We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1Δ18/Δ18), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5′ part of the ITPR1 gene, encompassing exons 1–10, 1–40, and 1–44 in three studied families, underlies SCA15 in humans.

The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from ~17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in β-pinealocytes and to a lesser degree in α-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.

Oligodendrocytes and their progenitors (0-2A) express functional kainate-and DL-a-amino-3-hydroxy-5-methyl-4-isoxazoleproplonlc acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied In pified rat cortical 0-2A progenitors and In the primary oligodendrocyte cell line Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to Intracellular Ca2+ concentration, as measured by fura-2 Ca+ imaging. Both in CG-4 and in cortical 0-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. In the forebrain and cerebellum, oligodendrocytes develop from progenitor cells that originate in the subventricular zone and migrate into gray and white matter in early postnatal life (1, 2). The developmental potential of oligodendrocyte progenitors (0-2A) is maintained when these cells are isolated, purified, and cultured in the presence of appropriate growth factors (3-5). 0-2A development depends on homologous and heterologous interactions with other cells in the central nervous system (6), including neurons, which may synthesize and release factors that are essential for 0-2A progenitor cell division and differentiation (7). Recently, a primary oligodendrocyte cell line (CG-4) has been described (5). CG-4 cells can be fully induced to differentiate into myelinating oligodendrocytes with the same time schedule as 0-2A progenitors purified and cultured from different parts ofthe brain (5). CG-4 progenitors sequentially express oligodendrocytespecific markers throughout their development (5, 8) and respond to the same growth factors that regulate 0-2A proliferation and differentiation (5).Cells of the oligodendrocyte lineage have been shown to express both ligand-and voltage-gated ionic channels (9-11). We have demonstrated, by electrophysiological and molecular analyses in CG-4 cells and cortical glia, that functional kainate-and DL-a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors are coexpressed in cells ofthe oligodendrocyte lineage (8,12). In 0-2A progenitor cells and mature oligodendrocytes, glutamate and structurally related compounds generated large depolarizing currents due to the opening of membrane cationic channels (8,12 Vellis (16). Both CG-4 and primary progenitors were grown in Dulbecco's modified Eagle's medium (DME)-N1, supplemented with 30% B-104 conditioned medium. Cultures enriched in 01+ oligodendrocytes were obtained by replacing the B-104 conditioned medium after 3 days with DME-N1 plus 0.5% fetal bovine Abbreviations: AMPA, DL-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione; 1S,3R-ACPD, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid;[Ca2+]i, intracellular calcium concentration; IEG, immediate early gene; NMDA, N-methyl-D-aspartate.

In astrocytes in primary culture, activation of neurotransmitter receptors results in intracellular calcium signals that propagate as waves across the cell. Similar agonist-induced calcium waves have been observed in astrocytes in organotypic cultures in response to synaptic activation. By using primary cultured astrocytes grown on glass coverslips, in conjunction with fluorescence microscopy we have analyzed agonist-induced Ca2+ wave initiation and propagation in individual cells. Both norepinephrine and glutamate elicited Ca2+ signals which were initiated focally and discretely in one region of the cell, from where the signals spread as waves along the entire length of the cell. Analysis of the wave propagation and the waveform revealed that the propagation was nonlinear with one or more focal loci in the cytoplasm where the wave was regeneratively amplified. These individual loci appear as discrete focal areas 7-15 microns in diameter and having intrinsic oscillatory properties that differ from each other. The wave initiation locus and the different amplification loci remained invariant in space during the course of the experiment and supported an identical spatiotemporal pattern of signalling in any given cell in response to multiple agonist applications and when stimulated with different agonists which are coupled via InsP3. Cytoplasmic Ca2+ concentration at rest was consistently higher (17 +/- 4 nM, mean +/- S.E.M.) in the wave initiation locus compared with the rest of the cytoplasm. The nonlinear propagation results from significant changes in signal rise times, amplitudes, and wave velocity in cellular regions of active loci. Analysis of serial slices across the cell revealed that the rise times and amplitudes of local signals were as much as three- to fourfold higher in the loci of amplification. A phenomenon of hierarchy in local amplitudes of the signal in the amplification loci was observed with the wave initiation locus having the smallest and the most distal locus having the largest amplitude. By this mechanism locally very high concentrations of Ca2+ are achieved in strategic locations in the cell in response to receptor activation. While the average wave velocity calculated over the length of the cell was 10-15 microns/s, in the active loci rates as high as 40 microns/s were measured. Wave velocity was fivefold lower in regions of the cell separating active loci. The differences in the intrinsic oscillatory periods give rise to local Ca2+ waves that show the properties of collision and annihilation. It is hypothesized that the wave front provokes regenerative Ca2+ release from specialized areas in the cell where the endoplasmic reticulum is endowed with higher density of InsP3 receptor channels.(ABSTRACT TRUNCATED AT 400 WORDS)