Name: imaging of intracellular atp in organotypic tissue slices of the mouse brain using the fret-based sensor ateam1.03yemk
Uploaded: 2019-12-19T13:00:00.000+00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK at JoVE.com

The authors wish to thank Claudia Roderigo and Simone Durry for expert technical assistance. We thank Dr. Niklas J. Gerkau and M.Sc. Joel Nelson for assistance in the preparation of the organotypic slice cultures. Research in the author&#39;s laboratory was funded by the German Research Association (DFG; FOR 2795: Ro 2327/13-1 and SPP 1757: Ro 2327/8-2 to CRR; and SPP 1757: Young Glia Start-Up funding to RL).

The authors declare no competing interests. The authors received financial support enabling open access publication by Nikon Microscope Solutions, D&#252;sseldorf, Germany, which produces instruments used in the video article. The company was neither involved in designing the experiments presented here, nor in their execution, nor in the data handling, nor in the manuscript writing.

Here, we demonstrate a procedure for the cell-type specific expression of ATeam1.03YEMK, a FRET-based, genetically encoded nanosensor14, for measurement of changes in ATP levels in astrocytes or neurons in organotypic tissue slice cultures of the mouse brain15. In exemplary recordings, we show that an increase in the extracellular potassium concentration does not result in a change in ATP concentrations in neurons, while astrocytic ATP levels rise in response to this manipulation. Moreover, our results demonstrate that upon inhibition of cellular metabolism, ATeam1.03YEMK FRET ratio rapidly drops in both cell types, indicating a rapid decrease in intracellular ATP.
Expression of ATeam1.03YEMK in organotypic slice cultures requires maintenance of the tissue in culture under controlled conditions for at least 7-10 days. Alternatively, ATeam1.03YEMK can also be employed for measurement of ATP in acutely isolated brain tissue slices and in optic nerves of mice13,15. Measurements in acutely isolated tissue, however, necessitate the generation of transgenic animals or a stereotactic application of viral vectors into the brain, involving animal experimentation and strict animal care protocols. In this regard, ATeam1.03YEMK expression in organotypic tissue slice cultures represents a useful and valuable alternative22,23. For many years now, organotypic tissue slice cultures serve as an established model system to study neural properties, connectivity and development24,25,26. They not only maintain the general tissue architecture and lamination (Figure 1), but also host the preferential properties of cell cultures such as superior accessibility and direct control of experimental conditions. Organotypic tissue slice cultures are also routinely employed to express foreign genes by using viral vectors27. Several types of viral vectors have been reported to deliver transgenes into brain tissue16,28. Adenoviral vectors induce high expression in glial cells, but not hippocampal neurons16, and might generate glial reactivity17. Adeno-associated viral vectors as used here emerge as a good alternative15, and their effectiveness has also been shown in vivo29.
While mainly being used for the study of neuronal properties, recent studies have established that organotypic tissue slice cultures can also be employed for the analysis of astrocytes. Cultured slices are usually covered by a layer of reactive astrocytes19,30 (Figure 5), but astrocytes exhibit a more native, non-reactive morphology and cytoarchitecture in deeper layers19,30 (Figure 5). In the present study, we describe a procedure for the mechanical removal of the outer glial scar, which results in a better experimental and optical accessibility of native astrocytes within the proper organotypic tissue layers. Moreover, its removal improves the expression efficacy in deeper layers of the organotypic slices; if the glial scar is not removed, transduction by AAVs might tend to be restricted to the superficial cell layers.
Several external mechanical factors need to be considered when performing experiments in tissue slices. A variation in bath perfusion velocity can induce movements of the entire preparation and/or induce changes in focus, resulting in artificial transient changes of the sensor signal. Moreover, both astrocytes and neurons have been reported to respond to mechanical deformation such as imposed by high perfusion rates32,33. In our hands, using a reliable peristaltic pump, together with maintaining small and stable volumes of saline between the tissue and the objective (meniscus) results in a stable FRET-signal under baseline conditions at the perfusion velocity used here (1.5-2.5 mL/min; Figure 8).
