Questo lavoro è stato sostenuto da NIH / NINDS concedere 4R00 NS 057940, e concedere SCBI 200-12 dal midollo spinale e lesioni fondo di Ricerca sul Cervello da parte dello Stato dell&#39;Indiana Department of Health.

1. Fare un semplice dispositivo per la chirurgia sottosquadro <ol><li> Il dispositivo sottosquadro abbiamo creato si compone di tre parti (Fig. 1): (1) una piastra di supporto in acciaio inox o in plastica che consente l&#39;attaccamento di un tubo di guida e un ago, e si siede sulla finestra cranico durante l&#39;intervento chirurgico, (2) un tubo di guida che contiene un ago in posizione e permette la rotazione dell&#39;ago, e (3) un ago che viene piegato a 90 gradi ~ 3 mm dalla punta, ed è mobile con rotazione e di inserimento. </li><li> Tagliare un pezzo di 1-1,5 mm di spessore, 7 ~ 10 x 30 mm in acciaio inox o rettangolare di plastica trasparente (1) di fare la piastra di sostegno. Preparare un 1,5 pollici da 22 gauge (BD società, # 305156) e un 1,5 pollici da 25 gauge (BD società, # 305127) ago della siringa. Per fare un tubo di guida, tagliare l&#39;estremità in plastica e la punta dell&#39;ago inferiore del 22-ago da siringa in modo che la sua lunghezza totale è di circa 31-32 mm (2). Sabbia fine di metallo per renderla piatta e liscia. La lunghezza finale di questo tubo guida dovrebbe essere di circa 5-6 mm più corto del 25-gauge siringa. </li><li> Utilizzare colla cianoacrilato per fissare il tubo guiding sulla piastra di supporto, facendo in modo che l&#39;ago sia perpendicolare al bordo del metallo. </li><li> Inserire l&#39;ago calibro 25 siringa nel tubo di guida, e piegare l&#39;ago a 90 gradi a 2,5-3 mm dalla punta (Fig. 1). </li><li> Regolare la portata verticale movimento dell&#39;ago incollando un tubo di plastica piccolo sul finire dell&#39;ago (fig. 1, fermata ago). La gamma di finale in movimento dell&#39;ago determina la profondità l&#39;ago può essere inserito nella corteccia, e dovrebbe essere 1,6-1,8 mm per P21 ratti e 1,3-1,5 mm per P21 topi. Questa lunghezza deve essere adattata per le varie specie ed età degli animali. </li><li> Collegare un filo sottile di rame o un piccolo pezzo di nastro sulla parte superiore dell&#39;ago nella stessa direzione, come la punta piegata (Fig.1 indicatore ad ago). Il filo o nastro indica l&#39;angolo di rotazione dell&#39;ago durante l&#39;intervento chirurgico sottosquadri (fig. 1). </li></ol> 2. Preparazione degli animali <ol><li> Tutti gli strumenti chirurgici devono essere sterilizzati in autoclave, sterilizzati con una perlina di vetro sterilizzatore, o disinfettata con etanolo al 70%. </li><li> Noi usiamo Sprague / Dawley all&#39;età postnatale 20-22 giorni (P20-22) o topi CD1 alla stessa età. Diversi ceppi di topi o ratti possono essere utilizzati per progetti specifici. </li><li> Anestetizzare il mouse con una iniezione ip di ketamina 80 mg / kg e xilazina 8 mg / kg. La procedura chirurgica inizia dopo che l&#39;animale non risponde alla pizzico coda. Durante l&#39;intervento, se necessario, una dose di richiamo di un terzo la dose originale del cocktail anestetico può essere somministrato a ripristinare lo stato originale anestetico. </li><li> Tagliare i capelli sul cuoio capelluto dell&#39;animale con un trimmer capelli elettrici. Applicare una piccola quantità di pomata oftalmica sul occhi dell&#39;animale per la protezione da secchezza durante l&#39;anestesia. Disinfettare il cuoio capelluto con un 10% di povidone-iodio soluzione, seguita dal 70% di etanolo. </li><li> Montare l&#39;animale su un apparato stereotassico per mantenere la testa in una posizione stabile fisso. In tutto l&#39;intervento, teniamo l&#39;animale su una piastra elettrica per prevenire l&#39;ipotermia. </li><li> Effettuare una linea mediana anteriore-posteriore incisione sul cuoio capelluto con un bisturi, che si estende da lambda per mezzo agli occhi. Usa hemostats per tirare la pelle da parte ed esporre il cranio sinistra a sufficienza. </li><li> Fare un taglio rettangolare sul cranio a sinistra, e usare il bisturi per togliere periostio. Questo passo sarà ridurre il sanguinamento e facilitare la foratura sul cranio. </li></ol> 3. Fare sottosquadri <ol><li> Aggiungere una piccola quantità di soluzione salina sterile al cranio zona esposta, quindi utilizzare alcuni dei cotton fioc per pulire il sangue e asciugare la zona. </li><li> Sotto un microscopio operatorio, cominciare ad esercitarsi una scanalatura rettangolare (~ 5 x 7 mm in ratti, 4 x 5 mm nei topi) sul centro del cranio a sinistra (circa al di sopra della sinistra sensomotorio corteccia). Applicando una goccia di soluzione salina sul cranio facilita la foratura e dissipa il calore. Dopo circa 2 / 3 di profondità l&#39;osso si è esercitato, rimuovere salina