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Background: Intracellular Na + (K + )/H + antiporters (NHXs) have pivotal functions in regulating plant growth, development, and resistance to a range of stresses. To gain insight into the molecular events underlying their actions in switchgrass (Panicum virgatum L.), we analyzed transcriptomic changes between PvNHX1-overexpression transgenic lines and wild-type (WT) plants using RNA sequencing (RNA-seq) technology., Results: The comparison of transcriptomic data from the WT and transgenic plants revealed a large number of differentially expressed genes (DEGs) in the latter. Gene ontology (GO) and KEGG pathway analyses showed that these DEGs were associated with a wide range of functions, and participated in many biological processes. For example, we found that PvNHX1 had an important role in plant growth through its regulation of photosynthetic activity and cell expansion. In addition, PvNHX1 regulated K + homeostasis, cell expansion and pollen development, indicating that it has unique and specific roles in flower development. We also found that transgenic switchgrass exhibited a higher level of transcription of defense-related genes, especially those involved in disease resistance., Conclusion: We showed that PvNHX1 had an important role in plant growth and development through its regulation of photosynthetic activity, cell expansion, K + homeostasis, and pollen development. Additionally, PvNHX1 overexpression activated a complex signal transduction network in response to various biotic and abiotic stresses. In relation to plant growth, development, and defense responses, PvNHX1 also had a vital regulatory role in the formation of a series of plant hormones and transcription factors (TFs). The reliability of the RNA-seq data was confirmed by quantitative real-time PCR. Our data provide a valuable foundation for further research into the molecular mechanisms and physiological roles of NHXs in plants.

Tongue squamous epithelial cells are the main component of tongue coating, with proliferation, differentiation and apoptosis being the root cause of the formation and maintenance of tongue coating. The present study aimed to explore the molecular mechanism by which serum influences tongue coating, to enable a better understanding for future investigations. Tongue carcinoma squamous cells were exposed to serum‑starvation in vitro. Cellular proliferation and apoptosis were observed by using 3‑[4,5‑dimethyl‑2‑thiazolyl]‑2,5‑diphenyl‑2‑H‑tetrazolium bromide (MTT) assay, flow cytometry, Hoechst staining, scanning electron microscope (SEM), transmission electron microscope (TEM), and by measuring the expression ratio of B‑cell lymphoma 2 apoptosis regulator (Bcl‑2)/Bcl‑2 associated protein X apoptosis regulator (Bax) mRNA and protein by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting, respectively. MTT assays revealed that serum‑starvation results in suppression of cellular proliferation, while flow cytometry data revealed that serum‑starvation induces cell cycle arrest at G1 phase and increases apoptosis. In addition, chromatin condensation and membrane blebbing were observed through Hoechst staining, TEM and SEM. The Bcl‑2/Bax ratio was found to be significantly decreased in cells that had undergone serum‑starvation by both RT‑qPCR and western blotting analysis, further indicating that serum‑starvation induces apoptosis. Therefore, tongue carcinoma squamous cells in a serum‑free medium can simulate apoptosis related to the formation of tongue coating, which may offer guidance for future investigations about other factors.

Conservation methods have been shown to affect forage nutrient composition and value, but little information is available about the effect of forage conservation on plant condensed tannins (CT). The objective of this study was to assess the effects of conservation method on the concentration, chemical composition and biological activity of CT. Whole-plant purple prairie clover (PPC, Dalea purpurea Vent.) was harvested at full flower and conserved as freeze-dried forage (FD), hay (HAY) or silage (SIL). Concentration of CT in conserved PPC was determined by the butanol-HCl-acetone method. Structural composition, protein-precipitation capacity and anti-bacterial activity of CT isolated from conserved forage were determined by in situ thiolytic degradation followed by HPLC-MS analysis, a protein precipitation assay using bovine serum albumin and ribulose 1,5-disphosphate carboxylase as model proteins and by an Escherichia coli ( E. coli ) growth test, respectively. Conservation method had no effect on concentration of total CT, but ensiling decreased ( p < 0.001) extractable CT and increased ( p < 0.001) protein- and fiber-bound CT. In contrast, hay-making only increased ( p < 0.01) protein-bound CT. Regardless of conservation method, epigallocatechin (EGC), catechin (C) and epicatechin (EC) were the major flavan-3-ol units, and gallocatechin (GC) was absent from both terminal and extension units of PPC CT. The SIL CT had the lowest ( p < 0.001) EGC, but the highest ( p < 0.01) EC in the extension units. Similarly, SIL CT exhibited a lower ( p < 0.001) mean degree of polymerization (mDP), but higher ( p < 0.001) procyanidins (PC) than FD or HAY CT. The protein-precipitating capacity of CT in conserved PPC ranked ( p < 0.001) as FD > HAY > SIL. E. coli growth n M9 medium was inhibited by 25-100 µg/mL of CT isolated from FD, HAY and SIL ( p < 0.05), but preservation method had no effect on the ability of CT to inhibit bacterial growth. The results demonstrated that ensiling decreased the extractability and protein-precipitating capacity of CT by increasing the proportions of PC. Purple prairie clover conserved as hay retained more biologically active CT than if it was conserved as silage., Competing Interests: The authors declare no conflict of interest.

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has developed as an important bridge between innate immune and infection recently, and has the ability to drive proteolytic procaspase-1 into bioactive caspase-1, then responsible for proteolytic processing of inflammatory cytokines IL-1β and IL-18. Fungal β-glucan, a major component of fungal cell wall, triggers inflammatory response in multiple immune cells, but rarely described in epithelial cells. Also, the relationship between fungal β-glucan and NLRP3 inflammasome is not clear yet. In this study, we first identified that curdlan, a large particulate β-glucan, could activate the NLRP3 inflammasome in LPS-primed human bronchial epithelial cells (HBECs). RT-PCR and Western Blot showed that curdlan upregulate the mRNA as well as intracellular protein expression of NLRP3 and IL-1β in HBECs, along with the activity of caspase-1, and the level of mature IL-1β in cell supernatants was higher by ELISA detection. Further studies demonstrated that the activation of NLRP3 inflammasome could be attenuated by NAC, an inhibitor of ROS. Thus, it indicated curdlan activate NLRP3 inflammasome through a pathway requiring ROS production in HBECs. These findings may provide a new therapeutic target, NLRP3 inflammasome, in invasive pulmonary fungal infections.