In the present study, we also demonstrate that FRET-based imaging with ATeam1.03YEMK can be employed to monitor ATP levels in neurons and astrocytes. An alternative means introduced earlier for measurement of cellular ATP is the so-called luciferin-luciferase assay34,35,36,37. This approach, however, is based on imaging of bioluminescence and only provides a rather low temporal and spatial resolution partly due to high background noise levels. Another method routinely employed in recent years was the imaging of changes in the intracellular magnesium concentration using the ion-sensitive fluorophore magnesium green38,39,40. This approach relates to the observation that a consumption of ATP results in the release of its co-factor magnesium. Imaging with magnesium green thereby only provides a secondary estimate of changes in ATP levels. Moreover, magnesium green is also sensitive to changes in intracellular calcium, introducing another difficulty when interpreting results obtained with this method.
The recent development of genetically-encoded nanosensors for direct imaging of cellular metabolites, therefore, represented a big step forward11,12. Several different sensors were generated that can be employed for measurement of intracellular ATP36,41,42,43. Among those are the ratiometric fluorescent ATP indicator &#34;QUEEN&#34;41 as well as PercevalHR, which senses the ATP:ADP ratio42. While the latter probe is a valuable tool for the study of the energy status of cells, it requires simultaneous measurement of changes in pH42.
ATeam is a nanosensor of which several variants exist, which &#8212; among others &#8212; differ in their binding affinity for ATP14. In vitro, ATeam1.03YEMK exhibits a Kd of 1.2 mM at 37 &#176;C, which is close to cellular ATP levels determined in different neuronal cell types, ranging from hypothalamus and cerebellum34 to hippocampus37,44,45. In cuvette measurements, lowering the temperature by 10 &#176;C resulted in a significant decrease in the binding affinity of ATeam1.03YEMK to ATP, suggesting that it might not be ideal for cellular imaging at room temperature14. Our earlier study15, however, demonstrated that the behavior and response of ATeam1.03YEMK expressed in neurons and astrocytes to different manipulations is similar at near physiological and at room temperature, indicating that the sensor allows reliable determination of intracellular ATP levels under both conditions. In addition, our previous experiments addressed the pH-sensitivity of ATeam1.03YEMK expressed inside cells15, showing that it is insensitive to changes in intracellular pH by about 0.1-0.2 pH units. If the Kd in the low mM range is a concern, alternative ATeam variants might be used14, among them red-shifted variants of ATeam (&#34;GO-ATeam&#34;)43.
Our experiments using ATeam1.03YEMK demonstrate that an increase in the extracellular potassium concentration by a few mM only (from 3 to 8 mM) results in a transient increase in the ATeam1.03YEMK ratio in astrocytes in organotypic slice culture. This observation confirms earlier studies15,46 and clearly indicates that astrocytes respond to the release of potassium by active neurons with an increase in their ATP production, mostly likely as a consequence of a stimulation of the Na+/K+-ATPase and the Na+/HCO3- cotransporter, respectively47,48. In contrast to this, neurons did not show a response, which is in line with previous work as well15. Both cell types, however, rapidly and strongly reacted to inhibition of cellular glycolysis and mitochondrial respiration as shown before15. Under conditions of chemical ischemia, ATeam FRET ratios fell to a new stable level, indicating a nominal depletion of cellular ATP. The latter result suggests that both neurons as well as astrocytes exhibit a relevant consumption of ATP also under steady-state conditions without additional stimulation by synaptic activation or application of neurotransmitters. Taken together, we conclude that FRET-based imaging with genetically encoded nanosensors, among them ATeam1.03YEMK, will provide a valuable approach to elucidate the cellular processes that are responsible for changes in intracellular ATP levels and cellular ATP consumption under different conditions.

