Rasd1 is an estrogen-responsive immediate early gene and modulates expression of late genes in rat anterior pituitary cells.

Dexamethasone-induced Ras-related protein 1 (Rasd1) is a member of the Ras superfamily of monomeric G proteins that have a regulatory function in signal transduction. Here we investigated the role of Rasd1 in regulating estrogen-induced gene expression in primary cultures of rat anterior pituitary cells. Rasd1 mRNA expression in anterior pituitary cells decreased after treatment with forskolin or serum and increased after treatment with 17ß-estradiol (E2). Increases in Rasd1 mRNA expression occurred as early as 0.5 h after E2 treatment, peaked at 1 h and were sustained for as long as 96 h. This rapid and profound increase in Rasd1 mRNA expression induced by E2 was also seen in GH4C1 cells, an estrogen receptor-positive somatolactotroph cell line. Among pituitary estrogen-responsive late genes studied, basal mRNA expression of Pim3 and Igf1 genes was decreased by RNA interference-mediated knockdown of Rasd1 expression, whereas basal expression of the Giot1 gene was increased. Moreover, Rasd1 knockdown enhanced stimulation of Pim3 mRNA expression and attenuated inhibition of Fosl1 mRNA expression 24 h after E2 treatment. These changes in mRNA expression were accompanied by enhanced activity of promoters containing CRE, AP-1 and SRE binding sequences. These results suggest that Rasd1 is an estrogen-responsive immediate early gene and modulates E2 induction of at least several late genes in anterior pituitary cells.

Activation of G protein-coupled estrogen receptor 1 mimics, but does not mediate, the anti-proliferative action of estradiol on pituitary lactotrophs in primary culture.

Estrogen binds to nuclear estrogen receptors (ERs) to modulate transcription of target genes in estrogen-responsive cells. However, recent studies have shown that estrogen also binds to cytoplasmic membrane ERs to modulate protein kinase signaling cascades, leading to non-genomic actions. We investigated whether either nuclear or membrane ERs, including G protein-coupled estrogen receptor 1 (Gper1), mediate the inhibitory action of estrogen on insulin-like growth factor-1 (IGF-1)-induced proliferation of pituitary lactotrophs in primary culture. The cytoplasmic membrane-impermeable bovine serum albumin-conjugated estradiol (BSA-E2) at 1 nM, an equimolar concentration at which 17ß-estradiol (E2) exerts anti-proliferative effects, did not inhibit IGF-1-induced lactotroph proliferation. In contrast, diethylstilbestrol, which is known to selectively activate nuclear ERs but not membrane ERs, inhibited IGF-1-induced proliferation and modulated mRNA expression of estrogen-responsive genes to a similar degree as E2. Activation of Gper1 by its agonist G-1 inhibited IGF-1-induced proliferation in a dose-dependent manner, but it had little effect on modulation of mRNA expression of estrogen-responsive genes. However, blockade of Gper1 by its antagonist G-15 did not affect the inhibitory action of E2 on IGF-1-induced proliferation. Here, we demonstrate that E2 inhibition of lactotroph proliferation is due to nuclear ER-mediated genomic action. Our results suggest that activation of Gper1 mimics, but does not mediate, the anti-proliferative action of E2 on lactotrophs.

Immunohistochemical and morphofunctional studies of skeletal muscle tissues with electric nerve stimulation by in vivo cryotechnique.

In this study, morphological and immunohistochemical alterations of skeletal muscle tissues during persistent contraction were examined by in vivo cryotechnique (IVCT). Contraction of gastrocnemius muscles was induced by sciatic nerve stimulation. The IVCT was performed immediately, 3 min or 10 min after the stimulation start. Prominent ripples of muscle fibers or wavy deformation of sarcolemma were detected immediately after the stimulation, but they gradually diminished to normal levels during the stimulation. The relative ratio of sarcomere and A band lengths was the highest in the control group, but it immediately decreased to the lowest level and then gradually recovered at 3 min or 10 min. Although histochemical intensity of PAS reaction was almost homogeneous in muscle tissues of the control group or immediately after the stimulation, it decreased at 3 min or 10 min. Serum albumin was immunolocalized as dot-like patterns within some muscle fibers at 3 min stimulation. These patterns became more prominent at 10 min, and the dots got larger and saccular in some sarcoplasmic regions. However, IgG1 and IgM were immunolocalized in blood vessels under nerve stimulation conditions. Therefore, IVCT was useful to capture the morphofunctional and metabolic changes of heterogeneous muscle fibers during the persistent contraction.