in eccesso e pulire la zona di foratura con un Q-tip. Lentamente e con attenzione foratura profonda fino a quando il pezzo centrale di osso è mobile su tocco gentile di una pinza. </li><li> Rimuovere con attenzione il pezzo centrale di osso inserendo una punta acuminata di una pinza sul bordo dell&#39;osso e lentamente sollevando il forcipe per esporre l&#39;emisfero sinistro. </li><li> Sotto il microscopio operatorio, tenere il dispositivo sottosquadro, e orientare l&#39;ago in senso perpendicolare parasagittale e la piastra di supporto alla linea mediana, inclinando il dispositivo leggermente caudalmente in modo da mantenere la visualizzazione della punta dell&#39;ago e della regione bersaglio corticale. Puntare la punta dell&#39;ago ad una zona 1-2 millimetri laterale alla sutura sagittale superiore, al centro della finestra del cranio, ed evitare la penetrazione diretta dei grossi vasi. </li><li> Inserire l&#39;ago in senso orizzontale attraverso la dura e sotto la pia in modo da risparmiare i vasi sanguigni. Sedersi nella parte inferiore del dispositivo sottosquadrosu entrambi i bordi della finestra del cranio in modo che l&#39;ago è in posizione normale alla superficie corticale (Fig. 1). A riposo il dispositivo su entrambi i bordi della finestra del cranio a mano in modo significativo ridurre o eliminare stretta di mano durante la seguente procedura. Sollevare l&#39;ago al di sotto della pia, quindi abbassare lentamente per creare un taglio transcorticale che l&#39;ago non può andare più in profondità. Ruotare ~ 135 ° di distanza dalla linea mediana per creare un semicerchio della sostanza bianca / strato profondo VI sottosquadro. Poi sollevare l&#39;ago di nuovo per sotto la pia. Inclinare il dispositivo indietro e ritirare l&#39;ago. </li><li> Opzionalmente, si può ripetere la procedura (passo 2,5), ma senza girare l&#39;ago in modo da creare un taglio ulteriore transcorticale ai bordi laterali della finestra. Questo creerà un isolamento più completo corticale. </li><li> Posizionare un pezzo di pellicola di plastica (6 x 6 mm) sulla finestra del cranio per la protezione e sutura al cuoio capelluto. Mettere l&#39;animale su un blocco riscaldato fino alla completa guarigione dall&#39;anestesia. </li></ol> 4. Rappresentante dei risultati: Coronale fette corticale può essere pronti a confermare il successo della chirurgia sottosquadro. A fette preparato&gt; 2 settimane dopo l&#39;intervento, tagli transcorticale e minano sono discernibili nell&#39;ambito dell&#39;obiettivo di bassa potenza di un microscopio (Fig. 2). La corteccia parzialmente isolata di solito diventa leggermente più sottile, e l&#39;attività epilettiformi possono essere rilevati nella maggior parte dei fettine corticali con registrazione campo potenziale (Fig. 3). Al contrario, la formazione di grandi buchi nella materia bianca o in profondità strati corticali, drammatico assottigliamento della corteccia lesione, o un taglio errante essere al di sopra o al di sotto della sostanza bianca del cervello renderà inutilizzabile per ulteriori esperimenti. <img src="/files/ftp_upload/2840/2840fig1.jpg" alt="Figura 1"/> Figura 1. Struttura e applicazione di un dispositivo di sottosquadro. Un tubo di guida (2) è incollato su una piastra di supporto (1) che è in acciaio inox o in plastica trasparente. Un ago della siringa (3) viene inserito attraverso il tubo di guida e piegate a ~ 3 mm a punta. Un ago sosta in tubo di plastica è incollato sulla parte superiore dell&#39;ago in modo che l&#39;intervallo verticale movimento dell&#39;ago è limitata a ~ ~ 1,2 mm e 1,5 mm per l&#39;utilizzo in P21 topi e ratti, rispettivamente. Un segmento di filo di rame è fissato sotto l&#39;impugnatura, come l&#39;ago indicatore per l&#39;orientamento dell&#39;ago piegato. Si noti che il dispositivo è inclinato e in contatto con entrambi i bordi della finestra del cranio in modo che un taglio può essere fatto in parallelo alla superficie piale. <img src="/files/ftp_upload/2840/2840fig2.jpg" alt="Figura 2"/> Figura 2. Un&#39;immagine rappresentante della fetta sottosquadro. A. immagine fluorescente di una fetta che è stato preparato due settimane dopo la lesione sottosquadro in un topo P48. La ferita da taglio è stato etichettato da DII colorante fluorescente. Le frecce bianche sulla destra indicano taglio transcorticale, e le frecce in basso indicano sottosquadro che passava anche se il confine tra strato IV e della sostanza bianca. <img src="/files/ftp_upload/2840/2840fig3.jpg" alt="Figura 3"/> Figura 3. Registrazione di un campo potenziale fetta sottosquadro. Campo di registrazione da una fetta di potenziali cerebrali sottosquadro esposti attività epilettiformi, suggerendo ipereccitabilità del tessuto danneggiato corticale.

