This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

NOT: Oregon Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi tarafından onaylanan aşağıdaki protokol sağlık kuralları National Institutes uygundur. 1. Akut Cerrahi <ol><li> Intraperitonal (ip) enjeksiyon (Tablo 1) ile, bir ketamin-medetomidin kokteyl ile hayvan anestezisi. Not: Bu deneylerde kullanılan fare sürücü hatları (, SST-iCre12 SOM +; * Cr iCre12, CR + Pvalb-iCre11 PV +) interneuron bir CRE bağımlı ChR2 EYFP transgenik line10 çaprazlama yoluyla üretilir. CHR2 veya ilgili opsins viral iletim benzer ekspresyon düzeyleri elde varsayarak, eşit derecede iyi çalışmasıdır. </li><li> Ameliyat başlamadan önce, emin olun hayvan sergiler nazik bir ayak tutam hiçbir yanıt olduğunu. Bu anestezi derinliği korumak için gerekli olarak deney boyunca anestezi yeniden yönetmek. Enjektabl anestezikler kullanıyorsanız, isteğe bağlı olarak bakım enjeksiyonlar için ip kateter implant. </li><li> Tuzlu su ya da bakım enjeksiyonlar (Tablo 1), uygun bir şekilde seyreltilir anestezik kokteyli kullanarak, örneğin, deney boyunca laktatlanmış Ringer çözeltisi (yaklaşık 3 ml / kg / saat) ile sulu hayvan tutun. </li><li> Sterotaksik veya diğer baş tutma cihazında hayvan yerleştirin. Kafatası iyi korunan emin olun. Bu kararlı tek hücre kayıtları muhafaza etmek için gereklidir. </li><li> Kuruluğunu önlemek için gözlerin için opthalamic merhem sürün. 36,5-37 ° C vücut ısısını korumak. </li><li> Ek kayıt istikrar için bir sisternal drenaj gerçekleştirin. Bir neşter bıçak kullanarak, sisterna magna maruz kafatasının arka yüzündeki doku kaldırmak. Serebrospinal sıvı boşaltmak için, dura içinde küçük bir çentik olun. Bıçağın sadece çok uç kullanın ve beyincik veya beyin sapıyla temas kaçının. </li><li> Bisturi kullanarak, işitsel için (ilgi kortikal alanı üzerinde küçük bir kranyotomiyi (~ 2 mm 2) gerçekleştirmekkorteks bregmaya kabaca -2,3 mm posterior, orta hatta 4,5 mm lateral). </li><li> Dura çıkarın ve sıcak 0.5 agaroz tabakası ile maruz korteksi kapak - 1 mm kalınlığında (tuzlu su içinde% 1.5 agaroz,% 0.9 NaCl, en geçerli ~ 37 ° C). Periyodik tuz birkaç damla damlatılarak deney boyunca agaroz nemli tutmak. </li></ol> 2. Kayıt Set-up <ol><li> Tungsten elektrot hazırlayın (7-14 MΩ, 127 mikron çapında, 12 ° konik ucu, epoksi kaplı). Bir cam kılcal tüp elektrodu superglue ve kavrama (Şekil 1) ısı ile büzülen boru ekleyin. </li><li> Kortikal yüzeye dik seyahat için ayarlanmış bir motorlu (veya hidrolik) mikromanipülatör elektrot, monte edin. Kafatası karşı, deri altında bir tel zemin kaydırın. Onlar elektromiyografik eserler üretmek gibi baş tarafı ve boyun arkasındaki kasları ile temastan kaçının. </li><li> Bir hücre dışı amfi kullanarak elektrik sinyali yükseltinTek ünite kayıt için uygun li fi er, tercihen bir empedans kontrol modu ile donatılmıştır. İki Osiloskoplara ve güçlendirilmiş hoparlör setini - devam eden spike aktivitesini (5000 Hz bant geçiren 300) izleyin. Burada, kaydedilen veriler 10 kHz örnekleme hızında sürekli sayısallaştırılmaktadır; ani çevrimdışı ayıklanır. </li><li> Ucu korteks yüzeyine ulaşıncaya kadar agaroz aracılığıyla elektrot ilerlemek. Bir mikroskop aracılığıyla bu adımı gözlemleyin. &quot;Sıfır&quot; mikromanipülatör