This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas (19H04753, 19H05219, and 25110732 to H. W.), Grants-in-Aid for Transformative Research Areas (A) (20H05899 to H. W., 20H05886 to O. M., and 21H05587 to D. K.), Fostering Joint International Research (B) (20KK0170 to H. W.), Grant-in-Aid for Scientific Research (B) (18H02598 to H. W.), Grant-in-Aid for Scientific Research (A) (21H04663 to O. M.), Grant-in-Aid for Early-Career Scientists (20K15193 to X. Q.), JST CREST Grant Number JPMJCR1755, Japan, and JST A-STEP Grant Number JPMJTR204C.

All experimental protocols were approved by the Animal Care and Use Committees of Nagoya University Graduate School of Medicine (approval number: M220295-003).
1. Head plate implantation (Figure 1A)
<ol>
	<li>Administer the anesthetic (a mixture of 74 mg/kg ketamine and 10 mg/kg xylazine) intraperitoneally to anesthetize the mouse. Check the anesthetic status of the mouse frequently by assessing the pedal reflexes.</li>
	<li>After anesthesia, place the mouse in a stereotaxic instrument. Apply an eye ointment (see Table of Materials) to prevent the cornea from drying out when the head plate is implanted.</li>
	<li>Shave the surgical area and disinfect the skin&#160;with three alternating rounds of povidone-iodine or chlorhexidine scrubs followed by 70% alcohol wipes. Carefully expose the skull and clean it with cotton swabs. 
	NOTE: All surgical instruments should be sterilized, and all procedures should be performed accordingly. Any remaining debris (e.g., hair or dried blood) causes inflammatory reactions. Therefore, any debris should be removed under a stereoscope using cotton swabs moistened with sterile water or 70% alcohol.</li>
	<li>Use the stereotactic coordinates-anterior and posterior = 0.5 mm, medial and lateral = 1.5 mm from bregma-to find the center of the craniotomy and label it with a marker pen.</li>
	<li>Place a custom-made head plate at the center of the skull. Next, apply dental cement (see Table of Materials) to firmly fix it to the skull. Apply light pressure until the head plate makes firm contact with the front and back of the skull. 
	NOTE: This step takes approximately 20 min to complete and is critical for reducing motion artifacts in the brain during two-photon imaging. The dimensions of the head-plate are 20 mm &#215; 40 mm &#215; 1 mm with a home plate shape opening with one edge 15 mm long, two adjacent sides 3 mm long, and the remaining two sides 10 mm long.</li>
	<li>Mix together an acrylic-based dental adhesive resin cement as follows: half a spoon of powder, three drops of liquid, and one drop of catalyst (see Table of Materials). To prevent drying, apply this mixed dental adhesive resin cement to the intact skull surface of the mousewith the head plate.</li>
	<li>Place the mouse in a warm cage until it has recovered from anesthesia. Do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
2. Surgery and adeno-associated virus (AAV) injection (Figure 1B)
<ol>
	<li>Perform craniotomy or viral injection without removal of the dental adhesive resin cement from the skull 1 day after head plate implantation. 
	NOTE: Administer anesthesia (a mixture of 74 mg/kg ketamine and 10 mg/kg xylazine)&#160;intraperitoneally to the mouse during this procedure.</li>
	<li>To avoid cerebral edema, administer dexamethasone sodium phosphate (1.32 mg/kg) intraperitoneally 1 h before surgery.</li>
	<li>Anesthetize the mouse with the head plate with 1% isoflurane anesthesia using a vaporizer (anesthesia delivery system) while maintaining body temperature with a heating pad. Apply an eye ointment to prevent corneal drying.</li>
	<li>Under a stereoscope, perform a circular craniotomy approximately 2 mm in diameter using a dental drill. To reduce brain damage, operate the dental drill carefully with constant slight movement and light downward pressure.</li>
	<li>Remove bone fragments several times using a suction system. After removing the bone fragments, use an artificial spinal fluid (ACSF) solution to remove and wash any debris remaining on the brain surface. Repeat this cleaning procedure several times to suppress inflammatory reactions. 
	NOTE: The ACSF solution (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 2.0 mM CaCl2, and 1.0 mM MgCl2) was stored at 4 &#176;C for 1 month after the reagent was dissolved and filtered (pore size = 0.22 &#181;m).</li>
	<li>Using a pressure injection system (see Table of Materials), set the appropriate pressure (about 10 PSI in pulses with a duration of 4 ms) to inject 500 nL of AAV solution through a glass capillary with a tip diameter of 10-20 &#181;m (prepared with a micropipette puller) for 10 min.</li>
	<li>Determine whether the AAV solution is being administered to the brain by checking whether the level of the AAV solution in the glass capillary gradually decreases.</li>
	<li>Leave the glass capillary in place for an additional 10 min to prevent backflow. Repeat three times to administer a total of 1.5 &#181;L of AAV solution into the brain.</li>
	<li>To evaluate and manipulate neural activity in layer 2/3 (L2/3) pyramidal cells, inject an AAV solution (for Ca2+ imaging: AAV2/1-Syn-jGCaMP8f-WPRE at 1.28 &#215; 1014 vector genomes/mL, diluted 1:1 in saline; for Ca2+ imaging with optogenetics: AAV2/8-CaMKII-GCaMP6m-P2A-ChRmine-Kv2.1-WPRE at 1.73 &#215; 1014 vector genomes/mL, diluted 1:1 in saline) into the hind paw area of the primary somatosensory cortex of wild-type mice (S1, centered at 0.5 mm posterior and 1.5 mm lateral from the bregma, 150 &#181;m depth from the surface). 