<ol>
	<li>Hoffman, S. N., Salin, P. A., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neocortical+epileptogenesis+in+vitro.">Chronic neocortical epileptogenesis in vitro.</a> J Neurophysiol. 71, 1762-1773 (1994).</li><li>Topolnik, L., Steriade, M., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperexcitability+of+intact+neurons+underlies+acute+development+of+trauma-related+electrographic+seizures+in+cats+in+vivo.">Hyperexcitability of intact neurons underlies acute development of trauma-related electrographic seizures in cats in vivo.</a> Eur J Neurosci. 18, 486-496 (2003).</li><li>Graber, K., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Models of Seizures and Epilepsy. , 477-493 (2005).</li><li>Nita, D. A., Cisse, Y., Timofeev, I., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+propensity+to+seizures+after+chronic+cortical+deafferentation+in+vivo.">Increased propensity to seizures after chronic cortical deafferentation in vivo.</a> J Neurophysiol. 95, 902-913 (2006).</li><li>Sharpless, S. K., Halpern, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrical+excitability+of+chronically+isolated+cortex+studied+by+means+of+permanently+implanted+electrodes.">The electrical excitability of chronically isolated cortex studied by means of permanently implanted electrodes.</a> Electroencephalogr Clin Neurophysiol. 14, 244-255 (1962).</li><li>Echlin, F. A., Battista, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+Seizures+from+Chronic+Isolated+Cortex.">Epileptiform Seizures from Chronic Isolated Cortex.</a> Arch Neurol. 9, 154-170 (1963).</li><li>Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptogenic+neurons+and+circuits.">Epileptogenic neurons and circuits.</a> Adv Neurol. 79, 665-684 (1999).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neuropathology+and+impact+of+perinatal+brain+damage.+II:+white+matter+lesions+of+the+neocortex.">Developmental neuropathology and impact of perinatal brain damage. II: white matter lesions of the neocortex.</a> J Neuropathol Exp Neurol. 56, 219-235 (1997).</li><li>Jin, X., Prince, D. A., Huguenard, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitatory+synaptic+connectivity+in+layer+v+pyramidal+neurons+of+chronically+injured+epileptogenic+neocortex+in+rats.">Enhanced excitatory synaptic connectivity in layer v pyramidal neurons of chronically injured epileptogenic neocortex in rats.</a> J Neurosci. 26, 4891-4900 (2006).</li><li>Li, H., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+in+chronically+injured,+epileptogenic+sensory-motor+neocortex.">Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex.</a> J Neurophysiol. 88, 2-12 (2002).</li><li>Salin, P., Tseng, G. F., Hoffman, S., Parada, I., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+sprouting+in+layer+V+pyramidal+neurons+of+chronically+injured+cerebral+cortex.">Axonal sprouting in layer V pyramidal neurons of chronically injured cerebral cortex.</a> J Neurosci. 15, 8234-8245 (1995).</li><li>Avramescu, S., Nita, D. A., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+post-traumatic+epileptogenesis+is+associated+with+loss+of+GABAergic+neurons.">Neocortical post-traumatic epileptogenesis is associated with loss of GABAergic neurons.</a> J Neurotrauma. 26, 799-812 (2009).</li><li>Avramescu, S., Timofeev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+strength+modulation+after+cortical+trauma:+a+role+in+epileptogenesis.">Synaptic strength modulation after cortical trauma: a role in epileptogenesis.</a> J Neurosci. 28, 6760-6772 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Il modello sottosquadro è un sistema altamente efficiente per lo studio dell&#39;epilessia post-traumatico. Un intervento chirurgico tipica richiede solo circa 20-30 minuti per finire, ed evocato o attività epilettiformi spontanee possono essere registrati in fette dalla maggior parte dei animali due settimane dopo l&#39;intervento 1-2. Ancora più importante, questo modello imita aspetti delle seguenti modifiche lesioni cerebrali traumatiche come sanguinamento, infiammazione, edema, assotomia e la morte ne...

