Tutti gli esperimenti sugli animali sono stati condotti in conformità con gli istituti nazionali di salute animale uso orientamenti. Questo lavoro è stato sostenuto in parte dalle concessioni dal National Institutes of Health (R01NS093073 e R21AA024873) a C. Gu.

<ol>
	<li>Niki&#263;, I., 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+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis.">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.</a> Nat. Med. 17, 495-499 (2011).</li><li>Yang, 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=Regulation+of+axon+degeneration+after+injury+and+in+development+by+the+endogenous+calpain+inhibitor+calpastatin.">Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin.</a> Neuron. 80 (5), 1175-1189 (2013).</li><li>Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+in+the+axon.">Information processing in the axon.</a> Nat. Rev. Neurosci. 5, 304-316 (2004).</li><li>Gu, Y., Jukkola, P., Wang, Q., Esparza, T., Zhao, Y., Brody, D., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+varicosity+initiation+in+central+neuron+mechanosensation.">Polarity of varicosity initiation in central neuron mechanosensation.</a> J Cell Biol. 216 (7), 2179-2199 (2017).</li><li>Chung, R. S., 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=Mild+axonal+stretch+injury+in+vitro+induces+a+progressive+series+of+neurofilament+alterations+ultimately+leading+to+delayed+axotomy.">Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy.</a> J. Neurotrauma. 22 (10), 1081-1091 (2005).</li><li>Staal, J. A., Dickson, T. C., Chung, R. S., Vickers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin-A+treatment+attenuates+delayed+cytoskeletal+alterations+and+secondary+axotomy+following+mild+axonal+stretch+injury.">Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury.</a> Dev. Neurobiol. 67 (14), 1831-1842 (2007).</li><li>Tang-Schomer, M. D., Johnson, V. E., Baas, P. W., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+interruption+of+axonal+transport+due+to+microtubule+breakage+accounts+for+the+formation+of+periodic+varicosities+after+traumatic+axonal+injury.">Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.</a> Exp. Neurol. 233 (1), 364-372 (2012).</li><li>Donkin, J. J., Vink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cerebral+edema+in+traumatic+brain+injury:+therapeutic+developments.">Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments.</a> Curr Opin Neurol. 23 (3), 293-299 (2010).</li><li>Gardner, A., Jukkola, P., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+of+rodent+hippocampal+neurons+in+culture.">Myelination of rodent hippocampal neurons in culture.</a> Nat Protoc. 7 (10), 1774-1782 (2012).</li><li>Wang, Q., Zhang, X., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+stimulator+for+localized+cell+loading:+fabrication+and+strain+analysis.">Micromechanical stimulator for localized cell loading: fabrication and strain analysis.</a> J. Micromech. Microeng. 23, 015002 (2013).</li><li>Lu, Y. B., 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=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17759-17764 (2006).</li><li>Elkin, B. S., Azeloglu, E. U., Costa, K. D., Morrison, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+heterogeneity+of+the+rat+hippocampus+measured+by+atomic+force+microscope+indentation.">Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation.</a> J. Neurotrauma. 24 (5), 812-822 (2007).</li><li>Franze, K., 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=Neurite+branch+retraction+is+caused+by+a+threshold-dependent+mechanical+impact.">Neurite branch retraction is caused by a threshold-dependent mechanical impact.</a> Biophys. J. 97 (7), 1883-1890 (2009).</li><li>Camp&#224;s, O., 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=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11 (2), 183-189 (2014).</li></ol>

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La procedura di questo test di microbiomechanical è dritto in avanti. Esso produrrà risultati affidabili, se tutti i suoi passaggi sono eseguiti con attenzione. Ci sono diversi passaggi chiave che, se correttamente eseguita, ostacolerà la raccolta dei dati di successo. I passaggi critici cominciano a monte dell'effettiva applicazione dello stimolo puffing. La dissezione attenta, coltura e cura della cultura primaria del neurone sono di primaria importanza. Se i neuroni coltivati non sono sani, non reagiranno in modo c...

