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At the nerve terminal, neurotransmitter release is triggered by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs). During postnatal development, VGCC subtypes in the nerve terminal switch at many synapses. In immature rodent cerebella, N-type and P/Q-type VGCCs mediate GABAergic neurotransmission from Purkinje cells (PCs) to deep nuclear cells, but as animals mature, neu-rotransmission becomes entirely P/Q-type dependent. We reproduced this developmental switch in rat cerebellar slice culture to address the underlying mechanism. Chronic block of cerebellar neuronal activity with tetrodotoxin (TTX) in slice culture, or in vivo, reversed the switch, leaving neurotransmission predominantly N-type channel-dependent. Brain-derived neurotrophic factor or neurotrophin-4 rescued this TTX effect, whereas pharmacological blockade of neurotrophin receptors mimicked the TTX effect. In PC somata, unlike in presynaptic terminals, TTX had no effect on the proportion of Ca2+ channel subtype currents. We conclude that neuronal activity activates the neurotrophin-TrkB signaling pathway, thereby causing the N-to-P/Q channel switch in presynaptic terminals. [ABSTRACT FROM AUTHOR]

The c-RET proto-oncogene encodes a receptor-tyrosine kinase. Loss-of-function mutations of RET have been shown to be associated with Hirschsprung disease and Down's syndrome (HSCR-DS) in humans. DS is known to involve cerebellar hypoplasia, which is characterized by reduced cerebellar size. Despite the fact that c-Ret has been shown to be associated with HSCR-DS in humans and to be expressed in Purkinje cells (PCs) in experimental animals, there is limited information about the role of activity of c-Ret/c-RET kinase in cerebellar hypoplasia. We found that a loss-of-function mutation of c-Ret Y1062 in PCs causes cerebellar hypoplasia in c-Ret mutant mice. Wild-type mice had increased phosphorylation of c-Ret in PCs during postnatal development, while c-Ret mutant mice had postnatal hypoplasia of the cerebellum with immature neurite outgrowth in PCs and granule cells (GCs). c-Ret mutant mice also showed decreased numbers of glial fibers and mitogenic sonic hedgehog (Shh)-positive vesicles in the external germinal layer of PCs. c-Ret-mediated cerebellar hypoplasia was rescued by subcutaneous injection of a smoothened agonist (SAG) as well as by reduced expression of Patched1, a negative regulator for Shh. Our results suggest that the loss-of-function mutation of c-Ret Y1062 results in the development of cerebellar hypoplasia via impairment of the Shh-mediated development of GCs and glial fibers in mice with HSCR-DS. [ABSTRACT FROM AUTHOR]

Neurons form dense neural circuits by connecting to each other via synapses and exchange information through synaptic receptors to sustain brain activities. Excitatory postsynapses form and mature on spines composed predominantly of actin, while inhibitory synapses are formed directly on the shafts of dendrites where both actin and microtubules (MTs) are present. Thus, it is the accumulation of specific proteins that characterizes inhibitory synapses. In this study, we explored the mechanisms that enable efficient protein accumulation at inhibitory postsynapse. We found that some inhibitory synapses function to recruit the plus end of MTs. One of the synaptic organizers, Teneurin- 2 (TEN2), tends to localize to such MT- rich synapses and recruits MTs to inhibitory postsynapses via interaction with MT plus- end tracking proteins EBs. This recruitment mechanism provides a platform for the exocytosis of GABAA receptors. These regulatory mechanisms could lead to a better understanding of the pathogenesis of disorders such as schizophrenia and autism, which are caused by excitatory/inhibitory (E/I) imbalances during synaptogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Resistant glucan mixture (RGM), a water-soluble dietary fiber produced by the random polymerization of glucose with activated carbon as a catalyst at a high temperature, has been recently developed by our group. There has been little physiological and safety research into RGM and therefore we now present our research into its safety. A reverse mutation assay indicated that RGM is not mutagenic either with or without metabolic activation. We conducted a 90-day subchronic oral toxicity study in rats. Male and female rats fed either a 3% or 5% w/w RGM diet had no muddy or watery stools, and there was no RGM-related death in any group. Although some parameters in the 3% and 5% w/w groups were significantly different from those in the control group, these changes were not due to any toxicity from RGM. The results indicated that the No Observed Adverse Effect Level (NOAEL) of RGM was 3.3 and 3.9 g/kg body weight (BW) per day in male and female rats, respectively. We then studied the gastrointestinal effects of RGM in healthy adult humans. Gastrointestinal symptoms, such as gurgling sounds, flatus and tenesmus, were mild and transient. In men and women, the maximum no-effect dose for diarrhea was more than 0.9 g RGM /kg BW. The results of our current safety assessment studies suggest that RGM is safe for human consumption. [ABSTRACT FROM AUTHOR]

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ- knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination. [ABSTRACT FROM AUTHOR]

