benzofurans and sulforhodamine-101

Fast synaptic transmission requires a rapid clearance of the released neurotransmitter from the extracellular space. Glial glutamate transporters (excitatory amino acid transporters, EAATs) strongly contribute to glutamate removal. In this work, we investigated the paired-pulse plasticity of synaptically activated, glutamate transporter-mediated currents (STCs) in cortical layer 2/3 astrocytes. STCs were elicited by local electrical stimulation in layer 4 in the presence of ionotropic glutamate (AMPA and NMDA), GABAA, and GABAB receptor antagonists. In experiments with low [Na(+)]i (5 mM) intrapipette solution, STCs elicited by paired-pulse stimulation demonstrated paired-pulse facilitation (PPF) at short (<250 ms) interstimulus intervals (ISIs) and paired-pulse depression at longer ISIs. In experiments with close to physiological, high [Na(+)]i (20 mM) intrapipette solution, PPF of STCs at short ISIs was significantly reduced. In addition, the STC kinetics was slowed in the presence of high [Na(+)]i. Exogenous GABA increased astrocytic [Na(+)]i, reduced the mean STC amplitude, decreased PPF at short ISIs, and slowed STC kinetics. All GABA-induced changes were blocked by NO-711 and SNAP-5114, GABA transporter (GATs) antagonists. In experiments with the low intrapipette solution, GAT blockade under control conditions decreased PPF at short ISIs both at room and at near physiological temperatures. Dialysis of single astrocyte with low [Na(+)]i solution increased the amplitude and reduced PPR of evoked field potentials recorded in the vicinity of the astrocyte. We conclude that (1) endogenous GABA via GATs may influence EAAT functioning and (2) astrocytic [Na(+)]i modulates the short-term plasticity of STCs and in turn the efficacy of glutamate removal.

Topics: Action Potentials; Amino Acid Transport System X-AG; Animals; Animals, Newborn; Anisoles; Aspartic Acid; Astrocytes; Benzofurans; Biophysics; Cadmium Chloride; Calcium Channel Blockers; Electric Stimulation; Ethers, Cyclic; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Intracellular Fluid; Mice; Mice, Inbred C57BL; Neocortex; Neuronal Plasticity; Nipecotic Acids; Oximes; Patch-Clamp Techniques; Rhodamines; Sodium; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31-7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24-27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na+ currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.

Topics: Animals; Animals, Newborn; Benzofurans; Calcium Signaling; Cell Membrane; Coloring Agents; Cytological Techniques; Electrophysiology; Ethers, Cyclic; Fluorescent Dyes; Fura-2; Glial Fibrillary Acidic Protein; Hippocampus; Immunohistochemistry; Membrane Potentials; Neuroglia; Neurophysiology; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Rhodamines; Sodium Channels; Staining and Labeling