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 utilizz...

<table><tbody><tr><td>Capillary glass tube</td><td>Wiretrol I</td><td>5-000-1001</td><td></td></tr><tr><td>Micropipette puller</td><td>Kopf Instruments</td><td>Model 730</td><td></td></tr><tr><td>Microforge</td><td>World Precision Instruments, Inc.</td><td>Model 48000</td><td></td></tr><tr><td>Mineral oil</td><td>MSDS</td><td>M7700</td><td></td></tr><tr><td>High-vacuum grease</td><td>Dow Corning</td><td>05054-AB</td><td></td></tr><tr><td>Anesthesia: 2.5% Avertin</td><td></td><td></td><td>2,2,2-tribrom&amp;#8211;thanol + tert-amyl alcohol, 1:1 w/v</td></tr><tr><td>Heater pad</td><td>Mastex</td><td>Model 900</td><td></td></tr><tr><td>Stereotactic device</td><td>Kopf Instruments</td><td>Model Kopf-900</td><td></td></tr><tr><td>Surgical scalpel blade # 15</td><td>Medi-Cut</td><td></td><td></td></tr><tr><td>Micro driller</td><td>Ideal Micro Drill</td><td>67-1000</td><td></td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>1-&amp;mu;L Micropipette</td><td>Rainin</td><td></td><td></td></tr><tr><td>Parafilm M.</td><td></td><td></td><td></td></tr><tr><td>Surgical microscope</td><td>Carl Zeiss, Inc.</td><td>Vasiorkop with Contraves system</td><td></td></tr><tr><td>Microinjector</td><td>Narishige International</td><td>Model MO-10</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

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

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Regioselettiva Biolistic Targeting in Organotipica Fette cervello Utilizzando un Gene Gun Modificato

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Analisi delle variazioni di espressione genica nel Rat Hippocampus Dopo stimolazione cerebrale profonda del nucleo anteriore talamiche

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microelettrodo guidata impianto di elettrodi nel subtalamico Nucleo di Ratti per lungo termine stimolazione cerebrale profonda

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Tratti della fibra di un neurone di targeting per la terapia di stimolazione cerebrale profonda utilizzando modelli interattivi, specifico per ogni paziente

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Bioingegneria

Modificazione genetica degli organoidi cerebrali dei primati

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Autore in primo piano: Microiniezione mirata ed elettroporazione di organoidi cerebrali di primati per la modificazione genetica  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

Microiniezione mirata ed elettroporazione di organoidi cerebrali di primati per la modificazione genetica

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

Biologia dello sviluppo

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Tecnica mouse Microchirurgia infusione per Targeted Sostanza consegna nel sistema nervoso centrale<i&gt; via</i&gt; Carotide interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

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