	NOTE: The AAV solution should be injected at 2-3 weeks and 1-2 weeks before imaging for jGCaMP8f and GCaMP6m-P2A-ChRmine expression, respectively.</li>
	<li>After an application of 2% (w/v) low-melting agarose to the brain surface of S1 using a micropipette, place a glass window over the craniotomy with two cover glasses. Attach the two cover glasses (small 2.0 mm diameter and large 4.5 mm diameter; see Table of Materials) with UV curable adhesive.</li>
	<li>Press the cover glass against the agarose while it is still liquid; this prevents the formation of air bubbles in the agarose. Seal the edges of the cranial window with dental and adhesive resin cement (Figure 1C).</li>
	<li>Remove the mouse from the stereotaxic instrument and return it to its cage. Place the mouse in a warm cage and do not return to the cage with other animals until it has fully recovered from anesthesia. Carefully maintain sterile conditions during survival surgery.</li>
	<li>For the first 72 h after surgery, check the health status of the mouse by observing general behavior. If there are any abnormalities in general behavior, subcutaneously inject anti-inflammatory and analgesic agents.</li>
</ol>
3. Preparation for the holographic stimulation or illumination system (Figure 3)
<ol>
	<li>Calibrate the holographic stimulation system by placing the surface of a red fluorescence slide (cast acrylic substrate) at the sample plane. Place the microscope in live imaging mode with a weak excitation light and run calibration_GUI.m file. Check&#160;the parameters pane and click on the Save button.</li>
	<li>Click the Z Scan button in the step 1 pane. It will automatically generate three random spots in all 21 axial planes, 2 &#181;m apart from each plane.</li>
	<li>Move the slide bar and check the live image. Find a perfect plane where the spots appear the smallest and the brightest, and then click the Save button. This will automatically generate an offset spherical wavefront for the digital hologram. 
	NOTE: If you fail to find the brightest fluorescence spots, change the minimum and maximum values of the scan range and try again.</li>
	<li>Click the Go button in the step 2 pane, and then click six spots on the left square. Check the live image. If there are six distinguishable fluorescence spots, then type their x and y axis to the edit boxes and click the Save button. This will automatically generate affine transform coefficients to coordinate calibration between the holographic stimulation and imaging system. 
	NOTE: The axis pair number and clicked spot number must be matched in order. If not sure, or if there are no spots in your image, please try again and generate a unique spot pattern or choose a smaller range around the center of the field of view (FOV).</li>
	<li>Click the Scan button in the step 3 pane. It will generate 441 digital holograms to perform single spot scanning across the FOV&#160;in 21 &#215; 21 steps.
	<ol>
		<li>First, check the images while changing patterns in the list box. Then, adjust the laser power to obtain spot images within the dynamic range of the imaging device (for instance, to avoid overly saturated images).</li>
		<li>Subsequently, adjust the interval time in the edit box; the interval time should be more than two times the recording interval time. Finally, put the imaging device in recording mode and click the Play button. If the play completes, there will be &#34;display OK&#34; strings shown in the command window. Stop recording and stack up recorded sequential images using the max intensity method.</li>
	</ol>
	</li>
	<li>Click Generate WM in the step 4 pane and choose a stacked image from above. Then close the calibration_GUI window. It will automatically generate a weight map to compensate for the unbalanced intensity in each spot. 
	NOTE: For a more detailed description, please refer to 2; the Matlab code can be downloaded from here (https://github.com/ZenKG/SLM_control).</li>
</ol>
4. Ca2+ imaging using an image sensor with holographic illumination (Figure 4)
<ol>
	<li>Place the AAV-injected mouse with a head plate under the microscope (Figure 1D). 
	NOTE: During this procedure, the mouse is restrained in the awake state, but is able to escape uncomfortable stimuli.</li>
	<li>Perform two-photon imaging (point scanning mode) using a holographic microscope and a mode-locked Ti:sapphire laser tuned to 920 nm with a 25x objective (see Table of Materials).</li>
	<li>Turn on the commercial imaging software (see Table of Materials). In the live imaging mode, adjust the voltage of the image detector (see Table of Materials) and the power of the imaging laser to optimize the brightness of the neurons expressing jGCaMP8f. Capture images of the neurons expressing this protein. 
	NOTE: The intensity of the imaging laser (920 nm) is 20-30 mW. The FOV was 512 &#181;m &#215; 512 &#181;m at a depth of 100-150 &#181;m from the cortical surface.</li>
	<li>To illuminate specific neurons expressing jGCaMP8f with holographic illumination, run the SLMcontrol.m script file. Click the Reference Image and choose the image acquired above. Then, click the Spot button to choose specific pixels on the neurons in the image by continuous mouse clicking (Figure 4A). If the selection is complete, push the Enter button on the keyboard to finalize it. 
