Article SPECIAL TOPIC: Huazhong University of Science and Technology Materials Science
October 2010 Vol.55 No.30: 3436–3440 doi: 10.1007/s11434-010-3376-z
Modulation of synchronous calcium oscillations in hippocampal neurons by photostimulation of astrocytes with femtosecond laser ZHAO Yuan, LIU XiuLi, ZHANG Yuan, ZHOU Wei & ZENG ShaoQun* Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China Received February 27, 2010; accepted June 30, 2010
A large body of evidence indicates that astrocytes play an important role in a range of brain functions through calcium (Ca2+) signaling. Experimentally evoking Ca2+ signaling is a useful technique for investigating the functions of astrocytes. However, conventional stimulation methods typically have poor spatio-temporal precision, and some are invasive. Our group has developed a technique to overcome these problems, in which astrocytes are photostimulated with a femtosecond laser. In the current study, we applied this method to a hippocampal neural network to explore astrocytic functions in detail. The results revealed that applying photostimulation to astrocytes in a cultured hippocampal astrocyte-neuron network caused the following changes: (i) Synchronous Ca2+ oscillations in neurons were induced; (ii) spontaneous Ca2+ synchrony instantaneously emerged; and (iii) high-frequency spontaneous Ca2+ synchrony was regulated. Thus, astrocytic Ca2+ signaling evoked by photostimulation was found to modulate synchronous Ca2+ oscillations in hippocampal neurons. We propose that photostimulation with a femtosecond laser will serve as a powerful tool in investigating astrocytic functions at the network level. femtosecond laser, photostimulation, astrocyte, hippocampal neuron, synchronous calcium oscillation Citation:
Zhao Y, Liu X L, Zhang Y, et al. Modulation of synchronous calcium oscillations in hippocampal neurons by photostimulation of astrocytes with femtosecond laser. Chinese Sci Bull, 2010, 55: 3436−3440, doi: 10.1007/s11434-010-3376-z
The neural cells comprising neural systems include neurons and neuroglial cells (glials). The neuron is the primary cell in the neural system. The number of glials in the brain is 10–50 times greater than that of neurons. There are several subtypes of glials, and, of these, astrocytes are the most numerous. Astrocytes are widely distributed in the mammalian brain. It was traditionally believed that the function of astrocytes was limited to supporting and nurturing neurons by clearing neurotransmitters and balancing ion homeostasis. With the development of new techniques, an increasing number of studies have demonstrated that astrocytes play a range of more complicated and important roles in the brain [1–5] than previously believed. Unlike excitable cells, such as the neuron, astrocytes cannot generate action potentials. However, they express *Corresponding author (email:
[email protected])
© Science China Press and Springer-Verlag Berlin Heidelberg 2010
various kinds of neurotransmitter receptors on the plasma membrane [6]. Therefore, they can respond to outside stimulation, especially synaptic neurotransmitters. As the astrocyte is activated, an intracellular Ca2+ increase is induced. This can subsequently induce Ca2+ increases in other astrocytes, generating a form of spatial propagation termed the Ca2+ wave [7]. During Ca2+ signaling, many physiological reactions are triggered in the astrocyte. The most important one is the release of transmitters, including neurotransmitters and vascular regulation factors. Neurotransmitters, such as ATP [8], Glu [9] and D-serine [10], can act on synapses, with diverse influences on synaptic electrical and Ca2+ activities. Thus, astrocytes reciprocally communicate with neurons [1]. On the other hand, these cells form endfeet around blood vessels. In response to neuronal activities, astrocytes release chemicals to regulate cerebral blood flow, thus playing a bridging role between the neuronal and cerecsb.scichina.com
