Search

A disintegrin and metalloproteinase 10 (ADAM10) plays an essential role in the regulation of survival, proliferation, migration, and differentiation of various neural cells. Nevertheless, the role of ADAM10 in oligodendrocyte precursors (OPCs) and myelination in the central nervous system (CNS) of developing and adult mouse brains is still unknown. We generated ADAM10 conditional knockout (ADAM10 cKO) mice lacking the ADAM10 gene primarily in OPCs by crossing NG2-Cre mice with ADAM10 loxp/loxp mice. We found that OPCs expressed ADAM10 in the mouse corpus callosum and the hippocampus. ADAM10 cKO mice showed significant loss of back hair and reduction in weight and length on postnatal (30 ± 2.1) day, died at (65 ± 5) days after birth and exhibited the “anxiety and depression-like” performances. Conditional knockout of ADAM10 in OPCs resulted in a prominent increase in myelination and a decrease in the number of OPCs in the corpus callosum at P30 owing to premyelination and lack of proliferation of OPCs. Moreover, the number of proliferating OPCs and mature oligodendrocytes (OLs) also decreased with age in the corpus callosum of ADAM10 cKO mice from P30 to P60. Western blot and RT-PCR results showed that the activation of Notch-1 and its four target genes, Hes1, Hes5, Hey1, and Hey2, was inhibited in the corpus callosum tissue of ADAM10 knockout mice. In our study, we provided experimental evidence to demonstrate that ADAM10 is essential for modulating CNS myelination and OPC development by activating Notch-1 signalling in the developing and adult mouse brain.

Social defeat stress (SDS) plays a major role in the pathogenesis of psychiatric disorders like anxiety and depression. Sleep is generally considered to involve recovery of the brain from prior experience during wakefulness and is altered after acute SDS. However, the effect of acute SDS on sleep/wake behavior in mice varies between studies. In addition, whether sleep changes in response to stress contribute to anxiety is not well established. Here, we first investigated the effects of acute SDS on sleep/wake states in the active period in mice. Our results showed that total sleep time (time in rapid eye-movement [REM] and non-REM [NREM] sleep) increased in the active period after acute SDS. NREM sleep increased mainly during the first 3 h after SDS, while REM sleep increased at a later time. Then, we demonstrated that the increased NREM sleep had an anxiolytic benefit in acute SDS. Mice deprived of sleep for 1 h or 3 h after acute SDS remained in a highly anxious state, while in mice with ad libitum sleep the anxiety rapidly faded away. Altogether, our findings suggest an anxiolytic effect of NREM sleep, and indicate a potential therapeutic strategy for anxiety.

Microglia constantly survey the brain microenvironment and rapidly adopt different phenotypes in response to environmental stimuli. Such dynamic functions require a unique metabolism and bioenergetics. However, little is known about the basic metabolism of microglia and how metabolic changes regulate microglia function. Here, we uncover that microglia activation is accompanied by extensive transcriptional changes in glucose and lipid metabolism-related genes. Using metabolic flux assays, we found that LPS, a prototype of the pathogen-associated molecular patterns (PAMPs), significantly enhanced glycolysis but suppressed oxidative phosphorylation (OXPHOS) in primary cultured microglia. By contrast, ATP, a known damage-associated molecular pattern (DAMPs) that triggers sterile activation of microglia, boosted both glycolysis and OXPHOS. Importantly, both LPS and ATP activated the mechanistic target of rapamycin (mTOR) pathway and enhanced the intracellular reactive oxygen species (ROS). Inhibition of mTOR activity suppressed glycolysis and ROS production in both conditions but exerted different effects on OXPHOS: it attenuated the ATP-induced elevation of OXPHOS, yet had no impact on the LPS-induced suppression of OXPHOS. Further, inhibition of mTOR or glycolysis decreased production of LPS-induced proinflammatory cytokines and ATP-induced tumor necrosis factor-α (TNF-α) and brain derived neurotrophic factor (BDNF) in microglia. Our study reveals a critical role for mTOR in the regulation of metabolic programming of microglia to shape their distinct functions under different states and shed light on the potential application of targeting metabolism to interfere with microglia-mediated neuroinflammation in multiple disorders.