Formazione del varicosity, o gonfiore/bordatura, lungo gli assoni, è una caratteristica prominente di neurodegenerazione osservata in molti disturbi o lesioni del sistema nervoso centrale, tra cui la sclerosi multipla, morbo di Alzheimer, morbo di Parkinson e traumatica brain injury1,2. Nonostante gli impatti fisiologici significativi dei varicosities axonal sulla propagazione del potenziale d'azione e trasmissione sinaptica3, come vengono generati i varicosities rimane sconosciuto. Recentemente, usando un'analisi di nuova costituzione microbiomechanical su colture di neuroni ippocampali da roditori, abbiamo trovato che stimoli meccanici possono indurre varicosità in questi neuroni con altamente interessanti caratteristiche. In primo luogo, l'induzione del varicosity è rapida (< 10 s) e questo processo è reversibile in modo imprevisto. In secondo luogo, l'inizio del varicosity dipende dalla forza sbuffando pressione: maggiore è la pressione, più veloce all'avvio. In terzo luogo, l'inizio del varicosity dipende dall'età neuronale. Gli assoni dei neuroni più giovani appaiono più reattivi alle sollecitazioni meccaniche, rispetto a quelle dei neuroni più anziani. In quarto luogo, il modulo di varicosità lungo gli assoni dei neuroni ippocampali, mentre i segmenti di assone iniziale di questi neuroni e dendriti non visualizzare nessun cambiamento nella stessa circostanza puffing. Così, il nostro studio ha rivelato una caratteristica novella di polarità di un neurone. Questi risultati con il sistema in vitro sono fisiologicamente rilevanti. Utilizzando un modello in vivo per trauma cranico lieve (mTBI), abbiamo mostrato che varicosities axonal sviluppato in modo multi-focale nella corteccia somatosensoriale dei topi immediatamente dopo l'impatto di Chiudi-cranio, coerenza con il nostro in vitro Risultati4. È importante notare che la nostra colorazione e imaging dei topi mTBI forniscono solo un'istantanea di un neurone cambiamenti morfologici, da eseguire in vivo imaging time-lapse della morfologia neuronale durante un impatto meccanico è ancora non è fattibile.
Questo sistema fluido-sbuffando ci ha permesso non solo di catturare caratteristiche uniche associate alla formazione di varici indotta da stress meccanico, ma anche per determinare il meccanismo di fondo. Provando diverse soluzioni extracellulare, bloccanti e apribottiglie di diversi candidati di canali ionici mechanosensitive ed elettrofisiologia cellulare, abbiamo identificato che il catione potenziale transitorio del ricevitore canale membro subfamily V 4 (TRPV4) canale che è permeabile per Ca2 + e Na+ e attivato da sbuffando è principalmente responsabile per la rilevazione iniziale stress meccanico durante la formazione di varici axonal4. Questo è stato ulteriormente confermato con un approccio di knockout di siRNA. Presi insieme, questo nuovo sistema di dosaggio che abbiamo sviluppato con la cultura di Neurone ippocampale, è altamente utile per studiare le proprietà di micromeccanica dei neuroni centrali, specialmente in combinazione con altre tecniche.
Questo sistema di micromeccanica che abbiamo stabilito è unico e si differenzia dai sistemi già esistenti in diversi aspetti importanti. In primo luogo, in questo sistema, i neuroni esperienza fuori del piano sollecitazioni meccaniche nelle forme di compressione e taglio. Durante l'impatto meccanico, processi neuronali rimangono attaccati alla superficie del vetrino coprioggetto e non si muovono. Questo è diverso da altri sistemi che si occupa principalmente di flessione e stretching in piano sperimentale (o tensione), per esempio, la deflessione degli assoni in bundle come stringhe5,6 in movimento o stretching assoni coltivati su bioerodibili canali e membrane estensibile7,8. Inoltre, sebbene varicosities axonal possono anche essere indotti questi saggi come nel nostro sistema fluido-sbuffando, il processo in queste impostazioni richiede molto più tempo (da 10 minuti a parecchie ore6,7,8) e viene visualizzato irreversibile. Infine, il nostro sistema utilizzando sbuffando fluido locale consente l'esame delle caratteristiche spaziali della formazione del varicosity (ad es., dendriti, spine dendritiche, soma, axonal segmenti iniziali e terminali axonal), oltre alle sue caratteristiche temporali. Utilizzando questo sistema, abbiamo scoperto diverse caratteristiche inaspettate e unici di formazione del varicosity assonale, particolarmente rapida insorgenza, lenta reversibilità e polarità di assone-dendrite.
