Expression profile of addicsin/GTRAP3-18 mRNA in mouse brain.

Addicsin is a murine homologue of rat glutamate-transporter-associated protein 3-18 (GTRAP3-18), a putative modulator of neural glutamate-transporter excitatory amino acid carrier 1 (EAAC1). The other molecular functions of addicsin, however, remain largely unknown. We analyzed here the regional and cellular distribution of addicsin transcript in the mature brain using in situ hybridization analysis, and examined the sequential addicsin mRNA expression levels in the developing brain using Northern blot analysis. In the mature brain, we found addicsin mRNA to be ubiquitously expressed in neuron-like cells, but not glial cells, in various brain regions including the olfactory bulbs, hippocampus and cerebral cortex. In the hippocampus, addicsin mRNA was expressed in the neuron-like cells of the CA1-CA3 pyramidal cell layer and the interneuron-like cells of the stratum oriens, stratum radiatum and stratum lacunosum-moleculare. Addicsin transcripts were also extremely abundant in trigeminal neurons such as the principal trigeminal nucleus, mesencephalic trigeminal nucleus and motor trigeminal nucleus. Our evidence suggests that addicsin mRNA is chiefly expressed in the excitatory and inhibitory neurons, and that addicsin may participate in the functional expression of the somatic sensory system by modulation of EAAC1-mediated glutamate transport. Northern blot analysis revealed that addicsin mRNA levels increased with maturation, reaching their maximum level at postnatal day 5 (P5), then significantly declined by about 50% until P14, suggesting that addicsin may contribute to embryogenesis and synaptogenesis in the developing brain. Thus, addicsin may participate in multiple physiological functions in the developing and mature brain.

Two-dimensional neural activity mapping of the entire population of hippocampal CA1 pyramidal cells responding to fear conditioning.

The hippocampus is involved in the encoding, storage, and retrieval of memory. Here, we have developed a novel mapping method for detecting the distribution of neural activity of the entire population of pyramidal cells in the hippocampal CA1 and subiculum regions, where expression profiles of Arc mRNA were used as an indicator of neural activity. The spherical pyramidal cell layer of the intact hippocampus was flattened into a two-dimensional plane, which was then serially sectioned in parallel with the plane to make tangential sections. Tangential sections were hybridized with an Arc cRNA probe and Arc signals from serial tangential sections were stacked and displayed on a two-dimensional plane, allowing one to easily visualize the neural activity of all pyramidal cells. We applied this method to the hippocampus of rats that had experienced contextual fear conditioning, which requires hippocampal function. We observed a net shift of Arc signals from dorsal to ventral CA1/subiculum with an interval prolongation to reconditioning after the initial conditioning. The reconditioning-revealed shift may reflect a reorganization process, which takes place during the period between initial conditioning and reconditioning, in the CA1/subiculum neural network that represents the neural storage and/or retrieval of the contextual fear conditioning.

Identification of addicsin/GTRAP3-18 as a chronic morphine-augmented gene in amygdala.

Using subtractive cloning, we identified a 1.4 kb mRNA that was ubiquitously expressed in various tissues; this mRNA was highly up-regulated in amygdala nuclei in mice when morphine was repeatedly administered but not when an opiate-receptor antagonist was co-administered. The mRNA encodes a 23 kDa protein, designated 'addicsin'. This contains two putative PKC-phosphorylation motifs and several hydrophobic regions, and was recovered in a soluble protein fraction of brain lysate. Its primary structure showed 98% identity with that of rat glutamate-transporter-associated protein 3-18 (GTRAP3-18), a putative modulator of neural glutamate-transporter EAAC1. Up-regulation of addicsin expression by morphine may affect glutamate uptake in the amygdala, causing mice to develop morphine tolerance and dependence.