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brovascular systems [2]. Taken together, astrocytes actively participate in brain functions through Ca2+ signaling in an important way, transferring information between different cells over a large spatial range. A key technique used in studies on astrocytic functions is selective activation to induce astrocytic Ca2+ signaling. Studies performed in the early 1990s involved the application of neurotransmitters and mechanical stimulation to activate astrocytes [11]. Later, electrical stimulation [12,13] and uncaging [14,15] techniques were developed. However, several problems are involved in these methods, including their complexity, the necessity of physical contact, the requirement of additional chemicals, and difficulties in precisely targeting them. In contrast, our group has developed photostimulation of the astrocyte using a femtosecond laser. It is a non-contact method, which is highly selective and does not require any additional agents [16]. In the locked mode, the pulse width of the femtosecond laser is shortened to ~10–15 s, while the laser energy is restricted to these ultrashort pulses. Thus, the femtosecond laser has high peak intensity and low laser energy. Due to the high peak intensity of the femtosecond laser, it can be used to induce nonlinear effects. Thus, this technique is efficient not only for multi-photon excitation or activation, but also for particular photon-tissue interactions [17,18]. In biology, the femtosecond laser has been used for nano-processing in subcellular structures, such as the disruption of single organelles [19,20], axotomy [21], stimulation of neuritis [22], cell fusion [23], optical transfection [24], and the dissection of chromosomes [25]. Utilizing nanosurgery with the femtosecond laser, photostimulation of the astrocyte was realized by introducing a transient poration in the upper plasma membrane of the astrocyte [16]. Extracellular Ca2+ entered into the cytoplasm through this poration due to a concentration gradient, leading to the release of internal Ca2+ stores. Intracellular Ca2+ elevation and intercellular Ca2+ waves were then induced. With laser targeting, we found that the noninvasive photostimulation could be selectively directed onto a single cell, with high spatio-temporal accuracy. Then in this paper, we applied photostimulation of astrocytes with the femtosecond laser in a hippocampal neural network, to examine astrocytic modulation of synchronous Ca2+ oscillations in neurons.
1 Materials and methods (i) Cell culture. Hippocampal astrocyte-neuron co-cultures were prepared as described [26] with minor modifications. Hippocampal tissue was dissected from embryonic (E18) Wistar rats. Cells were dissociated by incubation in 0.125% trypsin (Gibco) for 10 min followed by trituration. Suspended cells were then plated onto glass coverslips at an approximate density of 105 cells/cm2. They were maintained
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in a 5% CO2 incubator (Thermo Scientific) at 37°C for 8–14 d before experiments. (ii) Solution. The experimental solution contained (mmol/L): 145 NaCl, 3 KCl, 10 HEPES, 3 CaCl2·2H2O, 2 MgCl2·6H2O, 8 glucose. All chemicals were obtained from Sigma. (iii) Ca2+ imaging and photostimulation system. Before the experiment, cells were loaded with 3–5 μmol/L Fluo-3 AM (Invitrogen) at 37°C for 30 min, then washed in experimental solution. Experiments were carried out using a confocal laser scanning system (FluoView1000; Olympus). Fluo-3 fluorescence was excited using an argon laser, and collected with a photo-multiplier tube (PMT) through a dichroic mirror 405/488. Near-infrared (800 nm) femtosecond laser (90 fs, 80 MHz) for photostimulation was generated using a Ti: Sapphire laser (Spectra Physics), directed to FV1000, and focused onto the sample through the objective. The upper plasma membrane for photostimulation was determined by differential interference contrast (DIC) images at 800 nm. The laser power of the femtosecond laser after the objective was generally 15–50 mW, with a duration of 1–4 ms. The protocol of imaging and photostimulation was controlled by FluoView software (Olympus). Images were acquired at ~1 frame/s. (iv) Data analysis. Images were analyzed in Image-Pro Plus (Media Cybernetics) to obtain the relative fluorescence change (ΔF/F).
