OG-P è stato sostenuto da CONACyT s borsa (CB-2008-101476) e FRABA (686/10). AQ-H supportati dal National Institute of Health, l&#39;Howard Hughes Medical Institute, la Robert Wood Johnson Foundation e della Fondazione cellule staminali Maryland.

<ol><li> Fai aghi di vetro prima dell&#39;intervento del mouse: <ol class="lalpha"><li> Mettere il tubo capillare di vetro in un estrattore micropipetta. </li><li> Di calore al centro del tubo di vetro per ammorbidire il tubo di vetro nella zona localizzata. </li><li> Allungare il tubo di vetro lungo il suo asse longitudinale da una distanza iniziale sufficiente per causare una riduzione del diametro del tubo di vetro nella zona localizzata. </li><li> Tenere il tubo di vetro che si estende fino a quando non si rompe. In questo modo, due aghi identici canna singola si ottengono. </li></ol></li><li> Mettere l&#39;ago di vetro in un microforge. Un angolo di 30 ° è sufficiente per smussare la punta. </li><li> Controllare al microscopio che ogni ago è il diametro corretto interiore. Per la maggior parte dei farmaci idrofili uno 30-50 micron di diametro interno è l&#39;ideale (figura 1). </li><li> Riempire l&#39;ago con olio minerale dalla fine della più ampia ago di vetro. Lasciate che l&#39;olio minerale (scheda di sicurezza, cat. M7700) entra per capillarità fino a raggiungere circa il 50% della lunghezza del tubo capillare. Mettere alto vuoto grasso (Dow Corning, cat. 05.054-AB) attorno al pistone per sigillare la più ampia fine dell&#39;ago. </li><li> Inserire il pistone dalla più ampia fine dell&#39;ago e spingere delicatamente l&#39;olio minerale fino ad una piccola goccia di olio è visto uscire la punta di un ago di vetro. </li><li> Fissare l&#39;ago di vetro nel supporto del microinjector. </li><li> Anestetizzare il mouse con il 2,5% Avertin (2,2,2-tribromoethanol + terz-amilico, 1:1 w / v). Dosis 25-30μL per grammo intraperitoneale. </li><li> Mettere un pad riscaldatore sul dispositivo stereotassico per mantenere la temperatura del corpo dell&#39;animale a 37 ° C. </li><li> Posizionare e fissare il capo del mouse sul dispositivo stereotassica con barre orecchio e un supporto denti. </li><li> Pulire la testina mouse con soluzione di clorexidina gluconato 0,1%, seguito dal 70% etanolo e gluconato di clorexidina 0,1% per la seconda volta. </li><li> Incise la pelle con una lama chirurgica # 15 da orecchie del mouse per la linea cranio Lamba. </li><li> Lucidare il cranio con uno swap di cotone ad asciugare fuori e tirare la pelle fuori del campo chirurgico. </li><li> Utilizzare la punta di un ago di vetro per puntare al vertice della sutura bregma. Ci si deve impostare la posizione iniziale di iniettori (punto &quot;zero&quot;). </li><li> Impostare il punto da forare spostando la &quot;X&quot; e &quot;Y&quot; del dispositivo stereotassica sopra il cranio. </li><li> Praticare molto attentamente i fori al di coordinate selezionato. Microdrills e altri oggetti metallici, sono stati precedentemente sterilizzati a 250 ° C per 60 secondi con un secco perle di vetro sterilizzatore (Cole Palmer, cat. No. EW-10779-00). </li><li> Con una pinza sottile rimuovere con attenzione lo strato più profondo delle ossa e rompere la membrana duramatter (potrete vedere alcune fluido cerebro-spinale fuoriesce). </li><li> Verificare che l&#39;ago può passare attraverso fori. </li><li> Pipettare 1 ml di farmaco da iniettare e metterlo su un piccolo pezzo di parafilm. </li><li> Mettere il parafilm sul cranio. </li><li> Aspirare il farmaco facendo girare la ruota microinjector, mentre si verifica al microscopio che il fluido è in costante aumento nella ago di vetro. </li><li> Rimuovere il parafilm e re-impostare la coordinata zero. </li><li> Spostare l&#39;ago alle coordinate selezionato, in basso il supporto fino a quando la punta dell&#39;ago vetro toccare delicatamente la superficie del cervello e impostare la coordinata &quot;Z&quot; a zero. </li><li> Introdurre l&#39;ago di vetro nel parenchima cerebrale in profondità selezionata. </li><li> Iniettare il farmaco lentamente a ritmo di ~ 1 nl / sec. </li><li> Quando il volume desiderio viene iniettato, lasciare che il farmaco diffuso nel parenchima per 2 minuti. Quindi procedere per rimuovere l&#39;ago senza intoppi e lentamente. </li><li> Incollare la ferita chirurgica e rimuovere l&#39;animale dal dispositivo stereotassico e mettere l&#39;animale in un pre-riscaldato gabbia contenente un letto pulito per il recupero anestesia. </li><li> Per fornire analgesia post-operatoria tutti gli animali hanno ricevuto 5 mg / kg per via sottocutanea Ketorolac ogni 12 ore per 24 ore </li></ol> Rappresentante dei risultati: Quando si segue questo protocollo, un&#39;iniezione molto preciso e si ottiene una pista ago molto sottile minimizza lesioni cerebrali. Come risultato rappresentativo di questo metodo, abbiamo iniettato lysophosphatidyl colina (lisolecitina) nel corpo calloso che produce demielinizzazione dei tratti di sostanza bianca 1-4. Per minimizzare il danno prodotto dal cervello l&#39;ago di vetro, abbiamo iniettato solo 20 nl di lisolecitina nel corpo calloso, ma se i volumi di richieste superiori fino al 200 nl può essere iniettata con lo stesso metodo. Demielinizzazione viene rilevato dalla assenza di espressione della proteina basica della mielina nei tratti di sostanza bianca (Figura 2). <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="Figura 1" /> Figura 1. Un ago vetro con un diametro di 50 micron. Distanza tra due tick brevenella scala rappresenta una lunghezza di 50 micron. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="Figura 2" /> Figura 2. Iniezione Lysolecithin nel corpo calloso. Demielinizzazione è mostrato come un no espressione della proteina basica della mielina (area tratteggiata). Si noti la piccola dimensione del tratto ago di vetro (frecce). Bar = 100 micron

