Kenkichi Takase

Sex differences in the basolateral amygdala: the extracellular levels of serotonin and dopamine, and their responses to restraint stress in rats

The sex difference in the emotional response to stress suggests a sex‐specific stress response in the amygdala. To examine the sex difference in extracellular levels of serotonin (5HT) and dopamine (DA) in the basolateral amygdala (BLA) and their responses to restraint stress, in vivo microdialysis studies were performed in male and female rats. In experiment I, dialysates were collected from the BLA at 15‐min intervals under the freely moving condition. Mean extracellular levels of 5HT or DA in the BLA were higher in male rats than in female rats. In experiment II, rats were subjected to restraint stress for 60 min to examine the stress response of 5HT or DA levels. Although restraint stress significantly increased extracellular 5HT levels in both sexes of rats, female rats showed a greater response than male rats. Moreover, restraint stress significantly increased extracellular DA levels in female rats, but not in male rats. In experiment III, rats were subjected to restraint stress for 30 min to examine behavioral responses to restraint stress. Although no sex difference was observed in the number of audible vocalizations, male rats defecated a larger number of fecal pellets than female rats. In experiment IV, rats were tested for freezing behavior to examine contextual fear responses. Conditioned male rats showed a longer freezing time than conditioned female rats. We found sex differences in the extracellular levels of 5HT and DA in the BLA and their responses to restraint stress, which may be involved in the sex‐specific emotional response to stress in rats.

Gonadal Steroids Maintain 24 h Acetylcholine Release in the Hippocampus: Organizational and Activational Effects in Behaving Rats

Extracellular acetylcholine (ACh) levels in the dorsal hippocampus increases during learning or exploration, exhibiting a sex-specific 24 h release profile. To examine the activational effect of gonadal steroid hormones on the sex-specific ACh levels and its correlation with spontaneous locomotor activity, we observed these parameters simultaneously for 24 h. Gonadectomy severely attenuated the ACh levels, whereas the testosterone replacement in gonadectomized males or 17β-estradiol replacement in gonadectomized females successfully restored the levels. 17β-Estradiol-priming in gonadectomized males could not restore the ACh levels, and testosterone replacement in gonadectomized females failed to raise ACh levels to those seen in testosterone-primed gonadectomized males, revealing a sex-specific activational effect. Spontaneous locomotor activity was not changed in males by gonadectomy or the replacement of gonadal steroids, but 17β-estradiol enhanced the activity in gonadectomized females. Gonadectomy severely reduced the correlation between ACh release and activity levels, but the testosterone replacement in gonadectomized males or 17β-estradiol replacement in gonadectomized females successfully restored it. To further analyze the sex-specific effect of gonadal steroids, we examined the organizational effect of gonadal steroids on the ACh release in female rats. Neonatal testosterone or 17β-estradiol treatment not only increased the ACh levels but also altered them to resemble male-specific ACh release properties without affecting levels of spontaneous locomotor activity. We conclude that the activational effects of gonadal steroids maintaining the ACh levels and the high correlation with spontaneous locomotor activity are sex-specific, and that the organizational effects of gonadal steroids suggest estrogen receptor-mediated masculinization of the septo-hippocampal cholinergic system.

Disrupted cortical function underlies behavior dysfunction due to social isolation

Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.

Activational and Organisational Effects of Gonadal Steroids on Sex-Specific Acetylcholine Release in the Dorsal Hippocampus