bu pozisyon. </li><li> En iyi kayıt derinliğini tahmin etmek üzere görsel olarak penetrasyon sonunda elektrot geri çekildiği zaman, elektrot ucu sıfır derinlikte kortikal yüzeyinden çıkar olduğunu teyit etmektedir. Not: manipülatör okuma ve gözlenen elektrot konumu arasında bir farklılık bu dokuya göre sürüklenmiştir gösterir. Bu beyin ya da hayvan ya da manipülatör sürüklenme istikrarsızlık neden olabilir. <ol><li> Sağlamak için ilave bir kontrol olarakdoku ilk birkaç yüz mikrometre ile ilerletirken bu sıfır set-noktası korteks yüzeyine karşılık gelen, uyarıcı-uyarılmış alan potansiyelleri izlemek. Alan potansiyelleri izlerken, 10 Hz dışı amplifikatör alt bant geçiren filtre kesme indirin. Yerel alan potansiyelinin polaritesi tabakası I / II sınır 13 yakın, ~ 100 mikron derinliğinde etrafında tersine onaylayın. Bu işitsel korteks için güvenilir bir referans noktasıdır, ama diğer kortikal alanlar için farklı olabilir. </li></ol></li><li> Manuel bir mikromanipülatör üzerine bir mavi ışık kaynağı (Şekil 2) bağlı olan bir optik fiber monte edin. Mikroskop kullanarak, mümkün olan en agaroz yüzeyine yakın olarak, elyafın ucu yerleştirin. Elektrot dokuya girecek ışını Merkezlenmesi. </li><li> Belirtilen genişlik ve duratio bir TTL (transistör-transistör mantığı) darbe katarı sunma yeteneğine bir kontrol ünitesi ile ışık kaynağının çıkış düzenleyenn, örneğin, bir Arduino (Şekil 3), (gösterildiği gibi), bir bilgisayar I / O kartı ya da ticari olarak temin edilebilir darbe üreteci. <ol><li> Her iki osiloskoplarda sinyali izleyin. </li></ol></li><li> Bir güç ölçer kullanarak fiber optik ucunda toplam ışık gücü (mW) ölçün. Elyaf çekirdeğin kesit alanına bölünmesi ile ışınlama (mW / mm2) hesaplamak için bu değeri kullanın. 10 aralığı içinde bir yoğunluk değeri ile başlayın - 15 mW / mm 2 ve gerekirse eserler ortadan kalkıncaya kadar aşağı doğru ayarlanır. </li><li> Doku girmek için hazırlanıyor agaroz elektrot, hafif eserler için kontrol edin. Arama darbe tren başlatın (örneğin, 30 msn ışık darbeleri, 500 msn interstimulus aralığı (ISI)). <ol><li> Işığın açısını değiştirmek için elektrot için fiber optik göreceli konumlandırma geçici ışık eserler (Şekil 4) eleyin. Eserler devam ederse, ışık gücünü azaltmayı deneyin. The eserler tamamen (sadece minimize) elimine edilemez nadir durumda, arama darbenin süresini uzatmak. Bu doğru nöronal sivri tanınmasına yardımcı olabilir. </li></ol></li></ol> 3. Düz PINP-in &#39; <ol><li> Yaklaşık olarak 1 mm / sn bir hızda, beyin boyunca yavaşça elektrot ilerletin. Devam eden aktivitesini izlemek için bir osiloskop kullanın. Öte yandan, lazer impulslarından tetikleyebilir. </li><li> Elektrot bir ChR2 + hücre yaklaşıyor ve sık sık elektrik sinyali osiloskop belirgindir önce iyi algılanan edilebileceğini belirtir ses monitörde ışık uyarılmış başak hafif bir &#39;karma&#39;, dinleyin. Avans hızını yavaşlatabilir. NOT: Hücre elektrot doğrudan yolunda yatıyor olursa, ışık uyarılmış aktivite büyük (ve yüksek sesle) artacak. Elektrot tek bir interneuron yaklaştıkça, karma eşit boyut ve şekle sahip küçük ama iyi tanımlanmış sivri olarak çözülür. </li><li> En kısa sürede li gibiGHT-uyarılmış sivri ayrı osiloskop kapalı tetiklemek için yeterince büyük, bu yüzden yapmaya