NINDS tarafından finanse edilen, Amerikan Kalp Derneği, Amerikan Yaşlanma Araştırma Federasyonu, March of Dimes

1. Hazırlık Başlamadan önce, gerekli cerrahi aletler, diseksiyon ve kültür ortamı, ve 7 gün eski sıçan veya fare yavrular monte edin. Protokol, fare ve sıçan hazırlıkları için aynıdır. (I) Malzeme ve ekipman <ul><li> İşletim makas (düz uzunluğu 6 &quot;, kedi # RS6818 Roboz, Gaithersburg, MD) </li><li> Micro makas, diseksiyon (uzunluk 4 &quot;, kedi # RS5910. Roboz, Gaithersburg, MD) </li><li> Karbon Çelik Dumont Cımbız (kedi # RS-5045.Roboz, Gaithersburg, MD) </li><li> Modifiye cam &quot;transfer&quot; pipetler (kaldırıldı Pasteur pipeti alt ve kenar düzeltti) </li><li> Aclar plastik film meydanlarda (Honeywell, # 812.071, Pottsville, PA) &quot;x 2&quot; 2 kesilmiş </li><li> Kapak 3 sünger 4 (Kendall, kedi # 3157, Mansfield, MA) </li><li> 30 mm Millicell membran ekler (0.4 mikron; Millipore, Bedford, MA) </li><li> Iki ucu keskin bir jilet (McILwain doku kıyıcı, Vibratome Şirket, St Louis, MO) Mcllwain Doku kıyıcı </li><li> CCD kamera ve görüntü analiz yazılımları ile inverted mikroskop </li></ul> (Ii) Diseksiyon Orta (DM; pH 7,3 az 1 litre) <table border="0"><tbody><tr><td> Hank Kullanıcı Dengeli Tuz </td><td> 1 şişe (95.2 g/1L) (Sigma: H2387) </td></tr><tr><td> NHCO 3 (4.2 mM) </td><td> 350 mg </td></tr><tr><td> Hepes (10 mM) </td><td> 2.83 g </td></tr><tr><td> Glikoz (33.3 mM) </td><td> 6.0 g </td></tr><tr><td> Penisilin / streptomisin (100x) </td><td> 10 ml </td></tr><tr><td> BSA (% 0.3) </td><td> 300 mg </td></tr><tr><td> MgSO 4-7H 2 O </td><td> 1,44 g </td></tr></tbody></table> (Iii) Kültür ve orta (400 ml) <table border="0"><tbody><tr><td> MEM (Invitrogen 11.575-032) </td><td> 200 mL </td></tr><tr><td> *** Hank Dengesi Tuz (Invitrogen 24.020-117) </td><td> 100 mL </td></tr><tr><td> At serumu (Invitrogen 26.050-070) </td><td> 100 mL </td></tr><tr><td> HEPES tamponu (1 M Invitrogen 15.630-080) </td><td> 5 ml </td></tr><tr><td> Penisilin / Streptomisin (100x) </td><td> 4 mL </td></tr><tr><td colspan="2"> *** Kültür ortamı vermeden önce 12.8 gr glukoz 500 ml Hank Dengesi Salt ekle </td></tr></tbody></table> (Iv) Hayvanlar 7 gün eski sıçan veya fare postnatal yavrular, genellikle Deneme başına bir çöp. 2. Kültür Günü <ol><li> Tüm diseksiyon aletleri ve Aclar film başlamadan önce 30 dakika boyunca% 70 ETOH batırılmış olmalıdır. Diseksiyonu kaput, bıçak ve doku kıyıcı yüzey etanol ile dezenfekte edilmelidir. </li><li> (3-4) birkaç 6 sıra doku kültürü plakaları hazırlamak; ortalama 1 ml kültür ortamı ekleyin ve her kuyuda bir Millipore geçirgen zar takın. 