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 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.
There is growing evidence that the internal state of the body constantly influences the brain functions in animals1,2,3,4,5. Accordingly, to gain a deeper understanding of brain functions, it is crucial to elucidate how glial cells in the brain monitor biological conditions in the periphery and relay the information to neurons.
Microglia, immune cells in the brain, are involved in synaptic development and plasticity, which sculpt characteristics of neural circuits in the brain6,7,8,9. For instance, the pioneering work by Wake et al. demonstrated that microglial processes make contact with synapses in a neuronal activity-dependent manner in the mouse neocortex and that artificial ischemia induces synapse loss following prolonged contact with microglia10. Tremblay et al. found that alteration of visual experience changes the modality of microglial interaction with synapses. During the critical period when dendritic spine turnover in the primary visual cortex (V1) is increased by binocular deprivation, dark adaptation reduces the motility of microglial processes and increases both their contact frequency with synaptic clefts and the number of cellular inclusions in microglia11. These results suggest that microglial processes sense neural activity and their surrounding environment to remodel neural circuits. Furthermore, a recent study reported that microglial surveillance differs between awake and anesthetized conditions, suggesting the importance of experiments using awake mice for investigating neuron-microglia communication under physiological conditions12.
In vivo two-photon calcium imaging is a powerful tool for the recording of calcium dynamics, reflecting ongoing neuronal firing, in hundreds of neurons simultaneously in a living animal13,14,15. Calcium imaging in neurons generally requires a temporal resolution of more than a few Hz to track rapid neuronal responses16,17. In contrast, previous studies tracking microglial dynamics have sampled microglial structures at a relatively low temporal resolution of less than 0.1 Hz18,19,20. One recent study applied simultaneous two-photon imaging to understand neuron-microglia communication21. However, it is still unclear how dynamic microglial processes respond to the surrounding neural activity at a temporal resolution of more than a few Hz in awake mice. In order to address this issue, we describe a simultaneous in vivo two-photon imaging method of neural activity and microglial dynamics with a temporal resolution higher than 1 Hz in awake mice. This method allows us to achieve stable imaging in awake mice at a higher frame rate (maximum, 30 Hz with the pixel frame size of 512 x 512 pixels) and provides a more favorable way to investigate the surveillance behavior of microglia or their interaction with neuronal activity in awake mice.
All experiments were approved by the Animal Ethics Committee and the Genetic Recombinant Experiment Safety Committee of Graduate School of Medicine and Faculty of Medicine, the University of Tokyo, and performed according to their guidelines.
1. Preparation
2. AAV injection
3. Cranial window implantation
NOTE: Cranial window implantation follows the AAV injection on the same day.
4. In vivo two-photon imaging of microglia and neuronal calcium dynamics
We performed the AAV injection and cranial window implantation in V1 of an 8-week-old CX3XR1-EGFP transgenic mouse as described in this protocol. Four weeks after the surgery, we performed simultaneous in vivo two-photon imaging of R-CaMP-based neural activity and microglial dynamics in layer 2/3 of V1 (Figure 4 and Video 1). During the imaging, the mouse was placed on a treadmill and allowed to run freely. Images were acquired at the frame rate of 30 Hz with the spatial resolution of 0.25 µm/pixel using a 25x, 1.1 NA objective and GaAsP PMTs. Both EGFP and R-CaMP were excited at a wavelength of 1,000 nm, and EGFP fluorescence (525/50 nm emission filter) and R-CaMP fluorescence (593/46 nm emission filter) were collected simultaneously. After the registration of acquired data to minimize motion artifacts, every four frames were averaged into one frame to reduce background noise. Grating visual stimuli were presented to the mouse, and the visual responses of neurons and individual dendritic spines were analyzed (Figure 4D). Microglial processes showed fast dynamics and changed their morphology within 10 s (Figure 4E and Video 1). As detailed in the Discussion section, when the AAV injection failed, the expression of R-CaMP could not be detected. If the surgery was not successful, the signals of EGFP and R-CaMP could not be observed.
Figure 1: AAV injection. (A) A glass pipette was connected to a 26 G Hamilton syringe using a silicon tube. (B) The set-up for AAV injection. (C) AAV solution was injected via the glass pipette tilted 60° anteriorly from the vertical axis. (D) Schematic diagram of the procedure of open skull (described in step 3.3). Please click here to view a larger version of this figure.