Excitatory electrical activity of neurons is largely based on the flux of cations such as sodium (Na+) and potassium (K+) across their plasma membranes. Maintenance of the electrochemical gradients of these two ions is thus required for signaling. This is accomplished by the cellular Na+/K+-ATPase (NKA), an ubiquitously expressed electrogenic transmembrane pump, which extrudes 3 Na+ out of the cell in exchange for 2 K+ from the extracellular space, requiring the consumption of one molecule of ATP per transport cycle1. In addition to the NKA, there are several other ATP-consuming ion transporters including the plasma membrane Ca2+-ATPase, which is vital for intracellular Ca2+ homeostasis and its export following activity-induced influx2. In presynaptic vesicles, a vacuolar-type H+-ATPase (v-ATPase) creates the proton gradient necessary for neurotransmitter uptake into this compartment3.
While activity of neurons thus requires a substantial amount of ATP4, they do not exhibit a significant capacity for storage of energy. Instead, they seem to rely on metabolic interactions with neighboring astrocytes, the major glycogen stores in the brain5. It has been suggested that astrocytic glycogen indeed plays an important role in supporting neuronal energy needs; and a key phenomenon in this proposed neuro-metabolic coupling between the two cell types is the capacity of astrocytes to increase their ATP production in response to neuronal activity6,7,8. This hypothesis, known as astrocyte-neuron lactate shuttle (ANLS), is still under debate, because other work has provided evidence that neurons may also increase their own rate of glycolysis in response to stimulation9,10, reflecting the necessity of further methods and approaches to study neuro-glia interaction.
Investigation of cellular energy metabolism and ATP levels in neurons and astrocytes to elucidate neuro-glia metabolic interactions has long been hampered by the lack of suitable probes for the detection of changes in metabolite concentrations in living cells in brain tissue. The last decade, however, has provided a surge in the development of new tools and new genetically encoded fluorescent probes for different metabolites, including sensors for ATP, lactate, pyruvate and others11,12. Using these tools, it is now possible to directly address questions related to cellular ATP consumption and changes in cellular energy levels at the single cell level and in a cell-type specific manner in intact brain tissue13.
In the present work, we describe a procedure to visualize cytosolic ATP dynamics on neurons and astrocytes of cultured organotypic brain slices. We show how to employ adeno-associated viral vectors (AAV) for cell-type specific expression of the genetically encoded ATP-nanosensor ATeam1.03YEMK (14) in neurons and astrocytes of slices of mouse brain that can be maintained in cell culture for several weeks15. A procedure of how to remove the glial scar that covers cultured tissue slices is described, which improves optical accessibility and imaging of cells in the organotypic tissue layers underneath. Finally, we show how ATeam1.03YEMK can be used to perform FRET-based imaging of changes in cellular ATP levels in this preparation. This method hosts the major advantages that it does not require surgical brain procedures, provides high levels of expression of the sensor and cell type specificity in cultured brain slices, reducing invasiveness or stress in the cells as compared with other methods, like transfection by electroporation or transduction with other viral vectors10,16,17. In addition, this protocol can be applied to other FRET based nanosensors, among them variants of ATeam1.03 that provide lower binding affinity for ATP14.

