This research was supported by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2010-0022362) and (2012R1A2A2A02046812).

Etica Dichiarazione: Questo esperimento è stato approvato dalla Institutional Animal Care and Use Committee Kyungpook National University, ed è stata effettuata in base alla Guida per la cura e l&#39;uso di animali da laboratorio (Istituto Superiore di Sanità, 1996). Dopo esperimento, gli animali sono stati sacrificati sotto l&#39;anidride carbonica l&#39;eutanasia. 1. Progettazione del capo fissazione Bar per topi NOTA: Questo passaggio è un orientamento generale per un design bar fissazione. Qualsiasi modifica della dimensione e materiale è possibile a seconda animali o impianti. Progettare un bar testa fissazione utilizzando il software CAD è consigliato ma non necessario. <ol><li> Utilizzare il software CAD per la progettazione di un bar fissazione. <ol><li> Progettare una barra di fissaggio di forma rettangolare (123 × 35 × 6 mm) (Figura 1A). Realizzare un intaglio rettangolare (16 × 20 mm; porta) al centro della barra di fissaggio. Aggiungere pendii o rientranze which servire come una rotaia guida ai confini del porto (Figura 1A-a, Figura 1B -arrows). NOTA: Assicurarsi che la zona del porto copre il rettangolo previsto per il cranio di topo sopra il cervello per consentire accessi verticali utilizzando un manipolatore stereotassico alla maggior parte delle aree del cervello attraverso una craniotomia. </li><li> Montare le piastre (50 × 20 × 6 mm) con due fori di fissaggio allungati su entrambi i lati della barra fissaggio. Assicurarsi che questi fori passano viti 6 mm di diametro (Figura 1A-d). Effettuare un foro del diametro di 3 mm di fronte alla porta per un gancio di chiusura (Figura 1A-b). </li><li> Fare una scatola con un piano (135 × 305 mm) e pareti laterali (305 × 76 mm). Praticare due fori del diametro di 6 mm, come mostrato in Figura 1A (nel riquadro). Aggiungere muri di sostegno sulla barra di fissaggio (Figura 1A-e). </li></ol></li><li> Fabbricazione il bar progettato e box e assemblare ogni di loro usando cianoacrilato comemostrato nella Figura 1A (inserto). Montare la barra sulla scatola con viti di 6 mm di diametro. Applicare pista regolando la posizione della vite della barra per il fissaggio della testa ergonomico (Figura 1A-riquadro). NOTA: Per i topi, la dimensione della barra di fissaggio 60 × 20 × 4 mm (costituito da due strati di 2 mm di spessore) pannelli acrilici. La porta è quadrato di 8 mm di larghezza (superiore) e 9 x 10 mm (inferiore) come mostrato nella Figura 1C. Piastre di ancoraggio sono le stesse dimensioni dei ratti &#39;. Le pareti della piastra scatola e laterali sono 100 × 72 millimetri e 100 × 50 mm, rispettivamente. </li></ol> 2. Testa Mounter costruzione durante l&#39;impianto Chirurgia ATTENZIONE: Siate cauti quando si tratta di resina acrilica dentale. Il liquido può causare irritazione alla pelle. Indossare una maschera e indossare i guanti. Lavorare in un ambiente ben ventilato. <ol><li> Preparazione prima dell&#39;intervento chirurgico <ol><li> Strettamente avvolgere il bordo del porto della barra di fissaggio con appellicola araffin per impedire l&#39;adesione della resina acrilica applicata alla barra (vedi Figura 1B e Figura 2A). Coat confine del porto coperta dal film paraffina con uno strato sottile di resina acrilica. </li><li> Utilizzare strumenti chirurgici sterilizzati. Disinfettare accuratamente telaio stereotassico e il suo territorio con il 70% di alcol etilico. </li></ol></li><li> Eseguire un intervento chirurgico di impianto stereotassica <ol><li> Anestetizzare un animale mediante iniezione intraperitoneale di una miscela cocktail (2 ml / kg) di ketamina 10 ml (50 mg / ml), xilazina cloridrato 1,5 ml (23,32 mg / ml), e soluzione salina 2,5 ml. Verificare che l&#39;animale completamente anestetizzato e non risponde a coda e punta pizzica. Iniettare cocktail miscela di 1 ml / kg durante chirurgia quando necessario. </li><li> Fissare l&#39;animale al apparato stereotassico. Applicare pomata oftalmica sugli occhi per prevenire la disidratazione durante l&#39;intervento chirurgico. Shave e pulire il cuoio capelluto con il 70% di etanolo. Dare iniezione sottocutanea di lidocaina al 2% e incidere la scalp lungo la linea mediana del cranio. Completamente raschiare periostio. Risciacquare con saline diverse volte. </li><li> Rendere bava foratura vicino al bordo del cranio. Viti d&#39;impianto 4-5 giri (fino a 1 mm di profondità) dalla superficie del cranio per evitare una violazione