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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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

Research

JoVE Journal

Neuroscience

13.1K 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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

סידן הדמיה תפקודית בפיתוח רשתות קליפתיים

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

תגובות סידן הדמיה בנוירונים GFP-tagged של פרוסות מוח עכבר ההיפותלמוס

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

אלקטרו הקלטה בו זמנית ובסידן הדמיה של suprachiasmatic גרעין נוירונים

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

שינוי של Colliculo-thalamocortical עכבר המוח פרוסה, שילוב הדפסת 3-D של לשכת רכיבים ודימות אופטי רב בקנה מידה

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

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 ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

פרוסות היפוקמפוס אופקיות של מוח העכבר

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

הכנת פרוסות חריפות מהיפוקמפוס הגבי להקלטה של תא שלם ושחזור עצבי בGyrus דנטאטה של עכברים בוגרים

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

באתרו הדמיה של גדילה וצמיחה של דינמיקת חרוט אקסון בתרביות פרוסות מוח עובריות אקס-ויוו חריפות

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...