<table><tbody><tr><td>2-deoxyglucose</td><td>Alfa Aesar</td><td>L07338</td><td>Non-metabolizable glucose analog</td></tr><tr><td>36-IMA-410-019 Argon laser</td><td>Melles Griot</td><td></td><td>488 nm wavelength argon</td></tr><tr><td>Ascorbic acid</td><td>Carl Roth</td><td>3525.1</td><td>Antioxidant, Vitamin C</td></tr><tr><td>band pass filters 483/32</td><td>AHF Analysentechnik AG</td><td></td><td>Splitter compatible emmision filter</td></tr><tr><td>band pass filters 542/27</td><td>AHF Analysentechnik AG</td><td></td><td>Splitter compatible emmision filter</td></tr><tr><td>Beamsplitter T 455 LP</td><td>AHF Analysentechnik AG</td><td></td><td>Excitation dichroic mirror</td></tr><tr><td>Beamsplitter T 505 LPXR</td><td>AHF Analysentechnik AG</td><td></td><td>Splitter dichroic</td></tr><tr><td>Confocal laser scannig microscope C1</td><td>Nikon Microscope Solutions</td><td></td><td>Modular confocal microscope system C1</td></tr><tr><td>Data processing Origin Pro 9.0.0 (64-bit)</td><td>OriginLab corporation</td><td></td><td>Scientific graphing and data analysis software</td></tr><tr><td>D-glucose monohydrate</td><td>Caelo</td><td>2580-1kg</td><td></td></tr><tr><td>DPBS</td><td>GIBCO/Life</td><td>14190250</td><td>Dulbecco&#039;s phosphate-buffered saline</td></tr><tr><td>Eclipse E 600FN upright microscope</td><td>Nikon Microscope Solutions</td><td></td><td></td></tr><tr><td>Eclipse FN1 upright microscope</td><td>Nikon Microscope Solutions</td><td></td><td></td></tr><tr><td>Experimental chamber</td><td>custom build</td><td></td><td>Perfusion chamber for live-cell imaging</td></tr><tr><td>EZ-C1 Silver Version 3.91</td><td>Nikon Microscope Solutions</td><td></td><td>Imaging software for confocal microscope</td></tr><tr><td>Hanks&#039; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H9394</td><td>With Phenol Red for pH monitoring</td></tr><tr><td>HERAcell 150</td><td>Thermo Scientific</td><td></td><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator HERAcell &amp;reg; 150 with decontamination routine</td></tr><tr><td>HERAsafe KS/KSP</td><td>Thermo Scientific</td><td></td><td>Safety Cabinet</td></tr><tr><td>Horse serum</td><td>GIBCO/Life</td><td>26050088</td><td>Heat inactivated</td></tr><tr><td>Huygens Professional</td><td>SVI Imaging</td><td></td><td>Deconvolution software</td></tr><tr><td>Image J 1.52i</td><td>Wayne Rasban national Institute of Health</td><td></td><td>Image processing Software available in the public domain</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>Insulin from bovine pancreas</td></tr><tr><td>IP serie peristaltic pump</td><td>Ismatec</td><td></td><td>High-precisionmulti-channel pump</td></tr><tr><td>Layout software, Illustrator CS6</td><td>Adobe</td><td></td><td>Vector graphics editor</td></tr><tr><td>L-glutamine</td><td>GIBCO/Life</td><td>25030024</td><td></td></tr><tr><td>Microm HM 650 V</td><td>Thermo Scientific</td><td></td><td>Vibration microtome. Thermo scientific discontinued the production of the device in the meantime. Any other slicer or tissue chopper siutable for slicing living tissue is fine, too.</td></tr><tr><td>Microscope stage</td><td>custom build</td><td></td><td></td></tr><tr><td>Microsoft Excel 16</td><td>Microsoft</td><td></td><td>Spreadsheet software for basic data processing</td></tr><tr><td>Millicell culture insert</td><td>Merck Millipore</td><td>PICM0RG50</td><td>Hydrophilized PTFE, pore size 0.4 &amp;mu;m</td></tr><tr><td>Minimum Essential Medium Eagle</td><td>Sigma-Aldrich</td><td>M7278</td><td>Synthetic cell culture media</td></tr><tr><td>Monochromator Polychrome V</td><td>Thermo Scientific/FEI</td><td></td><td>Ultra fast switching monochromator</td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt; (Sodium Azide)</td><td>Sigma-Aldrich</td><td>S-8032</td><td>Mitochondrial inhibitor (complex IV inhibitor). CAUTION: Azide is toxic. Be aware not to accidentally ingest or inhale it, and prevent ist absoption through the skin.</td></tr><tr><td>Nikon Fluor 40x / 0.80 W DIC M &amp;infin;/0 WD 2.0</td><td>Nikon Microscope Solutions</td><td></td><td>Water Immersion Microscope Objective</td></tr><tr><td>NIS Elements 4.50 advanced Research</td><td>Nikon Microscope Solutions</td><td></td><td>Imaging software. Upgraded version for FRET imaging</td></tr><tr><td>ORCA-Flash4.0</td><td>Hamamatsu Photonics</td><td></td><td>Digital CMOS camera</td></tr><tr><td>Perfusion tubing</td><td>Pro Liquid GmbH</td><td></td><td>Tygon tubing, 1.52 x 322 mm (Wd: 0.85)</td></tr><tr><td>Photoshop CS 6 Version 13.0</td><td>Adobe</td><td></td><td>Image processing software</td></tr><tr><td>Sodium L-lactate</td><td>Sigma-Aldrich</td><td>71718-10G</td><td></td></tr><tr><td>ssAAV-2/2-hSyn1-ATeam1.03YEMK-WPRE-hGHp(A)</td><td>ETH Z&amp;uuml;rich</td><td>v244</td><td>Single-stranded AAV vector that induces the expression of ATeam1.03&lt;sup&gt;YEMK&lt;/sup&gt; under the control of the human synapsin 1 promoter fragment hSyn1.</td></tr><tr><td>ssAAV-5/2-hGFAP-hHBbI/E-ATeam1.03YEMK-WPRE-bGHp(A)</td><td>ETH Z&amp;uuml;rich</td><td>v307</td><td>Single-stranded AAV vector that induces the expression of ATeam1.03&lt;sup&gt;YEMK&lt;/sup&gt; under the control of the human glial fibrillary acidic protein promoter fragment ABC1D.</td></tr><tr><td>WVIEW GEMINI optic system</td><td>Hamamatsu Photonics</td><td></td><td>Emission Image Splitter</td></tr></tbody></table>

