עבודה זו נתמכה על ידי מענקים מהמכון הלאומי לבריאות (DC007681, א.ב.), CT תאי גזע המענק (א.ב.), פארדי היסוד (א.ב.), Predoctoral רות ל Kirschstein שירות מחקר של הפרסים הלאומיים (NRSA) (SZY), ובוגר NSF מחקר מלגה (BL). אנו מודים לחברי מעבדת Bordey על הערות מועילות על כתב היד. החומר זה מבוסס על העבודה הנתמכת בחלקו על ידי מדינת קונטיקט במסגרת התכנית לקונטיקט לתאי הגזע למחקר המענקים. התוכן שלה הוא באחריות בלעדית של הכותבים ואינם מייצג בהכרח את עמדתה הרשמית של מדינת קונטיקט, המחלקה לבריאות ציבור של מדינת קונטיקט או CT חידושים, Incorporated.

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C., Dupuis, A., Boisseau, S., Villaz, M., Albrieux, M., Brocard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrony+of+spontaneous+calcium+activity+in+mouse+neocortex+before+synaptogenesis.">Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis.</a> Eur. J. Neurosci. 25, 920-928 (2007).</li><li>Young, S. Z., Platel, J. C., Nielsen, J. V., Jensen, N. A., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA+increases+calcium+in+subventricular+zone+astrocyte-like+cells+through+L-+and+T-type+voltage-gated+calcium+channels.">GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels.</a> Front. Cell. Neurosci. 4, 8 (2010).</li><li>Bolteus, A. 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M., Henschel, O., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFAP-expressing+cells+in+the+postnatal+subventricular+zone+display+a+unique+glial+phenotype+intermediate+between+radial+glia+and+astrocytes.">GFAP-expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes.</a> Glia. 54, 394-410 (2006).</li><li>Wang, D. D., Krueger, D. D., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+and+ionic+signature+of+neuronal+progenitors+of+the+postnatal+subventricular+zone+in+situ.">Biophysical properties and ionic signature of neuronal progenitors of the postnatal subventricular zone in situ.</a> J. Neurophysiol. 90, 2291-2302 (2003).</li><li>Tian, L., Hires, S. A., Mao, T., Huber, D., Chiappe, M. E., Chalasani, S. H., Petreanu, L., Akerboom, J., McKinney, S. A., Schreiter, E. R., Bargmann, C. I., Jayaraman, V., Svoboda, K., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neural+activity+in+worms,+flies+and+mice+with+improved+GCaMP+calcium+indicators.">Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.</a> Nat. Methods. 6, 875-881 (2009).</li><li>Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expanded+palette+of+genetically+encoded+Ca(2)+indicators.">An expanded palette of genetically encoded Ca(2) indicators.</a> Science. 333, 1888-1891 (2011).</li><li>Shigetomi, E., Kracun, S., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+targeted+optical+sensor+to+monitor+calcium+signals+in+astrocyte+processes.">A genetically targeted optical sensor to monitor calcium signals in astrocyte processes.</a> Nat. Neurosci. 13, 759-766 (2010).</li></ol>

אין ניגודי האינטרסים הכריזו.

