Effects of Cu2O nanoparticles on the kinetic characteristics of rapid chlorophyll fluorescence induction and related genes in wheat seedlings.

Metal nanoparticles could be accumulated in soils, which threatens the ecological stability of crops. Investigating the effects of cuprous oxide nanoparticles (Cu2O-NPs) on photosystem Ⅱ (PSⅡ) of wheat seedling leaves holds considerable importance in comprehending the implications of Cu2O-NPs on crop photosynthesis. Following the hydroponic method, we investigated the effects of 0, 10, 50, 100, and 200 mg·L-1 Cu2O-NPs on chlorophyll fluorescence induction kinetics and photosynthetic-related genes in wheat seedlings of "Zhoumai 18". The results showed that, with the increases of Cu2O-NPs concentrations, chlorophyll contents in wheat leaves decreased, and the standardization of the OJIP curve showed a clearly K-phase (ΔK＞0). Cu2O-NPs stress increased the parameters of active PSⅡ reaction centers, including the absorption flux per active RC (ABS/RC), the trapping flux per active RC (TRo/RC), the electron transport flux per active RC (ETo/RC), and the dissipation flux per active RC (DIo/RC). Cu2O-NPs stress decreased the parameters of PSⅡ energy distribution ratio including the maximum quantum yield of PSⅡ (φPo), the quantum yield of electron transport from QA (φEo), and the probability that a trapped exciton moved an electron further than QA (Ψo), while increased the quantum ratio for heat dissipation (φDo). Moreover, there was a decrease in photosynthetic quantum yield Y(Ⅱ), photochemical quenching coefficient (qP), net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of leaves with the increases of Cu2O-NPs concentration. Under Cu2O-NPs stress, the expression levels of genes which included PSⅡ genes (PsbD, PsbP, Lhcb1), Rubisco large subunit genes (RbcL), cytochrome b6/f complex genes (PetD, Rieske), and ATP synthase genes (AtpA, AtpB, AtpE, AtpI) were downregulated. These results indicated that Cu2O-NPs stress altered the activity and structure of PSⅡ in wheat seedlings, affected the activity of PSⅡ reaction centers, performance parameters of PSⅡ donor and acceptor sides. PSⅡ related genes were downregulated and exhibited significant concentration effects.

Impacts of cuprous oxide nanoparticles on wheat root morphology and genotoxicity.

To explore the ecotoxicity of Cu2O nanoparticles (NPs) on plant roots, the effects of Cu2O-NPs with different concentrations of 10, 50, 100 and 200 mg·L-1 on the seedling growth, root morphology, and cytogenetic toxicity of wheat 'Zhoumai 18' (Triticum aestivum Zhoumai 18) were examined in a hydroponic experiment. The results showed that Cu2O-NPs inhibited the growth of wheat seedlings. Cu2O-NPs reduced root and shoot lengths, fresh weights of shoot and root, root relative activity and ratio of root to shoot of wheat seedlings, but increased primary root number. Furthermore, with the increases of Cu2O-NPs concentrations, the root elongation zone shortened and the root became hard and brittle, while the average diameter of roots increased. Under the concentration of 100 mg·L-1 Cu2O-NPs, the mitotic index significantly decreased, and vacuolization, plasma membrane detachment, chromosomal abnormality occurred in the root tip cell. In conclusion, Cu2O-NPs are genotoxic to wheat seedlings, with consequences on the growth and development of wheat seedlings and root morphology.

Baicalin ameliorates neuroinflammation-induced depressive-like behavior through inhibition of toll-like receptor 4 expression via the PI3K/AKT/FoxO1 pathway.