della dura madre. Assicurarsi che nessuna perdita di liquor e sangue sulla superficie del cranio dopo vite impianto. Sciacquare con soluzione salina e asciugare la superficie del cranio con aria-soffio, soprattutto intorno alle viti. </li><li> Cappotto superficie del cranio con un sottile strato di &quot;adesivo dentale (Super-Bond C &amp; B)&quot;. Non coprire interamente la superficie del cranio, compresi il bregma se aggiuntivo l&#39;impianto degli elettrodi è in programma. Utilizzare il bregma per la marcatura coordinate craniotomia. Elettrodi e / o microdrive implantari e fissarlo sul cranio applicando resina acrilica intorno ad esso. </li><li> Applicare ulteriore resina acrilica sopra il cemento dentale. Utilizzare adeguate quantità di resina acrilica di lasciare sufficiente spazio fill-out tra bar rivestito in resina di und resina montaggio iniziale quando fissarsi le viti e / o elettrodi (Figura 2A). </li></ol></li><li> Place Bar testa fissazione 5 mm o più al di sopra della superficie del cranio ed allineare il centro (Figura 2A -Sinistra). </li><li> Applicare resina acrilica nella fessura tra la base del cranio e resine montaggio iniziale. (Figura 2A -Center). Attendere che la resina diventare difficile. </li><li> Rimuovere orecchio-bar e tirare il montatore modellata dalla porta barra di fissaggio. (Figura 2A -Right). Applicare con cura supplementare resina acrilica intorno alle rotaie di bordo del montatore stampato, se necessario. </li><li> Lavare la superficie della ferita del cuoio capelluto con soluzione fisiologica sterile. Iniettare un antibiotico (enrofloxacina, 5 mg / kg sc) e (tartrato butorfanolo, 2 mg / kg sc) analgesico immediatamente dopo la chirurgia. Ospitare l&#39;animale individualmente in un&#39;unica gabbia. </li></ol> 3. abituare Animali da Head-fissaggio Nota: Questo passaggio describi orari assuefazione basate su valutazioni empiricamente successo degli autori. Per una panoramica di procedura di assuefazione per la testa-fissazione, vedere 11 opere di 10 Schwartz o di Guo. <ol><li> Dare sufficiente periodo di convalescenza (una settimana o più) per gli animali. Il giorno 1, Maniglia animali spesso in brevi sessioni divise. </li><li> Il giorno successivo (giorno 2), esporre l&#39;animale per l&#39;ambiente di fissazione (box). Non inserire il montatore testa nella porta della barra di fissaggio. Lasciare gli animali di abituarsi all&#39;ambiente. Trovare e afferrare il montatore testa con la mano per 1-2 secondi e rilasciarlo immediatamente. Ripetere la fissazione a breve termine a mano-grab più volte. </li><li> Il giorno 3, abituare gli animali a testa fissazione. <ol><li> Portare gli animali dalle gabbie casa e li stabilirsi nella casella di dirigere le teste verso il porto della barra di fissaggio. Afferra il montatore testa e inserirlo nella porta della barra di fissaggio. </li><li> Associare il montatore (animale ditesta) al gancio di bloccaggio della barra di fissaggio usando un elastico. Stare in piedi per 5 minuti. NOTA: Non toccare il tronco o la coda dell&#39;animale dopo la fissazione, che porta l&#39;animale a squittire e lottare aumentando lo stress dell&#39;animale. Quando l&#39;animale continua lotta più di 1 minuto, rilasciarlo e riprovare più tardi. Da questo punto, gli animali mostrano urine e feci l&#39;escrezione quando esposto a testa la fissazione, ma questo a poco a poco si riduce come procede assuefazione. </li><li> Recuperare l&#39;elastico di bloccaggio ed espellere il montatore dalla porta. Mettere gli animali torna alla gabbia a casa e di fornire un pezzo di cereali o di un seme di girasole nella gabbia di casa come ricompensa. Il processo successivo inizia in 30 min. Ripetere da 3.4.1 a 3.4.4 cinque o più in un giorno. </li></ol></li><li> Il giorno 4 a 7, esporre l&#39;animale a testa fissaggio come descritto nel protocollo 3.4. Aumentare gradualmente la durata di fissaggio testa (10 min, 20 min, 30 min e 50 min come procede assuefazione). NOTA: Se gli animali non fannot carica il premio cibo, può essere un segno di un alto livello di stress. Non aumentare il tempo di fissazione. Se gli animali mostrano ripetuti lottando comportamento, li abituare per un periodo più lungo. </li><li> Controllare il peso corporeo dell&#39;animale quotidiano. Considerare assuefazione infruttuoso quando il peso corporeo è in diminuzione. Procedere a compiti comportamentali con testa di fissaggio dopo assuefazione successo. NOTA: Riuscito assuefazione facilita la successiva formazione comportamentale (Figura 3E e F). </li></ol>

