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.

Hinweis: Alle Verfahren einschließlich Tier Themen wurden von Institutional Tiere Ethikkommission (IAEC) am Institut für Genomik und Integrative Biologie, Neu-Delhi genehmigt worden (IGIB / AEC / 10/2013). Dieses Protokoll ist speziell für die gezielte Abgabe Antagomir-29 im Gehirn und Knockdown von miR-29 eingestellt. 1. Antagomir Design Strategy <ol><li> Rufen Sie die reife miRNA-Sequenz aus miRBase 11 <a href="http://www.mirbase.org/">(http://www.mirbase.org/).</a> </li><li> Rufen Sie den Sequenzen verwandter miRNA Familienmitglieder über den Link &quot;Gene Family&quot; in der miRBase Rekord <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> Reverse Komplement der Sequenz: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> bietet ein komfortables Werkzeug zurreverse Komplement-Sequenzen. </li><li> Markieren Sie die Positionen, die von LNA geändert werden mit Hilfe der folgenden Richtlinien: <ol><li> Wählen Thyminreste 3-4 Basen auseinander für den LNA Modifikation platziert, da LNA Pyrimidine sind stabiler als Purine 12. </li><li> Wählen Cytosinreste um 3-4 Reste getrennt, wenn vorhergehenden Schritt erzeugt weniger als fünf Modifikationen. </li><li> Priorisieren der fünf Modifikationen am 5&#39;-Ende des antagomir, da die Saat-Sequenz vermieden wird. </li></ol></li></ol> 2. Antagomir-Neuropeptid Complex Vorbereitung Anmerkung: Dies ist der wichtigste Schritt im Protokoll und Standardisierung muss. Um das Protokoll zu jeder fluoreszenzmarkierte Oligonucleotid (FLO) kann anstelle des antagomir 9 verwendet werden standardisieren. Die Peptide sollten von hoher Reinheit sein (HPLC-Qualität, 98% Reinheit) .Sequences und Ladung des Peptids und antagomir sind in Tabelle 1 angegeben. <ol><li> Entscheiden Sie, die Anzahl der moles von antagomir bezogen auf molare Ladung auf antagomir, seiner Toxizität und Gewicht der Maus injiziert werden. Berechnen Sie die Anzahl der Mole des Peptids injiziert basieren auf molare Ladungsverhältnis von Antagomir: Peptid. HINWEIS: Standardisieren molaren Ladungsverhältnis von Antagomir: Peptid zur Toxizität und effektive Bereitstellung im Gehirn der Maus. Verwenden Sie unterschiedliche molare Ladungsverhältnissen von fluoreszenzmarkierten Oligonukleotid: Peptids zur Standardisierung. Wir fanden, dass Antagomir-29 und Kontrolle waren beide bei 4microgram / Gramm Körpergewicht der Maus giftig. Antagomir-29 und Kontrolle wurden bei 2microgram / Gramm Körpergewicht verwendet, aber das effektive Konzentration dürfte für jeden microRNA variieren. </li><li> Verdünnen des Peptids und antagomir in getrennten Mikrozentrifugenröhrchen aus Stammlösungen mit steriler 10% D-Glucose auf die gewünschte Endkonzentration in Schritt 1 berechnet. </li><li> Halten Sie die Mikrozentrifugenröhrchen mit dem Peptid-Lösung auf einem Vortex bei moderaten Vortexen Geschwindigkeit. </li><li> In 1/3 der einentagomir Lösung der Peptidlösung Rohr langsam tropfenweise, während es wird gründlich auf einem Vortex-Mischer gemischt. </li><li> Setze das Mischen der Lösungen auf dem Vortex für den nächsten 1 min und dann lassen Sie die Mischung für 1 min stehen. </li><li> Wiederholen Sie die Schritte 4 und 5 zwei weitere Male um den verbleibenden antagomir Lösung mit der Peptidlösung in die gleiche Mikrozentrifugenröhrchen gemischt. Langsame Zugabe ist kritisch für die Bildung von monodispersen Komplexe, die wirksam bei der Transfektion. Schnelle hinaus kann die Aggregation