This study was supported by the Lundbeck Foundation, the NOVO-Nordisk Foundation, the Danish Council for Independent Research | Medical Sciences, and the NORDEA Foundation Grant to the Center for Healthy Aging.

All procedures involving animals were approved by the Danish National Ethics Committee according to the guidelines set forth in the European Council&#8217;s Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and were in compliance with the ARRIVE guidelines.&#160;This is a terminal procedure with the mice being euthanized prior to anesthetic recovery.
1. Pre-surgical preparation
<ol>
	<li>Clean the surgical table and all the surrounding area with 70% ethanol. Thoroughly clean and dry the surgical tools before the surgery.</li>
	<li>Anesthetize an NG2DsRed mouse (Tg(Cspg4-DsRed.T1)1Akik/J; both genders; 4-8 months old) by a peritoneal injection of ketamine + xylazine (60 mg/kg + 10 mg/kg) dissolved in sterilized water, pH 7.4. Administer supplemental doses of ketamine (30 mg/kg) every 25 min until completion of the surgical procedure. Check tail and toe pinch reflexes regularly to verify the depth of anesthesia.</li>
	<li>Inject saline subcutaneously at four different locations on the back of the mouse for a total volume of 1 mL to prevent dehydration during the experiment. To protect the eyes from drying out, apply eye lubricant. Maintain body temperature at 37 &#176;C using a rectal temperature probe and heating blanket.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Tracheotomy 
	<ol>
		<li>Place the mouse on its back. Inject 0.04 mL of 0.5% lidocaine subcutaneously under the planned incision and wait for 2 min. Make a 10 mm long incision above the chest bone (manubrium). Separate the submandibular glands and sternothyroid muscles, and then expose the trachea.</li>
		<li>Make tracheostomy between two tracheal rings with scissors. Insert a small metal tube with a length of 16.2 mm and a diameter of 1 mm into the trachea. Connect the tube to a mechanical ventilator and capnograph to monitor the end-expiratory CO2 (Figure 1A).</li>
	</ol>
	</li>
	<li>Catheter insertion
	<ol>
		<li>Inject 0.5% lidocaine (0.04 mL) subcutaneously under the planned incision and wait for 2 min.</li>
		<li>Using fine tip forceps gently separate the artery from the vein. Stop the blood flow upstream with a microvascular clamp. Constrict the blood flow downstream with a 10-0 nylon suture ligating both blood vessels.</li>
		<li>Use micro-dissecting scissors to make a small incision in both the artery and the vein. Insert plastic catheters into both blood vessels. Secure the catheters by tightening the sutures (8-0 or 10-0 nylon sutures dependent on the vessel size) without compromising catheter lumens. Remove the microvascular clamp and close the skin.</li>
		<li>Monitor blood pressure via the arterial catheter. Measure levels of blood gases in arterial blood samples (50 &#956;L) before and after each experiment (normal values: pO2, 90-120 mmHg; pCO2, 35-40 mmHg; pH, 7.25-7.45) using a blood gas analyzer. Use the vein catheter for infusion of fluorescein and anesthesia.</li>
	</ol>
	</li>
	<li>Craniotomy
	<ol>
		<li>Shave the fur off the head of the mouse between the ears. Inject 0.04 mL of 0.5% lidocaine subcutaneously under the planned incision and wait for 2 min.</li>
		<li>Wipe the skull using a 10% iron-chloride solution to remove the periosteum. Rinse the skull thoroughly with saline. Glue a metal head bar to the skull with cyanoacrylate glue and activator (Figure 1A). 
		NOTE: The metal head bar is 75 mm long and 13.5 mm wide, with a round hole of 5 mm diameter in the center.</li>
		<li>Drill a 4-mm-diameter craniotomy, centered at 0.5 mm behind and 3 mm to the right of the bregma over the right sensory barrel cortex. Remove the dura with a fine-tip vessel dilator.</li>
		<li>Cover the exposed cortex with 0.75% agarose gel, dissolved in artificial cerebrospinal fluid (aCSF; pH = 7.4) and cooled to 35 &#176;C. Cover 80-90% of the craniotomy with a glass coverslip at an angle of 10-15&#176; to the metal head bar (Figure 1B) that permits insertion and placement of the glass micro-pipette.</li>
		<li>To minimize brain pulsation, glue the two corners of the glass coverslip onto the metal head bar with cyanoacrylate glue and activator. Apply the activator carefully to prevent getting onto the exposed brain. Rinse the glued glass coverslip thoroughly with saline afterwards.</li>
