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 an...

<ol>
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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 a...

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 border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Animal Strain: Mouse CD1&#160;</td>
			<td style="width:148px;">Charles River</td>
			<td style="width:159px;">022</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Isoflurane</td>
			<td style="width:148px;">AbbVie Corporation</td>
			<td>0B506-099</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CGP 55845 hydrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120337</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-(+)-Glucose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnesium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde powder, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium gluconate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1847</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium azide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma hydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium phosphate dibasic dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Sodium phosphate monobasic 
			monohydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Biocytin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:159px;">B4261</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 594 Hydrazide</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:159px;">A10438</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">SR95531 (Gabazine)&#160;</td>
			<td style="width:148px;">Abcam</td>
			<td style="width:159px;">ab120042</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Adenosine triphosphate (ATP)-Tris</td>
			<td>Sigma-Aldrich</td>
			<td>A9062</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:239px;"> 
			Guanosine (GTP)-Na+</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Oregon Green BAPTA-1</td>
			<td>ThermoFisher Scientific</td>
			<td>O6812</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Phosphocreatine di(tris) salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1937</td>
			<td></td>
		</tr>
		<tr height="42">
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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 t...

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+ ...

The overall goal of this experiment is to study calcium elevations in neuronal dendrites in response to different stimulation paradigms using whole-cell patch-clamp recordings in neuronal soma in parallel with two-photon calcium imaging in dendrites. This method can be employed to monitor calcium signaling in small dendritic compartments, allowing for close observation of the processes that occurs in dendritic branches during integration of synaptic inputs. The main advantage of this technique is that it enables calcium signaling patterns to be observed in high spatiotemporal resolution and specificity.Place the mouse brain onto a Petri dish containing continuously oxygenated, cold ACSF-sucrose. To prepare hippocampal slices from the extracted brain, let the brain chill for around two minutes. Afterward, place a piece of filter paper on an ice-packed Petri dish, and transfer the brain onto the filter paper.Then, dissect the brain into two hemispheres. Glue the dissected hemispheres onto a vibratome sectioning platform, and immerse it into the sectioning tray filled with ice-cold oxygenated ACSF-sucrose. Subsequently, section 300-micrometer-thick slices at one degree Celsius using a vibratome cooler or placing the ice around the vibratome tray.Using a paint brush, transfer the slices containing the hippocampus to a bath of oxygenated ACSF-recovery heated to 37 degrees Celsius. In this step, transfer a hippocampal slice to a bath under the objective of a laser-scanning two-photon microscope. Continuously perfuse the bath with oxygenated ACSF-normal.Then, use infrared differential interference contrast microscopy to locate a cell of interest using its size, shape, and position. After that, fill a stimulating pipette with an ACSF-normal solution containing the red fluorophore. Lower it on top of the slice so that the tip is in the same region as the cell of interest.Following that, fill the patch pipette with patch solution, and attach it to the head stage. Set it over the slice so that its tip is directly above the cell of interest. Subsequently, fit a syringe into the three-way stopcock, and connect it to the patch pipette through a plastic tube.Inject constant positive pressure into the patch pipette, and keep the stopcock in close position. Next, open the MultiClamp amplifier control software module, and switch to Voltage Clamp mode by clicking on the VC button. Open the electrophysiology Clampex data acquisition software for recording electrophysiological signals, and click on the Membrane Test icon to send out a constant square voltage pulse for monitoring changes in the pipette resistance.Now, 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 open position. Then, apply a slight negative pressure to the patch pipette using an empty syringe fitted into the stopcock until the pipette resistance reaches one gigaohm.In the Membrane Test window of the data acquisition software, clamp the cell at negative 60 millivolts. Continue applying negative pressure until the pipette resistance drops and the whole-cell configuration is achieved. In the MultiClamp amplifier control software module, set the patch configuration to current clamp by clicking on the IC button, and record membrane properties in response to somatic injections of hyperpolarizing and depolarizing currents.Wait for at least 30 minutes for the cell to be filled with the indicators present in the patch solution. To acquire images, locate a dendrite of interest using the red fluorescence signal. Ensure a visible response by choosing a proximal dendrite shorter than 50 micrometers, as the efficiency of AP backpropagation may significantly decline with distance in GABAergic interneurons, and switch to the xt mode.With the laser intensity controllers, set the two-photon laser at a minimal power level where baseline green fluorescence is just slightly visible, to avoid phototoxicity. Subsequently, in the Clampex electrophysiology data acquisition software Protocol window and image acquisition software, create a recording trial of the desired duration containing a somatic current injection of the desired amplitude. After that, click the Start Record button, and acquire the fluorescence continuously for one to two seconds.Repeat the image acquisition three to 10 times. Wait at least 30 seconds between single scans to avoid photodamage. To record dendritic calcium transients induced by electrical stimulation, locate a dendrite of interest using the red fluorescence signal.Set the stimulating pipette on the surface of the slice above the dendrite of interest. Slowly lower the stimulation pipette 10 to 15 micrometers from the dendrite, minimizing movement to avoid disturbing the whole-cell configuration. To visualize the location of synaptic microdomains in aspiny neurons, in the image acquisition software, switch to the xt mode and position the line along the dendritic branch of interest.In the Protocol window, create a recording trial that triggers the stimulation. Using the acquisition software, scan along the dendrite continuously for one to two seconds. Repeat acquisition three to five times, waiting 30 seconds between scans to prevent photodamage.To obtain preliminary information on the cell'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 xyz mode, set the upper and lower stack limits to image the entire cell with all processes included. Then, set the step size at one micrometer, and initiate the stack acquisition using the Start Record button.When the Z-stack acquisition is complete, use the Maximum Projection 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. To fix the slice for post hoc morphological identification, quickly remove the slice from the bath using a paint brush, and place it between two filter papers in an ACSF-filled Petri dish.Replace ACSF with a 4%paraformaldehyde solution, and leave the dish in a four degrees Celsius overnight. In this figure, the maximum projection of a two-photon Z-stack shows 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.This figure shows the dendritic branch near which the stimulating electrode was positioned. The dashed line shows the location of the line scan. The images here illustrate the result of a line scan in the red and green channels.The rise in green fluorescence at the time of stimulation indicates a rise in calcium concentration in that area. The images can be binned into smaller segments, allowing the analysis of the spread of the response. The traces seen here show the calcium transients elicited in different segments in response to electrical stimulation recorded at the somatic level during the imaging trial.The amplitude of the calcium transients declines with distance from the central hotspot, indicating that the response is spatially localized. Following this procedure, other methods like voltage-sensitive dye imaging or optogenetics can be performed in order to answer additional questions, like local changes in the memory potential within dendritic branches or integration of specific inputs. After watching this video, you should have a good understanding of how to monitor activity-dependent calcium fluctuations in neuronal dendrites using a combination of optophysiological techniques, which will be useful to explore the intricacies of dendritic input integration and plasticity.

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

Research

JoVE Journal

Neuroscience

10.5K visualizações.  Université Laval. The overall goal of this experiment is to study calcium elevations in neuronal dendrites in response to different stimulation paradigms using whole-cell patch-clamp recordings in neuronal soma in parallel with two-photon calcium imaging in dendrites. This method can be employed to monitor calcium signaling in small dendritic compartments, allowing for close observation of the processes that occurs in dendritic branches during integration of synaptic inputs. The main advantage of this techniqu...