	NOTE: The digital hologram is automatically calculated and displayed on the SLM. This pattern can also be revisited by clicking on a list box. The spatial resolution of a single spot generated by the SLM was approximately 1.2 &#181;m along the transverse direction and ~8.3 &#181;m along the optical axis. We used a high numerical aperture (1.1) objective lens to achieve more localized holographic stimulation. Table 1 summarizes previous reports and this system with respect to the spatial resolution of holographic stimulation.</li>
	<li>To detect neural activity with high temporal resolution using an image sensor (see Table of Materials), set the exposure time, imaging area, and binning (Figure 4B) before performing image acquisition (Figure 4C). 
	NOTE: The intensity of the holographic illumination laser (920 nm) that continuously stimulates one neuron is 2 mW, which is sufficient to detect neural activity. For example, to achieve a frame rate of 100 Hz for imaging, the exposure time is 9 ms, the imaging area is 400 &#181;m &#215; 400 &#181;m, and the binning is 4.</li>
	<li>After the experiment, return the mouse to its home cage.</li>
</ol>
5. Two-photon imaging (point scanning mode) with optogenetics using a holographic microscope (Figure 2)
<ol>
	<li>Repeat steps 4.1 and 4.2.</li>
	<li>Turn on the commercial imaging software (see Table of Materials). In the live imaging mode, adjust the voltage of the image detector (see Table of Materials) and the power of the imaging laser to optimize the brightness of the neurons expressing GCaMP6m-P2A-ChRmine. Capture images of the neurons expressing these proteins (Figure 1E).</li>
	<li>Repeat step 4.4.</li>
	<li>To investigate the functional connectivity within L2/3 neurons, use an SLM to generate holographic patterns of optogenetic stimulation (ChRmine; 1,040 nm) and combine it with two-photon Ca2+ imaging (GCaMP6m; 920 nm, 512 &#215; 512 pixels, 2 Hz or 30 Hz, 2x digital zoom, point scanning mode; Figure 4D-H).
	<ol>
		<li>For this protocol, set the intensity of the imaging laser to 920 nm, at 10-20 mW, and the FOV as 256 &#181;m &#215; 256 &#181;m measured at a depth of 100-150 &#181;m from the cortical surface. Set the pixel dwell time at 1.5 &#181;s for 2 Hz or 100 ns for 30 Hz.</li>
		<li>To see if a single holographic stimulus caused a calcium response in the neurons, use both 2 Hz and 30 Hz as the imaging frame rate. Set the intensity of the holographic stimulation laser (1,040 nm) that stimulates a single neuron at 10 mW, which is sufficient to induce neural activity (Figure 4D). 
		NOTE: The spatial resolution of a single spot generated by the SLM is approximately 1.2 &#181;m along the transverse direction and ~8.3 &#181;m along the optical axis. The range of accessible volume in the lateral direction is around 500 &#181;m &#215; 500 &#181;m and 100 &#181;m in the axial direction. We have further confirmed with Ca2+ imaging at 2 Hz or 30 Hz imaging frame rate that not only one neuron, but multiple neurons can be holographically stimulated simultaneously (Figure 4E).</li>
	</ol>
	</li>
	<li>Perform image acquisition with the following protocol: simultaneously image the Ca2+ response at&#160;920 nm with 10 holographic stimuli at 1,040 nm with 8 s intervals (0.125 Hz) for a duration of 50 ms after a baseline period of 10 s. After all the experiments are completed, the mice are euthanized. 
	NOTE: Ca2+ transients, if present, were evoked by holographic stimulation, with their peak appearing within 1 s after stimulation (Figure 4F-H).</li>
</ol>
6. Image analysis and assessment of functional connectivity (Figure 4)
<ol>
	<li>Open the raw images saved in steps 4.5 or 5.5 using ImageJ. To compensate for focal plane displacement, use the ImageJ plug-in TurboReg. 
	NOTE: If the correction with TurboReg is not sufficient, it is recommended to use CaImAn (http://github. com/simonsfoundation) to correct the focal plane displacement.</li>
	<li>To assess neural activity, determine the regions of interest (ROIs) in L2/3 using an automated algorithm (CaImAn). Detect and analyze Ca2+ transients after defining baseline fluorescence intensity (F0) and the threshold value. 
	NOTE: F0 is the 35th percentile value of fluorescence intensity acquired during the baseline imaging period. Ca2+ transients are denoted by &#916;F/F0 (&#916;F = F-F0), where F is the instantaneous fluorescence signal. If the &#916;F value is over 2 S.D. from F0, we evaluate a significant Ca2+ transient.</li>
	<li>To define functional connectivity in L2/3 neurons, stimulate target neurons and measure GCaMP6m responses in stimulated and surrounding neurons, accordingly (Figure 4F-H).</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+channel+conduction+in+the+pump-like+channelrhodopsin+ChRmine.">Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.</a> Cell. 185 (4), 672-689 (2022).</li><li>Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics:+10+years+of+microbial+opsins+in+neuroscience.">Optogenetics: 10 years of microbial opsins in neuroscience.</a> Nature Neuroscience. 18 (9), 1213-1225 (2015).</li><li>Ko, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+functional+microcircuits+in+visual+cortex.">The emergence of functional microcircuits in visual cortex.</a> Nature. 496 (7443), 96-100 (2013).</li><li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+two-photon+calcium+imaging+in+freely+moving+mice.">Large-scale two-photon calcium imaging in freely moving mice.</a> Cell. 185 (7), 1240-1256 (2022).</li><li>Villette, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ultrafast+two-photon+imaging+of+a+high-gain+voltage+indicator+in+awake+behaving+mice.">ultrafast two-photon imaging of a high-gain voltage indicator in awake behaving mice.</a> Cell. 179 (7), 1590-1608 (2019).</li></ol>

