epithelium

Research

JoVE Journal

Neuroscience

12.1K 조회수.  Washington University School of Medicine. Multielectrode 어레이 (MEA) 녹음 뉴런의 대규모 인구의 전기적 활동을 연구하는 방법을 제공합니다. 여기, 우리는 동시에 조직을 자극하는 동안 마우스 vomeronasal 상피에서 기록 MEA 준비의 세부 사항을 제시한다.

비디오: Multielectrode Array Recordings of the Vomeronasal Epithelium

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

단일 유닛 생체내에서</em암흑의 광학 Chiasm에서> 레코딩

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

의 거대한 섬유 패스에서 Electrophysiological 레코딩 D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Organotypic 문화에의 연결을 눈사태의 다중 전극 어레이 레코딩

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

이미징 페로몬은 마우스 Vomeronasal 급성 조직 슬라이스 준비에 감지

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

영상의 연결 응답 Vomeronasal 조직의 슬라이스 준비에 유전적으로 인코딩된 칼슘 센서를 표현

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

본래 Vomeronasal 기관 및 액세서리 후각 망울의 생체 준비

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

마우스 보습 코 기관의 급성 조직 조각에서 정의 된 세포 집단의 심층 생리 학적 분석

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

흑색질 도파민 뉴런의 Somatodendritic 도메인에서 세포 내 패치 클램프 녹음

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법<em&gt; Drosophila</em&gt; 광 수용체

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...