We thank Ms. Tess J. Lameyer, Ms. Ruth Frank, and Dr. Michael A. Muniak for edits and comments, and Dr. Ryohei Yasuda at Max Planck Florida for 2pFLIM acquisition software. This work was supported by two BRAIN Initiative awards U01NS094247 (H.Z. and T.M.) and R01NS104944 (H.Z. and T.M.), an R01 grant R01NS081071 (T.M.), and an R21 grant R21NS097856 (H.Z.). All awards are from the National Institute of Neurological Disorders and Stroke, United States.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon Health and Science University.
1. 2pFLIM Microscope Setup
<ol>
	<li>Install a photon timing counting module (PTCM, Table of Materials) and connect to the computer (Figure 1) according to the manufacturer&#8217;s manual. 
	NOTE: The PTCM is typically a computer board that receives a &#8220;sync&#8221; input for the laser pulse timing and a photon input from the photomultiplier tube (PMT). It also receives clock timing for pixels, lines, and frames, from two-photon imaging control software. The PTCM uses the clock signals to separate individual photons into different pixels and frames.</li>
	<li>Add a photodiode with &#62;200 MHz bandwidth to measure the laser timing. Place a standard glass coverslip in the light path to reflect a small fraction of the laser light into the photodiode placed perpendicular to the light path (Figure 1). Connect the photodiode output to the &#8220;sync&#8221; input of the PTCM. 
	NOTE: Many modern lasers also output the laser timing. For these lasers, the photodiode is not necessary, and one can directly connect the laser timing output to the sync input of the PTCM.</li>
	<li>Exchange the PMT, in case of the tAKAR&#945;, the green channel PMT, with a low-noise, fast GaAsP PMT (Figure 1). Connect the PMT output to the signal input of the PTCM.
	<ol>
		<li>Add an optional signal splitter (Figure 1) if simultaneous acquisition of intensity through the conventional two-photon imaging channel is desired. Connect the PMT output to the signal splitter and connect the splitter output to the PTCM and the conventional two-photon imaging module. 
		NOTE: GaAsP PMTs give faster single-photon signals than conventional bialkali PMTs and allow for a more precise determination of the photon timing. Certain models of GaAsP PMTs can be cooled to 10&#8722;35 &#176;C below ambient temperature, allowing the suppression of dark counts to a level below a few hundred per second (typically &#8804;200 counts/s). This low noise level is important for the precise measurement of fluorescence lifetimes because noise photon counts cannot be easily distinguished or subtracted from the fluorescence lifetime curve.</li>
	</ol>
	</li>
	<li>Add a band-pass fluorescence emission filter that minimizes the spectral contamination, if any, from the acceptor fluorophore. For example, for tAKAR&#945;, a 500 nm &#177; 20 nm barrier filter for the green channel is used to reduce the contamination from the acceptor sREACh, which is a dark (QY ~0.07) yellow fluorescent protein (YFP)29,30. Connect timing signals, such as the clocks for individual image pixels, lines, and frames, as appropriate to the control software and described in the PTCM user manual. Install the appropriate data control and acquisition software. 
	NOTE: Some PTCM manufacturers (Table of Materials) provide their software for 2pFLIM imaging. Here, custom software called FLIMimage is used, which was developed by the Yasuda Lab (Max Planck Florida, via personal communication). This software functions as an add-on user function to certain two-photon acquisition software (Table of Materials). It controls and communicates with the PTCM at the appropriate timing during two-photon imaging to acquire 2pFLIM images.</li>
</ol>
2. Construction of a Motorized Treadmill
NOTE: The design of the custom-built motorized treadmill is shown in Figure 2.
<ol>
	<li>Cut a foam roller (&#216; = 200 mm) to 150 mm in length with a fine hacksaw. Alternatively, glue the two halves of a foam ball together and place tape over the seam. Optionally, glue a rubber mat with profile on the roller to increase traction on the roller.</li>
	<li>Drill a &#188; inch diameter hole through the center of the roller at the flat side of the roller or drill a &#188; inch diameter hole through the middle of each half of the ball if the foam ball is used.</li>
