We thank the Cell and Tissue Imaging Facility of the Institut du Fer &#224; Moulin, where all image acquisition and analysis have been performed. This work has been supported in part by the Centre National de la Recherche Scientifique, the Institut National de la Sant&#233; et de la Recherche M&#233;dicale, the Sorbonne Universit&#233; Sciences, and by grants from Sorbonne Universit&#233;s-Pierre et Marie Curie University (Emergence-UPMC program 2011/2014), the Fondation pour la Recherche sur le Cerveau, the Fondation de France, the Fondation pour la Recherche M&#233;dicale &#34;Equipe FRM DEQ2014039529&#34;, the French Ministry of Research (Agence Nationale pour la Recherche ANR-17-CE16-0008 and the Investissements d&#39;Avenir programme &#34;Bio-Psy Labex&#34; ANR-11-IDEX-0004-02) and a Collaborative Research in Computational Neuroscience program, National Science Foundation/French National Agency for Research (number : 1515686). All the authors are affiliated to research groups which are members of the Paris School of Neuroscience (ENP) and of the Bio-Psy Labex. F.E. is a Ph.D. student affiliated with Sorbonne Universit&#233;, Coll&#232;ge Doctoral, F-75005 Paris, France, and is funded by the Bio-Psy Labex. V.M. is a post-doctoral fellow funded by the Collaborative Research in Computational Neuroscience program, National Science Foundation/French National Agency for Research (number: 1515686). The authors thank Marta Kolodziejczak who participated in the initiation of the project.

<ol>
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The authors have nothing to disclose.

By maintaining, unlike in dissociated or organotypic slice culture, a structural integrity with limited network adjustments, acute brain slices allow researchers to study microglia in their physiological environment. However, one of the major limitations is the fact that the slicing procedure creates injuries that can rapidly compromise the viability of neurons, particularly in the adult brain. As microglia are particularly reactive to cell damage, it is important to limit neuronal cell death as much as possible to prese...

Microglial cells are the brain&#39;s resident macrophages and play a role in both physiological and pathological conditions1,2. They have a highly branched morphology and are constantly extending and retracting their processes3,4. This &#34;scanning&#34; behavior is believed to be related and necessary to the survey of their surroundings. The morphological plasticity of microglia is expressed in three modes. First, some compounds rapidly modulate microglial morphology: the addition of ATP5,6 or NMDA5,7 in the medium bathing acute brain slices increases the complexity of microglial ramifications, whereas norepinephrine decreases it6. These effects either are directly mediated by microglial receptors (for ATP and norepinephrine) or require an ATP release from neurons (for NMDA). Second, the growth and retraction speed of microglial processes, called motility or &#34;surveillance&#34;, can be affected by extracellular factors8, homeostasis disruptions9,10, or mutations9,10,11. Third, in addition to these isotropic changes of morphology and motility, microglia have the capacity to extend their processes directionally toward a pipette delivering ATP3,5,12,13,14, in culture, in acute brain slices or in vivo, or delivering 5-HT in acute brain slices15. Such oriented growth of microglial processes, also called directional motility, was first described as a response to a local laser lesion3,4. Thus, physiologically, it may be related to the response to injury or required for targeting microglial processes toward synapses or brain regions requiring pruning during development15,16, or in physiological17,18,19 or pathological situations9,18,19,20 in adulthood. The three types of morphological changes rely on different intracellular mechanisms11,13,20, and one given compound does not necessarily modulate all of them (e.g., NMDA, which acts indirectly on microglia, has an effect on morphology but does not induce directional motility5,7). Therefore, when aiming to characterize the effect of a compound, a mutation or a pathology on microglia, it is important to characterize the three components of their morphological plasticity. Here, we describe a method to study the directional growth of microglial processes toward a local source of compound, which is, here, ATP or 5-HT.
There are several models to study microglia processes&#39; attraction: primary cultures in 3D environment6,18,19, acute brain slices6,13,15,&#160;and in vivo imaging3,13. The in vivo approach is the best to preserve the physiological state of microglia. However, intravital imaging of deep regions requires complex surgical procedures and, therefore, it is often limited to superficial cortical layers. The use of microglia primary culture is the easiest technique to test a large number of conditions with a limited number of animals. Nevertheless, it is impossible to obtain the same cell morphology as in vivo, and cells lose their physiological interactions with neurons and astrocytes. Acute brain slices represent a compromise between these two approaches. This model allows researchers to study brain structures which are otherwise difficult to reach and to image with high resolution in vivo, and to investigate slices from neonatal stages, whereas transcranial microscopy is mostly performed at adulthood. Finally, it makes it possible to observe in real-time the effects of local drug application, and to repeat experiments while using a limited number of animals. Nonetheless, an issue with acute brain slices is the limited time (a few hours) during which the cells remain alive, notably for slices from mice older than two weeks, and the potential change of microglia morphology over time21,22.
Here, we describe a protocol to prepare acute brain slices of young and adult Cx3cr1GFP/+ mice up to two months old, with the preservation of microglia morphology and motility for several hours. We, then, describe how to use these slices to study the attraction of microglial processes toward compounds like ATP or 5-HT.

