Masaki Kakeyama

Schema-Dependent Gene Activation and Memory Encoding in Neocortex

New hippocampal-dependent learning is in parallel consolidated with existing memories in the neocortex. When new learning occurs against the background of established prior knowledge, relevant new information can be assimilated into a schema and thereby expand the knowledge base. An animal model of this important component of memory consolidation reveals that systems memory consolidation can be very fast. In experiments with rats, we found that the hippocampal-dependent learning of new paired associates is associated with a striking up-regulation of immediate early genes in the prelimbic region of the medial prefrontal cortex, and that pharmacological interventions targeted at that area can prevent both new learning and the recall of remotely and even recently consolidated information. These findings challenge the concept of distinct fast (hippocampal) and slow (cortical) learning systems, and shed new light on the neural mechanisms of memory assimilation into schemas.

Upregulation of colonic luminal polyamines produced by intestinal microbiota delays senescence in mice

Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.

Executive Function Deficits and Social-Behavioral Abnormality in Mice Exposed to a Low Dose of Dioxin In Utero and via Lactation

An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.

Changes in expression of NMDA receptor subunit mRNA by perinatal exposure to dioxin

Since dioxin and related compounds are suspected of affecting permanently the brain function of offspring of human and experimental animals, effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of rat NMDA receptor NR2A and NR2B subunit mRNA were examined. The mRNA quantification by competitive RT-PCR clearly revealed that TCDD inhibited NR2B mRNA expression and enhanced NR2A mRNA expression in the neocortex and hippocampus on postnatal day (PND) 49, whereas these changes in mRNA expression were not found on PND 5. The results demonstrate for the first time that the perinatal exposure to TCDD can alter the molecular basis of brain of offspring in adulthood.

Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes.

Recent studies have suggested that astrocytes release gliotransmitters (i.e., ATP, L-glutamate, D-serine, and peptide hormones) and participate actively in synaptic functioning. Although ATP release from astrocytes modulates the activity of neurons, the mechanisms regulating the ATP release from astrocytes and the source of ATP in astrocytes are not well understood. Recently a vesicular nucleotide transporter (VNUT)/solute carrier family 17, member 9 (SLC17A9) has been identified as a mediator of the active accumulation of ATP into vesicles. Here we show by immunocytochemical analysis under confocal microscope and live cell imaging under total internal reflection fluorescence microscope that lysosome-associated VNUT is responsible for ATP release in astrocytes. VNUT was expressed in both primary cultured cortical astrocytes and glioma cell line C6 cells, and mainly localized on lysosome in the cells. We found that VNUT-associated secretory lysosomes do not fully collapse into the plasma membrane after lysosomal exocytosis. We also found that inhibition of VNUT function by Evans Blue decreased ATP uptake into secretory lysosomes. Depletion and inhibition of endogenous VNUT by small interference RNA and Evans Blue, respectively decreased the amount of ATP release from the cells, whereas overexpression of VNUT increased it. Taken together, these findings indicate that the participation of VNUT in ATP storage in secretory lysosomes during lysosomal exocytosis of ATP from astrocytes.

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters activity-dependent expression of BDNF mRNA in the neocortex and male rat sexual behavior in adulthood

Dioxin and its related compounds are suspected to cause neurological and nueroendocrinological disruption in human and laboratory animal offspring upon in utero and lactational exposure during growth and development. We tested the hypothesis by utilizing Long-Evans Hooded rats that perinatal exposure to dioxins affects the neocortical function and expression of sexual behavior in adulthood. In the sexual behavior test, perinatal exposure to TCDD significantly reduced the number of mounts and intromissions. The mRNA semi-quantification in in situ hybridization showed that the mating stimulus in control males induced c-fos mRNA expression in the preoptic area (POA) and the brain derived neurotrophic factor (BDNF) mRNA upregulation in the frontal cortex. In contrast, perinatal exposure to TCDD lowered the upregulation of BDNF mRNA in the frontal cortex but not that of c-fos mRNA in the POA. The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was not affected. The results suggest that perinatal TCDD affects the neocortical function independently from the brain sexual differentiation and alters the expression of sexual behavior.

