OG-P wurde durch CONACyT s zu gewähren (CB-2008 bis 101476) und FRABA (686/10) unterstützt. AQ-H durch das National Institute of Health, der Howard Hughes Medical Institute, der Robert Wood Johnson Foundation und der Maryland Stem Cell Foundation unterstützt.

<ol><li> Machen Glasnadeln bevor Maus-Chirurgie: <ol class="lalpha"><li> Legen Sie die Glaskapillare in einer Mikropipette Abzieher. </li><li> Erhitzen Sie die Mitte des Glasrohres mit dem Glasrohr in der lokalisierten Bereich zu erweichen. </li><li> Stretch das Glasrohr entlang seiner Längsachse durch eine anfängliche Abstand ausreicht, um eine Reduzierung der Durchmesser der Glasröhre in der lokalisierten Bereich verursachen. </li><li> Halten Dehnung der Glasröhre, bis er bricht. Auf diese Weise sind zwei identische Single Barrel Nadeln erhalten. </li></ol></li><li> Setzen Sie das Glas Nadel im microforge. Ein Winkel von 30 ° reicht zur Abschrägung der Spitze. </li><li> Überprüfen Sie unter dem Mikroskop, dass jeder Nadel die richtige innere Durchmesser hat. Für die meisten der hydrophilen Medikamenten eine 30-50 um Innendurchmesser ist ideal (Abbildung 1). </li><li> Füllen Sie die Nadel mit Mineralöl von der breitesten Ende Glasnadel. Lassen Sie das Mineralöl (MSDS, Cat. M7700) tritt durch Kapillarwirkung, bis es ~ 50% der Länge der Kapillare erreicht. Put Hochvakuum-Fett (Dow Corning, Cat. 05054-AB) um den Kolben, um die weiteste Ende der Nadel zu versiegeln. </li><li> Legen Sie den Kolben durch den breitesten Ende der Nadel und drücken Sie vorsichtig Mineralöl, bis ein kleiner Tropfen Öl gesehen geht aus der Spitze des Glases Nadel. </li><li> Sichern Sie die Glasnadel in der Inhaber der Mikroinjektor. </li><li> Anesthetize der Maus mit 2,5% Avertin (2,2,2-Tribromethanol + tert-Amylalkohol, 1:1 w / v). Dosis 25-30μL pro Gramm intraperitoneal. </li><li> Legen Sie eine Heizung Pad auf der stereotaktischen Gerät Tier seine Körpertemperatur auf 37 ° C zu halten </li><li> Ort und sichern die Maus den Kopf in den stereotaktischen Gerät mit Ohr Bars und ein Zähnen Inhaber. </li><li> Reinigen Sie die Maus Kopf mit 0,1% Chlorhexidingluconat-Lösung, um 70% Ethanol und 0,1% Chlorhexidingluconat ein zweites Mal an. </li><li> Inzision der Haut mit einem Skalpell Nr. 15 von Maus-Ohren, die Lamba Schädel Linie. </li><li> Polnische den Schädel mit einem Wattestäbchen zu trocknen und ziehen Haut aus dem OP-Feld. </li><li> Verwenden Sie die Spitze des Glases Nadel an der Spitze des Bregma Naht Punkt. Dort haben Sie die Ausgangsposition der Injektor (die Koordinate &quot;Null&quot;) gesetzt. </li><li> Stellen Sie den Punkt, indem Sie den &quot;X&quot; und &quot;Y&quot; Achse des stereotaktischen Gerät über den Schädel gebohrt werden. </li><li> Drill sehr vorsichtig Löcher an den ausgewählten koordinieren. Mikrobohrer und andere Werkzeuge aus Metall waren zuvor bei 250 ° C sterilisiert für 60 Sekunden mit einem trockenen Glasperlen Sterilisator (Cole Palmer, Cat. Nr. EW-10779-00). </li><li> Mit feinen Pinzette vorsichtig die tiefste Schicht des Knochens und brechen die duramatter Membran (Sie werden einige Rückenmarksflüssigkeit Austreten zu sehen). </li><li> Bestätigen Sie, dass die Nadel durch Bohrungen zu gehen. </li><li> Pipette 1 ul des Medikaments Sie injizieren und legte sie auf ein kleines Stück Parafilm. </li><li> Setzen Sie den Parafilm auf den Schädel. </li><li> Saugen bis das Medikament durch das Drehen der Mikroinjektor Rad, während Sie überprüfen unter dem Mikroskop, dass die Flüssigkeit wird bis in das Glas Nadel. </li><li> Entfernen Sie den Parafilm und re-set die Null zu koordinieren. </li><li> Bewegen Sie die Nadel auf die ausgewählten Koordinaten, auf der Halterung, bis die Spitze aus Glas Nadel sanft berühren der Hirnoberfläche und stellen Sie die &quot;Z&quot; zu koordinieren bei Null. </li><li> Führen Sie das Glas Nadel in das Gehirngewebe an ausgewählten Tiefe. </li><li> Spritzen Sie das Medikament langsam mit einer Geschwindigkeit von ca. 1 nl / sec. </li><li> Wenn das Begehren Volumen eingespritzt wird, lassen Sie das Medikament diffundieren in das Parenchym für 2 min. Dann fahren Sie mit der Nadel langsam und gleichmäßig zu entfernen. </li><li> Kleben Sie die OP-Wunde und entfernen Sie das Tier aus der stereotaktischen Gerät und setzte das Tier in einer vorgewärmten Käfig mit einem sauberen Bett zur Narkose Erholung. </li><li> Um die postoperative Analgesie alle Tiere erhielten 5 mg / kg subkutan Ketorolac jedem 12-Stunden für 24 h. </li></ol> Repräsentative Ergebnisse: Wenn nach diesem Protokoll ist eine sehr präzise Einspritzung erhalten und eine sehr enge Nadelbahn minimiert Hirn-Trauma. Als ein repräsentatives Ergebnis dieser Methode, injizierten wir Lysophosphatidylcholin (Lysolecithin) in das Corpus Callosum, dass Demyelinisierung der weißen Substanz Bahnen 1-4 produziert. Zur Minimierung der Hirnschädigung durch die Glasnadel produziert, injizierten wir nur 20 nl von Lysolecithin in das Corpus Callosum, sondern bei Bedarf höhere Volumina so viel wie 200 nl kann mit der gleichen Methode injiziert werden. Demyelinisierung wird durch die Abwesenheit von Myelin basisches Protein-Expression in der weißen Substanz (Abb. 2) nachgewiesen werden. <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein Glas Nadel mit einem 50-Mikrometer Durchmesser. Abstand zwischen zwei kurzen Zeckenin der Skala repräsentiert ein 50-um Länge. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Lysolecithin Injektion in den Corpus Callosum. Demyelinisierung wird als nicht Myelin basisches Protein-Expression (gepunktete Fläche) dargestellt. Beachten Sie die geringe Größe der Glas-Stichkanal (Pfeile). Bar = 100 pm