Acetylcholine (ACh) release in the dorsal hippocampus increases during stress, exploration or learning, exhibiting sex‐specific 24‐h release profile. We review the role of gonadal steroids on the ACh release in the dorsal hippocampus. In our studies, we found that male rats showed higher extracellular ACh levels than females, but gonadectomy decreased ACh levels in both sexes of rats and subsequently eliminated the sex difference. To examine the sex difference under comparable gonadal steroid levels, we implanted steroid capsules after gonadectomy. Oestradiol supplementation maintained circulating oestradiol to the levels in proestrous female rats, whereas testosterone capsules maintained circulating testosterone to the levels similar to intact male rats. Under comparable gonadal steroids levels, ACh levels were sex‐specific. Testosterone replacement in orchidectomised rats clearly restored ACh levels, which were greater than ovariectomised testosterone‐primed rats. Similarly, oestradiol replacement in ovariectomised rats successfully restored ACh levels, which were higher than orchidectomised oestradiol‐primed rats. These results suggest sex‐specific activational effects of gonadal steroids on ACh release. To further examine the organisational effect, female pups were neonatally treated with oil, testosterone, oestradiol, or dihydrotestosterone. These rats were bilaterally ovariectomised and a testosterone capsule was implanted at postnatal week 8. Neonatal treatment of either testosterone or oestradiol clearly increased ACh levels, whereas neonatal dihydrotestosterone treatment failed to change levels. These results suggest that: (i) gonadal steroids maintain the sex‐specific ACh release in the dorsal hippocampus and (ii) neonatal activation of oestrogen receptors is sufficient to mediate masculinisation of the septo‐hippocampal cholinergic system.

Monoaminergic and Neuropeptidergic Neurons Have Distinct Expression Profiles of Histone Deacetylases

Monoaminergic and neuropeptidergic neurons regulate a wide variety of behaviors, such as feeding, sleep/wakefulness behavior, stress response, addiction, and social behavior. These neurons form neural circuits to integrate different modalities of behavioral and environmental factors, such as stress, maternal care, and feeding conditions. One possible mechanism for integrating environmental factors through the monoaminergic and neuropeptidergic neurons is through the epigenetic regulation of gene expression via altered acetylation of histones. Histone deacetylases (HDACs) play an important role in altering behavior in response to environmental factors. Despite increasing attention and the versatile roles of HDACs in a variety of brain functions and disorders, no reports have detailed the localization of the HDACs in the monoaminergic and neuropeptidergic neurons. Here, we examined the expression profile of the HDAC protein family from HDAC1 to HDAC11 in corticotropin-releasing hormone, oxytocin, vasopressin, agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), orexin, histamine, dopamine, serotonin, and noradrenaline neurons. Immunoreactivities for HDAC1,-2,-3,-5,-6,-7,-9, and -11 were very similar among the monoaminergic and neuropeptidergic neurons, while the HDAC4, -8, and -10 immunoreactivities were clearly different among neuronal groups. HDAC10 expression was found in AgRP neurons, POMC neurons, dopamine neurons and noradrenaline neurons but not in other neuronal groups. HDAC8 immunoreactivity was detected in the cytoplasm of almost all histamine neurons with a pericellular pattern but not in other neuropeptidergic and monoaminergic neurons. Thus, the differential expression of HDACs in monoaminergic and neuropeptidergic neurons may be crucial for the maintenance of biological characteristics and may be altered in response to environmental factors.

Proteomic Identification of Protein Targets for 15-Deoxy-Δ12,14-Prostaglandin J2 in Neuronal Plasma Membrane

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimers disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Aβ. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.

Cytotoxicity of troglitazone through PPARγ-independent pathway and p38 MAPK pathway in renal cell carcinoma

Agonists of peroxisome proliferator-activated receptor gamma (PPARγ) have been examined as chemopreventive and chemotherapeutic agents. The aim was to investigate the cytotoxicity of troglitazone (TGZ) and its mechanisms in terms of PPARγ dependency and the p38 mitogen-activated protein kinase (MAPK) pathway in three human renal cell carcinoma (RCC) cell lines, 786-O, Caki-2 and ACHN cells. TGZ induced apoptosis and exerted cytotoxicity in a PPARγ-independent manner. We demonstrated that TGZ activated the p38 MAPK pathway and was involved in the cytotoxicity of TGZ. It was also revealed that TGZ induced G(2)/M cell cycle arrest through activation of p38 MAPK.