<ol>
	<li>Menn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+oligodendrocytes+in+the+subventricular+zone+of+the+adult+brain.">Origin of oligodendrocytes in the subventricular zone of the adult brain.</a> J Neurosci. 26, 7907-7918 (2006).</li><li>Gonzalez-Perez, O., Romero-Rodriguez, R., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor+Induces+the+Progeny+of+Subventricular+Zone+Type+B+Cells+to+Migrate+and+Differentiate+into+Oligodendrocytes.">Epidermal Growth Factor Induces the Progeny of Subventricular Zone Type B Cells to Migrate and Differentiate into Oligodendrocytes.</a> Stem Cells. 27, 2032-2043 (2009).</li><li>Hall, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+aspects+of+remyelination+after+demyelination+produced+by+the+intraneural+injection+of+lysophosphatidyl+choline.">Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline.</a> J Cell Sci. 13, 461-477 (1973).</li><li>Webster, G. R., Thompson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+lysolecithin+in+nervous+tissues.">Observations on the presence of lysolecithin in nervous tissues.</a> Biochim Biophys Acta. 63, 38-45 (1962).</li><li>Doetsch, F., Caille, I., Lim, D. A., Garc&#237;a-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+Zone+Astrocytes+Are+Neural+Stem+Cells+in+the+Adult+mammalian+Brain.">Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult mammalian Brain.</a> Cell. 97, 1-20 (1999).</li><li>Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+converts+transit-amplifying+neurogenic+precursors+in+the+adult+brain+into+multipotent+stem+cells.">EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.</a> Neuron. 36, 1021-1034 (2002).</li><li>Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106, 15472-15477 (2009).</li><li>Richardson, R. M., Barbaro, N. M., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+transplantation+for+temporal+lobe+epilepsy.">Developing cell transplantation for temporal lobe epilepsy.</a> Neurosurg Focus. 24, E17-E17 (2008).</li><li>Alvarez-Dolado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26, 7380-7389 (2006).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo mostrato in questo video è molto utile per fornire la maggior parte dei farmaci o vettori virali in luoghi molto preciso nel cervello. Alcuni dei principali vantaggi di questa tecnica sono l&#39;affidabilità del punto di misura, l&#39;accuratezza delle iniezioni e le piccole dimensioni della lesione cerebrale e danni tratto 1, 2, 5, 6. Una volta che la tecnica è standardizzata la gamma di mistargeting deve 50 micron o meno 1, 2. Trapianti di cellule può essere effettuata anche utilizzando più ampio, 100-150 micron 7-9, aghi. Consegna delle cellule quindi efficiente può essere depositato in molto specifiche lesione riducendo al minimo i danni collaterali indotti da trapianto di cellule. Ci sono tre passaggi fondamentali di questa tecnica: <ol><li> Raccomandiamo sempre graffiare dolcemente il cranio con un ago da insulina per etichettare le coordinate selezionato prima di forare. </li><li> Introdurre l&#39;ago di vetro con un rapido movimento di penetrare rapidamente la superficie del cervello. </li><li> Una volta che la profondità ottimale si raggiunge si dovrebbe introdurre l&#39;ago 0,1 millimetri più in profondità nel parenchima cerebrale per fare un &quot;canale&quot; che permettono di diffondere liberamente della droga nel cervello. </li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Capillary glass tube</td>
<td>&nbsp;</td>
<td>Wiretrol I</td>
<td>5-000-1001</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model 730</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Model 48000</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>MSDS</td>
<td>M7700</td>
<td>&nbsp;</td>
<tr>
<td>High-vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>05054-AB</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia: 2.5% Avertin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v</td>
</tr>
<tr>
<td>Heater pad</td>
<td>&nbsp;</td>
<td>Mastex</td>
<td>Model 900</td>
<td>&nbsp;</td>
<tr>
<td>Stereotactic device</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model Kopf-900</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scalpel blade # 15</td>
<td>&nbsp;</td>
<td>Medi-Cut</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro driller</td>
<td>&nbsp;</td>
<td>Ideal Micro Drill</td>
<td>67-1000</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Fine Scientific Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-&mu;L Micropipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Vasiorkop with Contraves system</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>Model MO-10</td>
<td>&nbsp;</td>		</tbody>
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targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

