XHTW WM Keck Tıp Araştırma Vakfı Seçkin Genç Bilim İnsanı ve aynı zamanda, Ulusal Sağlık Enstitüsü / NHLBI hibe R01-HL089598 ve R01HL091947 ve Müsküler Distrofi Birliği hibe # 69.238 tarafından destekleniyor. Bu çalışma aynı zamanda CaMKII Sinyalizasyon Kalp Vakfı Leducq Alliance tarafından kısmen destekleniyor. NL Kalp Pili Kalp Ritmi Derneği ve 2009-2012 Amerikan Kalp Derneği Doktora Sonrası Bursu ve Elektrofizyoloji 2009-2010 Michel Mirowski Uluslararası Bursu alıcı.

Bölüm 1. Cerrahi hazırlık <ol><li> Steril koşullarda, programlı elektrik stimülasyonu deneylerde farelerin hayatta kalma ameliyatları yapılmaktadır durumda gerekli olacaktır. Ancak, deney en yaygın türü olan düzenli açık cerrahi teknikler yeterli, doğa terminal. </li><li> Izofluran% 2, 0,5 L / dk% 100 O 2 kullanarak fare anestezi . </li><li> Saç kesme makineleri, kürk yaka orta göğüs seviyesine tıraş için kullanılır. </li><li> Bacaklarda ısıtma kartı (İndus Instruments, Houston, Teksas) dahil EKG elektrotları üzerine bantlanmış anestezi fare, yatar pozisyonda yerleştirilir. Ve cerrahi alanında% 10 Povidon İyot ile dezenfekte edilir. Önerilen sistem, vücut ısısı 37.0 tutulur böyle ° C ± 1.0 ° C bir thermoanalyzer sistem tarafından kontrol edilen bir ısıtma yastığı bağlı bir rektal ısı probu, </li></ol> Bölüm 2. Sağ atriyum ve ventrikül içine kanülasyonu ve EP kateter yerleştirilmesi <ol><li> Fare tamamen uyuşturulduktan sonra, bir ayak-tutam tarafından onayladıktan sonra, 1 / 2 inç bir kesi klavikula düzeyinde kaudal uçla orta hatta sağa doğru yapılır. </li><li> Subkutan doku, tükürük bezleri ve lenfatik dokular sağ juguler ven görselleştirmek için künt diseksiyon ile ayrılır. </li><li> Ven proksimal ucunu 6-0 sütür ile bağlıdır. Kateter takarken bu dikiş hafifçe çekerek, internal juguler ven düz devam edecektir. Başka bir dikiş görüntülenmiştir segmentin distal ucunda ven altına yerleştirilir. Bu dikiş kateter kalbin içinde en iyi konulduğunda, hemostaz sağlamak ve istenen pozisyona (Şekil 1) kateter korumak için, kateter çevresinde bağlı olacaktır. </li><li> Bilgisayar tabanlı veri toplama, yüzey EKG ve eş zamanlı intrakardiyak electrograms (yani, Iox-2 kazanım yazılım, Emka Teknolojileri, VA, USA) 4 yol açar kayıtlarına başladı. Intrakardiyak elektrogram kayıt için &quot;kayıt&quot; modunda (yani modeli STG3008, Çok Kanallı Sistemler, Reutlingen, Almanya) bir dış stimülatörü, kateter elektrotlar bağlanır. </li><li> Mikro-makas kullanarak, ven uzunlamasına yönde küçük bir kesi yapılır ve 1.1F octapolar kateter (EPR-800, Millar Instruments, Houston, Teksas), sağ atrium içine damar yoluyla ileri düzeydedir. Proksimal sütür hafifçe çekerek, internal juguler ven düz tutmaya yardımcı olacaktır ve sağ atrium ve ventrikül içine kateter kolay geçişine olanak sağlayacaktır. Doğru kateter pozisyonu sırasıyla sağ ventrikül, sağ ventrikül, atriyoventriküler düğüm ve sağ atrium temel (Şekil 2) zirvesine düzeyinde 4 intrakardiyak electrograms dalga görselleştirme tarafından doğrulandı. </li><li> Distal sütür kateter pozisyonu güvence altına almak ve olası kanama önlemek için kapalı bağlıdır. </li></ol> Bölüm 3. Programlı elektriksel uyarı <ol><li> Seçici atriyal pacing durumda, diğer elektrot çiftleri &quot;kayıt&quot; modunda devam ederken, atrium içinde bulunan elektrot çifti, &quot;recoding&quot; modundan &quot;stimülasyon&quot; modunda açılır. </li><li> Bir fare atriyal aritmiler için artmış bir güvenlik açığı olup olmadığını belirlemek için, sağ atrium programlı elektrik stimülasyonu yapılmaktadır. İlk olarak, atriyal pacing eşik uyaran yakalama tutarlılığını test etmek için farklı temel döngü uzunlukları (BCL) (en az 50) 2 ms akım darbeleri uygulayarak belirlenir. Ilk darbe tren BCL içsel BCL göre biraz daha düşük ve 10 ms (örneğin, 100 ms, 90 ms, 80 ms ve 70 ms) azalır. Uyaran yakalamak için gerekli tipik geçerli genliği 100-200 uA. </li><li> Sinüs düğümü toparlanma zamanı (SNRT) 100 ms BCL 15-atriyal pacing tren uyguladıktan sonra ölçülür. SNRT pacing tren son uyarıcı ve ilk spontan sinüs ritmi başlangıcı arasındaki zaman aralığı olarak tanımlanır. </li><li> Atriyal efektif refrakter dönemi (AERP) birleştiğinde kısa S2 erken uyaran bir sabit BCLs (yani, 100 ms) atriyal pacing tren dizi uygulayarak belirlenir. S1-S2 aralığı giderek 20ms 70ms gelen her Pacing tren 2-ms azalır. AERP S2 (düzenli