BACKGROUND: Baicalin, which is isolated from Radix Scutellariae, possesses strong biological activities including an anti-inflammation property. Recent studies have shown that the anti-inflammatory effect of baicalin is linked to toll-like receptor 4 (TLR4), which participates in pathological changes of central nervous system diseases such as depression. In this study, we explored whether baicalin could produce antidepressant effects via regulation of TLR4 signaling in mice and attempted to elucidate the underlying mechanisms. METHODS: A chronic unpredictable mild stress (CUMS) mice model was performed to explore whether baicalin could produce antidepressant effects via the inhibition of neuroinflammation. To clarify the role of TLR4 in the anti-neuroinflammatory efficacy of baicalin, a lipopolysaccharide (LPS) was employed in mice to specially activate TLR4 and the behavioral changes were determined. Furthermore, we used LY294002 to examine the molecular mechanisms of baicalin in regulating the expression of TLR4 in vivo and in vitro using western blot, ELISA kits, and immunostaining. In the in vitro tests, the BV2 microglia cell lines and primary microglia cultures were pretreated with baicalin and LY292002 for 1 h and then stimulated 24 h with LPS. The primary microglial cells were transfected with the forkhead transcription factor forkhead box protein O 1 (FoxO1)-specific siRNA for 5 h and then co-stimulated with baicalin and LPS to investigate whether FoxO1 participated in the effect of baicalin on TLR4 expression. RESULTS: The administration of baicalin (especially 60 mg/kg) dramatically ameliorated CUMS-induced depressive-like symptoms; substantially decreased the levels of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in the hippocampus; and significantly decreased the expression of TLR4. The activation of TLR4 by the LPS triggered neuroinflammation and evoked depressive-like behaviors in mice, which were also alleviated by the treatment with baicalin (60 mg/kg). Furthermore, the application of baicalin significantly increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and FoxO1. The application of baicalin also promoted FoxO1 nuclear exclusion and contributed to the inhibition of the FoxO1 transactivation potential, which led to the downregulation of the expression of TLR4 in CUMS mice or LPS-treated BV2 cells and primary microglia cells. However, prophylactic treatment of LY294002 abolished the above effects of baicalin. In addition, we found that FoxO1 played a vital role in baicalin by regulating the TLR4 and TLR4-mediating neuroinflammation triggered by the LPS via knocking down the expression of FoxO1 in the primary microglia. CONCLUSION: Collectively, these results demonstrate that baicalin ameliorated neuroinflammation-induced depressive-like behaviors through the inhibition of TLR4 expression via the PI3K/AKT/FoxO1 pathway.

Baicalin exerts neuroprotective effects via inhibiting activation of GSK3ß/NF-κB/NLRP3 signal pathway in a rat model of depression.

Chronic stress can provoke depressive-like behaviors through activation of inflammation and apoptosis, leading to a reduction of neurons. Antidepressant therapy may contribute to inhibiting inflammation responses and have neuroprotective effects. Baicalin (BA) has an antidepressant effect in the chronic unpredictable mild stress (CUMS) animal model and exerts anti-inflammation, anti-apoptosis, as well as neuroprotective effects in many central nervous system (CNS)-related diseases. But the effects of BA on neuroprotection, apoptosis, and neuroinflammation and the potential mechanisms in depression are unclear. Here, we focused on examining the therapeutic effects of BA in CUMS-induced depression rats and investigating the molecular mechanisms. Results showed that administration of BA improved depressive-like behaviors and significantly increased the levels of doublecortin (DCX), Neuron-specific enolase (NSE), and Brain-derived neurotrophic factor (BDNF) in hippocampus. Furthermore, administration of BA increased the cell survival by reducing the level of malondialdehyde (MDA) and increasing the level of superoxide dismutase (SOD). Finally, administration of BA significantly decreased CUMS-induced apoptosis and inflammatory cytokines (caspase-1 and IL-1ß) in hippocampus. These responses were mediated by Glycogen synthase kinase-3 (GSK3) ß/Nuclear factor-κB (NF-κB)/Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing protein 3 (NLRP3) signal pathway. Taken together, these results indicate that BA could promote neuronal maturation and rescue neurons from apoptosis via inhibiting activation of GSK3ß/NF-κB/NLRP3 signal pathway that is known to be associated with inflammation, thus exerting neuroprotective effects and preventing CUMS-induced depressive-like behaviors.

Astragaloside IV ameliorates neuroinflammation-induced depressive-like behaviors in mice via the PPARγ/NF-κB/NLRP3 inflammasome axis.

Major depressive disorder is a common but devastating mental disorder, and recent evidence shows that neuroinflammation may play a pivotal role in the etiology of depression. Astragaloside IV (AS-IV) is an active component purifed from Astragalus membranaceus (Fisch) Bge, which has shown anti-inflammatory, anti-oxidative and anti-apoptotic effects. In this study, we explored whether AS-IV produced antidepressant effects via its inhibition of neuroinflammation in mouse models of depression. Depressive-like behaviors including decreased sucrose consumption, reduced locomotor activity and increased immobility time were induced in mice using repeated restraint stress (RRS). We found that administration of AS-IV (16, 32 and 64 mg·kg-1·d-1, ig) significantly attenuated RRS-induced depressive-like behaviors. Furthermore, AS-IV administration significantly reduced the levels of TNF-α and IL-1ß, increased PPARγ expression and GSK3ß phosphorylation, decreased NF-κB phosphorylation, and reduced NOD-, LRR- and pyrin domain-containingprotein 3 (NLRP3) inflammasome and caspase-1 p20 generation in the hippocampus of the mice. LPS-induced depression-like behaviors were induced by LPS injection (1 mg·kg-1·d-1, ip), which were ameliorated by administration of AS-IV (20, 40 mg·kg-1·d-1, ig). The results of the LPS-induced mouse model were in accordance with those acquired from the RRS-induced mouse model: LPS injection significantly increased TNF-α and IL-1ß expression in the mouse hippocampus, which was reversed by administration of AS-IV. Moreover, administration of AS-IV significantly increased PPARγ expression and GSK3ß phosphorylation, and decreased NF-κB phosphorylation and NLRP3 inflammasome. These results suggest that AS-IV is a potential drug against depression, and its antidepressant effects are partially mediated by inhibition of neuroinflammation via the upregulation of PPARγ expression.