Il sistema che discutiamo in questa carta è compatibile con molte tecniche di molecolare e biologia cellulare. Per esempio, per studiare gli effetti dello stress meccanico sulla morfologia neuronale e funzione, può essere utilizzato insieme a coculture di mielina, time-lapse di imaging di fluorescenza di riflessione interna totale (TIRF) e di trasferimento di energia di risonanza fluorescente (FRET) formazione immagine del calcio e registrazione di patch clamp. In questa carta, ci concentriamo sui componenti di base del sistema. Cultura di neuroni ippocampali, il fluido-sbuffando installazione, imaging ad alta risoluzione time-lapse per trasporto assonale e calcio imaging sono illustrate passo passo qui di seguito.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">12 mm coverslips&#160;</td>
			<td style="width:139px;">Warner Instruments&#160;</td>
			<td style="width:119px;">64-0702</td>
			<td style="width:167px;">for 24-well plate&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">25 mm coverslips&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-545-102&#160;</td>
			<td>for 6-well plate&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acetic acid</td>
			<td>Fisher Scientific&#160;</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-D-lysine</td>
			<td>Sigma&#160;</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rat tail collagen&#160;</td>
			<td>Roche&#160;</td>
			<td>11 179 179 001&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10X PBS&#160;</td>
			<td>National Diagnostics&#160;</td>
			<td>CL-253</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">Na2SO4</td>
			<td>Fisher Scientific&#160;</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">K2SO4</td>
			<td>Fisher Scientific&#160;</td>
			<td>P304-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP410-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-glucose&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">MgCl2</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NaOH</td>
			<td>Fisher Scientific&#160;</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Protease enzyme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P4032</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FBS</td>
			<td>Gibco&#160;</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium pyruvate&#160;</td>
			<td>Gibco&#160;</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine&#160;</td>
			<td>Gibco&#160;</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin 100x (P/S)</td>
			<td>Gibco&#160;</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM Earle&#39;s Salts&#160;</td>
			<td>Gibco&#160;</td>
			<td>11090</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27 supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neurobasal&#160;</td>
			<td>Gibco&#160;</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Arabinosylcytosine (Ara-C)</td>
			<td>Sigma&#160;</td>
			<td>147-94-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM media&#160;</td>
			<td>Gibco&#160;</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Invitrogen&#160;</td>
			<td>1854313</td>
			<td>transfection reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate rods&#160;</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>PG52151-4</td>
			<td>for puffing pipette&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rubber tubing&#160;</td>
			<td style="width:139px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">14-169-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic syringe and plunger</td>
			<td>Becton Dickinson&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micromanipulator&#160;</td>
			<td>Sutter Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>S640-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific&#160;</td>
			<td>C70-500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:341px;">Cell culture dish (35 mm x 10 mm)</td>
			<td style="width:139px;">Corning&#160;</td>
			<td style="width:119px;">3294</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Fluo-4 AM</td>
			<td style="width:139px;">Molecular Probes</td>
			<td style="width:119px;">F14201</td>
			<td style="width:167px;">for calcium imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Mito-YFP construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">for cell transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">YFP-N1 construct&#160;</td>
			<td style="width:139px;">Takara Bio Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">for cell transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Model P-1000 Flaming/Brown Micropipette puller&#160;</td>
			<td style="width:139px;">Sutter Instruments&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Eclipse TE2000-U Mcroscope&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Plan Fluor ELWD 20x lens&#160;&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">062933</td>
			<td style="width:167px;">objective&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Apo TIRF 100x/1.49 oil lens&#160;</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">MRD01991</td>
			<td style="width:167px;">objective&#160;</td>
		</tr>
	</tbody>
</table>

