NIH hibe AG027297, bir ödül Kentucky Omurilik ve Kafa Travması Araştırma Güven, ve hediye Kléberg Vakfı tarafından desteklenen çalışın.

<ol>
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Bu protokolde belirtilen adımları beyin diseksiyonları, yaşlı, genç erişkin sıçan gibi en azından hızlı ve verimli bir şekilde yapılmaktadır sigorta yardımcı olacaktır. Biz de LTP ve LTD kendi dilim çalışmalar kurmak için yeni başlayanlar için yeterli ayrıntı sağlar. Yaşlanma ve sinaptik fonksiyon ve plastisite AD değişiklikleri daha fazla arama hedeflerinden biri ise, bir daha görüşülmek hak yukarıda ima en az iki metodolojik konular, vardır. İlk olarak, çeşitli laboratuvarlar gösterilen Ca 2 +: Mg2 + ACSF hipokampal dilim 2,10,24,25 indüksiyon sinaptik plastisite üzerinde belirgin bir etkisi olabilir kayıt oranı. Memeli BOS, Ca 2 +: Mg2 + oranı yaklaşık biri (örneğin 26 görmek). Ancak, ACSF Ca 2 +: Mg2 + yakın 2 oranları sinaptik fonksiyon ve plastisite dilim çalışmaları yaygın olarak kullanılmaktadır. Erken çalışmalarda, bu uygulama muhtemelen LTP indüksiyon optimize etmek için adapte oldu, ardından tüm plastisite çalışmaları için bir rutin haline geldi. Ancak, bu uygulama nöronal Ca 2 + düzenleme iyi karakterize farklılıklar nedeniyle yaşlanma ve AD çalışmaları sorunlu olabilir. Özellikle, Ca 2 + akını ve / veya Ca 2 +-indüklenen Ca 2 +-sürüm 3,27-31 nöronal aktivasyon sırasında yaşlı sıçan ve / veya AD modeli fareler yüksek. LTD İndüksiyon ACSF Ca 2 + düzeyleri ince değişiklikler özellikle duyarlıdır. Mg2 yakın iki + oran, erişkinlerde sağlam LTD gözlenen var içinde: 2mm Ca 2 + ve 2 mM Mg2 +,, yaşlı, ama 2 yetişkin hayvanların genç değil LTD yaygın sonuçları ise bir Ca 2 kullanarak çalışmalar + kullanan Bizim protokol, 32 yaşlı sıçanlarda azaltılmış LTD ile birlikte bir yaş farkı 2,10 ya da yokluğu. Bu gözlemler, yaşlı ve genç erişkin hayvanlarda Ca 2 + bağımlı plastisite karşılaştırırken dikkatle ACSF Ca 2 + ve Mg2 + düzeyleri dikkate almak gerektiğini vurgulayın. Ikinci metodolojik sorunu postsinaptik depolarizasyon 33 ve sinaptik gücü yaşlanma / genotip farklılıklar LTP güçlü bağımlılık ilgilidir. Tipik bir LTP deney, temel ve LTP stimülasyon yoğunluğu, genellikle bir buçuk maksimal (veya üç çeyrek) EPSP maksimum genlik üretmek için ayarlanır. Potansiyel bir sorun, yaşlı sıçanlar ve APP/PS1 fareler, genellikle bazal EPSP değerleri de yaşlı sıçanlar ve APP/PS1 fareler daha küçük olacağı anlamına, genç ve / veya yabani tip meslektaşları göre sinaptik gücü azalır göstermektedir. Daha küçük EPSPS LTP 33 indüklemesi için daha düşük bir olasılık, LTP stimülasyon sırasında daha az depolarizasyon çevirmek olabilir . Çünkü bu potansiyel bulandırabilir, bu hayvanların verim açığı, bir plastisite açığı ya da her iki sergi belirlemek zordur. Yani, yaşlı ve / veya APP/PS1 fareler LTP indüksiyon mekanizmaları (hayır plastisite açığı) işlevsel sağlam olabilir, ama yetersiz, bu koşullar altında (verim açığı) uyarılır. Bu ayrım, hacmi ve plastisite mekanizmaları mekanizmaları belirli bir farmakolojik tedavi çok farklı tepki olarak kritik öneme sahiptir. Biz LTP stimülasyon öncesinde tüm dilimleri boyunca aynı seviyede (örneğin 1 mV) EPSP genlik normalleştirilmesi LTP indüksiyon azaltılmış throughput etkisini en aza indirmek için çalışıyoruz. Diğer stratejiler de etkili olabilir (örneğin, voltaj ya da akım kelepçe kullanımı LTP stimülasyon sırasında gruplar arasında membran potansiyeli eşitlemek için) ve bu hayvan modellerinde LTP soruşturma zaman düşünülmelidir.

