The Polycomb group (PcG) gene products form complexes that regulate chromatin configuration to mediate cellular memory to postmitotic somatic cells and postmeiotic oocytes in Drosophila melanogaster. Structural and functional similarities of PcG proteins between invertebrates and vertebrates suggest mammalian PcG proteins may be involved to imprint transcriptional status at various loci into postmitotic and postmeiotic daughter cells. To address molecular mechanisms underlying PcG-mediated cellular memory, it might be a prerequisite to understand subcellular localization of PcG proteins during mitosis and meiosis. In this study, we analyzed subcellular localization of Rae28/Ph1 and Ring1B by using newly generated monoclonal antibodies in mitotic somatic cells and meiotic mouse oocytes. Results suggest that Rae28/Ph1 and Ring1B dissociate from the chromatin upon its condensation in mitotic prophase in the U2-OS human osteosarcoma cell line. During maturation of oocytes, significant alterations of Rae28/Ph1 and Ring1B localization are concordant with configuration changes of the chromatin at the germinal vesicle stage of meiotic prophase. Importantly, dissociation of Rae28/Ph1 and Ring1B from the chromatin temporally correlates with transcriptional arrest both in mitosis and meiosis. Present and previous observations suggest molecular mechanisms required for mitotic regulation of RNA polymerase II could be involved in dissociation of PcG proteins.

Mammalian Polycomb group (PcG) proteins are known to function during the maintenance of spatially restricted expression of Hox cluster genes and cellular proliferation. To understand the molecular basis of PcG functions, it is important to identify the components of mammalian PcG complexes. We isolated mouse YAF2 as a protein that interacts with Ring1B, a known constituent of mammalian PcG complexes. We show that the murine YAF2 locus generates two different transcripts, mYAF2-a and mYAF2-b by alternative splicing of the third exons which encode two YAF2 isoforms of 179 and conceptual 60 amino acids, respectively. At least five exons encoding mYAF2 transcripts are mapped on chromosome 15E3 region. Expression of mYAF2 mRNA was observed in both pre- and postimplantation embryos. In mid-gestation embryos, mYAF2 expression is strongly seen in the region close to the surface ectoderm. Finally, biochemical evidence and colocalization studies in tissue culture cells suggest that the product of the mYAF2 gene is involved in PcG complexes together with Ring1B and/or Ring1A.

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.

The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have identified Ring1B, the product of the Ring1B gene (Rnf2 - Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.

The expression level of type II adenylyl cyclase mRNA (ACII) was analyzed by northern blotting in amygdaloid kindled rats. Remarkable increases in ACII mRNA were observed in the bilateral cerebral cortex and hippocampus at 24 h after the last generalized seizure. The elevated expression level in the hippocampus persisted for 4 weeks on the stimulated side. There were no changes in expression level in single-stimulated and partially-kindled states. These results suggest that the involvement of ACII might have an effect on the mechanisms of seizure generalization and the maintenance of persistent epileptogenesis rather than on the acquisition process.