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).

AAV vectors are a reliable tool to selectively express foreign genes in cells within living tissue16. Direct application of AAVs containing the sequence cassette of ATeam1.03YEMK and a specific promoter results in a high expression of the sensor in the chosen cell type. At DIV 14 (~10 days after a transduction), neurons expressing ATeam under the human synapsin promoter are found at high density in the neocortex of cultured tissue slices at depths of up to 50 &#181;m below the slice surface (Figure 5A). Comparable results can be achieved in the hippocampus (Figure 5B).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60294/60294fig5a.jpg" /> 
Figure 5: Visualization of neurons expressing ATeam1.03YEMK in cultured parasagittal organotypic brain slices. Images on the left correspond to extended focus projections of 43 optical sections (1.05 &#181;m each) of cortical tissue (A) and (B) of 70 optical sections (0.6 &#181;m each) of hippocampal tissue. Images on the right represent the volume view of the same projection. Cells are color-coded according to their depth relative to the slice surface as indicated by the color scale on the right. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60294/60294fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For the measurement of ATP levels in astrocytes, ATeam1.03YEMK is expressed under the control of the human glial fibrillary acidic protein (GFAP) promoter. This results in efficient transduction of cells in both neocortex and hippocampus of cultured tissue slices (Figure 6). Notably, two different morphological phenotypes can be distinguished, depending on the depth relative to the surface of slice preparations. In the first, superficial layer, cells are characterized by thick primary processes that are predominately arranged in parallel to the surface. These cells exhibit strongly overlapping domains, creating a dense meshwork of apparently reactive astrocytes (Figure 6). In deeper layers (30-60 &#181;m from the surface), transduced astrocytes exhibit fine cellular processes that form largely spherical domains and their morphology resembles that of astrocytes in situ as reported earlier19,20,21 (Figure 6). To obtain better transduction of deeper-layer astrocytes as well as better optical access to these deeper layers, the glial scar tissue can be removed as described in Step 4.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60294/60294fig6a.jpg" /> 
Figure 6: Visualization of astrocytes expressing ATeam1.03YEMK in cultured parasagittal organotypic brain slices. The image on the left corresponds to an extended focus projection of 191 optical sections (0.45 &#181;m each). For illustration purposes, the glial scar was excluded from the projection of astrocytes. Images on the right represent the volume view of the same projection before and after removal of the glial scar. Cells are color-coded according to their depth relative to the slice surface as indicated by the color scales on the right. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60294/60294fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Successful expression of ATeam1.03YEMK allows the dynamic measurement of changes in ATP levels in neurons or astrocytes, depending on the promoter used (see above). Experiments were performed in an experimental bath constantly perfused with E-ACSF (bubbled with 95% O2/5% CO2). In organotypic slices expressing ATeam1.03YEMK in hippocampal neurons, regions of interest (ROIs) were selected before starting the recording, representing the somata of pyramidal cells (Figure 7A). Moreover, a region for background subtraction was chosen (Figure 7A). Emission of Venus as well as eCFP fluorescence was then collected for each of these ROIs separately and depicted as fluorescence emission level over time (Figure 7B). After recording fluorescence under control conditions for several minutes to ensure a stable baseline, cellular metabolism was inhibited by exposing the slice preparation to a glucose-free saline, to which 5 mM sodium azide (NaN3) was added for one minute (Figure 7B). This manipulation induced opposite changes in the emission intensity of the FRET pair (Figure 7B, left panels), with a decrease of Venus (527 nm) and an increase of eCFP (475 nm) emission. Calculating the FRET ratio by dividing the fluorescence emission of Venus by that of eCFP (FVenus/FeCFP) resulted in signals that reflect the relative changes in the intracellular ATP levels, the so-called &#34;ATeam FRET ratio&#34; (Figure 7B, right panel). In all recorded neurons (n = 70 cells in N = 5 slices), NaN3 caused a reversible decrease in the ATeam FRET ratio, indicating a reversible decrease in intracellular ATP levels upon inhibition of cellular metabolism.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60294/60294fig7a.jpg" /> 