ANG II modulates both slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rabbit rostral ventrolateral medulla.

This study investigated the effects of ANG II on slow and rapid baroreflex responses of barosensitive bulbospinal neurons in the rostral ventrolateral medulla (RVLM) in urethane-anesthetized rabbits to determine whether the sympathetic baroreflex modulation induced by application of ANG II into the RVLM can be explained by the total action of ANG II on individual RVLM neurons. In response to pharmacologically induced slow ramp changes in mean arterial pressure (MAP), individual RVLM neurons exhibited a unit activity-MAP relationship that was fitted by a straight line with upper and lower plateaus. Iontophoretically applied ANG II raised the upper plateau without changing the slope, and, thereby, increased the working range of the baroreflex response. An asymmetric sigmoid curve that was determined by averaging individual unit activity-MAP relationship lines became more symmetric with ANG II application. The characteristics of the average curves, both before and during ANG II application, were consistent with the renal sympathetic nerve activity-MAP relationship curves obtained under the same experimental conditions. ANG II also affected rapid baroreflex responses of RVLM neurons that were induced by cardiac beats, as application of ANG II predominantly raised the average unit activities in the downstroke phase of arterial pulse waves. The present study provides a possible explanation for the ANG II-induced sympathetic baroreflex modulation based on the action of ANG II on barosensitive bulbospinal RVLM neurons. Our results also suggest that ANG II changes both static and dynamic characteristics of baroreflex responses of RVLM neurons.

Differences between rat strains in the development of PRL-secreting pituitary tumors with long-term estrogen treatment: In vitro insulin-like growth factor-1-induced lactotroph proliferation and gene expression are affected in Wistar-Kyoto rats with low estrogen-susceptibility.

There are differences in the susceptibility of rat strains to pituitary growth and lactotroph proliferation caused by long-term treatment with estrogens. To investigate the pituitary mechanism for this strain difference in estrogen-induced lactotroph proliferation, we compared the abilities of 17-ß estradiol (E2) and insulin-like growth factor-1 (IGF-1) to modulate lactotroph proliferation and gene expression in vitro in Wistar and Wistar-Kyoto (WKY) rats. These two strains of rats have a high and very low susceptibility to estrogen, respectively. Long-term in vivo treatment with E2 was confirmed to markedly increase pituitary weight and lactotroph proliferation in ovariectomized Wistar, but not in WKY rats. Pituitary lactotrophs in primary cultures showed similar proliferative responsiveness to the culture condition-dependent, stimulatory and inhibitory actions of E2 in both strains. The only difference in lactotroph proliferation in vitro was a lower response to IGF-1 in WKY cells compared with Wistar cells. This difference in proliferation was associated with strain differences in IGF-1-induced gene expression in Wistar and WKY cultured cells. Of the genes tested, IGF-1-induced expression of the Wnt4, Stc1, Mybl1, and Myc genes was attenuated or abolished in WKY cells. These results suggest that the proliferative response to estrogen in lactotrophs in primary culture does not reflect the proliferative response to long-term estrogen treatment observed in vivo in Wistar and WKY rats. The strain difference in proliferation and gene expression to IGF-1 may be implicated in the variable degree of susceptibility for lactotroph proliferation observed in different strains of rats following long-term estrogen treatment.

Activation of D2 dopamine receptors inhibits estrogen response element-mediated estrogen receptor transactivation in rat pituitary lactotrophs.

Estrogen and dopamine are major opposing regulators of the endocrine functions of pituitary lactotrophs. Dopamine inhibits estrogen-induced changes in the synthesis and secretion of prolactin, and lactotroph proliferation. We studied the mechanism of the inhibitory effects of dopaminergic stimulation on estrogen-induced functional changes of rat lactotrophs in primary culture. The dopaminergic agonist, bromocriptine (BC), suppressed 17ß-estradiol-stimulated lactotroph proliferation, prolactin promoter activity, and mRNA expression of some estrogen-responsive genes. In lactotroph-enriched pituitary cells, BC treatment inhibited the estrogen response element (ERE) DNA sequence-mediated estrogen receptor (ER) transcriptional activity. Using a lactotroph-specific ERE transcriptional assay, we found that BC inhibition of the ERE-mediated ER transcriptional activity partly involved D2 dopamine receptor-mediated, pertussis toxin-sensitive G protein-coupled, cAMP/protein kinase A-dependent signaling. BC treatment had no effect on the cellular concentration of ERα or its phosphorylation status at Ser-118. Similar transcriptional inhibition by BC was also found in GH4ZR7 cells, a D2 dopamine receptor-expressing somatomammotrophic cell line. These results suggest that activation of the D2 dopamine receptors inhibits estrogen-dependent lactotroph functions in part via attenuation of ERE-mediated ER transactivation.

Inhibition of Bcl3 gene expression mediates the anti-proliferative action of estrogen in pituitary lactotrophs in primary culture.

In addition to their well-known stimulatory action, estrogens have an anti-proliferative effect. The present study was undertaken to investigate the mechanism by which 17ß-estradiol (E2) inhibits insulin-like growth factor-1 (IGF-1)-induced proliferation in vitro in the rat pituitary lactotroph, a typical estrogen-responsive cell. E2 treatment of pituitary cells did not change levels of IGF-1-induced phosphorylation of proliferation-related protein kinases such as Erk1/2 and Akt. We performed global gene expression profiling by DNA microarray analysis and identified 177 genes regulated by E2 in the presence of IGF-1. These results were verified by quantitative real time PCR. The estrogen-regulated genes included several NFκB family related genes. As pharmacological inhibition of the NFκB pathway blocked IGF-1-induced lactotroph proliferation, we chose to investigate whether one NFκB pathway gene, Bcl3, was involved in the anti-proliferative action of E2. RNA interference-mediated knockdown of Bcl3 expression attenuated IGF-1-induced lactotroph proliferation. Even minimal induced overexpression of Bcl3 blocked the anti-proliferative action of E2. In contrast, Nfkb2, another E2-downregulated protein, required maximal overexpression to block the anti-proliferative action of E2. These results suggest that inhibition of Bcl3 expression is involved in the anti-proliferative action of estrogens in pituitary lactotrophs in culture.

cAMP response element-binding protein (CREB) is required for epidermal growth factor (EGF)-induced cell proliferation and serum response element activation in neural stem cells isolated from the forebrain subventricular zone of adult mice.

Neurogenesis, which occurs not only in the developing brain but also in restricted regions in the adult brain including the forebrain subventricular zone (SVZ), is regulated by a variety of environmental factors, extracellular signals, and intracellular signal transduction pathways. We investigated whether the transcription factor cAMP response element (CRE)-binding protein (CREB) is involved in the regulation of cell proliferation of neural stem cells (NSCs) isolated from the SVZ of adult mice. Treatment of NSCs with the protein kinase A (PKA) inhibitors H89 and KT5720 inhibited epidermal growth factor (EGF)-stimulated NSC proliferation. Similar inhibition was observed when a dominant-negative mutant of CREB (MCREB) was expressed. EGF treatment increased CRE-mediated transcriptional activity, but this increase was much less than that caused by treatment with the adenylate cyclase activator forskolin, which changed neither basal nor EGF-stimulated proliferation of NSCs. Neither PKA inhibitors nor MCREB expression blocked EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), a protein kinase mediating EGF's mitogenic action. However, MCREB suppressed EGF-induced expression of several immediately early genes including c-fos, c-jun, jun-B, and fra-1 and subsequent AP-1 transcriptional activation. MCREB expression also inhibited the ability of EGF to stimulate transcriptional activation mediated by the serum response element (SRE), a promoter sequence regulating c-fos gene expression. These results suggest that basal activity of CREB is required for the mitogenic signaling of EGF in NSCs at a level between ERK activation and SRE-mediated transcriptional activation.

Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces expression of the chemokine genes Cxcl4 and Cxcl7 in the perinatal mouse brain.

Fetal exposure to dioxins affects brain development and influences behaviors in human and laboratory animals. However, the cellular target and mechanisms of the neurotoxic action of dioxins are largely unknown. To investigate the molecular basis for the neurotoxicity of dioxins, pregnant C57BL/6 mice were exposed to 5 µg kg(-1) body weight of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by a single gavage on gestational day 12.5 (GD 12.5), and gene expression of the whole fetal brain at GD 18.5 was profiled by DNA microarray analysis. The analysis revealed that the expression of two chemokine genes, Cxcl4 and Cxcl7, was up-regulated by TCDD exposure. Real-time PCR analysis verified that they were up-regulated by TCDD in both male and female brains, while the mRNA levels of a majority of other chemokines and their receptor genes were not affected. The up-regulation was TCDD dose-dependent and peaked at GD 15.5-18.5. In situ hybridization analysis showed that the Cxcl4 mRNA expression was localized in part of the surface of cerebral cortex and that the level was increased by TCDD treatment. These results suggest that Cxcl4 and Cxcl7 play a role in the development of neurobehavioral alterations that are triggered by in utero TCDD exposure and later surface in adults.