başlar. Başak dalga kesin şeklini gözlemlemek için yatay eksende ölçekleme ayarlayın. </li><li> Durdurmak ve doku yerleşmek için bekleyin. Yakın hücreye elektrodu taşımak için günaha karşı. Bu adım, sabit bir kayıt için çok önemlidir. Birkaç dakika sonra sinyal-gürültü oranı gelişmiş değilse - ki, doku yerleşmiş, ancak herhangi bir yakın elektrot ucu hücreyi getirmemiştir - avansı 5 ileri um ve tekrar bekleyin. <ol><li> Tepe ya da aksiyon potansiyelinin çukur ya güvenilir bir gürültü kat (örneğin, +/- 300 mV veya daha fazla) üzerinde iyi ayarlanmış bir gerilim eşik ile çekilebilir kadar bu işlemi tekrarlayın. NOT: inhibitör internöronlardan PINPing zaman yanlış pozitif veya belirsizlik az risk var. Çoğu hücreleri kolayca 30 msn süresi ve inter-pul ile bakliyat bir tren takip edebilirsinizse aralığı 2-5 msn (Şekil 5) güvenilir bir ilk başak gecikme ile 500 msn, veya daha hızlı. PV + hücrelerinin büyük bir çoğunluğu, özellikle, bir tam olarak 1 sn (Şekil 6) ateşleme sağlanabilmektedir. Bu inhibitör nöronlar Çünkü, disinaptik (dolaylı) aktivasyonu önemli bir endişe değildir. </li></ol></li><li> Kayıt sırasında, ilk osiloskop devam eden etkinliğini izlemek. Boyutu ve ikinci osiloskop kullanarak sivri şekline yakın bir göz tutmak. Hoparlör kendi ses kalitesine dikkat edin. <ol><li> Hücre ya (onu kazığa veya hasar görme riski) elektrot çok yakın çizim belirtmek ani değişiklikler için dikkatle dinleyin, ya da uzağa sürüklüyor. Ani, büyük ve bozuk hale gelirse, dışarı geri; daha küçük büyürse, elektrot ilerlemek. 2 mikron adımda yavaş hareket. </li><li> Zirve veya çukur için hizalanmış tüm başak dalga formları, atma tarafından kayıt sonrası hoc kalitesini onaylayın. Voltaj thres Varytutun. Eşik değerlerin geniş bir yelpazede, sadece hiç eşit yükseklikte (Şekil 5a) tutarlı bir başak şekli olmalıdır. </li></ol></li><li> Herhangi bir noktada sinyal bir komşu nöronun gelen sivri kirlenirse, hareket. Elektrot komşu hücre aralığının dışında geçer, ama hedef nöron aralığında kalacağını kapalı-şans, yavaş yavaş ilerlemek. (Bu genellikle başarısız olur.) </li><li> Her penetrasyonu korteks (900+ mikron) tüm derinliği boyunca elektrot hareket ettirin. Işık-duyarlı nöronlar tersine, kayıtlar rutin komşu ChR2- hücrelerinden sivri ile kontamine birçok sızmalar veya sonra karşılaşılan, yeni bir elektrot deneyin. NOT: Hatta tek bir parti içinde, bireysel elektrotlar empedans ve ucu geometri önemli ölçüde değişebilir. Her iki faktör wh-ışık uyarılmış ani algılamak için yeterli büyüklükte olmalıdır elektrodun etkili &quot;dinleme yarıçapı&quot;, katkıdaIle arama, henüz tek başına bir tek interneuron kayıt sağlayacak kadar sınırlı. </li><li> Istediğiniz gibi elektrot empedansı test edin. Dokuya zarar görmesini önlemek için, iyi beyin dışında, agaroz tabakasının üst kısmında elektrodun ucu ile bunu. 7-14 MΩ aralığı içinde, tam olarak empedans bu uygulama için performans, veya verim kesin bir belirleyici değildir. Yani dinleme yarıçapı için makul bir vekil var, dedi: alt-empedanslı elektrotlar daha birimleri pick up; yüksek empedans, az. </li><li> Deneyin sonunda, böyle anestezi derinliğinin düzlemi altında anestetik aşırı dozda, ya da servikal bozma gibi kurumsal onaylı vasıtasıyla, hayvan euthanize. </li></ol>

