Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma-infected mice 5days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin (5-HT) and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and 5-HT metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.

Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure inhibits the turning of cerebellar granule cells in vivo and in vitro. First, in vivo studies using P10 mice demonstrated that a single intraperitoneal injection of ethanol not only reduces the number of turning granule cells but also alters the mode of turning at the EGL-ML border of the cerebellum. Second, in vitro analysis using microexplant cultures of P0-P3 mouse cerebella revealed that ethanol directly reduces the frequency of spontaneous granule cell turning in a dose-dependent manner. Third, the action of ethanol on the frequency of granule cell turning was significantly ameliorated by stimulating Ca(2+) and cGMP signaling or by inhibiting cAMP signaling. Taken together, these results indicate that ethanol affects the frequency and mode of cerebellar granule cell turning through alteration of the Ca(2+) and cyclic nucleotide signaling pathways, suggesting that the abnormal allocation of neurons found in the brains of FASD and FSA patients results, at least in part, from impaired turning of immature neurons by alcohol.

The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5' to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly conserved regions within the transgene: 400 bp immediately 5' to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind basic helix-loop-helix (bHLH) transcription factors, including motifs activated by ATOH1. Cotransfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared with the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.

Nuclear distribution factor E homolog like 1 (NDEL1) plays an important role in mitosis, neuronal migration, and microtubule organization during brain development by binding to disrupted-in-schizophrenia-1 (DISC1) or lissencephaly (LIS1). Although some evidence has suggested that DISC1 expression is altered in epilepsy, few studies have reported the relationship between NDEL1 and the etiology of epilepsy. In present study, we first investigated the expression of NDEL1 and its binding protein DISC1 after pilocarpine-induced epilepsy in male C57BL/6 mice. Data revealed that the mRNA and protein expression of NDEL1 and DISC1 in the whole hippocampus increased during the spontaneous seizure period after status epilepticus (SE). Interestingly, however, the expression of NDEL1 was decreased in the cornu ammonis 3 (CA3) and dentate gyrus (DG) regions. Moreover, SE also increased the number of blood vessels that fed the CA3 and DG regions of the hippocampus and increased the incidence of abnormalities in capillary network formation where NDEL1 protein was expressed positively. Meanwhile, the expression of phosphorylated ERK (p-ERK) was also increased during the spontaneous seizure period, with a similar expression pattern as NDEL1 and DISC1. Based on these results, we hypothesize that NDEL1 might interact with DISC1 to activate ERK signaling and function as a potential protective factor during the spontaneous seizure period after pilocarpine-induced SE.

This meta-analysis aims to investigate the association between mutations of glucocerebrosidase (GBA) gene and susceptibility to Parkinson's disease (PD) in a European population. Several electronic databases were extensively searched. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association. In total, fourteen published papers screening L444P, N370S and other GBA variants were identified. The GBA mutations were significantly associated with PD in the European population. Subgroup analysis stratified by the age of onset (AAO) revealed that the association between GBA mutations and PD existed in the patients with age at onset ⩽50 years but did not exist in the patients with age at onset >50 years. Furthermore, the associations between N370S, and L444P with PD were also analyzed to explore the roles of the two most frequent GBA mutations in the development of PD. The results showed that significant associations between N370S, and L444P with PD were observed, respectively. Overall, the study supported that GBA mutations were a risk factor for PD in the European population. Patients with early-onset were more likely to carry GBA mutations than those with late-onset. Moreover, both L444P and N370S were associated with increased PD risk.

Transplantation of bone marrow stromal cells (BMSCs) improves animal neurological functional recovery after stroke. To obtain insight into the mechanisms underlying the therapeutic benefit, we directed our attention to the interaction of BMSCs with astrocytes. Astrocytes become reactive (astrogliosis) after a brain injury, such as stroke. Astrogliosis plays both beneficial and detrimental roles in brain recovery. Previously, we have shown that administration of BMSCs to animals with stroke significantly reduces the thickness of the scar wall formed by reactive astrocytes. We tested the influence of mouse bone marrow stromal cell (mBMSC) on astrogliosis under oxygen-glucose deprivation (OGD)/reoxygenation conditions in vitro, employing an anaerobic chamber. Our data indicate that mBMSCs down-regulate glial fibrillary acidic protein (GFAP) expression in astrocytes after 2 h of OGD and an additional 16 h reoxygenation. mBMSCs protected astrocytes from ischemia, maintaining morphological integrity and proliferation. The IL-6/IL-6R/gp130 pathway is associated with astrogliosis in response to CNS (disorders. Therefore, we examined the effects of mBMSC on the IL-6/IL-6R/gp130 pathway as an underlying mechanism of mBMSC-altered astrogliosis. Furthermore, IL-6 siRNA was used to block IL-6 expression in astrocytes to further investigate IL-6 involvement in mBMSC-altered astrogliosis. Our results indicate that the mBMSC-conferred decline of astrogliosis post-ischemia may derive from the down-regulation of the IL-6/IL-6R/gp130 pathway.