The authors have no conflicts of interest to declare.

To understand brain function, it is necessary to accurately assess the neural circuitry underlying the brain function by extracting the dynamics of neural activity. Furthermore, it is essential to identify how this neural circuitry is altered to elucidate the pathogenesis of neuropsychiatric disorders. Indeed, it is known that neural activity is elevated in mouse models of Alzheimer&#39;s disease4 and fragile X syndrome26 and mice with impaired white matter function3. Moreover, in a mouse model of inflammatory pain, increased synchronization of neural activity and functional connectivity between neurons is associated with symptoms24. Two-photon holographic microscopy allows us to simultaneously observe the activity of individual neurons and the functional connections between neurons, which is necessary for understanding neural circuitry. We used a 25x objective lens with numerical aperture = 1.1 with wavelengths of 1,040 nm. The theoretical point spread function is a Gaussian distribution with full width at half maximum of 0.5 &#181;m laterally and 1.7 &#181;m axially. However, the actual numerical aperture is less than 1.1, and the measured spot size on a fluorescence bead is 1.2 &#181;m laterally, and 8.3 &#181;m axially. Given that the neuron diameter is approximately 15 &#181;m and the calibration error is within 3 &#181;m, the targeting is generally good. However, cells in the axial direction may be affected by the longer spot size6. Here, we described the viral injection, surgery, calibration of holographic stimulation or illumination systems, and imaging protocols for evaluating and manipulating neural activity in live mice using our microscopy system.
It takes 2-4 weeks to complete all experimental procedures, from head plate implantation and virus injection to data acquisition for in vivo Ca2+ imaging using holographic microscopy. The process is complex and laborious, and the ultimate success of the experiment depends on multiple factors, including the condition of the cranial window, which is affected by postoperative inflammation, the proper choice of Ca2+ indicator and opsin, and whether motion artifacts in the acquired images can be corrected. In particular, two steps are important for a successful outcome. The first concerns head plate fixation and surgery; it is crucial that the head plate be firmly fixed to the mouse head with dental cement. Furthermore, it is important to repeatedly clean bone fragments and clotted blood using cold ACSF during surgery. Since adherence to this procedure reduces inflammation, we successfully observed the dynamics of microglia, the cells responsible for the brain&#39;s immune system, without activating their processes and spines or the microstructures of neurons27,28. The second issue is the evaluation and manipulation of neural activity. We chose AAV2/8-CaMKII-GCaMP6m-P2A-ChRmine-Kv2.1 to express the Ca2+ indicator and opsin in one neuron simultaneously. This is because it is difficult to efficiently infect one neuron with different AAV types. Another reason for this choice was that ChRmine can efficiently activate neural activity at 1,040 nm using a two-photon laser12. Recently, it has been reported that ChRmine, by mutating its structure based on structural information obtained by cryo-electron microscopy, improves its function29, which is considered useful for target function analysis in the field of neuroscience. In light of these issues, it is necessary to share effective methods for evaluating and manipulating neurons when reading neural activity and writing information using holographic microscopy.
Recent advances in imaging and optogenetics have revealed the detailed neural activity involved in brain functions such as learning and memory, and it is possible to artificially manipulate this neural activity to express brain functions30. However, conventional methods for the manipulation of neural activity are highly invasive on account of the insertion of optical fibers in the brain and because a group of opsin-expressing cells is simultaneously stimulated, making it impossible to manipulate neural activity with temporal and spatial precision. Our method can manipulate neural activity by stimulating only specific neurons in the brain, thus enabling manipulation of neural activity with specific stimulation patterns and high spatiotemporal resolution. Furthermore, it is important to note that functional connectivity between neurons can only be evaluated in a small number of neurons using brain slice experiments31; however, this technique allows simultaneous evaluation of multiple neurons in living animals.
One of the major limitations of current holographic microscopes is the need to fix the mouse head, which limits the mouse&#39;s behavior. Recently, a miniaturized two-photon microscope was developed32, and with the further miniaturization of the device, in vivo Ca2+ imaging with holographic stimulation may be possible in free-moving mice. In addition, the potential of this microscope could be expanded by improving the temporal resolution of imaging and combining it with highly sensitive voltage-sensitive fluorescent proteins33.

