Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

Time-dependent enhancement of hippocampus-dependent memory after treatment with memantine: Implications for enhanced hippocampal adult neurogenesis.

Adult hippocampal neurogenesis has been suggested to play modulatory roles in learning and memory. Importantly, previous studies have shown that newborn neurons in the adult hippocampus are integrated into the dentate gyrus circuit and are recruited more efficiently into the hippocampal memory trace of mice when they become 3 weeks old. Interestingly, a single high-dose treatment with the N-methyl-d-aspartate receptor antagonist memantine (MEM) has been shown to increase hippocampal neurogenesis dramatically by promoting cell proliferation. In the present study, to understand the impact of increased adult neurogenesis on memory performance, we examined the effects of a single treatment of MEM on hippocampus-dependent memory in mice. Interestingly, mice treated with MEM showed an improvement of hippocampus-dependent spatial and social recognition memories when they were trained and tested at 3-6 weeks, but not at 3 days or 4 months, after treatment with MEM. Importantly, we observed a significant positive correlation between the scores for spatial memory (probe trial in the Morris water maze task) and the number of young mature neurons (3 weeks old) in MEM-treated mice, but not saline-treated mice. We also observed that the young mature neurons generated by treatment with MEM were recruited into the trace of spatial memory similarly to those generated through endogenous neurogenesis. Taken together, our observations suggest that treatment with MEM temporally improves hippocampus-dependent memory formation and that the newborn neurons increased by treatment with MEM contribute to this improvement when they become 3 weeks old.

Towards a better understanding of cognitive behaviors regulated by gene expression downstream of activity-dependent transcription factors.

In the field of molecular and cellular neuroscience, it is not a trivial task to see the forest for the trees, where numerous, and seemingly independent, molecules often work in concert to control critical steps of synaptic plasticity and signalling. Here, we will first summarize our current knowledge on essential activity-dependent transcription factors (TFs) such as CREB, MEF2, Npas4 and SRF, then examine how various transcription cofactors (TcoFs) also contribute to defining the transcriptional outputs during learning and memory. This review finally attempts a provisory synthesis that sheds new light on some of the emerging principles of neuronal circuit dynamics driven by activity-regulated gene transcription to help better understand the intricate relationship between activity-dependent gene expression and cognitive behavior.

Fear learning induces synaptic potentiation between engram neurons in the rat lateral amygdala.

The lateral amygdala (LA) encodes fear memories by potentiating sensory inputs associated with threats and, in the process, recruits 10-30% of its neurons per fear memory engram. However, how the local network within the LA processes this information and whether it also plays a role in storing it are still largely unknown. Here, using ex vivo 12-patch-clamp and in vivo 32-electrode electrophysiological recordings in the LA of fear-conditioned rats, in combination with activity-dependent fluorescent and optogenetic tagging and recall, we identified a sparsely connected network between principal LA neurons that is organized in clusters. Fear conditioning specifically causes potentiation of synaptic connections between learning-recruited neurons. These findings of synaptic plasticity in an autoassociative excitatory network of the LA may suggest a basic principle through which a small number of pyramidal neurons could encode a large number of memories.

APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function.

Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo.

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.

Vitamin D facilitates trophoblast invasion through induction of epithelial-mesenchymal transition.

PROBLEM: Vitamin D deficiency increases the risk of developing pregnancy-related complications, including preeclampsia and small-for-gestational-age infants. Vitamin D was demonstrated to promote the invasiveness of human extravillous trophoblasts (EVTs). However, whether vitamin D induces the epithelial-mesenchymal transition (EMT) of EVTs remains unclear. Therefore, we investigated whether vitamin D promotes EMT and the related signaling pathways. METHOD OF STUDY: In this study, we performed EMT experiments using JAR cells based on the expression of the mesenchymal markers and vitamin D receptor. JAR cells were treated with calcitriol, the active form of vitamin D. Western blotting was performed to evaluate EMT markers and key molecules of signaling pathways. Invasion assays were conducted. Expression and secretion of MMPs were analyzed by real-time PCR and zymography. RESULTS: Calcitriol significantly enhanced EMT and the invasive capability of JAR cells, along with increased expression and secretion of MMP-2 and MMP-9. Moreover, ERK signaling pathway was activated by calcitriol. The effects of calcitriol were neutralized by ERK signaling blocker. CONCLUSION: Calcitriol facilitated EMT induction and expression of MMPs via ERK signaling pathway, which promoted the invasive capability of EVTs. Further studies are warranted to elucidate the potential application of vitamin D in the prevention of pregnancy complications.

Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses.

Odour information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons (primary neurons), to the posteroventral main olfactory bulb (PV MOB) in mice. Here we report that Nrp2-positive (Nrp2+) mitral cells (MCs, second-order neurons) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2+ MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2+ MCs and odour-induced attractive social responses are impaired. In utero, electroporation demonstrates that activation of the Nrp2 gene in MCs is sufficient to instruct their circuit formation from the PV MOB to the anterior MeA.

Activation of NOD-1/JNK/IL-8 signal axis in decidual stromal cells facilitates trophoblast invasion.

Decidual stromal cells (DSCs) are known to regulate trophoblast invasion via unveiled mechanism yet. And nucleotide-binding oligomerization domain-containing protein 1 (NOD1) may influence on this DSC-trophoblast interaction. We investigated the mechanism underlying the DSC-mediated regulation of trophoblast invasion and the effect of NOD1 on their cross talk. Using human primary DSCs, BeWo cell invasion was measured. Cytokine secretion and MAP kinase signaling were examined in DSCs following treatment with NOD1 agonist, Tri-DAP. DSCs secreted IL-8 and increased trophoblast invasion. Tri-DAP further increased IL-8 secretion from DSCs via JNK pathway and facilitated both MMP-2 production and trophoblast invasion compared with control. Upon cotreatment of IL-8 and anti-IL-8 antibody to BeWo cells, the number of invading trophoblasts and MMP-2 production decreased significantly. These results suggest that IL-8 from DSCs may play a role to increase the invasiveness of trophoblast cells into the decidua via NOD1/JNK pathway.

Synthesis and structure-activity studies of side chain analogues of the anti-tubercular agent, Q203.

The anti-tubercular activity of 6-chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)benzyl)imidazo [1,2-a]pyridine-3-carboxamide (Q203) is modified by varying its side chain. In this study, we synthesized Q203 analogues with different side chains and studied their effects on anti-tubercular activity. Many analogues showed good potency against M. tuberculosis replicating in liquid broth culture medium (extracellular activity) regardless of chain length and conformational changes. However, a polar character in the side chain region was unfavorable for anti-tubercular activity. The analogues, 25, 28, 35, and 36, displayed excellent activity against M. tuberculosis replicating inside macrophages (intracellular activity) and promising pharmacokinetic (PK) properties with high drug exposure level and long half-life.

A linear function for the approximation of accessible surface area of proteins.

Since the solvent accessible surface area of proteins has been utilized as an important factor in the prediction of their thermodynamic properties, a simple and analytical method for its determination would contribute to protein structure prediction and analysis methods. We report the development of a simple and analytical method for the estimation of the accessible surface area of protein, consisting of linear functions of distances between unified atoms. While our formulation was much simpler than previously developed methods, it showed comparable performance, with the mean relative error on total solvent accessible surface area of 0.49 and 2.16% for 89 denatured and native protein structures, respectively. Moreover, the partial derivatives of accessible surface area to the position of unified atoms could also be derived in a simpler form.

Effectiveness of Intraluminal Air Decompression on Postcolonoscopic Pain According to Reinsertion Site.

