Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Astrocytes were traditionally regarded as a passive glue that connects and supports neurons in the central nerve system
(CNS). It builds the micro-environment in which neurons fulfill their tasks and recover from injury. However, growing
evidence indicates that the role of astrocytes in the CNS may be underestimated, as bidirectional communication between
neurons and astrocytes at the site of synapse has been found in types of astroglia from different tissues, leading to the
concept of "tripartite
synapse"[1_3]. With glutamate or other factors diffusing out of the synapse, the activation of neurons
is able to affect astrocytes[4,5]. However, the activation of astrocytes can also affect
neurons[6], via releasing varieties of neurotransmitters, including glutamate and
ATP[7,8]. Although the mechanism of neurotransmitter secreting is unclear, this
process is believed to couple with intracellular
Ca2+ elevation[9,10].
Ca2+ is one of the most important second messengers and is thought to mediate communication between neurons and
astrocytes[3,11]. Astrocytes are described as non-excitable cells, for their lack of voltage-gated sodium channels;
however, they exhibit complicated intracellular
Ca2+ activity[12]. It has been reported that astrocytes express
voltage-gated Ca2+
channels[13] and the receptors of
neurotransmitters[11]. Intracellular
Ca2+ oscillation in astrocyte in
situ, coupled with neurotransmitter release, can drive NMDA receptor mediated neuronal
excitation[14]. Many studies have shown that the spontaneous
Ca2+ oscillations existed in astrocytes and suggested that such
Ca2+ events are essential for communication between neurons and
astrocytes[14_16]. These findings imply that astrocytes may act as command generators in neural regulation. Understanding
the initiation of spontaneous Ca2+ oscillations in astrocytes is thus substantial for better evaluation of the contribution of
astrocytes to the whole neural system.
The increase in plasmic Ca2+ concentrations during spontaneous
Ca2+ oscillations is either caused by the influx of
extracellular Ca2+ or the release from endoplasmic reticulum (ER)
Ca2+ store. In the present study, we sought to determine the
Ca2+ source in hippocampal astrocytes. With cultured hippocampal astrocytes from the neonatal rat, we investigated the
correlation between the spontaneous
Ca2+ oscillations and ER
Ca2+ store with some pharmacological experiments using a
confocal microscope. We found that the content of ER
Ca2+ store was necessary for the spontaneous
Ca2+ oscillations, and the activation of
InsP3 receptor (InsP3R) played a key role in the process of the oscillations. Our finding suggests that
InsP3R-induced ER Ca2+ release is an important cellular mechanism for the spontaneous
Ca2+ oscillation in hippocampal astrocytes.
Materials and methods
Cell cultures After the brain of neonatal rats (Sprague-Dawley rats, purchased from Vital River Lab Animal Technology,
China) were removed and placed into dissection solution (NaCl 137 mmol/L, KCl 5.4 mmol/L,
Na2HPO4·12H2O 0.67 mmol/L,
KH2PO4 0.22 mmol/L, HEPES 10 mmol/L, glucose 8.3 mmol/L and sucrose 11 mmol/L; pH
7.35), hippocampus were dissected and treated with 4 mL of 0.5% trypsin (Invitrogen, USA) at 37
oC for 30 min. Digestion was stopped by fetal bovine serum (FBS; HyClone, USA). Culture medium consisted of minimum essential medium (Invitrogen),
containing 26 mmol/L NaHCO3, 40 mmol/L glucose, 1 mmol/L pyruvate,
1×105 U/L penicillin, and 100 mg/L streptomycin,
supplemented with 10% FBS and 2 mmol/L glutamine immediately before use.
Cells were plated into 35-mm culture dishes for co-culture of neurons and astrocytes or 25
cm2 culture flasks for purification later at a density of approximately
5×108 cells/L. Cells in flasks were grown to confluence at 37 °C in a humidified 5%
CO2/95% air. In order to get the purity culture of
astrocytes, the flasks were shaken on a horizontal orbital shaker at 250 rpm for 18 h after 5_7 d.
The remaining adherent cells were enzymatically detached with trypsin (0.5%) plus EDTA (0.06%), resuspended in
culture medium, and plated onto poly-D-lysine-coated (12.5
mg/L) glass coverslips. Cells were fed every 3_4 d by replacing the medium with fresh medium. The cells
were used in experiments after 1_4 d, by which time they had grown to confluence.