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	<li>Mayrhofer, J. M., 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=Novel+two-alternative+forced+choice+paradigm+for+bilateral+vibrotactile+whisker+frequency+discrimination+in+head-fixed+mice+and+rats.">Novel two-alternative forced choice paradigm for bilateral vibrotactile whisker frequency discrimination in head-fixed mice and rats.</a> J Neurophysiol. 109, 273-284 (2013).</li><li>Bryant, J. L., Roy, S., Heck, 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=A+technique+for+stereotaxic+recordings+of+neuronal+activity+in+awake,+head-restrained+mice.">A technique for stereotaxic recordings of neuronal activity in awake, head-restrained mice.</a> J Neurosci Methods. 178, 75-79 (2009).</li><li>Ollerenshaw, D. 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=Detection+of+tactile+inputs+in+the+rat+vibrissa+pathway.">Detection of tactile inputs in the rat vibrissa pathway.</a> J Neurophysiol. 108, 479-490 (2012).</li><li>Lee, M. G., Manns, I. D., Alonso, A., Jones, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-wake+related+discharge+properties+of+basal+forebrain+neurons+recorded+with+micropipettes+in+head-fixed+rats.">Sleep-wake related discharge properties of basal forebrain neurons recorded with micropipettes in head-fixed rats.</a> J Neurophysiol. 92, 1182-1198 (2004).</li><li>Lutcke, H., 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=Optical+recording+of+neuronal+activity+with+a+genetically-encoded+calcium+indicator+in+anesthetized+and+freely+moving+mice.">Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice.</a> Front Neural Circuits. 4, 9 (2010).</li><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56, 43-57 (2007).</li><li>Kimura, 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=Reinforcing+operandum:+rapid+and+reliable+learning+of+skilled+forelimb+movements+by+head-fixed+rodents.">Reinforcing operandum: rapid and reliable learning of skilled forelimb movements by head-fixed rodents.</a> Journal of neurophysiology. 108, 1781-1792 (2012).</li><li>Hadlock, T., Kowaleski, J., Mackinnon, S., Heaton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+head+fixation+for+the+study+of+rodent+facial+function.">A novel method of head fixation for the study of rodent facial function.</a> Experimental neurology. 205, 279-282 (2007).</li><li>Roh, M., Jang, I. S., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Push-in+Head+Restraining+Apparatus+for+Intracranial+Self+Stimulation+Tasks+in+Rats.">Push-in Head Restraining Apparatus for Intracranial Self Stimulation Tasks in Rats.</a> Korean J Physiol Pharmacol. 18, 103-108 (2014).</li><li>Schwarz, C., 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=The+head-fixed+behaving+rat--procedures+and+pitfalls.">The head-fixed behaving rat--procedures and pitfalls.</a> Somatosensory &amp; motor research. 27, 131-148 (2010).</li><li>Guo, Z. V., 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=Procedures+for+Behavioral+Experiments+in+Head-Fixed+Mice.">Procedures for Behavioral Experiments in Head-Fixed Mice.</a> PLoS ONE. 9, (2014).</li><li>Osborne, J. E., Dudman, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIVETS:+a+mechanical+system+for+in+vivo+and+in+vitro+electrophysiology+and+imaging.">RIVETS: a mechanical system for in vivo and in vitro electrophysiology and imaging.</a> PLoS One. 9, e89007 (2014).</li></ol>

The authors have nothing to disclose.

