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Synaptically evoked glutamate transporter currents in Spinal Dorsal Horn Astrocytes.

Zhang H, Xin W, Dougherty PM - Mol Pain (2009)

Bottom Line: Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1.Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 - 1 Hz) repetitive stimulation.These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology and Pain Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. haijun.zhang@mdanderson.org

ABSTRACT

Background: Removing and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na+ ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents.

Results: Whole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG) area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd2+. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 - 1 Hz) repetitive stimulation.

Conclusion: These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

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Pharmacological characterization of glutamate transporter currents in spinal astrocytes evoked through dorsal root entry zone stimulation. A-B, bath application of DHK (300 μM), a selective antagonist for glial GLT-1 glutamate transporter, partially blocked the evoked glutamate transporter current (n = 6), while TBOA, a non-selective antagonist for glial glutamate transporter, almost completely blocked the evoked glutamate transporter current (n = 15). Notice that a slow-decaying inward current (arrow) was not blocked by TBOA. *P < 0.01, DHK or TBOA vs. Control, Student's paired t-test. C, DHK slowed the decay of the glutamate transporter current, which was shown when the response in DHK was scaled to the peak of the control response (n = 6).
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Figure 5: Pharmacological characterization of glutamate transporter currents in spinal astrocytes evoked through dorsal root entry zone stimulation. A-B, bath application of DHK (300 μM), a selective antagonist for glial GLT-1 glutamate transporter, partially blocked the evoked glutamate transporter current (n = 6), while TBOA, a non-selective antagonist for glial glutamate transporter, almost completely blocked the evoked glutamate transporter current (n = 15). Notice that a slow-decaying inward current (arrow) was not blocked by TBOA. *P < 0.01, DHK or TBOA vs. Control, Student's paired t-test. C, DHK slowed the decay of the glutamate transporter current, which was shown when the response in DHK was scaled to the peak of the control response (n = 6).

Mentions: The fast inward current remained following addition of DNQX [6,7-Dinitroquinoxaline-2, 3-dione (10 μM)], AP-5 [D-2-Amino-5-phosphonopentanoic acid (25 μM)], bicuculline methiodide (10 μM)] and strychnine hydrochloride (5 μM)] to the bath and thus was not mediated by AMPA, NMDA, gamma-amino-butyric acid-A (GABAA) or glycine receptors (n = 15, Fig. 2). These currents were also insensitive to the GABA transporter inhibitor SKF-89976A (25 μM, n = 3) and nipecotic acid (100 μM, n = 3), and glycine transporter inhibitor sarcosine (500 μM, n = 3). In contrast, this current was inhibited by glutamate transporter antagonists. Application of dihydrokainate (DHK, 300 μM), a nontransported inhibitor selective for the glial GLT-1 transporter (IC50 = 23 μM for GLT-1 vs. = 3 mM for GLAST & EAAC1) [20], inhibited the evoked response by 45.6% ± 6.4% (n = 6; Fig. 5A, B). While DL-threo-β-benzyloxyaspartate (TBOA, 100 μM), a nonselective glial transporter inhibitor (IC50= 19 μM for EAAC1 [21], 68 μM for GLAST, and 6 μM for GLT-1, [22]), inhibited the current by 82.7% ± 2.1% (n = 15; Fig. 5A, B). After application of DHK, the decay of the remaining current was slowed (11.6 ± 1.4 vs. 17.9 ± 2.6 ms before and after DHK, n = 6, P < 0.01, Student's paired t-test), visibly when the response with DHK was scaled to the peak of the control response (Fig. 5C). The prolongation of the response in the presence of DHK suggests that synaptically released glutamate remains elevated in the extracellular space longer when uptake into astrocytes is reduced [17]. Blockade of glutamate transporter currents with TBOA revealed a residual small slow-decaying inward current (Fig. 5A, arrow) confirm the fast inward current (Fig. 2) as produced by glutamate transporter activity in dorsal horn astrocytes [17,23,24].


Synaptically evoked glutamate transporter currents in Spinal Dorsal Horn Astrocytes.