	</ol>
	</li>
	<li>Perform insertion of the whisker pad stimulation probe. Insert a set of custom-made bipolar electrodes (8 mm length and 0.25 mm thickness) percutaneously into the left side of face to stimulate the ramus infraorbitalis of the trigeminal nerve contralateral to the craniotomy. Position the cathode close to the hiatus infraorbitalis, and insert the anode into the masticatory muscles7. Perform the stimulation with an electrical stimulator at an intensity of 1.5 mA for 1 ms in trains of 20 s at 2 Hz.</li>
	<li>Upon completion of surgery, administer a bolus of 0.05 mL fluorescein isothiocyanate-dextran (FITC-dextran, 4% w/v, molecular weight [MW] 50,000) into the femoral vein to label the blood plasma. Discontinue ketamine and switch the anesthesia to continuous intravenous infusion of &#945;-chloralose (17% w/v, final concentration) mixed with FITC-dextran (2% w/v, final concentration) at 0.02 mL/10 g/h. Wait for about half an hour for stabilization of the new anesthetic state. 
	NOTE: Both FITC-dextran and &#945;-chloralose are dissolved in 0.9% saline.</li>
</ol>
3. First two-photon imaging session
<ol>
	<li>Transfer the mouse to the stage of a commercial two-photon microscope (Table of Materials). Under both red fluorescent protein (RFP, Figure 1C) and green fluorescent protein (GFP, Figure 1C) modes of epi-fluorescent illumination, take a picture of the exposed cortex with a 5x objective. Use the RFP picture as a &#39;map&#39;&#160;for insertion of the glass micro-pipette in the next session. 
	NOTE: The GFP &#39;map&#39;&#160;is used to distinguish veins/venules from arteries/arterioles.</li>
	<li>Perform two-photon imaging using the two-photon microscope and a 25x 1.0 numerical aperture (NA) water-immersion objective with piezo motor. Set the excitation wavelength to 900 nm. Search the cortex and follow each penetrating arteriole and find its horizontal branches (1st order capillaries).</li>
	<li>Image penetrating arterioles and their 1st order capillaries at rest and during whisker pad stimulation. See the detailed imaging parameters in section 5.</li>
	<li>Mark locations of 1st order capillaries with &#62;5% vasodilation during whisker pad stimulation on the RFP &#39;map&#39;&#160;(Figure 1C). Locations with &#60;5% vasodilation are considered as being outside of the whisker barrel cortex region. 
	NOTE: If using wild-type mice instead of NG2DsRed mice in the experiment, the GFP image is used as &#8216;map&#8217; instead.</li>
</ol>
4. Insertion of the glass micro-pipette
<ol>
	<li>Prepare glass micro-pipettes for puffing using a pipette puller (Table of Materials). The glass micro-pipettes have a resistance of 3-3.5 M&#8486; and a taper length of 4.5-5 mm. Load a pipette with a mixture of 1 mM ATP and 10 &#181;M Alexa 594 in order to visualize the pipette tip under the epi-fluorescent lamp and two-photon microscope.</li>
	<li>Mount the glass micro-pipette onto a patch clamp holder and connect it to an air pump. Set the pump holding pressure to 0 psi.</li>
	<li>Using red epi-fluorescent illumination, focus on the pipette tip with the 5x objective. Place the glass micro-pipette into the aCSF above the agarose. Adjust the holding pressure of the air pump to 0.2 psi. A small red cloud can be observed ejecting out of pipette tip in the aCSF. This is to prevent clogging during pipette insertion.</li>
	<li>Choose one of the marked locations in the RFP &#39;map&#39;&#160;as destination. Move the pipette tip to the horizontal plane of the cover glass edge with 30 &#181;m distance, roughly pointing towards the destination in the x-y plane.</li>
	<li>Carefully lower the pipette tip in z-axis under the cover glass slip. Then start advancing the glass pipette towards the destination until ~500 &#181;m above the brain surface. Switch focus frequently between the cover glass edge, the pipette tip and the brain surface, making sure there is enough free space for moving the pipette.</li>
	<li>Switch the objective to 25x. Re-center the pipette tip and advance the pipette tip further towards the destination on the RFP &#39;map&#39;. Keep the pipette tip in the agarose layer.</li>
	<li>When the pipette tip is ~100 &#181;m above the brain surface, switch the imaging mode to two-photon microscopy. Focus on a target location at which to puff. Write down its x, y coordinates (x0, y0) and its depth below the surface (z0). Calculate the coordinates of the insertion point on the brain surface (xi, yi) as follows: 