	<li>Install a &#188; inch diameter steel axle through the hole. Glue the foam roller/ball to the axle using foam-compatible glue. Optionally, modify two flexible shaft couplings (&#188; inch inner diameter) to strengthen the coupling of the axle to the foam roller/ball. 
	NOTE: Please note that many common glues may dissolve foam.
	<ol>
		<li>For each shaft coupling, position the shaft coupling on its flat side and at the center of the rectangle metal plate (0.7 mm x 15 mm x 76 mm). Weld the plate to the shaft coupling. Drill a &#188; inch hole at the center of the plate to allow for installation of the modified shaft coupling on to the axle and two screw holes into the metal plate lateral from the center.</li>
		<li>Install the shaft coupling onto the axle against the foam roller/ball. If using the latter, slightly bend the plate to fit the curvature of the ball. Place screws into the lateral holes to fix the roller/ball on the axle.</li>
	</ol>
	</li>
	<li>Drill and tap a 3/8-32 thread at the center of a cage plate and install the rotary encoder. Drill a screw hole into the base of the right-angle motor bracket to allow attachment of the motor onto the post holder. Attach both the rotary encoder and the motor to the end of the axle using a flexible shaft coupling.</li>
	<li>Install the assembled treadmill on an aluminum bread board using posts for the motor and rotary encoder&#160;(Figure 2). Connect the motor inputs to the speed controller, and the rotary encoder&#160;output to an analog input of the computer data acquisition (DAQ) board. 
	NOTE: The rotation angular speed is encoded by the rotary encoder as voltage and is digitized using custom software called AnimalTracker written in MATLAB.</li>
	<li>Install the headplate-compatible holder to a right-angle&#160;bracket. Install a solid post on the bread plate in front of the treadmill and install the assembled headplate holder with the right-angle bracket on the post (Figure 2). Ensure that the headplate holder bars are aligned with the axle such that the mouse can adopt an adequate and comfortable walking position on the treadmill (Figure 2C).</li>
</ol>
3. Expression of tAKAR&#945; Sensor in the Mouse Cortex
<ol>
	<li>In utero electroporation
	<ol>
		<li>Prepare a DNA solution for IUE by adding 0.2% final concentration of fast green dye (for visualization during injection) to a plasmid DNA (3&#8722;4 &#181;g/&#181;L; the sensor constructs containing a CAG promotor, sensor sequence, and a woodchuck hepatitis virus post-transcriptional response element [WPRE] translational enhancer) dissolved/diluted in water or Tris-EDTA.</li>
		<li>Prepare a timed pregnant female mouse (e.g., C57BL/6) for IUE at E1624. Anesthetize the mouse with isoflurane (4% for induction and 1.5% for maintenance, on 95% O2 with 5% CO2) and apply subcutaneous injection of peri-operative analgesics containing 5 mg/kg Meloxicam and 4 mg/kg Bupivacaine. Cut open the abdominal cavity with a scalpel and a pair of scissors and carefully expose the uterine horns.</li>
		<li>Inject 1 &#181;L of DNA solution per embryo in the lateral ventricle of one hemisphere, as previously described24.</li>
		<li>Perform regular IUE24 for cortical neurons by placing the positive electrode end foot at the cortex and using five 100-ms square pulses (38 V) at 1 Hz with an electroporator. 
		NOTE: Different cortical regions can be targeted for electroporation by changing the placement of the electrode end foot relative to the lateral ventricle.</li>
	</ol>
	</li>
	<li>Stereotaxic injection 
	<ol>
		<li>Prepare a mouse at postnatal day 30 for stereotaxic surgery26.&#160;Anesthetize the mouse as described in step 3.1.2., and apply subcutaneous injection of peri-operative analgesics containing 5 mg/kg Carprofen.</li>
		<li>Dilute AAV serotype 2/1 (AAV2/1) expressing hSyn-tAKAR&#945;-WPRE to an empirically determined titer (~1 x 109&#8722;1 x 1010 genomes/&#181;L) in syringe-filtered (0.2 &#181;m cellulose acetate membrane) phosphate-buffered saline.</li>
		<li>Drill a ~500 &#956;m diameter hole using a handheld drill under a stereomicroscope at the following coordinates for the motor cortex: 0.5 mm anterior to bregma, 1.2 mm lateral to midline.</li>