Geraniol produces antidepressant-like effects in a chronic unpredictable mild stress mice model.

Geraniol (GE), which has neuroprotection and anti-inflammation activities, is mostly abundant in the essential oils of rose, ginger, lemon, orange, lavender etc. However, its antidepressant-like effect has not been reported before. The present study was designed to investigate whether GE confers an antidepressant effect in mice exposed to a chronic unpredictable mild stress (CUMS) model of depression and to explore its possible mechanisms. The results showed that GE treatments for 3 weeks significantly alleviated the depression-related behaviors of CUMS-exposed mice, as indicated by restored decreased sucrose preference and shortened immobile time in both the forced swimming tests (FST) and tail suspension tests (TST). And these ameliorative effects of GE treatment were involved with regulating CUMS-induced pro-inflammatory cytokine interleukin-1 beta (IL-1ß) related neuro-inflammation. We further found that GE treatment reversed CUMS-induced IL-1ß elevation, possibly by inhibiting nuclear factor kappa B (NF-κB) pathway activation and regulating nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome expression. Taken together, our findings suggested that GE exerted a potential antidepressant-like effect in CUMS mice model of depression, which may provide an insight into the potential of GE in therapeutic implications for depression.

Cardioprotective effects of timosaponin B II from Anemarrhenae asphodeloides Bge on isoproterenol-induced myocardial infarction in rats.

The aim of the present study was to investigate the cardioprotective effects of Timosaponin B II (TB), a main bioactive constituent from Anemarrhenae asphodeloides Bge, on an isoproterenol (ISO)-induced myocardial infarction model in rats and explore its underlying mechanisms. Rats were treated with TB (50 mg/kg, 100 mg/kg) or diltiazem hydrochloride (DH, 5 mg/kg) by gastric gavage for five days. At the 4th and 5th days, myocardial injury was induced by ISO injection (85 mg/kg) at an interval of 24 h for 2 consecutive days. After the induction, rats were anaesthetized with pentobarbital sodium (30 mg/kg) to record the electrocardiogram. Our research showed that ISO administration resulted in significant elevations in the ST-segment, the levels of cardiac injury biomarkers creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), and concentrations of serum proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Pretreatment with TB significantly reversed these alterations induced by ISO challenge. The cardioprotective effects of TB were further proved by the histopathological examination. Exploration of the underlying mechanisms of its actions revealed that TB pretreatment restored the ISO-induced decrease of super oxide dismutase (SOD) and the increase of malondialdehyde (MDA). Meanwhile, we found that the enhancement of antioxidant defense system might be associated with the increased heme oxygenase isoform 1 (HO-1) induction and activated nuclear respiratory factor 2 (Nrf-2) translocation. Furthermore, the present research also demonstrated that nuclear translocation of Nrf-2 and subsequent HO-1 expression might be associated with nuclear factor kappa B (NF-κB) pathway activation. Taken together, our finding demonstrated that TB might have a potential benefit in preventing ischemic heart diseases like myocardial infarction.

Antidepressant-like effects of salidroside on olfactory bulbectomy-induced pro-inflammatory cytokine production and hyperactivity of HPA axis in rats.

Salidroside (SA) is the primary bioactive marker compound in the standardized extracts from Rhodiola rosea. Although it has potential antidepressant activity in a rat behavioral despair model, the mechanisms of antidepressant effect for SA remain unclear. The objective of this study was to evaluate the antidepressant effects of SA and to discuss the potential mechanisms in olfactory bulbectomized (OBX) rats. SA of 20, 40 mg/kg (p.o.) for 2 weeks notably alleviated OBX-induced hyperactivity in open field test, decreased immobility time in TST and FST. Chronic treatment with SA could remarkably reduce TNF-α and IL-1ß levels in hippocampus. Western blot showed that SA could markedly increase glucocorticoid receptor (GR) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Besides, SA could also attenuate corticotropin-releasing hormone (CRH) expression in hypothalamus, as well as reducing significantly the levels of serum corticosterone. In conclusion, this study demonstrated that OBX rats treated with SA could significantly improve the depressive-like behaviors. The antidepressant mechanisms of SA might be associated with its anti-inflammatory effects and the regulation of HPA axis activity. Reversal of abnormalities of GR may be partly responsible for those effects. These findings suggested that SA might become a beneficial agent to prevent and treat the depression.

