Distribution of a brain-specific extracellular matrix protein in developing and adult zebrafish

A monoclonal antibody (IgG) that recognizes a 53-kDa zebrafishnext brain protein was isolated and used to characterize the distribution of this protein in zebrafish.next (1) The antigen was found only in the brain and not in any other tissues such as muscle, dermis and cartilage. Within the brain, the antibody recognized extracellular matrix (ECM) outside neuronal cells. (2) Digestion by hyaluronidase released the antigen from brain tissue, and the monoclonal antibody staining was also decreased by the digestion by hyaluronidase. (3) The pattern of antigen distribution is not perineuronal, as the density of the antigen at the periphery of the cells was practically identical to that of the empty intercellular spaces. Therefore, this monoclonal antibody does not recognize the perineuronal glycocortex. (4) The antigen is distributed only in limited areas of the brain, namely in the periphery of the forebrain, the hypothalamus, the optic tectum, the interpeduncular nucleus, the cerebellum and the ventricular rim of the medulla. In the optic tectum, the antibody strongly stained the most superficial layer, and in the cerebellum, it stained the molecular but not the granular layer. These patterns of distribution are very different from those of other typical brain ECM proteins and suggest that this protein may play quite distinct roles in brain development and maintenance.</p

Involvement of Sphingosine-1-Phosphate in Glutamate Secretion in Hippocampal Neurons▿ †

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for sphingosine-1-phosphate (S1P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S1P remain largely undefined. Here, we report two novel actions of S1P using primary hippocampal neurons as a model system: (i) as a secretagogue where S1P triggers glutamate secretion and (ii) as an enhancer where S1P potentiates depolarization-evoked glutamate secretion. Sphingosine kinase 1 (SK1), a key enzyme for S1P production, was enriched in functional puncta of hippocampal neurons. Silencing SK1 expression by small interfering RNA as well as SK1 inhibition by dimethylsphingosine resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK1 from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S1P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S1P1 receptor at the puncta was activated during depolarization and that depolarization-induced S1P1 receptor activation was inhibited in SK1-knock-down cells. Importantly, exogenously added S1P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na+-channel was blocked by tetrodotoxin, suggesting that S1P acts on presynaptic membranes. Furthermore, exogenous S1P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S1P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission