Glial contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippocampus

Abstract

Astrocytes in the hippocampus express high-affinity glutamate transporters that are important for lowering the concentration of extracellular glutamate after release at excitatory synapses. These transporters exhibit a permeability to chaotropic anions that is associated with transport, allowing their activity to be monitored in cell-fee patches when highly permeant anions are present. Astrocyte glutamate transporters are highly temperature sensitive, because L-glutamate-activated, anion-potentiated transporter currents in outside-out patches from these cells exhibited larger amplitudes and faster kinetics at 36 degreesC than at 24 degreesC. The cycling rate of these transporters was estimated by using paired applications of either L-glutamate or D-aspartate to measure the time necessary for the peak of the transporter current to recover from the steady-state level. Transporter currents in patches recovered with a time constant of 11.6 msec at 36 degreesC, suggesting that either the turnover rate of native transporters is much faster than previously reported for expressed EAAT2 transporters or the efficiency of these transporters is very low. Synaptically activated transporter currents persisted in astrocytes at physiological temperatures, although no evidence of these currents was found in CA1 pyramidal neurons in response to afferent stimulation. L-glutamate-gated transporter currents were also not detected in outside-out patches from pyramidal neurons. These results are consistent with the hypothesis that astrocyte transporters are responsible for taking up the majority of glutamate released at Schaffer collateral-commissural synapses in the hippocampus.

Fig. 1.

Temperature-dependent changes in astrocyte transporter currents. A, Outside-out patch removed from an astrocyte located in stratum radiatum of area CA1. Solid lines in A and B are the control responses recorded at room temperature, and dotted linesare the responses of the same patch recorded at 36°C.Inset, Response at 36°C has been scaled to that in control and shown at a faster time base to illustrate the time course of the two responses. B, Response of a different patch to d-aspartate (10 mm). Tracesare illustrated as in A. Traces are averages of 8–12 consecutive responses recorded at −90 mV. KSCN-based internal solution. The open tip response above eachtrace indicates the duration of the agonist application.

Fig. 2.

Recovery time course ofl-glutamate-evoked transporter currents. A,B, l-glutamate (10 mm) was applied for 30 msec to an outside-out patch from an astrocyte and then reapplied for 20 msec after a variable delay, at both 24°C (top traces) and 36°C (bottom traces).Traces are averages of six consecutive responses recorded at −90 mV. KSCN-based internal solution. C, Summary plot of the ratio of the peak amplitude of the second pulse (P2) over the peak amplitude of the control response (P1) for recordings made at both 24°C (n = 4) and 36°C (n = 11). The four patches used to measure the recovery at 24°C were also used to measure the recovery at 36°C.

Fig. 3.

Recovery time course ofd-aspartate-evoked transporter currents. A,B, d-aspartate (10 mm) was applied to an outside-out patch from astrocytes for 30 msec and then reapplied for 20 msec after a variable delay, at both 24°C (top traces) and 36°C (bottom traces).Traces are averages of five consecutive responses recorded at −90 mV. KSCN-based internal solution. C, Summary plot of the ratio of the peak amplitude of the second pulse (P2) over the peak amplitude of the control response (P1) for recordings made at both 24°C (n = 4) and 36°C (n = 10). The four patches used to measure the recovery at 24°C were also used to measure the recovery at 36°C.

Fig. 4.

Temperature-dependent changes in evoked responses.A, Synaptically activated transporter current (STC) recorded from a stratum radiatum astrocyte.V_m = −96 mV. K-methanesulfonate-based internal solution. B, AMPA receptor-mediated EPSCs recorded from a CA1 pyramidal neuron. V_m = −80 mV. Cs-methanesulfonate-based internal solution. C, Field EPSPs (fEPSP) recorded in stratum radiatum of area CA1. A–C, Solid lines are the responses recorded at room temperature, and the dotted lines are the responses recorded at 36°C.

Fig. 5.

Transporter currents are not associated with EPSCs in CA1 pyramidal neurons. The ionotropic glutamate receptor antagonists NBQX (10 μm) and d,l-CPP (10 μm) completely blocked evoked responses, at both 24°C and 36°C. The brief inward current observed in NBQX and CPP is attributable to the increased amplitude and slower decay of the stimulus artifact at 36°C. V_m = −90 mV. ACSF contained picrotoxin (100 μm) and SR-95531 (5 μm). KSCN-based internal solution.

Fig. 6.

Transporter currents are not detected in pyramidal cell patches. A, Outside-out patch from a CA1 pyramidal neuron. l-glutamate (10 mm) activates inward (V_m = −90 mV) and outward (V_m = 90 mV) (A₁) currents that are completely blocked by NBQX (10 μm), GYKI-52466 (25 μm), and d,l-CPP (10 μm) (A₂). B,l-glutamate (10 mm) activates transporter currents in outside-out patches from CA1 astrocytes (B₁), which were not affected by NBQX (10 μm), GYKI-52466 (25 μm), andd,l-CPP (10 μm) (B₂). Responses were recorded at −90 mV and +90 mV, as in A. All solutions contained 20 μmglycine. Traces are averages of 8–12 consecutive responses. A KSCN-based internal solution was used for both recordings.