37 koyun 6-kuyucuğu ° inkübatör dilim aktarmak için hazır olana kadar. </li><li> DM ve buz üzerinde yer ile birkaç (4-6) 60 mm tabak hazırlayın. </li><li> Hızla başını kesmek makas ile ve% 70 ETOH daldırma ve beyin, kafatasının bir saggital kesi ile hızlı bir şekilde ortaya çıkarmak. Dikkatle soğuk DM beyin yeri ve hemen çıkarın. Iki yarımküresinin içine ayırın ve 2-3 saniye için bir kapak sünger üzerinde, her yarımkürede medial yerde. </li><li> Yarıkürede doku kıyıcı Aclar film kare yer ve üzerine yavaşça kaldırın ve yeri; 350 mikron bölümlerde koronal beyin kesmek. Bittiğinde, yavaşça ALCAR kare kesitli soğuk DM yarımkürede ve batığın alır. </li><li> Diseksiyon mikroskop kullanarak, hipokampal bölümlere ayırın. Sıçan için, kaudal hipokampus rostral kapsayan 5 bölümden olmalıdır ve fare için 3-4 bölüm olması gerekir. </li><li> 37 ° doku kültürü inkübatör membran ekler ile 6-kuyucuğu çıkartın. Hipokampal dilimleri ayrı ayrı cam transferi pipet kullanarak geçirgen membranlar üzerine aktarın. Çok DM membran üzerine bırakılırsa, Pasteur pipeti ile DM fazla çıkarın. Her membran beş hipokampal dilimleri yerleştirin. Başına en az 3 kuyu, durum ya da 15 dilim izin verin. </li><li> Toksisite deney başlamadan önce 12-14 gün için% 5 CO nemlendirilmiş inkübatör 37 ° kültür plakaları inkübe edin. Kültür ortamı haftada iki kez değiştirilebilir. </li></ol> 3. Dilim Kültür Hipokampal Nöronal Yaralanma uyaran ve miktarının <ol><li> Kültür 12-14 gün sonra 5 mg / ml propidium iyodür (PI), kültür ortamı ekleyin. Bu adım, görselleştirme ve bazal hücre ölümü ölçümü sağlayacaktır. </li><li> Ertesi gün, PI ölçmekher hipokampal dilim CA1 subregion (ve / veya CA3 veya dentat istenirse) floresans ve elde PI floresans ölçümleri bazal hücre ölümü (zaman olarak adlandırılır = 0 saat, ya da T 0) temsil eden anlamına gelir. Görüntü elde etme ve ölçülmesi için, biz openlabın yazılımı (Doğaçlama, Birleşik Krallık), ancak diğer görüntü toplama ve analiz yazılımı floresan yoğunluğunu ölçmek için kullanılabilir. </li><li> T 0 PI floresan görüntü elde etme hemen sonra, seçtiğiniz yöntem (biz nöronal toksisite 2,5 ikna etmek için bir saat 10 mcM NMDA, oksijen-glikoz yoksunluk, veya LPS) nöronal hasara yol. Toksik uyaran sonra, 5 mg / ml PI içeren taze orta orta yerine ve geceleme dilim korumak. </li><li> 3 gün, ortalama PI 24 saat deneysel durumun floresan veya T 24 belirlenir. </li><li> Bu görüntü alımından sonra hemen orta değiştirmek ve 10 mcM NMDA ve 5 mg / ml PI ve maksimal nöron ölümü elde etmek için gece boyunca inkübe dilimleri eklemek. </li><li> 4 gün, ortalama Tmax olarak tanımlanan maksimum nöronal ölüm PI floresan ölçmek. Aşağıdaki gibi yüzde hücre ölümü hesaplayın: (T 24 - T 0) / (T max-T 0). </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2106/2106fig1.jpg" alt="Şekil 1" /> Şekil 1 (A) Zaman deneysel yaralanma ve görüntü alımı, hipokampus ve sonraki nesil için. (B) hipokampal dorsalde dilimleri (T 0), 24 saat 10 mcM NMDA (T 24) 1 saat maruz kalma ve NMDA maksimum nöronal hasarı (T max) elde etmek için daha ileri bir 24 saat ya da 10 mcM. Temsilcisi görüntüleri