Purpose: Human temporal lobe cpilepsy is frequently related to loss of hippocampal neurons, called hippocampal sclerosis. It is still controversial whether neuronal cell death is directly involved in the pathogenesis or epilepsy or whether it is a secondary phenomenon following recurrent seizures. The present study aims to clarify the contribution of neuronal apoptotic changes to the basic pathophysiology of epilepsy. We investigated apoptotic neural damage in the hippocampi of both a patient with intractable temporal lobe epilepsy and an amygdaloidkindling epilepsy model. Methods: The patient was a 30‐year old male who had a 15‐year history of drug‐resistant complex partial seizures. His seizure frequency increased to 5–10 seizures per week. Scalp EEG revealed right temporal‐dominant spikes, and spike and slow wave complexes. Frequent epileptic spike discharges from the right mid‐hippocampal region were detected by eicctrocorticography (ECoG) before temporal lobectomy. Samples of right hippocampal tissue were obtained during surgery. We cxamined apoptotic brain damage by i n situ terminal deoxynuclcotidyltraiisferase‐mediated dUTP nick‐end labeling of fragmented DNA (TUNEL) in the. hippocampal formation removed at surgery. We also examined the involvement of apoptotic brain damage in the kindling model of epilepsy. Sprague‐Dawley rats were kindled by a daily electrical stimulation in the left hasolateral amygdala with a SOHz biphasic square wave at an intensity just above the afterdischarge threshold for 2 seconds. The analysis was performed in the resected right hippocampus of the patient and in partially‐kindled (PK) and lully‐kindled (FK; 20 consecutive generalized convulsions) rats, 24 hours after the last seizure. After fixation i n 10% neutral‐buffered formalin, brain tissue was embedded i n paraffin. Coronal sections werc ording to ordinary TUNEL method. Immunoreactivity of Bax and the expression level of bax mRNA by in situ hybridination were alsn examined. Results: Apoptotic brain damage was observed in the spike‐detected and non spike‐detected areas of the patient's hippocampus. More DNA‐fragmented nuclei were seen i n non spike‐detected areas than in the spike‐detected areas. Only a few DNA‐fragmented nuclei were detected in the spike‐detected area. Compared with the spike‐detected area, an increased expression of Bax protein and bax mRNA levels was observed in non spike‐detected areas. In the kindling model, an increased number of TUNEL positive cells were secn in the CA3 and CA4 from the PK group rats. Only a few DNA‐fragmented nuclei were seen in the hippocampus from the FK group rats. The relative increase in expression levels of Bax protein and bax mRNA was observed in the CA3 and CA4 in both kindled groups. Conclusions: I1 we hypothesize that results from both human temporal lobe epilepsy and kindling models arise from a common basis in the pathophysiology of epilepsy, apoptotic cell death in hippocampus is induced not only by recurrent seizures, but also by factors rclated to the aquiaition process of epileptogenesis. The present results might be associated with the hippocainpal synaptic reorganization. However, clarification of this relationship requires further study. Our detection of changes in apoptosis‐related genes such as Bax and neuronal apoptotic cell death suggests their contribution to the pathogenesis of epilepsy.

The expressions of mRNAs encoding G protein α subunits were analyzed in the cerebral cortex of amygdaloid kindled rats. A remarkable increase in Gsα mRNA were observed on the bilateral cerebral cortex at 24 h after the last generalized seizure and persisted 3 weeks on the unstimulated side. Gi2α mRNA level was also increased on the stimulated side at 24 h and persisted 3 weeks. These result suggest that dysfunction of Gs and Gi2 might relate to the basic mechanisms of seizure generation and the maintenance of epileptogenesis.

YOSHIMOTO, K., T. YOSHIDA, Y. SORIMACHI, A. HIRANO, Y. TAKEUCHI, S. UEDA AND M. YASUHARA. Effects of Age and Ethanol on Dopamine and Serotonin Release in the Rat Nucleus Accumbens. PHYSIOL BEHAV 64(3) 347–351, 1998.—Neural functions in the nucleus accumbens (ACC) play an important role in alcohol drinking behavior. In the present study, we observed the effects of age and ethanol (EtOH) on dopamine (DA) and serotonin (5-HT) release in the ACC of freely moving 4-, 10-, and 16-month-old rats using brain microdialysis techniques. After co-perfusion with 200 mM ethanol, ACC DA, and 5-HT release were decreased significantly in 16-month-old rats compared to those at 4 months old. ACC DA and 5-HT neurons of aged rats were less sensitive to ethanol. On the other hand, both basal extracellular DA and 5-HT release in the ACC were significantly higher in 16-month-old than in 4-month-old rats. Therefore, aging results in opposite changes in basal and alcohol-induced DA and 5-HT release in the ACC.