Summary: Overeating is a serious issue in modern society, causing many health problems, including obesity. Although the hypothalamus has been previously identified as the key brain structure that regulates body weight homeostasis, the downstream pathways and non-canonical neural circuitry involved in feeding behavior remain largely uncharacterized. Here, we discover that suppressing the activity of GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG), whether directly or through long-projection GABAergic inputs from either the bed nucleus of the stria terminalis (BNST) or the lateral hypothalamus (LH), is sufficient to promptly induce feeding behavior in well-fed mice. In contrast, optogenetic activation of these cells interrupts food intake in starved mice. Long-term chemogenetic manipulation of vlPAG GABAergic cell activity elicits a corresponding change in mouse body weight. Our studies reveal distinct midbrain GABAergic pathways and highlight an important role of GABAergic cells in the anterior vlPAG in feeding behavior. : Hao et al. uncovered an important role of the GABAergic cells in the anterior ventrolateral periaqueductal gray (vlPAG) and two distinct midbrain GABAergic pathways (the lateral hypothalamus-vlPAG and BNST-vlPAG) in regulation of feeding behavior. Keywords: feeding, vlPAG, GABAergic pathway, lateral hypothalamus, mice

Protease cathD escapes from late endosomes to cytosol and accelerates actin dynamics., Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome–derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate–evoked microglia migration.

Cathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP(+)) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP(+) cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP(+) cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP(+) cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP(+) cells mainly generated NeuN(+) neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP(+) cells at the young adult stage is significantly different from that at the adult stage.

The main lysosomal protease cathepsin D (cathD) is essential for maintaining tissue homeostasis via its degradative function, and its loss leads to ceroid accumulation in the mammalian nervous system, which results in progressive neurodegeneration. Increasing evidence implies non-proteolytic roles of cathD in regulating various biological processes such as apoptosis, cell proliferation, and migration. Along these lines, we here showed that cathD is required for modulating dendritic architecture in the nervous system independent of its traditional degradative function. Upon cathD depletion, class I and class III arborization (da) neurons in Drosophila larvae exhibited aberrant dendritic morphology, including over-branching, aberrant turning, and elongation defects. Re-introduction of wild-type cathD or its proteolytically-inactive mutant dramatically abolished these morphological defects. Moreover, cathD knockdown also led to dendritic defects in the adult mushroom bodies, suggesting that cathD-mediated processes are required in both the peripheral and central nervous systems. Taken together, our results demonstrate a critical role of cathD in shaping dendritic architecture independent of its proteolytic function. Electronic supplementary material The online version of this article (10.1007/s12264-020-00479-6) contains supplementary material, which is available to authorized users.

The laterodorsal tegmentum (LDT) is a brain structure involved in distinct behaviors including arousal, reward, and innate fear. How environmental stimuli and top-down control from high-order sensory and limbic cortical areas converge and coordinate in this region to modulate diverse behavioral outputs remains unclear. Using a modified rabies virus, we applied monosynaptic retrograde tracing to the whole brain to examine the LDT cell type specific upstream nuclei. The LDT received very strong midbrain and hindbrain afferents and moderate cortical and hypothalamic innervation but weak connections to the thalamus. The main projection neurons from cortical areas were restricted to the limbic lobe, including the ventral orbital cortex (VO), prelimbic, and cingulate cortices. Although different cell populations received qualitatively similar inputs, primarily via afferents from the periaqueductal gray area, superior colliculus, and the LDT itself, parvalbumin-positive (PV(+)) GABAergic cells received preferential projections from local LDT neurons. With regard to the different subtypes of GABAergic cells, a considerable number of nuclei, including those of the ventral tegmental area, central amygdaloid nucleus, and VO, made significantly greater inputs to somatostatin-positive cells than to PV(+) cells. Diverse inputs to the LDT on a system-wide level were revealed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00397-2) contains supplementary material, which is available to authorized users.

