מארי קירי International Fellowship יוצא (FP/2007-2013 של האיחוד האירופי הסכמי גרנט # 221834 ו - 254780), JD קרן מקדונל, NSF גרנט SBE 0542013, המכונים הלאומיים לבריאות גרנט NS034994, המכון הלאומי לבריאות הנפש MH5467 גרנט לבין הווארד יוז רפואי במכון (Janelia חוות מחקר קמפוס המענק).

<ol>
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P., Luczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+Large-scale+Neuronal+Ensembles+with+Silicon+Probes+in+the+Anesthetized+Rat.">Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat.</a> J. Vis. Exp. (56), e3282-e3282 (2011).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rat+Brain.">The Rat Brain.</a> Stereotaxic Coordinates. , (1982).</li><li>Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+tetrode+spike+separation+as+determined+by+simultaneous+intracellular+and+extracellular+measurements.">Accuracy of tetrode spike separation as determined by simultaneous intracellular and extracellular measurements.</a> J. Neurophysiol. 84, 401-414 (2000).</li><li>Hazan, L., Zugaro, M., Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klusters,+NeuroScope,+NDManager:+a+Free+Software+Suite+for+Neurophysiological+Data+Processing+and+Visualization.">Klusters, NeuroScope, NDManager: a Free Software Suite for Neurophysiological Data Processing and Visualization.</a> J. Neurosci. Methods. 155, 207-216 (2006).</li><li>Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+neurotechnologies+for+chronic+neural+interfaces:+new+horizons+and+clinical+opportunities.">Advanced neurotechnologies for chronic neural interfaces: new horizons and clinical opportunities.</a> J. Neurosci. 28, 11830-11838 (2008).</li><li>Csicsvari, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+recording+of+unit+and+local+field+potentials+with+silicon-based+electrodes.">Massively parallel recording of unit and local field potentials with silicon-based electrodes.</a> J. Neurophysiol. 90, 1314-1323 (2003).</li><li>Sodagar, A. M., Wise, K. D., Najafi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fully+integrated+mixed-signal+neural+processor+for+implantable+multichannel+cortical+recording.">A fully integrated mixed-signal neural processor for implantable multichannel cortical recording.</a> IEEE Trans. Biomed. Eng. 54, 1075-1088 (2007).</li><li>O'Connor, D. H., Huber, D., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+engineering+the+mouse+brain.">Reverse engineering the mouse brain.</a> Nature. 461, 923-929 (2009).</li><li>Boyden, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-breakers:+optical+technologies+for+probing+neural+signals+and+systems.">Circuit-breakers: optical technologies for probing neural signals and systems.</a> Nat. Rev. Neurosci. 8, 577-581 (2007).</li><li>Royer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-array+silicon+probes+with+integrated+optical+fibers:+light-assisted+perturbation+and+recording+of+local+neural+circuits+in+the+behaving+animal.">Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal.</a> Eur. J. Neurosci. 31, 2279-2291 (2010).</li></ol>

אין ניגוד עניינים הצהיר.