This protocol demonstrates a simple and convenient head fixation method using materials at hand. For physically successful head fixation, the first critical steps during surgery is to rinse and dry the skull surface clearly before applying the dental adhesive resin cement. Second, this method does not uses screws implanting under temporal muscles and only depends on the screws on the skull, their perpendicular implantation against the surface of skull rim is crucial10. This reduces the possibility of accidental resin mounter separation from the skull, especially in rats.
The acrylic resin to build the head mounter is adhesive to the bar. We prevented this by covering the paraffin film over the port. However, this is an inconvenient procedure. One possible solution to improve this is to design the fixation bar with a metallic material. Furthermore, this should serve more stability in clamping than the acrylic bar by removing the gap occupied by the paraffin film. The described method might be inappropriate for brain imaging study because of small gap originated from paraffin film. This could also be resolved by metallic fixation bar or other method proposed elsewhere12.
One of benefits of this method is flexibility (or compatibility). Since the area of the port covers vertical access routes into nearly most of the brain areas. This indicates that experiment-specific head frame design is not required. Once the fixation bar is designed, it can be used without any modification. Also, this method is immediately applicable in general laboratory condition. The head fixation without metal head-posts is achieved by molding the acrylic resin which is available in general laboratory performing implantation surgery. Fixation procedure is convenient to investigators without using screws and plates assembly2,7,8.

Testa fissaggio o la testa di ritegno è una tecnica di immobilizzare testa dell&#39;animale collegando un frame o piatto sul cranio per il bloccaggio rigido. La tecnica è stata adottata per una precisa stimolazione sensoriale, rilevamento comportamentale con o senza registrazione neurali in animali svegli 1-3 e per l&#39;etichettatura juxtacellular durante il ciclo sonno-veglia 4. E &#39;utilizzato anche per l&#39;imaging in vivo cerebrale 5,6. Ora tecnica di fissazione testa è uno strumento comunemente usato nel campo delle neuroscienze e ricerca comportamentale. In sostanza, la fissazione testa tradizionale richiede attacco chirurgico di metallo testa-messaggi su parte superiore del cranio 6-8. Gli allegati vengono poi fissate ad una piattaforma fissa, con conseguente immobilizzazione della testa. Ricerca neurofisiologica in animali svegli, come neurale e / o la registrazione ottica, intervento circuiti cerebrali, imaging cerebrale è accompagnata da un intervento chirurgico di impianto utilizzando resine dentali. Dunque,sembra un compito semplice da implementare la fissazione testa nei laboratori quando è necessario sperimentalmente. I telai devono essere progettati per essere leggero e abbastanza piccolo da non disturbare il comportamento naturale dell&#39;animale nelle loro gabbie a casa. Inoltre fornisce rigidità meccanica quando ancorata al piattaforma fissa. Ha inoltre biocompatibilità seconda fini sperimentali. Pertanto, è tecnicamente difficile progettare e fabbricare tali testa-messaggi in apparecchiature da laboratorio. In questo studio, un metodo di fissaggio testa romanzo è descritto, fornendo rigidità meccanica attraverso conveniente serraggio senza usare metallo testa-messaggi. Usando il metodo di fissazione testa potrebbe essere fatto in condizioni di laboratorio generali, in cui la chirurgia di impianto è di solito eseguita nei roditori per lo studio del cervello. Gli head-perni in metallo vengono sostituiti con cemento resina acrilica stampata parzialmente da una barra di fissaggio testa su misura e il processo di stampaggio viene fatto durante implanchirurgia zione. La barra di fissaggio offre elevata compatibilità spaziale con altri impianti, quali sonde di registrazione neurali, elettrodi di stimolazione. Inoltre semplifica fasi di serraggio dell&#39;animale sulla piattaforma fissaggio rimuovendo vite di fissaggio. Abbiamo usato questo metodo nel precedente studio comportamentale e abbiamo concluso che questo metodo è facile da implementare e conveniente in sperimentazione pratica 9.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dental acrylic resin</td>
			<td style="width:199px;">Lang Dental Manufacturing</td>
			<td style="width:129px;">Ortho-Jet</td>
			<td style="width:343px;">to make molded mounter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Paraffin film</td>
			<td style="width:199px;">Bemis</td>
			<td style="width:129px;">Parafilm</td>
			<td>to prevent adhesion of acrylic resin to the bar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dental adhesive</td>
			<td style="width:199px;">Sun Medical</td>
			<td style="width:129px;">Super-Bond C&#38;B</td>
			<td>to bond between acrylic resin and skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Plexglass or other acrylic plate</td>
			<td style="width:199px;"></td>
			<td style="width:129px;"></td>
			<td>to make fixation bar and box platform</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cyanoacrylate</td>
			<td style="width:199px;"></td>
			<td style="width:129px;"></td>
			<td>to bond acrylic plates</td>
		</tr>
	</tbody>
</table>

