Department of Integrative Neurophysiology, VU University, Amsterdam
Dawitz, J., Kroon, T., Hjorth, J. J., Meredith, R. M. Functional Calcium Imaging in Developing Cortical Networks. J. Vis. Exp. (56), e3550, doi:10.3791/3550 (2011).
A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.
1. Making horizontal entorhinal-hippocampal brain slices
Entorhinal-hippocampal brain slices are made using anatomical guides, detailed in a 3D overview of the region according to Canto, Wouterlood & Witter (2008) Neural Plasticity ID 3812439.
|Slice solution (in mM) – 10|
Table 1. Recipe for slice solution.
|ACSF (in mM)|
Table 2. Recipe for both recovery (r-ACSF) and experimental (e-ACSF) solution.
2. Preparation of the staining chamber
To load the cells with the calcium-dependent indicator or cell-specific marker, slices need to be transferred to a chamber for the staining procedure. Although commercial chambers may be available, one can easily be assembled from standard lab equipment for very little cost. The key features of such a chamber are that the slices are warmed to between 30 and 35°C, incubated in a continuously oxygenated medium and that the chamber is shielded from light.
Methodology Figure 1. Cross-section of the staining chamber showing slice incubation (above) and application of calcium-sensitive indicator (pipetted green dye, below).
3. Staining of slices
Throughout any handling involving fluorescent dyes, avoid photobleaching by working with little light and keep the dye and the stained tissue in the dark between handling.
|Age (postnatal days)||Incubation time (min)|
Table 3. Incubation times for different ages, empirically-determined in the lab.
4. Other dyes and older tissue
Perhaps due to increased myelination in the brain tissue, slices from older rodents do not take up Fura 2-AM ester that easily. To facilitate uptake of this dye a preincubation step using Cremophor EL (Sigma) is used for brains of mice at P13 and older11. Cremophor is a non-ionic surfactant used as an excipient in many pharmaceutical applications. Without this step, we find that cell-specific labelling is extremely poor and inconsistent throughout the cortical slice.
Calcium dyes load both neurons and non-neurons in the slice preparation. To identify and distinguish between these cell types within the network, sulforhodamine 101 (SR101) can be used to label astrocytes within the slice.
Staining of slices for sulforhodamine 101
Follow steps 1-3 as before (section 3).
Take 1 μl of 10mM stock solution of sulforhodamine (Sigma) from its storage at -20°C and dissolve in 999 μl r-ACSF to give a 10 μM solution. Pipette the purple dye over the slices as before (steps 5-7), leaving the slices incubating for a period of 15 minutes.
Microglia and endothelial cells are labelled using FITC tomato lectin dye
Follow steps 1-3 as before.
Take 25 μl of 2mg/ml Lycopersicon esculentum (tomato) lectin FITC conjugate (L0401, Sigma) stock solution into 2.5ml r-ACSF to give 20 μg/ml concentration. Pipette the dye over the slices as before (steps 5-7), leaving the slices incubating for a period of 45 minutes.
Note, it is not possible to combine this dye with a calcium-sensitive indicator like Fura 2-AM or Oregon Green BAPTA-1 (OGB-1) that fluoresce in the green range of the spectrum since the photons from both dyes will be emitted at overlapping wavelengths. However, there are other calcium-indicator dyes such as Calcium orange, Fura Red that may be used in combination with appropriate filters or Texas-Red lectin conjugates to combine with calcium-indicator dyes in the green spectrum.
5. Attaching slices to recording chamber
During imaging slices need to be stable under the microscope. Usually a metal harp is placed to hold down the tissue but it can unevenly distort the surface of the slice, giving only part of the field of view for imaging in focus. To avoid this, slices are stuck to the recording chamber using Polyethylenimine (PEI).
|in 250ml boric buffer|
|10mM||sodium tetraborate decahydrate|
Table 4. Recipe PEI solution.
Calcium-dependent indicator dyes can be imaged using either one- or two-photon microscopy. Use of two-photon imaging only activates indicator dye within the focal volume of the region of interest, thus reducing the amount of light scatter in the tissue. Furthermore, it enables better depth penetration of the light into the slice.
