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The involvement of astrocytes in brain functions rather than support has been identified and widely concerned. However the lack of an effective stimulation of astrocytes hampers our understanding of their essential roles. Here, we employed 800-nm near infrared (NIR) femtosecond laser to induce Ca2+ wave in astrocytes. It was demonstrated that photostimulation of astrocytes with femtosecond laser pulses is efficient with the advantages of non-contact, non-disruptiveness, reproducibility, and high spatiotemporal precision. Photostimulation of astrocytes would facilitate investigations on information processing in neuronal circuits by providing effective way to excite astrocytes.
©2009 Optical Society of America
1. Introduction
Astrocytes are the major electrically non-excitable cells in the central nervous system, the role of which have long been constricted as supporting neurons. However, new functions of astrocytes have been identified in the past decades [1, 2], and become increasingly attractive to neuroscientists since early studies [3]. Astrocytes express various membrane receptors activated by neurotransmitters, in response of intracellular Ca2+ elevation and intercellular Ca2+ wave. In turn, they release chemical transmitters, regulating the synaptic transmission and strength, and influencing the cerebral microcirculation [4-6]. In terms of the distinct bidirectional communications between astrocytes and neurons, it has been proposed that the astrocyte is the third functional element of the synapse [5]. Recently, precise consistency of tuned responses in astrocytes and neurons was proved, and astrocytes were proposed to contribute to functional brain imaging [7], leading us to explore the delicate communications among these brain cells. While there are accumulative evidences that astrocytes are involved in brain functions through Ca2+ signaling, the essential roles have not been resolved. One of the key techniques is an effective stimulation.
Existing approaches developed for generating Ca2+ signaling in astrocytes, include mechanical stimulation [8], electrical stimulation [3], uncaging [9] and pharmacological applications [8, 10]. However, despite of complexity and poor efficiency in performing, none of these allow noncontact and non-disruptive stimulation with selective and precise targeting, especially for in vivo studies. Hence new techniques are obliged to be introduced.
As a novel tool for nanoprocessing, femtosecond laser, not only for its multi-photon imaging, has been previously performed in various biological fields. Compared to ultraviolet light, high spatial resolution is achieved by two-photon excitation of femtosecond laser, for selectively labeling single organelles with photoconvertible proteins [11, 12]. Besides, and more interestingly, femtosecond laser has been widely adopted in nanosurgery. The laser beam is directed through an objective of high numerical aperture (NA), and focused onto subcellular structures, such as a chromosome [13], single organelles [11, 14-16], or even neuronal processes [17, 18], resulting in ultra-precise disruption there. This makes it possible to inactivate subcellular functional regions, to examine the contributions and interactions of local structures. Besides, effects of femtosecond laser have also been studied at cell level [19-21]. Laser targets can either be larger, like blood vessels [22], or highly localized on cell membrane [15, 23, 24], enabling tissue dissection with sub-micron precision, as called photoporation. Tightly focused femtosecond laser, with high peak intensity and low pulse energies, is proved to hardly compromise cell viability [11, 14-21, 23, 24]. Therefore, in addition with its high efficiency and precision, femtosecond laser has been confirmed to be a preferred tool compared to previous approaches in the fields above.
In this paper, we introduced 800-nm NIR femtosecond laser pulses focused on cell membrane, to evoke Ca2+ increase in the stimulated astrocyte and subsequent Ca2+ wave across other cells, which was demonstrated to be resulted from photoporation. Similar responses in astrocytes were observed by repetitive photostimulation with well-controlled laser intensity and target. Noncontact photostimulation employing femtosecond laser was proved to be non-disruptive, reproducible, and with high spatiotemporal precision. This method has potentials to be applied to investigations on detailed communications between astrocytes and other brains cells, especially in vivo or in live animals.
2. Materials and methods
All chemicals were obtained from Sigma (St Louis, MO, USA) unless indicated.
2.1 Cell cultures
Pure cortical astrocytes were prepared as described [25] with some modifications. In brief, cortex was obtained from postnatal 1- to 3-day Wistar rats, dissociated by trypsin (0.125%), and cultured with Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FCS) (HyClone, USA). As forming a confluent layer, astrocytes were ready for experiments. Use of animals followed the guideline of Huazhong University of Science and Technology for Biological Sciences Animal Research Advisory Committee.
