Long-term depression-inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor.

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca2+ condition. Confocal calcium imaging revealed that dendritic Ca2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

Adult-onset hypothyroidism causes mechanical hypersensitivity due to peripheral nerve hyperexcitability based on voltage-gated potassium channel downregulation in male mice.

Thyroid hormones play an important role in the central and peripheral nervous system functions. Approximately 50% of adult-onset hypothyroid patients have sensory symptoms including pain, possibly caused by peripheral neuropathy. However, the mechanism causing the pain has not been clarified. We generated an adult-onset hypothyroid model animal by administering 50 ppm propylthiouracil (PTU) for 5 weeks to male mice. Female mice were not tested in this study. Mechanical hypersensitivity, determined by the von Frey hair test, was observed during the PTU exposure and recovered after the exposure termination. The sciatic nerve compound action potential was also analyzed. Under single-pulse stimulation, no significant change in the threshold and conduction velocity was observed in the PTU-administered group. On the other hand, under train-pulse stimulation, the latency delay in the Aδ-fiber component was less in the PTU-administered group in Week 4 of PTU exposure, indicating relative hyperexcitability. Fluticasone, which is the anti-inflammatory agent with an ability to activate the voltage-gated potassium channel subfamily A (Kv1), restored the decrease in the latency change ratio by PTU exposure under the train-pulse stimulation supporting our hypothesis that Kv1 may be involved in the conductivity change. Kv1.1 protein level decreased significantly in the sciatic nerve of the PTU-administered group. These results indicate that adult-onset hypothyroidism causes mechanical hypersensitivity owing to hyperexcitability of the peripheral nerve and that reduction of Kv1.1 level may be involved in such alteration.

Maternal prolactin during late pregnancy is important in generating nurturing behavior in the offspring.

Although maternal nurturing behavior is extremely important for the preservation of a species, our knowledge of the biological underpinnings of these behaviors is insufficient. Here we show that the degree of a mother's nurturing behavior is regulated by factors present during her own fetal development. We found that Cin85-deficient (Cin85-/-) mother mice had reduced pituitary hormone prolactin (PRL) secretion as a result of excessive dopamine signaling in the brain. Their offspring matured normally and produced their own pups; however, nurturing behaviors such as pup retrieval and nursing were strongly inhibited. Surprisingly, when WT embryos were transplanted into the fallopian tubes of Cin85-/- mice, they also exhibited inhibited nurturing behavior as adults. Conversely, when Cin85-/- embryos were transplanted into the fallopian tubes of WT mice, the resultant pups exhibited normal nurturing behaviors as adults. When PRL was administered to Cin85-/- mice during late pregnancy, a higher proportion of the resultant pups exhibited nurturing behaviors as adults. This correlates with our findings that neural circuitry associated with nurturing behaviors was less active in pups born to Cin85-/- mothers, but PRL administration to mothers restored neural activity to normal levels. These results suggest that the prenatal period is extremely important in determining the expression of nurturing behaviors in the subsequent generation, and that maternal PRL is one of the critical factors for expression. In conclusion, perinatally secreted maternal PRL affects the expression of nurturing behaviors not only in a mother, but also in her pups when they have reached adulthood.

Critical role of the astrocyte for functional remodeling in contralateral hemisphere of somatosensory cortex after stroke.

After ischemic stroke, the corresponding area contralateral to the lesion may partly compensate for the loss of function. We previously reported the remodeling of neuronal circuits in the contralateral somatosensory cortex (SSC) during the first week after infarction for processing bilateral information, resulting in functional compensation. However, the underlying processes in the contralateral hemisphere after stroke have not yet been fully elucidated. Recent studies have shown that astrocytes may play critical roles in synaptic reorganization and functional compensation after a stroke. Thus, we aim to clarify the contribution of astrocytes using a rodent stroke model. In vivo calcium imaging showed a significantly large number of astrocytes in the contralateral SSC responding to ipsilateral limb stimulation at the first week after infarction. Simultaneously, extracellular glutamine level increased, indicating the involvement of astrocytes in the conversion of glutamate to glutamine, which may be an important process for functional recovery. This hypothesis was supported further by the observation that application of (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy} aspartate, a glial glutamate transporter blocker, disturbed the functional recovery. These findings indicate the involvement of astrocytes in functional remodeling/recovery in the area contralateral to the lesion. Our study has provided new insights into the mechanisms underlying synaptic remodeling after cerebral infarction, which contributes to the development of effective therapeutic approaches for patients after a stroke.

