Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
21 Related JoVE Articles!
Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices
Institutions: Collège de France, Paris Diderot University.
Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum
astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.
Neuroscience, Issue 69, Physiology, Anatomy, Medicine, hippocampus preparation, acute brain slice, electrophysiology, patch-clamp, neurons, astrocytes, astroglial, neuroglial interactions, glutamate transporter current, potassium current, paired recordings, synaptic activity, synaptically-evoked responses
Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents
Institutions: National Institutes of Health.
The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+
and 1 H+
and the counter-transport of 1 K+
. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.
Neurobiology, Issue 78, Neuroscience, Biochemistry, Molecular Biology, Cellular Biology, Anatomy, Physiology, Biophysics, Astrocytes, Synapses, Glutamic Acid, Membrane Transport Proteins, Astrocytes, glutamate transporters, uptake, clearance, hippocampus, stratum radiatum, CA1, gene, brain, slice, animal model
Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System
Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences.
Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2
. Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo
tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e.
, axon, dendrite, or soma)3
. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.
Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture
Imaging of Estrogen Receptor-α in Rat Pial Arterioles using a Digital Immunofluorescent Microscope
Institutions: Uniformed Services University of the Health Sciences.
Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3
. Although two sub-types exist (estrogen receptor -α and β),estrogen receptor-α has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4
. Furthermore, ER-α is located in the nuclei and in the cytoplasm of rat basilar arteries 5
. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6
. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-α using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 μm cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.
Molecular Biology, Issue 57, digital immunofluorescent microscopy, brain, estrogen receptor-α, cerebral microvasculature, rat, immunohistochemistry
The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity
Institutions: West Virginia University .
The isolated microvessel preparation is an ex vivo
preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5
. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15
several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8
, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13
. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo
, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo
or in situ
tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables.
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.
Physiology, Issue 62, isolated microvessel preparation, skeletal muscle arterioles, resistance arteriole, microcirculation, arteriolar wall mechanics
Viability Assays for Cells in Culture
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number
Institutions: Duke University, Duke University.
One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, firstname.lastname@example.org) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.
Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte
Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices
Institutions: Purdue University.
Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis
oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1
and α6 L9′S mice 2
, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.
Neuroscience, Issue 68, Nicotinic, acetylcholine, neurotransmitter, neuron, patch clamp, brain slice, picospritzer
Intravital Microscopy of the Inguinal Lymph Node
Institutions: University of Northern British Columbia, University of Northern British Columbia.
Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response.
The ability to study the LN microvasculature in vivo
is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo
imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented.
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.
Immunology, Issue 50, Intravital vital microscopy, lymph node, arteriole, vasculature, cellular trafficking, immune response
Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation
Institutions: University of Missouri, University of Missouri.
Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1
by delivering neurotransmitters and neuropeptides onto neurons reproducibly2
. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3
has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5
. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6
. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7
, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be ˜ 1 μm or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8
. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below).
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+
) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC
-GCaMP2 transgenic mice9
enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.
Medicine, Issue 52, cremaster muscle, microcirculation, micropipettes, intravital microscopy
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators
Institutions: University of California Los Angeles, University of California Los Angeles.
Astrocytes display spontaneous intracellular Ca2+
concentration fluctuations ([Ca2+
) and in several settings respond to neuronal excitation with enhanced [Ca2+
signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+
imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+
signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo,
and confocal imaging approaches to record [Ca2+
signals in striatal astrocytes in situ,
are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+
imaging in striatal astrocytes in situ
. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+
signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+
signals in the striatal microcircuitry.
Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging
Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons
Institutions: Charité Universitätmedizin.
GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.
Neuroscience, Issue 91, electrophysiology, acute slice, whole-cell patch-clamp recording, neuronal morphology, immunocytochemistry, parvalbumin, hippocampus, inhibition, GABAergic interneurons, synaptic transmission, IPSC, GABA-B receptor
Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration
Institutions: University of Bonn, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE).
One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g.
the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.
Neuroscience, Issue 77, Neurobiology, Molecular Biology, Cellular Biology, Physiology, Biomedical Engineering, Biophysics, Biochemistry, biology (general), animal biology, Nervous System, Life Sciences (General), Neurosciences, brain slices, dendrites, inhibition, excitation, glutamate, GABA, micro-iontophoresis, iontophoresis, neurons, patch clamp, whole cell recordings
Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons
Institutions: Heinrich Heine University Düsseldorf.
Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis
subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.
Neuroscience, Issue 92, Neurosciences, two-photon microscopy, patch-clamp, UV-flash photolysis, mouse, hippocampus, caged compounds, glutamate, brain slice, dendrite, sodium signals
In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy
Institutions: Massachusetts General Hospital, and Harvard Medical School, The University of Tokyo.
BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT & CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.
Cellular Biology, issue 4, mouse, skeletal muscle, microscopy, circulation
Quantifying Agonist Activity at G Protein-coupled Receptors
Institutions: University of California, Irvine, University of California, Chapman University.
When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb
) is much greater than that for the inactive state (Ka
). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs
), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε
) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs
and relative efficacy of an agonist 1,2
. In this report, we show how to modify this analysis to estimate the agonist Kb
value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb
in absolute units of M-1
Our method of analyzing agonist concentration-response curves 3,4
consists of global nonlinear regression using the operational model 5
. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs
and a parameter proportional to efficacy (τ
). The estimate of τKobs
of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6
. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τsys
). In this case, the Kb
value of an agonist is equivalent to τKobs/τsys3
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.
Molecular Biology, Issue 58, agonist activity, active state, ligand bias, constitutive activity, G protein-coupled receptor