<table><tbody><tr><td>&lt;strong&gt;for pipettes preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Clark Borosilicate Thin Wall Capillaries</td><td>Harvard Apparatus</td><td>30-0065</td><td>Borosilicate Thin Wall without Filament, 1.5 mm OD, 1.17 mm ID, 75 mm L , Pkg. of 225</td></tr><tr><td>DMZ Universal Puller</td><td>Zeitz Instrumente</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;for solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Calcium Chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>Choline Chloride</td><td>Sigma</td><td>C7527</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>L-Ascorbic acid</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>Magnesium Chloride solution 1 M (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>63020</td><td></td></tr><tr><td>Potassium chloride SigmaUltra &gt;99.0% (KCl)</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>Ultrapure water</td><td>MilliQ</td><td></td><td>for all the solutions</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;for slice preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2x 200 mL crystalizing dishes</td><td></td><td></td><td></td></tr><tr><td>80 mL Pyrex beaker</td><td></td><td></td><td></td></tr><tr><td>Antlia-3C Digital Peristaltic pump</td><td>DD Biolab</td><td>178961</td><td>For mice perfusion and 2-photon chamber perfusion (aCSF)</td></tr><tr><td>Carbogen 5% CO&lt;sub&gt;2&lt;/sub&gt;/95% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Air Liquide France Industrie</td><td></td><td></td></tr><tr><td>Dolethal</td><td>Vetoquinol</td><td>Dolethal 50 mg/mL</td><td></td></tr><tr><td>Filter papers (Whatman)</td><td>Sigma</td><td>WHA1001042</td><td>Whatman qualitative filter paper, Grade 1 (Pore size: 11&amp;micro;M)</td></tr><tr><td>Fine Scissors - Sharp</td><td>Fine Science Tools</td><td>14060-60</td><td></td></tr><tr><td>Food box 10 cm diameter, 8 cm Height</td><td></td><td></td><td></td></tr><tr><td>glue (ethyl cyanoacrylate)</td><td>Loctite</td><td>super glue 3 power flex</td><td></td></tr><tr><td>Hippocampal Tool (spatula)</td><td>Fine Science Tools</td><td>10099-15</td><td>The largest extremity has to be angled at 90&amp;deg;</td></tr><tr><td>Ice</td><td></td><td></td><td></td></tr><tr><td>Iris Forceps (curved)</td><td>Moria</td><td>MC31</td><td></td></tr><tr><td>Lens cleaning tissue</td><td>THOR LABS</td><td></td><td></td></tr><tr><td>Nylon mesh strainer</td><td></td><td></td><td>diameter 7 cm</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td>For the slicer</td></tr><tr><td>scalpel blade</td><td></td><td></td><td></td></tr><tr><td>Slice interface holder</td><td></td><td></td><td>home-made, the file for 3D printing is provided in Supplemental Material</td></tr><tr><td>Surgical Scissors - Sharp</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>Vibrating slicer</td><td>Thermo Scientific</td><td>720-2709</td><td>Model: HM 650V (Vibrating blade microtome)</td></tr><tr><td>Water bath</td><td></td><td></td><td>Set at 32 &amp;deg;C (first recovery step)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;for slice imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;times; 25 0.95 NA water-immersion objective</td><td>Leica Microsystems (Germany)</td><td>HCX Irapo</td><td></td></tr><tr><td>2-photon MP5 upright microscope with resonant scanners (8 kHz) and two HyD Hybrid detectors</td><td>Leica Microsystems (Germany)</td><td></td><td></td></tr><tr><td>Antlia-3C Digital Peristaltic pump</td><td>DD Biolab</td><td>178961</td><td>For 2-photon chamber perfusion with aCSF</td></tr><tr><td>Carbogen 5% CO&lt;sub&gt;2&lt;/sub&gt;/95% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Air Liquide France Industrie</td><td>I1501L50R2A001</td><td></td></tr><tr><td>Chameleon Ultra2 Ti:sapphire laser</td><td>Coherent (Germany)</td><td></td><td></td></tr><tr><td>disposable transfer pipettes , wide mouth</td><td>ThermoFischer scientific</td><td>for example : 232-11</td><td>5.8 mL with fin tip, but we cut it (approx 7 cm) to have a 4 mm diameter mouth</td></tr><tr><td>emission filter SP680</td><td>Leica Microsystems (Germany)</td><td></td><td></td></tr><tr><td>fluorescent cube containing a 525/50 emission filter and a 560 dichroic filter (for fluorescence collection)</td><td>Leica Microsystems (Germany)</td><td></td><td></td></tr><tr><td>glass beaker with 50 mL of ACSF to maintain constant perfusion of the slice</td><td></td><td></td><td></td></tr><tr><td>Heating system</td><td>Warner Instrument Corporation</td><td>Automatic Heater Controller TC-324B</td><td>to maintain perfusion solution at 32 &amp;deg;C</td></tr><tr><td>perfusion chamber</td><td></td><td></td><td>home-made, the file for 3D printing is provided in Supplemental Material</td></tr><tr><td>slice holder (&quot;harp&quot;)</td><td></td><td></td><td>home made : hairpin made of platinum with the two branches joined by parallel nylon threads</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;for slice stimulation &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate (ATP)</td><td>Sigma</td><td>A-26209</td><td>to be prepared ex-temporaneously : 1 mg/mL (3 mM) stock solution prepared the day of the experiment, kept at 4 &amp;deg;C (a few hours) and diluted just before use</td></tr><tr><td>Fluorescein (optional)</td><td>Sigma</td><td>F-6377</td><td>use at 1 &amp;micro;M final</td></tr><tr><td>Micromanipulator</td><td>Luigs and Neumann</td><td>SM7</td><td>connected to the micropipette holde</td></tr><tr><td>Micropipette holder</td><td></td><td></td><td>same as for eletrophysiology</td></tr><tr><td>Serotonin hydrochloride</td><td>Sigma</td><td>H-9523</td><td>aliquots of 50mM stock solution in H&lt;sub&gt;2&lt;/sub&gt;0 kept at -20 &amp;deg;C. 500 &amp;micro;M solution prepared the day of the experiment.</td></tr><tr><td>Syringe 5 mL (without needle)</td><td>Terumo medical products</td><td>SS+05S1</td><td></td></tr><tr><td>Transparent tubing</td><td>Fischer Scientific</td><td>11750105</td><td>Saint Gobain Performance Plastics&amp;trade; Tygon&amp;trade; E-3603 Non-DEHP Tubing</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;for image analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fiji</td><td></td><td>https://fiji.sc</td><td>Schindelin, J. et al Nat. Methods (2012) doi 10.1038</td></tr><tr><td>Icy</td><td>Institut Pasteur</td><td>http://icy.bioimageanalysis.org</td><td>de Chaumont, F. et al. Nat. Methods (2012)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CX3CR1-GFP mice</td><td>Jung et al, 2000</td><td></td><td>male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.</td></tr><tr><td>CX3CR1creER-YFP mice</td><td>Parkhurst et al 2013</td><td></td><td>male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.</td></tr></tbody></table>