The globus pallidus (GP) is emerging as a critical locus of basal ganglia control of motor activity, but the exact role of GABAergic GP neurons remain to be defined. By targeted expression of channelrhodopsin 2 (ChR2) in GABAergic neurons using the VGAT-ChR2-EYFP transgenic mice, we showed that optogenetic stimulation of GABAergic neurons in the right GP produced hyperkinesia. Optogenetic stimulation of GABAergic GP neurons increased c-Fos-positive cells in GP, M1 cortex, and caudate-putamen (CPu), and decreased c-Fos-positive cells in entopeduncular nucleus (EPN), compared to the contralateral hemisphere. In agreement with the canonical basal ganglia model. Furthermore, we delivered AAV-CaMKIIα-ChR2-mCherry virus to the excitatory neurons of the subthalamic nucleus (STN) and selectively stimulated glutamatergic afferent fibers from the STN onto the GP. This optogenetic stimulation produced abnormal movements, similar to the behaviors that observed in the VGAT-ChR2-EYFP transgenic mice. Meanwhile, we found that the c-Fos expression pattern in the GP, M1, STN, EPN, and CPu produced by optogenetic activation of glutamatergic afferent fibers from the STN in GP was similar to the c-Fos expression pattern in the VGAT-ChR2-EYFP transgenic mice. Taken together, our results suggest that excess GP GABAergic neurons activity could be the neural substrate of abnormal involuntary movements in hyperkinetic movement disorders. The neural circuitry underlying the abnormal involuntary movements is associated with excessive GP, M1, CPu activity, and reduced EPN activity. Inhibition of GP GABAergic neurons represents new treatment targets for hyperkinetic movement disorder.

Endocytosis is a basic cellular process that describes a form of active transport across the plasma membrane into the cell. The endocytic pathway consists of distinct membrane compartments; internalized molecules are delivered to early endosomes, and some of them are recycled back to the surface, whereas other molecules are sent to late endosomes and lysosomes for degradation. However, little is known about how mitochondria are involved in the endocytic pathway. Here, we report that FM dyes, membrane-impermeant fluorescent lipid probes, can traffic to mitochondria directly from the plasma membrane by clathrin-mediated endocytosis. FM dye entry into mitochondria uses microtubule-dependent active transport, but the mechanism is different from the classical endocytic pathway. Hence, this study reveals a previously unrealized lipid trafficking pathway from the plasma membrane to mitochondria.

Innate fear has a critical role in survival of animals. Unlike conditioned fear, the neuronal circuitry underlying innate fear is largely unknown. We found that the laterodorsal tegmentum (LDT) and lateral habenula (LHb) are specifically activated by the mouse predator odorant trimethylthiazoline (TMT). Using optogenetics to selectively stimulate GABAergic neurons in the LDT immediately produced fear-like responses (freezing, accelerated heart rate and increased serum corticosterone), whereas prolonged stimulation caused anxiety-like behaviors. Notably, although selective stimulation of parvalbumin (PV)-positive interneurons similarly induced fear-like responses, stimulation of somatostatin-positive interneurons or inhibition of PV neurons in the LDT suppressed TMT-induced fear-like responses without affecting conditioned fear. Finally, activation of LHb glutamatergic inputs to LDT interneurons was sufficient to generate fear-like responses. Thus, the LHb-LDT pathway is important for regulating olfactory cue-induced innate fear. Our results provide a potential target for therapeutic intervention for anxiety disorder.

Intracellular Ca(2+) elevation resulting from different Ca(2+) pathways may play different roles in astrocyte functions. Channelrhodopsin-2 (ChR2), a light-gated cation channel, has been used to selectively stimulate astrocytes by inducing intracellular Ca(2+) ([Ca(2+)]i) elevation, but the exact underlying mechanism is still unclear. We found that in the absence of extracellular Ca(2+), light stimulation failed to induce [Ca(2+)]i elevation in astrocytes expressed ChR2. Pharmacological experiments excluded the involvement of Ca(2+)-induced Ca(2+) release from intracellular stores. Further experiments demonstrated that the ChR2-induced [Ca(2+)]i elevation was mainly mediated by reversal of the Na(+)-Ca(2+) exchanger following Na(+) influx through ChR2 channels. Since intracellular Na(+) homeostasis plays important roles in astrocytes, including the modulation of [Ca(2+)]i, neurotransmitter uptake and cell metabolism, our results indicate that ChR2 is a good candidate which could be used for mimicking the intracellular Na(+) disturbance in astrocytes that occurs in various physiological and pathological processes including the uptake of neurotransmitters and ischemia, as well as the activities of various cation channels, ion exchangers, and pumps.

