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, 140 (1), 24-36

Multiple Pathways for Elevating Extracellular Adenosine in the Rat Hippocampal CA1 Region Characterized by Adenosine Sensor Cells

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Multiple Pathways for Elevating Extracellular Adenosine in the Rat Hippocampal CA1 Region Characterized by Adenosine Sensor Cells

Kunihiko Yamashiro et al. J Neurochem.

Abstract

Extracellular adenosine in the brain, which modulates various physiological and pathological processes, fluctuates in a complicated manner that reflects the circadian cycle, neuronal activity, metabolism, and disease states. The dynamics of extracellular adenosine in the brain are not fully understood, largely because of the lack of simple and reliable methods of measuring time-dependent changes in tissue adenosine distribution. This study describes the development of a biosensor, designated an adenosine sensor cell, expressing adenosine A1 receptor, and a genetically modified G protein. This biosensor was used to characterize extracellular adenosine elevation in brain tissue by measuring intracellular calcium elevation in response to adenosine. Placement of adenosine sensor cells below hippocampal slices successfully detected adenosine releases from these slices in response to neuronal activity and astrocyte swelling by conventional calcium imaging. Pharmacological analyses indicated that high-frequency electrical stimulation-induced post-synaptic adenosine release in a manner dependent on L-type calcium channels and calcium-induced calcium release. Adenosine release following treatments that cause astrocyte swelling is independent of calcium channels, but dependent on aquaporin 4, an astrocyte-specific water channel subtype. The ability of ectonucleotidase inhibitors to inhibit adenosine release following astrocyte swelling, but not electrical stimulation, suggests that the former reflects astrocytic ATP release and subsequent enzymatic breakdown, whereas the latter reflects direct adenosine release from neurons. These results suggest that distinct mechanisms are responsible for extracellular adenosine elevations by neurons and astrocytes, allowing exquisite regulation of extracellular adenosine in the brain.

Keywords: adenosine; astrocyte; calcium channel; dendrite; water channel.

Conflict of interest statement

Conflicts of interest: The authors have no conflict of interest to declare.

The authors declare there are no conflicts of interest.

