Freshly isolated hippocampal CA1 astrocytes comprise two populations differing in glutamate transporter and AMPA receptor expression

Abstract

We have shown previously that process-bearing GFAP+ astrocytes freshly isolated from rat hippocampus CA1 and CA3 regions are heterogeneous in ion channel expression and K(+) uptake capabilities, such that two distinct populations of astrocytes can be described (Zhou and Kimelberg, 2000). In the present study, we report that glutamate transporter (GT) currents can only be measured from one type of these freshly isolated hippocampal CA1 astrocytes [variably rectifying astrocytes (VRAs)] but were not detectable in the second type of astrocyte [outwardly rectifying astrocytes (ORAs)]. The GT currents showed a strict Na(+) dependency and high affinity for glutamate (EC(50) of 4 +/- 1.1 microm). The astrocytes lacking GT currents (ORAs) showed an AMPA receptor current density (55 pA/pF) that was 42-fold higher than VRAs (1.3 pA/pF). In contrast, the GABA(A) currents were of comparable current density in both types. The specificity of these differences makes it unlikely that they are attributable to preparative damage. Therefore, these findings strongly indicate that, within a single region of the hippocampus, GFAP+ astrocytes comprise a functionally diverse population that are qualitatively different in their functional glutamate transporter and quantitatively different in their functional AMPA receptor expression. This heterogeneity implies that GFAP+ astrocytes may participate in or modulate glutamate synaptic transmission differently.

Fig. 1.

Morphology and current profiles of ORAs and VRAs.A, Imaging of a freshly isolated astrocyte during recording with Lucifer yellow dye (0.3%), in the pipette, showing bushy processes extending from the cell body. The processes are not very distinct because there is some folding back of the processes and because of the out-of-focus fluorescence halo, as this photograph was taken through the nonconfocal Nikon Diaphot microscope we used in the recording set up. B, E, Membrane currents induced by voltage steps (50 msec) from −160 to +60 mV (20 mV increments) with a NO₃⁻-based pipette solution (see Materials and Methods). ORAs (B) are characterized by a dominant expression of outwardIK_a and IK_dr plus small inward INa⁺ currents (seeinset below B). VRAs (E) are characterized by a symmetric expression of inward and outward potassium currents. C,F, When K⁺ channel-mediated currents were completely masked by the substitution of pipette K⁺ with Cs⁺, an ORA (C) and a VRA (F) were identified based on their identical bushy morphology but marked different membrane capacitances (10.5 pF in recording Cand 38 pF in F). TheINa⁺ is shown in higher resolution inC. INa⁺ currents were never observed in VRAs (F). D andG are recordings C and F, respectively, at higher resolution and after off-line compensation for leak and capacitance.

Fig. 2.

Differential expression of GT and AMPA-R currents by VRAs and ORAs. With NO₃⁻ as the major intracellular anion, 10 mm Glu (at −70 mV) evoked a fast activating and rapidly desensitizing inward current in ORAs (dashed traces in A). The response to the same Glu application in VRA showed the initial transient plus a substantial steady-state current during the 0.5 sec Glu pulse (dashed traces in B). The selective AMPA-R antagonists NBQX (10 μm) plus GYKI₅₂₄₆₆ (25 μm) completely blocked the Glu-induced current in the ORA (solid trace inA). In the VRA, however, the same antagonists reduced the initial peak current by only 34% and potentiated the steady-state current by 40% (solid trace in B). In both cases, NBQX–GYKI₅₂₄₆₆ was applied 250 msec before the Glu pulse to ensure a complete block of AMPA activation. The dashed traces in A and B are the superimposed Glu-evoked currents of the initial control and then after washout of the AMPA-R antagonists. Both recordings A andB were in cells from P11 rats.

Fig. 3.

NBQX–GYKI₅₂₄₆₆-resistant currents in VRAs are GT-associated currents. A, Substitution of extracellular Na⁺ by Li⁺completely abolished the Glu-induced NBQX–GYKI₅₂₄₆₆-insensitive current. B, THA, a transportable GT inhibitor, induced a sustained inward current and blocked any Glu-generated additional current in the same cells. Cells were clamped at −70 mV throughout. C, D, With 140 mm NO₃⁻ in the pipette and 150 mm Cl⁻ in the bath solution, the NBQX–GYKI-resistant inward current persisted until the voltage steps reached +70 to +80 mV, consistent with a primary NO₃⁻ identity of this current (E_NO3 ∼70 mV). RecordingsA–C were obtained in VRAs from P7, P12, and P10 animals, respectively.

Fig. 4.

The absence of GT currents in ORAs is independent of the permeant intracellular anion and animal age. A1and A2 show an ORA recording with SCN⁻ as the major anion in the pipette solution. Glu at 1 mm induced a fast activation and rapidly desensitizing inward current (A1), which was completely blocked by NBQX–GYKI₅₂₄₆₆ (A2).B1 shows an ORA from a P31 rat, characterized by the expression of INa⁺ (see theinset below B1) and also shows a smallIK_in in response to hyperpolarization voltage steps (same voltage commands as in Fig. 1). The Glu-evoked current from this cell (B2) had the same kinetics as inA1, and the current was completely abolished by NBQX–GYKI₅₂₄₆₆ (B3).

Fig. 5.

GT current blockade by the selective GLT-1 inhibitor DHK. All of the recording traces were obtained at an identical holding potential of −70 mV. NBQX–GYKI₅₂₄₆₆were present throughout the recording to block AMPA-R currents. Thedashed lines are the GT current in control and after washout of DHK. The solid line trace shows the effect of addition of 300 μm DHK. DHK was applied before the Glu pulse and continued throughout (as indicated by the dotted horizontal line at the bottom). DHK reduced the negative holding current (−70 mV) when Glu was absent (13 pA upward shift as indicated by arrow). DHK also inhibited the Glu-evoked peak current by 38% and steady-state current by 40% in this cell. Recording traces were obtained from a P13 rat. See Results for the variability in the magnitude of DHK blockade.

Fig. 6.

GT currents in VRAs show high Glu affinity for l-glutamate. A, Representative GT current traces induced by a series of Glu concentrations in a P10 VRA. B, Current amplitudes of the steady-state Glu-induced GT currents at different concentrations of Glu were normalized to the corresponding response evoked by 1 mm Glu in the same cell. Each data point represents the mean from three cells. Error bars show SEMs. Smooth linegives the best fit according to the Hill equation (see Materials and Methods), yielding an EC₅₀ of 4 ± 1.1 μm and n_H of 0.6.

Fig. 7.

GABA_A receptor currents are comparable in ORAs and VRAs. A1–A3 andB1–B3 are recordings from an ORA and a VRA, respectively, at holding potential of −70 mV. In A1 andB1, a 0.5 sec 1 mm GABA pulse induced robust inward currents in both cells with a similar desensitization time course. At the end of the GABA pulse, the initial peak current (A_P) desensitized 47% inA1 and 40% in B1. A2 andB2 show the similar inhibition of GABA-induced currents by the selective GABA_A antagonist bicuculline (10 μm). The GABA-induced currents were reduced by 52 and 64% in an ORA (A2) and a VRA (B2), respectively. A3 and B3 were the responses induced by the selective GABA_A receptor agonist muscimol (50 μm). In A3 andB3, the muscimol-induced peak current amplitudes amounted to 61 and 64% of the GABA induced currents. All recordings inA and B represent the same respective cells.