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> NaCl </td><td> Balıkçı </td><td> BP358-1 </td></tr><tr><td> KCl </td><td> Balıkçı </td><td> BP366-500 </td></tr><tr><td> KH 2 PO 4 (tekbazlı) </td><td> Sigma </td><td> P5379-100G </td></tr><tr><td> MgSO 4 </td><td> Sigma </td><td> M2643-500G </td></tr><tr><td> CaCl 2 (dihidrat) </td><td> Sigma </td><td> C3306-250G </td></tr><tr><td> NaHCO 3 </td><td> Balıkçı </td><td> S233-500 </td></tr><tr><td> C 6 H 12 O 6 (dekstroz) </td><td> Balıkçı </td><td> BP350-1 </td></tr></tbody></table> Tablo 1. Reaktifler gerekli <table border="1"><tbody><tr><td> Ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Erlenmeyer şişeler </td><td> Balıkçı </td><td> FB-500-2000 FB-500-1000 </td><td></td></tr><tr><td> Akvaryum bubbler </td><td></td><td></td><td> Oksijen medya için kullanılır. Pek çok evcil hayvan mağazasında </td></tr><tr><td> 50 ml Cam beher </td><td> Balıkçı </td><td> 02-540g </td><td> ACSF beyin storarge için </td></tr><tr><td> Parafilm </td><td> Balıkçı </td><td> 13-374-10 </td><td></td></tr><tr><td> Küçük Hayvan Giyotin </td><td> Dünya Hassas Aletler (TEFE) </td><td> DCAP-M </td><td></td></tr><tr><td> Düz kağıt havlu </td><td></td><td></td><td></td></tr><tr><td> # 11 Feather cerrahi bıçak </td><td> Balıkçı </td><td> 08-916-5B </td><td></td></tr><tr><td> Beebee kemik makas </td><td> Güzel Bilim Araçları (FST) </td><td> 16044-10 </td><td></td></tr><tr><td> Lempert Rongeurs </td><td> Roboz </td><td> RS-8321 </td><td> Sıçanlar için kullanın </td></tr><tr><td> Friedman-Pearson Rongeurs </td><td> FST </td><td> 16020-14 </td><td> Fareler ya da sıçanlar için kullanın. </td></tr><tr><td> Hippocampus aracı </td><td> FST </td><td> 10099-15 </td><td></td></tr><tr><td> Kaşık </td><td></td><td></td><td> Plastik bir çay kaşığı yapacak </td></tr><tr><td> Spatula </td><td> Balıkçı </td><td> 21-401-25A </td><td> Spatula </td></tr><tr><td> Cerrahi iris makası </td><td> FST </td><td> 14058-09 </td><td></td></tr><tr><td> plastik transferi pipetler </td><td> Balıkçı </td><td> 13-711-43 </td><td></td></tr><tr><td> 110mm Whatman filtre kağıdı </td><td> Balıkçı </td><td> 09-805E </td><td> Whatman kedi. 1001-110 </td></tr><tr><td> Cam petri </td><td> Balıkçı </td><td></td><td></td></tr><tr><td> Leica VT1000P Manuel Mikrotom Titreşimli </td><td> Vibratome </td><td></td><td></td></tr><tr><td> 0.1mm FA-10 Feather G bıçak </td><td> Ted Pella </td><td> 121-9 </td><td> 0.1mm FA-10 Feather G bıçak </td></tr><tr><td> Borosilikat Pasteur pipetleyin (kauçuk ampul) </td><td> Balıkçı </td><td> 13-678-20A </td><td> Dilimleri transfer için: İpucu büyük