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.

Remarque: Tous les sujets y compris la procédure d&#39;animaux ont été approuvés par le Comité institutionnel Animaux éthique (AIVE) à l&#39;Institut de Génomique et biologie intégrative, New Delhi (IGIB / AEC / 10/2013). Ce protocole est particulièrement adaptée pour l&#39;administration ciblée de antagomir-29 dans le cerveau et knock-down de miR-29. 1. Stratégie de conception antagomir <ol><li> Récupérer la séquence miARN mature à partir miRBase 11 <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li> Récupérez les séquences de membres de la famille liés miRNA en utilisant le lien &quot;Gene famille&quot; dans le dossier de miRBase <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> Inverser compléter la séquence: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> fournit un outil pratique pourinverser séquences complément. </li><li> Marquez les positions être modifiés par LNA en utilisant les lignes directrices suivantes: <ol><li> Choisissez résidus thymine placés 3-4 bases en dehors de la modification de LNA, depuis pyrimidines de LNA sont plus stables que les purines 12. </li><li> Choisissez résidus cytosine séparés par 3-4 résidus si l&#39;étape précédente produit moins de cinq modifications. </li><li> Prioriser les cinq modifications à l&#39;extrémité 5 &#39;de la antagomir, puisque cela évite la séquence de semences. </li></ol></li></ol> Préparation Complexe 2. antagomir-neuropeptide Note: Ceci est l&#39;étape la plus critique dans le protocole et il a besoin de normalisation. Pour standardiser le protocole tout oligonucléotide marqué par fluorescence (FLO) peut être utilisé à la place du antagomir 9. Les peptides devraient être de haute pureté (qualité HPLC, 98% de pureté) et .Sequences charge de peptide et antagomir sont donnés dans le tableau 1. <ol><li> Déterminez le nombre de Mode les antagomir à injecter sur la base de la charge molaire de antagomir, sa toxicité et de poids de la souris. Calculer le nombre de moles de peptide à être injecté basée sur rapport de charge molaire de antagomir: peptide. NOTE: Normaliser rapport de charge molaire de antagomir: peptide pour la toxicité et la prestation efficace dans le cerveau de la souris. Utilisez différents rapports de charge molaire de l&#39;oligonucléotide marqué par fluorescence: peptide de normalisation. Nous avons constaté que antagomir-29 et de contrôle étaient à la fois toxique à 4microgram / gramme de poids corporel de souris. Antagomir-29 et de contrôle ont été utilisés au poids corporel 2microgram / gramme, mais cette concentration efficace est susceptible de varier pour chaque microARN. </li><li> Diluer le peptide et antagomir dans des tubes de microcentrifugation séparer les solutions mères avec le D-glucose stérile à 10% de la concentration finale souhaitée, tel que calculé à l&#39;étape 1. </li><li> Maintenir le tube de centrifugeuse avec la solution de peptide sur un vortex à la vitesse de tourbillonnement modéré. </li><li> Ajouter 1/3 de l&#39;unntagomir solution dans le tube de solution de peptide, lentement, goutte à goutte, pendant qu&#39;il est bien mélangé sur un mélangeur vortex. </li><li> Continuer de mélanger des solutions sur le vortex pendant 1 min suivantes, puis laisser le mélange reposer pendant 1 min. </li><li> Répétez l&#39;étape 4 et 5 deux fois de plus à mélanger la solution restante antagomir avec la solution de peptide dans le même tube de microcentrifugation. L&#39;addition lente est critique pour la formation de complexes mono-dispersée qui sont efficaces pour la transfection. Plus rapide peut conduire à l&#39;agrégation des complexes et leur précipitation. </li><li> Incuber le complexe pendant 30 min à la température ambiante sans agitation au vortex. Pendant ce temps d&#39;incubation, les souris s&#39;acclimater au laboratoire où les injections seront réalisées. </li></ol> 3. L&#39;injection de la veine caudale Complexe antagomir neuropeptide- <ol><li> Pour retenir l&#39;animal, placer la souris dans un dispositif de retenue ou decapicone de la bonne taille. </li><li> Nettoyer la surface de la queue en utilisant un coton-tige pre-imbibée d&#39;eau tiède (environ 40 ° C). Cela permettra de promouvoir la vasodilatation et accroître la visibilité de la veine. </li><li> Approchez la queue avec un petit volume (0,5 à 1,0 cc) seringue à insuline à un angle de 15-20 °. Veillez à ne pas introduire de l&#39;air dans la seringue. Commencez par la partie distale de la queue. Injecter du complexe. Retirer l&#39;aiguille et appliquer un tampon antiseptique directement au site d&#39;injection (environ 5-10 sec) pour arrêter le saignement. </li><li> Remplacer la souris dans une cage ventilée individuellement dans le groupe de 3-5, à l&#39;épi de maïs et du papier de soie que l&#39;enrichissement. Ne gardez pas les souris non-injecté et injectés dans la même cage. </li></ol> 4. comportementales Assays Remarque: Conservez l&#39;intervalle de temps de 3-4 heures entre injection et comportementaux tests pour permettre la récupération après l&#39;injection. Apportez la souris dans une chambre calme. Ne pas déranger les animaux pendant cette période d&#39;acclimatation. <ol><li> Acclimater la souris vers lanouvelle ambiante pendant 30 min, puis commencer les tests comportementaux suivants. Gardez un espace de 10 min entre chaque test de comportement. </li><li> Accrochage postérieur: NOTE: Ce test indique un dysfonctionnement du moteur et des troubles neurologiques. <ol><li> Soulevez doucement la souris en maintenant près de la base de la queue dans le fond clair. </li><li> Vérifiez la position des membres postérieurs pour 10-15 sec. Ébrasements de souris de type sauvage membres postérieurs loin de l&#39;abdomen, alors qu&#39;une souris ataxique tend à rétracter un ou les deux membres postérieurs en direction de l&#39;abdomen de plus de 50% du temps mis en suspension 13. </li><li> Retour de la souris à la cage </li></ol></li><li> Test de Ledge: <ol><li> Soulevez doucement la souris en maintenant la queue et le garder sur le rebord d&#39;une cage. </li><li> Observez la souris spécifiquement à l&#39;angle de la cage. Souris de type sauvage peut passer facilement les coins sans peur et sans perdre l&#39;équilibre. Souris Ataxique perdre son équilibre, gel ou secoue tout en marchant le long de la corniche de la cage et dans les coins. </li></ol></li><li&gt; dosage pied souris d&#39;impression: NOTE: Pour cet essai garder la feuille absorbante prêt, à l&#39;étage d&#39;une piste étroite (~ 70 cm de long, environ 5 cm de large avec ~ 5 cm de hauts murs.) <ol><li> Maintenez la souris doucement une main et appliquer de l&#39;encre sur les membres postérieurs par une brosse. </li><li> Placez la feuille absorbante sur le plancher d&#39;une piste étroite. </li><li> Laisser la souris pour marcher ou courir sur une feuille absorbante en ligne droite dans une piste d&#39;un bout à l&#39;autre. </li><li> Répétez le processus deux fois de plus avec l&#39;aide de la souris feuille absorbante frais. </li><li> Mesurer la distance entre deux étapes consécutives dans le mouvement vers l&#39;avant. Ne pas inclure les premiers et derniers quelques empreintes où l&#39;animal est tout simplement lancer et terminer sa course, respectivement. </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...