BACKGROUND: Colonoscopy is a very effective and essential examination to diagnose colorectal cancer; however, many patients experience discomfort due to post-examination abdominal pain, which reduces colonoscopy compliance. This study was conducted to determine methods for reducing post-colonoscopic abdominal pain. METHODS: We conducted a randomized controlled study of 405 male and female adults who visited Hana General Hospital in Cheongju. We surveyed general characteristics, history of colonoscopy, and other related factors, then categorized examinees into 5 groups (0-5) according to the site of scope reinsertion. Pain was measured using a numeric rating scale (NRS). RESULTS: The mean age of examinees in this study was 47.8 years, and 210 participants had prior experience of colonoscopy. No significant difference was observed between variables, with the exception of reinsertion duration (P=0.005). Pain scores were different between performing physicians (P=0.006), and were higher when the subjective level of procedure difficulty was low (P=0.026) in univariate analysis. Pain scores decreased as the reinsertion site became closer to the proximal colon (P<0.001), but there was no significant difference between groups 3 and 4. The results of multiple logistic regression analysis, including univariate analysis, showed that group 1 had 0.48 times, group 2 had 0.38 times, group 3 had 0.09 times, and group 4 had 0.03 times odds ratio (moderate-to-severe pain, NRS ≥4) than control group 0. CONCLUSION: Air decompression by scope reinsertion is an effective way to reduce abdominal pain after colonoscopy. Removing air when the reinserted scope approaches the hepatic flexure seems to be the most effective method to reduce post-colonoscopic pain.

Low prealbumin levels are independently associated with higher mortality in patients on peritoneal dialysis.

BACKGROUND: Prealbumin, a sensitive marker for protein-energy status, is also known as an independent risk factor for mortality in hemodialysis patients. We investigated the impact of prealbumin on survival in incident peritoneal dialysis (PD) patients. METHODS: In total, 136 incident PD patients (mean age, 53.0 ± 15.8 years) between 2002 and 2007 were enrolled in the study. Laboratory data, dialysis adequacy, and nutritional parameters were assessed 3 months after PD initiation. Patients were classified into 2 groups according to prealbumin level: high prealbumin (≥ 40 mg/dL) and low prealbumin (< 40 mg/dL). RESULTS: The patients in the low-prealbumin group were older and had more comorbidities such as diabetes and cardiovascular diseases compared with the patients in the high-prealbumin group. Mean subjective global assessment scores were lower, and the high-sensitivity C-reactive protein levels were higher in the low-prealbumin group. Serum creatinine, albumin, and transferrin levels; percent lean body mass; and normalized protein catabolic rate were positively associated, whereas subjective global assessment scores and high-sensitivity C-reactive protein levels were negatively associated with prealbumin concentration. During the median follow-up of 49 months, patients in the lower prealbumin group had a higher mortality rate. Multivariate analysis revealed that prealbumin < 40 mg/dL (hazard ratio, 2.30; 95% confidence interval, 1.14-4.64) was an independent risk factor for mortality. In receiver operating characteristic curves, the area under the curve of prealbumin for mortality was the largest among the parameters. CONCLUSION: Prealbumin levels were an independent and sensitive predictor for mortality in incident PD patients, showing a good correlation with nutritional and inflammatory markers.

Shigella outer membrane protein PSSP-1 is broadly protective against Shigella infection.

In developing countries, Shigella is a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multiple Shigella serotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigella surface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection against Shigella flexneri serotypes 2a, 5a, and 6, Shigella boydii, Shigella sonnei, and Shigella dysenteriae serotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conserved Shigella protein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine against Shigella.

Carnitine palmitoyltransferase-1 (CPT-1) activity stimulation by cerulenin via sympathetic nervous system activation overrides cerulenin's peripheral effect.

