We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

Not: Hayvan konuları içeren tüm prosedürü Kurumsal Hayvanlar Etik Genomik Enstitüsü Komitesi (IAEC) ve Bütünleştirici Biyoloji, Yeni Delhi tarafından onaylanmış (IGIB / AEC / 10/2013). Bu protokol, spesifik olarak miR-29 beyin ve demonte olarak Antagomir-29 hedefli uygulama için ayarlanır. 1. Antagomir Tasarım Stratejisi <ol><li> MiRBase 11 olgun miRNA dizisi Al <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li> MiRBase kaydında &quot;Gen Aile&quot; bağlantısını kullanarak ilgili miRNA aile üyelerinin dizileri Al <a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">(Http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009).</a> </li><li> Diziyi tamamlayacak Ters: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> için uygun bir araç sağlarKompleman dizileri ters. </li><li> Aşağıdaki yönergeleri kullanarak LNA tarafından modifiye edilmesi pozisyonları işaretle: <ol><li> LNA pirimidinler pürin 12 daha stabil olduğu için, LNA değişiklik için 3-4 bazlar ayrı yerleştirildiğinde Timin artıkları seçin. </li><li> Önceki adımı az beş değişiklikler üretiyor eğer 3-4 artıkları ile ayrılmış Cytosine artıklarını seçin. </li><li> Bu tohum dizisini önler, çünkü antagomir 5 &#39;ucuna beş değişiklikler öncelik. </li></ol></li></ol> 2. Antagomir-nöropeptid Kompleks hazırlanması Not: Bu protokolde en kritik adımdır ve standardizasyon ihtiyacı vardır. Herhangi bir floresan etiketli oligonükleotid (FLO) antagomir 9 yerine kullanılabilir protokolü standardize etmek. Peptidler yüksek saflıkta olmalıdır (HPLC derece,% 98 saflık) .Sequences ve peptit ve antagomir yük Tablo 1 &#39;de verilmiştir. <ol><li> Mo sayısına kararantagomir les fare antagomir, toksisite ve ağırlığı molar ücret dayalı enjekte edilecek. Peptit: peptidin mol sayısını hesaplayın enjekte antagomir molar yük oranı dayalı olması. NOT: fare beyninde toksisite ve etkin verilme için bir peptit: antagomir molar yük oranı standart hale getirilmiştir. Standardizasyon peptid: floresan etiketli oligonükleotid farklı molar ücret oranları kullanın. Biz Antagomir-29 ve kontrol fare 4microgram / gram vücut ağırlığı toksik ikisi de bulundu. Antagomir-29 ve kontrol 2microgram / gram vücut ağırlığı kullanılmıştır, ancak bu etkili konsantrasyon, her microRNA için farklı muhtemeldir. </li><li> Aşama 1 &#39;de hesaplanan arzu edilen son konsantrasyona ve steril% 10 D-glükoz ile stok çözeltileri ayrı mikrofüj tüplerinde peptid ve antagomir seyreltilir. </li><li> Orta girdaplama hızında bir girdap üzerindeki peptid solüsyonu ile mikrofuge&#39;de tüp tutun. </li><li> A 1/3 rd eklebir vorteks karıştırıcıda iyice karıştırıldı ise peptid solüsyonu tüpe ntagomir çözeltisi yavaş yavaş, damla damla. </li><li> Bir sonraki 1 dakika boyunca vorteks çözümlerden karıştırmaya devam edin ve daha sonra karışım, 1 dakika boyunca bekletin. </li><li> Aynı mikrofuge&#39;de tüp peptid çözeltisi ile kalan antagomir çözüm karıştırmak için adım 4 ve 5 iki kez daha tekrarlayın. Yavaş ekleme, transfeksiyon etkili olan mono-dispers kompleksleri oluşumu açısından büyük önem