Microiniezioni nel parenchima cerebrale sono importanti procedure per la fornitura di farmaci, vettori virali o trapianti di cellule. La lesione del cervello che produce l&#39;iniezione di un ago durante la sua traiettoria è una delle principali preoccupazioni soprattutto nel cervello di topo non solo per il cervello è piccolo, ma a volte anche iniezioni multiple sono necessari. Mostriamo qui un metodo per produrre aghi capillare di vetro con un 50-micron lume che riduce significativamente i danni cerebrali e permette una mira precisa nel cervello dei roditori. Questo metodo permette una consegna di piccoli volumi (da 20 a 100 Nl), riduce i rischi di sanguinamento, e riduce al minimo la diffusione passiva dei farmaci nel parenchima cerebrale. Usando diverse dimensioni di tubi di vetro capillari, o cambiando il lume ago, diversi tipi di sostanze e cellule possono essere iniettato. Microiniezioni con un tubo capillare di vetro rappresentano un significativo miglioramento nelle tecniche di iniezione e cerebrale profonda targeting con danni collaterali minimi in piccoli roditori.

Watch this Scientific Journal Video about Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants at JoVE.com

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

In questo articolo, mostriamo un metodo per fare gli aghi capillare di vetro con un 50-micron lumen. Questa tecnica riduce in modo significativo il danno al cervello, riduce al minimo la diffusione passiva di farmaci e permette una mira precisa nel cervello dei roditori.

Targeting di strutture cerebrali profonde con microiniezioni di consegna dei farmaci, vettori virali, o trapianti di cellule

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

targeting-deep-brain-structures-with-microinjections-for-delivery

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.5K Views.  University of Colima. In questo articolo, mostriamo un metodo per fare gli aghi capillare di vetro con un 50-micron lumen. Questa tecnica riduce in modo significativo il danno al cervello, riduce al minimo la diffusione passiva di farmaci e permette una mira precisa nel cervello dei roditori.

Video: Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Gli array scalabile fluidico Iniettore per Viral targeting di intatto 3-D circuiti cerebrali

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Ricerca

Neuroscienze

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Iniezione intracranica di adeno-associato vettori virali

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

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 ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Non restrittivo EEG Radiotelemetry: epidurale e Deep intracerebrale Stereotassica EEG Posizionamento degli elettrodi

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Personalizzato aghi per microiniezioni nel cervello del roditore

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

Esplorazione dello spazio profondo - scoprendo l'anatomia delle strutture Periventricular per rivelare i ventricoli laterali del cervello umano

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

Biologia cellulare quantitativa della neurodegenerazione in Drosophila attraverso analisi imparziale delle proteine fluorescente contrassegnate usando ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Produzione, purificazione e controllo di qualità per i vettori basati su Virus Adeno-associato

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

Modellazione tridimensionale delle forme e analisi delle strutture cerebrali

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

Riprogrammazione diretta in vivo delle cellule gliali residenti negli interneuroni mediante iniezione intracerebrale di vettori virali

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...