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In the developing mammalian brain, gamma-aminobutyric acid (GABA) is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl(-) in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF) may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl(-) concentrations, and therefore a more depolarized GABA receptor (GABAR) reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments.
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22496804
???displayArticle.pmcLink???PMC3319581 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 1. A schematic view of the preparation, and examples of the data.A. A simplified scheme of the preparation: R – recording electrode; OT – middle third of the Optic Tectum; OCh – Optic Chiasm; St – Stimulating Electrode. B. Typical responses to the optic chiasm stimulation, recorded in loose-cell-attached current-clamp mode (s49, control ACSF, 10 responses superimposed); C. An example of RTS calculation for one of the cells (s49, pyruvate-containing ACSF). Average spikes/stimulus values are plotted against respective stimulation strengths, and are shown together with a fit curve. Each dot corresponds to the average number of spikes/stimulus observed over 10 consecutive responses.
Figure 2. Responsiveness to stimulation and inter-spike interval values for different developmental stages and ACSF compositions.A. Average responsiveness to stimulation (RTS). Each colored circle represents average responsiveness-to-stimulation value for an individual cell, with cells from younger animals (s45) shown in the left group, and those from older animals (s49) – in the right group. Data obtained in ACSF of different formulations is shown in columns of different color. ACSF types are encoded in the following way (left to right): c – standard (control) ACSF; x – standard ACSF+PTX; p – pyruvate-based ACSF; o – ACSF lacking energy sources; ox – no energy sources+PTX. Horizontal bars represent mean RTS values across all cells within each data group. B. Median inter-stimulus intervals (ISI). Each colored circle represents median inter-stimulus interval value for an individual cell; data from animals of different age, and recorded in different ACSF formulations are given in the same order and with same labels as in panel A. Horizontal bars show average ISI values across all cells within each data group.
Figure 3. Measurement of GABAR reversal potential.A. Example of currents evoked by local GABA application at different command potentials (stage 45, control ACSF, central third of OT). B. IV-curve of GABA-evoked currents from panel A, with amplitudes shown against the command potential, polynomial fit of these amplitudes, and an estimation for GABAR reversal potential EGABA. C. EGABA values observed in all cells recorded (n = 122), shown separately for stage 45 (left) and stage 49 (right) animals; control ACSF (“c”, blue) and pyruvate-containing ACSF (“p”, green). Horizontal bars show average EGABA values across all cells in a data group.
Figure 4. Spatial gradient of GABAR reversal potential in the OT.A. All cells that had their position within OT recorded (n = 105), for both stages and ACSF formulations, shown projected onto open right OT outline. Rostral direction is up, caudal is down, medial is left, lateral is right. Circles stand for cells recorded in control ACSF at stage s45; squares – control ACSF s49; down triangles – pyruvate-containing ACSF s45; up triangles – pyruvate-containing ACSF s49. Color of the marker encodes EGABA measured in each of the cells, with blue corresponding to more negative, and red – to more positive values (see color-bar on the right). Subset of cells further referred to as the “Rostral group” is encircled with a dashed circle. B. EGABA observed in OT cells as a function of distance from the center of the “Rostral group” shown on the left. Blue for control ACSF; green for pyruvate-containing ACSF; marker shapes follow same conventions as in the left panel. The threshold distance limiting the “Rostral group” is shown as a dashed line.
Figure 5. Comparison of GABAR reversal potentials in cells grouped by their location in the OT.Stage 45 cells are shown on the left; stage 49 – on the right. C – caudal group of cells (adjacent to the proliferative zone), R – rostral group of cells. Blue – control ACSF, green – pyruvate-containing ACSF. Statistically significant differences are marked with a square brake on top. Horizontal bars represent average EGABA values across all cells in a data group.
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