n?=?6 experiments; **p? ?0.01 versus (?) in the region of persistent [Ca2+]i increases, (?), Bonferronis multiple comparison test after one-way ANOVA. The effects of omitting extracellular Ca2+ on calcium waves were further analyzed. transient [Ca2+]i increases were found to propagate slowly via extracellular ATP in the distal region. Simultaneous imaging of astrocyte [Ca2+]i and extracellular ATP, the latter of which was measured by an ATP sniffing cell, revealed that ATP was released within the proximal region by volume-regulated anion channel in a [Ca2+]i impartial manner. This detailed analysis of a classical model is the first to address the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP. Introduction Deeper understanding of the mechanisms underlying the spatio-temporal diversity and complexity of [Ca2+]i increases in astrocytes is crucial for exploring the physiological and pathological functions of this glial cell population. Calcium waves are a remarkable aspect of [Ca2+]i dynamics in astrocytes, and a unique type of intercellular communication in astrocyte networks, intercalating neuronal circuitries and vasculature. Various pharmacologic and physical stimuli have been found to induce [Ca2+]i increases propagating between astrocytes in cell cultures1,2, in brain slices3,4, and in other preparations5,6. These calcium waves are regarded as transmitting physiologic and pathologic signals within the brain, because they influence the activities of adjacent neurons7,8, microglia9, and endothelial cells10. Furthermore, recent studies have demonstrated the involvement of astrocyte networks and calcium waves in regulating neuronal activities11 and neurological diseases12,13. Because calcium waves can propagate between astrocytes in the absence of physical contact14, they are likely induced by intracellular and extracellular signals in a synergistic manner. Astrocytes are intracellularly connected via connexin channels15, Prodipine hydrochloride and their transmission of Ca2+ and IP3 via gap junctions has been demonstrated experimentally and theoretically16,17. Moreover, astrocytes are equipped with ATP release mechanisms and ATP receptors inducing [Ca2+]i increases18, and purinergic signaling has been found to be involved in calcium waves19,20. Furthermore, gap junction and purinergic signaling are regulated in a supplementary manner to maintain calcium waves21. However, the contributions of these components to the dynamics and functions of calcium waves, and the mechanisms involved in initiating [Ca2+]i increases and release ATP in calcium waves are incompletely understood. Theoretical17 and experimental1,2 studies have shown that calcium waves can be mechanically induced by gently touching cultured astrocytes with tips of glass pipettes. The present analysis of this classical model pharmacologically and by using an ATP sniffing cell revealed distinct [Ca2+]i increases during calcium waves. This study was therefore designed to assess the distinct contributions of gap junction and extracellular ATP and the ATP release mechanism in calcium waves, revealing novel aspects of the diverse and complicated dynamics of astrocyte [Ca2+]i. Results Components of [Ca2+]i increases in calcium waves Figure?1a shows a representative calcium wave induced by mechanical stimulation of cultured astrocytes. The [Ca2+]i increase in the mechanically-stimulated cell (arrow) propagated to adjacent cells, and the area of [Ca2+]i increases reached a maximum at 24?sec. Then, [Ca2+]i in the distal region declined to the baseline by 120?sec, whereas that proximal to the stimulated cell remained elevated for longer than 120?sec. The distribution of [Ca2+]i increases was expressed as a maximum [Ca2+]i projection, in which each pixel represents the maximum 340/380 ratio during the calcium wave (Fig.?1b left). As shown in Fig.?1b center, we defined the peak [Ca2+]i increase (red) as [Ca2+]i increase in the stimulated cell, and the persistent (orange) and transient (blue) [Ca2+]i increases as [Ca2+]i increases sustained and declined until 120?sec, respectively. The appropriateness of 120?sec was clarified later. The histogram of maximum [Ca2+]i increases along a line in Fig.?1b right, shows that the peak [Ca2+]i increase was the largest [Ca2+]i increase during the calcium wave, and the persistent [Ca2+]i increases were larger than the transient [Ca2+]i increases (Fig.?1c). The [Ca2+]i increases of individual cells in the region of the peak and persistent [Ca2+]i increases (Cell 1C3) were persistent, whereas those in the region of transient [Ca2+]i increases (Cell 4C6) were transient (Fig.?1d). These findings suggested that the peak, persistent and transient [Ca2+]i increases were distinct components of the same calcium wave. Open in a separate window Figure 1 Distinct components of [Ca2+]i increases in an astrocyte calcium wave. (a) Representative Fura2 ratio (340/380) images of an astrocyte calcium wave 0, 3, 9, 24, 36 and 120?sec after mechanical stimulation (arrow). (b) Distribution of [Ca2+]i increases during the calcium wave. The maximum [Ca2+]i.A representative [Ca2+]i increase in an individual cell treated with Ca2+-free medium was transient similar to that in the distal region of control (Fig.?3c). within the proximal region by