نشكر اندريا موران مع أعضاء لحيوانات المختبر مرفق الخدمة (LASF) في معهد نيفيز لدعمهم ممتازة على تربية الحيوانات والخدمات التقنية. ويدعم هذا العمل بتمويل من المعاهد الوطنية للصحة والمعهد نيفيز في (NIDCD 008003) في البكاء. المحتوى هو فقط من مسؤولية الكتاب ولا تمثل بالضرورة وجهة النظر الرسمية للمعهد الوطني للصمم واضطرابات التواصل الأخرى أو المعاهد الوطنية للصحة. الولايات المتحدة لبراءات الاختراع في انتظار الفئران tetO - G - CaMP2 لمعهد نيفيز ، والبكاء وLM.

<ol>
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E., Dulac, C., Meister, M. <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>Chamero, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+pheromones+that+promote+aggressive+behaviour.">Identification of protein pheromones that promote aggressive behaviour.</a> Nature. 450, 899-902 (2007).</li><li>Leinders-Zufall, T., Ishii, T., Mombaerts, P., Zufall, F., Boehm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+requirements+for+the+activation+of+vomeronasal+sensory+neurons+by+MHC+peptides.">Structural requirements for the activation of vomeronasal sensory neurons by MHC peptides.</a> Nat. Neurosci. 12, 1551-1558 (2009).</li><li>Kimoto, H., Haga, S., Sato, K., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+peptides+from+exocrine+glands+stimulate+mouse+vomeronasal+sensory+neurons.">Sex-specific peptides from exocrine glands stimulate mouse vomeronasal sensory neurons.</a> Nature. 437, 898-901 (2005).</li><li>Holekamp, T. F., Turaga, D., 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=Fast+three-dimensional+fluorescence+imaging+of+activity+in+neural+populations+by+objective-coupled+planar+illumination+microscopy.">Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy.</a> Neuron. 57, 661-672 (2008).</li><li>Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I+peptides+as+chemosensory+signals+in+the+vomeronasal+organ.">MHC class I peptides as chemosensory signals in the vomeronasal organ.</a> Science. 306, 1033-1037 (2004).</li><li>He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+signals+conveyed+by+pheromone+concentrations+to+the+mouse+vomeronasal+organ.">Distinct signals conveyed by pheromone concentrations to the mouse vomeronasal organ.</a> J. Neurosci. 30, 7473-7483 (2010).</li><li>Haga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+male+mouse+pheromone+ESP1+enhances+female+sexual+receptive+behaviour+through+a+specific+vomeronasal+receptor.">The male mouse pheromone ESP1 enhances female sexual receptive behaviour through a specific vomeronasal receptor.</a> Nature. 466, 118-122 (2010).</li><li>Boschat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+detection+mediated+by+a+V1r+vomeronasal+receptor.">Pheromone detection mediated by a V1r vomeronasal receptor.</a> Nat. 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الإعلان عن أي تضارب في المصالح.

غالبية المستقبلات الميكعي (VRS) تبقى كما مستقبلات اليتيم منذ اكتشافه من قبل Dulac وأكسل 5. ليست ليغاندس فرمون هذه المستقبلات حسي كيميائي ودورها في التوسط في سلوكيات الحيوان مفهومة جيدا. حتى الآن ، تم التعرف زوج واحد فقط من يجند / مستقبلة ، والببتيد ESP1 ومستقبله وما شا?...

imaging neuronal responses in slice preparations of vomeronasal organ expressing a genetically encoded calcium sensor