Purpose: Changes in neural transmission via N‐methyl‐D‐aspartate (NMDA) receptors (NMDARs) play a crucial role in seizure generation and epileptogenesis. Our previous study suggested that NMDAR1 contributes to the acquisition and maintenance of epileptogenesis in the kindled brain. However, the factors involved in the changes of NMDAR1 expression are still unclear. To clarify the relation between the induction of afterdischarges at the primary stimulation site and NMDAR expression in other brain regions, we investigated the levels of gene transcripts (NMDAR1 and NMDAR2A‐2D) during several seizure stages in amygdaloid‐kindled rats by using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). Methods: Twenty male Sprague‐Dawley rats were used. The kindling stimulus was a 50‐Hz biphasic square wave at an intensity abovethe threshold for afterdischarges (fixed at 350 FA). The conditions for sham stimulation were the same as those for kindling, except that theintensity was fixed at 50 μA. The rats in the partially kindled group (K‐P; n = 4) received three stimulations and were analyzed 24 h after the last partial seizure (class 0–2, according to Racine's classification). The rats in the sham‐stimulated group (K‐S; n = 4) were analyzed 24 h after three stimulations without any local afterdischarges or behavioral changes. The rats in the fully kindled group showed 10 consecutive generalized seizures and were analyzed 24 h (K‐I; n = 4) and 4 weeks (K‐11; n = 4) after the last seizure. The rats in the control group (n = 4) were given only a sham operation. Total RNAs were extracted and purified from the cerebral cortex, hippocampus, and cerebellar cortex. The cDNAs were synthesized by using murine leukemia virus reverse transcriptase. PCR was performed with a [32P] deoxycytidine triphosphate (dCTP) and subunit‐specific primers that were reported previously (J Biol Chem 1994;269: 16989–92). P‐Actin was amplified in the same way as a reference. The PCR products were electrophoresed in polyacrylamide gels. The radioactivity of the radiopositive bands was measured with an image analyzer (Fujix Bas 2000). Statistical analysis was done with a Student's unpaired r test. Results: NMDARl mRNA increased significantly in the cerebral cortex bilaterally in the K‐P group (stimulated side, 41%, and unstimulated side, 33%; p < 0.05) in comparison with the control group. whereas no change was observed in the K‐S group. An increase of NMDARl mRNA also was observed bilaterally in the cerebral cortex in rats of both the K‐I (90 and 77%; p < 0.05) and K‐I1 (54 and 49%: p < 0.05) groups. No changes in the NMDARI mRNA levels were detected in other brain regions in any of the groups. NMDAR2C mRNA decreased in the cerebellar cortex of rats in the K‐I group. But in the K‐I1 group, it returned to the same level as that in the controls. The levels of other subunit mRNAs remained unaltered in all brain regions in all of the groups. Conclusions: Because the increase of NMDAR 1 gene transcripts was marked in the K‐P group, but not in the K‐S group, it is suggested that the induction of afterdischarges at the primary stimulation site accelerates the transcription of the NMDARI gene in distant regions transsynaptically. On the other hand, because the decrease in the level of NMDAR2C mRNA in the cerebellum was marked only during the early phase after generalized seizures, the decrease may be related to the seizure activity. As the increase of NMDARI mRNA appeared to be unaffected by any further electrical stimulation over a long period after achievement of full kindling, it is also suggested that NMDAR I plays an important role in the maintenance as well as the acquisition of epileptogenesis in the kindled brain.

The mechanisms which mediate cortical map plasticity and functional recovery following stroke remain a matter of debate. Readjustment of the excitatory-inhibitory balance may support cortical map plasticity in perilesional areas. Here we studied cortical net inhibition in the vicinity of photothrombotically-induced cortical lesions in young adult (3 months) and aged (24 months) male rats. Field potentials were recorded in cortical layer II/III following application of paired-pulse stimulation at layer VI/white matter in coronal brain slices. Additionally, we analyzed the regional distribution of 5 major gamma-aminobutyric acid A (GABAA) receptor subunits (α1, α2, α3, α5, and γ2) by immunohistochemistry. Paired-pulse inhibition in the perilesional parietal cortex was decreased in young rats but was increased in aged rats. As a consequence of the diminished intrinsic net inhibition in aged control animals, the excitatory-inhibitory balance was readjusted to an age-independent similar level in young and aged lesioned rats in a homeostatic-like fashion. These physiological changes in neuronal activity were accompanied by age-specific laminar alterations of the gamma-aminobutyric acid A (GABAA) receptor subunit composition, most prominently of the subunit α5. The present study suggests that the mechanisms underlying functional reorganization in aged animals may be distinctly different from those in young animals.