Figure 5. NMDA receptor-dependent NO release is αNOS1 dependent and basal NO levels are both αNOS1 and NOS3 dependent. A solution containing bicuculline and lacking magnesium (BMI/0Mg2+) results in spontaneous bursts of spikes in neocortex that activated NMDA receptors (Fig. 6) and generated NO. A, In wild types, the left-most two bars show that application of APV (50 μm; gray bar; n = 11) has no effect on basal levels of NO in untreated cortex (black bar; n = 11). The right-most two bars show that BMI/0Mg2+ causes an increase in NO production (black bar; n = 11) unless the cortex is pretreated with APV (gray bar; n = 11). B, Using the same convention as in A, in wild types BMI/0Mg2+ causes an increase in NO unless the cortex is pretreated with l-NAME (100 μm) 30 min beforehand (gray bar; n = 10). Thirty minutes of treatment with l-NAME did not produce a significant decrease in [NO] (gray bar; n = 10), but if the animals were injected with l-NAME 2 d previously, [NO] was significantly reduced (white bar; n = 3; p < 0.01). C, Basal levels of NO are significantly lower in αNOS1 knock-outs than wild types (n = 5 for both genotypes; p < 0.01). However, the increase in NO levels normally caused by BMI/0Mg2+ in wild types (black bar; n = 5) is not present in αNOS1 knock-outs (white bars; n = 5). D, Basal levels of NO are also significantly lower in NOS3 knock-outs (white bar; n = 5) than in wild types (black bar; n = 5; p < 0.01). However, the increase in NO production caused by BMI/0Mg2+ still occurs in NOS3 knock-outs (white bars; n = 5) and reaches similar levels to that seen in wild types (black bar; n = 5). NO values were normalized to the untreated wild-type condition in each case (indicated by the dashed line at unity). The amount of NO2− present in wild-type controls (over 30 min) was 15.4 ± 2.4 pm/mg (A), 7.4 ± 0.6 pm/mg (B), 9.1 ± 1.7 pm/mg (C), and 6.2 ± 1.2 pm/mg (D) (wet whole brain weight). *p < 0.05, **p < 0.01, NS, Not significant (p > 0.05). E, F, Calibration of the Griess assay for estimating NO concentration. Error bars indicate SEM. E, Concentrations of NO2− measured from solutions of increasing concentrations of NO donor (spermine NONOate; 0, 1, 10, and 100 μm). F, The same data as in E are plotted on logarithmic axes and show a linear relationship between [donor] and [NO2−] in the range of 1 to 10 μm. Our measures of [NO2−] from brain slices were within this range. Average basal levels of NO evolution (over 30 min) were equivalent to NO2− concentrations ranging from ∼2.8 to 7 μm, and stimulated values were between 69 and 128% greater than these. The point referring to a zero concentration of donor gave an NO2− concentration of 0.03 ± 0.03 μm.