tren darbe S1, S2 erken uyaran) ile yayılır yendi oluşturmak için başarısız kulakçıklar için uzun S1-S2 bağlantı aralığı olarak tanımlanır. Her stimülasyon protokolü arasında, en az 30 saniye bir iyileşme dönemi vardı. </li><li> Efektif refrakter dönemde atriyoventriküler (AV) düğüm (AVNERP) birleştiğinde S2 erken bir stimulus ile 100 ms BCL atriyal pacing tren dizi uygulayarak belirlenir. S1-S2 aralığı giderek azalır2 ms 70 ms 20 ms her Pacing tren. AVNERP atriyuma teslim erken uyarılma değil bir QRS kompleksi Onun potansiyeli olan en uzun S1-S2 bağlantı aralığı olarak tanımlanır. Her stimülasyon protokolü arasında, en az 30 saniye bir iyileşme dönemi vardı. </li><li> , Atriyal fibrilasyon (AF) de dahil olmak üzere atriyal aritmiler, Inducibility Verheule ve arkadaşları tarafından açıklanan burst pacing protokolünü kullanarak test edilecek. 2 2 saniyelik atışlarla bir dizi atriyal aritmi inducibility belirlemek için uygulanır . Ilk 2 ikinci patlama 40 ms siklus uzunluğu (CL), ve son CL 20 ms kadar birbirini izleyen 2 ikinci patlama, bir önceki patlamada kısa 2-ms CL. Aritmojenik substrat yokluğunda, kalp pacing protokolü hemen sinüs ritmi devam edecektir. Durumda atriyal flutter, ventriküler elektrogram görüldüğü gibi ventriküler yanıt frekans, tipik olarak yavaş ise atriyal elektrogram görülen hızlı A dalgaların düzenli bir desen var olacaktır. Atriyal fibrilasyon durumunda, yüzey EKG&#39;de P dalgalarının yokluğu düzensiz RR aralıkları gösterecektir. Ayrıca, atriyal elektrogram ventriküler elektrogram düzensiz ve yavaş ventriküler dalgaları (Şekil 3) ortaya çıkaracaktır ise, hızlı ve düzensiz dalgalar ortaya koyacaktır. Aritmi indüksiyon protokolleri genellikle üç nüsha olarak yapılır ve 3 çalışmaların en az 2 uyarılmış olabilir aritmi olarak kabul edilir. 3,4. </li></ol> Bölüm 4. Kateterin çıkarılması <ol><li> Tüm pacemaker protokolleri tamamladıktan sonra, veri toplama durdurulur. Kateterin distal ucunda dikiş yavaşça kateter serbest bırakmak amacıyla kesilir. </li><li> Terminali EP çalışmaları durumunda, düğüm kateter yavaşça serbest bırakmak için gevşetti. </li><li> Çalışmanın bitiminde, fare, izofluran altında servikal dislokasyon kullanırken insanca ötenazi olacaktır. </li></ol> Temsilcisi Sonuçlar Yüzey EKG ve intrakardiyak electrograms in vivo elektrofizyoloji çalışma boyunca aynı anda kayıt altına alınır ve tüm protokolleri tamamlanmasının ardından ayrıntılı olarak gözden geçirilmiştir . Bazal elektrofizyolojik parametreler, PR aralığı, PQ aralığı, QRS süresi, QT aralıkları ve QRS morfolojisi içerir. Bu parametreler, elle ölçülen ya da otomatik veri toplama yazılımı Iox-2 veya EKG-AUTO (Emka Teknolojileri, VA, USA) kullanılarak olabilir. SNRT, AERP ve AVNERP sinüs düğümü &quot;kalp pili&quot; fonksiyonu, atriyal, AV nodal iletim özellikleri, sırasıyla hakkında bilgi verir. Atriyal fibrilasyon indüklenen pacemaker bölüm örnekleri kağıt Chelu ve arkadaşları tarafından bulundu. 4 <img src="/files/ftp_upload/1730/1730fig1.jpg" alt="Şekil 1" /> Şekil 1 farelerde elektrofizyoloji çalışmaları için intrakardiyak kateterizasyon İllüstrasyon A. sağ internal juguler ven izole ve kanüle. Uygun kateter pozisyonu sağlandıktan sonra ven distal ucuna bağlıdır. B. Fare, bu işlem için bir yatar pozisyonda yerleştirilir. C. Cartoon kateter intrakardiyak konumunu resmeden . Elektrot çifti, sırasıyla, sağ ventrikül, sağ ventrikül tabanı, atriyoventriküler düğüm ve sağ atrium apeks düzeyinde konumlandırılmış . 1.1F octapolar kateter-up kapatın. Mathur et al değiştirilmiş Şekil. Circ Arrhtyhm Electrophys 5 izni ile. <img src="/files/ftp_upload/1730/1730fig2.jpg" alt="Şekil 2" /> Şekil 2. Fare Temsilcisi Yüzey EKG ve İntrakardiyak Electrograms. (A) 540 sağ atrium seviyesinde dakika (BE) Bipolar intrakardiyak elektrogram kayıtları başına atım (B), atrioventriküler düğüm (C), ve baz bir frekans ile normal sinüs ritmi gösteren kurşun II yapılandırma Yüzey EKG sağ ventrikül (D) ve (E) sağ ventrikül apeks, sırasıyla. Panel B intrakardiyak bir dalga yüzey EKG&#39;de P-dalga karşılık geldiğini unutmayın. Ventriküler elektrogram V-dalga yüzey EKG&#39;de QRS dalgası karşılık gelir. <img src="/files/ftp_upload/1730/1730fig3.jpg" alt="Şekil 3" /> Şekil 3. Atriyal fibrilasyon, atriyal burst pacing sonra geliştirilen bir fare Temsilcisi yüzey EKG ve intrakardiyak electrograms .