Early-life-stress affects the homeostasis of glutamatergic synapses.

Early-life stress induces several neuropsychological disorders in adulthood, including depression. Such disorders may be induced by functional alteration of the glutamatergic system. However, their underlying mechanisms have not yet been fully clarified. Furthermore, the involvement of glucocorticoids, which are representative stress hormones, has not yet been fully clarified. In this study, we used maternal deprivation (MD) mice as an early-life-stress model, and studied the changes in the glutamatergic system in adulthood. The glutamate concentration and neuronal activity in the somatosensory cortex (SSC) increased under basal conditions in MD mice. Stressful physical stimulation (SPS) increased the concentration of corticosterone, but not of glutamate, in the control mouse SSC. On the other hand, in the MD mice, although the basal concentration of corticosterone in the SSC increased, no SPS-induced increase was observed. In contrast, the concentration of glutamate increased greatly during SPS. It was significantly high for 30 min after stimulation. The expression level of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/N-methyl-d-aspartate receptors in the MD mice was also changed compared with that in the control mice after stimulation. These findings indicate that early-life stress disrupts the homeostasis of glutamatergic synapses.

Lactational exposure to hydroxylated polychlorinated biphenyl (OH-PCB 106) causes hyperactivity in male rat pups by aberrant increase in dopamine and its receptor.

Polychlorinated biphenyls (PCBs) are recognized as persistent environmental pollutants that may cause adverse health problems. Despite extensive investigations of PCB in neural function, little is known about behavioral traits by PCB exposure and its neurochemical mechanism. Here, we report the behavioral study of a rat pup that was exposed to hydroxylated-PCB 106 (OH-PCB 106; 4-hydroxy-2',3,3',4',5'-pentachlorobiphenyl) through maternal milk. The different groups of mothers received via gavage corn oil vehicle, 0.5, 5, or 50 mg/kg body weight of OH-PCB 106 every second day from day 3 to 13 after delivery. The exposure did not affect the body weight of the dams or the physical development of the newborn pups in both sexes. Male rats exposed to OH-PCB 106 showed hyperactivity that was characterized by increased locomotor activity in novel environment and circadian period. Interestingly, OH-PCB 106-exposed rat pups displayed abnormally high levels of dopamine and D2 dopamine receptor (D2DR), but not D1DR and D5DR, in the striatum, an important center for the coordination of behavior. These findings indicate that OH-PCB 106 has a significant neurotoxic effect on rat behavior, which may be associated with increased D2DR mediated signals.

External negative electric potential accelerates exocytosis of lamellar bodies in human skin ex vivo.

Exocytosis of lamellar bodies at the uppermost nucleated layer of the epidermis is a crucial process for epidermal permeability barrier homoeostasis. We have previously suggested that skin surface electric potential might be associated with barrier homoeostasis. Thus, we hypothesized that the potential might drive exocytosis of lamellar bodies. In this study, we tested this idea by applying negative electric potential (-0.5 V) to human skin samples ex vivo for 2 h and observing the ultrastructure of the uppermost layer. The secretion of lamellar bodies was accelerated in the potential-applied skin, compared to that in untreated control skin. Multiphoton observation indicated that extracellular lipid domains were more extensive in treated skin than in control skin. Moreover, the calcium ion gradient was greater at the uppermost layer of the epidermis of treated skin, compared to that in control skin. These results indicate that electric potential may regulate lamellar body secretion in healthy human skin.

Frontal medial cortex and angular gyrus functional connectivity is related to sex and age differences in odor sensitivity.

BACKGROUND AND PURPOSE: Odor preference is one of the key factors for the rehabilitation of the swallowing function. On the other hand, sensitivity to odor differs between sexes and decreases with age. These factors rely on brain neuronal circuits. However, it remains not fully clarified which neuronal circuit determines the sex and age differences in odor sensitivity. In this study, we carried out both the odor sensitivity test and functional MRI (fMRI) to find the key neuronal circuits determining sex and age differences in odor sensitivity. METHODS: Healthy volunteers (28 males, aged 27-62 years, and 30 females, aged 21-59 years) participated in this study. Some of them (seven males and seven females) underwent fMRI. We prepared five odorous test substances and presented each substance at 1 minute intervals. After 5 minutes of questioning about food intake, the subjects were asked to recall each of the test substances presented from the list. In the fMRI study, all the subjects underwent 15 minutes of the prestimulation, stimulation with peppermint odor, and poststimulation sessions. RESULTS: The odor test score was significantly higher in females than in males and showed an age-dependent decrease. We found four functional connectivities whose degrees were significantly different between males and females. One of them, the functional connectivity between the frontal medial cortex (MedFC) and the left angular gyrus (AG. l), showed an age-dependent change. CONCLUSIONS: The functional MedFC-AG.l connectivity is one of the important neuronal circuits that affect the sex- and age-dependent odor sensitivity.

Absence of Thyroid Hormone Induced Delayed Dendritic Arborization in Mouse Primary Hippocampal Neurons Through Insufficient Expression of Brain-Derived Neurotrophic Factor.

Thyroid hormone (TH) plays important roles in the developing brain. TH deficiency in early life leads to severe developmental impairment in the hippocampus. However, the mechanisms of TH action in the developing hippocampus are still largely unknown. In this study, we generated 3,5,3'-tri-iodo-l-thyronine (T3)-free neuronal supplement, based on the composition of neuronal supplement 21 (NS21), to examine the effect of TH in the developing hippocampus using primary cultured neurons. Effects of TH on neurons were compared between cultures in this T3-free culture medium (-T3 group) and a medium in which T3 was added (+T3 group). Morphometric analysis and RT-qPCR were performed on 7, 10, and 14 days in vitro (DIV). On 10 DIV, a decreased dendrite arborization in -T3 group was observed. Such difference was not observed on 7 and 14 DIV. Brain-derived neurotrophic factor (Bdnf) mRNA levels also decreased significantly in -T3 group on 10 DIV. We then confirmed protein levels of phosphorylated neurotrophic tyrosine kinase type 2 (NTRK2, TRKB), which is a receptor for BDNF, on 10 DIV by immunocytochemistry and Western blot analysis. Phosphorylated NTRK2 levels significantly decreased in -T3 group compared to +T3 group on 10 DIV. Considering the role of BDNF on neurodevelopment, we examined its involvement by adding BDNF on 8 and 9 DIV. Addition of 10 ng/ml BDNF recovered the suppressed dendrite arborization induced by T3 deficiency on 10 DIV. We show that the lack of TH induces a developmental delay in primary hippocampal neurons, likely caused through a decreased Bdnf expression. Thus, BDNF may play a role in TH-regulated dendritogenesis.

Neuronal circuit remodeling in the contralateral cortical hemisphere during functional recovery from cerebral infarction.

Recent advances in functional imaging of human brain activity in stroke patients, e.g., functional magnetic resonance imaging, have revealed that cortical hemisphere contralateral to the infarction plays an important role in the recovery process. However, underlying mechanisms occurring in contralateral hemisphere during functional recovery have not been elucidated. We experimentally induced a complete infarction of somatosensory cortex in right hemisphere of mice and examined the neuronal changes in contralateral (left) somatosensory cortex during recovery. Both basal and ipsilateral somatosensory stimuli-evoked neuronal activity in left (intact) hemisphere transiently increased 2 d after stroke, followed by an increase in the turnover rate of usually stable mushroom-type synaptic spines at 1 week, observed by using two-photon imaging in vivo. At 4 weeks after stroke, when functional recovery had occurred, a new pattern of electrical circuit activity in response to somatosensory stimuli was established in intact ipsilateral hemisphere. Thus, the left somatosensory cortex can compensate for the loss of the right somatosensory cortex by remodeling neuronal circuits and establishing new sensory processing. This finding could contribute to establish the effective clinical treatments targeted on the intact hemisphere for the recovery of impaired functions and to achieve better quality of life of patients.