der Komplexe und deren Niederschlag führen. </li><li> Inkubieren des Komplexes für 30 min bei RT ohne Verwirbelung. Während dieser Zeit der Inkubation akklimatisieren die Mäuse ins Labor, wo Injektionen durchgeführt. </li></ol> 3. Schwanzveneninjektion von Antagomir-Neuropeptid-Komplex <ol><li> Um das Tier zurückzuhalten, die Maus in einem Verzögerer oder decapicone der richtigen Größe. </li><li> Reinigen Sie die Oberfläche des Schwanzes mit Wattestäbchen pre getränkten in warmem Wasser (ca. 40 ° C). Dies wird Vasodilatation fördern und erhöhen die Sichtbarkeit der Vene. </li><li> Nähern Sie sich dem Schwanz mit einem kleinen Volumen (0,5 bis 1,0 cc) Insulinspritze in einem 15-20 ° -Winkel. Darauf achten, dass keine Luft in die Spritze einzuführen. Beginnen an dem distalen Abschnitt des Schwanzes. Spritzen Sie die Anlage. Entfernen Sie die Nadel und wenden eine antiseptische Tupfer direkt an der Injektionsstelle (etwa 5-10 sec), um Blutungen zu stoppen. </li><li> Ersetzen Sie die Maus in einem individuell belüfteten Käfig in der Gruppe von 3-5, mit dem Maiskolben und Tissue-Papier als Bereicherung. Injizierten und injizierten Mäusen halten Sie nicht in demselben Käfig. </li></ol> 4. Verhaltens Assays Hinweis: Bewahren Sie die Zeitspanne von 3-4 Stunden zwischen Injektion und Verhaltenstests zur Erholung nach der Injektion zu ermöglichen. Bringen Sie die Maus, um ein ruhiges Zimmer. In dieser Zeit der Eingewöhnung der Tiere nicht stören. <ol><li> Akklimatisieren Sie die Maus auf dieneuer Raum für 30 Minuten und starten Sie dann die folgenden Verhaltenstests. Halten Sie einen Abstand von 10 Minuten zwischen den einzelnen Verhaltenstest. </li><li> Hinterlauf clasping: HINWEIS: Dieser Test zeigt, motorische Störungen und neurologischen Störungen. <ol><li> Heben Sie die Maus sanft, indem in der Nähe Basis des Schwanzes im klaren Hintergrund. </li><li> Überprüfen Sie die hinteren Gliedmaßen Position für 10-15 Sekunden. Wildtyp-Maus spreizt Hinterbeine von Bauch, während eine ataktische Maus neigt dazu, eine oder beide Hinterbeine in Richtung des Bauches mehr als 50% der Zeit ausgesetzt 13 zurückzuziehen. </li><li> Bringen Sie die Maus, um den Käfig </li></ol></li><li> Ledge-Test: <ol><li> Heben Sie die Maus vorsichtig, indem Sie den Schwanz und halten Sie auf dem Sims eines Käfigs. </li><li> Beachten Sie die Maus, die speziell auf die Ecke des Käfigs. Wildtyp-Maus können Sie die Ecken problemlos ohne Angst und verlieren das Gleichgewicht. Ataktische Maus verliert sein Gleichgewicht, einfrieren oder schüttelt bei einem Spaziergang entlang des Käfigs Leiste und an den Ecken. </li></ol></li><li&gt; Mouse Fußdruck Assay: HINWEIS: Für diesen Test halten die Absorptionslage bereit, auf dem Boden einer schmalen Landebahn (~ 70 cm langen, ~ 5 cm breit mit ca. 5 cm hohe Wände.) <ol><li> Halten Sie die Maus sanft mit einer Hand und wenden Tinte auf den Hinterbeinen mit einem Pinsel. </li><li> Legen Sie die Absorptionsschicht auf dem Boden einer schmalen Landebahn. </li><li> Lassen Sie Maus zu gehen oder laufen über eine Absorptionsschicht in einer geraden Linie in einer Start- und Landebahn von einem Ende zum anderen. </li><li> Wiederholen Sie den Vorgang noch zweimal mit jeder Maus mit frischen Absorptionslage. </li><li> Den Abstand zwischen zwei aufeinanderfolgenden Schritten in der Vorwärtsbewegung. Fügen Sie keine ersten und letzten paar Fußspuren, wo das Tier ist nur die Einleitung und Beendigung ihrer Sicht sind. </li></ol></li></ol>