Les microARN ont émergé comme de nouvelles cibles thérapeutiques en raison de leur rôle universel dans la régulation de l&#39;expression des gènes et de preuve directe de l&#39;implication dans la maladie. MiRNAs sont activement explorées pour leur potentiel en tant que médicament cible 1,2. En outre, dans l&#39;expression des miARN altérations sont associées à plusieurs maladies 3 et simulation de ces changements par une perturbation artificielle d&#39;expression des miARN peuvent être utilisées pour étudier les voies cellulaires impliquées dans la manifestation de la maladie. Livraison spécifique tissulaire de médicaments ciblant miARN est actuellement un défi majeur pour le développement de médicaments à base de miARN. Antagomirs et imite miARN sont des agents prometteurs pour perturber les niveaux de miARN 4-6. Cependant, les caractéristiques spéciales qui améliorent leur spécificité et l&#39;efficacité doivent être intégrées dans la conception des antagomirs avant qu&#39;ils ne puissent être utilisés pour perturbation in vivo d&#39;expression des miARN. Les microARN sont particulièrement pertinentes<html> comme cibles dans neurodégénérative actuellement incurable et les maladies neuro-développementale. La barrière hémato-encéphalique constitue une restriction à la fourniture de antagomirs dans le cerveau. Injections stéréotaxiques sont largement utilisés dans les modèles de rongeurs pour délivrer des molécules à des endroits précis dans le cerveau 7. Il exige des compétences, d&#39;importants investissements dans l&#39;instrumentation et de l&#39;heure. Injections stéréotaxiques sont envahissantes, implique la chirurgie, provoquer au moins des blessures légères et sont limitées à la livraison locale. L&#39;utilisation de peptides de pénétration de cellules avec une préférence pour les neurones de ciblage peut contrer ces limitations, car ils peuvent être livrés par la voie trans-vasculaire, mais franchir la barrière hémato-encéphalique. Un tel peptide dérivé de la glycoprotéine virus de la rage (RVG), a déjà été utilisé pour délivrer siRNA contre le virus de l&#39;encéphalite japonaise chez des souris 8. Nous avons trouvé que l&#39;utilisation du peptide pour antagomir livraison, miARN peut être efficacement éliminé dans le cerveau de souris 9. ontenu &quot;&gt; Le deuxième défi majeur de miARN knock-down découle de la petite taille des miARN et la présence d&#39;isoformes de séquences étroitement liés. Nous prenons l&#39;exemple de la MMU-miR-29 famille qui se compose de trois isoformes étroitement liés, miR-29a , b et c. antagomirs sont aussi généralement modifié le long de l&#39;épine dorsale d&#39;augmenter leur stabilité et les rendre résistant à l&#39;attaque par des nucléases. Verrouillé Nucleic Acids (LNA) offrent un autre avantage à ce qu&#39;ils améliorent la stabilité thermique et même conduire à cibler la dégradation au cours et au-delà 10 encombrement stérique. Présentation des modifications tout le long du squelette peut être efficace mais coûteux. Nous avons vu précédemment que des modifications au-delà d&#39;un nombre optimal ne peuvent pas améliorer encore l&#39;efficacité. La conception de l&#39;antagomir implique donc la modification de la antagomir optimale. Pour le complexe antagomir non covalente avec le peptide neurotrophique, un hepta chargé de l&#39;extension de nona-arginine est utilisé. D-arginineles résidus sont utilisés car ils confèrent une meilleure stabilité car ils ne sont pas sensibles au clivage par des proteases. Hepta à tronçons de nona-arginine Loi sur les agents de pénétration de cellules comme efficaces, même si elles ne confèrent pas de spécificité de type cellulaire. Par liaison covalente du peptide RVG au lieur nona-arginine, un neurotrope, pénétrant dans les cellules peptide a été généré. Les résidus chargés positivement du peptide d&#39;interagir avec le squelette d&#39;acide nucléique chargé négativement, pour former des complexes. Ces complexes peuvent être utilisés pour transfecter efficacement ADN ou d&#39;ARN dans des cellules en culture et in vivo dans les tissus.