	xi = x0 + z0 / tan &#952; 
	yi = y0 
	where &#952; is the angle between the pipette holder and the horizontal plane.</li>
	<li>Position the pipette tip to the coordinates (xi, yi) on the brain surface. Gently and slowly insert the pipette until it reaches (x0, y0, z0). If a vessel is in the way of an insertion path, withdraw the pipette and re-calculate other insertion coordinates and path; or turn the stage plate slightly (as indicated in Figure 1A).</li>
	<li>Set the pump holding pressure at 0 psi and ejection pressure at 10-15 psi. Puff ATP for 200-400 ms at the target location during two-photon imaging. Adjust the pressure and duration of puffing for each pipette and over time as explained in the discussion section.</li>
</ol>
5. Hyperstack two-photon imaging 
<ol>
	<li>Perform experiments using the two-photon microscope and a 25 x 1.0 NA water-immersion objective with piezo motor. Set the excitation wavelength to 900 nm. Filter the emitted light to collect red light from DsRed (pericytes)/tetramethylrhodamine isothiocyanate-dextran (TRITC-dextran) staining blood plasma and green light from FITC-dextran (blood plasma)/GCaMP6 (GFP, astrocytic calcium).</li>
	<li>Produce a high-resolution z-stack at the location of interest containing a penetrating arteriole, a 1st order capillary, a 2nd order capillary and possibly neighboring fluorescent cells (e.g., pericytes, astrocytes). Determine the x*y*z volume of hyperstack imaging by including the 3D structure of the blood vessels as much as possible. The total height of each stack may vary from 30-50 &#956;m, while the x-y plane roughly covers an area of 60 &#181;m x 40 &#181;m.</li>
	<li>Set the image stack in the imaging software to be comprised of 8-10 planes with an inter-plane distance of 4-5 &#181;m. The piezo-motor objective stops at each level on the z-axis to acquire the image of each plane. The sampling rate is 1 s per stack and pixel resolution in the x-y plane is 0.2-0.3 &#181;m (Figure 2A). One recording normally includes 10 pre-puff image stacks and 150 post-puff image stacks.</li>
</ol>
6. Data processing
<ol>
	<li>Process data using custom-made analytical software. Flatten each image stack into one image by maximal intensity projection (Figure 2B). Draw a rectangular region of interest (ROI) with a width of 3 &#181;m perpendicularly across the vessel of interest (Figure 2C).</li>
	<li>To minimize interference from shadows of red blood cells and from minor vibrations of the cortex, average the rectangular ROI by projecting it into one line, which then represents the average profile of the vessel segment. Do this for each maximal intensity image.</li>
	<li>Plot the profile lines as a 2D image with the x-axis representing maximal intensity images in chronological order (Figure 2D, upper panel). Use an active contour algorithm (Chan-Vese segmentation) to find the edges of the vessel8,9 (indicated by red curves; Figure 2D, upper panel).</li>
	<li>Calculate the time course of the diameter changes as the difference between the edges of the blood vessel (vertical distance between the upper and lower red curves in Figure 2D, lower panel). Normalize diameter changes to pre-puffing diameter baseline and plot curves of diameter change over time. Do this for each ROI (Figure 2E).</li>
	<li>The vessel response amplitude is defined as the highest/lowest peak amplitude after puffing. The response latency is defined as the latency of half-max amplitude (Figure 2G). Manually trace the skeletonized vascular structure by placing nodes along the vessels (Figure 2F) and measuring geographical distance between each ROI and the penetrating arteriole. Calculate CVR speed by dividing the distances by latency differences.</li>
</ol>
7. Viral vector injection
<ol>
	<li>In order to simultaneously study astrocytic calcium responses, inject a volume of 0.6 &#181;L viral vector (pZac2.1 gfaABC1D-lck-GCaMP6f, Addgene) into the somatosensory cortex of NG2DsRed mice, following the standard stereotactic viral vector injection procedure10.</li>
	<li>After three weeks, astrocytes in mouse somatosensory cortex express the genetically encoded calcium indicator, GCaMP6f. Follow the aforementioned surgical procedure and two-photon imaging for mice. To distinguish between astrocytes and vessel lumina, TRITC-dextran instead of FITC-dextran is used to stain vessel lumina red.</li>
</ol>

