In vivo two‐photon calcium imaging using multi‐cell bolus loading
Physiologisches Institut, Ludwig-Maximilians Universität München, Pettenkoferstr. 12, 80336 München, Germany
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subcutaneously at the location were the skin is to be
This chapter describes an approach for in vivo
two-photon Ca2+ imaging of large neuronal circuits with
2. Remove the skin above the desired brain area. Perform
a small (~1 mm) craniotomy above an area devoid of
big blood vessels. Thin the skull near the craniotomy
Area of application
and polish it with a felt polisher (for example, from Dr.
The approach was developed for in vivo imaging
Ihde Dental, Munich, Germany). Use cyanoacryl glue
of the cortex. It can be easily adapted for imaging other
to adhere the custom-made recording chamber to the
brain regions, including the cerebellum and olfactory bulb.
skull, such that the middle of the chamber opening lies
Recently it was successfully used for in vivo recordings
from individual spinal cord neurons in zebrafish larvae
3. Transfer the animal into the set-up and place onto a
(Brustein et al., 2003). The staining technique can be also
warming plate (38° C). Perfuse the recording chamber
applied in brain slices of any developmental stage, from
with a warm (37°C) standard external saline. We used
a saline containing (in mM): 125 NaCl, 2.5 KCl, 26
NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 20 glucose,
pH 7.4, when bubbled with 95% O2 and 5% CO2.
1. Anesthesia unit including chamber for pre-anesthetic
4. Dissolve acetoxymethyl (AM)-ester of an indicator dye
medication and flow meter plus vaporizer (latter
in DMSO plus 20% Pluronic F-127 (e.g. 2 g Pluronic
items are for volatile anesthetic agents only). Consult
in 10 ml DMSO) to yield a dye concentration of 10
literature (for example, Flecknell, 2000) for the best
mM. Dilute this solution 1/10 or 1/20 with the standard
choice of anesthesia for your species. Anesthetic
pipette solution of the following composition (in mM):
procedures: for adult mice we used either
ketamine/xylazine or urethane (0.1/0.01 mg/g and 1.9
5. Fill a micropipette with this staining solution (pipette
mg/g body weight, respectively, i.p.). Alternatively,
resistance 6-9 MΩ). Insert the pipette into the cortex
the mice were anesthetized by inhalation of
and advance it along its axis until it reaches the desired
depth (see Fig.1A). Apply a pressure pulse (1 min, 70
2. Stereotaxic instrument, drill, warming plate to keep
kPa) to eject ~ 400 fl of the staining solution near the
animals body temperature constant (available from
cells of interest. Remove the pipette. Wait for an hour
many providers, for example, TSE-Systems, Bad
to obtain a stable maximal fluorescence level in stained
cells (Stosiek et al., 2003). This protocol yields a
3. Custom-made recording chamber with central access
stained area with a diameter of 200-400 µm.
4. Membrane-permeable calcium indicator dye (e.g.
Calcium Green-1 AM, Fura-2 AM, Fluo-4 AM, Indo-
We used 800 nm excitation light to image neurons
1 AM, etc.; Molecular Probes, Eugene, Oregon,
stained with all indicator dyes tested (see above). The
average power under the objective was <70 mW. With dye
5. Manipulator and a pressure application device for
application pipette located 150-200 µm below the cortical
injection of the staining solution into the brain. We
surface, all cortical cells between the surface and 400-µm
depth are stained (Stosiek et al., 2003). When imaging
&Neumann GmbH, Ratingen, Germany and through the thinned skull (thickness of 8-10 µm), individual
Picospritzer II, General Valve, Fairfield NJ, cells could be well resolved up to 200 µm below the
cortical surface. Removing the skull above the imaging
6. Two-photon laser-scanning microscope (see “Two-
field further improves depth resolution, allowing the
photon chloride imaging using the quinolinium-based
detection of individual cells up to 300 µm below the
indicator dye MQAE” chapter for details).
cortical surface. It should be stressed that the stability of
recordings depends critically on the diameter of the
Protocol and Procedures
craniotomy. Thus, openings larger than 1 mm in diameter
Staining neurons with a calcium indicator dye
are often accompanied by movement artefacts occurring at
1. Anesthetize the animal. Assure that the surgical level
of anesthesia is reached (for example, by testing the
pinch withdrawal and the eyelid reflex). Inject ~ 50
Example of application
µl of a local anesthetic agent (e.g. 2% lidocaine)
Fig. 1B-D shows examples of the in vivo two-
photon Ca2+ imaging experiments in the barrel cortex of
mice (modified from Stosiek et al., 2003). Fig.1B
experimental arrangement. (B) High-magnification images
illustrates the quality of imaging data, obtained at
of the barrel cortex of a 13-day-old mouse (P13) taken at
different depths. Fig.1C shows Ca2+ transients in layer 2/3
increasing depth. (C) Ca2+ transients (Lower) in 3
neurons evoked by ionophoretic glutamate application in
individual layer 2/3 neurons (as indicated in Upper) of
vivo. The glutamate-containing pipette was positioned
another P13 mouse evoked by five consecutive 500-ms
less then 50 µm apart from the imaged cells. Ca2+
ionophoretic glutamate applications. (D) Line-scan
transients in Fig. 1D were evoked by the deflection of the
recordings of Ca2+ transients (Lower) evoked in two layer
majority of whiskers on the contralateral side of the
2/3 neurons by a deflection of the majority of whiskers on
mouse’s snout. Note that the signal-to-noise ratio is
the contralateral side of the mouse’s snout (P13 mouse).