		<li>Mount an injector (e.g., oil hydraulic manipulator, with custom-made plunger/glass pipette holder) to a motorized manipulator. Place the injection needle at a 15&#176; angle relative to the bregma-lambda plane. Program a diagonal movement across the x- and z- axes equivalent to a 700 &#181;m and 200 &#181;m progression along the anterior-posterior and dorsal-ventral axes, respectively. 
		&#8203;NOTE: To avoid damage to the tissue directly above the intended imaging field, AAV particles are injected at an angle relative to the bregma-lambda plane.</li>
		<li>Position the tip of the injection needle at the pia in the center of the drill hole and slowly execute the diagonal movement (~25 &#181;m/s) described above. This procedure will position the center of injection at 1.2 mm anterior to bregma, 1.2 mm lateral to midline, 0.2 mm below the pia.</li>
		<li>Inject 20 nL of diluted viral particles (~10 nL/min). Wait at least 10 min and slowly retract the injection needle (~12.5 &#181;m/s).</li>
		<li>Finish the stereotaxic injection procedure and glue/suture the skin26.</li>
	</ol>
	</li>
</ol>
4. Installation of the Cranial Window
<ol>
	<li>Perform the placement of the cranial window on mice expressing tAKAR&#945; via IUE (section 3.1) or stereotaxic injection of viral particles (section 3.2), between postnatal days 30 and 60. For mouse infected with viral particles, implement the cranial window at least two weeks after the virus injection. Install the cranial window as previously described27,28, with the following details.&#160;Anesthetize the mouse as described in step 3.1.2., and apply subcutaneous injection of peri-operative analgesics 0.075 mg/kg Buprenex and anti-inflammatory agent Dexamethasone at 4 mg/kg.</li>
	<li>Remove the periosteum and retract the neck muscle. Glue the edge of the skin to the skull with tissue adhesive to avoid exposure of the neck musculature after surgery.</li>
	<li>Dry and remove any periosteum from the skull by gently scraping using a scalpel. Place the imaging headplate (8 mm inner diameter) to surround the intended imaging field. Glue the headplate to the skull using cyanoacrylate-based glue, followed by dental acrylic cement. For optimal adhesion, ensure that the headplate rests on the exposed and dried skull. Glue accelerator can be used to accelerate the hardening.</li>
	<li>Draw a circle of 5 mm in diameter above the intended imaging field (coordinates as specified in step 3.2.3) using a dental drill and expose the dura mater.</li>
	<li>Apply a thin layer of transparent polymer, also called artificial dura, to the dura surface to cover the entire cranial window. The polymer will protect and stabilize the dura mater. Place a sterile circular coverslip (5 mm diameter) on the dura mater. Secure the coverslip with cyanoacrylate glue applied around the edges of the window followed by dental acrylic cement.</li>
</ol>
5. In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
<ol>
	<li>Commence 2pFLIM imaging at or beyond 2 weeks post-installation of the cranial window (section 4). Minimize experimental interference due to stress by frequent handling and scruffing of the mouse prior to the start of the imaging study to habituate the mouse.</li>
	<li>Set the two-photon excitation laser wavelength to 960 nm using the software that controls the two-photon laser.</li>
	<li>Anaesthetize the mouse using 4% isoflurane. Confirm proper anesthetization by tail-pinch and observing breathing rates. That is, there should be no response to the tail-pinch and the breathing rate should be reduced to ~1 breath per second.&#160;To minimize unnecessary procedural time and because the anesthesia lasts only for two to three minutes, eye lubricants are not used.</li>
	<li>Transfer the anaesthetized mouse to the motorized treadmill (Figure 2C) and mount the headplate of the mouse to the headplate holder of the treadmill setup (see Figure 2 for details). Clean the surface of the cranial window coverslip on the mouse with 70% ethanol.</li>
	<li>Place the motorized treadmill with the mounted mouse under the 2pFLIM objective. Apply a drop of distilled water between the cranial window coverslip and the objective.</li>
	<li>Let the mounted mouse wake up from anesthesia and become acclimated to the treadmill and microscope environment for at least 10 min.&#160;Monitor respiration rate of the mouse while the mounted mouse wakes up from anesthesia.</li>