Two-photon Ca2+ imaging is a useful technique for the assessment of neural activity. It can be used to identify not only the neural activity required for behavior and memory in normal animals1,2 but also an abnormal neuronal activity that occurs in mouse models of neuropsychiatric disorders3,4. The technique has been used to elucidate the neural basis of brain functions. However, although it can provide high-resolution and high-quality images, its temporal resolution is lower than that of the electrophysiological method1,3.
Optogenetics has helped to innovate the way neuroscientists understand brain function5. Given the technical limitations, the majority of optogenetic research has used activation schemes with low spatial resolution, thus limiting the types of manipulations of neural activity that can be performed accordingly. However, manipulating neural activity at finer spatiotemporal scales can potentially be useful for a more complete understanding of neural computation and the pathogenesis of neuropsychiatric disorders. Spatially precise holographic technology that can shape femtosecond near infrared laser beams promises to overcome this challenge and opens up several new experimental classes that were previously impossible6,7. This technology enables neuroscientists to uncover the fundamental aspects and pathologies of sensory, cognitive, and behavioral neural codes that have been beyond reach.
Holographic projection involves the generation of desired light patterns to access individual cells and functional networks selectively. In vivo experiments require optimal light transmission to target cells in the living brain. Infrared light penetrates deeper into living tissue and can be used for nonlinear two-photon excitation (2PE)8,9,10. Thus, two-photon holographic microscopy, which combines holographic projection and 2PE, can be used to evaluate and manipulate neural activities to probe cellular and functional networks in vivo. Recent biological applications of two-photon holographic microscopy have elucidated the neural activity and circuitry required for learning in the visual cortex11,12, olfactory bulb13, and hippocampus14.
Numerous laboratories worldwide have reported exciting results and improvements using their holographic stimulation systems15,16,17,18,19,20,21,22,23. In the system described here, the holographic stimulation system can be built as an add-on device for a conventional microscope. The phase-only spatial light modulator (SLM) is the key device for modulating a plane wavefront to any shape, and the interference effect is used to control the intensity and location of the foci. Figure 2 shows holographic stimulation and imaging light paths. The first light path is for point-scanning imaging mode and consists of a scan head and image detectors.&#160;The second light path is for holographic stimulation with a 1040 nm wavelength and consists of an SLM1. The third light path is for holographic illumination with 920 nm wavelength and consists of an SLM2 and an image sensor. The holographic imaging mode can record intensities from multiple regions of interest by illuminating multiple points in the sample. In this way, the recording speed can be increased to few hundred of frames per second. To achieve point scanning imaging or holographic illumination imaging, the 920 nm laser was split into two paths by a beam splitter with a fixed ratio of 3:7. All the optical elements were aligned on an optical breadboard with dimensions of 600 mm &#215; 600 mm. The modulated light entered through the light port on the side of the microscopic body, while the point scanning imaging light entered through the scan head at the top of the microscopic body. These lights were integrated just above the objective lens and created foci in the sample plane. In addition, the custom-made software enabled the regular workflow to be simple and consistent.
In this article, a complete protocol is presented for the use of holographic stimulation or illumination to measure neural activity and assess the functional connectivity between neurons. For demonstration purposes, we describe here a brain surgery targeting the hindlimb area of the primary somatosensory cortex (S1HL) of the mouse brain and a method to assess and manipulate neural activity using two-photon holographic microscopy. The experimental procedure is divided into four parts. First, the head plate was fixed to the skull of the mouse using dental cement. Second, a viral vector expressing jGCaMP8f or GCaMP6m-P2A-ChRmine was stereotactically injected into the S1HL. Third, the holographic stimulation or illumination&#160;system was calibrated. Fourth, after postoperative recovery and expression of these two proteins, in vivo Ca2+ imaging was performed to assess the neural activity and functional connectivity between neurons with two-photon holographic microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25x Objective</td>