volume-regulated anion channel in a [Ca2+]i independent manner. This detailed analysis of a classical model is the first to address the different contributions of two major pathways of calcium waves, gap junctions and extracellular ATP. Introduction Deeper understanding of the mechanisms underlying the spatio-temporal diversity and complexity of [Ca2+]i increases in astrocytes is crucial for exploring the physiological and pathological functions of this glial cell human population. Calcium waves are a impressive aspect of [Ca2+]i dynamics in astrocytes, and a unique type of intercellular Prodipine hydrochloride communication in astrocyte networks, intercalating neuronal circuitries and vasculature. Numerous pharmacologic and physical stimuli have been found Prodipine hydrochloride to induce [Ca2+]i raises propagating between astrocytes in cell ethnicities1,2, in mind slices3,4, and in additional preparations5,6. These calcium waves are regarded as transmitting physiologic and pathologic signals within the brain, because they influence the activities of adjacent neurons7,8, microglia9, and endothelial cells10. Furthermore, recent studies have shown the involvement of astrocyte networks and calcium waves in regulating neuronal activities11 and neurological diseases12,13. Because calcium waves can propagate between astrocytes in the absence of physical contact14, they are likely induced by intracellular and extracellular signals inside a synergistic manner. Astrocytes are intracellularly connected via connexin channels15, and their transmission of Ca2+ and IP3 via space junctions has been shown experimentally and theoretically16,17. Rabbit Polyclonal to Fyn (phospho-Tyr530) Moreover, astrocytes are equipped with ATP launch mechanisms and ATP receptors inducing [Ca2+]i raises18, and purinergic signaling has been found to be involved in calcium waves19,20. Furthermore, space junction and purinergic signaling are controlled inside a supplementary manner to maintain calcium waves21. However, the contributions of these components to the dynamics and functions of calcium waves, and the mechanisms involved in initiating [Ca2+]i raises and launch ATP in calcium waves are incompletely recognized. Theoretical17 and experimental1,2 studies have shown that calcium waves can be mechanically induced by softly touching cultured astrocytes with suggestions of glass pipettes. The present analysis of this classical model pharmacologically and by using an ATP sniffing cell exposed unique [Ca2+]i raises during calcium waves. This study was therefore designed to assess the unique contributions of space junction and extracellular ATP and the ATP launch mechanism in calcium waves, revealing novel aspects of the varied and complicated dynamics of astrocyte [Ca2+]i. Results Components of [Ca2+]i raises in calcium waves Number?1a shows a representative calcium wave induced by mechanical activation of cultured astrocytes. The [Ca2+]i increase in the mechanically-stimulated cell (arrow) propagated to adjacent cells, and the area of [Ca2+]i raises reached a maximum at 24?sec. Then, [Ca2+]i in the distal region declined to the baseline by 120?sec, whereas that proximal to the stimulated cell remained elevated for longer than 120?sec. The distribution of [Ca2+]i raises was expressed like a maximum [Ca2+]i projection, in which each pixel represents the maximum 340/380 percentage during the calcium wave (Fig.?1b remaining). As demonstrated in Fig.?1b center, we defined the peak [Ca2+]i increase (reddish) as [Ca2+]i increase in the stimulated cell, and the prolonged (orange) and transient (blue) [Ca2+]i raises as [Ca2+]i increases sustained and declined Prodipine hydrochloride until 120?sec, respectively. The appropriateness of 120?sec was clarified later. The histogram of maximum [Ca2+]i raises along a collection in Fig.?1b right, demonstrates the maximum [Ca2+]i increase was the largest [Ca2+]i increase during the calcium wave, and the prolonged [Ca2+]i raises were larger than the transient [Ca2+]i raises (Fig.?1c). The [Ca2+]i raises of individual cells in the region of the peak and prolonged [Ca2+]i raises (Cell 1C3) were prolonged, whereas those in the region of transient [Ca2+]i raises (Cell 4C6) were transient (Fig.?1d). These findings suggested the peak, prolonged and transient [Ca2+]i raises were unique components of the same calcium wave. Open in a separate window Number 1 Distinct components of [Ca2+]i raises in an astrocyte calcium wave. (a) Representative Fura2 percentage (340/380) images of an astrocyte calcium wave 0, 3, 9, 24, 36 and 120?sec after mechanical activation (arrow). (b) Distribution of [Ca2+]i raises during the calcium wave. The maximum [Ca2+]i projection, in which each pixel signifies the maximum 340/380 during the calcium wave (remaining). The peak [Ca2+]i increase (reddish), which is the [Ca2+]i increase in the mechanically-stimulated cell, and the prolonged Prodipine hydrochloride (orange) and transient (blue) [Ca2+]i raises, in which the 340/380 percentage of each pixel sustained above and declined below 30% of the mean 340/380 percentage of pixels within the peak [Ca2+]i increase until 120?sec, respectively are shown overlaid on the maximum [Ca2+]i projection (center). A Collection and cells for the analyses in (c) and.