In utero and lactational exposure to low doses of chlorinated and brominated dioxins induces deficits in the fear memory of male mice

Environmental-level in utero and lactational exposures to dioxins have been considered to affect brain functions of offspring. Here, we determined whether in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD), at the dose that does not harm the dams, affects the acquisition and retention of fear memory in mouse offspring. Pregnant C57BL/6J mice were administered by gavages TCDD or TBDD at a dose of 0 or 3.0 microg/kg body weight on gestation day 12.5, and their male offspring were examined for their behavior in adulthood. In the fear conditioning, a paired presentation of tone and foot shock was repeated three times, and retention tests for contextual and auditory fear memory were carried out 1 and 24h after the fear conditioning. Groups of mice that were exposed to TCDD and TBDD in utero and via lactation showed deficits in the contextual and auditory retention tests at 1 and 24h retention intervals. The present results suggest that maternal exposure to a low dose of TCDD or TBDD disrupts the functions of memory and emotion in male mouse offspring, and that the developmental toxicities of these chemicals are similar to each other.

Inhibitory effect of baclofen on lordosis in female and male rats with dorsal raphe nucleus lesion or septal cut

The inhibitory effect of baclofen, a GABAB receptor agonist, on lordosis was examined in female and male rats with dorsal raphe nucleus lesions (DRL) or cut of the septal fibers (ARD). Both female and male DRL and ARD rats showed higher lordosis quotients (LQ) than corresponding controls without brain surgery. This indicates that the dorsal raphe nucleus and the septum exert lordosis-inhibiting influences in female and male rats. After treatment with 10 mg/kg baclofen, the mean LQs in female control and female ARD groups were significantly lower than those of vehicle-treated control and ARD females. In DRL females, however, LQs did not decrease, even after the injection of baclofen. In males, baclofen also diminished lordosis in ARD rats, but not in DRL rats. These results suggest that the GABAB receptor system plays an inhibitory role in regulating lordosis behavior not only in female but also in male rats. Furthermore, the function of the GABA neurons depends on the inhibitory mechanism of the dorsal raphe nucleus in the regulation of female sexual behavior.

Lordosis in male rats: The facilitatory effect of mesencephalic dorsal raphe nucleus lesion

The dorsal raphe nucleus was destroyed (DRL) by a radiofrequency lesion generator in castrated male rats, and lordosis behavior was observed. Five weeks after the surgery, all animals were implanted with two Silastic tubes containing estradiol (E2). Behavioral tests were started 2 days after implantation of E2 and carried out every other day for 16 days. Castrated control and sham-operated control males showed low incidence of lordosis and low lordosis quotient (LQ) throughout the tests, whereas all the DRL males displayed lordosis and the mean LQ of the group was significantly higher than that of control groups, but lower than that of an ovariectomized female group. These results suggested that the dorsal raphe nucleus plays an important role in inhibiting lordosis in male rat brain.

Perinatal exposure of female rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces central precocious puberty in the offspring

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during the fetal and neonatal periods has been indicated to alter the development of the offspring later in life. In this study, we determined whether perinatal exposure to a low dose of TCDD affects the onset of puberty in the female offspring of Long-Evans hooded rats. On day 15 of gestation, pregnant female rats were administered TCDD by gavage at a single dose of 0 (vehicle), 200, or 800 ng/kg b.w. In the female offspring born to dams administered with TCDD at either 200 or 800 ng/kg b.w., the vaginal opening and first estrus occurred approximately 4-7 days earlier than in the offspring born to vehicle-treated animals. The ovarian weight gain was also accelerated following exposure to TCDD in a dose-dependent manner. We next examined the ovarian compensatory hypertrophy (OCH) as an indicator of the maturation of the LH/GnRH-generating system in the pituitary and the hypothalamus. Exposure to TCDD accelerated the onset of OCH in the female offspring in a dose-dependent manner. In particular, in the offspring born to the dams exposed to TCDD at 800 ng/kg b.w., hypertrophy, which is characterized by hyperovulation and a marked increase in the weight of the remaining ovary after hemi-ovariectomy, was observed on postnatal days 27-30, which was 10 days earlier than in the offspring born to the vehicle-treated dams. These results indicate that perinatal exposure to a low dose of TCDD induces precocious puberty, including early maturation of the hypothalamic-pituitary axis, the gonads and genitals, in female Long-Evans hooded rats.