			<td>Nikon</td>
			<td>N25X-APO-MP</td>
			<td>Objective</td>
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			<td>A1MP</td>
			<td>Nikon</td>
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			<td>AnesII</td>
			<td>Bio machinery</td>
			<td>AnesII</td>
			<td>Anesthesia delivery system</td>
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			<td>C2 plus</td>
			<td>Nikon</td>
			<td>C2 plus</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>DECADRON&#160;Phosphate&#160;Injection</td>
			<td>Aspen</td>
			<td>21N024</td>
			<td>Avoid cerebral edema</td>
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		<tr>
			<td>Dental Drill</td>
			<td>Jota</td>
			<td>C1.HP.005</td>
			<td>Dental drill</td>
		</tr>
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			<td>Electric Microinjector</td>
			<td>NARISHIGE</td>
			<td>IM-31</td>
			<td>Pressure injection system</td>
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			<td>FEATHERS</td>
			<td>FEARGER</td>
			<td>FA-10</td>
			<td>Shaving</td>
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			<td>G-CEM ONE ADHESIVE ENHANCING PRIMER</td>
			<td>GC</td>
			<td>2110271</td>
			<td>Resin cement primer for dental adhesion</td>
		</tr>
		<tr>
			<td>G-CEM ONE neo</td>
			<td>GC</td>
			<td>43093</td>
			<td>Resin cement for dental adhesion</td>
		</tr>
		<tr>
			<td>Glass Capillary with Filament</td>
			<td>NARISHIGE</td>
			<td>GDC-1</td>
			<td>Glass capillary</td>
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		<tr>
			<td>Image Detector</td>
			<td>Hamamatsu</td>
			<td>H10770PA-40</td>
			<td>GaAsP photocathode photomultiplier tube</td>
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		<tr>
			<td>Imaging Software</td>
			<td>Nikon</td>
			<td>NISelements</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Isoflurane Inhalation Solution</td>
			<td>Pfizer</td>
			<td>229KAR</td>
			<td>Anesthetics</td>
		</tr>
		<tr>
			<td>iXon EMCCD Camera</td>
			<td>Andor</td>
			<td>iXon Life 888</td>
			<td>Image sensor</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>daiitisannkyou</td>
			<td>s9-018506</td>
			<td>Anesthetics</td>
		</tr>
		<tr>
			<td>Leica-M60</td>
			<td>Leica</td>
			<td>M60</td>
			<td>Stereoscope</td>
		</tr>
		<tr>
			<td>Linicon</td>
			<td>Linicon</td>
			<td>LV-125</td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Mode-locked Ti:sapphire Chameleon Ultra II laser&#160;</td>
			<td>Coherent</td>
			<td>Chameleon Discovery NX</td>
			<td>Femtosecond laser</td>
		</tr>
		<tr>
			<td>Mos-Cure</td>
			<td>U-VIX</td>
			<td>mini 365</td>
			<td>Portable LED UV Light Source</td>
		</tr>
		<tr>
			<td>PEN Bright</td>
			<td>SHOFU INC.</td>
			<td>PEN Bright</td>
			<td>Dental light curing unit</td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>SUTTER instaument</td>
			<td>P-97</td>
			<td>Puller</td>
		</tr>
		<tr>
			<td>Stereotaxic Instrument (for Mice)</td>
			<td>NARISHIGE</td>
			<td>SR-6M-H</td>
			<td>Stereotaxic instrument</td>
		</tr>
		<tr>
			<td>Stereotaxic Micromanipulator</td>
			<td>NARISHIGE</td>
			<td>SM-15R</td>
			<td>Stereotaxic micromanipulator</td>
		</tr>
		<tr>
			<td>Super-Bond CATALYST V</td>
			<td>SUN MEDICAL</td>
			<td>8070</td>
			<td>Dental adhesive resin cement</td>
		</tr>
		<tr>
			<td>Super-Bond Dental Adhesive Monomer</td>
			<td>SUN MEDICAL</td>
			<td>8071</td>
			<td>Dental adhesive monomer</td>
		</tr>
		<tr>
			<td>Super-Bond Teeth Color Polymer Powder</td>
			<td>SUN MEDICAL</td>
			<td>145052000</td>
			<td>Teeth color polymer powder</td>
		</tr>
		<tr>
			<td>Tarivid&#160;Ophthalmic&#160;Ointment 0.3%</td>
			<td>Santen Pharmaceutical&#160;</td>
			<td>TRN3952</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>UlTIMATE XL</td>
			<td>NSK</td>
			<td>Y141446</td>
			<td>Dental laboratory micromotor control unit</td>
		</tr>
		<tr>
			<td>UV Curing Optical Adhesives</td>
			<td>THORLABS</td>
			<td>NOA61</td>
			<td>UV Curing Optical Adhesives</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>KP0F2BK</td>
			<td>Anesthetics</td>
		</tr>
	</tbody>
</table>