Abnormal circadian rhythms and circadian genes are strongly associated with several psychiatric disorders. Neuronal PAS Domain Protein 2 (NPAS2) is a core component of the molecular clock that acts as a transcription factor and is highly expressed in reward- and stress-related brain regions such as the striatum. However, the mechanism by which NPAS2 is involved in mood-related behaviors is still unclear. We measured anxiety-like behaviors in mice with a global null mutation in Npas2 (Npas2 null mutant mice) and found that Npas2 null mutant mice exhibit less anxiety-like behavior than their wild-type (WT) littermates (in elevated plus maze, light/dark box and open field assay). We assessed the effects of acute or chronic stress on striatal Npas2 expression, and found that both stressors increased levels of Npas2 . Moreover, knockdown of Npas2 in the ventral striatum resulted in a similar reduction of anxiety-like behaviors as seen in the Npas2 null mutant mouse. Additionally, we identified Gabra genes as transcriptional targets of NPAS2, found that Npas2 null mutant mice exhibit reduced sensitivity to the GABAa positive allosteric modulator, diazepam and that knockdown of Npas2 reduced Gabra1 expression and response to diazepam in the ventral striatum. These results: (1) implicate Npas2 in the response to stress and the development of anxiety; and (2) provide functional evidence for the regulation of GABAergic neurotransmission by NPAS2 in the ventral striatum.

The basolateral amygdala (BLA) sends excitatory projections to the nucleus accumbens (NAc) and regulates motivated behaviors partially by activating NAc medium spiny neurons (MSNs). Here, we characterized a feedforward inhibition circuit, through which BLA-evoked activation of NAc shell (NAcSh) MSNs was fine-tuned by GABAergic monosynaptic innervation from adjacent fast-spiking interneurons (FSIs). Specifically, BLA-to-NAcSh projections predominantly innervated NAcSh FSIs compared with MSNs and triggered action potentials in FSIs preceding BLA-mediated activation of MSNs. Due to these anatomical and temporal properties, activation of the BLA-to-NAcSh projection resulted in a rapid FSI-mediated inhibition of MSNs, timing-contingently dictating BLA-evoked activation of MSNs. Cocaine self-administration selectively and persistently up-regulated the presynaptic release probability of BLA-to-FSI synapses, entailing enhanced FSI-mediated feedforward inhibition of MSNs upon BLA activation. Experimentally enhancing the BLA-to-FSI transmission in vivo expedited the acquisition of cocaine self-administration. These results reveal a previously unidentified role of an FSI-embedded circuit in regulating NAc-based drug seeking and taking., Competing Interests: The authors declare no conflict of interest.

Switchgrass ( Panicum virgatum L.) has been increasingly recognized as one of the most valuable perennial bioenergy crop. To improve its biomass production, especially under salt stress, we isolated a putative vacuolar Na + (K + )/H + antiporter gene from switchgrass and designated as PvNHX1 . Subcellular localization revealed that this protein was localized mainly on the vacuole membrane. The PvNHX1 was found to be expressed throughout the entire growth period of switchgrass, exhibited preferentially expressed in the leaf tissue, and highly induced by salt stress. Transgenic switchgrass overexpressing PvNHX1 showed obvious advantages with respect to plant height and leaf development compared to the wild-type (WT) and transgenic control (EV, expressing the empty vector only) plants, suggesting PvNHX1 may serve as a promoter in switchgrass growth and development. Moreover, transgenic switchgrass were more tolerant than control plants with better growth-related phenotypes (higher shoot height, larger stem diameter, longer leaf length, and width) and physiological capacities (increased proline accumulation, reduced malondialdehyde production, preserved cell membrane integrity, etc.) under high salinity stress. Furthermore, the genes related to cell growth, flowering, and potassium transporters in transgenic switchgrass exhibited a different expression profiles when compared to the control plants, indicating a pivotal function of PvNHX1 in cell expansion and K + homeostasis. Taken together, PvNHX1 is essential for normal plant growth and development, and play an important role in the response to salt stress by improving K + accumulation. Our data provide a valuable foundation for further researches on the molecular mechanism and physiological roles of NHXs in plants.

Melatonin serves pleiotropic functions in prompting plant growth and resistance to various stresses. The accurate biosynthetic pathway of melatonin remains elusive in plant species, while the N -acetyltransferase and O -methyltransferase were considered to be the last two key enzymes during its biosynthesis. To investigate the biosynthesis and metabolic pathway of melatonin in plants, the RNA-seq profile of overexpression of the ovine HIOMT was analyzed and compared with the previous transcriptome of transgenic oAANAT gene in switchgrass, a model plant for cellulosic ethanol production. A total of 946, 405, and 807 differentially expressed unigenes were observed in AANAT vs. control, HIOMT vs. control, and AANAT vs. HIOMT , respectively. Two hundred and seventy-five upregulated and 130 downregulated unigenes were detected in transgenic oHIOMT line comparing with control, including the significantly upregulated (F-box/kelch-repeat protein, zinc finger BED domain-containing protein-3) genes, which were potentially correlated with enhanced phenotypes of shoot, stem and root growth in transgenic oHIOMT switchgrass. Several stress resistant related genes (SPX domain-containing membrane protein, copper transporter 1, late blight resistance protein homolog R1A-6 OS etc.) were specifically and significantly upregulated in transgenic oHIOMT only, while metabolism-related genes (phenylalanine-4-hydroxylase, tyrosine decarboxylase 1, protein disulfide-isomerase and galactinol synthase 2 etc.) were significantly upregulated in transgenic oAANAT only. These results provide new sights into the biosynthetic and physiological functional networks of melatonin in plants.