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(76), e50170 (2013).</li><li>Kumar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448, 39-43 (2007).</li><li>Roshan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-specific+knockdown+of+miR-29+results+in+neuronal+cell+death+and+ataxia+in+mice.">Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice.</a> RNA. 20, 1287-1297 (2014).</li><li>Kaur, H., Wengel, J., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+DNA&#8722;RNA+Heteroduplex+Formation:+Effects+of+Locked+Nucleic+Acid+Nucleotides+Incorporated+into+the+DNA+Strand.">Thermodynamics of DNA&#8722;RNA Heteroduplex Formation: Effects of Locked Nucleic Acid Nucleotides Incorporated into the DNA Strand.</a> Biochemistry. 47, 1218-1227 (2008).</li><li>Griffiths-Jones, S., Grocock, R. 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J., et al. <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+Composite+Phenotype+Scoring+System+for+Evaluating+Mouse+Models+of+Cerebellar+Ataxia.">A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia.</a> J. Vis. Exp. (39), (2010).</li><li>Bergen, J. M., Park, I. -. K., Horner, P. J., Pun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+Approaches+for+Neuronal+Delivery+of+Nucleic+Acids.">Nonviral Approaches for Neuronal Delivery of Nucleic Acids.</a> Pharmaceutical Research. 25, 983-998 (2008).</li><li>Zou, L. -. L., Ma, J. -. L., Wang, T., Yang, T. -. B., Liu, C. -. 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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...

MicroRNAs sind als neue therapeutische Targets aufgrund ihrer universellen Rolle bei der Regulation der Genexpression und direkte Beweise für die Beteiligung an der Krankheit entstanden. MiRNAs werden aktiv auf ihr Potenzial untersucht, wie Drogen-Ziele 1,2. Ferner werden Veränderungen der miRNA-Expression mit verschiedenen Krankheiten 3 und Simulation dieser Veränderungen durch künstliche Störung der miRNA-Expression verbunden sind, können verwendet werden, um die zelluläre Signalwege in Krankheitsmanifestation beteiligt untersuchen. Gewebespezifische Lieferung von miRNA gezielt Medikamente ist derzeit eine der größten Herausforderungen für die miRNA-basierte Wirkstoffentwicklung. Antagomirs und miRNA-Mimetika sind vielversprechend Mittel zum Stören miRNA Ebenen 4-6. Spezielle Funktionen, die ihre Spezifität und Wirksamkeit erhöhen, weisen jedoch in der Gestaltung der Antagomirs eingebaut werden, bevor sie für die in vivo Störung der miRNA-Expression verwendet werden. MicroRNAs sind besonders relevant,<html> als Ziele in derzeit unheilbaren neurodegenerativen und neuroentwicklungs Krankheiten. Die Blut-Hirn-Schranke stellt eine Beschränkung für die Lieferung von Antagomirs im Gehirn. Stereotaktische Injektionen sind weit verbreitet in Nagetiermodellen verwendet werden, um Moleküle an bestimmten Stellen im Gehirn 7 zu liefern. Es erfordert Geschick, umfangreiche Investitionen in der Instrumentierung und Zeit. Stereotaktische Injektionen sind invasive, umfassen Chirurgie, führen zumindest leichten Verletzungen und an lokale Auslieferung beschränkt. Die Verwendung von Zelldringenden Peptide mit einer Vorliebe für die Ausrichtung Neuronen können diese Einschränkungen zu begegnen, da sie durch die trans-Kreislauf-Strecke geliefert werden, sondern durchbrechen die Bluthirnschranke. Solch ein Peptid aus der Rabies Virus Glycoprotein (RVG) abgeleitet ist, wurde zuvor verwendet, um siRNA gegen Japanische Enzephalitis-Virus bei Mäusen 8 zu liefern. Wir haben festgestellt, dass unter Verwendung des Peptids für Antagomir Lieferung, miRNAs kann wirksam sein niedergeschlagen im Gehirn der Maus 9. NHALT &quot;&gt; Die zweite große Herausforderung der miRNA knock-down ergibt sich aus der geringen Größe der miRNAs und das Vorhandensein von eng verwandten Sequenz Isoformen. Wir nehmen das Beispiel des MMU-miR-29-Familie, die aus drei eng verwandte Isoformen besteht, miR-29a , b und c. Antagomire sind auch allgemein entlang der Hauptkette modifiziert werden, um ihre Stabilität zu erhöhen und machen sie beständig gegenüber Nukleasen angreifen. Locked Nucleic Acids (LNAs) bieten einen weiteren Vorteil, dass sie verbessern die thermische Stabilität und sogar dazu führen, um den Abbau über und jenseits Ziel sterische Hinderung. 10 Einführung Änderungen entlang der Hauptkette kann wirksam, aber teuer sein. Wir haben bereits gesehen, daß Modifikationen über eine optimale Anzahl kann nicht weiter die Wirksamkeit. Die Gestaltung des antagomir beinhaltet daher die optimale Änderung der antagomir. Um die komplexen Antagomir nicht-kovalent mit dem neurotropen Peptid, einem geladenen Hepta- zu nona-Arginin-Erweiterung verwendet wird. D-ArgininRückstände werden verwendet, da sie eine höhere Stabilität zu verleihen, da sie nicht anfällig für eine Spaltung durch Proteasen. Hepta- zu nona-Arginin Strecken wirken als effiziente Zellpenetrationsmittel, obwohl sie Behandlungen nicht die Zelltyp-Spezifität. Durch kovalente Verknüpfung der RVG-Peptid an die nona-Arginin-Linker, einem neurotropen, Zelle eindringende Peptid generiert wurde. Die positiv geladenen Reste des Peptids in Wechselwirkung mit der negativ geladenen Nukleinsäure Rückgrat, um Komplexe zu bilden. Diese Komplexe können verwendet werden, um DNA oder RNA effektiv transfizieren in kultivierten Zellen und in vivo in das Gewebe wird.