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

Deneyler kopyalayan tekrarlanabilir ve tutarlı sonuçları elde etmek için izlemeniz gereken iki önemli nokta vardır. İlk olarak, 12-14 gün önce deney için hipokampal dilim yaş izin vermek önemlidir. Dilim yalıtımı sayesinde, önemli bir mikrogliyal yanıt vardır ve mikroglia in vitro 3,6 10 günde meydana gelen bir dinlenme dallanmış devlet bir dönüş, bir süre aktif kalır . İkincisi, PI floresan yoğunluğu, yaralı hücrelerinin sayısı 4 ile orantılıdır anlamı...

the organotypic hippocampal slice culture model for examining neuronal injury

Organotipik hipokampal kesit kültürü, temel mimari ve hipokampus bileşimi nispeten 1 korunduğu nöronal yaralanma mekanizmaları incelemek için bir in vitro yöntemdir . Organotipik kültür sistemi, nöronal astrositik ve mikroglia etkilerinin incelenmesi için izin verir, ama bir ex vivo hazırlama olarak, periferik inflamatuar hücrelerin kan akışı, ya da işe etkileri hitap etmez . Bu amaçla, bu kültür yöntemi hipokampus piramidal nöronlar eksitotoksik ve hipoksik hasarı incelemek için sık sık kullanılır, ama aynı zamanda inflamatuvar yanıt incelemek için kullanılan olmuştur. Bu yazıda, doğum sonrası kemirgen beyin hipokampal dilim kültürleri, nöronal hasara yol toksik uyaranlara yönetmek ve assaying hipokampal nöron ölümü ve miktarının belirlenmesi için yöntemler açıklanmaktadır.

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organoptypic hipokampal kesit kültürü modeli<em&gt; In vitro</em&gt; Model paradigmaların çeşitli nöronal hasarı incelemek için kullanılır. Bu makalede, biz dilim kültürler oluşturma ve nöronal yaralanma miktarının belirlenmesi için yöntemler açıklanmaktadır.

Nöronal Sakatlık incelenmesi için Organotipik Hipokampal Dilim Kültür Modeli

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

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JoVE Journal

Neuroscience

19.0K Görüntüleme.  Stanford University School of Medicine. Organoptypic hipokampal kesit kültürü modeli In vitro Model paradigmaların çeşitli nöronal hasarı incelemek için kullanılır. Bu makalede, biz dilim kültürler oluşturma ve nöronal yaralanma miktarının belirlenmesi için yöntemler açıklanmaktadır.

Video: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

Organotipik Dilim Kültür GFP-Periferik Sinir akıbet Gerçek zamanlı Görüntüleme Fare Embriyolar ifade

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Nörobilim

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipik Hipokampal Dilim Kültürler

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Doğum Sonrası Beyin Nöronal Göç Yüksek Çözünürlüklü Zaman Aşımı Görüntüleme için bir Organotipik Dilim Testi

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Embriyonik ventral orta beyin Organotipik Dilim Kültürler: dopaminerjik Nöronal Gelişim Eğitim Bir Sistem In vitro</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Görüntüleme Nöronal cevaplar Vomeronasal Organ Dilim hazırlıklar Genetiği Kodlanmış Kalsiyum Sensör ifade

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

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotipik Dilim Kültürleri Purkinje Hücre Dendritik Morfoloji Analizi

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Organotipik Hipokampal Dilimleri eşleştirilmiş Tüm Hücre Kayıtlar

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Içinde Neurovascular Yaptırma Analizi Entorhino-hipokampal Organotipik Dilim Kültürler

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hipokampal dilim kültürler çalışma Neuroprotection ve tümör hücrelerinin Invasiveness Model olarak

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Hipokampal teta salınımlarını fareler davranışlar içinde Optogenetic sürüklenme

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...