acrylic resin molding based head fixation technique in rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Questo metodo di fissazione testa è applicabile sia ai ratti e topi. Totale otto ratti hanno ricevuto impianto bilaterale di stimolare elettrodi nel fascio proencefalo mediale. Per quattro di loro, elettrodi a filo di tungsteno personalizzati (Microdrive) sono stati ulteriormente impiantate nella zona forelimb M1 (Figura 2C). Per i topi (n = 2), quattro elettrodi a filo di acciaio inossidabile sono stati impiantati in aree corticali frontali / occipitali. Durante l&#39;intervento, montatori di testa sono state stampate per la fissazione testa in entrambe le ratti e topi, come descritto nella sezione protocollo 2. Dopo il recupero da un intervento chirurgico, sono stati esposti alle sessioni giornaliere di fissaggio testa sia per assuefazione o attività comportamentale. I montatori testa modellati rimasti fino alla fine della sperimentazione (40.5 ± 1.73 (media ± DS) giorni per i ratti e 81 ± 5,66 (media ± DS) giorni per i topi dopo l&#39;intervento chirurgico). I ratti sono stati esposti quotidianamente 40-60 min fissaggio testa durante compito comportamento; topi sono stati esposti giornaliera da 20 a 40 min di esserecompito comporta-. In precedenza abbiamo utilizzato questa tecnica per allenare ratti spingere e tirare una leva sotto di fissaggio testa. La ricompensa era stimolazione elettrica diretta del fascio proencefalo mediale. Anche se suscita comportamento motorio esagerato, fissaggio testa era fermamente mantenuta senza separazione montatore accidentale. Dopo assuefazione, l&#39;attracco il montatore testa nella porta della barra di fissaggio è stata completata in pochi secondi bloccando elastico al gancio. Quando la prima volta alla fissazione testa, animali presenta lottando comportamenti come correre sul posto, raddrizzare le gambe (Figura 3A e B). Tuttavia, tali comportamenti diminuiscono con l&#39;assuefazione. Assuefazione successo può essere confermato che gli animali non mostrano un comportamento più che lotta mentre la testa fissa (Figura 3C e D). <img alt="Figura 1" src="/files/ftp_upload/53064/53064fig1.jpg" /> Figura 1. Progettazione della barra testa-fissazione. (A) i nomi dei componenti della barra di fissaggio testa. a) porto; b) gancio di bloccaggio; c) il corpo barra di fissaggio; d) di ancoraggio fori; e) il sostegno a parete; f) piastra di ancoraggio. Disegno della scatola di fissaggio è raffigurato nel riquadro. Due fori della parete laterale sono utilizzati per posizionare la barra ergonomicamente fissazione. (B) Vista di prospettiva di barra di fissaggio testa progettato per i ratti. Frecce nere indicano il limite di coprire film paraffina. (C) Il design della porta per il fissaggio della testa nei topi. (Modificata dalla figura 1 di Roh et al. 9) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53064/53064fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53064/53064fig2.jpg" /> Figura 2. Costruzione di stampaggio montatore durante l&#39;intervento chirurgico. (A) Critico<html> Al fasi di stampaggio il montatore testa. (Sinistra) Viti (T forme in nero) devono essere impiantati perpendicolari al cranio (grigio) di superficie. Un sottile strato di cemento dentale resina adesiva (verde) comprende viti e la superficie del cranio. (Center) Dopo l&#39;impianto a seconda fini sperimentali, resine acriliche addizionali (arancione) sono riempiti nello spazio tra resina iniziale rivestita sulle resine portuali e di base della superficie del cranio e diventa parte del montatore testa. (A destra) Dopo aver aggiunto resine acriliche sono difficili insieme, il montatore testa può essere rilasciato facendo scorrere fuori dal porto. Linee nere continue indicano film paraffina. (Modificato da figura 1 del Roh et al. 9) (B) Costruito testa montatore nei ratti è pronta per rilasciare (dopo la fase di A-Center). (CD) modellata resine acriliche che servono come montatore testa. Dopo l&#39;intervento chirurgico, ancora sotto anestesia, gli animali vengono rilasciati dalla barra di fissaggio. Frecce nere indicano guide corrispondenti alla forma della porta.<html> ps: //www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53064/53064fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 3" src="/files/ftp_upload/53064/53064fig3.jpg" /> Ratto e nel topo Figura 3. Head-fisso. (AB) Quando gli animali sono inizialmente esposti a testa la fissazione, emettono lottando comportamento. (CD) Come procede assuefazione, gli animali mostrano un comportamento meno che lotta e di solito sedersi sul pavimento della piattaforma fissa. (EF) Risultato delle prestazioni comportamentali attività di successo (E) e successo (F) assuefazione dell&#39;ambiente fissaggio testa (Modificato da figura 3 Roh et al., 9). Numero ricompensa abbraccia entrambi gratuiti (per scopi di plasmare) e contingente della leva di trazione. In entrambe le sessioni, non sufficientemente ratto abituata mostra scarso rendimento con premiata solo la leva accidentale tira dal comportamento non contingente.= &quot;https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53064/53064fig3large.jpg&quot; target = &quot;_ blank&quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

