This work was supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research council (NSERC Discovery Grant) and the Savoy Foundation. OC was supported by a Ph.D. fellowship from NSERC.

All experiments were performed in accordance with the animal welfare guidelines of the Animal Protection Committee of Universit&#233; Laval and the Canadian Council on Animal Care.
1. Preliminary Preparation (Optional: Prepare 1 Day in Advance)
<ol>
	<li>Prepare three types of artificial cerebrospinal fluid (ACSF) solution (Normal, Sucrose and Recovery Solutions, 1 L each; see Table 1). Adjust the osmolality of the solutions to 300 &#177; 10 mOsm25 and cool the ACSF-Sucrose down to a near-freezing point (0-4 &#176;C). 
	NOTE: The use of a low-sodium cutting solution (ACSF-Sucrose) helps preserve the viability of neurons in the superficial layers of acute slices26.</li>
	<li>Prepare 0.75 mL of patch-solution containing a red fluorophore (e.g., Alexa-594) and a green synthetic calcium indicator (e.g., Oregon Green BAPTA-1; see Table 1). Include biocytin (see Table 1) in the patch-solution if post hoc morphological identification of recorded cells is required.</li>
	<li>Adjust the osmolality of the solution to 280 &#177; 5 mOsm and pH to 7.35 &#177; 0.5. Keep the solution in the refrigerator or on ice at all times. 
	NOTE: Choice of the calcium indicator should be predicated on its dynamic range and on the nature of the investigated Ca2+ events.</li>
	<li>Using a micropipette puller, prepare patch pipettes from borosilicate glass capillaries (see Table of Materials) with a &#8776; 2 &#181;m tip (pipette resistance of 3-5 M&#937;) and stimulation pipettes from borosilicate theta-glass capillaries (see Table of Materials) with a 2-5 &#181;m tip. 
	NOTE: The puller settings to make pipettes of a given diameter and shape will be determined by the puller filament type and the ramp test results for a given type of glass.</li>
</ol>
2. Hippocampal Slice Preparation
<ol>
	<li>Anesthetize the mouse (P13-20; the strain and sex of the mouse is determined by the experimental question being addressed) with 1 mL of isoflurane placed onto a paper towel within an inhalation narcosis chamber. When the animal is deeply anesthetized, as evidenced by a lack of movement and slow respiration, remove it from the narcosis chamber and decapitate using a guillotine.</li>
	<li>Expose the skull by cutting the skin with a small pair of scissors from the back of the skull to the nose. Use a small pair of mini bone rongeurs to make a hole at the bregma level. Then use the scissors to make a caudal-to-rostral cut along the sagittal suture. With the rongeurs grasp the back of each skull flap and lift upward and outward to remove the skull covering each hemisphere.</li>
	<li>After the brain is fully exposed, undercut the optic nerves with a small spatula. Remove the brain from the skull and put it into a Petri dish containing continuously oxygenated, cold ACSF-Sucrose (see Table 1 for sucrose concentration).
	<ol>
		<li>If tissue from older mice is required, perform an intracardiac perfusion using a syringe (25G) filled with 20 mL of ice-cold ACSF-Sucrose prior to the brain extraction to obtain good quality slices.</li>
	</ol>
	</li>
	<li>Prepare hippocampal slices from the extracted animal brain.
	<ol>
		<li>Let the brain chill for around 2 min. Place a piece of filter paper on an ice-packed Petri dish, transfer the brain onto the filter paper. Dissect the two hemispheres.</li>
		<li>Glue the dissected hemispheres (the orientation is determined by the experimental goal to obtain coronal, transversal, or horizontal slices) onto a vibratome table and immerse it into the vibratome tray filled with ice-cold oxygenated ACSF-Sucrose. Make 300-&#181;m thick slices at a temperature of about 1 &#176;C by using a vibratome cooler or placing the ice around the vibratome tray.</li>
		<li>Using a flat paint brush, accumulate the slices containing the hippocampus (up to 6 slices per hemisphere) in a bath containing oxygenated ACSF-Recovery heated to 37 &#176;C.</li>
		<li>Leave the slices in the ACSF-Recovery bath for at least 45 min.</li>
	</ol>
	</li>
</ol>
3. Whole-cell Patch-clamp Recordings
<ol>
	<li>Set a hippocampal slice in place in a bath positioned under the objective (magnification: 40x, numerical aperture: 0.8) of a laser-scanning two-photon microscope. Continually perfuse the bath with oxygenated ACSF-Normal heated at 30-32 &#176;C at a rate of 2.5-3 mL/min.</li>
	<li>Use infrared differential interference contrast (IR-DIC) or Dodt-SGC microscopy to locate a cell of interest using its size, shape, and position. 