Background: Eplerenone (CAS 107724-20-9) is the first highly selective aldosterone receptor blocker and is used worldwide for treatment of hypertension and heart failure. Objective: The objective of this study was to investigate the eplerenone pharmacokinetics in healthy Chinese subjects and assess the dose proportionality over the therapeutic dose range. Methods: A single-dose, randomized, 6-sequence, 3-treatment, 3-period crossover, open label study was conducted in 12 healthy Chinese subjects, who received 3 doses of eplerenone in random order (25, 50, 100 mg). The power model was used to evaluate the dose proportionality of eplerenone. The pharmacokinetic study of multiple-dose of eplerenone was also conducted. Results: After single-dose oral administration, the mean C max value increased from 489 to 1 641 ng/mL, and the mean AUC 0-t value increased from 3 030 to 10 893 ng/mL·h with an increase in dose from 25 to 100 mg, respectively. The mean value for terminal T 1/2 was approximate 3 h with no significant differences among different dose groups. Though dose proportionality of eplerenone was inconclusive in Chinese subjects over the dose range of 25–100 mg, the maximal proportionality dose range (ρ 1 ) was 2.06 based on power model. Steady state could achieve within at least 4 days and no accumulation was observed after multiple-dose of eplerenone. Conclusion: Dose proportionality was inconclusive in over the dose range of 25–100 mg; however, linear pharmacokinetics could be considered when dose ratio is no more than 2.06.

Microglia are the resident immune cells in the CNS and play diverse roles in the maintenance of CNS homeostasis. Recent studies have shown that microglia continually survey the CNS microenvironment and scavenge cell debris and aberrant proteins by phagocytosis and pinocytosis, and that reactive microglia are capable to present antigens to T cells and initiate immune responses. However, how microglia process the endocytosed contents and evoke an immune response remain unclear. Here we report that a size-dependent selective transport of small soluble contents from the pinosomal lumen into lysosomes is critical for the antigen processing in microglia. Using fluorescent probes and water-soluble magnetic nanobeads of defined sizes, we showed in cultured rodent microglia, and in a cell-free reconstructed system that pinocytosed proteins become degraded immediately following pinocytosis and the resulting peptides are selectively delivered to major histocompatibility complex class II (MHC-II) containing lysosomes, whereas undegraded proteins are retained in the pinosomal lumen. This early size-based sorting of pinosomal contents relied on the formation of transient tunnel between pinosomes and lysosomes in a Rab7- and dynamin II-dependent manner, which allowed the small contents to pass through but restricted large ones. Inhibition of the size-based sorting markedly reduced proliferation and cytokine release of cocultured CD4+T cells, indicating that the size-based sorting is required for efficient antigen presentation by microglial cells. Together, these findings reveal a novel early sorting mechanism for pinosomal luminal contents in microglial cells, which may explain how microglia efficiently process protein antigens and evoke an immune response.

Summary Presynaptic endosomes reportedly participate in synaptic vesicle (SV) recycling. However, it remains unclear whether they differentially regulate SV biogenesis and synaptic transmission in different types of synapses and how they are implicated in diseases. Using cryo-electron tomography and endocytic tracing, we uncover different endocytic modes and dynamics associated with distinct SV morphology between glutamatergic and GABAergic synapses. We further find that cathepsin D (CatD), a lysosomal storage disease (LSD) protein, is selectively located in GABAergic presynaptic endosomes. Inactivation of CatD results in enlarged presynaptic endosomes, reduces the readily releasable pool, and impairs synaptic transmission in GABAergic, but not glutamatergic, synapses. Moreover, CatD-deficient mice exhibit hyperactivity and increased sensitivity to seizure, mimicking epileptic behavior in CatD-related LSD patients. These data reveal an important role for presynaptic endosomal CatD in regulating GABAergic SV biogenesis and provide mechanistic insights for understanding the synaptic pathology and behavioral defects in CatD-associated LSD.