2.2 Dyes and drugs
During experiments, cells were incubated in a Hepes-buffered solution (HBS, pH 7.2) containing (in mM): 145 NaCl, 3 KCl, 10 HEPES, 3 CaCl2·2H2O, 2 MgCl2·6H2O, 8 glucose. For Ca2+ imaging, cells were loaded with 4 μM Fluo-3-AM in HBS for 30 min at 37°C.
Propidium iodide (PI) was diluted in HBS at a final concentration of 50 μM. Ca2+-free solution contained 2 mM Ethylene glycol tetraacetic acid (EGTA) in HBS.
2.3 System for imaging and photostimulation
Experiments were carried out on a commercial confocal microscope system (FluoView1000, Olympus) equipped with multiphoton excitation settings, as well as a homebuilt random-access two-photon microscopy [26]. The confocal system consisted of an inverted microscope (IX71) employing 20× 0.75 NA, and 60× 0.95 NA water-immersion objectives and a FV1000 confocal system (Olympus, Japan). Fluorescence of Fluo-3 was excited by the 488 nm band of the argon ion laser, relayed through a dichroic mirror (DM) 405/488, and collected by photo-multiplier tubes (PMTs). For stimulating, mode-locked laser beam (800 nm, ~90 fs pulses at 80 MHz) produced from a Ti: Sapphire laser (Mai Tai, Spectra-Physics) was coupled to the confocal scan head of FV1000, and directed into IX71, with the maximum average power of 60 mW after the 20× objective. A custom–built prism pair was typically used as a pulse compressor to compensate the dispersion in an acousto-optic modulator (AOM, A-A, France), which is employed to regulate the laser power. Pulse width was eventually recovered to nearly 90 fs. Time sequences of imaging and stimulating were computer controlled by FluoView software (Olympus). Images with each frame of 512 × 512 pixels were acquired at 2 μs/pixel, that is ~1 s/frame, and then analyzed in Image-Pro Plus5.0 (Media Cybernetics, USA).
3. Results
3.1 Photogenerated Ca2+ wave in astrocytes
Femtosecond laser was focused onto the upper membrane of a selected cell. Intracellular Ca2+ concentration in astrocytes was monitored by the fluorescence of Fluo-3 before and immediately after laser irradiation with controlled energies and durations. As shown in Fig. 1, femtosecond laser pulses with average power of 30 mW and duration of 4 ms, were targeted onto one of the astrocytes “As” (white dash curve in Fig. 1(a)). Ca2+ elevations in the stimulated cell and neighboring cells were detected instantaneously (Fig. 1(b), “1 s”), followed by obvious propagation throughout the astrocyte network, called Ca2+ wave. In most cases, the wave was already initiated within 1 s after laser irradiation, spreading concentrically (red dashed curves in Fig. 1(b), and see also supplementary Media 1) as in other studies [27-29]. With average laser power of 30~50 mW and duration of 1~4 ms, Ca2+ wave was always photogenerated, covering a range of 166.5 ± 63.9 μm in radius and at the average velocity of 11.5 ± 2.5 μm/s.
Fig. 1. Photogeneration of Ca2+ wave in astrocytes. (a) DIC image of cultured astrocytes and corresponding fluorescence images before and after laser irradiation. Red arrowhead (and in b) points to femtosecond laser target on an astrocyte “As”, which is indicated by white dashed curve. (b) Image series of fluorescence change ΔF, representing only Ca2+ increases. The edges of the wave at corresponding time points are marked by red dashed curves. Scale bar, 100 μm
3.2 Photoporation
We next verified whether poration was formed on cell membrane by femtosecond laser pulses [15, 23, 24], resulting in astrocytic Ca2+ signaling. Virtually, local transformation of cell membrane was observed around the laser target directly in DIC images, suggesting the disruption there caused by femtosecond laser irradiation (Fig. 2(a), and see also supplementary Media 2). Astrocytes were bathed in HBS containing FM1-43, a membrane-staining dye, and photostimulation was carried out (see Media 3). Fluorescent spot appeared afterwards and was highly localized at the irradiated point initially, indicating a tiny pore open for the dye to fuse with cell membrane and emit fluorescence. This pore seemed to be closed soon, as fluorescence spot no longer extended after a while, suggesting FM1-43 hardly diffused any more intimately.