açılış kopmuş ve ısı parlatılır </td></tr><tr><td> 35 mm Polysterine Kültür çanağı </td><td> Corning </td><td> 430588 </td><td> Diseksiyonu sonra dilimleri toplamak için kullanılır </td></tr></tbody></table> Tablo 2. Araçlar ve diseksiyon için malzeme <table border="1"><tbody><tr><td> Yapı / Malzeme </td><td> Şirket </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Holding kamara </td><td> Özel Yapım </td><td></td></tr><tr><td> P-97 Yatay Pipet Çektirme </td><td> Sutter Instrument Co. </td><td></td></tr><tr><td> Titreşim izolasyonu tablosu </td><td> Teknik Manufacturing Corporation (TMC) </td><td></td></tr><tr><td> Faraday kafesi </td><td> Özel inşa </td><td></td></tr><tr><td> Alt tabancası ile Pyrex Aspiratör Şişe (Ürün # 1220-1L) </td><td> Corning </td><td></td></tr><tr><td> Regülatör ile Gravite-contolled IV seti (Ürün # 2C8891) </td><td> Baxter </td><td></td></tr><tr><td> Merkezi Vakum Hattı </td><td></td><td> En modern laboratuvarları </td></tr><tr><td> % 95 O 2 /% 5 CO 2 Gaz Mix </td><td> İskoçyalıt-Brüt Co. </td><td></td></tr><tr><td> TygonTM Lab boru O 2 / CO 2 teslimat için </td><td> Fisher Scientific </td><td> Non-toksik, non-oksitleyici, çeşitli boyutlarda gelir. </td></tr><tr><td> Eclipse E600FN Mikroskop </td><td> Nikon </td><td> infared filtresi, GFP, DS-Red2 filtreler yakın 10x ve 40x hedefleri, </td></tr><tr><td> Cool Ek Bileşen ES Dijital Fotoğraf Makinesi </td><td> Photometrics </td><td> Cool Ek Bileşen ES Dijital Fotoğraf Makinesi </td></tr><tr><td> X-Cite Floresan Aydınlatıcı </td><td> EXFO </td><td> X-Cite Floresan Aydınlatıcı </td></tr><tr><td> Mikroskop Platformu </td><td> Siskiyou </td><td> Özel toplandı </td></tr><tr><td> RC-22 Dalgıç kayıt odası (Ürün # 64-0228) </td><td> Warner Instruments (WI) </td><td> P-1 platformu ve sahne adaptörü (Warner itibaren Ürün # 64-0277) gerektirir </td></tr><tr><td> TC2BIP 2/3Ch sıcaklık kontrol </td><td> Hücre Microcontrols </td><td> TC2BIP 2/3Ch sıcaklık kontrol </td></tr><tr><td> 4 Eksen Kılavuzu Minyatür manipülatör </td><td> Siskiyou </td><td></td></tr><tr><td> Platin Iridium Tel (0.002 in) (Madde PTT0203) </td><td> TEFE </td><td></td></tr><tr><td> A365 Stimulus Isolator </td><td> TEFE </td><td> A365 Stimulus Isolator </td></tr><tr><td> 700b Amplifikatör Multiclamp </td><td> Akson Aletleri </td><td></td></tr><tr><td> Digidata 1322A A / D dönüştürücüsü </td><td> Akson Aletleri </td><td></td></tr><tr><td> PClamp yazılım </td><td> Akson Aletleri </td><td></td></tr><tr><td> Kişisel Bilgisayar (Pentium 4) </td><td> Vadi </td><td></td></tr></tbody></table> Tablo 3 Elektrofizyoloji ekipman ve malzemeleri

preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology

Kemirgen hipokampal dilim hazırlık memeli sinaptik fonksiyon ve plastisite araştırmak için belki de en çok kullanılan araçtır. Hipokampus sıçan ve fareler hızlı ve kolay bir şekilde ayıklanır ve dilim oksijenli yapay beyin omurilik sıvısı saat canlı kalır olabilir. Ayrıca, temel electrophysisologic teknikleri hipokampal dilimleri sinaptik fonksiyon soruşturma kolayca uygulanır ve bazı bilişsel bozukluklar için en iyi biyobelirteç sağladı. Hipokampal dilim, öğrenme ve hafıza yer sinaptik plastisite mekanizmalarının çalışma için özellikle popüler. Hipokampal dilimleri (veya eksikliği bunların), sinaptik etkinliği uzun vadeli potansiyasyonu ve depresyon (LTP ve LTD) indüksiyon değişiklikler, bilişsel-engelli hayvan ve / nörolojik fenotip tanımlamak için ya da eylem mekanizması değerlendirmek için sık sık kullanılır nootropic bileşikler. Bu makale hipokampal dilim sıçan ve transgenik farelerin beyin yaşlanması ve Alzheimer hastalığı (AH) 1-3 ile ilişkili sinaptik değişiklikler çalışma için hazırlamak için kullanılan prosedürler sıralanıyor. Yaşlı sıçanlarda ve AD modeli fareler kullanımı, araştırma genç sıçan ve / ya da fare kullanarak alışık araştırmacılara özel bir takım zorluklar ortaya çıkabilir. Yaşlı sıçanlarda beyin çekimi ve / veya diseksiyonu gecikme ve dolayısıyla sinaptik fonksiyon ve plastisite gerçek yaşı farklılıklar inkâr veya abartmak genç sıçanlar ve fareler, daha kalın kafatasları ve sert bağ dokusu var. Yaşlanma ve amiloid patoloji yine fizyolojik değerlendirme alınan herhangi bir çıkarımlar komplike diseksiyon işlemi sırasında sürekli hipokampal hasar alevlendirebilir. Burada, biz bu problemleri en aza indirmek için diseksiyon işlemi sırasında atılan adımları tartışacağız. Örnekler, &quot;sağlıklı&quot; ve &quot;sağlıksız&quot; bir dilim sıçan ve fareler elde sinaptik yanıtlarının yanı sıra temsili sinaptik plastisite deneyler sağlanmaktadır. Bu hayvan modellerinde (örneğin, kayıt çözümü bileşenleri, stimülasyon parametreleri) sinaptik fonksiyon diğer metodolojik faktörlerin olası etkileri de tartışılmıştır. Bu makalenin odak noktası, yaşlı sıçanlar ve transgenik fareler, acemiler fizyolojisi dilim kullanımı olsa da, burada çeşitli kemirgen modelleri kullanarak, kendi çalışmalarına başlamak için yeterli detay bulmalısınız.

Watch this Scientific Journal Video about Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology at JoVE.com

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Bu makale, sıçan ve transgenik farelerin hipokampal dilimler sinaptik değişiklikler, beynin yaşlanma ve Alzheimer hastalığı gibi nörodejeneratif hastalıklar, yaşa bağlı ile ilgili çalışma için hazırlamak için prosedürler sıralanıyor.

Yaşlanma ve Amiloid Patoloji sırasında sinaptik değişiklikler Çalışması Sıçanlar ve Transgenik Farelerde akut Hipokampal Dilimleri hazırlanması

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

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Research

JoVE Journal

Neuroscience

93.7K Görüntüleme.  University of Kentucky College of Public Health. Bu makale, sıçan ve transgenik farelerin hipokampal dilimler sinaptik değişiklikler, beynin yaşlanma ve Alzheimer hastalığı gibi nörodejeneratif hastalıklar, yaşa bağlı ile ilgili çalışma için hazırlamak için prosedürler sıralanıyor.

Video: Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Β-amiloid Oligomerik hazırlanması 1-42</subHipokampal Dilimleri Sinaptik Plastisite değer düşüklüğü> İndüksiyon

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Nörobilim

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Kemirgen Beyin Dilimleri Nöronlar Vibrodissociation Sinaptik İletim ve Görüntü Presinaptik Terminalleri Eğitim

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Kalsiyum Görüntüleme için akut subventricular dilimi Dilimleri hazırlanması

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Akut Hipokampal Dilimler içinde Synaptically-uyarılmış astroglial ve Nöronal Cevaplarının İkili Elektrofizyolojik Kayıtlar

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Fare Hippocampus içine Oligomerik Amiloid-beta Stereotaksik İnfüzyon

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Kemirgenler gelen akut Hipokampal Dilimleri kullanarak Synaptic Etiketleme / yakalama ve Çapraz yakalama İncelenmesi

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

En iyi duruma getirilmiş bir Nkullanarak akut beyin dilimleri hazırlanması-metil-D-glucamine koruyucu kurtarma yöntemi

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Sinaptik plastisite akut Hipokampal dilimleri bir küçük hacimli geri dönüşüm-, perfüzyon- ve batma-tipi oda sistemi içinde tutulan kayıt

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Normal ve Huntington Farelerden Akut Beyin Dilimlerinde Glutamat Salınımı ve Alımının Tek Sinaps Göstergeleri

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Elektrofizyolojik Kayıtlar için Akut İnsan Hipokampal Dilimlerinin Hazırlanması

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...