sufficient to allow individual, non-averaged somatic Ca2+
The position of the scanned line and the cells analyzed are
transients to be distinguished clearly from the background
Advantages and limits
The approach described, named multi-cell bolus
loading (MCBL; Stosiek et al., 2003), allows References
simultaneous monitoring of Ca2+ levels in many
Brustein, E., Marandi, N., Kovalchuk, Y., Drapeau, P.
individual neurons. The major difference between MCBL
and Konnerth, A. (2003). 'In vivo' monitoring of neuronal
and other staining methods utilizing AM indicator dyes is
network activity in zebrafish by two-photon Ca2+ imaging.
that the indicators are delivered for a short period directly
Pflugers Arch. in press.
to the target cells. In particular, this approach improves
Christie, R. H., Bacskai, B. J., Zipfel, W. R., Williams, R.
the staining of neurons in the adult brain, which are, in
M., Kajdasz, S. T., Webb, W. W. and Hyman, B. T.
general, not stained by AM indicator dyes bath-applied to
(2001). Growth arrest of individual senile plaques in a
brain slices. Additional advantages of MCBL include the
model of Alzheimer's disease observed by in vivo
necessity of a minor surgery and the possibility to re-stain
multiphoton microscopy. J. Neurosci.21, 858-864.
neurons and thus to conduct long-lasting, perhaps even
Flecknell, P. (2000). Laboratory animal anaesthesia. San
chronic (Christie et al., 2001), recordings.
Diego, San Francisco, New York,Boston, London, Sydney,
Although MCBL allows to image many cells
simultaneously, the resolution of subcellular structures is
Helmchen, F., Fee, M. S., Tank, D. W. and Denk, W.
lower, as compared to in vivo Ca2+ imaging of individual,
(2001). A miniature head-mounted two-photon microscope.
microelectrode-loaded cells (Svoboda et al., 1997). This
high-resolution brain imaging in freely moving animals.
is due to two obvious reasons. Firstly, the image contrast
is reduced due to the staining of many fine processes in
Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J.
the surrounding neuropil. Secondly, the dye concentration
and Charpak, S. (2001). Two-photon microscopy in brain
in MCBL-loaded cells is lower, on average 20 µM
tissue: parameters influencing the imaging depth. J
indicator dye, instead of <3-6 mM when stained using a
Neurosci. Methods111, 29-37.
microelectrode (Stosiek et al., 2003; Svoboda et al., 1997).
Stosiek, C., Garaschuk, O., Holthoff, K. and Konnerth,
These limitations restrict the use of MCBL to analyses of
A. (2003). In vivo two-photon calcium imaging using
somatic Ca2+ transients and make in vivo imaging of
multi-cell bolus loading (MCBL). Proc. Natl. Acad. Sci.
neuronal dendrites at present difficult. Furthermore, they
reduce the depth resolution of our recordings (200-300
Svoboda, K., Denk, W., Kleinfeld, D. and Tank, D. W.
µm compared with 500 µm when imaging cell dendrites
(1997). In vivo dendritic calcium dynamics in neocortical
of microelectrode-loaded cells). Future strategies for
pyramidal neurons. Nature385, 161-165.
improving the quality of recordings include the use of
longer wavelengths of the excitation light, larger
numerical apertures of the objective lens, better
transmittance of the optics, higher photon sensitivity of
the PMT, etc. Because the proportion of scattered photons
in the emitted fluorescence signal increases markedly
with increasing imaging depth, a larger craniotomy and a
larger effective angular acceptance of the detection optics
(Oheim et al., 2001) should also significantly improve
depth resolution by enabling the collection of larger
In conclusion, the approach described here is
applicable for Ca2+ imaging of intact neurons both in vivo
and in brain slices. It enables staining of adult neurons
and, if combined with a miniature head-mounted two-
photon microscope (Helmchen et al., 2001), it may allow in vivo two-photon imaging in freely moving animals. Figure legend. Fig.1. In vivo Ca2+ imaging of neuronal populations in the
barrel cortex of mice. (A) Schematic drawing of the
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