<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>

Die Methode zeigte in diesem Video ist sehr nützlich für die meisten Medikamente oder viralen Vektoren in sehr genauen Orte in das Gehirn liefern. Einige der wichtigsten Vorteile dieser Technik sind die Zuverlässigkeit der Targeting-Punkt, die Richtigkeit der Injektionen und die geringe Größe der Hirnschädigung und Darm-Trakt Schaden 1, 2, 5, 6. Sobald die Technik ist standardisiert Bereich von mistargeting sollte 50 um oder weniger 1, 2. Zelltransplantate kann auch mit weiteren, 100-150 um <...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
		</table>
		</div>

targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

Mikroinjektionen in das Hirnparenchym sind wichtige Verfahren, die Drogen, virale Vektoren oder Zelltransplantaten liefern. Die Hirnverletzung, dass eine Injektionsnadel produziert während ihrer Flugbahn ist ein wichtiges Anliegen vor allem im Gehirn der Maus nicht nur für das Gehirn ist klein, aber manchmal auch mehrere Injektionen erforderlich sind. Wir zeigen hier eine Methode zur Glaskapillare Nadeln mit einem 50-Lumen um die erheblich reduziert die Hirnschäden und ermöglicht eine genaue Ausrichtung in die Nager Gehirn zu produzieren. Diese Methode erlaubt eine Lieferung von kleinen Mengen (20 bis 100 nl), reduziert Blutungen Risiken und minimiert passive Verbreitung von Drogen in das Hirnparenchym. Durch die Verwendung unterschiedlicher Größe der Kapillare Glasröhren oder dem Wechseln der Nadel Lumen, können verschiedene Arten von Substanzen und Zellen injiziert werden. Mikroinjektionen mit einer Glaskapillare eine erhebliche Verbesserung in Injektionstechniken und Tiefenhirnstimulation Targeting mit minimalen Kollateralschäden in kleinen Nagern.