Zhang H, Xin W, Dougherty PM - Mol Pain (2009)

Pharmacological characterization of glutamate transporter currents in spinal astrocytes evoked through dorsal root entry zone stimulation. A-B, bath application of DHK (300 μM), a selective antagonist for glial GLT-1 glutamate transporter, partially blocked the evoked glutamate transporter current (n = 6), while TBOA, a non-selective antagonist for glial glutamate transporter, almost completely blocked the evoked glutamate transporter current (n = 15). Notice that a slow-decaying inward current (arrow) was not blocked by TBOA. *P < 0.01, DHK or TBOA vs. Control, Student's paired t-test. C, DHK slowed the decay of the glutamate transporter current, which was shown when the response in DHK was scaled to the peak of the control response (n = 6).
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Figure 5: Pharmacological characterization of glutamate transporter currents in spinal astrocytes evoked through dorsal root entry zone stimulation. A-B, bath application of DHK (300 μM), a selective antagonist for glial GLT-1 glutamate transporter, partially blocked the evoked glutamate transporter current (n = 6), while TBOA, a non-selective antagonist for glial glutamate transporter, almost completely blocked the evoked glutamate transporter current (n = 15). Notice that a slow-decaying inward current (arrow) was not blocked by TBOA. *P < 0.01, DHK or TBOA vs. Control, Student's paired t-test. C, DHK slowed the decay of the glutamate transporter current, which was shown when the response in DHK was scaled to the peak of the control response (n = 6).
Mentions: The fast inward current remained following addition of DNQX [6,7-Dinitroquinoxaline-2, 3-dione (10 μM)], AP-5 [D-2-Amino-5-phosphonopentanoic acid (25 μM)], bicuculline methiodide (10 μM)] and strychnine hydrochloride (5 μM)] to the bath and thus was not mediated by AMPA, NMDA, gamma-amino-butyric acid-A (GABAA) or glycine receptors (n = 15, Fig. 2). These currents were also insensitive to the GABA transporter inhibitor SKF-89976A (25 μM, n = 3) and nipecotic acid (100 μM, n = 3), and glycine transporter inhibitor sarcosine (500 μM, n = 3). In contrast, this current was inhibited by glutamate transporter antagonists. Application of dihydrokainate (DHK, 300 μM), a nontransported inhibitor selective for the glial GLT-1 transporter (IC50 = 23 μM for GLT-1 vs. = 3 mM for GLAST & EAAC1) [20], inhibited the evoked response by 45.6% ± 6.4% (n = 6; Fig. 5A, B). While DL-threo-β-benzyloxyaspartate (TBOA, 100 μM), a nonselective glial transporter inhibitor (IC50= 19 μM for EAAC1 [21], 68 μM for GLAST, and 6 μM for GLT-1, [22]), inhibited the current by 82.7% ± 2.1% (n = 15; Fig. 5A, B). After application of DHK, the decay of the remaining current was slowed (11.6 ± 1.4 vs. 17.9 ± 2.6 ms before and after DHK, n = 6, P < 0.01, Student's paired t-test), visibly when the response with DHK was scaled to the peak of the control response (Fig. 5C). The prolongation of the response in the presence of DHK suggests that synaptically released glutamate remains elevated in the extracellular space longer when uptake into astrocytes is reduced [17]. Blockade of glutamate transporter currents with TBOA revealed a residual small slow-decaying inward current (Fig. 5A, arrow) confirm the fast inward current (Fig. 2) as produced by glutamate transporter activity in dorsal horn astrocytes [17,23,24].

Bottom Line: Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1.Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 - 1 Hz) repetitive stimulation.These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology and Pain Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. haijun.zhang@mdanderson.org

ABSTRACT

Background: Removing and sequestering synaptically released glutamate from the extracellular space is carried out by specific plasma membrane transporters that are primarily located in astrocytes. Glial glutamate transporter function can be monitored by recording the currents that are produced by co-transportation of Na+ ions with the uptake of glutamate. The goal of this study was to characterize glutamate transporter function in astrocytes of the spinal cord dorsal horn in real time by recording synaptically evoked glutamate transporter currents.

Results: Whole-cell patch clamp recordings were obtained from astrocytes in the spinal substantia gelatinosa (SG) area in spinal slices of young adult rats. Glutamate transporter currents were evoked in these cells by electrical stimulation at the spinal dorsal root entry zone in the presence of bicuculline, strychnine, DNQX and D-AP5. Transporter currents were abolished when synaptic transmission was blocked by TTX or Cd2+. Pharmacological studies identified two subtypes of glutamate transporters in spinal astrocytes, GLAST and GLT-1. Glutamate transporter currents were graded with stimulus intensity, reaching peak responses at 4 to 5 times activation threshold, but were reduced following low-frequency (0.1 - 1 Hz) repetitive stimulation.

Conclusion: These results suggest that glutamate transporters of spinal astrocytes could be activated by synaptic activation, and recording glutamate transporter currents may provide a means of examining the real time physiological responses of glial cells in spinal sensory processing, sensitization, hyperalgesia and chronic pain.

Show MeSH
Related in: MedlinePlus