الجهاز الميكعي (VNO) بالكشف عن إشارات حسي كيميائي التي تحمل معلومات عن الحالة الاجتماعية والجنسية والإنجابية للأفراد داخل 1،2 نفس النوع. هذه الإشارات إختلافات بين الأنواع ، والفيرومونات ، فضلا عن إشارات من بعض الحيوانات المفترسة 3 ، تنشيط الخلايا العصبية الحسية الميكعي (VSNs) مع مستويات عالية من الدقة والحساسية 4. وأعرب ما لا يقل عن ثلاث عائلات مختلفة من المستقبلات بالإضافة G - البروتين ، V1R ، وV2R FPR 14/05 ، في الخلايا العصبية VNO للتوسط في الكشف عن العظة حسي كيميائي. أن نفهم كيف يتم ترميز المعلومات من قبل فرمون VNO ، من المهم جدا لتحليل الأوضاع VSNs استجابة الفرد للمؤثرات المختلفة والتعرف على المستقبلات الخاصة التي تتوسط هذه الاستجابات. يتم تضمين neuroepithelia من VNO في زوج من العظام الميكعة. هيكل شبه أعمى أنبوبي واحد من VNO نهاية مفتوحة (القناة الميكعي) الاتصالتجويف الأنف. VSNs تمديد التشعبات على جزء من التجويف VNO ، حيث العظة فرمون على اتصال مع المستقبلات التي أعرب عنها في المقابض شجيري. الهيئات خلية من VSNs شكل شبه طبقية طبقات مع V1R V2R وأعرب في الطبقات القاعدية وقمية على التوالي 6-8. وقد تم استخدام عدة تقنيات لرصد ردود VSNs للمؤثرات الحسية 4،12،15-19. من بين هذه التقنيات ، وإعداد شريحة الحاد يوفر مزايا عدة. الأولى ، بالمقارنة مع 3،17 VSNs فصلها ، والأعمال التحضيرية شريحة حفاظ على الخلايا العصبية في التشكل وطنهم والتشعبات للخلايا البقاء متماسكا نسبيا. الثانية ، والهيئات خلية من VSNs يمكن الحصول عليها بسهولة في شريحة الاكليلية من VNO للسماح للدراسات وتجارب التصوير الكهربية بالمقارنة مع ظهارة كله وكامل الاستعدادات لشن 12،20. الثالثة ، يمكن الجمع بين هذه الطريقة مع تقنيات الاستنساخ الجزيئي للسماح بتحديد مستقبل. تثير الحواس كا 2 + تدفق قوي في VSNs ذلك يدل على تنشيط مستقبلات 4،21. نحن نطور بالتالي الفئران المعدلة وراثيا التي تعبر عن G - CaMP2 في الخلايا العصبية الحسية الشمية ، بما في ذلك VSNs 15،22. حساسية وطبيعة وراثية للمسبار يسهل إلى حد كبير كا 2 + تجارب التصوير. وقد ألغت هذه الطريقة عملية التحميل الصبغة المستخدمة في الدراسات السابقة 4،21. نحن نوظف أيضا نظام التسليم التي تمكن من تطبيق يجند من المحفزات المختلفة لشرائح VNO. مزيج من الطريقتين يسمح لنا لرصد الخلايا العصبية متعددة في نفس الوقت استجابة لأعداد كبيرة من المحفزات. أخيرا ، أنشأنا خط أنابيب التحليل شبه الآلي لمساعدة ومعالجة الصور.

Watch this Scientific Journal Video about Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor at JoVE.com

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

الجهاز الميكعي (VNO) بالكشف عن اشارات كيميائية إختلافات بين الأنواع التي تنقل المعلومات الاجتماعية والإنجابية. لقد أجرينا تجارب التصوير + CA2 الفئران المعدلة وراثيا باستخدام تعبير عن G - CaMP2 في الأنسجة VNO. هذا الأسلوب يتيح لنا لتحليل أنماط استجابة معقدة من الخلايا العصبية الميكعي لأعداد كبيرة من المحفزات فرمون.

الردود تصوير الخلايا العصبية في الجهاز الاستعدادات لقطات للالميكعي تعرب عن جهاز استشعار الكالسيوم مشفرة وراثيا

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

imaging-neuronal-responses-slice-preparations-vomeronasal-organ

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Neuroscience

10.7K Views.  Stowers Institute for Medical Research. الجهاز الميكعي (VNO) بالكشف عن اشارات كيميائية إختلافات بين الأنواع التي تنقل المعلومات الاجتماعية والإنجابية. لقد أجرينا تجارب التصوير + CA2 الفئران المعدلة وراثيا باستخدام تعبير عن G - CaMP2 في الأنسجة VNO. هذا الأسلوب يتيح لنا لتحليل أنماط استجابة معقدة من الخلايا ال...

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

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

التصوير الكالسيوم من الردود، رائحة أثار في ذبابة الفاكهة لوب Antennal

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

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.

التصوير فرمون الاستشعار في الميكعي ماوس الحادة الأنسجة إعداد شريحة

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. ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

الكالسيوم في الجسم الحي التصوير الخلايا العصبية في C. ايليجانس

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

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.

فيفو السابقين الاستعدادات للالميكعي الجهاز سليمة والإكسسوار الشم لمبة

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

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

التصوير الكالسيوم داخل الخلايا<sup&gt; 2+</sup&gt; إشارات في الجسم المخطط النجمية من الفئران الكبار باستخدام مؤشرات الكالسيوم المشفرة وراثيا

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

تسجيل الخفيفة أثار-الردود بعد المشبكي في الخلايا العصبية في الظلام تكييفها، الاستعدادات ماوس الشبكية شريحة عن طريق تقنيات المشبك التصحيح

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

التصوير غشاء المحتملة مع اثنين من أنواع مشفرة وراثيا نيون الجهد مجسات

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

مراقبة البصرية من البروتين العصبية باستخدام حمض أميني مصطنعة مشفرة وراثيا في الخلايا العصبية

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

الحضانة لفترات طويلة الحادة العصبية الأنسجة للالكهربية والكالسيوم التصوير

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

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.