Figure 2: Cranial window and head-plate. (A) Schematic diagram of cranial window implantation. (B) Custom-made head-plates were used. For imaging in the right hemisphere, the shorter arm should be used on the right side. (C) Dorsal view of a mouse with a cranial window and an implanted head plate. (D) High magnification view of the cranial window shown in Figure 2C. (E) Dorsal view of a mouse fixed with the custom-made stereotaxic instrument. Please click here to view a larger version of this figure.
Figure 3: The set-up for in vivo two-photon imaging. (A) Dorsal view of shading device. (B) Side view of shading device. (C) View of a mouse with the shading device on the head plate under the objective lens. The mouse is placed on the treadmill. (D) View under the two-photon excitation microscope. A monitor presenting visual stimuli is placed on the right side of the figure. The objective lens is covered with the shading device and black aluminum foil to avoid light from the monitor to the objective lens. Please click here to view a larger version of this figure.
Figure 4: Microglial dynamics and visual responses in a dendritic spine. (A–C) Time-averaged images of EGFP(+) microglia and R-CaMP(+) neurons in layer 2/3 of V1 in a 12-week-old CX3XR1-EGFP transgenic mouse. Signal intensity in each channel was normalized independently. (D) Visual responses in a dendritic spine. Stripe diagrams with black arrows indicate the direction of the grating stimuli presented in the timing indicated by gray columns. In calcium traces, gray lines indicate individual responses, and a black line indicates their average. In the right inset, a yellow arrowhead indicates the dendritic spine in which the region of interest (ROI) was generated to acquire the calcium traces. (E) Microglial process (cyan arrowhead) showed rapid retraction in 10 s. Please click here to view a larger version of this figure.
Video 1: Two-photon imaging of neuronal activity and microglial dynamics. Imaging data of EGFP(+) microglia and R-CaMP(+) neurons in layer 2/3 of V1 in a 12-week-old CX3XR1-EGFP transgenic mouse. On the left side of the movie, magenta indicates R-CaMP in neurons, and green indicates EGFP in microglia. The center of the movie corresponds to the EGFP signal, and the right side corresponds to the R-CaMP signal. Signal intensity in each channel was normalized independently. The frame rate of the movie is 40 times faster than the actual rate. Scale bar = 10 µm Please click here to download this Video.
We describe the protocol of AAV injection and craniotomy for simultaneous imaging of microglial dynamics and neuronal activity in awake mice as well as data processing. This technique can uncover the coordination between microglial dynamics and neuronal activity on time scales ranging from sub-second to tens of seconds.
The surgery protocol involves several technically demanding steps. AAV injection is one of the critical steps. Unsuccessful AAV injection may cause a significant reduction in the expression of R-CaMP. There are two main reasons: clogged glass pipettes and tissue damage. The clogging of glass pipettes reduces or completely blocks the ejection of AAV solution. This situation can be avoided by coloring the AAV solution with a dye such as fast green and visually confirming the success of the injection. Tissue damage caused by AAV injection prevents exogenous gene expression near the center of the injection site. The damage induced by AAV injection is likely to be caused by a sudden increase in the efflux from the tip of the glass pipette after the accumulation of the pressure inside the pipette. Therefore, the outflow of AAV solution from the glass pipette should be constant during the injection. Selecting glass pipettes with a slightly wider diameter at their tips would solve this problem. In cranial window implantation, the speed of surgery is critical. If the surgery takes too long, the brain tissue can be severely damaged. Accordingly, repeated practice is necessary to ensure the speed and smoothness of surgery. In addition, during the 4 weeks from surgery to imaging, the quality of imaging may be reduced by tissue regeneration between the cranial window and the brain tissue by the conventional method17. The method here overcomes this issue by applying thick glasses for the inner glass disc of the cranial windows to inhibit tissue regeneration and keep the window clear. In the system described here, this effect was evident by using an inner glass disc with a thickness of 0.525 ± 0.075 µm.
The method can be applied successfully to mice older than 4 weeks, but the application to younger mice may be problematic. In juvenile mice, the skull shows rapid and prominent growth, which induces a mismatch between the cranial bone and the glass window.
In some cutting-edge studies, in vivo two-photon imaging was used to study microglia-neuron interactions12,21,23. Especially in the pioneering work by Merlini et al., they performed simultaneous in vivo imaging of microglial dynamics and neural activity within the cell bodies21. In this method, by using thicker inner glasses for cranial windows, we could suppress motion artifacts in the depth orientation and achieve the stable measurement of neuronal activity in microstructures, such as dendritic spines. This method would help investigate synapse-microglial process interactions in awake mice.