Figure 7: Demonstration of time lapse ATeam FRET ratio imaging. (A) Top left: Wide-field fluorescence image of the pyramidal layer and stratum radiatum of the CA1 region of a cultured organotypic hippocampal slice expressing ATeam1.03YEMK in neurons. Top right: Enlarged view of boxed section as indicated on the left. White lines delineate regions of interest (ROIs) 1-3 representing cell bodies of CA1 pyramidal neurons chosen for analysis in (B). BG represents the ROI chosen for background correction. Bottom: Pseudo-colored images representing fluorescence emission of Venus (green), eCFP (purple) and the ratio of Venus/eCFP. (B) Time lapse recording in ROIs 1-3, representing neuronal cell bodies (see A). Traces on the left show normalized fluorescence emission of Venus (green) and eCFP (magenta). Traces on the right show the corresponding ATeam FRET ratio. Note that perfusion with 5 mM NaN3 in the absence of extracellular glucose for 1 minute (grey bar) induces a reversible decrease in the ATeam FRET ratio, indicating a decrease in intracellular ATP concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60294/60294fig7alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To ensure the stability of the preparation and the sensor under long-term experiment conditions, slices expressing ATeam in neurons or astrocytes were constantly perfused with ACSF for prolonged periods (&#62;50 min; n = 12 cells each, N = 3 OTCs from 3 brains). Under these conditions, the ATeam FRET ratio did not change (Figure 8). Exposing the preparations to CIS containing metabolic inhibitors (&#34;Chemical ischemia&#34;, see Table 1), in contrast, again resulted in the expected drop in the ATeam FRET ratio as observed above.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60294/60294fig8a.jpg" /> 
Figure 8: Baseline experiments employing ATeam. Long-term ATeam FRET ratio imaging in 14 different cells under baseline conditions in neurons (top) and astrocytes (bottom). Data were taken under comparable conditions as other experimental data. At the end of each measurement, chemical ischemia was elicited by perfusion with CIS as indicated by the arrow. NOTE: Baseline ATeam FRET ratios are stable over time under baseline conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60294/60294fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, we analyzed the responses of neurons and astrocytes expressing ATeam1.03YEMK to an increase in the extracellular potassium concentration. After establishing a stable baseline, neurons were perfused with a saline in which the potassium concentration was increased from 3 to 8 mM for 3 minutes (Figure 9A). This manipulation did, however, not result in a detectable change in the ATeam FRET ratio (n = 56 cells in N = 5 slices). To ensure that the sensor reacted to a change in ATP levels, slices were then again exposed to a sustained period of chemical ischemia elicited by replacing E-ACSF by CIS. Chemical ischemia resulted in a rapid decrease in the ATeam FRET ratio to a new, stable level, indicating nominal depletion of ATP after 2-3 min (Figure 9A).
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60294/60294fig9a.jpg" /> 
Figure 9: Representative experiments illustrating changes in ATP levels in neurons and astrocytes. (A,B): Images on the left show ATeam fluorescence from neurons and astrocytes located in the hippocampal CA1 region of organotypic slices. Traces on the right represent time lapse recordings of the ATeam FRET ratio obtained from a ROI positioned over a single cell body. In both experiments, slices were first subjected to an increase in the extracellular potassium concentration for 3 minutes (see bar), followed by a final exposure to chemical ischemia. Note that while neurons do not respond to the elevation of extracellular potassium (A), astrocytes react with an increase in ATP (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60294/60294fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The same experimental protocol was performed with slices, in which ATeam1.03YEMK was expressed in astrocytes. In contrast to what was observed in neurons, astrocytes reacted to the increase in extracellular potassium by a reversible increase in the ATeam FRET ratio, indicating an increase in intracellular ATP levels (n = 70 cells in N = 5 slices) (Figure 9B). Subsequent exposure to chemical ischemia resulted, as expected, in a large drop in the ATeam FRET ratio, indicative of nominal depletion of intracellular ATP (Figure 9B).