Ca2+ imaging The bathing solution consisted of NaCl 141 mmol/L, KCl 2.5 mmol/L,
MgCl2 1.3 mmol/L, CaCl2 2.4 mmol/L,
NaH2PO4 1.25 mmol/L, glucose 11 mmol/L, HEPES 10 mmol/L, pH 7.35. Cells were loaded with the
Ca2+ indicator, Fluo-4-AM (Invitrogen), at a concentration of 1.82 µmol/L in bathing solution for 5 min at room temperature. Confocal series-scan
imaging was performed by using a Zeiss LSM 510 confocal microscope equipped with an argon laser (488 nm) and 40×, 1.3 NA
oil immersion objectives. Series-image scanning was used to record the
Ca2+ oscillation in cells. The sampling rate was 1 Hz,
and the optical slice was approximately 3 µm.
Results
Spontaneous Ca2+ oscillations in hippocampal astrocytes
To evaluate the effects of neurons on spontaneous
Ca2+ oscillations of astrocytes, the co-cultured hippocampal neurons
and astrocytes were loaded with Fluo-4 AM first, and the intracellular
Ca2+ oscillations in astrocytes were investigated with
confocal-laser-scanning microscope. The cells were then exposed to 1 µmol/L TTX (Figure 1B), a selective antagonist of
voltage-gated Na+ channels, which can effectively block the action potential of neurons, in order to examine whether this
activity comes from neurons or originates from astrocytes themselves. We found that the robust activity of astrocytes was
not impacted (Figure 1C; n=30). These results indicated that the process of the oscillations was neuronal action
potential-independent.
Considering the possibility that the treatment of TTX may not completely occlude neurons’ effects, eg, via spontaneous
transmitter release, purified astrocytes (Figure 1A) from the co-cultured system were employed in all other
experiments. To determine the purity of astrocytes, we labeled astrocytes with anti-GFAP antibody and cell nuclei with Hoechst 33258, after
the culture was fixed by 4% paraformaldehyde in PBS. Immunofluorescence analysis showed that approximately 97% (132 in
136) cells were GFAP marked astrocytes (Figure 1E_1G). We observed the
Ca2+ activity in purity cultured astrocytes as
above, and there was no significant change in the activity when compared with the cells in co-culture (Figure 1D).
This result implied that the spontaneous
Ca2+ oscillations in astrocytes did not require the participation of neurons.
Spontaneous Ca2+ oscillation does not depend on extracellular
Ca2+ The results mentioned above suggested an
astrocytic-originated Ca2+ signal in the network, we then sought the cellular mechanism of such spontaneous action. The
elevation of intracellular Ca2+ may result either from the
Ca2+ influx of the extracellular environment, or from the
Ca2+ release of intracellular
Ca2+ stores. We first tested the former, and found that the spontaneous
Ca2+ oscillations in astrocytes were insensitive to the treatment of nifedipine, which can selectively block the L-type
Ca2+ channels. Neither the frequency nor
the amplitude of the intracellular Ca2+ oscillation was altered (Figure 2A, 2B). This data indicated that the elevation of
intracellular Ca2+ was not resulted from
Ca2+ influx through L-type
Ca2+ channels.
Apart from L-type Ca2+ channels, there might be other
Ca2+-permeable channels on plasma membrane. So we bathed the
cells in 20 mmol/L EDTA, which eliminated the source of extracellular
Ca2+. Even under this condition, the spontaneous
Ca2+ oscillations in astrocytes were not impacted (Figure 2C, 2D). This evidence does not support the extracellular origination of
the spontaneous Ca2+ oscillations.
ER Ca2+ store is necessary in spontaneous
Ca2+ oscillations The results above implied that an intracellular mechanism
must be responsible for spontaneous
Ca2+ oscillations in astrocytes. In order to realize the role of ER
Ca2+ store in the process, the cells were exposed to low
Na+ (70 mmol/L) solution (Figure 3A), which could increase the content of ER
Ca2+ store without significant change of intracellular
Ca2+ concentration[17]. The frequency of spontaneous
Ca2+ oscillations was enhanced to 237%±17% of control by low
Na+ solution treatment (Figure 3B;
P<0.01, n=51 and n=82 in control and low
Na+ group respectively). High
Ca2+ solution (5 mmol/L) was also applied (Figure 3C), and a similar result was observed (Figure 3D;
enhanced to 172%±25% of control;
P<0.05, n=30 in both the control and high
Ca2+ group). We next blocked the sarcoendoplasmic reticulum
Ca2+-ATPase (SERCA) on ER with its specific antagonist, thapsigargin, and found that 2
µmol/L thapsigargin completely eliminated the spontaneous
Ca2+ oscillations in astrocytes (Figure 3E). The frequency of
oscillations was 1.06±0.12 time/min (n=35) before and no event after thapsigargin treatment. These results suggest that the content
of ER Ca2+ store is necessary for generating spontaneous
Ca2+ oscillations.