For functional calcium imaging we use a Titanium sapphire laser supplied by Coherent coupled to an Olympus microscope with a 20x objective (NA 0.95) and a Trimscope system by LaVision Biotec. The Trimscope system enables frame scanning with 64 beamlets simultaneously and is coupled with a Hamamatsu C9100 EM-CCD camera for fast frame-scanning rates.
7. Representative results:
Successful loading of calcium indicators, Fura 2-AM are shown in Figure 1 in developing neocortical and entorhinal cortex networks using multiphoton imaging. Some dye is still present as background staining in the tissue but cell soma and in some cases, proximal dendrites are clearly visible and separate from surrounding neuropil. If loading has not been successful, very little cell-specific staining is observed and small clusters of dye spots are often visible on the slice surface in dead membrane debris.
In these screenshots, the network of cells is clearly visible in a single plane of focus for simultaneous cell imaging. Use of a metal harp or incomplete sticking of the slice to the recording chamber can result in an uneven slice surface to be imaged.
Figure 1. Fura 2-AM ester-loaded developing neocortical (A, left) and entorhinal (B, right) networks. Scale bars 100 μm.
To separate neurons from astrocytes, co-application of sulforhodamine 101 with Fura 2-AM ester enables separation of cell types within the network.
Figure 2. Astrocyte labeling with sulforhodamine 101. A Co-labelling of Fura 2-AM ester and sulforhodamine 101. Excitation wavelength: 820nm. Image collection on PMTs with dichroic mirror at 560/70nm for wavelength separation. B Representative fluorescence traces from a neuron (above) and an astrocyte (below). Scale bars 60 sec, ΔF 10 (au fluo).
Figure 3. FITC-conjugated Lycopersicon esculentum (tomato) lectin staining. Labelling of microglia and endothelial cells in developing mouse hippocampus (A) and superficial entorhinal cortex (B).
Calcium indicator dyes are used to read-out activity from multiple cells simultaneously in developing cortical and hippocampal networks.
Figure 4. Dynamic calcium transients in hippocampal and cortical networks.
Movie 1: Fura 2-AM ester loading of mouse entorhinal cortex during second postnatal week.
Click here to view movie 1.
Movie 2: Fura 2-AM ester loading of mouse hippocampus during the first postnatal week.
Click here to view movie 2.
Movie 3: Fluo-4 loading of mouse cortex, during the first postnatal week.
Click here to watch movie 3.
In the case of Fura 2-AM ester, cell activation involving a depolarization-induced influx of calcium, decreases dye fluorescence. For dyes such as Fluo-4, the opposite is true and cell depolarization is observed as an increase in photon emissions. Somatic calcium transients are mainly measured but activity in larger proximal dendrites can also be seen in some preparations, as shown in Movie 2.
Read-out of network activity can be quantified using commercial or in-house software scripts. In our lab, cell identification and network activity is measured and analyzed in a semi-automated manner using in-house code for Matlab (Mathworks).
Figure 5. Representative analysis of synchronized calcium transients from a developing cortical network. 3D representation of one neuron (A) used to automatically create a neuron mask (B) from a z-stack for automated neuron detection. Measurements of calcium transients include amplitude, frequency and # of active cells that can be read out from single neuron data (C). Synchrony between different traces can be visualized using a raster plot (D).
The methods we demonstrate here show suitable protocols for calcium imaging of network dynamics from identifiable cells within developing cortical and hippocampal networks in the mouse and also in the rat brain. These methods provide optimal spatial resolution to visualize a local network of cell somas simultaneously for measuring suprathreshold activity. Temporal resolution of network activity can be varied, depending on frame acquisition CCD camera settings, to optimize signal:noise measurements for long duration suprathreshold events, typically found in the developing nervous system. These protocols are not restricted to hippocampal and cortical networks but also label cells throughout the developing nervous system. The limitation of this method is that only suprathreshold activity can be recorded.
LaVision Biotec GmBH (Bielefeld, Germany) sponsored the submission fees of this manuscript article.
Work in the lab is supported by Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) (917.10.372) to RMM. JD is affiliated to the EU FP7 BrainTrain programme (www.brain-train.nl). We thank Pieter Laurens-Baljon and Sabine Schmitz (both CNCR, VU University Amsterdam) for images of FP multielectrode array waveforms and neuronal cultures used in the video sequence.