evaluation and manipulation of neural activity using two-photon holographic microscopy

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular activities, in living animals. In the field of neuroscience, detailed neural activity related to brain functions, such as learning and memory, has now been revealed, and it has become feasible to artificially manipulate this activity to express brain functions. However, the conventional evaluation of neural activity by two-photon Ca2+ imaging has the problem of low temporal resolution. In addition, manipulation of neural activity by conventional optogenetics through the optic fiber can only simultaneously regulate the activity of neurons with the same genetic background, making it difficult to control the activity of individual neurons. To solve this issue, we recently developed a microscope with a high spatiotemporal resolution for biological applications by combining optogenetics with digital holographic technology that can modify femtosecond infrared laser beams. Here, we describe protocols for the visualization, evaluation, and manipulation of neural activity, including the preparation of samples and operation of a two-photon holographic microscope (Figure 1). These protocols provide accurate spatiotemporal information on neural activity, which may be useful for elucidating the pathogenesis of neuropsychiatric disorders that lead to abnormalities in neural activity.

Representative recordings obtained using the method described here are presented. In vivo Ca2+ imaging using holographic microscopy requires 2-4 weeks to complete from head plate implantation and AAV injection to data acquisition. Therefore, to obtain stable results, it is important to reduce motion artifacts in the brain. The head plate implantation (step 1.5) and the placement of the cranial window (step 2.9) are very important steps in this process. Furthermore, it is also important to select an AAV (AAV2/8-CaMKII-GCaMP6m-P2A-ChRmine-Kv2.1) that simultaneously expresses calcium indicator and opsin in a single neuron (step 2.8).
Figure 4A shows a spot for the holographic illumination of an acquired neuron image using a custom-made MATLAB script. If holographic illumination successfully illuminated the neurons expressing jGCaMP8f, Ca2+ traces could be obtained with an image sensor (Figure 4B), as shown in Figure 4C.
Functional connectivity between neurons was evaluated using holographic stimulation (Figure 4D), as shown in Figure 4E. Because functional connectivity between neurons is one of the neural circuitry properties that is altered in the pathogenesis of a pain model mouse24, we describe a simple procedure to evaluate it. Figure 4F shows a typical image of L2/3 neurons in S1HL visualized using GCaMP6m. When one neuron (orange circle) was holographically stimulated, another neuron (red circle) was simultaneously active; thus, the number of functional connectivities between neurons was one (Figure 4G).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64205/64205fig01v2.jpg" /> 
Figure 1: Schematic outline of the experimental procedure. (A) Fixation of the head plate to the skull. (B) Stereotactic injection of AAV into the hind paw area of the primary somatosensory cortex (S1HL). (C) Implantation of the cranial window. To assess and manipulate neural activity, in vivo Ca2+ imaging is performed in awake mice (D) with holographic stimulation (E). Flash marks indicate holographic stimulation or illumination. <a href="https://www.jove.com/files/ftp_upload/64205/64205fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64205/64205fig02.jpg" /> 
Figure 2: System used for the holographic microscope. (A) Images of the holographic stimulation and illumination light paths (left) near the microscope (middle) with an image sensor (right). (B) These are magnified images of holographic stimulation and illumination light paths around respective SLMs (left and right) and a point scanning light path around a scan head (middle and right). (C) A schematic of the stimulation and imaging optical paths. Phase-only SLMs are used to display digital holograms, and a beam expander (a combination of L1 and L2) and a 4f relay system (a combination of L3 and L4 for holographic stimulation and L4 and L5 for holographic illumination) are placed before and after respective SLMs to make sure each digital hologram is imaged at the exit pupil of a water immersion objective lens, with slightly under-filled image size. To suppress residual zero-order components, a beam block is placed at the intermediate plane. <a href="https://www.jove.com/files/ftp_upload/64205/64205fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64205/64205fig03.jpg" /> 