סרט זה ממחיש את ההליך השתלת סיליקון עבור בדיקות בקנה מידה גדול הקלטות כרוניות בחולדה מתנהג. שלבים קריטיים כדי להבטיח הקלטות באיכות של הפעילות העצבית נובעים השבריריות של שניהם ביולוגי (רקמת המוח) ו טכניים (סיליקון בדיקה) חומרים. זהירות יש לנקוט בזמן הטיפול בדיקה כדי ל?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> סוג </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> הסיליקון בדיקה Buzsaki32, 4 ובמוטות מושחזים x 8 אתרים. אריזה: כבל גמיש פוליאמיד </td><td> חומר </td><td> NeuroNexus </td><td> בדיקה: buzsaki32 אריזה: HC32 </td><td> הקלטה בדיקה </td></tr><tr><td> בורג נחושת עגול, 00-90 x 1/2 ברגי פליז עגול </td><td> חומר </td><td> JIMorris </td><td> R0090B500 </td><td> כונן חלק </td></tr><tr><td> הקס אגוזים פליז, 00-90 </td><td> חומר </td><td> JIMorris </td><td> N0090B </td><td> כונן חלק </td></tr><tr><td> פליז C260 עזה, ASTM-B36 עובי: 0.025 &quot;, אורך: 12&quot;, רוחב: 1/2 &quot; </td><td> חומר </td><td> חלקים קטנים </td><td> B000FMYU72 </td><td> כונן חלק </td></tr><tr><td> מחבר כותרת, גובה 2 מ&quot;מ, מ &#39;אייל, שורה אחת, straigt, 36 עמדות </td><td> חומר </td><td> Digikey </td><td> 2163S-36-ND </td><td> כונן חלק </td></tr><tr><td> 2 חלקים Sylgard אלסטומר סיליקון </td><td> חומר </td><td> Precision של מכשירים בעולם </td><td> SYLG184 </td><td> כדי לבודד במיוחד בדיקה </td></tr><tr><td> Decon Contrad 70 בנוזל כביסה </td><td> מגיב </td><td> פישר סיינטיפיק </td><td> 04-355 Decon מעבדות מספר: 1002 </td><td> כדי לנקות את האתרים הקלטה </td></tr><tr><td> מיזוג עכבה מודול </td><td> ציוד </td><td> Fhc בע&quot;מ </td><td> 55-70-0 </td><td> עכבה מטר </td></tr><tr><td> niPOD - 32 ערוצים </td><td> ציוד </td><td> Neuronexus </td><td> niPOD -32 </td><td> עכבה מטר </td></tr><tr><td> מלט גריפ תעשייתי כיתה </td><td> חומר </td><td> קולק Dentsply </td><td> 675,571 (אבקה) 675,572 (ממס) </td><td> גריפ לספירהment </td></tr><tr><td> 1,1 &quot;, dioctadecyl-3, 3,3&quot;, 3&#39;-tetramethylindocarbocyanine פרכלורט (&quot;DiI &#39;; DiIC18 (3)) </td><td> מגיב </td><td> Invitrogen </td><td> D282 </td><td> להכתים את המסלול בדיקה במוח </td></tr><tr><td> מכונת נירוסטה בורג פלדה, ראש איגוד, כונן מחוררת, # 00-90, 1/8 &quot; </td><td> חומר </td><td> חלקים קטנים </td><td> MX-0090-02B </td><td> הקרקע ברגים התייחסות </td></tr><tr><td> חוט מגנט, 20 גר &#39;, ניילון, פוליאוריטן ציפוי, MW80 </td><td> חומר </td><td> חלקים קטנים </td><td> B000IJYRP2 </td><td> הקרקע חוט התייחסות </td></tr><tr><td> מכונת נירוסטה בורג פלדה, ראש איגוד מחוררת דרייב, # 000-120, 1/16 &quot; </td><td> חומר </td><td> חלקים קטנים </td><td> MX-000120-01B </td><td> עוגן ברגים </td></tr><tr><td> N-3 כל נוזלי שטף מטרה </td><td> מגיב </td><td> La-Co (Markal) </td><td> 23512 </td><td> מאפשר לרתך נירוסטה </td></tr><tr><td> MicroGrid Precision מורחב נחושת </td><td> חומר </td><td> Dexmet </td><td> 3 CU6 FA-050 </td><td> נחושת רשת עבור על ראש כלוב פאראדיי </td></tr><tr><td> C &amp; B-METABOND מהר! מלט מערכת - דנטין Activator </td><td> חומר </td><td> Parkell </td><td> S380 </td><td></td></tr><tr><td> C &amp; B-METABOND מהר! מערכת מלט - צמנט שיניים </td><td> חומר </td><td> Parkell </td><td> S380 </td><td></td></tr><tr><td> נקודה חדה טונגסטן המחט בעל </td><td> כלי </td><td> Roboz מכשירי ניתוח </td><td> RS-6064 ו-RS-6061 </td><td> כדי להפוך את הקרס להרים את דורה </td></tr><tr><td> קרביד Bur HP 1/4 </td><td> כלי </td><td> הנרי שיין </td><td> 9990013 </td><td></td></tr><tr><td> פרפין (גרגרי) </td><td> חומר </td><td> פישר סיינטיפיק </td><td> P31-500 </td><td></td></tr><tr><td> שמן מינרלי, אור (NF / FCC) </td><td> חומר </td><td> פישר סיינטיפיק </td><td> O121-1 </td><td></td></tr><tr><td> GC אלקטרוניקה 10-114 2 חלקים דבק אפוקסי </td><td> חומר </td><td> ניוארק </td><td> 00Z416 </td><td></td></tr><tr><td> 1 סוג LITZ 21 AWG 40/36 אדומה אחת פוליאוריתן, ניילון (MW80-C) .041 &quot;+ / -0.002&quot; OD </td><td> חומר </td><td> ניו אינגלנד Wire Technologies Corporation </td><td> N28-36E-400-2 </td><td> כדי להפוך את הכבל בין headstage ו מגבר </td></tr><tr><td> 32 ערוצים הגדול מאוד שילוב headstage קנה מידה, 20x רווח </td><td> ציוד </td><td> Plexon </td><td> HST/32V-G20 </td><td> Headstage </td></tr></tbody></table>