To clarify the paradoxic effects of cerulenin, namely its in vitro inhibitory effects on fat catabolism and its in vivo reduction of fat mass, we studied the in vivo and in vitro effects of cerulenin on carnitine palmitoyltransferase-1 (CPT-1) activity, the rate-limiting enzyme of fatty acid oxidation. A single ip injection of cerulenin significantly reduced body weight and increased core temperature without significantly reducing food intake. In situ hybridization study revealed that a single injection of cerulenin did not affect the expression of orexigenic neuropeptide mRNA. Cerulenin's effect on CPT-1 activity was biphasic in the liver and muscle: early suppression during the first 1 h and late stimulation in the 3-5 h after ip treatment. In vitro cerulenin treatment reduced CPT-1 activity, which was overcome by cotreating with catecholamine. Intracerebroventricular injection of cerulenin increased CPT-1 activity significantly in soleus muscle, and this effect was sustained for up to 3 h. Pretreatment with alpha-methyl-p-tyrosine inhibited the cerulenin-induced increase in core temperature and the late-phase stimulating effect of cerulenin on CPT-1 activity. In adrenalectomized mice, cerulenin also increased the activity. In vivo cerulenin treatment enhanced muscle CPT-1 activity in monosodium glutamate-treated arcuate nucleus lesioned mice but not in gold thioglucose-treated ventromedial hypothalamus lesioned mice. These findings suggest that cerulenin-induced late-phase stimulating effects on CPT-1 activity and energy expenditure is mediated by the activation of innervated sympathetic nervous system neurons through the firing of undefined neurons of the ventromedial hypothalamus, rather than the arcuate nucleus.

Enzyme adsorption, precipitation and crosslinking of glucose oxidase and laccase on polyaniline nanofibers for highly stable enzymatic biofuel cells.

Enzymatic biofuel cells have many great features as a small power source for medical, environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers (PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the maximum power density output of 37.4 µW/cm(2), while the power density output of 3.1 µW/cm(2) was shown without mediators. Under room temperature and 4°C for 28 days, enzymatic biofuel cells maintained 54 and 70% of its initial power density, respectively.

Untangling the two-way signalling route from synapses to the nucleus, and from the nucleus back to the synapses.

During learning and memory, it has been suggested that the coordinated electrical activity of hippocampal neurons translates information about the external environment into internal neuronal representations, which then are stored initially within the hippocampus and subsequently into other areas of the brain. A widely held hypothesis posits that synaptic plasticity is a key feature that critically modulates the triggering and the maintenance of such representations, some of which are thought to persist over time as traces or tags. However, the molecular and cell biological basis for these traces and tags has remained elusive. Here, we review recent findings that help clarify some of the molecular and cellular mechanisms critical for these events, by untangling a two-way signalling crosstalk route between the synapses and the neuronal soma. In particular, a detailed interrogation of the soma-to-synapse delivery of immediate early gene product Arc/Arg3.1, whose induction is triggered by heightened synaptic activity in many brain areas, teases apart an unsuspected 'inverse' synaptic tagging mechanism that likely contributes to maintaining the contrast of synaptic weight between strengthened and weak synapses within an active ensemble.

Region-specific activation of CRTC1-CREB signaling mediates long-term fear memory.

CREB is a pivotal mediator of activity-regulated gene transcription that underlies memory formation and allocation. The contribution of a key CREB cofactor, CREB-regulated transcription coactivator 1 (CRTC1), has, however, remained elusive. Here we show that several constitutive kinase pathways and an activity-regulated phosphatase, calcineurin, converge to determine the nucleocytoplasmic shuttling of CRTC1. This, in turn, triggered an activity-dependent association of CRTC1 with CREB-dependent regulatory elements found on IEG promoters. Forced expression of nuclear CRTC1 in hippocampal neurons activated CREB-dependent transcription, and was sufficient to enhance contextual fear memory. Surprisingly, during contextual fear conditioning, we found evidence of nuclear recruitment of endogenous CRTC1 only in the basolateral amygdala, and not in the hippocampus. Consistently, CRTC1 knockdown in the amygdala, but not in the hippocampus, significantly attenuated fear memory. Thus, CRTC1 has a wide impact on CREB-dependent memory processes, but fine-tunes CREB output in a region-specific manner.