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In vivo two‐photon calcium imaging using multi‐cell bolus loading 
Physiologisches Institut, Ludwig-Maximilians Universität München, Pettenkoferstr. 12, 80336 München, Germany Tel: +49-89-5996 584, Fax: +49-89-5996 512 E-mail: [email protected] 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, Materials
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 Neuron 31, 903-912.
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. Methods 111, 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 USA 100, 7319-7324.
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. Nature 385, 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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