Figures

Figure 1
Figure 1
Adenosine- and inosine-induced calcium elevations in adenosine sensor cells. A. Concentration dependent effects of adenosine and inosine. (a) Representative calcium increases in response to 0.1-100 μM adenosine and 1000 μM inosine. (c) Average calcium increases (n=29–40 cells from 3–-4 experiments). Cells were stimulated with adenosine or inosine at the concentrations indicated. The relative area under the curve during stimulation (relative AUC) was calculated by normalizing to the calcium increase achieved by 100 μM adenosine. B. Effect of an A1 receptor antagonist. Cells were stimulated with 10 μM adenosine or 1000 μM inosine in the presence or absence of 1 μM 8CPT. 8CPT treatment was started 5 min prior to stimulation with adenosine and continued during stimulation (pretreatment; 5 min). (a) Representative adenosine-induced calcium increases in the presence or absence of 8CPT. (b) Average calcium increases (n= 30–40 cells from 3–4 experiments). Relative AUC was calculated by normalizing to the calcium increase to adenosine in the absence of blocker. **p < 0.01, t-test.
Figure 2
Figure 2
Electrically-induced adenosine release by hippocampal slices. A. Experimental diagram. SC, Schaffer collateral; SO, Striatum oriens; IM, Imaging area of the inverted microscope (gray circular area). The stimulating electrode was placed on the SC for synaptic stimulation (Synaptic) or on the SO for antidromic stimulation (Antidromic). B. Calcium responses of adenosine sensor cells to synaptic stimulation. Bright field image of a hippocampal slice above the adenosine sensor cells (Slice). PN; Pyramidal neurons in the CA1 region; SR, Stratum radiatum; LM, Stratum lacunosum moleculare. The stimulating glass electrode was placed as indicated by the arrowhead. Fura2 fluorescence image of the adenosine sensor cells (Fura2). Ratio of the image (340 nm/380 nm) before stimulation (-), and 15 sec after high-frequency electrical stimulation (30 Hz for 5 sec) at 0.3 mA or 3 mA. C. Representative calcium increases in adenosine sensor cells in response to synaptic or antidromic stimulation (3 mA, 30 Hz for 5 sec, arrow) in the absence or presence of 8CPT (pretreatment 20 min). D. Average calcium increases in adenosine sensor cells in response to synaptic or antidromic stimulation, with results normalized to response to synaptic stimulation in the absence of 8CTP. Each n= 29–47 cells from 3–5 experiments, **p < 0.01, t-test.
Figure 2
Figure 2
Electrically-induced adenosine release by hippocampal slices. A. Experimental diagram. SC, Schaffer collateral; SO, Striatum oriens; IM, Imaging area of the inverted microscope (gray circular area). The stimulating electrode was placed on the SC for synaptic stimulation (Synaptic) or on the SO for antidromic stimulation (Antidromic). B. Calcium responses of adenosine sensor cells to synaptic stimulation. Bright field image of a hippocampal slice above the adenosine sensor cells (Slice). PN; Pyramidal neurons in the CA1 region; SR, Stratum radiatum; LM, Stratum lacunosum moleculare. The stimulating glass electrode was placed as indicated by the arrowhead. Fura2 fluorescence image of the adenosine sensor cells (Fura2). Ratio of the image (340 nm/380 nm) before stimulation (-), and 15 sec after high-frequency electrical stimulation (30 Hz for 5 sec) at 0.3 mA or 3 mA. C. Representative calcium increases in adenosine sensor cells in response to synaptic or antidromic stimulation (3 mA, 30 Hz for 5 sec, arrow) in the absence or presence of 8CPT (pretreatment 20 min). D. Average calcium increases in adenosine sensor cells in response to synaptic or antidromic stimulation, with results normalized to response to synaptic stimulation in the absence of 8CTP. Each n= 29–47 cells from 3–5 experiments, **p < 0.01, t-test.
Figure 3
Figure 3
Effects of glutamate receptor antagonists on electrically-induced adenosine release. Adenosine release in response to synaptic or antidromic stimulation was assessed in the absence or presence of 10 μM CNQX and 100 μM D,L-APV. n= 30–49 cells from 3–5 experiments, **p < 0.01, t-test
Figure 4
Figure 4
Involvement of L-type calcium channels and CICR in electrically-induced adenosine release. A. Pharmacologic inhibition of calcium channels. Adenosine release in response to synaptic or antidromic stimulation in the absence or presence of 100 μM Cd2+ (pretreatment; 10 min) or 50 μM nimodipine (Nimo, pretreatment; 30 min). n= 23–30 cells from 3–4 experiments, **p < 0.01, Dunnett's multiple comparison test with control. B. Pharmacologic inhibition of CICR. (a) Adenosine release by normal and thapsigargin-treated (Tg, 1 μM) slices in response to antidromic stimulation. Slices were treated with 1 μM thapsigargin for 15 min, washed three times with normal aCSF, and placed onto adenosine sensor cells in normal aCSF for electrical stimulation. n= 30 cells from 3 experiments, **p < 0.01, t-test. (b) Effects of ryanodine and thapsigargin on adenosine release in response to KCl stimulation. Representative calcium increases in adenosine sensor cells. Adenosine release was induced by bath application of KCl (final concentration, 25 mM) as indicated by solid lines, and the lack of effect of ryanodine and thapsigargin on the adenosine response of adenosine sensor cells was confirmed by bath applications of adenosine (final concentration, 10 μM) at the end of the experiment (dashed line). Ryanodine treatment (Ry, 20 μM) was started 30 min prior to KCl stimulation. Thapsigargin was used as in Fig 4B(a). (c) Average KCl-induced calcium increases in adenosine sensor cells in the presence or absence of ryanodine or thapsigargin. Each n= 30 cells from 3 experiments, **p < 0.01, Dunnett's multiple comparison test with control.
Figure 5
Figure 5
Lack of involvement of ectonucleotidases and equilibrate nucleoside transporter (ENT) in electrically-induced adenosine release. (a) KCl-induced adenosine release in the absence or presence of 100 μM ARL67156 and 100 μM AOPCP (pretreatment; 15 min). As the inhibition of ectonucleotidases may cause extracellular ATP build-up and ATP-induced calcium increase in adenosine sensor cells, a P2 receptor antagonist, 30 μM PPADS, was added to the extracellular solution. Each n= 30 cells from 3 experiments, **p < 0.01, t-test. (b) Adenosine release in response to antidromic stimulation in the absence or presence of 5 μM NBTI and 10 μM DIPY (pretreatment; 30 min). As DIPY has autofluorescence blocking ultraviolet excitation of Fura2AM, Rhod4AM was used. Each n=28–34 cells from 3–4 experiments. **p < 0.01, t-test.
Figure 6
Figure 6
Adenosine release by treatments known to swell astrocytes. A. Calcium increases in adenosine sensor cells below hippocampal slices following hypoosmotic treatment (HO) in the absence or presence of 100 μM Cd2+ or 1 μM 8CPT. (a) Representative calcium increases in adenosine sensor cells. (b) Average calcium increases. n= 18–30 cells from three experiments. B. Adenosine release in response to potassium channel blockers. Slices were treated with 25 mM TEA, 100 μM 4-AP or 6 mM Ba2+ for 10 min in the presence or absence of 100 μM Cd2+ or 1 μM 8CPT. (a) Representative calcium increases in adenosine sensor cells following treatment with potassium channel blockers. (b) Average calcium increases. n= 29–39 cells from 3–4 experiments. *p < 0.05, **p < 0.01, Dunnett's multiple comparison test with control.
Figure 6
Figure 6
Adenosine release by treatments known to swell astrocytes. A. Calcium increases in adenosine sensor cells below hippocampal slices following hypoosmotic treatment (HO) in the absence or presence of 100 μM Cd2+ or 1 μM 8CPT. (a) Representative calcium increases in adenosine sensor cells. (b) Average calcium increases. n= 18–30 cells from three experiments. B. Adenosine release in response to potassium channel blockers. Slices were treated with 25 mM TEA, 100 μM 4-AP or 6 mM Ba2+ for 10 min in the presence or absence of 100 μM Cd2+ or 1 μM 8CPT. (a) Representative calcium increases in adenosine sensor cells following treatment with potassium channel blockers. (b) Average calcium increases. n= 29–39 cells from 3–4 experiments. *p < 0.05, **p < 0.01, Dunnett's multiple comparison test with control.
Figure 7
Figure 7
Involvement of AQP-4 and ectonucleotidases in hypoosmotically-induced adenosine release. Adenosine release induced by hypoosmotic conditions or antidromic stimulation was measured in the presence or absence of 100 μM TGN-020 (pretreatment; 20 min) or the mixture of ARL67156, AOPCP and PPADS (pretreatment; 15 min). (a) Representative calcium increases in the adenosine sensor cells. (b) Effects of TGN-020 on hypoosmotically- and antidromically-induced adenosine release. Average calcium increases. n= 20–30 cells from 3 experiments. **p < 0.01, t-test. (c) Effect of ARL67156 and AOPCP on hypoosmotically-induced adenosine release. Average calcium increases. n= 20–30 cells from 3 experiments. **p < 0.01, t-test.

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