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Foredom micromotore kit </td><td> attrezzatura </td><td> Foredom </td><td> K.1070 </td></tr><tr><td> 1,5 pollici 22-gauge siringa </td><td> materiale </td><td> BD società </td><td> 305156 </td></tr><tr><td> 1,5 pollici da 25 gauge siringa </td><td> materiale </td><td> BD società </td><td> 305127 </td></tr><tr><td> Collante cianoacrilato </td><td> materiale </td><td> Ted Pella </td><td> 14450 </td></tr></tbody></table>

preparing undercut model of posttraumatic epileptogenesis in rodents

Parzialmente isolato corteccia (&quot;sottosquadro&quot;) è un modello animale di epilettogenesi post-traumatico. La procedura chirurgica coinvolge tagliare attraverso la corteccia sensomotoria e sotto la materia bianca (sottosquadro) in modo che una specifica regione della corteccia cerebrale è in gran parte isolato dalla corteccia e le regioni confinanti subcorticali 1-3. Dopo una latenza di due o più settimane dopo l&#39;intervento, scariche epilettiformi possono essere registrate in fettine di cervello di roditori 1; e convulsioni elettriche o comportamento si può osservare in vivo da altre specie come il gatto e la scimmia 4-6. Questo modello animale ben consolidata è efficiente per generare e imita molte caratteristiche importanti traumi cerebrali. Tuttavia, è tecnicamente difficile tentativo di rendere precise lesioni corticali del cervello piccolo roditore con una mano libera. Sulla base della procedura stabilita inizialmente nel laboratorio del Dr. David Principe presso l&#39;Università di Stanford 1, qui vi presentiamo una tecnica migliore per eseguire un intervento chirurgico per la preparazione di questo modello in topi e ratti. Dimostriamo come fare un semplice dispositivo chirurgico e usarla per ottenere un miglior controllo della profondità di taglio e l&#39;angolo per generare risultati più precisi e coerenti. Il dispositivo è facile da fare, e la procedura è veloce da imparare. La generazione di questo modello animale fornisce un efficiente sistema per lo studio sui meccanismi di epilettogenesi post-traumatico.

Watch this Scientific Journal Video about Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents at JoVE.com

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Parzialmente isolato corteccia (&quot;sottosquadro&quot;) è un modello animale efficiente epilettogenesi post-traumatico. Qui mostriamo come fare un nuovo dispositivo chirurgico e usarlo per fare le lesioni più precisi e concordanti per generare questo modello.

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 ...

preparing-undercut-model-of-posttraumatic-epileptogenesis-in-rodents

Research

JoVE Journal

Neuroscience

9.3K Views.  Indiana University School of Medicine. Parzialmente isolato corteccia ("sottosquadro") è un modello animale efficiente epilettogenesi post-traumatico. Qui mostriamo come fare un nuovo dispositivo chirurgico e usarlo per fare le lesioni più precisi e concordanti per generare questo modello.

Video: Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

Inibizione assuefazione e prepulse di shock acustici in roditori

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

Ricerca

Neuroscienze

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

Su larga scala di registrazione dei neuroni da sonde mobili in silicio Comportarsi Roditori

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

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, ...

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 ...

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.

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 ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Estrusione idraulica del midollo spinale e isolamento dei gangli dorsali nei roditori

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Impianto di cannula in Cisterna Magna di roditori

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

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 ...