MgTiO 3 nanoplatelets were successfully synthesized by combining the sol–gel method with the molten salt method at low temperatures. The results show that the calcined process of the mixture sample (Mg–Ti gel and the eutectic salt NaCl–KCl at a molar ratio of 1:10) consists of a series of oxidation and combustion reactions in air. High purity MgTiO 3 without other crystal phase can be obtained over 600 °C for 2 h. MgTiO 3 synthesized at 700 and 800 °C is regular in shape with crystal size of about 200 nm in diameter and 50 nm in thickness. The present work offers us a new route to synthesize well-dispersed MgTiO 3 nanoplatelets with regular crystal morphology.

Huntington’s disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder caused by an unstable expansion of CAG repeats (>35 repeats) within exon 1 of the interesting transcript 15 (IT15) gene. This gene encodes a protein called Huntingtin (Htt), and mutation of the gene results in a polyglutamine (polyQ) near the N-terminus of Htt. The N-terminal fragments of mutant Htt (mHtt), which tend to aggregate, are suffi cient to cause HD. Whether these aggregates are causal or protective for HD remains hotly debated. Dysfunctional mitochondrial axonal transport is associated with HD. It remains unknown whether the soluble or aggregated form of mHtt is the primary cause of the impaired mitochondrial axonal transport in HD pathology. Here, we investigated the impact of soluble and aggregated N-terminal fragments of mHtt on mitochondrial axonal transport in cultured hippocampal neurons. We found that the N-terminal fragment of mHtt formed aggregates in almost half of the transfected neurons. Overexpression of the N-terminal fragment of mHtt decreased the velocity of mitochondrial axonal transport and mitochondrial mobility in neurons regardless of whether aggregates were formed. However, the impairment of mitochondrial axonal transport in neurons expressing the soluble and aggregated N-terminal fragments of mHtt did not differ. Our fi ndings indicate that both the soluble and aggregated N-terminal fragments of mHtt impair mitochondrial axonal transport in cultured hippocampal neurons. We predict that dysfunction of mitochondrial axonal transport is an early-stage event in the progression of HD, even before mHtt aggregates are formed.

Multipolar-to-bipolar transition (MBT) is crucial for the neuronal migration and positioning in the neocortex. Reelin-Disabled-1 (Dab1) signaling plays a pivotal role in neuronal migration, yet how Dab1 coordinatively regulates downstream molecules to affect MBT remains unclear. We have previously found that alternative splicing produces multiple Dab1 isoforms with different tyrosine motifs and differential ability to recruit downstream effectors. Here, we report that splicing of Dab1 exons 7 and 8 and 9bc dynamically regulates the inclusion and activities of Dab1 tyrosine motifs in the neocortex. By in utero electroporation, we show that expression of Dab1 isoforms missing exons 7 and 8 or retaining exons 9bc in WT neurons resulted in neuronal migration defects with attenuated Dab1 tyrosine phosphorylation, disrupted leading process extension, and disorientated multipolar neurons in the multipolar accumulation zone. Introducing the canonical Dab1 form, but not those missing exons 7 and 8 or retaining exons 9bc, into Dab1-deficient neurons promoted MBT and rescued neuronal migration defects, suggesting that alternative splicing of Dab1 modulates the tyrosine motif switch and mediates MBT of cortical neurons. Our study reveals a critical mechanism by which Dab1 alternative splicing coordinately controls MBT and neuronal migration in a spatiotemporal manner.

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum–Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body–exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.

A specific, sensitive and rapid liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of azithromycin in human plasma. After deproteinizing the plasma sample with methanol, azithromycin and internal standard (IS: roxithromycin) were separated using a mobile phase comprised of acetonitrile : ammonium acetate buffer (50 mM, containing 0.05% acetic acid)=85:15 on a Hypersil GOLD C 18 column (50 mm×2.1 mm ID, dp 1.9 μm). Detection was performed with a tandem mass spectrometer by selective reaction monitoring (SRM) through electrospray ionization. Target ions were monitored at [M+H] + m / z 749.5→591.5 and 837.7→679.5 in positive electrospray ionization (ESI) mode for azithromycin and IS respectively. Linearity was established for the range of concentrations 2–800 ng/mL with a coefficient of correlation ( r ) of 0.9996. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2.0 ng/mL. Both intra- and inter-batch standard deviations were less than 15%. The validated method was successfully applied to study the comparative bioavailability of azithromycin for suspension in test vs. reference in healthy Chinese volunteers through the statistical comparison of pharmacokinetic parameters obtained with the two formulations.

