Glutamate transporter currents in bergmann glial cells follow the time course of extrasynaptic glutamate

Abstract

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate flux. Although a complete cycle of transport may require 50-100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of L-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.

Figure 1

Identification of a Bergmann glial cell. (A) Voltage responses of a Bergmann glia cell to 50 ms current steps (−200 pA to 700 pA) from a resting potential of −87 mV. Fitting the rising phases of these responses with two exponentials gave a fast time constant of 0.36 ms (47.3% amplitude) and a slower time constant of 3.3 ms. The time constants for the rise and decay phases were not significantly different (P > 0.05). This cell had an input resistance of 27 MΩ. (B) The cell was recorded with a patch pipette filled with 1.5 mM Cy5-EDA in KNO₃ internal solution and simultaneously imaged on a confocal microscope. Image is a composite of 11 optical sections taken at 5 μm steps. Bar = 25 μm.

Figure 2

Glutamate transporter currents and AMPA receptor currents can be evoked in outside-out patches from Bergmann glia. (A) l-glutamate (10 mM) activates a transient current that is blocked by NBQX and GYKI-52466 and a smaller biphasic current in patches using a KSCN internal solution. V_m = −90 mV. (B) Complete substitution of extracellular Li⁺ for Na⁺ blocks the transporter current evoked by 10 mM l-glutamate. V_m = −90 mV. (C and D) The current-voltage relationship of the transporter current is inwardly rectifying and does not reverse. Responses in C were recorded at membrane potentials of −10, −30, −50, −70, and −90 mV. In D, each point is an average of responses from six patches normalized to the peak response at −110 mV. (E) In the presence of NBQX (10 μM) and GYKI (25 μM), THA (300 μM) produces a steady-state inward current and occludes the response to a pulse of 10 mM l-glutamate. The outward current elicited by l-glutamate in the presence of THA probably reflects the replacement of THA with l-glutamate, which is likely to occur at these concentrations. V_m = −110 mV. (F) l-aspartate (2 mM) activates a transporter current but not an AMPA receptor current. The control responses and those recorded in NBQX (10 μM) and GYKI (25 μM) are superimposed. This result also indicates that at these concentrations neither NBQX nor GYKI alter transporter function. KSCN-based internal solutions were used in all recordings. The uppermost traces in each figure are the “open-tip” response obtained by solution exchanges after disrupting the membrane patch. In each case the concentrations listed were used, except in F, where ACSF was diluted with 50% dH₂O to increase the size of the junction current. Traces are averages of 5–15 responses.

Figure 3

CF stimulation elicits both glutamate transporter currents and AMPA receptor currents in Bergmann glia. (A) Bergmann glia response to CF stimulation is all-or-none and blocked by a combination of NBQX (10 μM), D-CPP (5 μM), SR-95531 (5 μM), and THA (300 μM). Each point represents the average of three consecutive responses. Holding potential was −90 mV. At the first asterisk the stimulation intensity was decreased to 10 μA, below the threshold for the response. The stimulation intensity was then increased in steps of 20 μA, with three responses recorded at each intensity. The response returned at 70 μA and did not increase in amplitude when the intensity was increased further (up to 130 μA). A stimulation intensity of 110 μA was maintained during the rest of the experiment except at the second asterisk where the threshold was again tested. (B) Averaged Bergmann glial responses to stimuli of different intensities. (C) The rapid component of the current is blocked by NBQX (10 μM) and the remaining slow component is blocked by THA (300 μM). (D) Comparison of the kinetics of the AMPA receptor and transporter currents evoked by CF stimulation. The AMPA receptor current was obtained by subtracting the response in the presence of NBQX from the control response. (E) CdCl₂ (30 μM), but not dihydrokainate (300 μM), blocked the NBQX-insensitive current. All traces are averages of 3–5 consecutive responses and were recorded from the same Bergmann glial cell. KNO₃-based internal solution.

Figure 4

Concentration-dependence of the kinetics of glutamate transporter and AMPA receptor currents in patches from Bergmann glial cells. (A) Currents evoked in an outside-out patch by 30 ms applications of l-glutamate at 250 μM, 500 μM, 1 mM and 10 mM. Cs₂SO₄-based internal solution. (B) Currents evoked in an outside-out patch in the presence of NBQX (10 μM) and GYKI (25 μM) by applications of l-glutamate at 10 μM, 100 μM, 1 mM and 10 mM. KSCN internal solution. Traces are averages of 15–50 responses. V_m = −90 mV.