This experiment was conducted to investigate the effects of dietary arginine levels on growth performance, body composition, serum biochemical indices and resistance ability against ammonia-nitrogen stress in juvenile yellow catfish ( Pelteobagrus fulvidraco ). Five isonitrogenous and isolipidic diets (42% protein and 9% lipid) were formulated to contain graded levels of arginine (2.44%, 2.64%, 2.81%, 3.01% and 3.23% of diet), by supplementing L -Arginine HCl. Seven hundred juvenile yellow catfish with an initial average body weight of 1.13 ± 0.02 g were randomly divided into 5 groups with 4 replicates of 35 fish each and each group was fed one of the diets. After 56 d feeding, fish were exposed to 100 mg/L of ammonia-nitrogen for 72 h. The results showed that weight gain (WG) and specific growth rate (SGR) in 2.64% and 2.81% groups were significantly higher than those in 3.23% group ( P  < 0.05). The feed conversation ratio (FCR) in 2.64%, 2.81% and 3.01% groups was significantly decreased when compared with 3.23% group. The protein efficiency ratio (PER) in 2.64% group was significantly higher than that in 2.44% and 3.23% groups ( P  < 0.05). The condition factor (CF) of fish was significantly higher in 2.81% group than that in 2.44% group ( P  < 0.05). Dietary arginine levels had no significant effect on hepatosomatic index (HSI), viscerosomatic index (VSI), and whole-body dry matter, crude protein, crude lipid, ash contents, as well as serum total protein (TP), triglyceride (TG), glucose (GLU), urea nitrogen (UN) contents and aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities ( P  > 0.05). After the fish were challenged to ammonia-nitrogen for 72 h, their cumulative mortality rate in 2.81% group was significantly lower than that in 2.44% group ( P  < 0.05). The results suggested that dietary arginine level at 2.81% could optimize anti-ammonia-nitrogen stress ability of juvenile yellow catfish and a level of 3.23% arginine seemed to depress the growth performance of fish and decreased their tolerance to the ammonia-nitrogen stress under current study. A quadratic regression analysis based on WG indicated that the optimal dietary arginine requirement of juvenile yellow catfish was estimated to be 2.74% of the diet (6.45% of dietary protein) under current culture conditions.

Melatonin serves important functions in the promotion of growth and anti-stress regulation by efficient radical scavenging and regulation of antioxidant enzyme activity in various plants. To investigate its regulatory roles and metabolism pathways, the transcriptomic profile of overexpressing the ovine arylalkylamine N-acetyltransferase (oAANAT) gene, encoding the penultimate enzyme in melatonin biosynthesis, was compared with empty vector control using RNA-seq in switchgrass, a model plant of cellulosic ethanol conversion. The 85.22 million high quality reads that were assembled into 135,684 unigenes were generated by Illumina sequencing for transgenic oAANAT switchgrass with an average sequence length of 716 bp. A total of 946 differentially expression genes in transgenic line comparing to control switchgrass, including 737 up-regulated and 209 down-regulated genes, were mainly enriched with two main functional patterns of melatonin identifying by gene ontology analysis: the growth regulator and stress tolerance. Furthermore, KEGG maps indicated that the biosynthetic pathways of secondary metabolite (phenylpropanoids, flavonoids, steroids, stilbenoid, diarylheptanoid, and gingerol) and signaling pathways (MAPK signaling pathway, estrogen signaling pathway) were involved in melatonin metabolism. This study substantially expands the transcriptome information for switchgrass and provides valuable clues for identifying candidate genes involved in melatonin biosynthesis and elucidating the mechanism of melatonin metabolism.

Exposure to cocaine induces addiction-associated behaviors partially through remodeling neurocircuits in the nucleus accumbens (NAc). The paraventricular nucleus of thalamus (PVT), which projects to the NAc monosynaptically, is activated by cocaine exposure and has been implicated in several cocaine-induced emotional and motivational states. Here we show that disrupting synaptic transmission of select PVT neurons with tetanus toxin activated via retrograde trans-synaptic transport of cre from NAc efferents decreased cocaine self-administration in rats. This projection underwent complex adaptations after self-administration of cocaine (0.75 mg/kg/infusion; 2 h/d × 5 d, 1d overnight training). Specifically, 1d after cocaine self-administration, we observed increased levels of AMPA receptor (AMPAR)-silent glutamatergic synapses in this projection, accompanied by a decreased ratio of AMPAR-to-NMDA receptor (NMDAR)-mediated EPSCs. Furthermore, the decay kinetics of NMDAR EPSCs was significantly prolonged, suggesting insertion of new GluN2B-containing NMDARs to PVT-to-NAc synapses. After 45-d withdrawal, silent synapses within this projection returned to the basal levels, accompanied by a return of the AMPAR/NMDAR ratio and NMDAR decay kinetics to the basal levels. In amygdala and infralimbic prefrontal cortical projections to the NAc, a portion of cocaine-generated silent synapses becomes unsilenced by recruiting calcium-permeable AMPARs (CP-AMPARs) after drug withdrawal. However, the sensitivity of PVT-to-NAc synapses to CP-AMPAR-selective antagonists was not changed after withdrawal, suggesting that CP-AMPAR trafficking is not involved in the evolution of cocaine-generated silent synapses within this projection. Meanwhile, the release probability of PVT-to-NAc synapses was increased after short- and long-term cocaine withdrawal. These results reveal complex and profound alterations at PVT-to-NAc synapses after cocaine exposure and withdrawal.

