L-cysteine, a component of Neurobasal, elicits a D-APV-sensitive current similar to Neurobasal.

<p><b>A</b>. Representative currents from a hippocampal neuron showing the response to acute application of 130 µM L-cysteine (L-cys) and 50% Neurobasal (NB). The current was blocked by co-application of 50 µM D-APV. Acute application of L-cysteine in BBS and Neurobasal generated similar D-APV-sensitive current amplitude and kinetics. The calibration bar applies to all traces. <b>B</b>. Summary of steady-state current amplitude from 7 cells. Asterisk designates p<0.05 compared with current amplitude in the absence of D-APV (unpaired, two-tailed t-tests with Bonferroni correction for multiple comparisons). N.S. designates no statistical significance (p>0.05 using paired, two-tailed <i>t</i> tests).</p

L-cysteine and Neurobasal generate similar toxicity.

<p><b>A</b>. Photomicrographs from a single experiment showing toxicity as a result of L-cysteine (260 µM) incubation. Upper left: brightfield image from a sham medium exchange control. Upper right: A field from a dish incubated for 4 h in BBS (saline). Lower left: a field from a dish exposed to BBS plus 260 µM L-cysteine for 4 h. Trypan-positive nuclei with remnants of cell bodies are present. Lower right: 50 µM D-APV protected against L-cysteine-induced toxicity. <b>B</b>. Summary of experiments like that of panel A compared with Neurobasal (NB) incubation in sibling cultures. Neurotoxicity of 260 µM L-cysteine in BBS is similar to Neurobasal toxicity, and both are blocked by co-incubation with 50 µM D-APV (N = 4 independent experiments on independent platings). Asterisk designates p<0.05 compared with sham (unpaired, two-tailed t-tests with Bonferroni correction for multiple comparisons).</p

Acute Neurobasal application (diluted 50% in recording saline) generates a D-APV-sensitive current in synaptically isolated hippocampal neurons grown in microculture.

<p><b>A</b>. Representative currents from a cell demonstrating the response to 50% Neurobasal (NB) in the absence and presence of 50 µM D-APV. MEM application (50% dilution) produced minimal current. In this and subsequent figures, cells were voltage-clamped at −30 mV to relieve Mg²⁺ block of the NMDA receptor channel. Scale bar applies to all traces. <b>B</b>. Summary of the steady-state current amplitudes from 5 cells. Asterisk designates p<0.05 compared with sham (unpaired, two-tailed t-tests with Bonferroni correction for multiple comparisons). <b>C</b>. Representative traces from a cell demonstrating similar current amplitude generated by acutely applied Neurobasal (NB) diluted to 50% in standard saline and 0.5 µM glutamate (Glu) added to BBS. The scale bar applies to both traces. The faster onset kinetics and offset kinetics of Neurobasal-gated currents compared with glutamate-gated currents were a consistent finding (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025633#s2" target="_blank">Results</a>). These calibrations suggest that undiluted Neurobasal contains a low-affinity agonist, equivalent to 1 µM glutamate.</p

Fresh Neurobasal is neurotoxic to cultured hippocampal neurons.

<p><b>A</b>. Photomicrographs from a single experiment showing toxicity resulting from fresh Neurobasal (NB) incubation. Upper left: brightfield image from a control sham medium exchange stained with trypan blue 24 h after the challenge. Upper right: a field from a dish incubated in fresh Neurobasal for 4 h. Pyknotic, trypan positive nuclei with remnants of cell bodies are present. Lower left: 50 µM D-APV, a competitive NMDA receptor antagonist, was included in the fresh Neurobasal medium and protected neurons from Neurobasal toxicity. Lower right: A field from a dish incubated for 4 h in MEM, matched to Neurobasal for inorganic salts, osmolarity, pH, glucose, and glycine concentrations. <b>B</b>. Summary of experiments like that of panel A showing the effects of Neurobasal (NB) exposure over various incubation times. MEM incubation had minimal effect (N = 4 independent platings, asterisk indicates a main effect of Neurobasal compared with sham condition; p<0.01, one-way ANOVA).</p

Protein synthesis is not required for depolarization-induced muting. A.

