Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

Abstract

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na + and 1 H + and the counter-transport of 1 K + . The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease. Video LinkThe video component of this article can be found at

PAR1 activation induces rapid changes in glutamate uptake and astrocyte morphology

“…1E). In contrast, TFLLR significantly reduced the slow component of the fSTC decay (Ctrl 18.3 ± 4.3 ms, TFLLR 12.0 ± 2.3 ms (n = 12) * p =  0.04), leading to a change of its centroid (<t>)2028 (Ctrl 12.1 ± 1.4 ms, TFLLR 9.7 ± 1.4 ms (n = 12) * p =  0.02; Fig. 1E).…”

Defective glutamate and K + clearance by cortical astrocytes in familial hemiplegic migraine type 2

“…The rapidly decaying component was completely inhibited by the GluTs inhibitor TFB‐TBOA (TBOA), identifying it as the STC (Fig B). The STC can thus be obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a‐b in Fig B) (Scimemi & Diamond, ). The STC can also be obtained by subtracting from the total inward current an exponential waveform (trace c in Fig C) that approximates the average TBOA‐insensitive current (Fig C, top trace) obtained as described in Materials and Methods (Devaraju et al , ; Scimemi & Diamond, ).…”

“…The STC can thus be obtained by subtracting the residual current remaining in the presence of TBOA from the total inward current (trace a‐b in Fig B) (Scimemi & Diamond, ). The STC can also be obtained by subtracting from the total inward current an exponential waveform (trace c in Fig C) that approximates the average TBOA‐insensitive current (Fig C, top trace) obtained as described in Materials and Methods (Devaraju et al , ; Scimemi & Diamond, ). As in hippocampal slices (Diamond & Jahr, ; Diamond, ), also in barrel cortex slices the decay kinetics of the STC slowed down when the density of GluTs was reduced by subsaturating concentrations of DL‐TBOA (), as expected for a reduced rate of Glu clearance and a longer lifetime of synaptically released Glu in the extracellular space.…”

“…Recordings were made from layer 1 astrocytes deeper than 45 μm from the slice surface. The cell bodies of astrocytes were visualized using an upright microscope equipped with infrared light and DIC optics and identified by their small soma size (< 10 μm), low input resistance (< 20 MΩ; WT: 13.2 ± 0.7 MΩ, n = 42; KI: 14.2 ± 1.2 MΩ, n = 24; P = 0.42), very negative resting membrane potential (WT: −91 ± 0.6 mV, n = 42; KI: −91 ± 0.6 mV, n = 24, after LJP correction of −10 mV), inability to generate action potentials and linear current–voltage relationships, typical of so‐called passive astrocytes (Bergles & Jahr, ; Bernardinelli & Chatton, ) (Scimemi & Diamond, ).…”

An astrocytic basis of caloric restriction action on the brain plasticity

“…5a). The residual IK was pharmacologically isolated with 100 µM DL-TBOA, a glutamate transporter blocker, and subtracted from the combined synaptically induced current to reveal pure IGluT Scimemi and Diamond, 2013). The response to the fifth stimulus (IGluT(5)) in the train (5 x 50 Hz) was compared to the response to a single stimulus (IGluT(1), Fig.…”