Background White globe appearance (WGA) is a small white lesion with a globular shape identified during magnifying endoscopy with narrow-band imaging. However, the association between WGA and synchronous multiple gastric cancer (SMGC) remains unclear. Methods Consecutive patients who underwent endoscopic submucosal dissection for gastric cancer (GC) between July 2013 and April 2015 at our institution were eligible for this study. We excluded patients with a history of gastric tumor or gastrectomy. Patients who had more than 2 GCs in their postoperative pathological evaluation were classified as SMGC-positive, and patients who had at least 1 WGA-positive GC were classified as WGA-positive patients. The primary outcome was a comparison of the prevalence of WGA in patients classified as SMGCpositive and SMGC-negative. Univariate and multivariate analyses were performed using the following variables: WGA, age, sex, atrophy, and Helicobacter pylori (H. pylori) status. Results There were 26 and 181 patients classified as SMGC-positive and SMGC-negative, respectively. Univariate analysis revealed that WGA-positive classification (50% vs. 23%, P=0.008) and male sex (88% vs. 66%, P=0.02) were significant factors associated with SMGC classification, while age ≥65 years (81% vs. 81%, P>0.99), severe atrophy (46% vs. 46%, P>0.99), and H. pylori positivity (69% vs. 65%, P=0.8) were not. In the multivariate analysis, only WGA-positive classification (odds ratio 2.78, 95% confidence interval 1.16-6.67; P=0.02) was a significant independent risk factor for SMGC. Conclusions Our exploratory study showed the possibility of WGA as a predictive factor for SMGC. In cases of WGA-positive gastric cancer, careful examination might be needed to diagnose SMGC. [ABSTRACT FROM AUTHOR]

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1,2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4 +/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4 1 /ARF5 /mice ofboth sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-damp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of poreforming voltage-dependent NaH channel subunit Navi.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Navi.6 protein localized in the AIS and the consequent tremors in ARF4+/- ARF5 mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class IIARF proteins in ARF4+/-/ARF5 mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Navl.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Navl.6, to the AIS. [ABSTRACT FROM AUTHOR]

Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. TheOBpossesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors. [ABSTRACT FROM AUTHOR]

Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation. [ABSTRACT FROM AUTHOR]

Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination. [ABSTRACT FROM AUTHOR]

Background: Mammalian sires participate in infant care. We previously demonstrated that sires of a strain of nonmonogamous laboratory mice initiate parental retrieval behavior in response to olfactory and auditory signals from the dam during isolation in a new environment. This behavior is rapidly lost in the absence of such signals when the sires are caged alone. The neural circuitry and hormones that control paternal behavior are not wellunderstood. CD38, a membrane glycoprotein, catalyzes synthesis of cyclic ADP-ribose and facilitates oxytocin (OT) secretion due to cyclic ADP-ribose-dependent increases in cytosolic free calcium concentrations in oxytocinergic neurons in the hypothalamus. In this paper, we studied CD38 in the nucleus accumbens (NAcc) and the role of OT on paternal pup retrieval behavior using CD38 knockout (CD38-/-) mice of the ICR strain. Results: CD38-/- sires failed to retrieve when they were reunited with their pups after isolation together with the mate dams, but not with pup, in a novel cage for 10 min. CD38-/- sires treated with a single subcutaneous injection of OT exhibited recovery in the retrieval events when caged with CD38-/- dams treated with OT. We introduced human CD38 in the NAcc of CD38-/- sires using a lentiviral infection technique and examined the effects of local expression of CD38. Pairs of knockout dams treated with OT and sires expressing CD38 in the NAcc showed more retrieval (83% of wild-type sire levels). Complete recovery of retrieval was obtained in sires with the expression of CD38 in the NAcc in combination with OT administration. Other paternal behaviors, including pup grooming, crouching and huddling, were also more common in CD38-/- sires with CD38 expression in the NAcc compared with those in CD38-/- sires without CD38 expression in the NAcc. Conclusions: CD38 in the NAcc and OT are critical in paternal behavior. [ABSTRACT FROM AUTHOR]

Cerebellar Purkinje cells (PCs) express a large amount of the γ isoform of protein kinase C (PKCγ) and a modest level of PKCα. The PKCγ is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKCα plays a critical role in long-term depression (LTD) at parallel fiber (PF)-PC synapses. Moreover, the PKC signaling in PCs negatively modulates the nonselective transient receptor potential cation channel type 3 (TRPC3),the opening of which elicits slow EPSCs at PF-PC synapses. Autosomal dominant spinocerebellar ataxia type 14 (SCA14) is caused by mutations in PKCγ. To clarify the pathology of this disorder, mutant (S119P) PKCγ tagged with GFP was lentivirally expressed in developing and mature mouse PCs in vivo, and the effects were assessed 3 weeks after the injection. Mutant PKCγ-GFP aggregated in PCs without signs of degeneration. Electrophysiology results showed impaired pruning of CF synapses from developing PCs, failure of LTD expression, and increases in slow EPSC amplitude. We also found that mutant PKCγ colocalized with wild-type PKCγ, which suggests that mutant PKCγ acts in a dominant-negative manner on wild-type PKCγ. In contrast, PKCα did not colocalize with mutant PKCγ. The membrane residence time of PKCα after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKCγ construct. These results suggest that mutant PKCγ in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type γ and α PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission. [ABSTRACT FROM AUTHOR]