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.

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			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
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			<td>Type III-A, High EEO</td>
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			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
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			<td height="20" style="height:20px;width:263px;">Scalpel</td>
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			<td>10003-12 (handle), 10011-00 (blades)</td>
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			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
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			<td height="20" style="height:20px;width:263px;">Homeothermic Blanket</td>
			<td style="width:171px;">Harvard Apparatus</td>
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			<td>Use with standard Super Glue.&#160;</td>
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</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 ...

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Research

JoVE Journal

Neuroscience

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

19.0K 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

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Kerevit Yansıma Davranış Yanıtları kaydedilmesi

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Tek Birim Faaliyet ve elektrokortikografik Sinyalleri Kayıt için Kronik Sinir Elektrotların Cerrahi İmplantasyonu

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

Belirlenen Nöronlar ve Beslenme Davranışı Kalsiyum sinyaller aynı anda kayıt Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograd Floresan Etiketleme Belirlenen Nöronlar gelen Hedefli Hücre dışı Tek ünitesi Kayıt izin verir<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Transgenik Balık Sağlam Beyin Tespit Edilen nöronlar Elektrik Aktivitesi Kayıt

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Hücre içi Kaydı, Duyu Alan Haritalama ve Sülük de Kültüre Belirlenen Nöronlar Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Kortikal internöronlar alt tip-selektif Elektroporasyon

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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 ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Eşzamanlı Elektrofizyolojik Kayıt ve kemirgen Beyindeki inhibitör Ajanların Mikro-enjeksiyon

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...