	NOTE: Depending on the targeted neuron population, a transgenic mouse model can be used to easily locate cells of interest. In this case, this step can be substituted with use of a fluorescent light source.</li>
	<li>Fill a stimulation pipette with an ACSF-Normal solution containing the red fluorophore (e.g., Alexa-594).</li>
	<li>Set the stimulation pipette (connected to an electrical stimulation unit) on top of the slice so that the tip is in the same region as the cell of interest.</li>
	<li>After filling the patch pipette with patch solution and attaching it to a head stage, set it over the slice so that its tip is directly above the cell of interest.</li>
	<li>Fit a syringe into a three-way stopcock and connect it to the patch-pipette through the plastic tube. Inject constant positive pressure into the patch pipette (&#8776; 0.1 mL) using the syringe. Keep the stopcock in &#34;close&#34; position.</li>
	<li>Activate the amplifier control software module and switch to voltage-clamp mode by clicking on the &#34;VC&#34; button. Open the electrophysiology data acquisition software (e.g., clampex) for acquisition of electrophysiological signals and click on the &#34;Membrane Test&#34; icon to have it continually send out a square voltage pulse (5 mV, 10 ms), which is necessary to monitor changes in the pipette resistance.</li>
	<li>Lower the patch pipette until it is right on top of the targeted cell. Upon making contact with the cell membrane, remove the pressure by turning the stopcock into the &#34;open&#34; position. 
	NOTE: Contact with the cell membrane is detected when a small increase in the pipette resistance (&#8776; 0.2 M&#937;) is seen and a small indentation on the cell is caused by positive pressure.</li>
	<li>Apply a slight negative pressure to the patch pipette using an empty syringe fitted into the stopcock until the pipette resistance provided by the software reaches 1 G&#937;. In the &#39;Membrane Test&#39; window of the data acquisition software, clamp the cell at -60 mV.</li>
	<li>Continue applying negative pressure until the cell is opened and the whole-cell configuration is achieved. Detection of this cell state is based upon the sudden change in the pipette resistance (from 1 G&#937; to 70-500 M&#937; depending on the interneuron type) and the appearance of large capacitive transients in the &#39;Membrane Test&#39; window.</li>
</ol>
4. Two-photon Ca2+ Imaging
<ol>
	<li>If interested in the excitatory postsynaptic responses, add the GABAA receptor blocker gabazine (10 &#181;M) and the GABAB receptor blocker CGP55845 (2 &#181;M) in the ACSF bath.</li>
	<li>In amplifier control software module, set the patch configuration to current-clamp by clicking on the &#34;CC&#34; button and record the cell&#39;s firing pattern in response to somatic injections of depolarizing current (0.8-1.0 nA, 1 s). Wait for at least 30 min for the cell to be filled with the indicators present in the patch solution. 
	NOTE: The cell&#39;s firing pattern and active properties can be used to identify the cell&#39;s subtype; e.g., fast-spiking cells. It is important for the indicator concentration to stabilize through diffusion before starting to record CaTs (see Table 2 for troubleshooting).</li>
	<li>AP-evoked CaTs
	<ol>
		<li>Using the image acquisition software, start acquiring images. Locate a dendrite of interest using the red fluorescence signal. To ensure a visible response, first, choose a proximal dendrite (&#8804; 50 &#181;m) as the efficiency of AP backpropagation may significantly decline with distance in GABAergic interneurons22.</li>
		<li>Using the &#39;rectangular tool&#39; in the image acquisition software, zoom in on the targeted region and switch to the &#34;xt&#34; (linescan) mode. Position the scan across the dendritic branch of interest. With the laser intensity controllers in the acquisition software, set the two-photon laser at a minimal power level where baseline green fluorescence is just slightly visible, to avoid phototoxicity.</li>
		<li>In the electrophysiology data acquisition software &#39;Protocol&#39; window and image acquisition software, create a recording trial of the desired duration containing a somatic current injection of the desired amplitude.
		<ol>
			<li>Using the image acquisition software, click the &#34;Start record&#34; button and acquire the fluorescence continuously for 1-2 s. Repeat the image acquisition 3-10 times. Wait at least 30 s between single scans to avoid photodamage. If acquiring linescans at multiple points along a dendrite, take them in random order to avoid order effects.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synaptically-evoked CaTs