Watch this Scientific Journal Video about Acrylic Resin Molding Based Head Fixation Technique in Rodents at JoVE.com

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Acrilico Resina Molding Based Testa tecnica di fissazione in Roditore

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

acrylic-resin-molding-based-head-fixation-technique-in-rodents

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.7K Views.  Kyungpook National University. Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

Video: Acrylic Resin Molding Based Head Fixation Technique in Rodents

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

Preparazione del modello di sottosquadro epilettogenesi Post-Traumatico in roditori

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Ricerca

Neuroscienze

Whole Animal Perfusion Fixation for Rodents

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate 5,7. Often, changes in response to hypoxia begin before the tissue can be preserved 12. The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures 3. It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.

Tutta la perfusione di fissaggio degli animali per i roditori

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

Un programma di installazione a basso costo per audiometria comportamentale nei roditori

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registrazione e analisi dei ritmi circadiani in Running-ruota Attività nei roditori

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Il trapianto di cellule olfattive ensheathing per valutare il recupero funzionale dopo Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La stimolazione cerebrale profonda con simultanea fMRI in Roditori

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Stimolazione elettrica cerebrale transcranica in roditori avvisi

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Dosaggi di dolore nei roditori e farmacoterapia intracranica

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Il potere di Interstimulus intervallo per la valutazione di elaborazione temporale in roditori

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) remain unclear. In order to adequately study the attributes of endolymphatic hydrops, such as the origins of low-frequency hearing loss, a reliable model is needed. The guinea pig is a&#160;good model because it hears in the low-frequency regions that are putatively affected by endolymphatic hydrops. Previous research has demonstrated that endolymphatic hydrops can be induced surgically via intradural or extradural approaches that involve drilling on the endolymphatic duct and sac. However, whether it was possible to create an endolymphatic hydrops model using an extradural approach that avoided dangerous drilling on the endolymphatic duct and sac was unknown. The objective of this study was to demonstrate a revised extradural approach to induce experimental endolymphatic hydrops at 30 days post-operatively by obliterating the endolymphatic sac and injuring the endolymphatic duct with a fine pick. The sample size consisted of seven guinea pigs. Functional measurements of hearing were made and temporal bones were subsequently harvested for histologic analysis. The approach had a success rate of 86% in achieving endolymphatic hydrops. The risk of cerebrospinal fluid leak was minimal. No perioperative deaths or injuries to the posterior semicircular canal occurred in the sample. The presented method demonstrates a safe and reliable way to induce endolymphatic hydrops at a relatively quick time point of 30 days. The clinical implications are that the presented method provides a reliable model to further explore the origins of low-frequency hearing loss that can be associated endolymphatic hydrops.

This article demonstrates an extradural approach to obliterate the guinea pig endolymphatic sac and injure the endolymphatic duct with a fine pick in order to induce experimental endolymphatic hydrops.

Un approccio chirurgico rivisto per indurre idramici endolymphatic in Guinea Pig

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...