Neurotoxic effects of lactational exposure to perfluorooctane sulfonate on learning and memory in adult male mouse.

The present study aims to examine the effect of early lactational perfluorooctane sulfonate (PFOS) exposures on learning and memory in male mice and reveal the underlying mechanisms involved. PFOS solution was orally administered to dams from the postpartum days 1-14, so that pups would be exposed through breast milk. At 8-10 weeks of age, we performed object location test (OLT), object recognition test (ORT), and pairwise visual discrimination (VD) task. We also performed in vivo microdialysis, and mRNA and protein analysis of genes responsible for hippocampal development and function. In both OLT and ORT, the performance of mice in the PFOS-exposed group was significantly lower than those in the control group. In the VD task, the PFOS-exposed group learned significantly slower than the control group. Concentrations of glutamate and gamma-aminobutyric acid in the dorsal hippocampus were significantly higher in the PFOS-exposed group than in the control group. No notable differences were shown in our mRNA and protein studies. The present study showed that lactational PFOS exposure has a profound, long-lasting neurotoxic effect in the hippocampus and consequently leads to learning and memory deficits.

PDGFR-ß restores blood-brain barrier functions in a mouse model of focal cerebral ischemia.

Although platelet-derived growth factor receptor beta (PDGFR-ß) mediates the recruitment of vascular pericytes into ischemic lesion to restore the blood-brain barrier (BBB) dysfunction, its mechanisms still remain elusive. Compared with control PDGFR-ßfloxed/floxed mice (Floxed), postnatally induced systemic PDGFR-ß knockout mice (Esr-KO) not only showed severe brain edema, neurologic functional deficits, decreased expression of tight junction (TJ) proteins, abundant endothelial transcytosis, and deformed TJs in the BBB, but also showed reduced expression of transforming growth factor-ß (TGF-ß) protein after photothrombotic middle cerebral artery occlusion (MCAO). In endothelial-pericyte co-culture, an in vitro model of BBB, the increment in the barrier function of endothelial monolayer induced by pericyte co-culture was completely cancelled by silencing PDGFR-ß gene expression in pericytes, and was additively improved by PDGFR-ß and TGF-ß receptor signals under hypoxia condition. Exogenous PDGF-BB increased the expression of p-Smad2/3, while anti-TGF-ß1 antibody at least partially inhibited the phosphorylation of Smad2/3 after PDGF-BB treatment in vitro. Furthermore, pre-administration of TGF-ß1 partially alleviated edema formation, neurologic dysfunction, and TJs reduction in Esr-KO mice after MCAO. Accordingly, PDGFR-ß signalling, via TGF-ß signalling, may be crucial for restoration of BBB integrity after cerebral ischemia and therefore represents a novel potential therapeutic target.

Sustained depolarizing shift of the GABA reversal potential by glutamate receptor activation in hippocampal neurons.