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 diesem Artikel zeigen wir eine Methode zur Glaskapillare Nadeln mit einem 50-um Lumen zu machen. Diese Technik reduziert die Schädigungen des Gehirns, minimiert passive Verbreitung von Drogen und ermöglicht eine genaue Ausrichtung in die Nager Gehirn.

Targeting von Deep Brain Strukturen mit Mikroinjektionen für die Lieferung von Drogen, virale Vektoren oder Zelltransplantaten

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

Research

JoVE Journal

Neuroscience

12.4K Aufrufe.  University of Colima. In diesem Artikel zeigen wir eine Methode zur Glaskapillare Nadeln mit einem 50-um Lumen zu machen. Diese Technik reduziert die Schädigungen des Gehirns, minimiert passive Verbreitung von Drogen und ermöglicht eine genaue Ausrichtung in die Nager Gehirn.

Serie: 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.

Skalierbare Fluidic Injector Arrays für Viral Targeting von Intact 3-D Schaltkreise im Gehirn

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

Forschung

Neurowissenschaften

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.

Intrakranielle Injektion von Adeno-assoziierte virale Vektoren

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.

Deep Brain Stimulation mit gleichzeitiger fMRT in Nagetiere

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.

Nicht beschränkende EEG Radiotelemetrie: epidurale und Deep Intrazerebrale Stereotaktische EEG Elektrodenplatzierung

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.

Personalisierte Nadeln für Mikroinjektionen im Nagetier Gehirn

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.

Erkunden Deep Space - Aufdeckung der Anatomie der periventrikulären Strukturen, die seitlichen Ventrikel des menschlichen Gehirns zu offenbaren

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.

Quantitative Zellbiologie der Neurodegeneration in Drosophila durch unvoreingenommene Analyse der Gewebekulturen Tagged Proteine mit ImageJ

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

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.

In Vivo Direkte Reprogrammierung von Resident Glial Cells in Interneurons durch 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 ...

Brain Morphology of Cannabis Users With or Without Psychosis: A Pilot MRI Study

Cannabis is the illicit drug most commonly used worldwide, and its consumption can both induce psychiatric symptoms in otherwise healthy subjects and unmask a florid psychotic picture in patients with a prior psychotic risk. Previous studies suggest that chronic and long-term cannabis exposure may exert significant negative effects in brain areas enriched with cannabinoid receptors. However, whether brain alterations determined by cannabis dependency will lead to a clinically significant phenotype or to a psychotic outbreak at some point of an abuser&#8217;s life remains unclear. The aim of this study was to investigate morphological brain differences between chronic cannabis users with cannabis-induced psychosis (CIP) and non-psychotic cannabis users (NPCU) without any psychiatric conditions and correlate brain deficits with selective socio-demographic, clinical and psychosocial variables.
3T magnetic resonance imaging (MRI) scans of 10 CIP patients and 12 NPCU were acquired. The type of drug, the frequency, and the duration, as well socio-demographic, clinical and psychosocial parameters of dependency were measured. CIP patients had extensive grey matter (GM) decreases in right superior frontal gyrus, right precentral, right superior temporal gyrus, insula bilaterally, right precuneus, right medial occipital gyrus, right fusiform gyrus, and left hippocampus in comparison to chronic cannabis users without psychosis. Finally, in CIP patients, the results showed a negative correlation between a domain of the Brief Psychiatric Rating Scale (BPRS), BPRS-Activity, and selective GM volumes. Overall, the results suggest that cannabis-induced psychosis is characterized by selective brain reductions that are not present in NPCU. Therefore, neuroimaging studies may provide a potential ground for identifying putative biomarkers associated with the risk of developing psychosis in cannabis users.

This is a 3T magnetic resonance imaging study aiming to investigate grey matter volume differences between cannabis-induced psychosis patients and non-psychotic chronic cannabis users.

Gehirnmorphologie von Cannabiskonsumenten mit oder ohne Psychose: Eine MrT-Pilotstudie

Cannabis is the illicit drug most commonly used worldwide, and its consumption can both induce psychiatric symptoms in otherwise healthy subjects and ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Fokussierte ultraschallinduzierte Öffnung der Blut-Hirn-Schranke zur gezielten Behandlung von Hirnstrukturen und zur Beurteilung der chemogenetischen Neuromodulation