taşımaktadır. Hızlı ilaveli kompleksleri ve çökelme topaklanmasına yol açar olabilir. </li><li> Vorteks RT&#39;de 30 dakika boyunca bir kompleks inkübe edin. Inkübasyon Bu süre boyunca, enjeksiyonlar yapılacaktır laboratuara fareler ACCLIMATIZE. </li></ol> Antagomir-nöropeptid Kompleksi&#39;nin 3. Kuyruk Ven Enjeksiyon <ol><li> Hayvan dizginlemek için, uygun büyüklükte bir süzgeç ya da decapicone fare yerleştirin. </li><li> Pamuklu çubukla pr kullanarak kuyruk yüzeyini temizleyinE-batırılmış, sıcak su (yaklaşık 40 ° C). Bu vazodilatasyon teşvik etmek ve ven görünürlüğünü artıracak. </li><li> 15-20 ° açıyla - (1.0 cc 0.5) insülin şırınga küçük bir hacme sahip kuyruk yaklaşın. Şırınganın içine herhangi bir hava tanıtmak için dikkatli olun. Kuyruk distal kısmında başlatın. Karmaşık enjekte edilir. İğneyi çıkarın ve herhangi bir kanamayı durdurmak için enjeksiyon yerinde (yaklaşık 5-10 sn) doğrudan bir antiseptik bez uygulayın. </li><li> Zenginlik olarak corncob ve kağıt mendil ile 3-5 grubundaki bir tek havalandırılan bir kafes içinde fare değiştirin. Aynı kafeste uninjected ve enjekte fareler tutmayınız. </li></ol> 4. Davranışsal Tahliller Not: enjeksiyondan sonra iyileşme sağlamak için enjeksiyon ve davranışsal tahliller arasında 3-4 saat zaman aralığını tutun. Sessiz bir odada fare getirin. Alışma sürecinden bu dönemde hayvanların rahatsız etmeyin. <ol><li> Fareyi iklime alıştırmakYeni oda 30 dakika boyunca ve daha sonra da aşağıdaki davranış testleri başlar. Her bir davranış tahlilinde arasında 10 dakikalık bir boşluk bırakın. </li><li> Arka bacak kopça: NOT: Bu test, motor disfonksiyon ve nörolojik bozukluğu gösterir. <ol><li> Net arka planda kuyruk yakın tabanını tutarak yavaşça fareyi kaldırın. </li><li> 10-15 saniye hindlimb pozisyonunu kontrol edin. Bir fare ataksik zaman% 50&#39;den fazla 13 süspansiyon haline karın yönelik bir veya her iki arka ayaklarının geri gelme eğilimi Yaban tip fare komponenti uzak karın arka ayaklarının. </li><li> Kafes fare dönün </li></ol></li><li> Ledge testi: <ol><li> Kuyruk tutarak yavaşça kaldırın ve fareyi bir kafes çıkıntıya tutmak. </li><li> Kafesin köşesinde özellikle fare gözlemleyin. Yabani tip fare korku ve dengesini kaybetmeden kolayca köşeleri geçebilir. Kafes çıkıntı boyunca ve köşelerde yürürken ataksik fare kendi denge, dondurma kaybetmek ya da sallıyor. </li></ol></li><li&gt; Fare ayak izi deneyi: NOT: Bu tahlil için dar bir pist katta hazır emici tabakanın tutmak (~ 70 cm uzunluğunda, ~ 5 cm genişliğinde ~ 5 cm yüksekliğinde duvarlarla.) <ol><li> Tek elle nazikçe fareyi tutun ve bir fırça ile arka bacaklarda mürekkep uygulayın. </li><li> Dar bir pist katta emici yaprak yerleştirin. </li><li> Fare yürümek ya da diğer bir ucundan bir pist düz bir çizgide bir emici tabaka üzerinde çalışmasına izin verir. </li><li> Taze emici sayfasını kullanarak her fare ile sürecini iki kez daha tekrarlayın. </li><li> Ileri hareketinde iki ardışık adımlar arasındaki mesafeyi ölçün. Hayvan sadece sırasıyla başlatılması ve koşmak bitirme nerede ilk ve son birkaç ayak izleri dahil etmeyin. </li></ol></li></ol>