	<li>Navigate to the injection location under epi-illumination. Document fiducial features (i.e., blood vessels) under brightfield to aid imaging of the same region of interest (ROI) during subsequent imaging sessions.</li>
	<li>Eliminate any incoming light other than the emitted light from the brain tissue. Switch off the&#160;epi-illumination light source and close the enclosure of the 2pFLIM rig. Activate the 2pFLIM PMT by switching on the hardware command voltage control.</li>
	<li>Acquire a z-stack 2pFLIM image using the 2pFLIM acquisition software FLIMimage with the following recommended settings for imaging tAKAR&#945;-positive somata in awake mice. Set frame averaging to 3 frames, scanning speed to 2 ms/line, image size to 128 x 128 pixels, and field of view to 90&#8722;100 &#956;m. Adjust imaging settings based on the preparation and hardware configuration.</li>
	<li>Inspect the acquired image in FLIMview (in-house developed custom software; see section 6). Adjust imaging settings following step 5.9 to optimize photon count and minimize photobleaching. 
	NOTE: A workable integrated photon count in an ROI for lifetime imaging of a tAKAR&#945;-positive soma in vivo is ~1,000&#8722;10,000 photons depending on the signal amplitude that results from a particular stimulus (see Discussion).
	<ol>
		<li>Where needed, use a decreased field of view, decreased scanning speed, increased laser power, and increased number of frames to be averaged to increase the integrated photon counts and reduce the lifetime estimation error. At the same time, be sure to use the minimal essential laser power, frame averaging, and scanning speed to minimize photobleaching.</li>
	</ol>
	</li>
	<li>Image at a regular time interval (e.g., every 30&#8722;60 s) by repeating the z-stack acquisition using settings determined in step 5.10. Acquire baseline 2pFLIM images for at least 15 min at zero treadmill speed.</li>
	<li>Set the treadmill rotation speed to ~15 cm/s for 15 min while acquiring 2pFLIM images. Continue imaging for &#8805;20 min after switching off the treadmill rotation, to assess the duration of PKA activity after cessation of forced locomotion.</li>
</ol>
6. Analysis of 2pFLIM Images
<ol>
	<li>Open the acquired images in FLIMview and set the following parameters in the FLIMview. 
	NOTE: Parameter details are described in DISCUSSION.
	<ol>
		<li>Click on the single photon counting (SPC) minimum and maximum range fields in FLIMview. Enter the appropriate minimum and maximum SPC range value, typically ranging between 1.2&#8722;2 and 10&#8722;12 ns, respectively.</li>
		<li>Click on the t0 value field in FLIMview and enter the t0 value (typically ~2 ns). Click on the lifetime luminance minimum threshold value field in FLIMview and enter the desired threshold value to 5&#8722;30 photons.</li>
	</ol>
	</li>
	<li>Click on the new group button (N) and assign an experiment group name. This will generate a group that combines data from each added FLIM image.</li>
	<li>Click on the ROI button in the Roi Controls module of FLIMview and draw an ROI around a tAKAR&#945;-positive soma. Reduce the z-stack range, by moving the lower and upper z-limit in the z-stack Control sliders in FLIMview, to minimize signal contamination originating from background photons in other z depths.</li>
	<li>Click on the + button to add the FLIM image to the group (step 6.2). Click on the Calc button to calculate the mean lifetime (LT, also called mean photon emission time [MPET]), for the ROI and the lifetime estimation error (&#948;&#964;).</li>
	<li>Open the next file in the chronical 2pFLIM imaging series. Repeat step 6.4. Be sure to adjust the position of the ROI and z-stack range to measure the same tAKAR&#945;-positive soma over time, because there can be tissue drift over time.</li>
	<li>Select the deltaMPET/MPET0 in the drop-down menu of the Group Controls module. Click on the baseline# field and enter the index(es) (e.g., 1 2 3 4 5 for the first five images in the group created in step 6.3). This will define the image(s) used to calculate baseline lifetime (LT0).</li>
	<li>Click on Plot to generate a graph containing the FLIM response (&#916;LT/LT0) of tAKAR&#945; during the experiment in the defined ROIs. Normalized changes in lifetime (&#916;LT) of individual ROIs by the corresponding baseline lifetime (LT0) allow for comparison of PKA activity during locomotion across different ROIs.</li>
</ol>