Absence of ligand-independent transcriptional activation of the estrogen receptor via the estrogen response element in pituitary lactotrophs in primary culture.

The estrogen receptor (ER) is a ligand-activated transcription factor that enhances gene expression by binding to specific regulatory DNA sequences called estrogen response elements (EREs). In some cell lines, the ER is also activated in a ligand-independent manner by multiple signaling pathways. In this study, we developed a novel adenovirus-mediated assay for promoter activation, termed LASETA, which we then used to examine whether ligand-independent activation of the ER occurred in normal pituitary lactotrophs in primary culture. In the LASETA adenovirus vector, the loxP-flanked stop sequence was deleted by prolactin (PRL) promoter-regulated expression of Cre recombinase. This led to lactotroph-specific expression of a reporter gene driven by an ERE-containing promoter. Estrogen-induced expression of the reporter protein luciferase in LASETA was specific for lactotrophs and was ER-dependent. LASETA was shown to be reliable even with varying Cre recombinase expression levels, which were caused by changes in PRL promoter activity. Using LASETA, we observed no change in ERE-mediated ER activity in the absence of estrogen after treatment of normal lactotrophs with agents such as insulin-like growth factor-1, epidermal growth factor, the adenylate cyclase activator forskolin, the extracellular signal-regulated kinase kinase inhibitor U0126, and the protein kinase A inhibitor H89. The ERE-mediated ligand-independent ER activity was induced by the growth factors and forskolin in the somatolactotroph tumor cell line GH4C1 cells. These results suggest that ERE-mediated ligand-independent activation of ER does not occur in normal lactotrophs in primary culture, and is a phenomenon likely restricted to transformed cells.

The cAMP response element-binding protein in the bed nucleus of the stria terminalis modulates the formalin-induced pain behavior in the female rat.

Abstract Differences in male and female responses to pain are widely recognized in many species, including humans, but the cerebral mechanisms that generate these responses are unknown. Using the formalin test, we confirmed that proestrus female rats showed nociceptive behavior, modulated by estrogen that was distinct from male rats, particularly during the interphase period. We then explored the brain areas, which were involved in the female pattern of nociceptive behavior. We found that, after a formalin injection and at the time corresponding to the behavioral interphase, the number of phosphorylated cAMP response element-binding protein (pCREB)-immunoreactive neurons observed by immunocytochemistry increased in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD) in female but not male rats. There were no significant sex differences in pCREB expression following formalin in any region other than the BSTLD. The increased pCREB in female rats was eliminated after an ovariectomy and restored with 17beta-estradiol treatment. Neither an orchidectomy nor 17beta-estradiol treatment affected the pCREB response in male rats. The increase in pCREB expression in the BSTLD in female rats after formalin injection was confirmed with immunoblotting. To determine the role of CREB in the BSTLD, adenovirus-mediated expression of a dominant-negative form of CREB (mCREB) was carried out. The nociceptive behavior during interphase was significantly attenuated by injection of virus carrying mCREB into the BSTLD in female rats but not in male rats. These results suggest a novel role for CREB in the BSTLD as a modulator of the pain response in a female-specific, estrogen-dependent manner.

Adenovirus vectors differentially modulate proliferation of pituitary lactotrophs in primary culture in a mitogen and infection time-dependent manner.

Adenoviruses are powerful, widely utilized vectors for gene transfer. Limitations to their application, however, have not been well described. We used rat pituitary lactotrophs in primary culture as a model for studying how adenovirus vector infection modulates mitogen-induced proliferation and the activities of mitogen signaling pathways. Infection with adenovirus vectors expressing beta-galactosidase (betagal) raised basal proliferative levels and blocked fetal bovine serum (FBS)-induced proliferation of lactotrophs, but did not influence the changes in proliferation induced by forskolin, IGF-I, and bromocriptine. The betagal-expressing adenoviruses did not alter the inhibitory action of 17beta-estradiol (E(2)) in the presence of IGF-I; however, they blocked the stimulatory action of E(2) in the presence of dextran-coated charcoal-striped serum or forskolin. An adenovirus expressing no protein failed to block FBS-induced proliferation, but was effective in modulating basal proliferative levels and the stimulatory actions of E(2). The increased basal proliferative level and the blockade of FBS-induced proliferation were transient, and lost 5 days after infection while the blockade of the stimulatory action of E(2) in the presence of forskolin persisted. Adenovirus infection raised basal protein levels of the phosphorylated forms of cAMP response element-binding protein (pCREB) and ERK1/2 and increased the proportion of pCREB-immunoreactive lactotrophs. Adenoviruses also altered estrogen-induced responses in mRNA expression of several estrogen-responsive genes in a gene-specific manner. The results demonstrate that an adenovirus vector differentially interferes with lactotroph proliferation in response to various mitogens. Our results suggest that the effects of the adenovirus that are independent of the genes transferred must be considered when performing adenoviral gene transfer in the primary cultures of normal cells.