The inhibitory action of GABA is a consequence of a relatively hyperpolarized Cl(-) reversal potential (E(Cl)), which results from the activity of K(+)-Cl(-) cotransporter (KCC2). In this study we investigated the effects of glutamate and glutamatergic synaptic activity on E(Cl). In dissociated culture of mature hippocampal neurons, the application of glutamate caused positive E(Cl) shifts with two distinct temporal components. Following a large transient depolarizing state, the sustained depolarizing state (E(Cl)-sustained) lasted more than 30 min. The E(Cl)-sustained disappeared in the absence of external Ca(2+) during glutamate application and was blocked by both AP5 and MK801, but not by nifedipine. The E(Cl)-sustained was also induced by NMDA. The E(Cl)-sustained was blocked by furosemide, a blocker of both KCC2 and NKCC1, but not bumetanide, a blocker of NKCC1. On the other hand, in immature neurons having less expression of KCC2, NMDA failed to induce the sustained depolarizing E(Cl) shift. In organotypic slice cultured neurons, repetitive activation of glutamatergic afferents also generated a sustained depolarizing E(Cl) shift. These results suggest that Ca(2+) influx through NMDA receptors causes the down-regulation of KCC2 and gives rise to long lasting positive E(Cl) shifts, which might contribute to hyperexcitability, LTP, and epileptiform discharges.

High prolactin concentration during lactation period induced disorders of maternal behavioral in offspring.

Early-life stress during the perinatal period induces several neuropsychological disorders in adulthood. In animal studies, early-life stress during the perinatal period induces not only behavioral disorders but also other neurofunctional disorders, such as somatosensory functional disorder in adulthood. Furthermore, the offspring of an early-life-stressed parent also show disturbance of brain function in humans. Behavioral and neurological alterations in the offspring of a stressed parent have also been shown in animal studies. However, the mechanisms underlying such behavioral/neurological alterations are not yet fully understood. In this study, we found a disorder of maternal behavior in the offspring of early-life-stressed mothers. The stressed mothers showed high concentrations of serum prolactin (PRL) during pregnancy and lactation. The concentration on the day of weaning the offspring significantly correlated with the changes in the concentration of corticosterone and the neurological function of offspring. These findings indicate that PRL may be involved in the induction of transgenerational effects of early-life stress on the brain function of offspring. In addition, maternal PRL can be a good biomarker for predicting the potential risk of neurofunctional alterations in the offspring.

Role of dopamine on functional recovery in the contralateral hemisphere after focal stroke in the somatosensory cortex.

Functional recovery after a stroke is important for patients' quality of life. Not only medical care during the acute phase, but also rehabilitation during the chronic phase after a stroke is important. However, the mechanisms underlying functional recovery, particularly the chronic phase after stroke, are still not fully understood. Thus, further basic study on brain after focal stroke is necessary. In this study, we found that the concentration of dopamine (DA) increased during first week after a stroke in the hemisphere contralateral in the site of stroke by in vivo microdialysis. When we applied haloperidol (HPD), a potent DA receptor blocker, functional recovery was inhibited. Interestingly, administration of aripiprazole (ARP), a novel partial agonist of the DA receptor, during the chronic phase improved the remodeling of neuronal circuits in somatosensory cortex (SSC). These findings indicate that the DAergic system play a critical role in functional compensation by the non-infarcted hemisphere after a focal stroke in SSC. It is also revealed that administration of HPD/ARP to stroke patients affects functional recovery after a stroke, and stimulation of the DAergic system during the chronic phase of stroke potentially benefits stroke patients.

Early-life stress induces motor coordination dysfunction in adult mice.

Although child abuse has become a serious social problem in most countries, the neural mechanisms by which it induces adulthood mental disorders is not yet fully understood. Mice exposed to early-life stresses, such as maternal deprivation (MD) during lactation, are a good model for studying the effects of neglect of humans in early life. Early-life stress induces structural/functional changes of neurons in the hippocampus, prefrontal cortex, and amygdala, and causes mental disorders in adulthood. In this study, we found motor coordination dysfunction in male MD mice. We also found that the expression levels of the aminomethylphosphonic acid receptor subunits GluA1 and GluA3 were high in the cerebellum of male MD mice. The basal activity of the cerebellum detected by field-potential analysis was higher in male MD mice than in male control mice. Caloric stimulation increased the activity of the cerebellum of control mice, but it did not significantly increase the activity of the cerebellum in male MD mice. We concluded that early-life stress induced a functional change in the cerebellum of MD mice and that this change induced motor coordination dysfunctions.