הדמית סיד של תאי SVZ נעשתה שימוש כדי ללמוד את הדפוסים בפעילות ספונטנית בneuroblasts 10, ערוץ ביטוי הקולטן בשני neuroblasts והאסטרוציטים 4,6,8 וגלי סיד astrocytic 3. מאחר ותאים בSVZ הם או לא בוגרים או בעלי תכונות גליה, הם לא יירו פוטנציאלי פעולה 11,12, כלומר שינויים באלפ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מומס </td><td> חברה </td><td> מספר קטלוגים </td><td> דיסקציה (מ&quot;מ) </td></tr><tr><td> סוכרוז </td><td> סיגמא </td><td> S0389 </td><td> נתיחה: 147 מ&quot;מ ACSF: 0 מ&quot;מ </td></tr><tr><td> NaCl </td><td> סיגמא </td><td> S9888 </td><td> נתיחה: 42 מ&quot;מ ACSF: 126 מ&quot;מ </td></tr><tr><td> KCl </td><td> סיגמא </td><td> P3911 </td><td> נתיחה: 2.5 מ&quot;מ ACSF: 2.5 מ&quot;מ </td></tr><tr><td> MgCl 2 0.6 H 2 O </td><td> סיגמא </td><td> M9272 </td><td> נתיחה: 4.33 מ&quot;מ ACSF: 1 mM </td></tr><tr><td> Nah 2 PO 4. H 2 O </td><td> סיגמא </td><td> S8282 </td><td> נתיחה: 1.25 מ&quot;מ ACSF: 1.25 מ&quot;מ </td></tr><tr><td> גלוקוז </td><td> סיגמא </td><td> G8270 </td><td> Dissection: 10 מ&quot;מימ ACSF: 10 מ&quot;מימ </td></tr><tr><td> 3 NaHCO </td><td> סיגמא </td><td> S6014 </td><td> נתיחה: 26 מ&quot;מ ACSF: 26 מ&quot;מ </td></tr><tr><td> CaCl 2 0.2 H 2 O </td><td> סיגמא </td><td> C3306 </td><td> נתיחה: 1.33 מ&quot;מ ACSF: 2 מ&quot;מ </td></tr></tbody></table> טבלה 1. רשימה כימית ומתכונים של פתרון נתיחה וACSF. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td></td><td></td><td> [כותרת] </td></tr><tr><td> שם </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> Vibratome </td><td> ליקה </td><td> VT 1000s </td><td></td></tr><tr><td> Super Glue </td><td> Surehold או 3M </td><td> Surehold 3G Super Glue או 3M וטרינר בונד </td><td></td></tr><tr><td> כלי נתיחה </td><td> Roboz או טד פלה </td><td></td><td></td></tr><tr><td> Fluo-04:00 לצבוע בסידן רגיש </td><td> Invitrogen </td><td> F14201 </td><td></td></tr><tr><td> אורגון גרין BAPTA-01:00 לצבוע בסידן רגיש </td><td> Invitrogen </td><td> O6807 </td><td></td></tr><tr><td> ה-F-127 20% פתרון Pluronic בDMSO </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> confocal מיקרוסקופ הזקוף </td><td> אולימפוס </td><td> FV300 או FV1000 </td><td></td></tr><tr><td> מטרות מי טבילה </td><td> אולימפוס </td><td> LUMPlanFl 40 x W / IR (NA 0.80); LUMPlanFl 60 x W / אני (NA 0.90) </td><td></td></tr><tr><td> Micromanipulators </td><td> סאטר </td><td> MPC-200/MPC-325/MPC-385 </td><td></td></tr><tr><td> &lt;בקר לחץ/ Td&gt; <td> פארקר Hannifin </td><td> Picospritzer </td><td> &lt;3 PSI במהלך יישום </td></tr><tr><td> פיפטה חולץ </td><td> סאטר או Narshige </td><td> סאטר P-97 או Narshige PP-830 </td><td></td></tr><tr><td> pipettes זכוכית </td><td> סאטר </td><td> BF150-110-10 </td><td> ID: 1.10, OD: 1.50 </td></tr><tr><td> משאבת peristaltic </td><td> הרווארד Apparatus </td><td> דגם 720 </td><td> קצב זרימה: 1 מ&quot;ל / דקה </td></tr><tr><td> אמבטיה קאמרית </td><td> וורנר מכשירים </td><td> RC-26 GLP </td><td> פרופיל נמוך מאפשר לאישור אובייקטיבי </td></tr><tr><td> צנור </td><td> Tygon </td><td></td><td></td></tr><tr><td> בקר טמפרטורה </td><td> וורנר מכשירים </td><td> TC-324B/344B </td><td></td></tr></tbody></table> טבלה 2. חומרים / רשימת ציוד.