Unlabelled: Sleep profoundly affects the emotional and motivational state. In humans and animals, loss of sleep often results in enhanced motivation for reward, which has direct implications for health risks as well as potential benefits. Current study aims at understanding the mechanisms underlying sleep deprivation (SDe)-induced enhancement of reward seeking. We found that after acute SDe, mice had an increase in sucrose seeking and consumption but not food intake, suggesting a selective enhancement of motivation for reward. In the nucleus accumbens (NAc), a key brain region regulating emotional and motivational responses, we observed a decrease in the ratio of the overall excitatory over inhibitory synaptic inputs onto NAc principle neurons after SDe. The shift was partly mediated by reduced glutamatergic transmission of presynaptic origin. Further analysis revealed that there was selective reduction of the glutamate release probability at the medial prefrontal cortex (mPFC)-to-NAc synapses, but not those from the hippocampus, thalamus, or the basal lateral amygdala. To reverse this SDe-induced synaptic alteration, we expressed the stabilized step function opsin (SSFO) in the mPFC; optogenetic stimulation of SSFO at mPFC-to-NAc projection terminals persistently enhanced the action potential-dependent glutamate release. Intra-NAc optogenetic stimulation of SSFO selectively at mPFC-to-NAc terminals restored normal sucrose seeking in mice after SDe without affecting food intake. These results highlight the mPFC-to-NAc projection as a key circuit-based target for sleep to regulate reward-motivated behaviors., Significance Statement: Sleep loss, a costly challenge of modern society, has profound physiological and psychological consequences, including altered reward processing of the brain. The current study aims at understanding the mechanisms underlying sleep deprivation-induced enhancement of reward seeking. We identify that the medial prefrontal cortex (mPFC)-to-nucleus accumbens (NAc) glutamatergic transmission is selectively weakened following acute sleep deprivation, whose restoration normalizes reward seeking in sleep-deprived mice. These results suggest a possibility of normalizing sleep deprivation-induced abnormal reward seeking by targeting specific neural projections, and they demonstrate the mPFC-to-NAc glutamatergic projection as a key circuit-based target for sleep to regulate reward-motivated behaviors., (Copyright © 2016 the authors 0270-6474/16/367897-14$15.00/0.)

Exposures to cocaine and morphine produce similar adaptations in nucleus accumbens (NAc)-based behaviors, yet produce very different adaptations at NAc excitatory synapses. In an effort to explain this paradox, we found that both drugs induced NMDA receptor-containing, AMPA receptor-silent excitatory synapses, albeit in distinct cell types through opposing cellular mechanisms. Cocaine selectively induced silent synapses in D1-type neurons, likely via a synaptogenesis process, whereas morphine induced silent synapses in D2-type neurons via internalization of AMPA receptors from pre-existing synapses. After drug withdrawal, cocaine-generated silent synapses became 'unsilenced' by recruiting AMPA receptors to strengthen excitatory inputs to D1-type neurons, whereas morphine-generated silent synapses were likely eliminated to weaken excitatory inputs to D2-type neurons. Thus, these cell type-specific, opposing mechanisms produced the same net shift of the balance between excitatory inputs to D1- and D2-type NAc neurons, which may underlie certain common alterations in NAc-based behaviors induced by both classes of drugs.

Environmental enrichment (EE) has long been postulated as a behavioral treatment for drug addiction based on its preventive effects in animal models: rodents experiencing prior EE exhibit increased resistance to establishing drug taking and seeking. However, the therapeutic effects of EE, namely, the effects of EE when applied after drug exposure, are often marginal and transient. Using incubation of cue-induced cocaine craving, a rat relapse model depicting progressive intensification of cocaine seeking after withdrawal from cocaine self-administration, our present study reveals that after cocaine withdrawal, in vivo circuit-specific long-term depression (LTD) unmasks the therapeutic power of EE to achieve long-lasting anti-relapse effects. Specifically, our previous results show that cocaine self-administration generates AMPA receptor (AMPAR)-silent excitatory synapses within the basolateral amygdala (BLA) to nucleus accumbens (NAc) projection, and maturation of these silent synapses via recruiting calcium-permeable (CP) AMPARs contributes to incubation of cocaine craving. Here, we show that after cocaine withdrawal and maturation of silent synapses, the BLA-to-NAc projection became highly resistant to EE. However, optogenetic LTD applied to this projection in vivo transiently re-silenced these silent synapses by removing CP-AMPARs. During this transient window, application of EE resulted in the insertion of nonCP-AMPARs, thereby remodeling the "incubated" BLA-to-NAc projection. Consequently, incubation of cocaine craving was decreased persistently. These results reveal a mechanistic basis through which the persistent anti-relapse effects of EE can be unleashed after drug withdrawal.

Researchers are enthusiastically concerned about neural stem cell (NSC) therapy in a wide array of diseases, including stroke, neurodegenerative disease, spinal cord injury, and depression. Although enormous evidences have demonstrated that neurobehavioral improvement may benefit from NSC-supporting regeneration in animal models, approaches to endogenous and transplanted NSCs are blocked by hurdles of migration, proliferation, maturation, and integration of NSCs. Electrical stimulation (ES) may be a selective non-drug approach for mobilizing NSCs in the central nervous system. This technique is suitable for clinical application, because it is well established and its potential complications are manageable. Here, we provide a comprehensive review of the emerging positive role of different electrical cues in regulating NSC biology in vitro and in vivo, as well as biomaterial-based and chemical stimulation of NSCs. In the future, ES combined with stem cell therapy or other cues probably becomes an approach for promoting brain repair.

A transient but prominent increase in the level of "silent synapses"--a signature of immature glutamatergic synapses that contain only NMDA receptors without stably expressed AMPA receptors--has been identified in the nucleus accumbens (NAc) following exposure to cocaine. As the NAc is a critical forebrain region implicated in forming addiction-associated behaviors, the initial discoveries have raised speculations about whether and how these drug-induced synapses mature and potentially contribute to addiction-related behaviors. Here, we summarize recent progress in recognizing the pathway-specific regulations of silent synapse maturation, and its diverse impacts on behavior. We provide an update of the guiding hypothesis--the "neural rejuvenation hypothesis"--with recently emerged evidence of silent synapses in cocaine craving and relapse., (© The Author(s) 2015.)

