Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Summary

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.

Abstract

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.

Introduction

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 dendriti....

Protocol

All experiments were performed in accordance with the animal welfare guidelines of the Animal Protection Committee of Université Laval and the Canadian Council on Animal Care.

1. Preliminary Preparation (Optional: Prepare 1 Day in Advance)

1. 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 ± 10 mOsm²⁵ and cool the ACSF-Sucrose down to a near-freezing point (0-4 °C).
   NOTE: The use of a low-sodium cutting solution (ACSF-Sucrose) helps preserve the viability ....

Results

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.......

Discussion

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's somatic response. This makes it an excellent tool to study how various parts of the dendritic tree integrate inputs a.......

Materials

List of materials used in this article

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