	<ol>
		<li>Locate a dendrite of interest using the red fluorescence signal.</li>
		<li>Set the stimulation pipette on the surface of the slice above the dendrite of interest. Slowly lower the stimulation pipette into place, minimizing movement to avoid disturbing the whole-cell configuration. Position the pipette at 10-15 &#181;m from the dendrite.</li>
		<li>To visualize the location of synaptic microdomains in aspiny dendrites, in the image acquisition software switch to the &#39;xt&#39; (linescan) mode and position the line along the dendritic branch of interest.</li>
		<li>In the &#39;Protocol&#39; Window (of electrophysiology data acquisition software) and image acquisition software, create a recording trial that triggers the stimulation unit after the trial start.</li>
		<li>Using the acquisition software, click the &#34;Start record&#34; button to scan along the dendrite continuously for 1-2 s. Repeat acquisition 3-5 times, waiting 30 s between scans to prevent photodamage. 
		NOTE: The length of scan can be adjusted depending on the evoked event&#39;s kinetics, but should be minimized to prevent photodamage (see Table 2 for troubleshooting).</li>
		<li>Repeat step 4.5.5 in different conditions (e.g., different intensity/length of stimulation, introduction of a pharmacological blocker, etc.) depending on the specific question being addressed.</li>
		<li>Stop the acquisition when the cell shows signs of deteriorating health: depolarization below -45 mV, increase in the baseline Ca2+ level, morphological deterioration of dendrites (e.g., blebbing, fragmentation; see Table 2 for troubleshooting).</li>
		<li>To obtain preliminary information on the cell&#39;s morphology and keep a record of the location of the stimulation pipette, acquire a Z-stack of the cell in the red channel. Using the acquisition software in &#39;xyz&#39; mode, set the upper and lower stack limits to image the entire cell with all processes included. Set the &#39;step size&#39; at 1 &#181;m and initiate the stack acquisition using the &#34;Start record&#34; button.</li>
		<li>When the Z-stack acquisition is complete, use the &#34;Maximum projection&#34; option in the software to superimpose all focal plans of the stack and verify the quality of acquisition. Then, slowly retract the patch pipette out of the slice.</li>
		<li>To fix the slice for post hoc morphological identification, quickly remove the slice from the bath using a flat paint brush, and place it between two filter papers, in an ACSF-filled Petri dish. Replace the ACSF with a 4% paraformaldehyde (PFA) solution and leave the dish in a 4 &#176;C room or refrigerator overnight.</li>
	</ol>
	</li>
</ol>
5. Immunohistochemistry for Post Hoc Morphological Identification of Recorded Cells
NOTE: After 1 night of fixation in PFA, the slice can be stored in phosphate buffer (PB) with sodium azide (0.5%) for up to 1 month.
<ol>
	<li>Put the slice in Tris-buffered saline solution (TBS) with Triton X-100 (0.3%) and perform 4 washes (5-10 min each).</li>
	<li>Put the slice in TBS-Triton 0.3% with 10% normal goat serum (NGS) for 1 h.</li>
	<li>Put the slice in TBS-Triton 0.3% with 1% NGS and a Streptavidin-conjugated fluorophore (e.g., Alexa Fluor 546-conjugated Streptavidin, 1:200) for overnight incubation.</li>
	<li>Wash 4 times (5-10 min) in TBS.</li>
	<li>Mount slices on microscope slides and image using a confocal microscope. To have a complete cell reconstruction, acquire a Z-stack (step size: 1 &#181;m) using a 20x objective. For imaging an Alexa-546-conjugated label, use a 543 nm laser and a 515-560 nm detection filter set.</li>
</ol>
6. Analysis of CaTs
<ol>
	<li>Extract the fluorescence values from the regions of interest (ROIs) in the linescan images from the red and green channels and average the values of the multiple repeats for each condition to reduce noise. 
	NOTE: When linescan acquisition is performed along the dendrite, it is possible to extract data from multiple ROIs, which may correspond to dendritic microdomains (2-5 &#181;m), and thus allowing the study of the Ca2+ signal compartmentalization.</li>
	<li>For each ROI, express the changes in Ca2+ amplitude as: 
	<img alt="Equation" src="/files/ftp_upload/56776/56776eq1.jpg" /> 
	NOTE: Here G is the fluorescence value from the green channel; G0 is the baseline fluorescence value from the green channel during the pre-stimulation period; R is the fluorescence value from the red channel18. As two-photon excitation is highly localized and does not generate a significant background, subtraction of background fluorescence is not required.</li>
</ol>