Sex-specific 24-h acetylcholine release profile in the medial prefrontal cortex: Simultaneous measurement of spontaneous locomotor activity in behaving rats

The difference in visual object recognition by males and females suggests a sex-specific function in the medial prefrontal cortex (mPFC). In the present study, we performed an in vivo microdialysis study in three groups of rats (males, diestrous females, and proestrous females) to examine the potential sex difference in acetylcholine (ACh) release in the mPFC. The dialysate was automatically collected from the mPFC every 20 min for 24 h under freely moving conditions and the spontaneous locomotor activity was simultaneously monitored. Although ACh release in the mPFC during the dark phase was significantly greater than during the light phase in both sexes, the female rats consistently exhibited a significantly greater mean ACh release than the males. Spontaneous locomotor activity during the dark phase was also significantly greater than during the light phase in both sexes, but the females exhibited significantly greater spontaneous locomotor activity than the males. In addition, both sexes of rats were found to have significant positive correlations between ACh release and spontaneous locomotor activity, but females were found to have significantly greater correlation coefficients than males. Stereological methods were used to examine the number of choline acetyltransferase immunoreactive cells in the nucleus basalis magnocellularis and the horizontal diagonal band of Broca. The number of choline acetyltransferase immunoreactive cells in the nucleus basalis magnocellularis was also greater in females than males, suggesting a contribution to the higher ACh release in females. In contrast, no sex difference in the choline acetyltransferase immunoreactive cells was observed in the horizontal diagonal band of Broca. This is the first report to show a sex difference in the 24-h ACh release profile in the mPFC of behaving rats.

Maternal deprivation in the middle of a stress hyporesponsive period decreases hippocampal calcineurin expression and causes abnormal social and cognitive behaviours in adult male Wistar rats: Relevance to negative symptoms of schizophrenia

Adverse experiences in early life profoundly influence the developing nervous, endocrine, and immune systems, and also affect human behaviour during adult life and are considered in the pathogenesis of psychiatric disorders. Numerous studies have provided evidence that maternal deprivation in the middle of a stress hyporesponsive period (SHRP) causes multiple behavioural and physiological abnormalities that mimic positive symptoms of schizophrenia in humans. To investigate the neurochemical characteristics of maternal deprivation in the middle of the SHRP in the context of a possible animal model of the symptoms of schizophrenia, we examined calcineurin expression in the hippocampus of maternally deprived rats. To investigate other behavioural characteristics, we behaviourally phenotyped the rats by applying a comprehensive behavioural test battery. The results indicate that maternal deprivation in the middle of the SHRP has no effects on general health, neurological reflexes, sensory function, or motor function, but does have sex-specific effects on a type of anxiety-related behaviour in the open field test and male-specific effects on hippocampal calcineurin expression, social behaviour, and objective memory function. An interpretation of our results and previous studies in the context of the neurodevelopmental hypothesis of schizophrenia suggests that maternal deprivation in the middle of the SHRP in rats models some positive and negative aspects of schizophrenia. The findings regarding the sex-specific effects of maternal deprivation in the middle of the SHRP may become a strong tool for investigating sex differences in the pathogenesis and pathology of schizophrenia in humans.

Sex difference in the 24-h acetylcholine release profile in the premotor/supplementary motor area of behaving rats

The sex differences in various motor functions suggest a sex-specific neural basis in the nonprimary or primary motor area. To examine the sex difference in the 24-h profile of acetylcholine (ACh) release in the rostral frontal cortex area 2 (rFr2), which is equivalent to the premotor/supplementary motor area in primates, we performed an in vivo microdialysis study in both sexes of rats fed pelleted or powdered diet. The dialysate was automatically collected from the rFr2 for 24 h under freely moving conditions. Moreover, the number of cholinergic neurons in the nucleus basalis magnocellularis (NBM) was examined. Further, to confirm the relation between ACh release in the rFr2 and motor function, the spontaneous locomotor activity was monitored for 24 h. Both sexes showed a distinct 24-h rhythm of ACh release, which was high during the dark phase and low during the light phase. Female rats, however, showed a greater ACh release and more cholinergic neurons in the NBM than male rats. Similarly, spontaneous locomotor activity also showed a 24-h rhythm, which paralleled the changes in ACh release in both sexes, and these changes were again greater in female rats than in male rats. In addition, feeding with powdered diet significantly increased the ACh release and spontaneous locomotor activity. The present study is the first to report the sex difference in the 24-h profile of ACh release in the rFr2 in rats. The sex specific ACh release in the rFr2 may partly contribute to the sex difference in motor function in rats.