two-photon imaging of microglial processes' attraction toward atp or serotonin in acute brain slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

This protocol describes a method to induce, observe, and quantify the oriented growth of microglial processes toward a locally applied compound, for example, ATP or 5-HT, in acute brain slices from young or adult (at least up to two-month-old) mice. Among the factors that contribute to maintaining brain slices from adult animals in a healthy state for several hours is the use of two tools designed to optimize cell survival at two steps of the protocol. First, the interface slice holder in...

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Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

two-photon-imaging-microglial-processes-attraction-toward-atp-or

Research

JoVE Journal

Neuroscience

9.8K Views.  Institut National de la Santé et de la Recherche Médicale (INSERM), UMR-S 1270 Paris, France. Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Video: Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

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.

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

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Volumetric Imaging and Axonal Projection Mapping in the Marmoset Brain

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. We successfully implemented this technique for tracing the axonal projections in the marmoset brain. Here, the detailed experimental procedures that resulted in reliable volumetric imaging of the whole marmoset brain are described. A key process for successful imaging was the removal of meninges surrounding the brain, which interferes with slicing. A big advantage of this methodology is that the sliced sections can be used for additional staining. In the original set-up, the sliced sections are scrambled in the water bath. These sections can be correctly aligned in their original order according to the blood vessel patterns in the cortex. An example of effective histology is the visualization of myelin structure by simple light reflection, which can be combined with Nissl staining to define anatomical borders. These sections can also be used for immunological detection of non-fluorescent anterograde and retrograde tracers, which can be registered to the STPT data for layering of multiple data.

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic ...

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

<a href='https://app.jove.com/v/58788/two-photon-imaging-microglial-processes-attraction-toward-atp-or'>Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. (2019)</a>This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Summary

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Chapters in this video

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03^YEMK

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Spatiotemporal Analysis of Microglial Ca²⁺ Activity at Single-Cell Resolution

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice