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The authors previously demonstrated that isoflurane, a widely used volatile anesthetic, induced depolarization and increased the frequency of spontaneous action potentials in principal dopamine neurons in rat substantia nigra pars compacta. We studied the effects of isoflurane on voltage-dependent K channels to clarify the mechanisms of the increase in excitability in these neurons. Voltage-clamp whole-cell recordings were made in rat midbrain slices. We recorded the outward membrane currents in response to depolarizing voltage steps from -120 mV and -25 mV and isolated the transient outward current mediated through A-type K channels by subtraction. Isoflurane at clinically relevant concentrations accelerated the decay of the A-type K current and delayed the recovery from inactivation without changing the steady-state inactivation curves. Isoflurane did not affect the non-inactivating outward current. Addition of 4-aminopyridine partially occluded the excitatory effects of isoflurane in current-clamp recordings. These results demonstrate that isoflurane accelerated the inactivation and delayed the recovery from inactivation of A-type K channels in principal neurons in rat substantia nigra pars compacta without affecting delayed rectifier K channels. These effects may contribute in part to excitation of these neurons and the isoflurane-induced increases in dopamine release reported in vitro and in vivo.

Thyroid hormones (THs) are important for brain development, and polychlorinated biphenyl (PCB) accumulation in humans is a serious problem because PCBs may affect TH functions. To determine the effects of hydroxylated metabolites of PCBs (OH-PCBs) on brain development, we performed mouse cerebellar culture assays. 4-OH-2',3,3',4',5'-pentachlorobiphenyl and 4-OH-2',3,3',4',5,5'-hexachlorobiphenyl significantly inhibited the TH-dependent extension of Purkinje cell dendrites even at 5 x 10(-11) M and 5 x 10(-12) M, respectively. OH-PCBs may disturb TH-dependent brain development.

Using a well-defined medium with insulin, transferrin and selenium but without serum and albumin, we quantitatively determined the effect of thyroid hormones on the development of Purkinje cells in mouse cerebellar monolayer cultures. Addition of a thyroid hormone, T3 or T4, to the serum-free medium resulted in a highly elaborate dendritic development of Purkinje cells. The cultured Purkinje cells in the presence of T4 even showed similarities in shape and in synapse formation to normal Purkinje cells in vivo. Such effect of T4 on the dendritic arborization of Purkinje cells was dose dependent and significantly sensitive to a low dose of T4 even at 50 pM. The effect of T4 was confirmed by an inhibition experiment using amiodarone, which was reported to induce thyroid dysfunction. Furthermore, T4 affected not only Purkinje cell development but also the shape of other neural cells such as small interneurons (mainly granule cells) and astrocytes in cerebellar cultures. T4 induced development of both interneurons and astrocytes having long processes. These results indicate that thyroid hormones play a pivotal role in the development of mouse Purkinje cell dendrites acting on Purkinje cells directly and/or indirectly via the close interaction with interneurons and astrocytes.

To elucidate the molecular mechanisms that regulate the maturation of action potential, we began by examining voltage-dependent K+ currents, known to contribute to the maturation of action potential, of developing granule cells in mouse cerebellar microexplant cultures. The migration of developing granule cells in this culture is reported to mimic the in vivo process, but their specific identification is still incomplete. In this study, we identified and characterized granule cells in this culture. Immunocytochemical analysis found that granule cells migrated radially out from explants and subsequently formed small clusters and also that their morphology changed from a bipolar to a T shape during migration. Moreover, in the electrophysiological study, the GABA response of granule cells in this culture clarified that the electrophysiological properties of granule cells were normally maintained. We therefore have concluded, that this culture system is a powerful tool for investigating the differentiation of cerebellar granule cells. Based on these findings, we recorded voltage-dependent K+ currents of developing granule cells in this culture, while concurrently observing their morphology. Our results show that voltage-dependent K+ currents of developing granule cells change from delayed rectifier to A current in parallel with their morphological changes from bipolar to T-shaped cells.

In order to study roles of the extracellular matrix (ECM) in the cerebellar granule cell migration, cerebellar microexplants of neonatal to postnatal 11-day-old mice were cultured on 3 kinds of substrata, poly- l -lysine (PL), PL/fibronectin and PL/laminin. A prominent outgrowth of small granule cells, which did not uptake GABA, was observed only on the PL/laminin substratum. The granule cells showed the following sequence of events: (1) Many polygonal undifferentiated cells migrated out from the microexplants. These blast cells differentiated into small bipolar neurons with long fine neurites which extended radially from the explants. (2) These cells then changed their orientation perpendicular to their radial neurites, by protruding a short process from the cell body at right angles. (3) Finally, cell bodies of these granule cells adhered to each other to form cell aggregates. Quantitative labelings by bromodeoxyuridine revealed that there were less mitotic cells in explants from the later postnatal cerebella compared to the earlier postnatal ones. Anti-MAP2 immunoreactivity was localized in short perpendicular processes of the aggregated granule cells. Thus, this unique cell behavior exhibited on the PL/laminin substratum provides the first defined experimental system for studying the granule cell differentiation in vitro.