Figure 3: Flowchart of how to calibrate the holographic stimulation or illumination system. This flowchart describes steps to calibrate a holographic stimulation or illumination system to the sample space and imaging system. Please visit step 3, preparation for the holographic stimulation or illumination system, for detailed instructions and download a sample program. <a href="https://www.jove.com/files/ftp_upload/64205/64205fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64205/64205fig04.jpg" /> 
Figure 4: Representative results of imaging and functional connectivity using a holographic microscope. (A) To illuminate specific neurons expressing jGCaMP8f or GCaMP6m-P2A-ChRmine, the image of neurons is captured, and then a spot is formed on the neuron using a custom-made MATLAB script. (B) The setting of the image sensor (exposure time, imaging area, and binning). (C) Representative image and traces of neurons expressing jGCaMP8f in 100 Hz imaging with holographic illumination and an image sensor. (D) This graph shows the neural response to holographic stimulation (1,040 nm) at each laser power (data from GCaMP6m-P2A-ChRmine expressing neurons at 2 Hz imaging frame rate [n = 16]). Error bars indicate the standard error of the mean. (E) Representative Ca2+ traces during holographic stimulation (blue vertical lines) of 10 different neurons at 2 Hz (left) and 30 Hz (right) imaging frame rates. In 2 Hz and 30 Hz Ca2+ traces, the same color indicates the same neuron. (F) Schematic diagram evaluating functional connections between neurons. When the orange neuron is stimulated, the red neurons respond at the same time, indicating that there is functional connectivity between these neurons. (G) A typical image of S1HL neurons expressing GCaMP6m in WT. Scale bar = 10 &#181;m. (H) Typical Ca2+ traces during holographic stimulation (blue vertical lines) at 2 Hz (upper) and 30 Hz (lower) imaging frame rates. The stimulated neuron is circled in orange, responding neurons are circled in red, and non-responding neurons are circled in grey. Neural responsiveness to holographic stimulation can be detected at both 2 Hz and 30 Hz imaging speeds. <a href="https://www.jove.com/files/ftp_upload/64205/64205fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Our set-up 6&#160;</td>
			<td>Prakash, R. et al. 25&#160;</td>
			<td>Marshel, J. H. et al. 12</td>
			<td>Robinson, N. T. M. et al. 14</td>
		</tr>
		<tr>
			<td>Lateral resolution</td>
			<td>1.2 &#956;m</td>
			<td>1.27 &#956;m</td>
			<td>&#8213;</td>
			<td>2.22 &#956;m</td>
		</tr>
		<tr>
			<td>Axial resolution</td>
			<td>8.3 &#956;m</td>
			<td>56.86 &#956;m</td>
			<td>15.5 &#956;m</td>
			<td>10.26 &#956;m</td>
		</tr>
		<tr>
			<td>Objective lens/Numerical Aperture</td>
			<td>25x/1.1</td>
			<td>20x/0.5</td>
			<td>16x/0.8</td>
			<td>16x/0.8</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of previous reports and this system for the spatial resolution of holographic stimulation. The lateral resolution, axial resolution, and the objective lens used during the measurement are described.

Watch this Scientific Journal Video about Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy at JoVE.com

Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Recent advances in optical bioimaging and optogenetics have enabled the visualization and manipulation of biological phenomena, including cellular ...

evaluation-manipulation-neural-activity-using-two-photon-holographic

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Neuroscience

2.3K Views.  Nagoya University Graduate School of Medicine. We developed a two-photon holographic microscope that can visualize, assess, and manipulate neural activity using high spatiotemporal resolution, with the aim of elucidating the pathogenesis of neuropsychiatric disorders that are associated with abnormal neural activity.

Video: Evaluation and Manipulation of Neural Activity Using Two-Photon Holographic Microscopy

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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 ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

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

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

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

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

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used ...