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The authors have nothing to disclose.

One challenge for vascular studies is the precise measurement of vessel diameters. The method we describe here used a motorized piezo objective to make fast and repetitive hyperstack imaging by two-photon microscopy. Firstly, this method allows repeated examinations of the penetrating arteriole, 1st order and 2nd order capillaries without loss of focus and led to the discovery of slowly conducted vascular responses in capillaries in vivo. Secondly, in combination with a viral vector injection techni...

The brain has a high energy consumption rate. About 20% of the oxygen and 25% of the glucose consumed by the human body are dedicated to brain function, while the brain only occupies 2% of the total body mass. Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by blood flow in a complex network of vessels. Local brain activity and cerebral blood flow (CBF) are robustly coupled, depending on the functional properties of neurons, astrocytes, pericytes, smooth muscle cells (SMCs) and endothelial cells (ECs)1. Recently, the first few orders of capillaries branching from penetrating arterioles have emerged as a &#39;hotspot&#39;2, showing active regulation of capillary blood flow. A slow conducted vascular response (CVR) was discovered at this &#39;hotspot&#39;&#160;in mouse somatosensory cortex during both whisker stimulation and local ejection (puffing) of ATP with a glass micro-pipette3.
Although in vivo imaging by two-photon laser scanning fluorescent microscopy has been widely used for studying neurovascular responses in cerebral cortex, most of the studies measured blood vessel diameters and investigated their regulation in a two-dimensional (2D) x-y plane. The challenges are: Firstly, cerebral blood vessels and their embracing astrocytes, pericytes and SMCs construct branches in three dimensions (3D). It is therefore crucial to study their interactions in 3D. Secondly, even a small amount of drift in focus will affect the precise measurement of both vessel diameters and cellular fluorescent signals. Finally, CVRs are fast and far-reaching in three dimensions. 3D volume scanning is optimal for detecting CVRs and unveiling their mechanisms. We implemented a piezo motor objective in a two-photon microscope to study mouse somatosensory cortex in vivo, allowing precise diameter measurements by maximal intensity projections of the obtained images.
Glass micro-pipettes have frequently been used for in vivo brain studies, e.g., to bulk-load organic dyes4, record EEGs5 and for patch clamping6. Nonetheless, limitations remain. Commonly, the tip of the glass micro-pipette is imprecisely placed, or the micro-pipette is not used for local interventions. Here, we have optimized the procedure of micro-pipette insertion and local ejection.
Furthermore, the combination of 3D two-photon microscopy and genetically-encoded fluorescent indicators offers an unprecedented opportunity to investigate neurovascular coupling in a 3D scope. In this study, we took advantage of this and injected viral vectors carrying astrocyte specific genetically-encoded calcium indicators into the mouse somatosensory cortex. Astrocytes as well as vessel diameters were imaged simultaneously by combining different fluorescent markers.
Overall, we present an optimized method of local ejection (puffing) by glass micro-pipette and fast two-photon hyperstack imaging, which allows precise measurement of capillary diameter changes. In addition, our method provides a novel tool to simultaneously study 3D profiles of Ca2+ responses in astrocytes and vascular diameter responses.