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T., Sieneart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+laser+scanning+microscopy+using+an+acoustic+optical+deflector.">Multi-photon laser scanning microscopy using an acoustic optical deflector.</a> Biophys J. 83, 2292-2299 (2002).</li></ol>

The authors have no competing financial interests or other conflicts of interest.

The method shown here demonstrates how the combination of two-photon Ca2+ imaging and patch-clamp electrophysiology can be used for studying dendritic Ca2+ signaling in neuronal dendrites in acute brain slices. This method allows for monitoring of both the local Ca2+ elevations evoked by electrical stimulation or backpropagating AP in dendritic segments, and the cell&#39;s somatic response. This makes it an excellent tool to study how various parts of the dendritic tree integrate inputs and communicate with the soma. But given the length of the experiment, particular attention needs to be paid to the cell health by limiting the use of laser power for imaging so as to avoid the photodamage of dendrites. This can be also achieved by decreasing the zoom level, decreasing the duration of scan, and increasing the scan speed during image acquisition. Imaging interneuron dendrites introduces additional challenges. As dendrites of these cells have no spines, scanning along the dendrite is recommended to facilitate the localization of postsynaptic Ca2+ microdomains. In addition, given a high endogenous Ca2+ binding capacity, Ca2+ elevations in interneuron dendrites are smaller and slower than those generated in the dendrites of pyramidal cells. This requires acquiring several linescan images for averaging as well as a sufficiently long duration of individual linescans in order to describe the Ca2+ signal kinetics appropriately.
Local electrical stimulation using bipolar theta-glass electrode represents a useful tool for characterization of dendritic postsynaptic Ca2+ microdomains. However, it may still activate different &#34;en passant&#34; axons located in a given region. The number of inputs activated in this case is unknown and can be only estimated using additional computational tools. Two-photon glutamate uncaging27, which allows for simultaneous stimulation of multiple individual synapses, represents a good alternative for eliciting local dendritic Ca2+ responses. This method is widely used for study of principal cells, which have clearly defined excitatory postsynaptic structures (spines), but is not well-suited to the study of interneurons, which overall have a low spine density. Uncaging glutamate along the interneuron dendrite as it is applied to principal cells would unavoidably activate multiple extrasynaptic mechanisms of Ca2+ signaling9,10, making it difficult to interpret the observations.
In the last decade, advances in optical technologies offered improved temporal resolution for two-photon Ca2+ imaging. One of the most promising is random-access microscopy (RAMP) through acousto-optic deflectors (AODs)28,29. Unlike conventional raster scanning, RAMP scans selected points of interest at high frequency, which is enabled by the fast speed of inertia-free AODs. It is ideal for the simultaneous study of multiple dendritic branches, as it is not limited to points laid out in a linear pattern. However, the higher temporal resolution offered by the AODs may be unnecessary when imaging Ca2+ events lasting hundreds of milliseconds within the same focal plan. In addition, the location of scanning points within the same structure of interest may be difficult to control due to micro-drifts in slice preparations associated with a high perfusion rate, which is necessary for good slice quality. Hence, the high temporal resolution (500-700 Hz) linescan-based acquisition method presented in this article is still preferable when imaging Ca2+ signals in individual dendritic branches.