<ol><li>Nattel, S., Shiroshita-Takeshita, A., Brundel, B. J., Rivard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+atrial+fibrillation%3A+lessons+from+animal+models%5BTitle%5D)+AND+%22Prog+Cardiovasc+Dis%22%5BJournal%5D)">Mechanisms of atrial fibrillation: lessons from animal models.</a> Prog Cardiovasc Dis. 48, 9-9 (2005).</li>
<li>Verheule, S., Sato, T., Everett, T. t, Engle, S. K., Otten, D., Rubart-von der Lohe, M., Nakajima, H. O., Nakajima, H., Field, L. J., Olgin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+vulnerability+to+atrial+fibrillation+in+transgenic+mice+with+selective+atrial+fibrosis+caused+by+overexpression+of+TGF-beta1%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1.</a> Circ Res. 94, 1458-1458 (2004).</li>
<li>Sood, S., Chelu, M. G., Oort, R. J. van, Skapura, D., Santonastasi, M., Dobrev, D., Wehrens, X. H., H, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+calcium+leak+due+to+FKBP12.6+deficiency+in+mice+facilitates+the+inducibility+of+atrial+fibrillation%5BTitle%5D)+AND+%22Heart+Rhythm%22%5BJournal%5D)">Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.</a> Heart Rhythm. 5, 1047-1047 (2008).</li>
<li>Chelu, M. G., Sarma, S., Sood, S., Wang, S., van Oort, R. J., Skapura, D. G., Li, N., Santonastasi, M., Muller, F. U., Schmitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calmodulin+kinase+II-mediated+sarcoplasmic+reticulum+Ca2%2B+leak+promotes+atrial+fibrillation+in+mice%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.</a> J Clin Invest. 119, 1940-1940 (2009).</li>
<li>Mathur, N., Subeena, S., Wang, S., van Oort, R. J., Sarma, S., Li, N., Skapura, D., Bayle, J. H., Valderrabano, M., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sudden+Infant+Death+Syndrome+in+Mice+With+an+Inherited+Mutation+in+RyR%5BTitle%5D)+AND+%22Circ+Arrhythmia+Electrophysiol%22%5BJournal%5D)">Sudden Infant Death Syndrome in Mice With an Inherited Mutation in RyR.</a> Circ Arrhythmia Electrophysiol. , Forthcoming (2009).</li>
</ol>