After withdrawal from cocaine, chronic cocaine users often experience persistent reduction in total sleep time, which is accompanied by increased sleep fragmentation resembling chronic insomnia. This and other sleep abnormalities have long been speculated to foster relapse and further drug addiction, but direct evidence is lacking. Here, we report that after prolonged withdrawal from cocaine self-administration, rats exhibited persistent reduction in nonrapid-eye-movement (NREM) and rapid-eye-movement (REM) sleep, as well as increased sleep fragmentation. In an attempt to improve sleep after cocaine withdrawal, we applied chronic sleep restriction to the rats during their active (dark) phase of the day, which selectively decreased the fragmentation of REM sleep during their inactive (light) phase without changing NREM or the total amount of daily sleep. Animals with improved REM sleep exhibited decreased incubation of cocaine craving, a phenomenon depicting the progressive intensification of cocaine seeking after withdrawal. In contrast, experimentally increasing sleep fragmentation after cocaine self-administration expedited the development of incubation of cocaine craving. Incubation of cocaine craving is partially mediated by progressive accumulation of calcium-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). After withdrawal from cocaine, animals with improved REM sleep exhibited reduced accumulation of CP-AMPARs in the NAc, whereas increasing sleep fragmentation accelerated NAc CP-AMPAR accumulation. These results reveal a potential molecular substrate that can be engaged by sleep to regulate cocaine craving and relapse, and demonstrate sleep-based therapeutic opportunities for cocaine addiction. Significance statement: Sleep abnormalities are common symptoms in chronic drug users long after drug withdrawal. These withdrawal-associated sleep symptoms, particularly reduction in total sleep time and deteriorating sleep quality, have been speculated to foster relapse and further drug addiction, but direct evidence is lacking. Here we show in rats that the sleep pattern was persistently changed long after withdrawal from cocaine self-administration, and demonstrate that sleep interventions can bidirectionally regulate cocaine craving and seeking after withdrawal. We further demonstrate that glutamatergic synapses in the nucleus accumbens are potential neuronal targets for sleep intervention to influence cocaine craving after withdrawal. These results provide a strong rationale supporting sleep-based therapies for cocaine addiction., (Copyright © 2015 the authors 0270-6474/15/3513300-11$15.00/0.)

Background: The substitution of Ca(2+) in Ca-hydroxylapatite by toxic Cd(2+) can cause the forming of Cd-hydroxylapatite and is a significant issue in a great variety of research areas, which hence needs an understanding of the essential physicochemical characteristics. Unfortunately, the solubility product and thermodynamic data for Cd-hydroxylapatite in water under a variety of conditions now are lacking. Little information has been reported by previous researchers. Additionally, the dissolution mechanism of Cd-hydroxylapatite has never been studied., Results: Dissolution of the synthetic cadmium hydroxylapatite [Cd-HAP, Cd5(PO4)3OH] in HNO3 solution (pH = 2), ultrapure water (pH = 5.6) and NaOH solution (pH = 9) was experimentally studied at 25, 35 and 45°C. Characterization by XRD, FT-IR and FE-SEM proved that Cd-HAP solids showed no recognizable change during dissolution. For the Cd-HAP dissolution in aqueous acidic media at initial pH 2 and 25°C, the solution cadmium and phosphate concentrations increased rapidly and reached the peak values after 20-30 days and 10 days reaction, respectively. Thereafter, the Cd-HAP dissolution rate decreased slowly, whereas the solution Cd/P molar ratio increased constantly from 1.65-1.69 to 6.61-6.76. The mean K sp values for Cd5(PO4)3OH were determined to be 10(-64.62) (10(-64.53)-10(-64.71)) at 25°C, 10(-65.58) (10(-65.31)-10(-65.80)) at 35°C and 10(-66.57) (10(-66.24)-10(-66.90)) at 45°C. Based on the obtained solubility data from the dissolution at initial pH 2 and 25°C, the Gibbs free energy of Cd5(PO4)3OH forming [Formula: see text] was determined to be -3,970.47 kJ/mol (-3,969.92 to -3,970.96 kJ/mol). Thermodynamic parameters, ΔG (0), ΔH (0), ΔS (0), and [Formula: see text] for the dissolution process of Cd-HAP in aqueous acidic media at initial pH 2 and 25°C were calculated 368,710.12 J/K mol, -158,809.54 J/mol, -1,770.20 and -869.53 J/K mol, respectively., Conclusions: Based on the experimental results of the present work and some previous researches, the cadmium hydroxylapatite (Cd-HAP) dissolution in aqueous media is considered to have the following coincident processes: the stoichiometric dissolution coupled with protonation and complexation reactions, the non-stoichiometric dissolution with Cd(2+) release and PO4 (3-) sorption and the sorption of Cd(2+) and PO4 (3-) species from solution backwards onto Cd-HAP surface. The obtained solubility products (K sp) 10(-64.62) (10(-64.53)-10(-64.71)) for Cd-HAP was approximately 7.62-5.62 log units lower than 10(-57)-10(-59) for calcium hydroxylapatite (Ca-HAP).Graphical abstractDissolution of cadmium hydroxylapatite [Cd5(PO4)3OH].

Four kinds of green deep eutectic solvents (DESs) based on choline chloride (ChCl) have been synthesized and coated on the surface of magnetic graphene oxide (Fe3O4@GO) to form Fe3O4@GO-DES for the magnetic solid-phase extraction of protein. X-ray diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FTIR), field emission scanning electron microscopy (FESEM) and thermal gravimetric analysis (TGA) were employed to characterize Fe3O4@GO-DES, and the results indicated the successful preparation of Fe3O4@GO-DES. The UV-vis spectrophotometer was used to measure the concentration of protein after extraction. Single factor experiments proved that the extraction amount was influenced by the types of DESs, solution temperature, solution ionic strength, extraction time, protein concentration and the amount of Fe3O4@GO-DES. Comparison of Fe3O4@GO and Fe3O4@GO-DES was carried out by extracting bovine serum albumin, ovalbumin, bovine hemoglobin and lysozyme. The experimental results showed that the proposed Fe3O4@GO-DES performs better than Fe3O4@GO in the extraction of acidic protein. Desorption of protein was carried out by eluting the solid extractant with 0.005 mol L(-1) Na2HPO4 contained 1 mol L(-1) NaCl. The obtained elution efficiency was about 90.9%. Attributed to the convenient magnetic separation, the solid extractant could be easily recycled., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Since the generation of induced pluripotent stem cells in 2006, cellular reprogramming has attracted increasing attention as a revolutionary strategy for cell replacement therapy. Recent advances have revealed that somatic cells can be directly converted into other mature cell types, which eliminates the risk of neoplasia and the generation of undesired cell types. Astrocytes become reactive and undergo proliferation, which hampers axon regeneration following injury, stroke, and neurodegenerative diseases. An emerging technique to directly reprogram astrocytes into induced neural stem cells (iNSCs) and induced neurons (iNs) by neural fate determinants brings potential hope to cell replacement therapy for the above neurological problems. Here, we discuss the development of direct reprogramming of various cell types into iNs and iNSCs, then detail astrocyte-derived iNSCs and iNs in vivo and in vitro. Finally, we highlight the unsolved challenges and opportunities for improvement.