Essential role of InsP3R in the spontaneous
Ca2+ oscillations The ER
Ca2+ store may generate intracellular
Ca2+ signal through two types of
Ca2+ release channels, the
InsP3Rs and ryanodine receptors (RyRs). To test the role of RyRs in
spontaneous Ca2+ oscillations, the cells were treated with 50 µmol/L ryanodine or 400 µmol/L tetracaine, two specific
antagonists of RyRs. The spontaneous
Ca2+ oscillations were still robust after blockers application (Figure 4A, 4B). How-ever,
treating the cells with 100 µmol/L 2-APB (Figure 4C), the blocker of
InsP3Rs, depressed the spontaneous
Ca2+ oscillations by approximately 90% (Figure 4D,
n=115). Furthermore, the inhibition of 2-APB behaved in a dose-dependent manner (Figure
4E). In the presence of tetracaine, the effect of 2-APB was more potent than in the absence of tetracaine (Figure 4E),
suggesting a potential interaction between
InsP3Rs and RyRs. The above evidence suggests
that, in the process of spontaneous
Ca2+ oscillations, InsP3Rs plays an essential role while RyRs may play an assistant role.
Discussion
Astrocytes might have a much more essential role than has been revealed in CNS. Thereby understanding the initiation
of spontaneous Ca2+ oscillations in astrocytes becomes very important. Using confocal laser-scanning microscopy we
found that: 1) the content of ER Ca2+ store was necessary for the spontaneous
Ca2+ oscillations; and 2) the activation of
InsP3R played a key role in the process of spontaneous
Ca2+ oscillation. Our results suggest that
InsP3R-induced ER Ca2+ release is an important cellular mechanism for the spontaneous
Ca2+ oscillation in hippocampal astrocytes.
Although there is still an argument that the intracellular
Ca2+ oscillations in astrocytes comes from
neurons[18], most believe the existence of spontaneous
Ca2+ oscillations in astrocytes. Many investigators have been trying to probe the
mechanism for the initiation of spontaneous
Ca2+ oscillations, but the results have been rather inconsistent. Some reports
showed that the spontaneous Ca2+ oscillations in astrocytes required extracellular
Ca2+[14,16], while others supported the
contribution of intracellular Ca2+[19]. This disagreement may be the result of different subtypes of astrocytes and different
preparations conditions. Our results support the view that the spontaneous
Ca2+ oscillations in astrocytes originate via
intracellular mechanism, and the ER
Ca2+ store is necessary for the process.
Most of studies supported that the
InsP3Rs played an essential role in the process of spontaneous
Ca2+ oscillations in
astrocytes[19_21], and our results is consistent with these reports. However, RyRs are also richly expressed on the ER of
astrocytes[12], and a functional
Ca2+ sensitive store has been
reported[22]. All these findings lead to an open question of what
the role is of RyRs in spontaneous Ca2+ of astrocytes. In the present study, we compared the property of the spontaneous
Ca2+ oscillations in astrocytes before and after blocking RyRs. Although RyRs blockers-perfusion could not block the
spontaneous Ca2+ oscillations, we found that tetracaine had some depressing effect when coapplied with 2-APB. Therefore,
there may be some interaction between RyRs and
InsP3Rs. The crosstalk between
RyRs and InsP3Rs has been
reported[20], but its physiological significance needs to be investigated further.
Recently studies have revealed that
Ca2+ signaling in astrocytes-mediated control of cerebral blood flow, is a mechanism
of neurovascular coupling[23,24]. It has been shown also that the activity of astrocytes may lead to synchronized
Ca2+ oscillation in
neurons[6]. We believe that the spontaneous
Ca2+ oscillations in astrocytes play a substantial role in the
process of information transferring from astrocytes to neurons. In summary, we demonstrated that the spontaneous
Ca2+ oscillations in astrocytes were dependent on release from ER
Ca2+ stores through InsP3Rs, and there was interaction between
RyRs and InsP3Rs in the process of spontaneous
Ca2+ oscillation. Our findings present a new aspect for understanding the
Ca2+ signal in astrocytes and the essential role of astrocytes in CNS.