Kateterizasyon sırasında yoğun kanama, kardiyak kateterizasyon sırasında hipovolemi nedeniyle kalp hızı artırabilir. Bu durumda, (0.3 1.0 mL) steril serum fizyolojik intraperitoneal dolum basıncı normale olabilir ve fare hemodinamik stresi azaltmak. Izofluran 2 saatten daha uzun ya da izofluran, yüksek konsantrasyonlarda (&gt;% 2) maruz kalmak kalp ve solunum fonksiyonları fare bastırmak. Bu nedenle, tüm çalışmalar, en az 2 saat içinde tamamlanmış olması tavsiye edilir. ...

programmed electrical stimulation in mice

Genetiği değiştirilmiş farelerde, iletim bozuklukları, atriyal ve ventriküler aritmiler ve ani kardiyak ölüm altında yatan moleküler mekanizmaları çalışmak için tercih edilen bir hayvan modeli olarak ortaya çıkmıştır.<sup&gt; 1</sup&gt; İntrakardiyak pacemaker çalışmaları, juguler ven içine yerleştirilen bir 1.1F octapolar kateteri kullanarak farelerde yapılan, sağ atrium ve ventrikül içine gelişmiş olabilir. Burada, farelerde programlı elektrik stimülasyonu yerine dahil olan adımları göstermektedir. Yüzey EKG ve intrakardiyak electrograms atrium intrakardiyak pacemaker harici stimülatörü kullanılarak gerçekleştirilir ise, kulakçıklar, atriyoventriküler kavşak ve ventriküler miyokardın eş zamanlı olarak kaydedilir. Böylece, farelerde programlı elektrik stimülasyonu iletim kusurları ve kardiyak aritmiler altında yatan moleküler mekanizmalarını keşfetmek için eşsiz fırsatlar sağlar.

Watch this Scientific Journal Video about Programmed Electrical Stimulation in Mice at JoVE.com

Programmed Electrical Stimulation in Mice

Programlı elektrik stimülasyonu, kalp iletim özellikleri belirlemek için yeteneği ve çeşitli pacemaker protokolleri kullanarak kardiyak aritmiler neden ve sona erdirme imkanı sağlar. Transvenöz bir kateter yardımıyla, intrakardiyak elektrogram kayıtları, programlı elektrik stimülasyonu protokolleri aritmojenik substratlar tanımlamak için aşağıdaki farelerde elde edilebilir.

Farelerde Programlı Elektrik Stimülasyon

Genetically-modified mice have emerged as a preferable animal model to study the molecular mechanisms underlying conduction abnormalities, atrial and ...

programmed-electrical-stimulation-in-mice

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Medicine

20.4K Görüntüleme.  Baylor College of Medicine (BCM). Programlı elektrik stimülasyonu, kalp iletim özellikleri belirlemek için yeteneği ve çeşitli pacemaker protokolleri kullanarak kardiyak aritmiler neden ve sona erdirme imkanı sağlar. Transvenöz bir kateter yardımıyla, intrakardiyak elektrogram kayıtları, programlı elektrik stimülasyonu protokolleri aritmojenik substratlar tanımlamak için aşağıdaki farelerde elde edilebilir.

Video: Programmed Electrical Stimulation in Mice

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

Serbestçe Fareler davranmak içinde delici yol-kıvrımlarının Synapse Uzun vadeli Potentiation

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

Davranış

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Nöromüsküler Elektriksel Stimülasyon tarafından Kas Eğitimi A Mürin Modeli

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Tıp

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Farenin Ventral Tegmental Alanı İçinde Optogenetik Kullanarak İki Farklı Gerçek Zamanlı Yer Tercihi Paradigması

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

SerbestÇe Hareket Eden Farelerde Davranışı Modüle Etmek Için Nöronal Aktivitenin Optogenetik Manipülasyonu

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

Kemirgen Santral Sinir Sistemi vivo optogenetic Stimülasyon içinde

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Nörobilim

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Kayıt ve modülasyon Epileptiform aktivite elektrot dizilerin birleştiğinde kemirgen beyin dilimleri içinde

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Vivo Bileşik kas aksiyon potansiyeli Motor nöron hastalığı, fare modellerinde ön ayakları üzerinden elektrofizyolojik ölçümü

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Yüksek frekanslı stimülasyon tarafından fare dentat Gyrus aktivasyonu için invaziv bir yöntem

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

Farelerde Transkraniyal doğru akım stimülasyon (tDCS)

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...

Serebral Kan Akışının ve Elektrik Alanlarının Haritalanması: Farelerde Elektrot Yerleştirme

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Yazar Spotlight: Alzheimer Modelinde Serebral Kan Akışını İyileştirmek için Stimülasyona Dayalı Yaklaşım 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

Anestezi Uygulanmış Farelerde Ekstrakraniyal Uyarıcı Elektrotların Yerleştirilmesi ve Serebral Kan Akımı ve İntrakraniyal Elektrik Alanlarının Ölçülmesi

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...