A novel adsorbent based on silica-coated magnetic multiwall carbon nanotubes (MWCNTs) surface modified by dual hydroxy functional ionic liquid (FIL) ([OH]-FIL-m-MWCNTs@SiO2) has been designed and used for the purification of lysozyme (Lys) by magnetic solid-phase extraction (MSPE). Fourier transform infrared spectroscopy (FTIR), a vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) were employed to characterize [OH]-FIL-m-MWCNTs@SiO2. After extraction, the concentration of Lys was determined by a UV-Vis spectrophotometer at 278 nm. A series of single-factor experiments were carried out to identify the optimal conditions of the extraction and the extraction amount could reach up to 94.6 mg g(-1). The RSD of the precision, the repeatability and the stability experiments were 0.37% (n = 3), 0.47% (n = 3) and 0.52% (n = 3), respectively. Comparison of [OH]-FIL-m-MWCNTs@SiO2 with silica-coated magnetic Fe3O4 (Fe3O4@SiO2), silica-coated magnetic multiwall carbon nanotubes (m-MWCNTs@SiO2) and alkyl quaternary ammonium ionic liquid-modified on m-MWCNTs@SiO2 was carried out by extracting Lys. The extraction of bovine serum albumin (BSA), trypsin (Try) and ovalbumin (OVA) was also done by the proposed method. Desorption of Lys was carried out by 0.005 mol L(-1) Na2HPO4-1 mol L(-1) NaCl as the eluent solution and the desorption ratio reached 91.6%. Nearly 97.8% of the [OH]-FIL-m-MWCNTs@SiO2 could be recovered from each run, and the extraction amount decreased less after five runs. The circular dichroism spectral experiment analysis indicated that the secondary structure of Lys was unchanged after extraction.

Background: The lateral habenula is a brain region that has been critically implicated in modulating negative emotional states and responses to aversive stimuli. Exposure to addictive drugs such as cocaine negatively impacts affective states, an effect persisting longer than acute drug effects. However, the mechanisms of this effect are poorly understood. We hypothesized that drugs of abuse, such as cocaine, may contribute to drug-induced negative affective states by altering the firing properties of lateral habenula neurons, thus changing the signaling patterns from the lateral habenula to downstream circuits., Methods: Using whole-cell current-clamp recording of acutely prepared brain slices of rats after various periods of withdrawal from cocaine self-administration, we characterized an important heterogeneous subregion of the lateral habenula based on membrane properties., Results: We found two major relevant neuronal subtypes: burst firing neurons and regular spiking neurons. We also found that lateral habenula regular spiking neurons had higher membrane excitability for at least 7 days following cocaine self-administration, likely due to a greater membrane resistance. Both the increase in lateral habenula excitability and membrane resistance returned to baseline when tested after a more prolonged period of 45 days of withdrawal., Conclusion: This is the first study to look at intrinsic lateral habenula neuron properties following cocaine exposure beyond acute drug effects. These results may help to explain how cocaine and other drugs negatively impact affect states., (© The Author 2015. Published by Oxford University Press on behalf of CINP.)

In this work, molecular dynamics simulations are performed to investigate the influence of material properties on the nanometric cutting of single crystal copper and aluminum with a diamond cutting tool. The atomic interactions in the two metallic materials are modeled by two sets of embedded atom method (EAM) potential parameters. Simulation results show that although the plastic deformation of the two materials is achieved by dislocation activities, the deformation behavior and related physical phenomena, such as the machining forces, machined surface quality, and chip morphology, are significantly different for different materials. Furthermore, the influence of material properties on the nanometric cutting has a strong dependence on the operating temperature.

The influence of all-plant-based diet on fingerling blunt snout breams (Megalobrama amblycephala) was tested by examining growth performance, anti-stress properties and related gene expression. Healthy fish were randomly divided into triplicate groups per dietary treatment and fed with different formulated diets. The results showed that both weight gain, specific growth rate and protein efficiency ratio of all-plant-based diet group were significant higher than those of the control (p < 0.05). In contrast, FCR of all-plant-based diet group was significantly lower than that of the control (p < 0.05). Therefore, all-plant-based diets could not affect the growth performance of blunt snout breams. Compared to the control group, the lysozyme levels in serum and mucus, and glutamic-oxaloacetic transaminase activities in serum and liver decreased significantly (p < 0.05). In contrast, the glutamic-pyruvic transaminase activities in serum and liver increased significantly (p < 0.05). For blunt snout breams fed with all-plant-based diets, the superoxide dismutase activities in mucus, serum and liver as well as catalase activity in serum and liver were decreased significantly (p < 0.05) comparing with that of the control group. But malondialdehyde contents were higher (p < 0.05) in serum and liver than that of control group. The expression of HSC70 mRNA increased significantly (p < 0.05) in blunt snout breams fed with all-plant-based diet, whereas the HSP70 mRNA expression decreased significantly (p < 0.05) when compared with control group. In conclusion, all these results indicated that the application of all-plant-based diet could decrease the anti-stress properties (non-specific immunity, stress resistance and antioxidant ability) and HSP70 mRNA expression in blunt snout breams fingerling. Although all-plant-based diets could not affect the growth performance of blunt snout breams, the application of all-plant-based diet should be discreet in the production practice.

In the present work, we investigate the minimum wear depth of single crystalline Cu(111) under single asperity friction by means of molecular dynamics simulations. The atomistic mechanisms governing the incipient plasticity are elucidated by characterizing specific defect structures and are correlated to the observed mechanical and frictional responses of the material. Furthermore, the effect of probe radius on the friction process is studied. Our simulations indicate that the formation of wear impression is closely associated with defect nucleation and the minimum wear depth is equivalent to the critical penetration depth at which plasticity initiates. It is found that the probe radius has a strong influence on the formation of defect structures and the observed mechanical responses.

