Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with β-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

Although connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by Western blotting and RT-PCR. All the data obtained were validated using Cx36 knockout mice. Taken together the present data on localization of Cx36 gap-junctions in two tissues of neural crest-derived neuroendocrine organs may provide an anatomical basis for future functional investigations.

Stress is the most frequently self-reported seizure precipitant in patients with epilepsy. Moreover, a relation between ear stress and epilepsy has been suggested. Although ear stress and stress hormones are known to influence seizure threshold in rodents, effects on the development of epilepsy (epileptogenesis) are still unclear. Therefore, we studied the consequences of ear corticosteroid exposure for epileptogenesis, under highly controlled conditions in an animal model. Experimental febrile seizures (eFS) were elicited in 10-day-old mice by warm-air induced hyperthermia, while a control group was exposed to a normothermic condition. In the following 2 weeks, mice received either seven corticosterone or vehicle injections or were left undisturbed. Specific measures indicative for epileptogenesis were examined at 25 days of age and compared with vehicle injected or untreated mice. We examined structural [neurogenesis, dendritic morphology, and mossy fiber sprouting (MFS)] and functional (glutamatergic postsynaptic currents and long-term potentiation) plasticity in the dentate gyrus (DG). We found that differences in DG morphology induced by eFS were aggravated by repetitive (mildly stressful) vehicle injections and corticosterone exposure. In the injected groups, eFS were associated with decreases in neurogenesis, and increases in cell proliferation, dendritic length, and spine density. No group differences were found in MFS. Despite these changes in DG morphology, no effects of eFS were found on functional plasticity. We conclude that corticosterone exposure during early epileptogenesis elicited by eFS aggravates morphological, but not functional, changes in the DG, which partly supports the hypothesis that ear stress stimulates epileptogenesis.

Stress is among the most frequently self-reported factors provoking epileptic seizures in children and adults. It is still unclear, however, why some people display stress-sensitive seizures and others do not. Recently, we showed that young epilepsy patients with stress-sensitive seizures exhibit a dysregulated hypothalamic-pituitaryadrenal (HPA)-axis. Most likely, this dysregulation gradually develops, and is triggered by stressors occurring early in life (early-life stress [ELS]). ELS may be particularly impactful when overlapping with the period of epileptogenesis. To examine this in a controlled and prospective manner, the present study investigated the effect of repetitive variable stressors or control treatment between postnatal day (PND) 12 and 24 in male mice exposed on PND10 to hyperthermia (HT)-induced prolonged seizures (control: normothermia). A number of peripheral and central indices of HPAaxis activity were evaluated at pre-adolescent and young adult age (ie, at PND25 and 90, respectively). At PND25 but not at PND90, body weight gain and absolute as well as relative (to body weight) thymus weight were reduced by ELS (vs control), whereas relative adrenal weight was enhanced, confirming the effectiveness of the stress treatment. Basal and stress-induced corticosterone levels were unaffected, though, by ELS at both ages. HT by itself did not affect any of these peripheral markers of HPA-axis activity, nor did it interact with ELS. However, centrally we did observe age-specific interaction effects of HT and ELS with regard to hippocampal glucocorticoid receptor mRNA expression, neurogenesis with the immature neurone marker doublecortin and the number of hilar (ectopic) granule cells using Prox1 staining. This 2 of 15 | UMEOKA Et Al. | INTRODUC TI ONApproximately 50% of all children and adults with epilepsy acknowledge stress as a seizure-provoking factor, 1,2 ranking stress among the most frequently self-reported precipitants of seizures. 2-10 Why some individuals show stress-sensitivity in seizure activity and others do not still remains unclear. One explanation could be that the former as opposed to the latter group responds differently to stress as, for example, reflected by aberrant hypothalamic-pituitary-adrenal (HPA) activation or downstream targets after stress. In accordance, recent evidence obtained in a cohort of children and adolescents (6-17 years of age) with epilepsy indicated that those with stress-sensitive compared to individuals with stress-insensitive seizures or healthy controls show a blunted cortisol response to experimentally induced stress. 11Possibly, this renders hormonal negative-feedback actions on the brain less adequate, which could result in increased levels of proexcitatory central stress hormones such as corticotrophin-releasing hormone. 12 Which elements of the stress system become aberrant at specific developmental stages is not easy to investigate in humans with stress-sensitive seizures because this would require prospective longitudinal investigation of both centr...