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

Summary

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

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.

Introduction

Astrocytes are one of the most abundant cell types in the brain with star-shaped morphology and fine membrane protrusions that extend throughout the neuropil and reach neighboring synaptic contacts ^1,2. The astrocytes’ cell membrane is densely packed with glutamate transporter molecules ³. Under physiological conditions, glutamate transporters rapidly bind glutamate at the extracellular side of the membrane and transfer it to the cell cytoplasm. By doing so, the transporters maintain low the basal concentration of glutamate in the extracellular space ⁴. Glutamate transporters in fine astrocytic processes adjacent to excitatory synapses are ideally positioned to bind glutamate released during synaptic events as it diffuses away from the synaptic cleft. By doing so, the transporters also limit glutamate spillover towards peri- and extra-synaptic regions and onto neighboring synapses, reducing the spatial spread of excitatory signals in the brain ^5-7.

Glutamate transport is an electrogenic process stoichiometrically coupled to the movement of 3 Na⁺ and 1 H⁺ along their electrochemical gradient and to the counter-transport of 1 K^{+ 8}. Glutamate transport is associated with (but not stoichiometrically coupled to) an anionic conductance permeable to SCN^– (thiocyanate) > NO₃^– (nitrate) ≈ ClO₄^– (perchlorate) > I^– > Br^– > Cl^– > F^–, not to CH₃SO₃^– (methane sulfonate) and C₆H₁₁O₇^– (gluconate) ^9-11. Both currents (stoichiometric and non-stoichiometric) can be recorded by obtaining whole-cell patch-clamp recordings from astrocytes, visually identified under Dodt illumination or infra-red differential interference contrast (IR-DIC) in acute brain slices ¹². The stoichiometric component of the current associated with glutamate transport across the membrane can be isolated by using CH₃SO₃^–, or C₆H₁₁O₇^– based intracellular solutions and can be evoked by flash-uncaging glutamate on astrocytes ^13,14, or by activating glutamate release from neighboring synapses, either electrically ¹² or with a targeted optogenetic control.

The time course of the stoichiometric component of the transporter current is shaped by the lifetime of the glutamate concentration profile at astrocytic membranes (i.e. glutamate clearance), the kinetics of glutamate transporters, the passive membrane properties of astrocytes, and during synaptic stimulations, by the synchronicity of glutamate release across the activated synapses ¹³. Here we describe in full detail: (1) an experimental approach to isolate the stoichiometric component of glutamate transporter currents from whole-cell patch-clamp recordings from astrocytes using acute mouse hippocampal slices as an example experimental preparation; (2) an analytical approach to derive the time course of glutamate clearance from these recordings ^13,14. These methods can be used to record and analyze glutamate transporter currents from astrocytes in any region of the central nervous system.

Protocol

1. Slice Preparation Prepare 500 ml slicing/storage solution containing (in mM): 119 NaCl, 2.5 KCl, 0.5 CaCl2, 1.3 MgSO4·7H2O, 4 MgCl2, 26.2 NaHCO3, 1 NaH2PO4, and 22 glucose, 320 mOsm, pH 7.4 Use a 250 ml beaker to prepare a submersion chamber for the slices, fill it with 200 ml slicing/storage solution, warm it in a water bath at 34 °C and bubble it with 95% O2, 5% CO2. Keep the …

Representative Results

The success of the analytical approach described here critically depends on obtaining high-quality electrophysiological recordings of transporter currents from astrocytes in any region of the central nervous system. In acute mouse hippocampal slices, astrocytes can be readily identified under Dodt illumination or IR-DIC because of their small cell body (Ø = 10 μm) and prominent nucleus (Figure 1). Their distinctive star-shaped morphology can be appreciated with epifluorescence, confocal, or two…

Discussion

Here we describe an experimental approach to obtain electrophysiological recordings from astrocytes, an analytical protocol to isolate glutamate transporter currents in astrocytes and a mathematical method to derive the time course of glutamate clearance from astrocytic transporter currents.

The success of the analysis relies on the ability to obtain high-quality patch clamp recordings from astrocytes and on the accuracy of the fitting algorithms used to describe the transporter currents. The …

Materials

Material Name	Company	Catalogue Number	Comments
CGP52432	Tocris	1246
(R,S)-CPP	Tocris	173
DPCPX	Tocris	439
LY341495 disodium salt	Tocris	4062
MSOP	Tocris	803
NBQX disodium salt	Tocris	1044
D,L-TBOA	Tocis	1223
Picrotoxin	Sigma	P1675
MNI-L-glutamate	Tocris	1490
Alexa 594	Life Technologies	A10438	Optional
Matrix electrodes	Frederick Haer Company	MX21AES(JD3)
Borosilicate glass capillaries	World Precision Instruments	PG10165-4
Dual-stage glass micro-pipette puller	Narishige	PC-10
Loctite 404 instant adhesive	Ted Pella	46551
Xe lamp	Rapp OptoElectronic	FlashMic
Igor Pro 6	Wavemetrics