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The authors have no competing financial interests.

PINP kavramsal olarak basit olmasına rağmen, pratikte zor olabilir. Başarının önemli bir belirleyici elektrot seçimdir. Elektrik dinleme çapı kritik bir parametredir. Bu ucu bazı mesafe uzakta bir ChR2 + hücreden hala zaman bir doğrultuda ilerleme hızını ayarlayabilirsiniz, böylece ışık-uyarılmış ani algılamak için yeterince büyük olmalıdır. Aynı zamanda, iyi bir tek birim izolasyonu sağlayacak kadar kısıtlanmalıdır. Bu elektrot da komşu ChR2- birimlerinden ani almak, olmamalıdır. Di...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table><tbody><tr><td>ChR2-EYFP Line</td><td>Jackson Colonies</td><td>12569</td><td></td></tr><tr><td>Pvalb-iCre (PV) Line</td><td>Jackson Colonies</td><td>8069</td><td></td></tr><tr><td>Sst-iCre (SOM) Line</td><td>Jackson Colonies</td><td>13044</td><td></td></tr><tr><td>Cr-iCre (CR) Line</td><td>Jackson Colonies</td><td>10774</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9793</td><td>Type III-A, High EEO</td></tr><tr><td>Micro Point (dural hook)</td><td>FST</td><td>10066-15</td><td></td></tr><tr><td>Surgical Scissors</td><td>FST</td><td>14084-09</td><td></td></tr><tr><td>Scalpel</td><td>FST</td><td>10003-12 (handle), 10011-00 (blades)</td><td></td></tr><tr><td>Puralube Ophthalmic Ointment</td><td>Foster &amp;amp; Smith</td><td>9N-76855</td><td></td></tr><tr><td>Homeothermic Blanket</td><td>Harvard Apparatus</td><td>&lt;a name=&quot;RANGE!C11&quot;&gt;507220F&lt;/a&gt;</td><td></td></tr><tr><td>Tungsten Microelectrodes</td><td>A-M Systems</td><td>577200</td><td>12 M&amp;Omega; AC resistance, 127 &amp;mu;m diameter, 12&amp;deg; tapered tip, epoxy-coated</td></tr><tr><td>Capillary Glass Tubing</td><td>Warner Instruments</td><td>G150TF-3</td><td></td></tr><tr><td>Heat Shrink Tubing</td><td>DigiKey</td><td>A332B-4-ND</td><td></td></tr><tr><td>Zapit Accelerator</td><td>DVA</td><td>SKU ZA/ZAA</td><td>Use with standard Super Glue.&amp;nbsp;</td></tr><tr><td>Microelectrode AC Amplifier 1800</td><td>AM Systems</td><td>700000</td><td></td></tr><tr><td>MP-285 Motorized Micromanipulator</td><td>Sutter</td><td>MP-285</td><td></td></tr><tr><td>4-channel Digital Oscilloscopes</td><td>Tektronix</td><td>TDS2000C</td><td></td></tr><tr><td>Powered Speakers</td><td>Harman</td><td>Model JBL&amp;nbsp;Duet</td><td></td></tr><tr><td>Manual Manipulator</td><td>Scientifica</td><td>LBM-7</td><td></td></tr><tr><td>800 &amp;micro;m Fiber Optic Patch Cable</td><td>ThorLabs</td><td>FC/PC BFL37-800</td><td></td></tr><tr><td>Power Meter</td><td>ThorLabs</td><td>PM100D (Power Meter), S121C (Standard Power Sensor)</td><td></td></tr><tr><td>475 nm Cree XLamp XP-E</td><td>DigiKey</td><td>XPEBLU-L1-R250-00Y01DKR-ND</td><td>LED power and efficiency are continually increasing, so we recommend checking for the latest products (www.cree.com).</td></tr><tr><td>Arduino UNO</td><td>DigiKey</td><td>1050-1024-ND</td><td></td></tr></tbody></table>