<p>Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control) or 30 mM KCl (depolarized) with or without 1 µg/ml cycloheximide 30 min pretreatment and co-incubation (cyh). <b>B.</b> Summary of EPSC amplitudes from neurons treated as in panel A (<i>n</i> = 10 neurons). *p<0.05, Bonferroni corrected Student’s unpaired <i>t</i> test.</p

PKA signaling is required for recovery of Rim1 levels after depolarization-induced muting. A.

<p>Western blot analysis of whole-cell lysates from hippocampal mass cultures treated with 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+KT5720) or without (recovered) 2 µM KT5720. KT5720 was applied 0.5 h prior to and during recovery. <b>B.</b> Summary of Rim1 levels from Western blots as shown in A (<i>n</i> = 3). Rim1 protein levels for each condition were normalized to SV2 and control treatment. *p<0.05, Newman-Keuls post hoc test vs. control after one-way ANOVA.</p

Unmuting of basally mute synapses by forskolin, but not PDBu, requires protein synthesis.

<p><b>A.</b> Representative FM1-43FX (green) and vGluT-1 (red) merged images after 4 h DMSO (control), 4 hr DMSO plus 1 µg/ml cycloheximide (cyh), 4 h 50 µM forskolin (FSK 4 h), 4 h 50 µM forskolin plus 1 µg/ml cycloheximide (FSK 4 h+cyh), 2 min 1 µM PDBu (PDBu 2 min), or 2 min 1 µM PDBu plus 1 µg/ml cycloheximide (PDBu 2 min+cyh) treatment. Cycloheximide was applied 0.5 h prior to and during treatments. Scale bar represents 5 µm. <b>B.</b> Quantification of the percentage of vGluT-1-defined synapses labeled with FM1-43FX (active synapses) after treatments described in panel A (<i>n</i> = 20 fields). *p<0.05, Bonferroni corrected Student’s unpaired <i>t</i> test vs. control.</p

Protein synthesis is required for functional recovery from depolarization-induced muting.

<p><b>A.</b> Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+cyh) or without (recovered) 1–5 µg/ml cycloheximide. Cycloheximide was applied 0.5–2 h prior to and during recovery. <b>B.</b> Summary of EPSC amplitudes from neurons treated as in panel A (<i>n</i> = 14 neurons). <b>C.</b> Representative autaptic EPSCs from hippocampal neurons after 16 h 30 mM NaCl (control), 16 h 30 mM KCl followed by 3 h recovery in fresh medium with 5 µg/ml cycloheximide (recovered+cyh), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with 5 µg/ml cycloheximide followed by an additional 3 h recovery in fresh medium without cycloheximide (recovered+cyh+recovery). <b>D.</b> Summary of autaptic EPSC amplitudes from neurons treated as in panel C (<i>n</i> = 25 neurons). *p<0.05, Bonferroni corrected Student’s unpaired <i>t</i> test.</p

Nuclear phosphorylated CREB (pCREB) levels remain elevated after recovery from depolarization-induced muting. A.

<p>Merged image of pCREB (red), GABA (blue), and MAP2 (green) immunofluorescence in a mass hippocampal culture. <b>B.</b> pCREB immunostaining after the following treatments: 30 min DMSO (control), 30 min 50 µM forskolin (forskolin 30 min), 30 min 30 mM KCl (depolarized 30 min), 16 h 30 mM KCl (depolarized O/N), or 16 h 30 mM KCl followed by 3 h in fresh medium (recovered). <b>C.</b> Quantification of pCREB intensity in GABA-negative nuclei after treatments as described in panel A (<i>n</i> = 327–586 neurons). Intensity values were normalized to the average control value for each experiment. *p<0.05, Student’s unpaired <i>t</i> test vs. control after Bonferroni correction. <b>D.</b> Quantification of pCREB intensity in GABA-negative nuclei after 16 h 30 mM NaCl (control), 16 h 30 mM KCl (depolarized O/N), or 16 h 30 mM KCl followed by 3 h recovery in fresh medium with (recovered+KT) or without (recovered) 2 µM KT5720 (<i>n</i> = 45–145 neurons). KT5720 was applied 0.5 h prior to and during recovery. Intensity values were normalized to the average control value for each experiment. *p<0.05, Bonferroni corrected Student’s unpaired <i>t</i> test.</p