Consumption of commercial whole and non-fat milk increases the incidence of 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats.

BACKGROUND: Breast cancer has become the most common cancer among women worldwide. Although the consumptions of milk and dairy products were considered to be a risk factor for breast cancer in some epidemiological studies, the results were inconsistent. METHODS: In the present study, female Sprague-Dawley rats received a single oral dose of 5mg 7,12-dimethylbenz(a)anthracene (DMBA). One week later, the animals were divided into four groups: whole milk (WM), artificial whole milk (A-WM), non-fat milk (NFM) or artificial non-fat milk (A-NFM) mixed with commercial powder chow. Rats were palpated weekly to monitor tumor development. At week 20 after DMBA administration, rats were decapitated and the volume and weight of mammary tumor were recorded. RESULTS: Tumor incidence, the cumulative number of tumors and the sums of tumor volume were higher in the WM and NFM groups than in the A-WM and A-NFM groups both at palpation and at autopsy. CONCLUSION: Combining our previous studies, we found the consumption of milk promoted the development of DMBA-induced mammary tumors in rats independent of the fat level.

Involvement of cAMP response element-binding protein in the regulation of cell proliferation and the prolactin promoter of lactotrophs in primary culture.

Hypothalamic hormones, including dopamine, regulate critical functions of pituitary cells via the cAMP-protein kinase A (PKA) pathway. The PKA-downstream transcription factor cAMP response element (CRE)-binding protein (CREB) is an integrating molecule that is also activated by many other protein kinase pathways. We investigated the involvement of CREB in the regulation of cell proliferation and the PRL promoter of rat lactotrophs in primary cell culture. Recombinant adenoviruses were used for efficient gene delivery into pituitary cells. Bromocriptine, a dopaminergic agonist known to decrease intracellular cAMP concentrations, caused inhibition of PRL promoter activity and lactotroph proliferation, which was accompanied by decreases in CRE-mediated transcription and CREB phosphorylation in lactotrophs. Expression of a dominant-negative form of CREB (MCREB), which was effective in suppressing CRE-mediated transcription induced by the adenylate cyclase activator forskolin, inhibited basal and forskolin-induced PRL promoter activity and PRL mRNA expression. MCREB expression lowered basal proliferative levels and blocked forskolin-induced proliferation of lactotrophs. Insulin-like growth factor I (IGF-I), a potent mitogen in lactotrophs, did not affect intracellular cAMP concentrations but transiently increased lactotroph CREB phosphorylation. MCREB expression also inhibited IGF-I-induced lactotroph proliferation. These results suggest that CREB is involved in the regulation of cell proliferation and the PRL promoter in normal lactotrophs and that dopamine inhibition of these lactotroph functions is at least in part due to inhibition of the cAMP-PKA-CREB pathway.

Estrogen actions on lactotroph proliferation are independent of a paracrine interaction with other pituitary cell types: a study using lactotroph-enriched cells.

The mitogenic action of estrogen on estrogen-responsive tissues is suggested to be mediated by paracrine growth factors secreted from neighboring estrogen receptor-positive cells. Using pituitary lactotrophs in primary culture, on which estrogen exerts both mitogenic and antimitogenic actions in a cell context-dependent manner, we investigated whether a paracrine cell-to-cell interaction with other pituitary cell types was required for estrogen action. In pituitary cells, enriched for lactotrophs by 85% using differential sedimentation on a discontinuous Percoll gradient, 17beta-estradiol (E2) showed an antimitogenic action on lactotrophs in the presence of IGF-I, which was similar to that in control unenriched cells. Mitogenic actions were also seen in lactotroph-enriched cells when E2 was administered alone, in combination with serum, or in combination with the adenylate cyclase activator forskolin. Similar results were obtained in 90% lactotroph-enriched cells collected by fluorescence-activated cell sorting from transgenic rats expressing enhanced green fluorescent protein under the control of the prolactin promoter. The putative role of basic fibroblast growth factor (bFGF) as a paracrine factor mediating the mitogenic action of estrogen was not supported by the results that: 1) bFGF inhibited lactotroph proliferation; 2) immunoneutralization of bFGF failed to block E2-induced proliferation; and 3) cellular bFGF levels were not altered by E2 treatment. These results suggest that the antimitogenic and mitogenic actions of estrogen on lactotrophs do not require paracrine signals from other pituitary cell types and that estrogen directly influences lactotroph proliferation.

