BDNF secretion from primary hippocampal neurons. (A) Representative time course of BDNF spontaneously secreted from primary hippocampal neurons. BDNF levels were evaluated during a 70-min period in 10-min fractions collected from neurons cultured at 37°C as described in Materials and Methods. (B) BDNF secretion was evaluated in the absence (Basal) or presence of a mixture of GluR-ant comprising 100 μM 6-cyano-7-nitroquinoxaline-2,3-dione, 50 μM d(−)-2-amino-5-phosphonopentanoic acid, 1 mM 1-aminoindan-1,5-dicarboxyl acid added to the incubation buffer for 20 min at 37°C or 15°C. GluR-ant administration induces a reduction of BDNF levels at both temperatures. Values are given as mean ± SE (n = 12).

Time course of BDNF secretion from primary hippocampal neurons treated with the NO donors SNP or NOR3. BDNF secretion was evaluated after the administration of 10 μM SNP (A) and 300 μM NOR3 (B) to cultured neurons. BDNF secretion was detected in the absence (0 min) or presence (10 and 20 min) of the indicated donor (Left). Values are given as mean ± SE (n = 10). * indicates statistical significance of the difference from the control at P < 0.02. In a representative experiment (Right), the respective NO donor administrated for 20 min caused a rapid down-regulation of BDNF secretion that partially recovered to baseline on withdrawal of the donor. (C) BDNF secretion evaluated after the administration of 10 μM SNP and 300 μM NOR3 at the reduced temperature of 15°C. Lowering the temperature from 37°C to 15°C caused a reduction of BDNF from basal levels that was further inhibited upon administration of SNP or NOR3 for 20 min. Values are given as mean ± SE (n = 6). * indicates statistical significance of the difference from the basal at P < 0.05.

(A) Western blots of NOS isoforms (neuronal NOS, endothelial NOS, and inducible NOS) in extracts of hippocampal neurons performed by using selective antibodies. Only nNOS was significantly expressed at the predicted molecular weight (arrow). (B) Expression of nNOS in hippocampal primary neurons evaluated by immunocytochemistry. Shown is a representative image from a confocal scan along the x-y plane. nNOS displays a uniform distribution in the cell body and in the dendritic processes of most of the neurons. (C) NO synthesis evaluated by monitoring L-[³H]citrulline formation from L-[2,3-³H]arginine. The level of NO production was evaluated in the absence (Control) or presence of 1 mM L-NAME, a NOS inhibitor. Data were expressed as picomoles of NO per sample per 30 min. (D) BDNF secretion evaluated in the absence (0 min) or presence (10 and 20 min) of L-NAME. Values are given as mean ± SE (n = 10). * indicates statistical significance of the difference from the control at P < 0.05.

Time course of BDNF secretion mediated by the administration of YC1, 8Br-cGMP, and KT5823. (A) BDNF secretion evaluated in the absence (0 min) or presence (10 and 20 min) of 100 μM YC1, an agonist of sGC (Left). Values are given as mean ± SE (n = 8). * indicates statistical significance of the difference from the control at P < 0.02. In a representative experiment (Right), the administration of YC1 induced a rapid down-regulation of BDNF secretion that slowly recovered to basal level after YC1 removal. (B) BDNF secretion was evaluated in the absence (0 min) or presence (10 min and 20 min) of 1 mM 8Br-cGMP, a permeable form of cGMP (Left). The values given are the mean ± SE (n = 12). * indicates statistical significance of the difference from the control at P < 0.02. In a representative experiment (Right), the administration of 8Br-cGMP decreased the BDNF secretion following the kinetics described in A. (C) BDNF secretion evaluated in the absence (0 min) or presence (10 and 20 min) of 40 μM KT5823, a PKG inhibitor. Values are given as mean ± SE (n = 10). * indicates statistical significance of the difference from the control at P < 0.05. (D) BDNF secretion evaluated in the presence of 300 μM NOR3 and 40 μM KT5823. NOR3 was administered to hippocampal neurons either alone (10 min) or in combination with KT5823 (20 min). NOR3 induced a down-regulation of BDNF secretion, which partially recovered to basal levels after KT5823 administration. Values are given as mean ± SE (n = 6). * indicates statistical significance of the difference from the control at P < 0.02.