large-scale recording of neurons by movable silicon probes in behaving rodents

אתגר גדול במדעי המוח מקשרת התנהגות לפעילות הקולקטיבית של מכלולים עצביים. הבנה של קלט פלט יחסים של נוירונים ומעגלים דורש שיטות עם סלקטיביות מרחבי ברזולוציה הזמני המתאים לניתוח מכניסטית של הרכבים עצביים בעלי חיים מתנהגים, כלומר הקלטה של ​​דגימות גדולות representatively של נוירונים בודדים. אנסמבל ניטור של הפעילות העצבית התקדמה במידה ניכרת אצל בעלי חיים קטנים וגדולים כאחד בעל המוח, כולל בבני אדם 1-11 בעשור האחרון. מרובת אתרים עם הקלטה מבוססי סיליקון מכשירים יעילים במיוחד הודות ליכולת ההרחבה שלהם, נפח קטן עיצוב גיאומטרי. כאן, נתאר שיטות הקלטה נוירונים בודדים מרובים פוטנציאליים בתחום המקומית מתנהג מכרסמים, תוך שימוש זמינים מסחרית מיקרו מכונות בדיקות סיליקון עם מחוייט רכיבים אביזר. יש 2 אפשרויות בסיסיות ואו התממשקות בדיקות סיליקון preamplifiers: מעגלים מודפסים, כבלים גמישים. חברות המספקות בדיקה ( <a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a> ; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a> ; <a href="http://www.acreo.se/">http://www.acreo.se/</a> ) בדרך כלל לספק את השירות מליטה לספק בדיקות מלוכדות מעגלים מודפסים או כבלים גמישים. כאן, אנו מתארים את ההשתלה של החללית 4-32-שוק באתר, מצורף כבל polyimide גמיש, רכוב על Microdrive מטלטלין. כל שלב בהכנת בדיקה, בנייה Microdrive ניתוח מתוארת כך משתמש הקצה יכול בקלות לשכפל את התהליך.

Watch this Scientific Journal Video about Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents at JoVE.com

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

אנו מתארים שיטות הקלטה בקנה מידה גדול של יחידות בודדות מרובות פוטנציאל בתחום המקומית מתנהג מכרסמים עם בדיקות סיליקון. כונן ייצור, מצורף בדיקה לכונן ואת ההשתלה תהליכי בדיקה באים לידי ביטוי מספיק פרטים עבור שכפול קל.

בקנה מידה גדול הקלטה של ​​הנוירונים על ידי בדיקות מטלטלין סיליקון מתנהג מכרסמים

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

large-scale-recording-neurons-movable-silicon-probes-behaving

Research

JoVE Journal

Neuroscience

34.5K Views.  University of New Jersey. אנו מתארים שיטות הקלטה בקנה מידה גדול של יחידות בודדות מרובות פוטנציאל בתחום המקומית מתנהג מכרסמים עם בדיקות סיליקון. כונן ייצור, מצורף בדיקה לכונן ואת ההשתלה תהליכי בדיקה באים לידי ביטוי מספיק פרטים עבור שכפול קל.

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

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

הקלטה בקנה מידה גדול הרכבים העצבית עם בדיקות הסיליקון ב עכברוש הרדים

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

תיוג של פלורסנט מדרדר מאפשר להקלטה ממוקדת חד תאית מיחידת נוירונים שזוהו<em&gt; בvivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

הקלטה וניתוח של מקצבי יממה בריצת גלגל פעילות במכרסמים

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

תנודות Band גמא הקלטה Pedunculopontine Nucleus נוירונים

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

שחזור בקנה מידה גדול ועצמאי, Clustering משוחדת בהתבסס על מדדי מורפולוגי לסווג נוירונים באוכלוסיות סלקטיבי

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

כימות של Endosome ו Lysosome Motilities ב נוירונים תרבותיים באמצעות בדיקות פלואורסצנטי

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

השרשה של הגששים סיליקון כרונית ורישום של תאים בהיפוקמפוס המקום מכשיר הליכון מועשר

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

סידן רציומטרי הדמיה של נוירונים בודדים ב מתנהג Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

בניית הנעת Tetrode רב משופרת עבור הקלטת עצבית בקנה מידה גדול ב מתנהג חולדות

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

ההכנות ופרוטוקולים עבור כל תא, תיקון קלאמפ הקלטה של נוירונים Tectal צפרדע רפואית זריזה

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

בקנה מידה גדול הקלטה של ​​הנוירונים על ידי בדיקות מטלטלין סיליקון מתנהג מכרסמים

בקנה מידה גדול הקלטה של הנוירונים על ידי בדיקות מטלטלין סיליקון מתנהג מכרסמים