We posit a bottom-up sleep-regulatory paradigm in which state changes are initiated within small networks as a consequence of local cell activity. Bottom-up regulatory mechanisms are prevalent throughout nature, occurring in vastly different systems and levels of organization. Synchronization of state without top-down regulation is a fundamental property of large collections of small semi-autonomous entities. We posit that such synchronization mechanisms are sufficient and necessary for whole-organism sleep onset. Within the brain we posit that small networks of highly interconnected neurons and glia, for example cortical columns, are semi-autonomous units oscillating between sleep-like and wake-like states. We review evidence showing that cells, small networks and regional areas of the brain share sleep-like properties with whole-animal sleep. A testable hypothesis focused on how sleep is initiated within local networks is presented. We posit that the release of cell activity-dependent molecules, such as ATP and nitric oxide, into the extracellular space initiates state changes within the local networks where they are produced. We review mechanisms of ATP induction of sleep-regulatory substances and their actions on receptor trafficking. Finally, we provide an example of how such local metabolic and state changes provide mechanistic explanations for clinical conditions, such as insomnia., (© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Endocannabinoid signaling critically regulates emotional and motivational states via activation of cannabinoid receptor 1 (CB1) in the brain. The nucleus accumbens (NAc) functions to gate emotional and motivational responses. Although expression of CB1 in the NAc is low, manipulation of CB1 signaling within the NAc triggers robust emotional/motivational alterations related to drug addiction and other psychiatric disorders, and these effects cannot be exclusively attributed to CB1 located at afferents to the NAc. Rather, CB1-expressing neurons in the NAc, although sparse, appear to be critical for emotional and motivational responses. However, the cellular properties of these neurons remain largely unknown. Here, we generated a knock-in mouse line in which CB1-expressing neurons expressed the fluorescent protein td-Tomato (tdT). Using these mice, we demonstrated that tdT-positive neurons within the NAc were exclusively fast-spiking interneurons (FSIs). These FSIs were electrically coupled with each other, and thus may help synchronize populations/ensembles of NAc neurons. CB1-expressing FSIs also form GABAergic synapses on adjacent medium spiny neurons (MSNs), providing feed-forward inhibition of NAc output. Furthermore, the membrane excitability of tdT-positive FSIs in the NAc was up-regulated after withdrawal from cocaine exposure, an effect that might increase FSI-to-MSN inhibition. Taken together with our previous findings that the membrane excitability of NAc MSNs is decreased during cocaine withdrawal, the present findings suggest that the basal functional output of the NAc is inhibited during cocaine withdrawal by multiple mechanisms. As such, CB1-expressing FSIs are targeted by cocaine exposure to influence the overall functional output of the NAc.

Background: Inhibitor of growth 1b (ING1b) is considered to be a class II tumor suppressor gene. Although reduced expression of p33(ING1b) has been reported in many human malignancies, including gastric cancers, the effect of p33(ING1b) on gastric cancer cells has yet to be investigated., Methods: Expression of p33(ING1b) in gastric adenocarcinoma tissues and their adjacent non-malignant gastric mucosa, as well as in gastric adenocarcinoma cell lines and normal gastric epithelial cells, was detected by using Western blotting. Recombinant adenoviruses were prepared to mediate the ectopic expression of p33(ING1b) (Ad-ING1b) and green fluorescent protein (GFP)(Ad-GFP) in the gastric adenocarcinoma cell lines, SGC-7901, MKN28, and MKN45 and the normal gastric epithelial cell line GES-1. Alterations in the proliferation and apoptosis of the cells after adenoviral infection were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively, and cell cycle distribution was analyzed in a fluorescence-activated cell sorter., Results: Western blotting confirmed the reduced expression of p33(ING1b) in gastric adenocarcinoma tissues and gastric adenocarcinoma cell lines. The ectopic expression of p33(ING1b) mediated by Ad-ING1b resulted in decreased growth, increased apoptosis, and cell cycle arrest at the G1 phase in both benign and malignant gastric epithelial cells regardless of their p53 status. Addition of a p53 inhibitor, pifithrin-α, did not abolish the pro-apoptotic and cell cycle-arresting effects of p33(ING1b) in p53 wild-type cells., Conclusions: Down-regulation of p33(ING1b) might play an important role in the development of gastric adenocarcinoma. Targeted local expression of p33(ING1b) may offer a promising alternative therapeutic measure for gastric cancer.

The nucleus accumbens shell (NAc) is a key brain region mediating emotional and motivational learning. In rodent models, dynamic alterations have been observed in synaptic NMDA receptors (NMDARs) within the NAc following incentive stimuli, and some of these alterations are critical for acquiring new emotional/motivational states. NMDARs are prominent molecular devices for controlling neural plasticity and memory formation. Although synaptic NMDARs are predominately located postsynaptically, recent evidence suggests that they may also exist at presynaptic terminals and reshape excitatory synaptic transmission by regulating presynaptic glutamate release. However, it remains unknown whether presynaptic NMDARs exist in the NAc and contribute to emotional and motivational learning. In an attempt to identify presynaptically located NMDARs in the NAc, the present study uses slice electrophysiology combined with pharmacological and genetic tools to examine the physiological role of the putative presynaptic NMDARs in rats. Our results show that application of glycine, the glycine-site agonist of NMDARs, potentiated presynaptic release of glutamate at excitatory synapses on NAc neurons, whereas application of 5,7-dichlorokynurenic acid or 7-chlorokynurenic acid, the glycine-site antagonists of NMDARs, produced the opposite effect. However, these seemingly presynaptic NMDAR-mediated effects could not be prevented by application of d-APV, the glutamate-site NMDAR antagonist, and were still present in the mice in which NMDAR NR1 or NR3 subunits were genetically deleted. Thus, rather than suggesting the existence of presynaptic NMDARs, our results support the idea that an unidentified type of glycine-activated substrate may account for the presynaptic effects appearing to be mediated by NMDARs.