The contribution of a neuron to network activity is largely determined by the dynamic nature of the synaptic inputs it receives. Traditionally, the predominant method of characterizing synaptic activity in neurons relied on somatic whole-cell patch-clamp recordings of postsynaptic currents evoked by electrical stimulation of axons of passage. However, only activity of the proximally located synapses is truthfully reported in this case1. In addition, to assess the synapse-specific mechanisms, recordings from pairs of neurons and from dendrites at a specific location have been used to target the synapses of interest and the mechanisms of dendritic integration, respectively. A major breakthrough in the field of synaptic physiology was achieved through a marriage of optical and electrophysiological techniques. Two-photon excitation laser scanning microscopy (2PLSM) in combination with Ca2+ imaging and optogenetic tools can reveal tremendous details of the dynamic organization of synaptic activity at specific neuronal connections in brain slices in vitro and in vivo.
Several key advantages made 2PLSM stand out from the conventional, one-photon excitation microscopy2: (1) due to a nonlinear nature of two-photon excitation, the fluorescence is generated only in the focal volume, and all emitted photons represent useful signals (no need for pinhole); (2) longer wavelengths, used in 2PLSM, penetrate the scattering tissue more efficiently; in addition, scattered photons are too dilute to produce two-photon excitation and background fluorescence; (3) photodamage and phototoxicity are also limited to the focal plane. Therefore, despite the high cost of 2-photon systems in comparison with conventional confocal microscopes, the 2PLSM remains a method of choice for high-resolution investigation of neuronal structure and function in thick living tissue.
The first application of 2PLSM in scattering tissue was to image the structure and function of dendritic spines3. 2PLSM in combination with Ca2+ imaging has revealed that spines function as isolated biochemical compartments. Since in many neuronal types there is a one-to-one correspondence between spines and individual synapses4, two-photon Ca2+ imaging soon became a useful tool reporting the activity of individual synapses in intact tissue5,6,7,8. Furthermore, 2PLSM-based Ca2+ imaging was successfully used to monitor the activity of single calcium channels and the nonlinear interactions between the intrinsic and synaptic conductances, as well as to assess the state- and activity-dependent regulation of Ca2+ signaling in neuronal dendrites5,6,7,8,9,10,11.
Calcium is a ubiquitous intracellular second messenger, and its subcellular spatiotemporal organization determines the direction of physiological reactions, from changes in synaptic strength to the regulation of ion channels, dendrite and spine growth, as well as cell death and survival. Dendritic Ca2+ elevations occur via activation of multiple pathways. Action potentials (APs), backpropagating to the dendrites, open voltage-gated calcium channels12 and produce relatively global Ca2+ transients (CaTs) in dendrites and spines13. Synaptic transmission is associated with activation of postsynaptic Ca2+-permeable receptors (NMDA, Ca2+-permeable AMPA and kainate), triggering synaptic CaTs6,14,15. Finally, supralinear Ca2+ events can be generated in dendrites under certain conditions11,12,13,16.
Two-photon Ca2+ imaging in combination with patch-clamp electrophysiological recordings employs synthetic Ca2+-sensitive fluorescent indicators, which are typically delivered through the patch electrode during whole-cell recordings. A standard method for quantification of Ca2+ dynamics is based on the dual indicator method17,18. It uses two fluorophores with well separated emission spectra (e.g., a combination of a red Ca2+-insensitive dye with green Ca2+ indicators, such as Oregon Green BAPTA-1 or Fluo-4) and has several advantages when compared to the single indicator method. First, a Ca2+-insensitive dye is used to locate small structures of interest (dendritic branches and spines) where Ca2+ imaging will be performed. Second, the ratio between the change in green and red fluorescence (&#916;G/R) is calculated as a measure of [Ca2+], which is largely insensitive to changes in baseline fluorescence due to fluctuations in [Ca2+]017,18. Furthermore, fluorescence changes can be calibrated in terms of absolute Ca2+ concentrations19.
A general concern when running two-photon Ca2+ imaging experiments in acute slices is cell health and stability of the image acquisition due to the high laser power typically used. Additionally, in Ca2+ imaging experiments, there is concern about the perturbation and substantial overestimation of subcellular Ca2+ dynamics due to the fact that Ca2+ indicators act as highly mobile exogenous Ca2+ buffers. Thus, the choice of the Ca2+ indicator and its concentration depends on the neuronal type, the anticipated amplitude of CaTs, and on the experimental question.
We adapted the method of two-photon Ca2+ imaging for investigation of Ca2+ fluctuations in dendrites of GABAergic interneurons9,10,11,20,21,22. While the bulk of early Ca2+ imaging studies were done in principal neurons, inhibitory interneurons showcase a large variety of functional Ca2+ mechanisms that are distinct from those in pyramidal cells20,23,24. These interneuron-specific mechanisms (e.g., Ca2+ permeable AMPA receptors) may play specific roles in regulating cell activity. While dendritic Ca2+ signaling in interneurons is a tempting target for further investigation, two-photon Ca2+ imaging in dendrites of these cells presents additional challenges, from a thinner diameter of dendrites and lack of spines to a particularly high endogenous Ca2+ binding capacity. As our research interests focus on the study of hippocampal interneurons, the following protocol, while applicable to different neuronal populations, was adapted to deal with those challenges.
This protocol was executed with a commercial confocal two-photon microscope, which was equipped with two external, non-descanned detectors (NDDs), electro-optical modulator (EOM), and a Dodt scanning gradient contrast (SGC), and installed on an optical table. The microscope was coupled with a Ti:Sapphire multiphoton laser mode-locked at 800 nm (&#62; 3 W, 140 fs pulses, 80 Hz repetition rate). The imaging system was equipped with a standard electrophysiology rig, including a perfusion chamber with temperature control, a translating platform with two micromanipulators, a computer-controlled microelectrode amplifier, a digitizer, a stimulation unit, and data acquisition software.