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<li>Baumgart, J., Baumgart, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortex-%2C+Hippocampus-%2C+Thalamus-%2C+Hypothalamus-%2C+Lateral+Septal+Nucleus-+and+Striatum-specific+In+utero+Electroporation+in+the+C57BL%2F6+Mouse%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In utero Electroporation in the C57BL/6 Mouse.</a> Journal of Visualized Experiments. (107), e53303(2016).</li>
<li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracranial+Injection+of+Adeno-associated+Viral+Vectors%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Intracranial Injection of Adeno-associated Viral Vectors.</a> Journal of Visualized Experiments. (45), 1-4 (2010).</li>
<li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> Journal of Visualized Experiments. (12), 18-19 (2008).</li>
<li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nature Protocols. 4 (8), 1128-1144 (2009).</li>
<li>Murakoshi, H., Lee, S. J., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+sensitive+and+quantitative+FRET-FLIM+imaging+in+single+dendritic+spines+using+improved+non-radiative+YFP%5BTitle%5D)+AND+%22Brain+Cell+Biology%22%5BJournal%5D)">Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP.</a> Brain Cell Biology. 36 (1-4), 31-42 (2008).</li>
<li>Murakoshi, H., Shibata, A. C. E., Nakahata, Y., Nabekura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+dark+green+fluorescent+protein+as+an+acceptor+for+measurement+of+F%C3%B6rster+resonance+energy+transfer%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.</a> Scientific Reports. 5, 1-11 (2015).</li>
<li>Guo, Z. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procedures+for+behavioral+experiments+in+head-fixed+mice%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Procedures for behavioral experiments in head-fixed mice.</a> PLoS ONE. 9 (2), (2014).</li>
<li>Yu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+central+amygdala+controls+learning+in+the+lateral+amygdala%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">The central amygdala controls learning in the lateral amygdala.</a> Nature Neuroscience. 20 (12), 1680-1685 (2017).</li>
<li>Harvey, C. D., Collman, F., Dombeck, D. A., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+dynamics+of+hippocampal+place+cells+during+virtual+navigation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Intracellular dynamics of hippocampal place cells during virtual navigation.</a> Nature. 461 (7266), 941-946 (2009).</li>
<li>Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+intracellular+signaling+using+two-photon+fluorescent+lifetime+imaging+microscopy%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Imaging intracellular signaling using two-photon fluorescent lifetime imaging microscopy.</a> Cold Spring Harbor Protocols. 7 (11), 1121-1128 (2012).</li>
<li>Mehta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-fluorophore+biosensors+for+sensitive+and+multiplexed+detection+of+signalling+activities%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities.</a> Nature Cell Biology. 20 (10), 1215-1225 (2018).</li>
</ol>