Acknowledgement
The authors thank Ms Shu-hua BAI for technical assistance.
References
1 Newman EA. New roles for astrocytes: regulation of synaptic transmission. Trends Neurosci 2003; 26: 536_42.
2 Fields DR, Stevens-Graham B. New insights into neuron-glia communication. Science 2002; 298: 556_62.
3 Haydon GP. Glia: listening and talking to the synapse. Nat Rev Neurosci 2001; 2: 185_93.
4 Porter JT, McCarthy KD. Hippocampal astrocytes
in situ respond to glutamate released from synaptic terminals. J Neurosci 1996; 16:
5073_81.
5 Araque A, Martin ED, Perea G, Arellano JI, Buno W. Synaptically released acetylcholine evokes
Ca2+ elevations in astrocytes in hippocampal slices. J Neurosci 2002; 22: 2443_50.
6 Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G. Neuronal synchrony mediated by astrocytic glutamate through
activation of extrasynaptic NMDA receptors. Neuron 2004; 43: 729_43.
7 Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL,
et al. Prostaglandins stimulate calcium-dependent glutamate release in
astrocytes. Nature 1998; 391: 281_85.
8 Guthrie PB, Knappenberger J, Segal M, Bennett MV, Charles AC, Kater SB. ATP released from astrocytes mediates glial calcium waves. J
Neurosci 1999; 19: 520_8.
9 Parpura V, Haydon PG. Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. Proc Natl Acad
Sci USA 2000; 97: 8629_34.
10 Bezzi P, Gundersen V, Galbete JL, Seifert G, Steinhauser C, Pilati E,
et al. Astrocytes contain a vesicular compartment that is competent
for regulated exocytosis of glutamate. Nat Neurosci 2004; 7: 613_20.
11 Volterra A, Meldolesi J. Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neursci 2005; 6:
626_40.
12 Verkhratsky A, Orkand RK, Kettenmann H. Glial calcium: homeostasis and signaling function. Physiol Rev 1998; 78: 99_141.
13 D'Ascenzo M, Vairano M, Andreassi C, Navarra P, Azzena GB, Grassi C. Electrophysiological and molecular evidence of L-(Cav1), N-
(Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes. Glia 2004; 45: 354_63.
14 Parri HR, Gould TM, Crunelli V. Spontaneous astrocytic
Ca2+ oscillations in situ drive NMDAR mediated neuronal excitation. Nat
Neurosci 2001; 4: 803_12.
15 Nett WJ, Oloff SH, McCarthy KD. Hippocampal astrocytes
in situ exhibit calcium oscillations that occur independent of neuronal
activity. J Neurophysiol 2002; 87: 528_37.
16 Aguado F, Espinosa-Parrilla JF, Carmona MA, Soriano E. Neuronal activity regulates correlated network properties of spontaneous
calcium transients in astrocytes in situ. J Neurosci 2002; 22: 9430_44.
17 Golovina VA, Bambrick LL, Yarowsky PJ, Krueger BK, Blaustein MP. Modulation of two functionally distinct
Ca2+ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger. Glia 1996; 16: 296_305.
18 Rizzoli S, Sharma G, Vijayaraghavan S. Calcium rise in cultured neurons from medial septum elicits calcium waves in surrounding glial cells.
Brain Res 2002; 957: 287_97.
19 Parri HR, Crunelli V. The role of
Ca2+ in the generation of spontaneous astrocytic
Ca2+ oscillations. Neuroscience 2003; 120: 979_92.
20 Haak LL, Song LS, Molinski TF, Pessah IN, Cheng H, Russell JT. Sparks and puffs in oligodendrocyte progenitors: cross talk between
ryanodine receptors and inositol trisphosphate receptors. J Neurosci 2001; 21: 3860_70.
21 Beck A, Nieden RZ, Schneider HP, Deitmer JW. Calcium release from intracellular stores in rodent astrocytes and neurons
in situ. Cell Calcium 2004; 35: 47_58.
22 Golovina VA, Mambrick LL, Yarowsky PJ, Krueger BK, Blaustein MP. Modulation of two functionally distinct
Ca2+ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger. Glia 1996; 16: 296_305.
23 Takano T, Tian GF, Pen W, Lou N, Libionka W, Han
X, et al. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci 2006;
9: 260_7.
24 Metea MR, Newman EA. Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling. J Neurosci 2006; 26:
2862_70.
|
