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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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Identification of astrocytes in spinal dorsal horn. An astrocyte (arrow) with attached patch pipette (P) is shown when viewed in DIC (A) and under epifluorescence when filled with Lucifer Yellow (B). C, Anti-GFAP antibody labeling (red) superimposed on the cell loaded with Lucifer Yellow (green) shown in B. D, under voltage clamp, inward and outward currents (upper left) were evoked in the same cell, by depolarizing and hyperpolarizing voltage steps from a holding potential of -80 mV (lower left). I/V curve (upper right) shows the nearly linear current-voltage relationship with slight rectification during hyperpolarization from the recorded cell. Whole-cell membrane potential changes (lower right) induced by 200 ms steps of current (-500 to 1200 pA) recorded from the same cell, noticing there is no action potential induced. Scale bar = 10 μm in A and B; 20 μm in C.
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Figure 1: Identification of astrocytes in spinal dorsal horn. An astrocyte (arrow) with attached patch pipette (P) is shown when viewed in DIC (A) and under epifluorescence when filled with Lucifer Yellow (B). C, Anti-GFAP antibody labeling (red) superimposed on the cell loaded with Lucifer Yellow (green) shown in B. D, under voltage clamp, inward and outward currents (upper left) were evoked in the same cell, by depolarizing and hyperpolarizing voltage steps from a holding potential of -80 mV (lower left). I/V curve (upper right) shows the nearly linear current-voltage relationship with slight rectification during hyperpolarization from the recorded cell. Whole-cell membrane potential changes (lower right) induced by 200 ms steps of current (-500 to 1200 pA) recorded from the same cell, noticing there is no action potential induced. Scale bar = 10 μm in A and B; 20 μm in C.

Mentions: Whole-cell recordings were obtained from 45 glial cells in the SG of spinal cord dorsal horn. Glial cells were first visually identified as small, rounded cells with a soma diameter less than 10 μm (Fig. 1A). We further distinguished glial cells from neurons by their electrophysiological properties after obtaining the whole-cell recording configuration. Glial cells were characterized by absence of action potentials to injection of depolarizing currents steps up to 1200 pA while in current clamp mode and by the demonstration of passive current responses to voltage steps while in voltage clamp mode (Fig. 1D). In addition, recorded cells had a characteristically low mean input resistance of 24.4 ± 2.4 MΩ (n = 45), negative resting membrane potential of -74.1 ± 0.5 mV (n = 45), and mean membrane capacitance of 11.4 ± 1.0 pF (n = 45).


Synaptically evoked glutamate transporter currents in Spinal Dorsal Horn Astrocytes.

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

Identification of astrocytes in spinal dorsal horn. An astrocyte (arrow) with attached patch pipette (P) is shown when viewed in DIC (A) and under epifluorescence when filled with Lucifer Yellow (B). C, Anti-GFAP antibody labeling (red) superimposed on the cell loaded with Lucifer Yellow (green) shown in B. D, under voltage clamp, inward and outward currents (upper left) were evoked in the same cell, by depolarizing and hyperpolarizing voltage steps from a holding potential of -80 mV (lower left). I/V curve (upper right) shows the nearly linear current-voltage relationship with slight rectification during hyperpolarization from the recorded cell. Whole-cell membrane potential changes (lower right) induced by 200 ms steps of current (-500 to 1200 pA) recorded from the same cell, noticing there is no action potential induced. Scale bar = 10 μm in A and B; 20 μm in C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2713213&req=5

Figure 1: Identification of astrocytes in spinal dorsal horn. An astrocyte (arrow) with attached patch pipette (P) is shown when viewed in DIC (A) and under epifluorescence when filled with Lucifer Yellow (B). C, Anti-GFAP antibody labeling (red) superimposed on the cell loaded with Lucifer Yellow (green) shown in B. D, under voltage clamp, inward and outward currents (upper left) were evoked in the same cell, by depolarizing and hyperpolarizing voltage steps from a holding potential of -80 mV (lower left). I/V curve (upper right) shows the nearly linear current-voltage relationship with slight rectification during hyperpolarization from the recorded cell. Whole-cell membrane potential changes (lower right) induced by 200 ms steps of current (-500 to 1200 pA) recorded from the same cell, noticing there is no action potential induced. Scale bar = 10 μm in A and B; 20 μm in C.
Mentions: Whole-cell recordings were obtained from 45 glial cells in the SG of spinal cord dorsal horn. Glial cells were first visually identified as small, rounded cells with a soma diameter less than 10 μm (Fig. 1A). We further distinguished glial cells from neurons by their electrophysiological properties after obtaining the whole-cell recording configuration. Glial cells were characterized by absence of action potentials to injection of depolarizing currents steps up to 1200 pA while in current clamp mode and by the demonstration of passive current responses to voltage steps while in voltage clamp mode (Fig. 1D). In addition, recorded cells had a characteristically low mean input resistance of 24.4 ± 2.4 MΩ (n = 45), negative resting membrane potential of -74.1 ± 0.5 mV (n = 45), and mean membrane capacitance of 11.4 ± 1.0 pF (n = 45).

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