PKC and CaMKII dependent synaptic potentiation in cultured cerebral neurons.

We have reported that the long-lasting potentiation of spontaneous excitatory postsynaptic currents (SEPSCs) was induced by a Mg(2+)-free treatment in cultured chick cerebral neurons and a factor(s) extracellularly released during the treatment could induce the potentiation by itself. In this paper, protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase type II (CaMKII) but not protein kinase A (PKA) were reported to contribute to the potentiation mechanism during a step between the activation of the N-methyl-D-aspartate receptors by the Mg(2+)-free treatment and the secretion of the protein factor(s).

Chick muscle-derived protein 62: a novel neurite outgrowth promoting protein.

A 3.2 kb chick cDNA clone that coded for a novel muscle-derived protein, MDP62, was isolated from a cDNA library of the denervated crus muscles using an antibody which inhibited the neurite (dendritic and axonal processes) outgrowth activity. MDP62 consisted of 539 aa with a calculated molecular mass of 62 k. The predicted protein sequence was hydrophilic and exhibited an extended coiled-coil domain and a leucine zipper motif. A recombinant protein promoted the neurite outgrowth from the cultured chick neurons of the telencephalon in a dose dependent manner. Northern blotting revealed that MDP77 was ubiquitously expressed. In the transfected COS-7 cells with the cDNA of the epitope-tagged MDP62, the expressed protein was detected in the culture medium, suggesting that the MDP62 might be secreted.

MDP77: A novel neurite-outgrowth-promoting protein predominantly expressed in chick muscles.

A 4.7 kb chick cDNA clone that coded for the novel muscle-derived protein, MDP77, was isolated from a cDNA library of the denervated crus muscles using an antibody which inhibited the neurite outgrowth activity. MDP77 consisted of 676 aa with a calculated molecular mass of 77 k. The deduced amino acid sequence exhibited an extended coiled-coil domain and a leucine zipper motif. A recombinant protein promoted the neurite-outgrowth from the cultured chick neurons of the spinal cord in a dose-dependent manner. Northern blotting and in situ hybridization revealed that MDP77 was predominantly expressed in the cardiac and the skeletal muscles. In the COS-7 cells transfected with the cDNA of the epitope-tagged MDP77, the expressed protein was detected in the culture medium, suggesting that the MDP77 was secreted.

Synaptic potentiation induced by a protein factor in cultured cerebral neurons.

1. We reported in a previous paper that long-lasting enhancement of spontaneous excitatory post synaptic currents (SEPSCs) in cultured chick cerebral neurons was induced by exposure to a conditioned medium (CM) prepared by Mg(2+)-free treatment of neurons. This suggested that the CM contained a diffusible factor(s) for the potentiation. 2. In this paper, the factor(s) was shown to be a protein(s) by heat and trypsin treatment of the CM. 3. The factor induced the potentiation within 5 min, but it was not required for maintenance of increased SEPSCs. 4. The factors in CM induced the potentiation without protein synthesis. 5. Protein synthesis at least in postsynaptic neurons, was indispensable to induce the potentiation by the Mg(2+)-free condition.

Long-lasting enhancement of synaptic activity in dissociated cerebral neurons induced by brief exposure to Mg2+-free conditions.

The long-lasting enhancement of periodic clusters of spontaneous excitatory postsynaptic currents (SEPSCs) was examined in dissociated chick cerebral neurons that had been transiently exposed to Mg2+-free solution for 15 min. Since the enhancement was diminished by blockade of synaptic transmission, it clearly depended on synaptic activities. A specific antagonist of N-methyl-D-aspartate receptors (NMDARs) also inhibited the potentiations. Furthermore, the presence of inhibitors of protein and RNA synthesis in the Mg2+-free solution blocked the potentiation. In the potentiated neurons, the frequency of miniature excitatory postsynaptic currents (mEPSPs) increased. In addition, a diffusible molecule(s) that promoted the potentiation appeared to be involved in this phenomenon, since the conditioned medium of Mg2+-free treated neurons enhanced synaptic activity in other dish.

Selective formation of silent synapses on immature postsynaptic cells in cocultures of chick neurons of different ages.

Glutamatergic synapses usually contain two types of ionotropic glutamate receptor, N-methyl-D-aspartate receptors (NMDARs) and non-NMDA receptors (non-NMDARs), and the ratio of these receptors is thought to be critical for synaptic plasticity. To determine whether or not the ratio of these receptors at synaptic sites is controlled by the developmental stage of postsynaptic neurons, we applied a dual whole-cell recording technique to a culture of dissociated chick cerebral neurons of different ages. We found that formation of synapses that contained both types of receptor required maturation of postsynaptic neurons. Moreover, during the early development of postsynaptic neurons, NMDARs were selectively present at synaptic sites prior to the presence of non-NMDARs, even though both types of receptor were expressed in functional form in the neuronal membranes.

Two modes of activity-dependent synaptogenesis of cerebral neurons in vitro.

Effects of transduction activity and transmission activity on synaptogenesis of chick cerebral neurones in dissociated cell culture were studied electrophysiologically using two blockers for these activities, tetrodotoxin (TTX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. CNQX inhibited the increase of evoked EPSCs more effectively than TTX, whereas both blockers similarly reduced the increase of miniature EPSCs (Minis). These data indicated that not only transduction-dependent transmission activity but also transduction-independent spontaneous activity regulate the synaptic efficiency. These two activities are suggested to change the quantal amplitude and the number of synaptic sites, respectively.

Okadaic acid gives concentration-dependent reciprocal effects on the fluid phase endocytosis activated by Ca2+ and phorbol 12-myristate 13-acetate.

Incubation of a human fibrosarcoma cell line HT-1080 in increasing concentration of Ca2+ was found to enhance endocytic internalization of a fluid phase marker, horseradish peroxidase. At 16.8 mM Ca2+, generation of the effect required incubation for more than 45 min. The effect was reversed by removal of the excess ion for 30 min. Monitoring the intracellular concentration showed that the incubation induced a transient large Ca2+ influx followed by a recovery to 230 +/- 50 nM instead of the normal level of 83 +/- 5 nM. The activation was not inhibited by inhibitors of protein kinases nor a cAMP antagonist. In contrast, the effect was prevented by okadaic acid (OKA) at 100 nM without detectable effect on the basal activity. Fluid phase uptake by HT-1080 cells was also enhanced by phorbol 12-myristate 13-acetate (PMA). In contrast to the case with Ca2+, OKA at 100 nM did not prevent the PMA effect but further enhanced the endocytosis. The effect of OKA was concentration-dependent, as the reagent at 1 microM inhibited not only both the activation but also the basal activity. In Ca(2+)- or PMA-stimulated cells, FITC-dextran was delivered to endosomes that had been labeled with TRITC-transferrin. In contrast, following treatment with a combination of PMA and 100 mM OKA, fluid phase was internalized in vesicular compartments devoid of transferrin labeling. These results suggest that, through differential modifications of protein phosphorylation, endocytosis can be enhanced distinctively either by employing conventional receptor-bearing compartments or generating a new endosomal population.

Synapse formation in dissociated cell cultures of embryonic chick cerebral neurons.

The development of synapses was confirmed in the primary cultures of dissociated cerebral cortex neurons from chick embryos. Whole-cell patch clamp recording applied to dissociated neurons from 6- to 12-day-old embryos revealed that these neurons form functional synapses. In these cultures, both excitatory and inhibitory postsynaptic responses were observed. Synaptogenesis in our cultures seemed to be in proportion to the embryonic equivalent days, which are the sum of the days in incubation and culture.