<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.

Unter Verwendung der hier vorgestellten Verfahren, Komplexe von 50microgram fluoreszenzmarkierten Oligonukleotid (FLO) und ~ 850microgram RVG-Peptid von 1:15 molaren Ladungsverhältnis: wurden (FLO Peptid) hergestellt und nur einmal durch Schwanzvene injiziert. Komplexe von nicht-neurotropen Rabies Virus Matrix (RVM) Peptid und FLO wurde als Lieferkontrolle verwendet. Am nächsten Tag, Mäuse Gehirn und Leber wurden isoliert und Einzelzellsuspensionen wurden hergestellt. Die Zellen wurden unter dem Mikroskop für grüne...

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.

Eine einfache Alternative zur stereotaktische Injektion für Hirnspezifische Knockdown von miRNA

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

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JoVE Journal

Neuroscience

12.6K Aufrufe.  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.

Serie: 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.

Die Vermicelli und Capellini Handhabung Tests: Einfache quantitative Maßnahmen der geschickten Vorderpfote Funktion bei Ratten und Mäusen

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

Forschung

Neurowissenschaften

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.

Gene Lieferung in Postnatale Rattengehirn von Non-ventrikuläre Plasmid Injektion und Elektroporation

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.

Stereotaktischen Chirurgie für Exzitotoxische Läsion Spezifische Hirnareale in der erwachsenen Ratte

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.

Kombinierte optogenetische und Gefrierbruch Replica Immunmarkierung Eingang spezifische Anordnung der Glutamate Receptors in der Maus Amygdala zu untersuchen

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.

Eine Alternative und Validated Injektionsmethode für den Zugriff auf den subretinalen Raum<i&gt; über</i&gt; A Transcleral dorsalen Zugang

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.

Ein einfacher Weg zu messen Veränderungen in Reward-seeking Verhalten verwenden<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.

Stereotaktische Atlas-geführte Laser Capture Microdissection Hirnregionen von traumatischen Verletzungen betroffen

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.

Einfache Erstellung einer High-Yield-Kultur der induzierten Neuronen aus Menschenhaut Erwachsenen Fibroblasten

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.

Kombination von Optogenetik mit künstlichen Mikro-RNAs, die Effekte gen Knockdown präsynaptischen Funktion innerhalb intakte neuronale Schaltkreise zu charakterisieren

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.

Ein alternativer Ansatz zur primären Ereignissen in Neurodegeneration mit Ex Vivo Ratte Gehirnscheiben studieren

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