preparation of acute subventricular zone slices for calcium imaging

אזור subventricular (SVZ) הוא אחד משני האזורים במוח neurogenic לאחר הלידה. SVZ מכיל תאים צפופים, כולל תאי אב עצביים עם תכונות astrocytic (הנקרא האסטרוציטים SVZ), neuroblasts, ותאי ביניים. Neuroblasts נולד בSVZ משיק לנדוד מרחק רב כדי נורת חוש הריח, שבו הם להתמיין interneurons. איתות אינטר באמצעות מולקולות דבקות ואותות diffusible ממלאת תפקידים חשובים בneurogenesis שליטה. רבים מאותות אלו לגרום לפעילות הסידן אינטר שמעבירה מידע בתוך ובין תאים. סידן היא פעילות ובכך משקף את הפעילות של אותות תאיים והיא דרך אופטימלית להבין איתות אינטר פונקציונלית בין תאי SVZ. פעילות סידן נחקרה באזורים רבים אחרים וסוגי תאים, כוללים תאי גליה ונוירונים בוגרים. עם זאת, השיטה המסורתית לואהתאי D עם סיד מחוון צבע (טעינת אמבטיה כלומר) לא היו יעילים בטעינה כל סוגי תאי SVZ. ואכן, בצפיפות הסלולרית SVZ מונעת התפשטות צבע בתוך הרקמה. בנוסף, הכנת פרוסות sagittal יהיה טוב יותר לשמור על ההסדר תלת הממדי של תאים, במיוחד SVZ זרם הגירת neuroblast על הציר מקורי-הזנב. כאן, אנו מתארים שיטות להכנת סעיפי sagittal המכילים SVZ, הטעינה של תאי SVZ עם צבע מחוון סידן, ורכישת פעילות סידן עם סרטי זמן לשגות. אנחנו השתמשנו Fluo-04:00 לצבוע לטעינת האסטרוציטים SVZ באמצעות יישום לחץ בתוך הרקמה. פעילות סידן נרשמה באמצעות מיקרוסקופ confocal סריקה המאפשר רזולוציה מדויקת לתאים בודדים מזהים. הגישה שלנו היא ישימה לאזורים אחרים, כולל neurogenic האזור המבוגר היפוקמפוס subgranular ואזורי neurogenic עובריים. בנוסף, סוגים אחרים של צבעים יכוליםלהיות מיושם בשיטה שתוארה.

Watch this Scientific Journal Video about Preparation of Acute Subventricular Zone Slices for Calcium Imaging at JoVE.com

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

שיטה לטעון אזור תאי subventricular (SVZ) עם צבעי סיד חיווי לפעילות הסידן הקלטה מתוארת. SVZ לאחר הלידה מכיל תאים צפופים, כולל תאי אב עצביים וneuroblasts. במקום להשתמש בטעינת אמבטיה אנו מזריקים צבע בלחץ בתוך הרקמה המאפשרת דיפוזיה צבע טובה יותר.

הכנת Slices האזור Subventricular החריף להדמית סיד

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

preparation-of-acute-subventricular-zone-slices-for-calcium-imaging

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Neuroscience

13.0K Views.  Yale University School of Medicine. שיטה לטעון אזור תאי subventricular (SVZ) עם צבעי סיד חיווי לפעילות הסידן הקלטה מתוארת. SVZ לאחר הלידה מכיל תאים צפופים, כולל תאי אב עצביים וneuroblasts. במקום להשתמש בטעינת אמבטיה אנו מזריקים צבע בלחץ בתוך הרקמה המאפשרת דיפוזיה צבע טובה יותר.

Video: Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

אזור Subventricular En פנים: מכתים Wholemount ושפל Ependymal

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Electroporation האזור Subventricular ילודים

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo electroporation ואחרי הלידה וזמן לשגות הדמיה של neuroblast הגירה בעכבר חריפה פרוסות המוח

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

מיון תא של תאי גזע ועצביים ממבוגרי עכבר Subventricular האזור וחי-הדמיה של Dynamics תא המחזור שלהם

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

הכנת פרוסות המוח חריפה באמצעות אופטימיזציה N-מתיל-D-שיטת שחזור מגן glucamine

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

דגירה ממושכת של רקמות חריפות עצביות עבור Electrophysiology ו-הדמית סידן

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

שני הפוטונים סידן הדמיה של דנדריטים עצביים במוח פרוסות

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

הכנה של חוט השדרה חריפה פרוסות תאים כל תיקון-קלאמפ הקלטה בנוירונים הסובסטנציה Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

הכנת פרוסות היפוקמפוס אנושיות חריפות להקלטות אלקטרופיזיולוגיות

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

<meta charset="utf8"></head><body>נוקלאופקציה יעילה של NSCs מבודדים מהאזור התת-חדרי של מורין

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>זרקור המחבר: נוקלאופקציה משופרת להעברת גנים ביעילות גבוהה בתרביות תאי גזע עצביים שמקורם באזור תת-חדרי של מורין

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

בידוד, התרחבות ונוקלאופקציה של תאי גזע עצביים מאזור תת-חדרי מורין בוגרים

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...