The authors have nothing to disclose.

This protocol demonstrates the use of FRET-FLIM sensor tAKAR&#945; to visualize neuromodulation-triggered PKA activity in head-fixed behaving mice. This application is based on extensive testing and characterizations of tAKAR&#945; in vitro and in vivo to demonstrate that the FLIM signal obtained is relevant to physiological neuromodulatory events17. Here, one in vivo application, locomotion-induced PKA activity in the motor cortex, is used to describe the procedures for delivering the sensor to t...

Neuromodulation, also known as slow synaptic transmission, imposes strong control over brain function during different behavioral states, such as stress, arousal, attention, and locomotion1,2,3,4. Despite its importance, the study of when and where neuromodulatory events take place is still in its infancy. Neuromodulators, including acetylcholine, dopamine, noradrenaline, serotonin, and many neuropeptides, activate G protein-coupled receptors (GPCRs), which in turn trigger intracellular second messenger pathways with a wide window of timescales ranging from seconds to hours. While each neuromodulator triggers a distinct set of signaling events, the cAMP/protein kinase A (PKA) pathway is a common downstream pathway for many neuromodulators1,5. The cAMP/PKA pathway regulates neuronal excitability, synaptic transmission, and plasticity6,7,8,9, and therefore, tunes the neuronal network dynamics. Because different neurons or neuronal types express different types or levels of neuromodulator receptors10, the intracellular effects of the same extracellular neuromodulator may be heterogeneous across different neurons, and thus, have to be studied with cellular resolution. To date, it remains challenging to monitor neuromodulatory events in individual neurons in vivo during behavior.
To study the spatiotemporal dynamics of neuromodulation, a suitable recording modality is required. Microdialysis and fast-scan cyclic voltammetry are frequently used to study release of neuromodulators, but they lack the spatial resolution to monitor cellular events11,12. Analogous to calcium dynamics being used as a proxy for neuronal electrical activity in population imaging13, PKA imaging may be used to read out neuromodulatory events across a neuronal population at cellular resolution. The present protocol describes the use of an improved A-kinase activity reporter (AKAR) to monitor PKA activities in vivo during animal behavior. The method described here allows for simultaneous imaging of neuronal populations at subcellular resolution with a temporal resolution that tracks physiological neuromodulatory events.
AKARs are composed of a donor and an acceptor fluorescent proteins linked by a PKA phosphorylation substrate peptide and a forkhead-associated (FHA) domain that binds to the phosphorylated serine or threonine of the substrate14,15. Upon activation of the PKA pathway, the substrate peptide of AKAR is phosphorylated. As a result, the FHA domain binds to the phosphorylated substrate peptide, thereby bringing the two fluorophores into close proximity, referred to as the closed state of AKAR. The closed state of a phosphorylated AKAR results in increased F&#246;rster resonance energy transfer (FRET) between the donor and acceptor fluorophores. Since the proportion of phosphorylated AKARs is related to the level of PKA activity16, the amount of FRET in a biological sample can be used to quantify the level of PKA activity16,17,18,19,20.
Early versions of AKARs were primarily designed for two-color ratiometric imaging14. When imaging deeper into brain tissue, the ratiometric method suffers from signal distortion due to wavelength-dependent light scattering17,18,21. As discussed below, fluorescence lifetime imaging microscopy (FLIM) eliminates this problem because FLIM only measures photons emitted by the donor fluorophore18,21. As a result, FLIM quantification of FRET is not affected by the tissue-depth17. In addition, a &#8220;dark&#8221; (i.e., low quantum yield [QY]) variant of the acceptor fluorophore can be used. This frees a color channel to facilitate multiplexed measurement of orthogonal neuronal properties via simultaneous imaging of a second sensor or a morphological marker17,19,20.
FLIM imaging quantifies the time that a fluorophore spends in the excited state, i.e., the fluorescence lifetime18. The return of a fluorophore to the ground state, thus the end of the excited state, often concomitates with the emission of a photon. Although the emission of a photon for an individual excited molecule is stochastic, in a population the mean fluorescence lifetime is a characteristic of that particular fluorophore. When a pure population of fluorophores are excited simultaneously, the resulting fluorescence will follow a single exponential decay. The time constant of this exponential decay corresponds to the mean fluorescence lifetime, which typically ranges from one to four nanoseconds for fluorescent proteins. The return of an excited donor fluorophore to the ground state can also occur by FRET. In the presence of FRET, the fluorescence lifetime of the donor fluorophore is reduced. The unphosphorylated AKARs exhibit a relatively longer donor fluorescence lifetime. Upon phosphorylation by PKA, the sensor exhibits a shorter lifetime because the donor and acceptor fluorophores are brought near each other and FRET is increased. The quantification of the fluorescence lifetime in a population of AKARs therefore represents the level of PKA activity.
Early versions of AKARs have not been successfully used for in vivo imaging at single-cell resolution. This is mainly due to the low signal amplitude of the AKAR sensors to physiological activations17. Recently, by systematically comparing available AKAR sensors for two-photon fluorescence lifetime imaging microscopy (2pFLIM), a sensor called FLIM-AKAR was found to outperform alternative sensors. Furthermore, a series of FLIM-AKAR variants called targeted AKARs (tAKARs) were developed to visualize PKA activity at specific subcellular locations: microtubules (tAKAR&#945;), cytosol (tAKAR&#946;), actin (tAKAR&#948;), filamentous actin (tAKAR&#949;), membrane (tAKAR&#947;), and postsynaptic density (tAKAR&#950;). Among tAKARs, tAKAR&#945; increased the signal amplitude elicited by norepinephrine by 2.7-fold. This is consistent with the knowledge that the majority of PKA in neurons are anchored to microtubules at the resting state22,23. tAKAR&#945; was the best performer among existing AKARs for 2pFLIM. Furthermore, tAKAR&#945; detected physiologically-relevant PKA activity elicited by multiple neuromodulators, and the expression of tAKAR&#945; did not alter neuronal functions17.
Recently, tAKAR&#945; was successfully used to visualize PKA activities in head-fixed behaving mice17. It was shown that enforced locomotion triggered PKA activity in the soma of superficial layer neurons (layer 1 through 3, up to a depth of ~300 &#956;m from pia) in the motor, barrel, and visual cortices. The locomotion-triggered PKA activity was in part dependent on signaling via &#946;-adrenergic receptors and D1 dopamine receptors, but was not affected by a D2 dopamine receptor antagonist. This work illustrates the ability of tAKARs to track neuromodulation events in vivo using 2pFLIM.
In the current protocol, the entire method for PKA activity imaging in head-fixed awake mice during an enforced locomotion paradigm is described in six steps. First, the addition of 2pFLIM capabilities to a conventional two-photon microscope (Figure 1). Second, the construction of a motorized treadmill (Figure 2). Third, the expression of the tAKAR&#945; sensor in the mouse cortex by in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). Excellent protocols for surgeries for IUE24,25 and stereotaxic injection of viral particles26 have been previously published. The key parameters we used are described below. Forth, the installation of a cranial window. Excellent protocols have been previously published for cranial window surgery27,28. Several steps that have been modified from the standard protocols are described. Fifth, performing in vivo 2pFLIM. Sixth, the analyses of 2pFLIM images (Figure 3 and Figure 4). This approach should be readily applicable to many other head-fixed behavioral paradigms and brain areas.