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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specifi...

MikroRNA&#39;lar, gen ekspresyonu ve hastalık dahil olmak için doğrudan kanıt düzenlenmesinde, evrensel rolüne yeni tedavi yaklaşımları olarak ortaya çıkmıştır. İlaç 1,2 hedef olarak MiRNA aktif olarak potansiyeli araştırılmaktadır. Dahası, miRNA ifade değişiklikler çeşitli hastalıkların 3 ve miRNA ifade yapay pertürbasyon bu değişikliklerin simülasyonu ile ilişkili hastalık tezahürü katılan hücresel yollar incelemek için kullanılabilir. MiRNA hedef ilaçların dokuya özgü dağıtım şu anda miRNA tabanlı ilaç gelişimi için önemli bir sorundur. Antagomirs ve miRNA taklit miRNA düzeyleri 4-6 perturbing için umut verici maddelerdir. Bununla birlikte, bunların özgüllük ve etkinliğini arttırmak vasıflar da MiRNA ekspresyonu in vivo olarak bir pertürbasyon için kullanılmadan önce antagomirs tasarımına dahil edilmesi gerekir. MikroRNA&#39;lar alakalı olan<html> Şu anda tedavi edilemeyen nörodejeneratif ve nöro-gelişimsel hastalıklarda hedef olarak. Kan-beyin bariyeri beyindeki antagomirs teslim bir kısıtlama teşkil etmektedir. Stereotaksik enjeksiyonlar beyne 7 belirli yerlere molekülleri sağlamak için kemirgen modelleri kullanılmaktadır. Bu beceri, enstrümantasyon ve zamanla geniş yatırım gerektirir. Stereotaktik enjeksiyonları en azından ufak yaralanmalara neden, ameliyat dahil ve yerel teslimat sınırlıdır, invaziv bulunmaktadır. Hedefleme nöronlar için bir tercih hücre delici peptidler kullanımı trans-damar yoldan teslim edilebilir çünkü bu sınırlamaları karşı ancak kan beyin bariyerini ihlal edebilir. Kuduz virüsü glikoproteini (RVG) elde edilen bu tür bir peptid, daha önce farelerde 8 Japon Ensefaliti Virüs karşı siRNA teslim etmek için kullanılmıştır. Biz antagomir teslimat için peptid kullanarak, miRNA&#39;lar etkin olarak fare beyninde 9 devrilen bulmuşlardır. ontent &quot;&gt; miRNA ikinci büyük zorluk knock-down miRNA&#39;lar küçük boyutu ve yakından ilişkili dizi izoformlarının varlığı ortaya çıkar. Biz üç yakından ilişkili izoformlannın oluşur-miR-29 MMU ailesi örnek almak, miR-29a Nükleik asitler (LNA&#39;lar) bile termal stabilitesini arttırmak ve bir avantajı da kilitli, b ve c. Antagomirs ayrıca genel olarak stabiliteyi artırmak ve nükleazlar tarafından saldırılara karşı dirençli kılmak için omurgası boyunca modifiye edilir. üzerinde ve ötesinde bozunmasını hedef yol sterik engelleme 10. tüm omurga boyunca değişiklikler Tanıtımı etkili ama pahalı olabilir. Biz daha önce optimal sayıda ötesinde değişiklikler ayrıca etkinliğini artırmak olmayabilir gördük. antagomir tasarımı dolayısıyla antagomir optimal değişiklik içerir. Karmaşık için antagomir kovalent olmayan nona-arginin uzantısı nörotropik peptid, bir yüklü hepta kullanılır. D-ArgininOnlar proteazlar tarafından bölünmesi kolay olan olmadığında daha yüksek stabilite kazandırabilir yana kalıntıları kullanılır. Nona-arginin uzanıyor hepta- ve hücre tipine spesifısitesi olmayan, ancak verimli hücre nüfuz etkenler olarak hareket etmektedir. Kovalent nona-arginin linker, bir nörotropik, hücre delici peptid oluşturulan için RVG peptid bağlayarak. Peptidin pozitif yüklü tortuları, kompleksler oluşturmak için, negatif yüklü nükleik asit omurgasıyla ilişki kurar etkileşim. Bu kompleksler, etkili bir şekilde kültürlenmiş hücrelere ve dokulara in vivo olarak DNA veya RNA transfekte etmek için kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

Burada yer alan prosedür kullanılarak, 50microgram floresan etiketli oligonükleotid (FLO) ve • 01:15 mol yük oranı 850microgram RVG peptid kompleksleri (FLO: peptid) hazırlanmış ve kuyruk damarı yoluyla sadece bir kez enjekte edilmiştir. Olmayan nörotropik Kuduz Virüsü Matrisi (RVM) peptid ve FLO komple bir besleme kontrol olarak kullanılmıştır. Bir sonraki gün, fareler, beyin ve karaciğer izole edildi ve tek hücre süspansiyonları hazırlandı. Hücreler, yeşil floresan mikroskop altında gözle...

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MiRNA Beyin Özgül demonte için Stereotaktik Injection Basit Alternatif

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

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Research

JoVE Journal

Neuroscience

12.6K Görüntüleme.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Video: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Şehriye ve Capellini İşleme Testleri: sıçan ve fareler hünerli forepaw fonksiyonu Basit kantitatif tedbirler

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Nörobilim

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Non-ventriküler Plazmid Enjeksiyon ve Elektroporasyon Postnatal Rat Beyin Gen Teslimat

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Yetişkin Rat spesifik Beyin Alanlarının eksitotoksik Lezyon için Stereotaksik Cerrahi

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Optogenetic ve donma-fraktür çoğaltma ile immün Fare amigdalada glutamat reseptörlerinin giriş-özel bir düzenleme incelemek için bir araya

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal Space erişim için bir alternatif ve Onaylı Enjeksiyon Yöntemi<i&gt; yoluyla</i&gt; Bir transkleral Posterior Yaklaşım

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

Kullanımı Ödül arayan davranış değişiklikleri Tedbir Basit Yolu<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

Stereotaksik Atlas güdümlü lazer yakalama mikrodiseksiyon travma yaralanma tarafından etkilenen beyin bölgeleri

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

İnsan yetişkin cilt fibroblastlar indüklenen Neurons yüksek verimli kültürünün basit üretimi

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Optogenetics gen nakavt etkileri olduğu gibi nöronal devreler içinde Presynaptic işlevi karakterize etmek için yapay mikroRNA'lar birleştirme

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Ateş Ex Vivo sıçan beyin dilimleri kullanarak birincil olayları incelemek için alternatif bir yaklaşım

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...