Tomiko Yakura

Short-term exposure to ethanol causes a differential response between nerve growth factor and brain-derived neurotrophic factor ligand/receptor systems in the mouse cerebellum.

Alcohol ingestion affects both neuropsychological and motor functions. We hypothesized that one of the key factors involved in such functions are neurotrophins and their receptors. We have therefore examined the effects of short-term ethanol exposure on the mRNA expression and protein levels of neurotrophin ligands and receptors in the cerebellum using real-time RT-PCR and Western blotting techniques. Male BALB/C mice were fed a liquid diet containing 5% (v/v) ethanol. The pair-fed control mice were fed an identical liquid diet except that sucrose was substituted isocalorically for ethanol. The cerebellum of mice exhibiting intoxication signs of stage 1 or 2 were used in the present study. We found that exposure to ethanol resulted in elevated levels of nerve growth factor (NGF) and TrkA mRNA expression but a decreased level of brain-derived neurotrophic factor (BDNF) mRNA expression. The expression of TrkB and p73 mRNA was unchanged. Changes in the level of these proteins were found to mirror these mRNA expression levels. We conclude that exposure to ethanol for a short period can cause a differential responsive in the various neurotrophin ligand/receptor systems. The functional consequences of these changes are unknown at present.

Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats.

AIMS This study was carried out to examine the effects of early postnatal maternal separation stress on the development of the cerebral cortex with respect to time-dependent fluctuations of neurotrophic factor ligand and receptor expression. MAIN METHODS Wistar rats were separated from their mothers for 3h per day during postnatal days (PND) 10 to 15. The cerebral cortex was analyzed by real-time RT-PCR for the evaluation of the expression of mRNA for brain-derived neurotrophic factor (BDNF), TrkB, insulin-like growth factor-1 (IGF-1), and type 1 IGF receptor (IGF-1R) on PND16, 20, 30, and 60. KEY FINDINGS The expression of these neurotrophic factor ligands and receptors in the cerebral cortex was enhanced on PND16 and PND20, and then it returned to baseline levels on PND30. By PND60, however, the expression levels were attenuated. SIGNIFICANCE The important implication of this study is the persistent abnormal fluctuation of neurotrophic factor expression for a prolonged period, triggered even after the brain growth spurt. Given that neurotrophic factors play important roles in brain development, it can be speculated that the altered expression of these factors induced by maternal separation may interrupt normal brain development and ultimately lead to functional disruption. However, the possibility of such changes leading to various functional disruptions and the underlying mechanisms involved require further study.

Prolonged maternal separation disturbs the serotonergic system during early brain development.

Early life stress interrupts brain development through the disturbance of various neurotransmitter and neurotrophic factor activities, but the details remain unclear. In the current study, we focused on the serotonergic system, which plays a critical role in brain development, and examined the time‐dependent influence of prolonged maternal separation on male Sprague‐Dawley rats. The rats were separated from their dams for 3 h twice‐daily during postnatal days (PDs) 2–20. The influence of prolonged maternal separation was analyzed on PDs 7, 14, 21, and 28 using HPLC to assess concentrations of serotonin and 5‐hydroxyindoleacetic acid and using real‐time RT‐PCR to measure mRNA expression of the serotonin 1A and 2A receptors in various brain regions. HPLC revealed imbalance between serotonin and 5‐hydroxyindoleacetic acid in midbrain raphe nuclei, the amygdala, the hippocampus, and the medial prefrontal cortex (mPFC) on PDs 7 and 14. Furthermore, real‐time RT‐PCR showed attenuation of mRNA expression of the serotonin 1A receptor in the hippocampus and the mPFC and of the serotonin 2A receptor only in the mPFC on PDs 7 and 14. The observed alterations returned to control levels after maternal separation ended. These findings suggest that the early life stress of prolonged maternal separation disturbs the serotonergic system during a crucial period of brain development, which might in part be responsible for emotional abnormalities later in life.

Early postnatal repeated maternal deprivation causes a transient increase in OMpg and BDNF in rat cerebellum suggesting precocious myelination

Repetitive maternal deprivation (MD) of neonatal rats during early life is known as one of the strongest stressors to pre-weaned animals. There is increasing evidence that the cerebellum is involved in cognition and emotion. In the present study, we examined how neurotrophic factors and myelin-associated molecules and their receptors (NGF, BDNF, OMgp, TrkA, TrkB, p75 NTR, and NgR) in the cerebellum are affected by early postnatal maternal separation. Rat pups were separated from their mothers for 3h/day during postnatal days (PND) 10-15. At PND 16 and 30, the levels of mRNA and protein in the cerebellum were determined using real-time PCR and Western blot analysis. Cerebellar mRNA and protein levels of BDNF, TrkB, and OMgp were significantly increased in MD rats at PND 16. However, by PND 30 these variables normalized to control levels. In contrast, the levels of mRNA and protein for NGF, TrkA, p75 NTR, and NgR were unchanged at both ages examined. Transient enhancement of neurotrophic system and myelin-associated molecule expression may cause interference of normal development of the cerebellum such as precocious myelination, which may lead to functional and cognitive deficits later in life.