a microbiomechanical system for studying varicosity formation and recovery in central neuron axons

Varicosities axonal sono allargate strutture lungo i fasci di assoni con un elevato grado di eterogeneità. Sono presenti non solo nei cervelli con malattie neurodegenerative o lesioni, ma anche nel cervello normale. Qui, descriviamo un sistema di nuova costituzione micromeccanici per in modo rapido, affidabile ed reversibilmente indurre varicosities axonal, permettendoci di comprendere i meccanismi che regolano la formazione del varicosity e la proteina eterogenea composizione. Questo sistema rappresenta un mezzo romanzo per valutare gli effetti dello sforzo di compressione e taglio su diversi compartimenti cellulari dei neuroni, diversi da altri sistemi in vitro che si concentrano principalmente sull'effetto di stretching. Cosa importante, a causa delle caratteristiche uniche del nostro sistema, abbiamo recentemente effettuato una scoperta di nuovi risultati che l'applicazione del fluido pressurizzato può indurre rapidamente e reversibilmente varicosities axonal attraverso l'attivazione di un canale potenziale transitorio del ricevitore. Il nostro sistema biomeccanico può essere utilizzato comodamente in combinazione con aspersione di droga, live cell imaging, imaging del calcio e registrazione di patch clamp. Pertanto, questo metodo può essere adottato per lo studio di canali ionici meccanosensibili, regolamento di trasporto assonale, dinamica del citoscheletro assonale, segnalazione calcio e cambiamenti morfologici correlati a traumatico cranico.

Prima sbuffando, assoni normalmente mostrano poca formazione di varici. Seguente sbuffando con la nostra pressione standard (190 mmH2O altezza), l'inizio di assoni di sviluppare molti tallone-come varicosità. La formazione di varicosità è parzialmente reversibile, come mostrato dalle regioni dell'assone tornando al loro stato pre-soffiato in seguito a un periodo di recupero di 10 min (Figura 2A-B). Dopo un periodo di recupero pi...

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A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Questo protocollo descrive un approccio fluido pressurizzato, fisiologicamente rilevante per induzione rapida e reversibile di varicosità in neuroni.

Un sistema di Microbiomechanical per lo studio della formazione del Varicosity e del recupero in centrale del neurone assoni

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

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6.2K Views.  The Ohio State University. Questo protocollo descrive un approccio fluido pressurizzato, fisiologicamente rilevante per induzione rapida e reversibile di varicosità in neuroni.

Video: A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Drug Delivery sistemica e locale per la cura delle malattie del Sistema Nervoso Centrale in modelli di roditori

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

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Neuroscienze

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Resezione del nervo ottico: un modello di apoptosi Neuron adulti nel Sistema Nervoso Centrale

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Analisi morfologica del Drosophila Larvale dendriti sensoriali e periferico Neuron assoni utilizzo Mosaici genetici

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In elettroporazione ovo nel mesencefalo Chick per lo Studio la funzione dei geni nello sviluppo dopaminergici Neuron

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Produzione di vettori lentivirali per Celle di trasduzione del Sistema Nervoso Centrale

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Un leggero, Cuffie-based per la gestione di sistema feedback uditivo in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Consegna diretta intraventricolare di farmaci per il sistema nervoso centrale, Roditore

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Analisi immunoistochimica nel Sistema Nervoso Centrale e Periferico Rat linfa nodo del tessuto Sezioni

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Laser nanochirurgia di cerebellari Assoni<em&gt; In Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo optogenetic stimolazione del sistema nervoso centrale Roditore

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...