<table><tbody><tr><td>Animal Strain: Mouse CD1</td><td>Charles River</td><td>022</td><td></td></tr><tr><td>Isoflurane</td><td>AbbVie Corporation</td><td>0B506-099</td><td></td></tr><tr><td>CGP 55845 hydrochloride</td><td>Abcam</td><td>ab120337</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Magnesium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Paraformaldehyde powder, 95%</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium gluconate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1847</td><td></td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S8875</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Trizma hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>&lt;br/&gt; Sodium phosphate monobasic&lt;br/&gt; monohydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Biocytin</td><td>Sigma-Aldrich</td><td>B4261</td><td></td></tr><tr><td>Alexa Fluor 594 Hydrazide</td><td>ThermoFisher Scientific</td><td>A10438</td><td></td></tr><tr><td>SR95531 (Gabazine)&amp;nbsp;</td><td>Abcam</td><td>ab120042</td><td></td></tr><tr><td>&lt;br/&gt; Adenosine triphosphate (ATP)-Tris</td><td>Sigma-Aldrich</td><td>A9062</td><td></td></tr><tr><td>&lt;br/&gt; Guanosine (GTP)-Na&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>G8877</td><td></td></tr><tr><td>&lt;br/&gt; Oregon Green BAPTA-1</td><td>ThermoFisher Scientific</td><td>O6812</td><td></td></tr><tr><td>&lt;br/&gt; Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td></td></tr><tr><td>&lt;br/&gt; Streptavidin-conjugated Alexa-546</td><td>ThermoFisher Scientific</td><td>S11225</td><td></td></tr><tr><td>&lt;br/&gt; Patch Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>&lt;br/&gt; Theta Borosilicate Glass Capillaries</td><td>Sutter Instrument</td><td>BT-150-10</td><td></td></tr><tr><td>&lt;br/&gt; P-97 Flaming/Brown Micropipette puller</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>&lt;br/&gt; TCS SP5 Confocal Multiphoton Microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Chameleon Ultra II Ti:Sapphire multiphoton laser&amp;nbsp;</td><td>Coherent</td><td></td><td></td></tr><tr><td>LAS AF Imaging Acquisition Software</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Temperature Controller TC-324B</td><td>Warner Instruments</td><td></td><td></td></tr><tr><td>&lt;br/&gt; MultiClamp 700B Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Digidata 1440A Digitizer</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Confocal Translator</td><td>Siskiyou</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Micromanipulator</td><td>Siskiyou</td><td></td><td></td></tr><tr><td>&lt;br/&gt; pClamp Data Acquisition Software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; A365 Constant Current Stimulus Isolator</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Vibraplane Optical Table</td><td>Kinetic Systems</td><td></td><td></td></tr></tbody></table>