To further confirm it, astrocytes preloading Fluo-3 were imaged in the bath of PI, a membrane-impermeable nuclear stain. Fluorescence images for the two dyes were acquired sequentially. After photostimulation, fluorescence of PI (ΔF/F) was observed in the irradiated cell (dashed curve in Fig. 2(b)) with a slight increase (45.4 ± 3.2%), whereas there were no changes in other cells at all (Fig. 2(b), “PI”). Comparatively, fluorescence of Fluo-3 increased remarkably in both the irradiated cell (264.7 ± 160.0%) and surrounding cells, indicating the Ca2+ wave (Fig. 2(b), “Fluo-3”). Therefore, extracellular PI entered only into the stimulated cell, to which femtosecond laser was pointed with consequent Ca2+ signaling in astrocytes. Moreover, chelating extracellular Ca2+ with EGTA, we discovered that photogenerated Ca2+ elevation in the stimulated astrocyte disappeared (Fig. 2(c), “control” and “EGTA”). After washout of EGTA-containing (Ca2+-free) HBS, the cells were put back into normal solution, and response of the same astrocyte upon photostimulation recovered (Fig. 2(c), “washout”). Notably, the amplitude of photogenerated Ca2+ elevation reduced after washout, probably due to EGTA, which entered into cytoplasm during the trial in Ca2+-free solution. Moreover, Ca2+ wave would diminish as well, without Ca2+ elevation in the stimulated cell (data not shown). Thus we proposed that localized transient photoporation was available on cell membrane caused by femtosecond laser irradiation, through which extracellular Ca2+ entry was necessary for Ca2+ elevation in the stimulated cell and subsequent propagation across others.
Fig. 2. Photoporation on astrocytes caused by femtosecond laser pulses. (a) Transformation of cell membrane. The right two images are enlargements of boxed area. Arrowhead points to the femtosecond laser target. (b) PI-test. (Left) ΔF Images show fluorescence changes of Fluo-3 and PI after photostimulation. The stimulated cell is marked by dashed curves. (Right) Comparison of ΔF/F between Fluo-3 and PI. (c) EGTA-test. Time courses of fluorescence change (ΔF/F) in the same stimulated astrocyte under different conditions are shown. Arrows indicate the onset of laser irradiation. Scale bar, 20 μm.
3.3 Reproducible photostimulation
In virtue of the facility of manipulating laser, an astrocyte was photostimulated for several times, as shown in Fig. 3. During the five trials, femtosecond laser was focused at a certain plane, onto the same point of cell membrane, with average power of 24 mW and duration of 2 ms. Time courses of Ca2+ elevation showed similar responses in the stimulated cell, suggesting the reproducibility of photostimulation.
Fig. 3. Responses to repetitive photostimulation. (a) ΔF images show fluorescence changes after laser irradiation. Arrowheads point to the femtosecond laser targets. (b) Corresponding responses (ΔF/F) are plotted. Arrows indicate the onset of laser irradiation. Scale bar, 50 μm.
4. Discussions and conclusion
As suggested in section 1, existing approaches have limits for defined targeting to effectively induce Ca2+ signaling in astrocytes. Electrical devices and micropipettes are necessary to achieve local selective stimulation by mechanical [27-29], electrical [3, 27, 28] and pharmacological methods [28, 30], which make it complex and invasive. Uncaging of uncaged Ca2+ [31] or uncaged IP3 [9, 31] using ultraviolet lights seems more favorable, though similarly to pharmacological way, additional agents are involved. Moreover, multiphoton photolysis, especially of caged Ca2+, have been examined [32, 33], which allows in vivo studies. However, the effectiveness of this kind of application in astrocytes is controversial due to poor Ca2+-specificity of the multiphoton uncaged chemical compounds [34, 35]. And basically, uncaged compounds enter into all cells by bath loading, whereas for selectively delivering them into target cells, micropipette is required. In this paper, the idea of photostimulation was motivated by successful biological applications of femtosecond laser, even in neural cells [20, 21, 36]. The advantages over conventional stimulating approaches are summarized as following. (1) Laser beam is focused at a sub-femtolitre volume, and the target location, laser power and duration, onset of irradiation can be finely controlled, enabling high spatiotemporal accuracy of stimulation. (2) Photostimulation is noncontact, noninvasive, without any chemical compounds and easy to perform. (3) The facility of manipulating laser makes it feasible to carry out repetitive stimulation.
