Name: two-photon calcium imaging in neuronal dendrites in brain slices
Uploaded: 2018-03-15T13:30:00
Description: Watch this Scientific Journal Video about Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices at JoVE.com

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.

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			<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>
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			<td height="40" style="height:40px;width:239px;">Isoflurane</td>
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			<td>0B506-099</td>
			<td></td>
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			<td height="40" style="height:40px;">CGP 55845 hydrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120337</td>
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			<td height="40" style="height:40px;">Calcium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
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			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
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			<td height="40" style="height:40px;">Paraformaldehyde powder, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
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			<td height="40" style="height:40px;">Potassium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
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			<td height="40" style="height:40px;">Potassium gluconate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1847</td>
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			<td height="40" style="height:40px;">Sodium azide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
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			<td height="40" style="height:40px;">Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
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			<td height="40" style="height:40px;">Sodium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
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			<td height="40" style="height:40px;">Sucrose&#160;</td>
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			<td>T9284</td>
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			<td height="40" style="height:40px;">Trizma base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
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			<td height="40" style="height:40px;">Trizma hydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
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			<td height="40" style="height:40px;">Sodium phosphate dibasic dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71643</td>
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			<td height="40" style="height:40px;width:239px;"> 
			Sodium phosphate monobasic 
			monohydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
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			<td height="40" style="height:40px;width:239px;">Biocytin</td>
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			<td height="40" style="height:40px;">Alexa Fluor 594 Hydrazide</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:159px;">A10438</td>
			<td></td>
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			<td height="40" style="height:40px;width:239px;">SR95531 (Gabazine)&#160;</td>
			<td style="width:148px;">Abcam</td>
			<td style="width:159px;">ab120042</td>
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			<td height="42" style="height:42px;width:239px;"> 
			Adenosine triphosphate (ATP)-Tris</td>
			<td>Sigma-Aldrich</td>
			<td>A9062</td>
			<td></td>
		</tr>
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			<td height="45" style="height:45px;width:239px;"> 
			Guanosine (GTP)-Na+</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;"> 
			Oregon Green BAPTA-1</td>
			<td>ThermoFisher Scientific</td>
			<td>O6812</td>
			<td></td>
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			<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">
			<td height="42" style="height:42px;width:239px;"> 
			Streptavidin-conjugated Alexa-546</td>
			<td>ThermoFisher Scientific</td>
			<td>S11225</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Patch Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Theta Borosilicate Glass Capillaries</td>
			<td>Sutter Instrument</td>
			<td>BT-150-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			P-97 Flaming/Brown Micropipette puller</td>
			<td style="width:148px;">Sutter Instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			TCS SP5 Confocal Multiphoton Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			Chameleon Ultra II Ti:Sapphire multiphoton laser&#160;</td>
			<td>Coherent</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">LAS AF Imaging Acquisition Software</td>
			<td>Leica Microsystems</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Temperature Controller TC-324B</td>
			<td style="width:148px;">Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			MultiClamp 700B Amplifier</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Digidata 1440A Digitizer</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Confocal Translator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Micromanipulator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			pClamp Data Acquisition Software</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			A365 Constant Current Stimulus Isolator</td>
			<td style="width:148px;">World Precision Instruments</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Vibraplane Optical Table</td>
			<td style="width:148px;">Kinetic Systems</td>
			<td style="width:159px;"></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 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

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

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.

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 ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...