Figure 1.
Fresh Neurobasal is neurotoxic to cultured hippocampal neurons.
A. 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. 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).
Figure 2.
Acute Neurobasal application (diluted 50% in recording saline) generates a D-APV-sensitive current in synaptically isolated hippocampal neurons grown in microculture.
A. 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. 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). C. 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 Results). These calibrations suggest that undiluted Neurobasal contains a low-affinity agonist, equivalent to 1 µM glutamate.
Figure 3.
L-cysteine, a component of Neurobasal, elicits a D-APV-sensitive current similar to Neurobasal.
A. 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. 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 t tests).
Figure 4.
L-cysteine and Neurobasal generate similar toxicity.
A. 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. 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).