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+ ...

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 ...

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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 ...

In vivo Neuronal Calcium Imaging in C. elegans

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, ...

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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

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 ...

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 ...

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 ...

In vivo Calcium Imaging in Mouse Inferior Olive

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 ...

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

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 ...