Pup removal suppresses estrogen-induced surges of LH secretion and activation of GnRH neurons in lactating rats.

During lactation, the suckling stimulus exerts profound influences on neuroendocrine regulation in nursing rats. We examined the acute effect of pup removal on the estrogen-induced surge of LH secretion in ovariectomized lactating rats. Lactating and nonlactating cyclic female rats were given an estradiol-containing capsule after ovariectomy, and blood samples were collected through an indwelling catheter for serum LH determinations. In lactating, freely suckled ovariectomized rats, estrogen treatment induced an afternoon LH surge with a magnitude and timing comparable to those seen in nonlactating rats. Removal of pups from the lactating rats at 0900, 1100, or 1300 h, but not at 1500 h, suppressed the estrogen-induced surge that normally occurs in the afternoon of the same day. The suppressive effect of pup removal at 0900 h was completely abolished when the pups were returned by 1400 h. In contrast, pup removal was ineffective in abolishing the stimulatory effect of progesterone on LH surges. Double immunohistochemical staining for gonadotropin-releasing hormone (GnRH) and c-Fos, a marker for neuronal activation, revealed a decrease, concomitantly with the suppression of LH surges, in the number of c-Fos-immunoreactive GnRH neurons in the preoptic regions of nonsuckled rats. An LH surge was restored in nonsuckled rats when 0.1 microg oxytocin was injected into the third ventricle three times at 1-h intervals during pup removal. These results suggest that the GnRH surge generator of lactating rats requires the suckling stimulus that is not involved in nonlactating cyclic female rats.

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin suppresses contextual fear conditioning-accompanied activation of cyclic AMP response element-binding protein in the hippocampal CA1 region of male rats.

We investigated the effect of in utero and lactational exposures to dioxin on adult offspring with contextual fear conditioning, a sex- and hippocampus-dependent learning paradigm; and we measured the conditioning-accompanied activation of cyclic AMP response element-binding protein (CREB) in the hippocampal CA1 region. Pregnant rats were treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gestation day 15. TCDD treatment decreased freezing time in conditioning tests of adult male offspring but not of female offspring. A similar, male-specific decrease was observed in the percentage of phosphorylated CREB-immunoreactive neurons in the CA1 region following conditioning in TCDD-treated rats. These results suggest that perinatal TCDD exposure impairs hippocampus-dependent learning in male offspring by suppressing CREB activation.

Age-related disturbance of memory and CREB phosphorylation in CA1 area of hippocampus of rats.

In the early process of long-term memory formation, cyclic AMP response element-binding protein (CREB), a transcription factor on which multiple signal transduction pathways converge, has been implicated. We examined whether the age difference in the performance of contextual fear conditioning (CFC) is associated with a change in activation of CREB in the hippocampus which is an important neural structure for long-term memory. The activation of CREB in the hippocampus in young (15 weeks old) and old (120 weeks old) male rats was determined immunohistochemically with an antibody that specifically recognizes the phosphorylated form of CREB (pCREB). Young rats exhibited better performance than old rats with respect to the freezing time in CFC. Phosphorylation of CREB as revealed by the ratio of the pCREB-immunoreactive cell number to the CREB-immunoreactive cell number was increased in the CA1 region, but not in other hippocampal regions following training for CFC. The close relationship between behavioral performance and CREB phosphorylation in the CA1 region suggests that hippocampal CREB is involved in age-related decline of learning and memory.

Phoneme classification for speech synthesiser using differential EMG signals between muscles.

This paper proposes the use of differential electromyography (EMG) signals between muscles for phoneme classification, with which a Japanese speech synthesiser system can be constructed using fewer electrodes. In distinction from traditional methods using differential EMG signals between bipolar electrodes on the same muscle, an EMG signal is derived as differential between monopolar signals on two different muscles in the proposed method. Then, frequency-based feature patterns are extracted with filter banks, and classification of phonemes is realized by using a probabilistic neural network, which combines feature reduction and pattern classification processes in a single network structure. Experimental results show that the proposed method can achieve considerably high classification performance with fewer electrodes.