<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.

En utilisant la procédure présentée ici, des complexes d&#39;oligonucléotide 50microgram marqué par fluorescence (FLO) et ~ 850microgram RVG peptide de 1:15 rapport de charge molaire (FLO: peptide) ont été préparés et injectés qu&#39;une seule fois par veine de la queue. Complexe de non-neurotrope virus de la rage Matrix (RVM) peptide et FLO a été utilisé comme un contrôle de livraison. Le jour suivant, les souris cerveau et le foie ont été isolés et des suspensions cellulaires simples ont été prépar...

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.

Une alternative simple à injection stéréotaxique pour Brain Knockdown spécifique de miARN

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

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Research

JoVE Journal

Neuroscience

12.6K vues.  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.

Vidéo: 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.

Les vermicelles et tests de manipulation Capellini: Simple mesures quantitatives de la fonction patte habile dans les rats et les souris

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

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Neurosciences

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.

Livraison de gènes au cerveau de rat postnatal par des non-ventriculaire injection Plasmide et électroporation

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.

Chirurgie stéréotaxique pour lésion excitotoxique des zones spécifiques du cerveau chez le rat adulte

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.

Combiné optogénétiques et Cryo-fracture Replica immunomarquage examiner l&#39;aménagement spécifique à l&#39;entrée de Glutamate Receptors dans l&#39;amygdale Souris

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.

Une méthode alternative et Validé Injection pour l&#39;accès à l&#39;espace sous-rétinien<i&gt; via</i&gt; Une approche transclérale Postérieur

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.

Un moyen simple pour mesurer Transformations en récompense le comportement de recherche Utilisation<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.

Stéréotaxique Atlas guidée Laser Microdissection de saisie des régions cérébrales touchées par lésion traumatique

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.

Génération simple d’une Culture de haut rendement de neurones induits des fibroblastes de la peau humaine adulte

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.

Alliant Optogenetics artificiel microARN pour caractériser les effets du gène Knockdown fonction présynaptique dans les Circuits de neurones intacts

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.

Une autre approche pour étudier les événements primaires dans la neurodégénérescence utilisant des coupes de cerveau de Rat Ex Vivo

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