Extracellular Ca2+ influx through photoporation on cell membrane was proved underlying Ca2+ elevation evoked by femtosecond laser. This process could be regarded as simulating physiological Ca2+ entry from voltage-gated or ligand-operated channels in plasma membrane [5]. Intracellular Ca2+ increase was then evoked probably due to the well-known Ca2+-induced Ca2+ release (CICR) from intracellular stores, leading to intercellular Ca2+ wave [37, 38]. The spreading velocity and covering range were comparable with previous studies [8, 29] by mechanical stimulation, in which the latter two were 11.25 μm/s in average and 250 μm in maximum.
Cell viability upon laser irradiation is a vital consideration, which has been confirmed in previous studies (see section 1). Because of the high peak intensity of femtosecond laser pulses to reduce the energy threshold [39, 40] , tissue dissection is achieved with low-energy source [13, 16, 17, 24]. Here, minimum laser energy (0.225~0.75 nJ/pulse in the sample) was adopted, relatively low compared to studies on other cultured cells [16, 20, 21, 23, 24]. The focus plane of femtosecond laser was carefully identified to be at the upper cell membrane, leading to formation of photoporation there. This process has been demonstrated to be mediated by nonlinear effects based on multi-photon ionization and plasma formation [39, 40]. Thus photo-chemistry including bond breaking is involved, hardly related to mechanical or thermal stresses due to the ultrashort pulses. Therefore, laser-membrane interaction was supposed to be highly localized in the focal volume, with little extended damage to circumstances or the entire cell. Actually, the tiny cut on biological structures by femtosecond laser has been observed and measured to be no more than hundreds of nanometers in width [13, 15]. In our study, transformation of cell membrane appeared after laser irradiation (Fig. 2(a)). Moreover, when laser power as high as 60 mW was applied, a cavity could, sometimes, be observed on cell membrane (supplementary Media 2). Restricted fluorescence spot of FM1-43 (supplementary Media 3), as well as slight increase in the fluorescence of PI (Fig. 2(b)), implied the localized transient poration. Further, the viability and function of the stimulated astrocyte were supported by observations: (1) restoration by membrane fluidity immediately after pore formation (supplementary Media 2); (2) no stain of PI in the nuclear 0.5~2 h after photostimulation (data not shown); (3) similar Ca2+ responses by repetitive laser irradiation (Fig. 3). Note that, laser intensity should not be too high (at average power of over 60 mW for several milliseconds), otherwise large holes on the membrane and cell blebbing would be generated, due to accumulative heating [40], resulting in cell death (data not shown).
In conclusion, noncontact photostimulation of astrocyte employing femtosecond laser is confirmed to be non-disruptive, reproducible, and with high spatiotemporal precision. It is efficient and feasible for the research on Ca2+ signaling in astrocytes and communications with other brain cells. Associated with other technologies such as uncaging and transgenics, this versatile optical tool is promising and helpful for resolving problems that traditional approaches are difficult to achieve, especially in studies looking into the brain in live animals.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grant No. 30727002, 30700215) and National High-Tech Research and Development Program of China (863 Program: 2006AA02Z343).
Supplementary movies
Supplementary Media 1. Photogenerated Ca2+ wave in astrocytes by femtosecond laser. Laser target is marked by white arrow. Scale bar, 100 μm.
Supplementary Media 2. Photogenerated pore on cell membrane and subsequent restoration after femtosecond laser irradiation. Laser target is marked by white arrow. Scale bar, 5 μm.
Supplementary Media 3. Photoporation on cell membrane labeled by FM 1-43. Laser target is marked by white arrow. Scale bar, 5 μm.
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