a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

Nörofizyolojideki büyük bir meydan okuma serebral kortekste çeşitli inhibitör hücre tiplerinin yanıtı özellikleri ve işlevini karakterize etmek olmuştur. Biz burada Lima ve 1 meslektaşları tarafından geliştirilen bir yöntem kullanılarak anestezi fare kortekste tespit inhibitör itibaren istikrarlı, iyi izole edilmiş tek ünite kayıtları elde etmek için strateji paylaşıyoruz. Kayıtlar spesifik sinir hücresi alt gruplarda channelrhodopsin-2 (CHR2) ifade eden farelerde gerçekleştirilmiştir. Nüfusun üyeleri mavi ışık kısa bir flaş kendi tepkisi ile tanımlanır. &quot;PINP&quot; olarak adlandırılan, ya da Nöronal Popülasyonlarının photostimulation destekli Kimlik - - Bu teknik, standart dışı kayıt ekipmanı ile uygulanabilir. Bu genetik olarak tanımlanan hücreler, hücre dışı kayıtları hedefleme amacıyla, kalsiyum görüntüleme veya görme güdümlü yama için ucuz ve erişilebilir bir alternatif olarak kullanılabilir. Here gündelik pratikte yöntemini optimize etmek için bir ilkeler kümesi sağlar. Bu parvalbumin pozitif (PV +) hücre hedeflenmesi için özel olarak bu strateji rafine edilmiş, ancak bu tür somatostatin salgılayan (som +) ve kalretinin salgılayan (CR +) internöronlar gibi, hem de diğer interneuron türler için geçerlidir bulduk.

Biz burada &quot;Lima ve ark. 1. Tablo 1 ayrıntıları önerilen anestezik kokteyl, ketamin-Medetomidin-acepromazine (tarafından geliştirilmiş bir Optogenetic yöntemi kullanılarak, anestezi fare kortekste genetik ilan inhibitör gelen tek birim kayıtları elde etmek için stratejimizi paylaşmak KMA &quot;). Şekil 1 Şekil 4. 3 Arduino mikrodenetleyici ile ışık çıkışı yolluk için yapılandırma ve kodu içeren Şekil. 2 basit bir LED...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide<em&gt; In vivo</emOptogenetically Belirlenen Kortikal inhibitör dan&gt; Tek birim Kayıt

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Universidad de Cartagena UDEC

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Neuroscience

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

19.1K Görüntüleme.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

Video: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

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.

Tek birim In vivo</emSıçan, optik kiazma> Kayıtlar

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 ...

Nörobilim

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties1-4. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited3,5,6. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons3,7-9. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis.
Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex3,10, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.

Lazer Tarama Photostimulation tarafından inhibitör nöronal devreler Haritalama

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Bireysel piramidal nöronlar High Fidelity optogenetic Kontrolü için Bir Yöntem<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- ve nörokimyasal tanımlanan hipokampal internöronlardan gelen tüm hücre Patch-kelepçe Kayıtlar

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Beyin Dilimleri Korku Devre Ex Vivo optogenetic Diseksiyon

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

Bir cam Optrode uyanık farelerde nöronal türüyle Optogenetics tanımlaması

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

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Fare Beyin Dilimlerinde Uzun Menzilli Girdilerin Optogenetik Stimülasyonu için Vivo İntraserebral Stereotaksik Enjeksiyonlarda

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Beyin Dilimlerinde Afferent Yolakların Optogenetik Aktivasyonu ve Uçucu Anesteziklerle Yanıtların Modülasyonu

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Eski Vivo Medial Prefrontal Korteksten Lateral Entorinal Kortekse Uzun Menzilli Sinaptik İletim ve Plastisitenin Optogenetik Sorgulanması

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

Fare hipokampal dilimlerindeki nöronların fotostimülasyon ve tam hücre yama kelepçesi kayıtları

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

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