<table><tbody><tr><td>0.2 &amp;mu;m cellulose acetate syringe filter</td><td>Nalgene</td><td>190-2520</td><td>Step 3.2.2.</td></tr><tr><td>16x 0.8 NA water-immersion objective</td><td>Nikon</td><td>MRP07220</td><td>Step 5.5.</td></tr><tr><td>3-pin cable</td><td>US digital</td><td>CA-MIC3-SH-NC</td><td>Step 2.5. To connect rotation sensor to the DAQ input of the microscope</td></tr><tr><td>Aluminum bread board</td><td>Thorlabs</td><td>MB1012</td><td>Step 2.5.</td></tr><tr><td>AnimalTracker MATLAB software</td><td>N/A</td><td>N/A</td><td>Step 2.5 and sections 5 - 6. Will be provided upon request to the lead author</td></tr><tr><td>Band-pass barrier filter</td><td>Chroma</td><td>ET500-40m</td><td>Step 1.4.</td></tr><tr><td>Cage plate</td><td>Thorlabs</td><td>CP01</td><td>Step 2.4. Used as mount for rotation sensor</td></tr><tr><td>Carbon steel burrs for micro drill, 0.5 mm tip diameter</td><td>FST</td><td>19007-05</td><td>Steps 3.2.3. and 4.4.</td></tr><tr><td>Circular coverslip (5 mm diameter)</td><td>VWR</td><td>101413-528</td><td>Step 4.5.</td></tr><tr><td>Custom-made injection needle holder</td><td>N/A</td><td>N/A</td><td>Step 3.2.4. Technical details provided upon request to the lead author</td></tr><tr><td>Dental acrylic</td><td>Yates Motloid</td><td>44114</td><td>Steps 4.3. and 4.5.</td></tr><tr><td>Dental drill; Microtorque ii</td><td>Ram products</td><td>66699</td><td>Steps 3.2.3. and 4.4.</td></tr><tr><td>Dowsil transparent polymer</td><td>The Dow Chemical Company</td><td>3-4680</td><td>Step 4.5. Artificial dura</td></tr><tr><td>Electroporation electrode</td><td>Bex</td><td>LF650P5</td><td>Step 3.1.4.</td></tr><tr><td>Electroporator</td><td>Bex</td><td>CUY21</td><td>Step 3.1.4.</td></tr><tr><td>Fast green FCF</td><td>Sigma-aldrich</td><td>F7258-25G</td><td>Step 3.1.1.</td></tr><tr><td>FLIMimage MATLAB software</td><td>N/A</td><td>N/A</td><td>Section 5. Kindly provided by Dr. Ryohei Yasuda, Max Planck Florida</td></tr><tr><td>FLIMview MATLAB software</td><td>N/A</td><td>N/A</td><td>Sections 5. and 6. Will be provided upon request to the lead author</td></tr><tr><td>Foam-compatible glue (Gorilla White Glue)</td><td>Gorilla</td><td>5201204</td><td>Step 2.3.</td></tr><tr><td>Headplate</td><td>N/A</td><td>N/A</td><td>Step 4.3. Technical details provided upon request to the lead author</td></tr><tr><td>Headplate holder</td><td>N/A</td><td>N/A</td><td>Step 2.6. Technical details provided upon request lead author, used in combination with mounting post bracket and right-angled bracket</td></tr><tr><td>Hydraulic micromanipulator</td><td>Narishige</td><td>MO-10</td><td>Step 3.2.4.</td></tr><tr><td>Krazy glue</td><td>Krazy glue</td><td>KG82648R</td><td>Step 4.3. Cyanoacrylate-based glue</td></tr><tr><td>Low-noise fast photomultiplier tube</td><td>Hamamatsu</td><td>H7422PA-40 or H10769PA-40</td><td>Step 1.3.</td></tr><tr><td>MATLAB 2012b</td><td>Mathworks</td><td>N/A</td><td>Steps 2.6, and sections 5, and 6. Used to run microscope acquisition and data analysis software</td></tr><tr><td>Motor</td><td>Zhengke</td><td>ZGA37RG</td><td>Step 2.4.</td></tr><tr><td>Motor speed controller</td><td>Elenker</td><td>EK-G00015A1-1</td><td>Step 2.5.</td></tr><tr><td>Motorized micromanipulator</td><td>Sutter</td><td>MP-285</td><td>Step 3.2.4.</td></tr><tr><td>Mounting base</td><td>Thorlabs</td><td>BA1S</td><td>Step 2.5. Used for posts for motor and sensor in combination with PH4 and TR2</td></tr><tr><td>Mounting post</td><td>Thorlabs</td><td>P14</td><td>Step 2.6. Used for headplate holder post in combination with PB2</td></tr><tr><td>Mounting post base</td><td>Thorlabs</td><td>PB2</td><td>Step 2.6. Used for headplate holder post in combination with P14</td></tr><tr><td>Mounting post bracket</td><td>Thorlabs</td><td>C1515</td><td>Step 2.6. Used in combination with right-angle bracket and headplate holder</td></tr><tr><td>Optical post</td><td>Thorlabs</td><td>TR2</td><td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and PH4</td></tr><tr><td>Phosphate-buffered saline</td><td>&amp;Nu;/&amp;Alpha;</td><td>&amp;Nu;/&amp;Alpha;</td><td>Step 3.2.2. Protocol: Cold Spring Harbor Protocols 2006, doi: 10.1101/pbd.rec8247</td></tr><tr><td>Photodiode</td><td>Thorlabs</td><td>FDS010</td><td>Step 1.2.