Recently, in vitro study demonstrated that thin filopodia-like microglial processes have faster motility than thick processes, suggesting the importance of their faster motility in surveillance19. The system here can track the motility of thin microglial processes with a temporal resolution from a sub-second to a few tens of seconds. This property helps elucidate the functional significance of their motility for surveillance in vivo.
In the future, the combination of this imaging technique with interventions, such as optogenetics24 or chemogenetics25, applied to local neural circuits or interregional neural connections would shed light on novel microglial functions in synaptic development and plasticity which sculpt characteristics of neural circuits. Also, further integration of imaging, intervention, and behavioral tasks would contribute to revealing the coordination of microglia and neurons underlying specific behaviors.
The authors have nothing to disclose.
We thank Dr. Masashi Kondo and Dr. Masanori Matsuzaki for providing virus vectors. This work was supported by Grants-in-Aid for Scientific Research (20H00481, 20A301, 20H05894, 20H05895 to S.O.), the Japan Agency for Medical Research and Development (JP19gm1310003 and JP17gm5010003 to S.O. and JP19dm0207082 to H.M.), the UTokyo Center for Integrative Science of Human Behavior (CiSHuB), the Japan Science and Technology Agency Moonshot R&D (JPMJMS2024 to H.M.), Brain Science Foundation (to H. M.).
10-inch LCD monitor | EIZO | DuraVision FDX1003 | For presenting grating visual stimuli |
26G Hamilton syringe | Hamilton | 701N | |
27G needle | Terumo | NN-2719S | |
AAV-hSyn-R-CAMP1.07 | N/A | N/A | Kindly gifted from Prof. Matsuzaki's laboratory |
Activated charcoal powder | Nacalai tesque | 07909-65 | |
Black aluminum foil | THORLABS | BKF12 | |
CX3CR1-EGFP mouse | Jackson laboratory | IMSR_JAX: 005582 | CX3CR-1EGFP/+ knock-in/knock-out mice expressing EGFP in microglia in the brain under the control of the endogenous Cx3cr1 locus. |
DENT SILICONE-V | Shofu Inc | N/A | For the attachment of a shading device to a head-plate |
Dental cement (quick resin, liquid) | Shofu Inc | N/A | AB |
Dental cement(quick resin, powder) | Shofu Inc | N/A | B Color 3 |
Drill | Toyo Associates | HP-200 | |
Glass capillary pipette | Drummond Scientific Company | 2-000-075 | |
Glass disc (large) | Matsunami | N/A | 4mm in diameter, 0.15±0.02mm in thickness |
Glass disc (small) | Matsunami | N/A | 2mm in diameter, 0.525±0.075mm in thickness |
Head-plate | Customized | N/A | Material: SUS304, thickness: 0.5mm, see Figure2B for the shape |
Hemostatic fiber | Davol Inc | 1010090 | |
ImageJ Fiji software | Free software | For data registration | |
Instant glue (Aron alpha) | Daiichi Sankyo | N/A | |
Isoflurane | Pfizer | N/A | |
Lidocaine | Astrazeneca | N/A | |
MATLAB 2017b | MathWorks | N/A | For data registration and processing |
Meloxicam | Tokyo Chemical Industry | M1959 | |
Microinjector | KD scienfitic | KDS-100 | |
Micropipette puller | Sutter Instrument Company | P-97 | |
Multi-photon excitation microscope | NIKON | N/A | The commercial name is "A1MP+". |
Objective lens | NIKON | N/A | The commercial name is "CFI75 apochromat 25xC W". |
Paraffin Liquid | Nacalai tesque | 26132-35 | |
Psychtoolbox | Free software | For presenting grating visual stimuli | |
Shading device | Customized | N/A | |
Stereomicroscope | Leica | M165 FC | |
Stereotaxic instrument | Narishige Scientific Instrument | SR-611 | For the surgery |
Stereotaxic instrument | Customized | N/A | For fixing mice under the two-photon microscope |
Stopcock | ISIS | VXB1079 | |
Surgical silk | Ethicon | K881H | |
Treadmill | Customized | N/A | |
UV light curing agent | Norland Products Inc | NOA 65 | |
Vaporizer | Penlon | Sigma Delta | Anesthetic machine |