Effects of Mild Perinatal Hypothyroidism on Cognitive Function of Adult Male Offspring.

Mild perinatal hypothyroidism may result from inadequate iodine intake, insufficient treatment of congenital hypothyroidism, or exposure to endocrine-disrupting chemicals. Because thyroid hormones are critical for brain development, severe hypothyroidism that is untreated in infancy causes irreversible cretinism. Milder hypothyroidism may also affect cognitive development; however, the effects of mild and/or moderate hypothyroidism on brain development are not fully understood. In this study, we examined the behavior of adult male mice rendered mildly hypothyroid during the perinatal period using low-dose propylthiouracil (PTU). PTU was administered through drinking water (5 or 50 ppm) from gestational day 14 to postnatal day 21. Cognitive performance, studied by an object in-location test (OLT), was impaired in PTU-treated mice at postnatal week 8. These results suggest that, although the hypothyroidism was mild, it partially impaired cognitive function. We next measured the concentration of neurotransmitters (glutamate, γ-aminobutyric acid, and glycine) in the hippocampus using in vivo microdialysis during OLT. The concentrations of neurotransmitters, particularly glutamate and glycine, decreased in PTU-treated mice. The expression levels of N-methyl-d-aspartate receptor subunits, which are profound regulators of glutamate neurotransmission and memory function, also were decreased in PTU-treated mice. These data indicate that mild perinatal hypothyroidism causes cognitive disorders in adult offspring. Such disorders may be partially induced secondary to decreased concentrations of neurotransmitters and receptor expression.

Early-life stress induces cognitive disorder in middle-aged mice.

Early-life stress can induce several neuropsychological disorders in adulthood. However, the underlying mechanisms inducing such disorders are still not fully understood. Furthermore, the effects of early-life stress on the changes in cognitive function with age are still not clarified. In this study, we used maternal deprivation (MD) to examine the cognitive function in middle-aged mice using a touchscreen-equipped operant chamber. In the visual-discrimination task, the aged (∼1.4 years old) control mice could accurately learn to discriminate between different visual stimuli. In contrast, the correct response rate of aged MD mice increased to ∼60% by day 10; it was still significantly lower than that of the control mice (85%). In the hippocampus of aged MD mice, the expression level of the N-methyl-d-aspartate receptor subunit GluN1 decreased significantly as compared to that in control mice. On the other hand, no significant difference in GluN1 expression level was detected in young (2.5 months old) mice. These findings indicate that early-life stress accelerates cognitive impairment in middle-aged mice.

In Utero and Postnatal Propylthiouracil-Induced Mild Hypothyroidism Impairs Maternal Behavior in Mice.

Thyroid hormones (THs) play crucial roles in general and brain development. Even if the hypothyroidism is mild, it may alter brain function, resulting in irreversible behavioral alterations. Although various behavioral analyses have been conducted, the effects of propylthiouracil (PTU) treatment during in utero and postnatal periods on maternal behavior have not yet been studied. The present study examined in mice whether THs insufficiency during development induce behavioral changes. Pregnant C57BL/6j mice were divided into three groups, and each group was administered different dosages of PTU (0, 5, or 50 ppm) in drinking water during in utero and postnatal periods (from gestational day 14 to postnatal day 21). First, locomotor activity and cognitive function were assessed in the offspring at 10 weeks. Next, female offspring were mated with normal mice and they and their offspring were used to assess several aspects of maternal behavior (identifying first pup, returning all pups to nest, time spent nursing, and licking pups). As expected, locomotor and cognitive functions in these mice were disrupted in a PTU dose-dependent manner. On postpartum day 2, dams who had been exposed 50 ppm PTU during in utero and postnatal periods displayed a significantly longer time identifying the first pup and returning all three pups back to the nest, less time nursing, and decreased licking behavior. The decrease in maternal behavior was significantly correlated with a decrease in cognition. These results indicate that insufficiency of THs during in utero and postnatal periods impairs maternal behavior, which may be partly induced by impaired cognitive function.