Astrocytes have been identified to actively contribute to brain functions through Ca<sup>2+</sup> signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca<sup>2+</sup>) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca<sup>2+</sup> signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca<sup>2+</sup> wave throughout the network with reduced speed is induced, and intracellular Ca<sup>2+</sup> oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca<sup>2+</sup> elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca<sup>2+</sup> signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca<sup>2+</sup> signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.
Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca<sup>2+</sup>]<sub>i</sub> transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca<sup>2+</sup>]<sub>i</sub>i transient under different concentrations of glutamate. Two different types of kinetic of the [Ca<sup>2+</sup>]<sub>i</sub> transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca<sup>2+</sup>]<sub>i</sub> transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca<sup>2+</sup>]<sub>i</sub> transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca<sup>2+</sup>]<sub>i</sub> transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.
Changes in the intracellular Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca<sup>2+</sup>]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca<sup>2+</sup>. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca<sup>2+</sup>-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca<sup>2+</sup>-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca<sup>2+</sup>]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca<sup>2+</sup>]i in response to a variety of stimuli. These [Ca<sup>2+</sup>]i increases provide a possible means for information coding.