An open-label, single-dose, randomized, crossover study was carried out in 20 Chinese healthy male subjects to compare the pharmacokinetics of 2 cefaclor (CAS 53994-73-3) formulations after administration of a single 250 mg dose of each drug with a 1-week wash-out period. Blood samples were collected before and with 6 h after drug administration. Plasma concentrations were determined by high-performance liquid chromatography (HPLC) with UV detector. 2 formulations were evaluated using the following pharmacokinetic parameters: AUC0-t, Cmax and tmax was analyzed nonparametrically. The 90% confidence interval (CI) of the ratios (teat/reference) of log-transformed AUC0-t and Cmax fell within the bioequivalence acceptance range of 80-125%. The results showed that the 90% CI of the ratios of AUC0-t and Cmax were 105.1% (101.0-109.4%) and 92.4% (82.5-103.4%), respectively, which therefore could conclude 2 oral cefaclor capsule formulations of cefaclor are bioequivalent. Both treatments showed similar tolerability and safety.

A simple and sensitive HPLC method was developed to determine cefdinir (CAS 91832-40-5) in human plasma. The method was validated by investigating the accuracy and precision for intra- and inter-day runs in a linear concentration from 0.05–2.0 µg/ml. The object of this study was to compare the bioavailability of cefdinir capsule (reference) and cefdinir granule (test) containing 100 mg of cefdinir. A randomized, open-label, single-dose, 2-way crossover bioequivalence study in 20 healthy, Chinese, male subjects was conducted. A 1-week wash-out period was applied. Blood samples were collected before and with 10 h after drug administration. The formulations were compared using the following pharmacokinetic parameters: AUC0-t, AUC0-∞ and C max. The 90% confidence interval (CI) of the ratios of log-transformed AUC0-t and AUC0-∞ were used to assess bioequivalence between the 2 formulations using the equivalence interval of 80 and 125%. The results showed that the 90% CI of the ratios of AUC0-t, AUC0-∞ and C max were 102.5% (94.7–111.0%), 103.4% (94.8–112.7%) and 106.4% (97.0–116.7%), respectively, which indicated 2 formulations of cefidinir are bioequivalent. Both treatments showed similar tolerability and safety.

hybridization; Bactericidal permeability-increasing protein; Lipopolysaccharide; Pc-BPI-like gene; Eukaryotic expression Abstract One expressed sequence tag (EST 64LF004 clone), which is from the subtracted cDNA library of the head kidney of large yellow croaker (Pseudosciaena crocea) stimulated with peptidoglycan (PG) by suppression subtractive hybridization (SSH) method, was cloned us- ing RACEePCR. The full length cDNA, which possesses typical structural features of a signal peptide, a conserved LPS binding domain and two bactericidal permeability-increasing (BPI) motifs as in higher vertebrates, was identified as a novel homologue, namely of the large yel- low croaker BPI-like molecule (Pc-BPI-L). Phylogenetic analysis showed this Pc-BPI-L of large yellow croaker as the most ancestral branch in bony fish clade. The recombinant Pc-BPI-L pro- tein expressed in the Tn-5B1-4 insect cells was successfully produced and confirmed to have the predicted size of 52 kDa by Western blot analysis. At the message level, Pc-BPI-L mRNA was ubiquitously expressed in all tissues examined. Following formalin-inactivated Vibrio algi- nolyticus and Nocardia seriolae treatment, Pc-BPI-L message was differentially up-regulated in primary immune organs. These results indicate that Pc-BPI-L might be involved in the im

Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2−/−) mice to specifically disturb somatic Ca2+ signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2−/− mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1−/− mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2−/− mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination. DOI: http://dx.doi.org/10.7554/eLife.15043.001, eLife digest Neighbouring neurons connect to each other and share information through structures known as synapses. As the brain develops, many synapses turn out to be redundant. Just like trees in a garden that need to be trimmed, these redundant synapses must be pruned in order to form the right pattern of connections between different neurons. Brain cells called astrocytes play a key role in synaptic pruning, but it is unclear exactly how astrocytes coordinate this process. One important way in which astrocytes communicate with neurons is through a process called calcium signaling, in which the movement of calcium ions into or out of the cell sets off a cascade of activity inside the astrocytes. Yang et al. have now studied developing mice that lacked a gene that is essential for calcium signaling in astrocytes. Two weeks after they were born, these mice still had redundant synapses that are normally lost after birth. However, injecting the developing brain with a substance called ATP prevented this defect and allowed synapses to be correctly pruned. This is likely to be because astrocytes also use ATP to communicate with neurons, and ATP compensated for the missing calcium signaling. The experiments also uncovered the specific structure – called the P2Y1 receptor – on the outer surface of a neuron that ATP latches on to in order to help remove synapses. Further work is now needed to reveal how activating the P2Y1 receptor coordinates synaptic removal. DOI: http://dx.doi.org/10.7554/eLife.15043.002