Watch this Scientific Journal Video about Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK at JoVE.com

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.

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 ...

imaging-intracellular-atp-organotypic-tissue-slices-mouse-brain-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.6K Views.  Heinrich Heine University Düsseldorf. Hier laten we zien hoe we de ATP-gevoelige FRET-sensor ATeam 1.03 YEMK kunnen gebruiken voor dynamische metingen van veranderingen in neuronale en astrocytische ATP-niveaus in organotypisch gekweekte muis hippocampal plakjes. Het belangrijkste voordeel van deze techniek is dat men energiemetabolisme in levend hersenweefsel in een gecontroleerde omgeving kan bestuderen, een omgeving met hoge sensorexpressieniveaus. Deze techniek kan helpen bij het verkrijgen van inzichten in pathomechanisms, o...

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Bioengineering

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.

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

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biology

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

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.

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 ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

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 &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Imaging Intracellular ATP Dynamics Using FRET-Based Sensors in an Organotypic Mouse Hippocampal Slice

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/60294/imaging-intracellular-atp-organotypic-tissue-slices-mouse-brain-using'>Source: Lerchundi, R., et. al. Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK. J. Vis. Exp. (2019)</a>This video demonstrates the use of FRET(Fluorescence Resonance Energy Transfer)-based sensors to measure intracellular ATP levels in organotypic mouse hippocampal slices, enabling real-time visualization of ATP dynamics in neurons and astrocytes under physiological and experimental conditions.

Source: Lerchundi, R., et. al. Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK. J. ...

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

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