ErbB1 receptor ligands attenuate the expression of synaptic scaffolding proteins, GRIP1 and SAP97, in developing neocortex.

Scaffolding proteins containing postsynaptic density-95/discs large/zone occludens-1 (PDZ) domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. Little is known, however, about what regulates the expression of PDZ proteins and how the levels of these proteins influence synaptic development. Here, we show that ligands for epidermal growth factor receptors (ErbB1) decrease a particular set of PDZ proteins and negatively influence synaptic formation or maturation. In short-term neocortical cultures, concentrations of epidermal growth factor and amphiregulin (2-9 pM) decreased the expression of glutamate receptor interacting protein 1 (GRIP1) and synapse-associated protein 97 kDa (SAP97) without affecting postsynaptic density-95 (PSD-95) levels and glial proliferation. In long-term cultures, epidermal growth factor treatment resulted in a decrease in the frequency of pan-PDZ-immunoreactive aggregates on dendritic processes. A similar activity on the same PDZ proteins was observed in the developing neocortex following epidermal growth factor administration to rat neonates. Immunoblotting revealed that administered epidermal growth factor from the periphery activated brain ErbB1 receptors and decreased GRIP1 and SAP97 protein levels in the neocortex. Laser-confocal imaging indicated that epidermal growth factor administration suppressed the formation of pan-PDZ-immunoreactive puncta and dispersed those structures in vivo as well. These findings revealed a novel negative activity of ErbB1 receptor ligands that attenuates the expression of the PDZ proteins and inhibits postsynaptic maturation in developing neocortex.

Brain-derived neurotrophic factor enhances depolarization-evoked glutamate release in cultured cortical neurons.

Brain-derived neurotrophic factor (BDNF) has been reported to play an important role in neuronal plasticity. In this study, we examined the effect of BDNF on an activity-dependent synaptic function in an acute phase. First, we found that short-term treatment (10 min) with BDNF enhanced depolarization-evoked glutamate release in cultured cortical neurons. The enhancement diminished gradually according to the length of BDNF treatment. The BDNF-enhanced release did not require the synthesis of protein and mRNA. Both tetanus toxin and bafilomycin abolished the depolarization-evoked glutamate release with or without BDNF, indicating that BDNF acted via an exocytotic pathway. Next, we investigated the effect of BDNF on intracellular Ca(2+). BDNF potentiated the increase in intracellular Ca(2+) induced by depolarization. The Ca(2+) was derived from intracellular stores, because thapsigargin completely inhibited the potentiation. Furthermore, both thapsigargin and xestospongin C inhibited the effect of BDNF. These results suggested that the release of Ca(2+) from intracellular stores mediated by the IP(3) receptor was involved in the BDNF-enhanced glutamate release. Last, it was revealed that the enhancement of glutamate release by BDNF was dependent on the TrkB-PLC-gamma pathway. These results clearly demonstrate that short-term treatment with BDNF enhances an exocytotic pathway by potentiating the accumulation of intracellular Ca(2+) through intracellular stores.

Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca(2+) and Na(+) in cultured cerebellar neurons.

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.

Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments.

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.