Mutations in cathepsin D (CTSD), an aspartic protease in the endosomal-lysosomal system, underlie congenital neuronal ceroid-lipofuscinosis (cNCL, also known as CLN10), a devastating neurodegenerative disease. CLN10 patients die within the first few days of life, and in the few patients who live into adulthood psychopathological symptoms have not been reported. Extensive neuropathology and altered neurotransmission have been reported in CTSD-deficient mice; however signs of neuropsychiatric behavior in these mice are not well characterized due to the severe movement disorder and premature death of the animal. In the present study, we show that heterozygous CTSD-deficient (CTSD HET) mice display an overall behavioral profile that is similar to human mania, including hyperlocomotion, d-amphetamine-induced hyperactivity, sleep-disturbance, and reduced anxiety-like behavior. However, under stressful conditions CTSD HET mice manifest depressive-like behavior, including anhedonia, behavioral despair, and enhanced learned helplessness. Chronic administration of lithium chloride or valproic acid, two clinically effective mood stabilizers, reverses the majority of these behavioral abnormalities. In addition, CTSD HET mice display stress-induced hypersecretion of corticosterone. These findings suggest an important role for CTSD in the regulation of mood stabilization.

Microglial cells have important roles in maintaining brain homeostasis, and they are implicated in multiple brain diseases. There is currently interest in investigating microglial migration that results in cell accumulation at focal sites of injury. Here we describe a protocol for rapidly triggering and monitoring microglial migration by using a micropipette assay. This protocol is an adaptation of the axon turning assay using microglial cells. Chemoattractants released from the micropipette tip produce a chemotactic gradient that induces robust microglial migration. In combination with microscopic imaging, this assay allows simultaneous recording of cell movement and subcellular compartment trafficking, along with quantitative analysis. The actual handling time for the assay takes ∼2-3 h in total. The protocol is simple, inexpensive and convenient to set up, and it can be adopted to examine cell migration in multiple cell types, including cancer cells with a wide range of chemical signals.

Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a "drink me" signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimer's disease.

Corrigendum: Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear

The cerebellum has a conserved foliation pattern and a well-organized layered structure. The process of foliation and lamination begins around birth. beta-catenin is a downstream molecule of Wnt signaling pathway, which plays a critical role in tissue organization. Lack of beta-catenin at early embryonic stages leads to either prenatal or neonatal death, therefore it has been difficult to resolve its role in cerebellar foliation and lamination. Here we used GFAP-Cre to ablate beta-catenin in neuronal cells of the cerebellum after embryonic day 12.5, and found an unexpected role of beta-catenin in determination of the foliation pattern. In the mutant mice, the positions of fissure formation were changed, and the meninges were improperly incorporated into fissures. At later stages, some lobules were formed by Purkinje cells remaining in deep regions of the cerebellum and the laminar structure was dramatically altered. Our results suggest that beta-catenin is critical for cerebellar foliation and lamination. We also found a non cell-autonomous role of beta-catenin in some developmental properties of major cerebellar cell types during specific stages.

Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system. Attracted by factors released from damaged cells, microglia are recruited towards the damaged or infected site, where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris. ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury. However, the mechanisms of the long-range migration of microglia remain to be clarified. Here, we found that lysosomes in microglia contain abundant ATP and exhibit Ca(2+)-dependent exocytosis in response to various stimuli. By establishing an efficient in vitro chemotaxis assay, we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia, a response that was significantly inhibited in microglia treated with an agent inducing lysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice), a small GTPase required for the trafficking and exocytosis of secretory lysosomes. These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis, thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.