Homeostatic response is an endowed self-correcting/maintaining property for living units, ranging from subcellular domains, single cells, and organs to the whole organism. Homeostatic responses maintain stable function through the ever-changing internal and external environments. In central neurons, several forms of homeostatic regulation have been identified, all of which tend to stabilize the functional output of neurons toward their prior "set-point." Medium spiny neurons (MSNs) within the forebrain region the nucleus accumbens (NAc) play a central role in gating/regulating emotional and motivational behaviors including craving and seeking drugs of abuse. Exposure to highly salient stimuli such as cocaine administration not only acutely activates a certain population of NAc MSNs, but also induces long-lasting changes in these neurons. It is these long-lasting cellular alterations that are speculated to mediate the increasingly strong cocaine-craving and cocaine-seeking behaviors. Why do the potentially powerful homeostatic mechanisms fail to correct or compensate for these drug-induced maladaptations in neurons? Based on recent experimental results, this review proposes a hypothesis of homeostatic dysregulation induced by exposure to cocaine. Specifically, we hypothesize that exposure to cocaine generates false molecular signals which misleads the homeostatic regulation process, resulting in maladaptive changes in NAc MSNs. Thus, many molecular and cellular alterations observed in the addicted brain may indeed result from homeostatic dysregulation. This review is among the first to introduce the concept of homeostatic neuroplasticity to understanding the molecular and cellular maladaptations following exposure to drugs of abuse., (Copyright © 2010 Elsevier B.V. All rights reserved.)

There are more than 50 class I MHC (MHCI) molecules in the mouse genome, some of which are now known to be expressed in neurons; however, the role of classical MHCI molecules in synaptic plasticity is unknown. We report that the classical MHCI molecules, H2-K(b) and H2-D(b), are co-expressed by Purkinje cells (PCs). In the cerebellum of mice deficient for both H2-K(b) and H2-D(b) (K(b)D(b-/-)), there is a lower threshold for induction of long-term depression (LTD) at parallel fiber to PC synapses. This change may be a result of additional glutamate release observed at K(b)D(b-/-) CF to PC synapses, which are thought to "train" the cerebellar circuit. A behavioral correlate of cerebellar LTD is motor learning; acquisition and retention of a Rotarod behavioral task is significantly better in K(b)D(b-/-) mice than in WT cohorts. These physiological and behavioral phenotypes in K(b)D(b-/-) mice reveal a surprising role for classical MHCI molecules in synaptic plasticity and motor learning.

Nucleus accumbens (NAc) medium spiny neurons cycle between two states, a functionally inactive downstate and a functionally active upstate. Here, we show that activation of the transcription factor cAMP-response element-binding protein (CREB), a common molecular response to several drugs of abuse, increases both duration of the upstate and action potential firing during the upstate. This effect of CREB is mediated by enhanced N-methyl-d-aspartate glutamate receptor (NMDAR) function: increased CREB activity increases both NMDAR-mediated synaptic currents and surface level of NMDARs, while inhibition of NMDARs abolishes the effect of CREB on upstate duration. Furthermore, mimicking the effect of CREB by pharmacological enhancement of NMDAR function in the NAc in vivo suppressed novelty- and cocaine-elicited locomotor activity. These findings suggest that by enhancing NMDAR-mediated synaptic transmission, CREB activation promotes the proportion of time NAc neurons spend in the upstate. This effect, along with the CREB enhancement of NAc membrane excitability (Dong, Y., Green, T., Saal, D., Marie, H., Neve, R., Nestler, E. J., and Malenka, R. C. (2006) Nat. Neurosci. 9, 475-477), may counteract drug-induced maladaptations in the NAc and thus ameliorate the addictive state.

KChIPs coassemble with pore-forming Kv4 alpha subunits to form a native complex in the brain and heart and regulate the expression and gating properties of Kv4 K(+) channels, but the mechanisms underlying these processes are unknown. Here we report a co-crystal structure of the complex of human Kv4.3 N-terminus and KChIP1 at a 3.2-A resolution. The structure reveals a unique clamping action of the complex, in which a single KChIP1 molecule, as a monomer, laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, forming an octamer. The proximal N-terminal peptide of Kv4.3 is sequestered by its binding to an elongated groove on the surface of KChIP1, which is indispensable for the modulation of Kv4.3 by KChIP1, and the same KChIP1 molecule binds to an adjacent T1 domain to stabilize the tetrameric Kv4.3 channels. Taken together with biochemical and functional data, our findings provide a structural basis for the modulation of Kv4 by KChIPs.

The D-isomer of aspartate is both a substrate for glutamate transporters and an agonist of N-methyl-D-aspartate (NMDA) receptors. To monitor the behavior of these receptors and transporters in intact tissue we synthesized a new photo-labile analogue of D-aspartate, N-[(6-nitrocoumarin-7-yl)methyl]-D-aspartic acid (Ncm-D-aspartate). This compound was photolyzed rapidly (t(1/2)=0.11 micros) by UV light with a quantum efficiency of 0.041 at pH 7.4. In acute hippocampal slices, photolysis of Ncm-D-aspartate by brief (1 ms) exposure to UV light elicited rapidly activating inward currents in astrocytes that were sensitive to inhibition by the glutamate transporter antagonist DL-threo-beta-benzyloxyaspartic acid (TBOA). Neither Ncm-D-aspartate nor the photo-released caging group exhibited agonist or antagonist activity at glutamate transporters, and Ncm-D-aspartate did not induce transporter currents prior to photolysis. Glutamate transporter currents were also elicited in cerebellar Purkinje cells in response to photolysis of Ncm-D-aspartate. Photo-release of D-aspartate from Ncm-D-aspartate did not induce alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor or metabotropic glutamate receptor (mGluR) currents, but triggered robust NMDA receptor currents in neurons; Ncm-D-aspartate and the photolzyed caging group were similarly inert at NMDA receptors. These results indicate that Ncm-D-aspartate can be used to study NMDA receptors at excitatory synapses and interactions between transporters and receptors in brain tissue.

Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that many beta-lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene. beta-Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways. When delivered to animals, the beta-lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation.