. Ca 2ϩ channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 M), -conotoxin GVIA (Ctx, 1 M), or -agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca 2ϩ channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca 2ϩ channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N-or P-type Ca 2ϩ channels. N-and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca 2ϩ -dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca 2ϩ channels may exhibit different thresholds for activation in different types of neurons. I N T R O D U C T I O NNumerous studies have indicated that Ca 2ϩ entry is important in regulating the firing patterns of dopaminergic (DA) neurons in the substantia nigra. The pacemaker-like slow depolarizations (PLSD), spontaneous oscillatory potentials (SOPs), and slow afterhyperpolarizations involved in regulation of DA neuron firing activity have all been shown to be Ca 2ϩ dependent (Fujimura and Matsuda 1989; Grace and Onn 1989; Harris et al. 1989; Kang and Kitai 1993a,b; Mercuri et al. 1994; Nedergaard et al. 1993; Ping and Shepard 1996; Yung et al. 1991). A previous study suggests that DA neurons express L-, N-, P/Q-, and R-type Ca 2ϩ channels (Cardozo and Bean 1995). However, the Ca 2ϩ channel subtype(s) involved in regulation of DA neuron firing behavior have not been conclusively determined. In two studies, DA neuron spontaneous firing and SOPs were abolished by dihydropyridines but not -conotoxin GVIA (Ctx), suggesting a prominent role for L-, but not N-type Ca 2ϩ channels in these phenomena (Mercuri et al. 1994; Nedergaard et al. 1993). However, Kang and Kitai (1993b) provided evidence that DA neuron pacemaker activity was underlain by N-but not L-type Ca 2ϩ channels. Finally, a study by Fujimura and Matsuda (1989) suggested that neither N-nor L-channels were involved in DA neuron pacemaker activity.The present study investigated the relative contribution of L-, N-, and P-type Ca 2ϩ channels to whole cell Ca 2ϩ channel current at different membrane potentials, to see which of the three channels opened at membrane potentials that would be commensurate with a role in the PLSD. We also compared the voltage dependence of DA neuron Ca 2ϩ channel activation to that of dorsal root ganglion (DRG) neurons. Our data indicate that a larger proportion of the whole cell Ca 2ϩ current observed near activation threshold is conducted through L-channels versus N-or P-chann...

This study was designed to determine the cellular distribution and pattern of expression for the mitochondria-associated protein, prohibitin, during the transitional stages of follicular differentiation within the rat ovary. Immunohistochemical staining techniques were used on frozen sections to examine the localization of prohibitin to preantral, antral, preovulatory, and atretic follicles. Prohibitin localization was also determined in corpus luteum from adult rats, in addition to those from infant and juvenile ovaries, before and after gonadotropin stimulation. Western and Northern blotting techniques were used for qualitative and quantitative assessment of prohibitin expression levels within the ovary. Prohibitin was localized within granulosa cells of infant and juvenile ovaries in a relatively heterogeneous staining pattern. The oocyte also exhibited robust prohibitin expression at all stages of follicular development. In addition, strong prohibitin expression was evident in the corpus luteum as well as in follicles undergoing atresia. Additional data derived from studies involving a GnRH-agonist indicate that increases in prohibitin protein expression correlate with the initial events of apoptosis. Collectively, these results support a growth regulatory role for prohibitin within the rat ovary. Therefore, we propose that prohibitin may serve as an important regulator of granulosa cell fate during follicular development.

During cell division, chromosome segregation is orchestrated by the interaction of spindle microtubules with the centromere. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, the chromosomal passenger protein complex, and microtubule bundling protein PRC1. PRC1 is phosphorylated by Cdc2 at Thr470 and Thr481 during mitosis. However, the functional relevance of PRC1 phosphorylation at Thr470 has remained elusive. Here we show that expression of the non-phosphorylatable mutant PRC1(T470A) but not the phospho-mimicking mutant PRC1(T470E) causes aberrant organization of the central spindle. Immunoprecipitation experiment indicates that both PRC1(T470A) and PRC1(T470E) mutant proteins associate with wild-type PRC1, suggesting that phosphorylation of Thr470 does not alter PRC1 self-association. In addition, in vitro co-sedimentation experiment showed that PRC1 binds to microtubule independent of the phosphorylation state of Thr470. Gel-filtration experiment suggested that phosphorylation of Thr470 promotes oligomerization of PRC1. Given the fact that prevention of the Thr470 phosphorylation inhibits PRC1 oligomerization in vitro and causes an aberrant organization of central spindle in vivo, we propose that this phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle.

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S U M M A R Y This study was designed to determine the cellular and ultrastructural distribution of the gonadotropin-releasing hormone (GnRH) and the relative expression of its mRNA in the oviduct of rats during different time points (days 7, 9, 16, and 20) of pregnancy. Immunofluorescent localization and confocal microscopic techniques were used to determine the cellular distribution of GnRH in the oviduct. Immunogold electron microscopy indicated its localization at the ultrastructural level, and real-time PCR was used to study the expression pattern of GnRH mRNA in the oviduct during pregnancy. In general, GnRH was localized within the epithelial cells lining the oviductal lumen at each selected time point. A strong correlation between the fluorescence intensity of GnRH-immunoreactive cells and the relative expression of GnRH mRNA was noted on days 7 and 16, followed by a plateau by day 20. At the ultrastructural level, uniform labeling of colloidal gold particles was observed in secretory vesicles and lamella of the luminal epithelium as well as the lumen of the oviduct.Collectively, these results demonstrate for the first time that the oviductal epithelium synthesizes and secretes the decapeptide GnRH during pregnancy in rats, which may have a possible role in postimplantation embryonic development and the maintenance of pregnancy. (J Histochem Cytochem 55:525-534, 2007)

The effect of muscarine on Ca2+ dependent electrical activity was studied in dopamine (DA) neurons located in the substantia nigra pars compacta (SNc) in brain slices from young rats, using sharp electrodes. In most DA neurons tested, muscarine (50 microM) reduced the amplitude of spontaneous oscillatory potentials and evoked Ca2+-dependent potentials recorded in the presence of TTX. Muscarine also reduced the amplitude of the slow afterhyperpolarization (sAHP) following action potentials in most DA neurons. These data suggest that muscarine reduces Ca2+ entry in SNc DA neurons. The reduction of the amplitude of the sAHP by muscarine in DA neurons may facilitate bursting initiated by glutamatergic input by increasing the frequency at which DA neurons can fire. The reduction of the sAHP via activation of muscarinic receptors in vivo may provide a mechanism whereby cholinergic inputs to DA neurons from the tegmental peduncular pontine nucleus could modulate dopamine release at dopaminergic targets in the brain.

Human infection with Trypanosoma brucei may result in meningo-encephalitis, neuronal demyelination, blood-brain-barrier dysfunction, peri-vascular infiltration, astrocytosis and neuronal apoptosis. Prevention of the short- or long-term, parasite-induced, neuronal assault requires a better understanding of the host's responses to the infection at the molecular level. Northern analysis, cDNA micro-arrays, reverse-transcriptase-PCR (RT-PCR), SDS-PAGE and immunohistology were therefore used to investigate global gene and protein expression in the brains of mice infected with T. brucei. Temporal and spatial expression of neuroleukin (NLK), a predominant neurotrophin which is associated with neuronal protection and regeneration during neuronal assault in the brain, was then assessed. Expression of 20 of the 588 genes investigated (representing pro- and anti-inflammatory immuno-modulators, growth factors, neurotransmitters, and pro- and anti-apoptosis factors) was significantly altered (P < 0.05). TUNEL analysis revealed extensive apoptosis at peak parasitaemia, mainly in the cerebellum. RT-PCR analysis of two regulators of apoptosis, Bcl-x(L) (anti-apoptotic) and Bax (pro-apoptotic), revealed equivalent increases in levels of expression. NLK expression was up-regulated in punctated fashion in brain and was mainly localized to abnormal (stellate) catecholamine neurons (CN) in the locus coeruleus (LC) of infected [and, to a lesser degree, the normal (polygonal) cells of uninfected] brainstem. Expression of NLK receptor (NLK-R) was inversely correlated with that of NLK. At peak parasitaemia, trypanosome infection apparently induces cerebellar apoptosis and a corresponding increase in NLK expression. NLK may be modulating inflammation and is probably involved in protecting CN and the cerebellum against apoptosis.

Prohibitin is an evolutionary conserved protein that is associated with cellular differentiation, atresia, and luteolysis in the rat ovary. However, the specific cellular location and function of prohibitin in ovarian cells has not been clearly elucidated. To characterize the expression of prohibitin during cell proliferation, differentiation, and cell death, we have successfully established a temperature-sensitive granulosa cell line, designated RGA-1. At a permissive temperature of 33 C, RGA-1 cells proliferate, but revert to a differentiated phenotype at a nonpermissive temperature of 39 C. Significant inductions of prohibitin mRNA and protein expression were observed in the differentiated phenotype when compared with proliferating cells. Differentiated RGA-1 cells were found to express inhibin alpha- and beta-transcripts, as well as steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor proteins in a manner reminiscent of steroidogenic functional responses observed in primary differentiated granulosa cells. Prohibitin expression correlated well with the expression of these steroidogenic proteins. At 39 C, RGA-1 cells also displayed increases in p53 protein levels, indicative of growth arrest in the nonproliferating cells. Confocal and electron microscopic examinations revealed increased prohibitin localization to the mitochondria at 39 C, along with changes in mitochondrial size and shape. These changes were accompanied by marked reductions in cytochrome c oxidase subunit II levels and in unit mitochondrial transmembrane potential. In addition, cell fractionation studies demonstrated that the prohibitin protein was mainly localized to the mitochondrial membrane. Collectively, these findings suggest a role for prohibitin in mitochondrial structure and function during growth and differentiation in ovarian granulosa cells. Prohibitin expression may also be indicative of mitochondrial destabilization during apoptosis-related events.