<table border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1162">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Agarose</td>
			<td style="width:263px;">Sigma&#8211;Aldrich</td>
			<td style="width:152px;">A6138</td>
			<td style="width:524px;">Apply upon exposed cortex for protection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 594</td>
			<td>Life Technologies</td>
			<td>A-10438</td>
			<td>Stain puffing compound to red fluorescent color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Vasodilator and vasoconstrictor, puffing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanoacrylate glue and activator</td>
			<td>Loctite</td>
			<td>Adhesives and SF7452</td>
			<td>Glue for metal piece and coverglass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye lubricant</td>
			<td style="width:263px;">Neutral Ophtha, Ophtha A/S, Denmark</td>
			<td style="width:152px;"></td>
			<td>Keep the mouse eyes moisterized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-dextran</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">FD500S</td>
			<td>Blood serum dye, green fluorescent color</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NG2DsRed mice</td>
			<td style="width:263px;">Jackson Laboratory</td>
			<td style="width:152px;">8241</td>
			<td style="width:524px;">These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pZac2.1 gfaABC1D-lck-GCaMP6f</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:152px;">52924-AAV5</td>
			<td>Astrocyte specific viral vectors carrying genetically encoded calcium indicators</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC-dextran</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">52194</td>
			<td>Blood serum dye, red fluorescent color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">List of Equipments</td>
			<td style="width:263px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Air pump</td>
			<td>WPI</td>
			<td>PV830</td>
			<td>Give air pressure to pipette puffing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Blood gas analyzer</td>
			<td style="width:263px;">Radiometer</td>
			<td>ABL 700</td>
			<td>Measure levels of blood gases&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Blood pressure monitor</td>
			<td>World Precision Instruments</td>
			<td>BP-1</td>
			<td>Monitor aterial blood pressure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Body temperature controller</td>
			<td>CWE</td>
			<td>Model TC-1000</td>
			<td>Keep the mouse body temperature in physiological range</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">Capnograph</td>
			<td>Harvard Apparatus</td>
			<td>Type 340</td>
			<td>Monitor the end-expiratory CO2 from the mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Electrical stimulator</td>
			<td style="width:263px;">A.M.P.I.</td>
			<td>ISO-flex</td>
			<td>Apply whisker pad stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td>D-79232</td>
			<td>Mechanically ventilate the mouse in physiological range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Micropipette puller</td>
			<td style="width:263px;">Sutter Instrument</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Two-photon microscope</td>
			<td>Femtonics Ltd</td>
			<td>Femto3D-RC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">List of Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anatomical tweezer&#160;</td>
			<td>Lawton</td>
			<td>09-0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Angled and balanced tweezer</td>
			<td>S&#38;T AG</td>
			<td>00595 FRAS-18 RM-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris scissor</td>
			<td>Lawton</td>
			<td>05-1450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro vascular clamp</td>
			<td>S&#38;T AG</td>
			<td>462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse vascular catheters</td>
			<td>Verutech</td>
			<td>100828</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Once the surgery was complete, mice were transported to two-photon microscope (Figure 1A). A glass micro-pipette containing 1 mM ATP was inserted in proximity of the destination blood vessel at the target location (Figure 1B).
We performed hyperstack imaging while giving a puff of 1 mM ATP (Figure 2A, Supplementary Video 1). Each image st...

Watch this Scientific Journal Video about In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette at JoVE.com

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

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 ...

This protocol is designed to study the slow conducted vascular responses in cerebral capillaries elicited by local ejection of ATP.The advantage of this technique is to simulate local synaptic activities by local pipette ejection and to keep the scant vessel in focus by 3D two-photon microscopy.This method can also be used for studying cerebral neuroma and glial activity at rest and upon stimulation in three dimensions.Demonstrating the procedure will be Michael L

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7.4K Views.  University of Copenhagen. This protocol is designed to study the slow conducted vascular responses in cerebral capillaries elicited by local ejection of ATP.The advantage of this technique is to simulate local synaptic activities by local pipette ejection and to keep the scant vessel in focus by 3D two-photon microscopy.This method can also be used for studying cerebral neuroma and glial activity at rest and upon stimulation in three dimensions.Demonstrating the procedure will be Michael L

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

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

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

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

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

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

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

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

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

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Two-photon Imaging of Microglial Processes&#039; Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

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. ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...