Ma Huang Tang ameliorates asthma though modulation of Th1/Th2 cytokines and inhibition of Th17 cells in ovalbumin-sensitized mice.

AIM: Ma Huang Tang (Ephedra decoction, MHT) is a famous classical formula from Shang Han Lun by Zhang Zhongjing in the Han Dynasty. The anti-asthmatic effects of MHT and the possible mechanisms were tested. METHOD: An asthma model was established by ovalbumin (OVA)-induction in mice. A total of forty-eight mice were randomly assigned to six experimental groups: control, model, dexamethasone (2 mg·kg(-1)) and MHT (5, 10, and 20 mg·kg(-1)). Airway resistance (Raw) was measured by the forced oscillation technique, histological studies were evaluated by hematoxylin and eosin (HE) staining, Th1/Th2 and Th17 cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA), and Th17 cells were evaluated by flow cytometry (FCM). RESULTS: This study demonstrated that MHT inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4 and IL-17 levels were recovered in bronchoalveolar lavage fluid, increased IFN-γ level in bronchoalveolar lavage fluid. Histological studies demonstrated that MHT substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that MHT substantially inhibited Th17 cells. CONCLUSION: These findings suggest that MHT may effectively ameliorate the progression of asthma, and could be further investigated for potential use as a therapy for patients with allergic asthma.

Asiaticoside attenuates memory impairment induced by transient cerebral ischemia-reperfusion in mice through anti-inflammatory mechanism.

Asiaticoside (AS) is isolated from Centella asiatica (L.) which has been using for a long time as a memory enhancing drug in India. This study was to investigate the effects of AS on memory impairment and inflammatory cytokines expression induced by transient cerebral ischemia and reperfusion in mice, as well as the potential signaling pathway. Transient bilateral common carotid artery occlusion (tBCCAO) induced severe memory deficits in mice according to the Morris water maze task and the step-down passive avoidance test. Meanwhile the microglial activation and the gene expression of inflammatory cytokines including interleukin (IL)-1ß, interleukin (IL)-6 and tumor necrosis factor (TNF)-α were increased in the hippocampus of the mice with cerebral ischemia and reperfusion. Oral administration of AS (40 and 60 mg/kg, once per day, started the day after surgery and lasted for 7 days) significantly ameliorated the memory impairment and the inflammation. Moreover, AS (20, 40 and 60 mg/kg) markedly reduced the microglial overactivation and the phosphorylation of p38 MAPK in hippocampus compared with the transient cerebral ischemia and reperfusion group. These results suggested that AS showed the neuroprotective effect against transient cerebral ischemia and reperfusion in mice, and this effect might be associated with the anti-inflammation effect of AS via inhibiting overactivation of p38 MAPK pathway.

Effects of perillaldehyde on alternations in serum cytokines and depressive-like behavior in mice after lipopolysaccharide administration.

Perillaldehyde (PAH), a major component of essential oil of Perilla Frutescens, has antidepressant-like effects and anti-inflammatory effects. The present study was designed to determine whether PAH is effective in treating lipopolysaccharide (LPS)-induced depression-like behavior in mice and to explore the possible mechanism between its antidepressant-like effect and anti-inflammatory activity. PAH (60 and 120 mg/kg) and fluoxetine (20mg/kg) were administered intragastrically once daily for 7 consecutive days. In the 7th day, LPS (0.5mg/kg) was injected intraperitoneally 30 min after drug administration. Blood samples were collected 90 min after LPS injection to evaluate serum interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels by enzyme-linked immunosorbent assay (ELISA). Behavioral tests were measured 24h after LPS injection. After the behavioral tests the prefrontal cortex was rapidly dissected from the brain of the sacrificed mice, then the 5-hydroxytryptamine (5-HT) and norepinephrine (NE) levels in prefrontal cortex were determined by HPLC-MS, and IL-6 and TNF-α mRNA expression was measured using quantitative real-time PCR. Our results showed that a single administration of LPS significantly increased the levels of TNF-α and IL-6 in both the serum and the prefrontal cortex and decreased 5-HT and NE levels in the prefrontal cortex in mice. Pretreatment with fluoxetine (20mg/kg) or PAH (60 and 120 mg/kg) could effectively reverse the alterations in the concentrations of 5-HT and NE, and attenuate LPS-induced increases in TNF-α and IL-6 levels. Besides, LPS administration increased the immobility time in tail suspension test (TST) and forced swimming test (FST) without affecting spontaneous locomotor activity. Fluoxetine (20mg/kg) or PAH (60 and 120 mg/kg) significantly shortened LPS-induced increases of immobility time in both TST and FST. In conclusion, PAH exhibited significant antidepressant-like effects in mice with LPS-induced depression. The antidepressant activity of PAH might be related to the alteration of monoaminergic responses and the anti-inflammatory effects.

Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules.

The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4-60 ºC for 15 min, had a wide range pH tolerance of 6.0-11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (µg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1-0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plant-growth promoting agent.

Colonization and plant growth promoting characterization of endophytic Pseudomonas chlororaphis strain Zong1 isolated from Sophora alopecuroides root nodules

The endophytic strain Zong1 isolated from root nodules of the legume Sophora alopecuroides was characterized by conducting physiological and biochemical tests employing gfp-marking, observing their plant growth promoting characteristics (PGPC) and detecting plant growth parameters of inoculation assays under greenhouse conditions. Results showed that strain Zong1 had an effective growth at 28 ºC after placed at 4-60 ºC for 15 min, had a wide range pH tolerance of 6.0-11.0 and salt tolerance up to 5% of NaCl. Zong1 was resistant to the following antibiotics (µg/mL): Phosphonomycin (100), Penicillin (100) and Ampicillin (100). It could grow in the medium supplemented with 1.2 mmol/L Cu, 0.1% (w/v) methylene blue and 0.1-0.2% (w/v) methyl red, respectively. Zong1 is closely related to Pseudomonas chlororaphis based on analysis the sequence of 16S rRNA gene. Its expression of the gfp gene indicated that strain Zong1 may colonize in root or root nodules and verified by microscopic observation. Furthermore, co-inoculation with Zong1 and SQ1 (Mesorhizobium sp.) showed significant effects compared to single inoculation for the following PGPC parameters: siderophore production, phosphate solubilization, organic acid production, IAA production and antifungal activity in vitro. These results suggest strains P. chlororaphi Zong1 and Mesorhizobium sp. SQ1 have better synergistic or addictive effect. It was noteworthy that each growth index of co-inoculated Zong1+SQ1 in growth assays under greenhouse conditions is higher than those of single inoculation, and showed a significant difference (p < 0.05) when compared to a negative control. Therefore, as an endophyte P. chlororaphis Zong1 may play important roles as a potential plantgrowth promoting agent.

Characterization of a copper-resistant symbiotic bacterium isolated from Medicago lupulina growing in mine tailings.

A root nodule bacterium, Sinorhizobium meliloti CCNWSX0020, resistant to 1.4 mM Cu2+ was isolated from Medicago lupulina growing in mine tailings. In medium supplied with copper, this bacterium showed cell deformation and aggregation due to precipitation of copper on the cell surface. Genes similar to the copper-resistant genes, pcoR and pcoA from Escherichia coli, were amplified by PCR from a 1.4-Mb megaplasmid. Inoculation with S. meliloti CCNWSX0020 increased the biomass of M. lupulina grown in medium added 0 and 100 mg Cu2+ kg(-1) by 45.8% and 78.2%, respectively, and increased the copper concentration inside the plant tissues grown in medium supplied with 100 µM Cu2+ by 39.3%, demonstrating that it is a prospective symbiotic system for bioremediation purposes.

Ameliorative and neuroprotective effect in MPTP model of Parkinson's disease by Zhen-Wu-Tang (ZWT), a traditional Chinese medicine.

AIM OF THE STUDY: Traditional Chinese medicine Zhen-Wu-Tang (ZWT) is a well-known PentaHerbs formula from "Treatise on Febrile Disease". This study is to elucidate its neuroprotective effect and mechanism of ameliorative effect of the syndrome of Parkinson's disease (PD). MATERIALS AND METHODS: The ameliorative effect of ZWT on symptom of PD through behavior tests including: swimming test, the tail suspension test and open-field test was investigated. The neuroprotective effect of dopaminergic neurons from the striatum and frontal cortex of brain was detected by high performance liquid chromatography with electrochemical detection (HPLC-ECD). RESULTS: This study proved that ZWT could ameliorate the typical symptom of PD and protect dopaminergic system. CONCLUSION: These results suggested that ZWT possessed protective and ameliorative properties of dopaminergic neurons.