Early postnatal maternal separation causes alterations in the expression of β3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity

The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague-Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved by separating the rat pups from their mothers for 3h each day during the 10-15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), β3-adrenergic receptor (β3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through β3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the β3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.

Anterograde Synaptic Transport of Neuronal Tracer Enzyme (WGA-HRP): Further Studies with Rab3A-siRNA in Rats

Neuronal tracer enzyme of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was employed to elucidate the detailed morphology of anterograde synaptic transport. After injection of WGA-HRP into the vagus nerve, sensory terminals in the nucleus of solitary tract (NST) were observed at the electron microscopic level using the tetramethyl benzidine and diaminobenzidine methods. In neuropil of the NST, electron-dense HRP-reaction product (HRP-RP) showed various types of lysosomallike structures. The RP characterized by containing membranous substance crossed synapses forming a mass without membrane surrounding the RP. Additionally, phenomena of anterograde synaptic transport of the RP and exocytosis of synaptic vesicles never occurred simultaneously. These findings raised the possibility of inducing synaptic transport of WGA-HRP at the stage of no neurotransmitter release, i.e. no activation of neuron. Therefore, further experiments were performed after co-injection of Rab3A-siRNA with WGA-HRP into the vagus nerve. This co-injection frequently resulted in not only suppression of vesicle trafficking to active zones and docking to pre-synaptic membranes but also abnormal aggregation of synaptic vesicles at terminals. Furthermore, synaptic transport of WGA-HRP, including secretion, followed by endocytosis of post-synaptic neurons was better seen in the experiments.

Diacrine- and apocrine-like secretions from the hypothalamo–pituitary terminals in the rat

amplitude of task-related power changes of EEG during motor imagery was recorded to assess the downstream modulation of primary sensorimotor cortex. Our result depicts clear gating of both N20 and N30 components by motor imagery. Inter-subject analysis with healthy nine participants revealed that amplitude of N30 gating was correlated with the amplitude of alpha/beta band (8–35 Hz) EEG attenuation. This result indicates that alpha/beta band EEG attenuation during motor imagery represents the binding process of kinesthetic sensory and motor information, and thus is influenced by supplementary motor area or/and premotor cortex, known as possible actors of N30. Research fund: the Ministry of Education, Culture, Sports, Science and Technology Strategic Research Program for Brain Science (H19-24).

Neuronal transcytosis of protein: Application for Amyloid-β in vivo

The synthesizing enzyme of acetylcholine, the choline acetyltransferase (ChAT) has two isoforms. In rat, immunoreactivity for the preferentially observed isoform in the peripheral nervous system, the peripheral ChAT (pChAT), was observed in enteric nervous system and also in neurons of the dorsal root ganglion (DRG). The purpose of the present study is to examine pChAT immunohistochemical staining of these two structures in the human. Human specimens, large or small intestine and DRG, obtained within 24 h postmortem at autopsy in Shiga University of Medical Science, were fixed by immersion in 4% paraformaldehyde in 0.1M phosphate buffer for 4 days at 4 ◦C, and then cryoprotected. Free-floating cryostat sections of 24 m in thickness were used for immunohistochemistry using an avidin-biotinperoxidase complex method. In human intestines and DRG, the pattern of distribution of pChAT immunostaining was similar to the pattern observed previously in rat. Positive staining was exclusively localized in neuronal cell bodies and fibers. In human large intestine, for instance, pChAT-positive somata were observed in the submucosal and intramural ganglionated plexuses. Networks of positive nerve fibers were found within the submucosa, the longitudinal and the circular muscle; varicoses could be observed. Co-localisation of pChAT staining with other cholinergic markers, the high affinity choline transporter and the vesicular acetylcholine transporter, was observed in nerves fibers. In human DRG, all neuronal somata appeared immunoreactive though varying in staining intensity. Small neurons were often stained very intensely, while medium-sized to large neurons exhibited moderate staining. Immunoreactive fibers were observed throughout the ganglion and extending toward the dorsal root. This study demonstrates a pChAT-like immunoreactivity in the human peripheral nervous system.

Anterograde synaptic transport of neuronal tracer enzyme (WGA-HRP): approach of Rab3A-siRNA injection into the vagus nerve in the rat

Most nerve terminals are small, thus electrical activity may easily change their ionic compositions. Moreover, synaptic transmission is easily modulated by ionic compositions within the terminal where some molecular machinery is present to maintain ionic balances in such as Na+, K+, Ca2+ and Cl-. However, exact roles of the molecular machinery are not well understood. We therefore investigated the effects of dihydro-ouabain, an antagonist of Na+/K+ ATPase (NKAs), on synaptic transmission in the nucleus magnocellularis in slice preparation of the chick. We found that the block of NKAs significantly reduced quantal content without affecting release probability. The results suggest that the presynaptic activity of NKAs is necessary for the maintenance of readily releasable pool size.