</td></tr><tr><td>Photon timing counting module</td><td>Becker and Hickl</td><td>SPC-150</td><td>Step 1.1.</td></tr><tr><td>Plasmid: tAKAR&amp;alpha; (CAG-tAKAR&amp;alpha;-WPRE)</td><td>Addgene</td><td>119913</td><td>Step 3.1.3.</td></tr><tr><td>Post holder</td><td>Thorlabs</td><td>PH4</td><td>Step 2.5. Used for posts for motor and sensor in combination with BA1S and TR2</td></tr><tr><td>Right-angle bracket</td><td>Thorlabs</td><td>AB90</td><td>Step 2.6 Used in combination with mounting post bracket and headplate holder</td></tr><tr><td>Rotation encoder</td><td>US digital</td><td>MA3-A10-250-N</td><td>Step 2.4.</td></tr><tr><td>Rubber mat</td><td>Rubber-Cal</td><td>B01DCR5LUG</td><td>Step 2.1.</td></tr><tr><td>Shaft coupling (1/4 inch x 1/4 inch)</td><td>McMaster</td><td>6208K433</td><td>Steps 2.3. and 2.4.</td></tr><tr><td>ScanImage 3.6</td><td>Svoboda Lab/Vidrio Technology</td><td>N/A</td><td>Steps 5.9. and 6.1.</td></tr><tr><td>Signal splitter</td><td>Becker and Hickl</td><td>HPM-CON-02</td><td>Step 1.3.1.</td></tr><tr><td>Stainless steel axle (diameter 1/4 inch, L = 12 inch)</td><td>McMaster</td><td>1327K66</td><td>Step 2.3.</td></tr><tr><td>Stereotaxic alignment systsem</td><td>David kopf</td><td>1900</td><td>Steps 3.2. and 4.1. modified; Sutter micromanipulator, custom-made injection needle holder, hydraulic micromanipulator</td></tr><tr><td>Two-photon microscope</td><td>N/A</td><td>N/A</td><td>Section 5. Built based on Modular in vivo multiphoton microscopy system (MIMMS) from HHMI Janelia Research Campus (https://www.janelia.org/open-science/mimms)</td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>14006</td><td>Step 3.2.6.</td></tr><tr><td>Virus: tAKAR&amp;alpha; (AAV2/1 hSyn-tAKAR&amp;alpha;-WPRE)</td><td>Addgene</td><td>119921</td><td>Step 3.2.2.</td></tr><tr><td>White PE foam roller (8 inch x 12 inch)</td><td>Fabrication enterprises INC.</td><td>30-2261</td><td>Step 2.1.1.</td></tr><tr><td>White polystyrene fom ball halves</td><td>GrahamSweet</td><td>200mm diameter 2 hollow halves</td><td>Step 2.1.1.</td></tr><tr><td>Zipkicker</td><td>PACER</td><td>PT29</td><td>Step 4.3. Hardening accelerator</td></tr></tbody></table>

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.

FRET-FLIM sensors allow for the visualization of many different signaling pathways, including the cAMP/PKA pathway involved in neuromodulation. The current protocol utilizes the recently-developed tAKAR&#945; sensor in combination with 2pFLIM to visualize PKA activities in head-fixed behaving mice. Most existing two-photon microscopes can be upgraded with 2pFLIM capabilities by adding three to four components, as illustrated in Figure 1 (see also section 1). ...

Watch this Scientific Journal Video about Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy at JoVE.com

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

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-protein-kinase-activity-head-fixed-behaving-mice-using

Research

JoVE Journal

Neuroscience

8.5K Views.  Oregon Health & Science University. A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKARα, 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.

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

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

Behavior

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

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 ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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

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

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

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

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

Summary

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In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

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

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse