Hatsuki Shiga

Taste discrimination in conditioned taste aversion of the pond snail Lymnaea stagnalis

SUMMARY Conditioned taste aversion (CTA) in the pond snail Lymnaea stagnalis has been widely used as a model for gaining an understanding of the molecular and behavioral mechanisms underlying learning and memory. At the behavioral level, however, it is still unclear how taste discrimination and CTA interact. We thus examined how CTA to one taste affected the feeding response induced by another appetitive food stimulus. We first demonstrated that snails have the capacity to recognize sucrose and carrot juice as distinct appetitive stimuli. We then found that snails can become conditioned (i.e. CTA) to avoid one of the stimuli and not the other. These results show that snails can distinguish between appetitive stimuli during CTA, suggesting that taste discrimination is processed upstream of the site where memory consolidation in the snail brain occurs. Moreover, we examined second-order conditioning with two appetitive stimuli and one aversive stimulus. Snails acquired second-order conditioning and were still able to distinguish between the different stimuli. Finally, we repeatedly presented the conditional stimulus alone to the conditioned snails, but this procedure did not extinguish the long-term memory of CTA in the snails. Taken together, our data suggest that CTA causes specific, irreversible and rigid changes from appetitive stimuli to aversive ones in the conditioning procedure.

GAP junctional channel inhibition alters actin organization and calcium propagation in rat cultured astrocytes.

Astrocytes are connected by gap junctions, which provide intercellular pathways that allow a direct exchange of ions and small metabolites including second messengers and the propagation of electric currents. The roles of gap junctional communication on whole-cell morphology, cytoskeletal organization, and intercellular communication in astrocytes are not yet clear even in vitro, though there are many studies that have examined the active relation between gap junctions and actin filaments in astrocytes. Here we examined the effects of gap junction inhibitors, which do not interrupt the formation but rather the function of gap junctions, on whole-cell morphology, cytoskeletal organization, and intercellular communication in rat cultured astrocytes. Functional blockade of gap junctions during the formation of an astrocytic monolayer resulted in discordance of actin stress fibers between neighboring cells, even though whole-cell morphology of these cells did not change by such treatment. Mechanical stimulation-induced calcium wave propagation was significantly reduced in these actin-discordance cells even after thorough wash out. Differentiation of astrocytes in the presence of gap junction inhibitors was associated with morphological disarrangement among neighboring cells due to disordered alignment of actin stress fibers between cells.Our results indicate that gap junctional communication enables cell-to-cell coordination of actin stress fibers in astrocytes, thus enhancing intercellular communication through calcium spread.

Ca2+ signaling regulated by an ATP-dependent autocrine mechanism in astrocytes

Although the mechanisms of Ca2+ wave propagation in astrocytes induced by mechanical stimulation have been well studied, it is still not known how the [Ca2+]i increases in the stimulated cells. Here, we have analyzed the mechanisms of [Ca2+]i increase in single, isolated astrocytes. Our results showed that there was an autocrine mechanism of Ca2+ regulation mediated by ATP in mechanically stimulated astrocytes. This autocrine mechanism induced the activation of phospholipase C via a G-protein, resulting in Ca2+ release from intracellular Ca2+ stores. A second pathway mediating a [Ca2+]i increase was via a Ca2+ influx from the extracellular space, which, interestingly, suppressed an intracellular Ca2+ oscillation. These two different Ca2+ cascades are involved in signal transduction and may function separately during intercellular communication.

Advancement of differentiation of oligodendrocyte progenitor cells by A cascade including protein kinase a and cyclic AMP-response element binding protein

A transcription factor, cyclic AMP-response element binding protein (CREB), which is phosphorylated by protein kinases (PKA and PKC), is known to be involved in the regulation of oligodendrocyte differentiation. However, it is still unclear whether protein kinase A (PKA) and protein kinase C (PKC) are used simultaneously or at different time points to phosphorylate CREB in oligodendrocytes and whether CREB phosphorylation advances oligodendrocyte differentiation or vise versa. Our previous experiments have shown that in the differentiation process from immature to mature cells, CREB phosphorylation depends on PKC activity and leads to the progression of differentiation. In order to gain a better understanding of the process of differentiation from progenitor to immature cells, we identified which protein kinase, i.e., PKA or PKC, regulates CREB phosphorylation and we determined whether CREB phosphorylation advances differentiation or the reverse. Our results showed that CREB phosphorylation is principally regulated by PKA activity in progenitor cells but not by PKC activity, and that this phosphorylation advances the differentiation of progenitor cells to immature cells in oligodendrocytes.

Mechanical Properties of Membrane Surface of Cultured Astrocyte Revealed by Atomic Force Microscopy

In order to examine the mechanical properties of the membrane surface of astrocytes, we observed living astrocytes by atomic force microscopy (AFM) both in contact mode and force-mapping mode. Ridge-like structures reflecting actin filaments were observed in the topographic images in contact mode, but not in force-mapping mode, using a zero-loading force. When we measured the elasticity of astrocytes, we observed that the cell membrane above the nucleus was soft and the cell membrane above the cytosol was stiff. In particular, the parts reflecting actin filaments were very stiff. This effect of actin filaments on the elasticity of astrocytes was confirmed by the loss of actin filaments after application of actin-polymerization inhibitor.

Glutamate release from astrocytes is stimulated via the appearance of exocytosis during cyclic AMP‐induced morphologic changes

Recent studies have shown that astrocytes release various transmitters including glutamate and thus directly affect synaptic neurotransmission. The mechanisms involved in the release of glutamate from astrocytes remain unclear, however. In the present study, we examined differences in 1) the amount of glutamate released, 2) the appearance of exocytosis, and 3) the expression of SNARE (soluble N‐ethylmaleimide sensitive fusion protein attachment protein receptor) proteins between cyclic AMP‐treated and non‐treated astrocytes in culture. Extracellular glutamate was detected in the recording solution of cyclic AMP‐treated astrocytes after stimulation with ATP by high‐performance liquid chromatography and NADH imaging. Exocytosis, which was observed by FM1‐43 imaging, appeared in cyclic AMP‐treated astrocytes in a punctiform fashion, but not in non‐treated cells, after stimulation with ATP and glutamate. Immunocytochemistry and Western blotting showed that the amount of SNARE proteins increased during cAMP‐induced morphologic changes, and in particular, a v‐SNARE, synaptobrevin, appeared as punctiform staining in the cytosol of cyclic AMP‐treated astrocytes. These findings show that astrocytes acquire SNARE proteins during cyclic AMP‐induced differentiation, and suggest that glutamate is released by exocytosis in cyclic AMP‐treated astrocytes in response to ATP released from neighboring neurons and astrocytes.

CELL CULTURES ON A SUPER WATER-REPELLENT ALKYLKETENE DIMER SURFACE

The fractal alkylketene dimer (AKD) surface is an artificial super water-repellent one with a high contact angle of 174°, therefore, may provide special surface circumstances for studies of biological cells such as cell cultures. The experimental results indicated that the distribution of F-actin in the astrocytes cultured on the fractal AKD-coated dishes showed the stellate shape, while that in the astrocytes cultured on the poly-L-lysine-coated coverslips showed the formation of long alignment. The morphological change of astrocytes is induced by the fractal AKD surface, and the result suggests that astrocyte differentiation is stimulated by the fractal AKD surface.