Changes in TRPC channel expression during postnatal development of cerebellar neurons

In the brain, classical (canonical) transient receptor potential (TRPC) channels are thought to be involved in different aspects of neuronal development. We investigated the developmental expression profile of TRPC channels in rat cerebellum during the first 6 weeks after birth. TRPC3 expression is significantly up-regulated whereas TRPC4 and TRPC6 expression are significantly down-regulated over this period of time. TRPC3 expression is mainly found on Purkinje cells and their dendrites, suggesting that the increase in TRPC3 expression reflects development of the dendritic tree of Purkinje cells. TRPC4 expression was restricted to granule and their precursor cells. TRPC6 expression is found on Purkinje cell bodies, on mature granule cells in the internal granule cell layer (but not their precursors) and interneurons in the molecular layer. The decrease in TRPC4 expression suggests that it is required for proper granule cell development whereas the decrease in TRPC6 expression is presumably correlated with interneuron development. Moreover, we demonstrate the presence of functional TRPC channels on Purkinje cell dendrites that are activated following stimulation of metabotropic glutamate receptors. Our results reveal cell-specific expression patterns for different TRPC proteins and suggest that developmental changes in TRPC protein expression may be required for proper postnatal cerebellar development.     

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.     

Highly restricted expression of Cre recombinase in cerebellar Purkinje cells

The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.     

Electromechanical characterization of the effects of racemic sotalol and its optical isomers on isolated canine ventricular trabecular muscles and Purkinje strands

In both isolated canine ventricular trabecular muscle and Purkinje strand preparations, dl-sotalol and its two optical isomers d- and l-sotalol produced a concentration-dependent increase in action potential duration while other transmembrane electrical characteristics were not significantly affected. The magnitude of the increase in action potential durations was greater in Purkinje strand preparations. In Purkinje strand preparations, the effect was rate dependent (i.e., the increase in duration was proportionately greater when stimulation frequency was slowed). From the concentration of each compound calculated to produce a 50% maximal increase in Purkinje fiber action potential durations, d-sotalol appeared to be one to three times more potent than either l-sotalol or the racemate. Each compound appeared to increase force development in ventricular trabecular muscle preparations stimulated at a frequency of 2 Hz. Increased force development was only observed in Purkinje strand preparations stimulated at slower rates (0.5-0.33 Hz). These results are unlike those produced by other beta-adrenergic blockers and suggest that the antiarrhythmic effects of sotalol are related primarily to its effect of action potential duration. The estimated potency ratios established for the effect of dl-sotalol and its optical isomers on both trabeculae and Purkinje fiber action potential durations (d greater than dl-l) may indicate that these effects are unrelated to the beta-adrenergic blocking properties of these compounds. The differential effect of sotalol on isolated trabeculae and Purkinje strand preparations may help to explain the clinically reported phenomenon of sotalol-induced torsade de pointes.     

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.     

Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures

Purkinje cells are vulnerable to a number of physical, chemical, and genetic insults during development and maturity. Normal development of these cells depends on the cell-cell interactions between granule and astroglial cell populations. Apoptotic death in Purkinje neurons had been shown to be associated with cell cycle activation, and new DNA synthesis is associated with Purkinje cell death in staggerer and lurcher mutant mice. Here using an in vitro organotypic slice culture model from 9 (P9) and 4 days (P4) old postnatal rats we show that the cyclin dependent kinase (cdk) inhibitors (roscovitine, olomoucine, and flavopiridol) protect the Purkinje cells from cell death. The results are more pronounced in the cerebellar sections from P4 rats. Analysis of Purkinje neurons in sections from P4 rats after 1 week of culturing showed that while there were very limited calbindin positive neurons in the untreated sections the cdk inhibitor treated sections had a notably higher number. Although treatment with cdk inhibitors inhibited Purkinje cell loss significantly, the morphology of these neurons was abnormal, with stunted dendrites and axons. Since the retinoblastoma protein (Rb) is the major pocket protein involved in determining the differentiated state of neurons we examined the effect of over-expressing Rb in the organotypic cultures. Rb overexpression significantly inhibited the Purkinje cell death and these neurons maintained their normal morphology. Thus our studies show that the cell death in Purkinje neurons observed in organotypic cultures is cell cycle dependent and the optimal survival requires Rb.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.     

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2^+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2^+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2^+/+ and GluRδ2^+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2^+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2^+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2^+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2^+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009