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

Summary

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.

Abstract

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 tAKARα, is used, which is based on Förster 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, tAKARα 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.

Introduction

Neuromodulation, also known as slow synaptic transmission, imposes strong control over brain function during different behavioral states, such as stress, arousal, attention, and locomotion^1,2,3,4. 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 neuromodulators^1,5. The cAMP/PKA pathway regulates neuronal excitability, synaptic transmission, and plasticity^6,7,8,9, 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 events^11,12. 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 substrate^14,15. 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 Förster 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 activity^{16,17,18,19,20}.

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 scattering^17,18,21. As discussed below, fluorescence lifetime imaging microscopy (FLIM) eliminates this problem because FLIM only measures photons emitted by the donor fluorophore^18,21. As a result, FLIM quantification of FRET is not affected by the tissue-depth¹⁷. In addition, a “dark” (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 marker^17,19,20.

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 (tAKARα), cytosol (tAKARβ), actin (tAKARδ), filamentous actin (tAKARε), membrane (tAKARγ), and postsynaptic density (tAKARζ). Among tAKARs, tAKARα 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 state^22,23. tAKARα was the best performer among existing AKARs for 2pFLIM. Furthermore, tAKARα detected physiologically-relevant PKA activity elicited by multiple neuromodulators, and the expression of tAKARα did not alter neuronal functions¹⁷.

Recently, tAKARα 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 μm from pia) in the motor, barrel, and visual cortices. The locomotion-triggered PKA activity was in part dependent on signaling via β-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 tAKARα 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 IUE^24,25 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 surgery^27,28. 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.

Protocol

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

1. Install a photon timing counting module (PTCM, Table of Materials) and connect to the computer (Figure 1) according to the manufacturer’s manual.
   NOTE: The PTCM is typically a computer board that receives a “sync” 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.
2. Add a photodiode with >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 “sync” 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.
3. Exchange the PMT, in case of the tAKARα, the green channel PMT, with a low-noise, fast GaAsP PMT (Figure 1). Connect the PMT output to the signal input of the PTCM.
   1. 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 10−35 °C below ambient temperature, allowing the suppression of dark counts to a level below a few hundred per second (typically ≤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.
4. Add a band-pass fluorescence emission filter that minimizes the spectral contamination, if any, from the acceptor fluorophore. For example, for tAKARα, a 500 nm ± 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.

2. Construction of a Motorized Treadmill

NOTE: The design of the custom-built motorized treadmill is shown in Figure 2.

1. Cut a foam roller (Ø = 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.
2. Drill a ¼ inch diameter hole through the center of the roller at the flat side of the roller or drill a ¼ inch diameter hole through the middle of each half of the ball if the foam ball is used.
3. Install a ¼ 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 (¼ 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.
   1. 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 ¼ 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.
   2. 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.
4. 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.
5. Install the assembled treadmill on an aluminum bread board using posts for the motor and rotary encoder (Figure 2). Connect the motor inputs to the speed controller, and the rotary encoder 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.
6. Install the headplate-compatible holder to a right-angle 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).

3. Expression of tAKARα Sensor in the Mouse Cortex

1. In utero electroporation
   1. Prepare a DNA solution for IUE by adding 0.2% final concentration of fast green dye (for visualization during injection) to a plasmid DNA (3−4 µg/µL; 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.
   2. 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.
   3. Inject 1 µL of DNA solution per embryo in the lateral ventricle of one hemisphere, as previously described²⁴.
   4. 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.
2. Stereotaxic injection
   1. Prepare a mouse at postnatal day 30 for stereotaxic surgery²⁶. Anesthetize the mouse as described in step 3.1.2., and apply subcutaneous injection of peri-operative analgesics containing 5 mg/kg Carprofen.
   2. Dilute AAV serotype 2/1 (AAV2/1) expressing hSyn-tAKARα-WPRE to an empirically determined titer (~1 x 10⁹−1 x 10¹⁰ genomes/µL) in syringe-filtered (0.2 µm cellulose acetate membrane) phosphate-buffered saline.
   3. Drill a ~500 μm 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.
   4. 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° angle relative to the bregma-lambda plane. Program a diagonal movement across the x- and z- axes equivalent to a 700 µm and 200 µm progression along the anterior-posterior and dorsal-ventral axes, respectively.
      ​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.
   5. Position the tip of the injection needle at the pia in the center of the drill hole and slowly execute the diagonal movement (~25 µm/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.
   6. Inject 20 nL of diluted viral particles (~10 nL/min). Wait at least 10 min and slowly retract the injection needle (~12.5 µm/s).
   7. Finish the stereotaxic injection procedure and glue/suture the skin²⁶.

4. Installation of the Cranial Window

1. Perform the placement of the cranial window on mice expressing tAKARα 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 described^27,28, with the following details. 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.
2. 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.
3. 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.
4. 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.
5. 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.

5. In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

1. 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.
2. Set the two-photon excitation laser wavelength to 960 nm using the software that controls the two-photon laser.
3. 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. To minimize unnecessary procedural time and because the anesthesia lasts only for two to three minutes, eye lubricants are not used.
4. 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.
5. 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.
6. Let the mounted mouse wake up from anesthesia and become acclimated to the treadmill and microscope environment for at least 10 min. Monitor respiration rate of the mouse while the mounted mouse wakes up from anesthesia.
7. 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.
8. Eliminate any incoming light other than the emitted light from the brain tissue. Switch off the epi-illumination light source and close the enclosure of the 2pFLIM rig. Activate the 2pFLIM PMT by switching on the hardware command voltage control.
9. Acquire a z-stack 2pFLIM image using the 2pFLIM acquisition software FLIMimage with the following recommended settings for imaging tAKARα-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 90−100 μm. Adjust imaging settings based on the preparation and hardware configuration.
10. 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 tAKARα-positive soma in vivo is ~1,000−10,000 photons depending on the signal amplitude that results from a particular stimulus (see Discussion).
    1. 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.
11. Image at a regular time interval (e.g., every 30−60 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.
12. Set the treadmill rotation speed to ~15 cm/s for 15 min while acquiring 2pFLIM images. Continue imaging for ≥20 min after switching off the treadmill rotation, to assess the duration of PKA activity after cessation of forced locomotion.

6. Analysis of 2pFLIM Images

1. Open the acquired images in FLIMview and set the following parameters in the FLIMview.
   NOTE: Parameter details are described in DISCUSSION.
   1. 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 1.2−2 and 10−12 ns, respectively.
   2. 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 5−30 photons.
2. 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.
3. Click on the ROI button in the Roi Controls module of FLIMview and draw an ROI around a tAKARα-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.
4. 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 (δτ).
5. 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 tAKARα-positive soma over time, because there can be tissue drift over time.
6. 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).
7. Click on Plot to generate a graph containing the FLIM response (ΔLT/LT₀) of tAKARα during the experiment in the defined ROIs. Normalized changes in lifetime (ΔLT) of individual ROIs by the corresponding baseline lifetime (LT₀) allow for comparison of PKA activity during locomotion across different ROIs.

Results

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 tAKARα 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). To vi...

Discussion

This protocol demonstrates the use of FRET-FLIM sensor tAKARα to visualize neuromodulation-triggered PKA activity in head-fixed behaving mice. This application is based on extensive testing and characterizations of tAKARα 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 the brain, ...

Materials

List of materials used in this article

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