two-photon calcium imaging in neuronal dendrites in brain slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

Using the protocol presented here, we obtained CaTs evoked by somatic current injection and by electrical stimulation in dendrites of oriens/alveus interneurons in the CA1 area of the hippocampus. After patching a neuron, identified based on its shape and position, we acquired linescans across a proximal dendrite at multiple points at given distances from the soma (Figure 1A). We observed a decrease in the amplitude of CaTs induced by backpropagating APs as the distance from the soma increased (indicative of reduced AP amplitude or a distance-dependent decline in calcium channel distribution, Figure 1B). After immunohistochemistry (using Streptavidin-conjugated Alexa Fluor 546), we were able to retrieve the biocytin labeling of the cell and identify the recorded neuron as a bistratified cell with an axon occupying stratum oriens and stratum radiatum (Figure 1C). In the second experiment, a stimulation pipette was brought to a distal dendritic site. Scanning along the dendrite, we were then able to assess the amplitude of the postsynaptic Ca2+ signals in a given dendritic branch (Figure 2A-C) in response to electrical stimulation of axons of passage. The postsynaptic CaTs differed between neighboring dendritic microdomains (segment 1 to 6; Figure 2D), which could be associated with a spread of Ca2+ signal from the zone of initiation and/or different postsynaptic mechanisms involved.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56776/56776fig1.jpg" /> 
Figure 1: Representative example of AP-evoked Ca2+ transients. (A) Maximum projection of a two-photon Z-stack (13 &#181;m) of a patched interneuron filled with Alexa-594 (20 &#181;M). White lines denote the location and length of linescans. (B) Traces showing the CaTs elicited in the locations shown in A. Top trace shows the short AP train generated through somatic current injection during the recording trial. Time scale is the same on all traces shown. (C) Maximum projection of a confocal Z-stack showing the biocytin labeling of the cell. O/A: Oriens/Alveus; PYR: Stratum Pyramidale; RAD: Stratum Radiatum. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56776/56776fig1large.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/56776/56776fig2.jpg" /> 
Figure 2: Postsynaptic Ca2+ transients evoked by a burst of electrical stimulation (3 stimuli at 100 Hz). (A) Maximum projection of a two-photon Z-stack (148 &#181;m) showing a patched interneuron filled with Alexa-594. White arrowheads point to the location of axonal terminals within the pyramidal layer, suggesting the interneuron is a basket cell. (B) Single focal plane illustrating the dendritic branch where the stimulation electrode was positioned. Dashed line shows the location of the linescan shown in (C1 and C2). (C) Images illustrating the result of a linescan in the red (1; Alexa-594; 20 &#181;M) and green (2; Oregon Green Bapta-5N; 300 &#181;M) channels. The images were binned into smaller segments to analyze the spread of the response over 30 &#181;m. (D) Traces showing the Ca2+ transients (CaTs) elicited in the segments displayed in C2. Top trace shows the voltage response to electrical stimulation recorded at the somatic level during the imaging trial. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56776/56776fig2large.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>Solution</td>
			<td>Component</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td rowspan="7">ACSF-Normal</td>
			<td>NaCl</td>
			<td>124</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>2</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2</td>
		</tr>
		<tr>
			<td rowspan="7">ACSF-Recovery</td>
			<td>NaCl</td>
			<td>124</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="7">ACSF-Sucrose</td>
			<td>KCl</td>
			<td>2</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>1.25</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>7</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>26</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>219</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="9">K+-based Patch solution</td>
			<td>K+-gluconate</td>
			<td>130</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Phosphocreatin di(tris)salt</td>
			<td>10</td>
		</tr>
		<tr>
			<td>ATP-Tris</td>
			<td>2</td>
		</tr>
		<tr>
			<td>GTP-Tris</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>Biocytin</td>
			<td>72</td>
		</tr>
		<tr>
			<td>Alexa-594</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>Oregon Green-BAPTA-1</td>
			<td>0.2</td>
		</tr>
	</tbody>
</table>
Table 1: Solution recipes. Compounds and concentrations for solutions used during the protocol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Unable to patch healthy neuron</td>
			<td>Check for signs that the slices are unhealthy: shrunken or swollen cells, visible nuclei, etc. If so, discard the slices. Also verify the patch-pipette resistance and the patch-solution&#8217;s osmolality; replace them if the values are not in the appropriate range.</td>
		</tr>
		<tr>
			<td>Fluorescence signal from dendrites is low</td>
			<td>Wait longer for the cell to fill. If an obstruction is keeping the patch-solution from diffusing into the cell, try to apply a small amount of negative pressure in the patch-pipette.</td>
		</tr>
		<tr>
			<td>Electrical stimulation is not evoking a Ca2+ response</td>
			<td>Check for the presence of an artefact in the electrophysiological recording. If absent, check for a short-circuit/stimulating unit malfunction. If present, raise the stimulation intensity or move the stimulation pipette closer to the dendrite. It is to be noted that the distance between the stimulation electrode and the dendrite should not exceed 8 um to prevent direct depolarization of dendrites when studying synaptic responses.</td>
		</tr>
		<tr>
			<td>Evoked Ca2+ signal is too high/saturates the Ca2+ indicator</td>
			<td>Reduce laser power. If the problem persists in multiple cells, use a different Ca2+ indicator, 
			with a lower Ca2+ affinity.</td>
		</tr>
		<tr>
			<td>Baseline Ca2+ signal is gradually increasing during the experiment</td>
			<td>Wait for a longer period between individual scans. If the baseline is still increasing, stop 
			acquisition; it indicates that the cell&#8217;s health is likely declining.</td>
		</tr>
		<tr>
			<td>Amplitude of Ca2+ signal decreases during scans</td>
			<td>Reduce laser power or zoom out (if applicable).</td>
		</tr>
		<tr>
			<td>Dendrite is fragmenting (&#8220;blebbing&#8221;) after scanning</td>
			<td>Reduce the laser power or zoom out. If blebbing is limited to the targeted dendrite, choose another dendrite and reduce laser power/ zoom out. If multiple dendrites are blebbing, stop acquisition.</td>
		</tr>
		<tr>
			<td>Fluorescence signals are &#8220;drifting&#8221; out of the scan line after sweeps</td>
			<td>Reduce movement in the slice by reducing the speed of ACSF perfusion. Before patching, make 
			sure that the slice is strongly fixed in place by a net.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting table. Solutions to common problems that may arise during a Ca2+ imaging experiment.

Watch this Scientific Journal Video about Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices at JoVE.com

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

two-photon-calcium-imaging-in-neuronal-dendrites-in-brain-slices

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.7K Views.  Université Laval. Presenteren we een methode geheel-cel patch-clamp opnamen en twee-foton imaging om vast te leggen van Ca2 + transiënten in neuronale dendrites in acute hersenen segmenten combineren.

Video: Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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

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

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

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

Biology

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Behavior

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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 ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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