2 Results 2.1
Inducing synchronous Ca2+ oscillations in neurons
Hippocampal astrocyte-neuron co-culture was shown in Figure 1(a). An astrocyte “As” (white arrow) was photostimulated. Synchronous Ca2+ oscillations were observed immediately following stimulation (Figure 1(b)) in 5 neurons (black numbers in Figure 1(a)) in this region. During the initial 50 s after photostimulation, the neurons showed burst firing at a high frequency. The oscillations slowed down as time passed, and were entirely diminished after about 200 s. Responses in neurons 1, 2 and 5 were strongly linked. Nevertheless, the number of Ca2+ firings in neurons 3 and 4 was less, although the firings were still coupled with those in the other 3 neurons. In addition, there was a spontaneous Ca2+ spike in neuron 2 in the control condition, while the other neurons were silent. 2.2 Instantaneous intervening of spontaneous Ca2+ synchrony in neurons After 10–14 d in culture, hippocampal neurons showed spontaneous synchronous oscillations [27], suggesting that these cells were forming a neuronal circuit with a particular function. Our experiments revealed that astrocytic signaling instantaneously induced these spontaneous activities. In the area shown in Figure 2(a), astrocytic signaling was evoked
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Figure 1 Photostimulation of the astrocyte induced synchronous Ca2+ oscillations in hippocampal neurons. (a) DIC image (left) and fluorescence image (right) of the hippocampal astrocyte-neuron co-culture. White arrowhead indicates the stimulated astrocyte “As”. Black numbers label the neurons showing synchronous Ca2+ responses. Scale bar, 50 μm; (b) Plots of relative fluorescence changes in cells labeled in (a). Arrow and dashed line indicate the onset of photostimulation.
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neurons 1–6 (boxed numbers and black circles in Figure 3(a)) showed additional Ca2+ activities, which occurred simultaneously (arrowheads in figure 3(b)), as well. Thus, a smaller synchronous circuit consisting of neurons 1–6 co-existed within the larger circuit. Figure 3 showed the alterations in this network after photostimulation of the astrocyte. The stimulated astrocyte “As” is labeled by a white arrowhead in Figure 3(a). After photostimulation, synchronous oscillations in the two circuits remained, but became regulated (Figure 3(b)). Following this regulation, Ca2+ activity was reduced in both large (25.2% ± 5.5%, Mean ± SEM) and small (23.1% ± 5.0%) synchronous circuits over time. This suggests that the astrocytes, after activation, depressed spontaneous activity in the neuronal network. Next, in the small circuit, neuronal Ca2+ oscillations converted to burst firing, in contrast to the discrete Ca2+ spikes that were exhibited before photostimulation. Within the recording period, there were three periods of bursting (dashed boxes in Figure 3(b)) with long intervals. During these three bursts, other neurons except neuron 10 in the large circuit also exhibited Ca2+ firing, although to
(the first two images in Figure 2(b), and lines 1–6 in Figure 2(c)) after photostimulation of astrocyte “1” (white arrowhead). Meanwhile, Ca2+ spikes were immediately elicited in neurons 7 and 8 (lines 7 and 8 in Figure 2(c)). In the following several minutes, these two neurons fired three Ca2+ spikes, simultaneously with neurons 9–17 (arrowheads in Figure 2(c), the image at the first synchronous Ca2+ spike was shown in Figure 2(b)). This indicates that after responding to astrocytic Ca2+ signaling, neurons 7 and 8 took part in the activity of the synchronous circuit they were involved in. It should be noted that their Ca2+ spikes during the first synchrony were much larger than those in the next two. We propose that this is related to the initial response to photostimulation of astrocytes. To summarize, we found that single neurons could be instantaneously activated by environmental stimulation, while maintaining their functions in a neuronal circuit. In addition, the results showed that astrocytes communicated with the neuronal circuit without breaking its structural and functional connections. 2.3 Regulation of high-frequency spontaneous Ca2+ synchrony in neurons 2+
In addition, spontaneous high-frequency Ca oscillations in hippocampal neurons were also observed, and astrocytes were found to regulate this activity. As shown in Figure 3(a), hippocampal neurons labeled 1–12 comprised a large circuit of synchronous activity. Two synchronous Ca2+ firings were noted by asterisks in Figure 3(b). Particularly,
Figure 2 Photostimulation of the astrocyte instantaneously intervened spontaneous Ca2+ synchrony in hippocampal neurons. (a) DIC image of the hippocampal astrocyte-neuron co-culture. White and black numbers label astrocytes and neurons, respectively. White arrowhead notes the stimulated astrocyte 1. Scale bar, 50 μm; (b) time serials of fluorescence images after photostimulation. Scale bar, 50 μm; (c) relative fluorescence changes in astrocytes (“As”, 1–6) and neurons (“Neu”, 7, 8, 9–17) labeled in (a). Arrows indicate the onset of photostimulation. Arrowheads indicate the synchronous Ca2+ spikes in neurons.
