Two types of neurons in the rat cerebellar nuclei as distinguished by membrane potentials and intracellular fillings.

Classically, three classes of neurons in the cerebellar nuclei (CN), defined by different projection targets and content of transmitters, have been distinguished. However, evidence for different types of neurons based on different intrinsic properties is lacking. The present study reports two types of neurons defined mainly by their intrinsic properties, as determined by whole-cell patch recordings. The majority of cells (type I, n = 63) showed cyclic burst firing whereas a small subset (type II, n = 7) did not. Burst firing was used to distinguish the two types of neurons because, as it turned out, pharmacological interference could not be used to convert the non-bursting cells to bursting ones. Some of the membrane potentials exclusively present in type I neurons, such as sodium and calcium plateau potentials, low-threshold calcium spikes, and a slow calcium-dependent afterhyperpolarization, were found to contribute to the generation of burst firing. Other membrane potentials of type I neurons were not obviously related to the generation of bursts. These were 1) the lower amplitude and width of the action potential during spontaneous activity, 2) a sequence of afterhyperpolarization-afterdepolarization-afterhyperpolarization following each spike, and 3) the high spontaneous firing rate. In contrast, type II neurons lacked slow plateau potentials and low threshold spikes. Their action potentials showed higher amplitude and width and were followed by a single deep afterhyperpolarization. Furthermore, they showed a lower firing rate at rest. In both types of neurons, a delayed inward rectification was present. Neurons filled with neurobiotin revealed that the sizes of the somata and dendritic fields of type I neurons comprised the whole range known from Golgi studies, whereas those of the few type II neurons recovered were found to be in the lowest range. In view of their size and scarcity, we propose that type II neurons may correspond to CN interneurons.

Characterization of nanoscaled heterogeneities in mechanically alloyed and compacted CuFe.

Cu80Fe20 and Cu50Fe50 were mechanically alloyed from the pure elements by ball milling for 36 h. The alloy powder was compacted into tablets at room temperature by applying a pressure of 5 GPa. Characterization of the Cu80Fe20) and Cu50Fe50 alloys was carried out by high-resolution transmission electron microscopy (HREM), atom probe field ion microscopy and three-dimensional atom probe (3DAP). The grain size of the nanocrystalline microstructure of the ball-milled alloys observed with HREM varies between 3 and 50 nm. Atom probe and 3DAP measurements indicate that the as-prepared state is a highly supersaturated alloy, in which the individual nanocrystals have largely varying composition. Fe concentration in Cu was found to range from about 8 to 50 at%. It is concluded that by ball milling and compacting an alloy is produced which on a nanometer scale is heterogeneous with respect to morphology and composition.

Desensitization of P2U receptor in neuronal cell line. Different control by the agonists ATP and UTP, as demonstrated by single-cell Ca2+ responses.

The neuronal cell line NG108-15 (180CC15) responds to extracellular stimuli of ATP or UTP with a transient increase in the level of cytosolic Ca2+. Desensitization was investigated by recording single-cell Ca2+ responses induced by consecutive, regularly spaced (100 s intervals), brief pulses of the nucleotides. The two natural ligands of the P2U receptor, ATP and UTP, were applied at a concentration that evoked responses of a comparable size. With two pulses of UTP (10 microM), a substantial decrease (of 43%) was observed in the size of the second response. The magnitude of response was determined by measuring either the maximal amplitude or the total response, represented by the area of the Ca2+ transient. The analogous studies with ATP pulses showed a much smaller decrease (of 12%). Comparable experiments performed to investigate the mutual interaction between ATP and UTP revealed that after stimulation with ATP the response to UTP was slightly (12%) diminished, whereas the response to ATP after UTP was greatly (52%) decreased. The different degree of desensitization by either UTP or ATP of P2U receptors could be due to (1) a difference in the mode of activation of the receptor by the two ligands or (2) recruitment of another effector mechanism besides elevated Ca2+. Our results indicate the existence of a novel mechanism of receptor control, involving different modes of the receptor, that are induced by two different, activating ligands. We also investigated the crosstalk between the bradykinin B2 receptor and the nucleotide receptor. ATP and UTP, even when eliciting responses of comparable size in the neuronal cell line, affect the desensitization of the bradykinin receptor differently. This suggests regulatory binding sites for the nucleotides on either the nucleotide receptor or the peptide receptor.

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH.

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca2+]i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca2+]i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca2+]i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca2+]i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca2+]i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca2+ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca2+]i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca2+]i oscillations. During [Ca2+]i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400-500 s) causes a large initial response superimposed on regular [Ca2+]i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca2+]i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pHi by application of NH4Cl. [Ca2+]i oscillations stop after addition of NH4Cl. Recovery of NH4Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca2+]i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca2+]i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction.

[Ca2+]i oscillations in single rat glioma cells induced by thrombin through activation of cell surface receptors.

Thrombin at nanomolar concentrations induces rapid changes in the second messenger Ca2+ in a glial astrocyte-type cell line. Continuous application of the protease thrombin causes regular [Ca2+]i oscillations (amplitude 109 nM, spike length 48 s) which are suppressed by hirudin. Reduction of [Ca2+]ex (from 1.8 mM to 50 microM) reversibly abolishes the oscillations indicating the contribution of Ca2+ influx to generation of the oscillations. Thrombin receptor-activating peptide (TRAP, 1-10 microM) causes similar Ca2+ oscillations which depend, like the oscillations induced by thrombin, on the continuous presence of agonist. Thus, we can deduce that cell surface receptors are responsible for the effect of thrombin on glioma cells.