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Figure 3 Photostimulation of the astrocyte regulated high-frequency spontaneous Ca2+ synchrony in hippocampal neurons. (a) DIC image (left) and fluorescence image (right) of hippocampal astrocyte-neuron co-culture. Two neuronal groups are labeled, one with boxed white numbers 1–6 (left image) or black circles (right image), the other with white numbers (left image) or white circles (right image). Scale bar, 50 μm; (b) Relative fluorescence changes in the neurons labeled in (a). Arrows indicate the onset of photostimulation. Asterisks note examples of activities in the large synchronous circuit (neurons 1–12). Arrowheads note examples of additional activities in the small synchronous circuit (neurons 1–6). Dashed boxes indicate synchronous bursting in neurons 1–6 after photostimulation. On the plot of neuron 10, the small arrow notes a long-term minor Ca2+ elevation immediately after photostimulation.
a lesser extent, indicating that synchronous oscillations in the small circuit changed to burst firing from discrete firing. Moreover, these bursts were temporally coupled with Ca2+ firing in other neurons in the large circuit. Therefore, after photostimulation, additional activity in the small circuit disappeared, and activity in the whole large circuit became more uniform. In addition, neuron 10, closest to the stimulated astrocyte, was dramatically affected. In the control condition, this neuron fired with weak synchrony with the others. Immediately after “As” was stimulated, a long-term, minor Ca2+ elevation was detected in neuron 10 (small arrow in Figure 3(b)). And, its Ca2+ firing in the last 200 s was not synchronous with other neurons at all.
3 Discussion and conclusions We previously developed a method of photostimulation of astrocytes with a femtosecond laser. In this paper, this technique was applied in a neural network to study astrocytic functions. It is important for brain research to elucidate the characteristics of neuronal activity at the network level [27,28], and synchronous oscillations in neuronal groups are of particular interest. Synchronous neuronal oscillations play im-
portant roles in neural development [29] and information processing in the brain [30]. For example, sensation and cognition are both dependent on synchronous activity in different brain regions [31]. Astrocytes have been reported to induce synchronous activities in hippocampal neurons through Glu release acting on NMDA receptors [32]. The present findings revealed that, in a cultured hippocampal neural network, photostimulation of the astrocyte, (i) induced synchronous Ca2+ oscillations in neurons (Figure 1), (ii) instantaneously intervened (Figure 2) or (iii) regulated (Figure 3) spontaneous Ca2+ synchrony in neurons. We propose that this new method is more efficient than conventional stimulation methods for examining astrocyte-to-neuron signaling. Applications of drugs and neurotransmitters influence many cells and are difficult to target. Mechanical stimulation using a micropipette to touch the cell can be directed to single astrocytes, but this contact is nevertheless disruptive. To perform electrical stimulation, a microelectrode is typically placed tens of μm away from the cell, without physical contact. However, the electricity can spread to other cells or neurites nearby. Uncaging requires a caged compound. This method depends on the cellular uptake of the agents, and cells may suffer from phototoxicity of the ultraviolet light used. Comparatively, photostimulation with a femtosecond laser provides a noncontact method
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that does not involve any chemicals, and can be selectively and precisely directed to efficiently activate the astrocyte [16]. The present findings revealed that this method can be applied to a neuronal network in culture. Moreover, we found evidence of modulation of synchronous Ca2+ oscillations in neurons by astrocytic Ca2+ signaling. In conclusion, we propose that photostimulation with a femtosecond laser will serve as a powerful tool in studies of astrocytic functions in neural networks.
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This work was supported by the National Natural Science Foundation of China (30927001), the National Science Fund for Distinguished Young Scholars (30925013), and the “111” Project.
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