Dendrites and spines undergo dynamic changes in physiological conditions, such as learning and memory, and in pathological conditions, such as Alzheimer's disease and epilepsy. Long-term dendritic plasticity has also been reported after ischemia/hypoxia, which might be compensatory effects of surviving neurons for the functional recovery after the insults. However, the dendritic changes shortly after ischemia, which might be associated with the pathogenesis of ischemic cell death, remain largely unknown. To reveal the morphological changes of ischemia-vulnerable neurons after ischemia, the present study investigated the alteration of dendritic arborization of CA1 pyramidal neurons in rats after transient cerebral ischemia using intracellular staining technique in vivo. The general appearance of dendritic arborization of CA1 neurons within 48 h after ischemia was similar to that of control neurons. However, a dramatic increase of dendritic disorientation was observed after ischemia with many basal dendrites coursed into the territory of apical dendrites and apical dendrites branched into the region of basal dendrites. In addition, a significant increase of apical dendritic length was found 24 h after ischemia. The increase of dendritic length after ischemia was mainly due to the dendritic sprouting rather than the extension of individual dendrites, which mainly occurred in the middle segment of the apical dendrites. These results reveal a plasticity change in dendritic arborization of CA1 neurons shortly after cerebral ischemia.

In the brain, CB1 cannabinoid receptors primarily mediate the effects of cannabinoids, but CB2 cannabinoid receptors (CB2Rs) have recently been discovered in the nervous system and also implicated in neuromodulatory roles. To understand the mechanisms of CB2R functions in the brain, it is essential to localize CB2Rs, but the types of cells expressing CB2Rs have been controversial. Unequivocal localization of CB2Rs in the brain has been impeded in part by the low expression levels of CB2Rs and poor specificity of detection methods. Here, we used an ultrasensitive and specific in situ hybridization method called the RNAscope to determine the spatial pattern of CB2R mRNA expression in the mouse hippocampus. CB2R mRNAs were mostly expressed in a subset of excitatory and inhibitory neurons in the CA1, CA3 and dentate gyrus areas, but rarely in microglia. CB2R knock-out mice were used as a negative control. Using the quantitative real-time polymerase chain reaction, we also found that the temporal pattern of CB2R mRNA expression was stable during postnatal development. Consistent with previous reports, the immunological detection of CB2Rs was not reliable, implying extremely low levels of the protein expression and/or insufficient specificity of the current anti-CB2R antibodies. Our findings of the expression patterns of CB2R mRNAs may help determine the cell types involved in, and hence the mechanisms of, the CB2R-mediated neuromodulation.

A 10 min exposure of rat hippocampal slices to hypoxic/hypoglycemic medium decreased tissue adenosine 5'-triphosphate (ATP) levels. Hypoxia/hypoglycemia also caused an anoxic depolarization and essentially no recovery of the synaptically evoked population spike from CA1 region recorded 30 min after re-introduction of normoxic/normoglycemic medium. Removal of Ca2+ or the addition of either the non-competitive N-methyl-D-aspartate antagonist dizocilpine maleate, the inorganic Ca2+ channel antagonist Co2+; or the Na+ channel blocker tetrodotoxin to hypoxic/hypoglycemic medium improved recovery of the evoked population spike upon re-oxygenation. Dizocilpine maleate, Co2+, and tetrodotoxin spared ATP during exposure to hypoxia/hypoglycemia. In contrast, Ca(2+)-free medium facilitated recovery of the population spike but did not preserve ATP during hypoxia/hypoglycemia. Dizocilpine maleate, Co2+ or dantrolene, when added to Ca(2+)-free medium, did not preserve ATP. Tetrodotoxin, when added to Ca(2+)-free medium, was effective in sparing ATP in hypoxic/hypoglycemic medium. To determine the effect of anoxic depolarization on ATP levels, hippocampal slices were collected just before and after the depolarization. There appeared to be an abrupt drop in ATP associated with the anoxic depolarization. We conclude that Na+ influx plays a relatively larger role in ATP consumption during hypoxia/hypoglycemia than Ca2+ influx. In addition, the anoxic depolarization imposes a large and rapid drop in ATP levels.

Transplantation of bone marrow stromal cells improves animal neurological functional recovery after stroke. Astrocytes are known to provide structural, trophic and metabolic support for neurons. Thus astrocytes are critical for neural survival during post-ischemia. However, information on the effects of bone marrow stromal cells on astrocytic survival post-ischemia is unavailable. We investigated the influence of rat bone marrow stromal cells on rat astrocytic apoptosis and survival post-ischemia employing an anaerobic chamber. Our data indicate that rat bone marrow stromal cells reduce cell death and apoptosis, and increase the DNA proliferation rate in astrocytes post-ischemia. Mitogen-activated protein kinase kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways are involved in cell survival. Western blot showed that rat bone marrow stromal cells activate these two pathways in astrocytes post-ischemia, and upregulate total extracellular signal regulated kinase 1/2 and threonine protein kinase. Since astrocytes produce various neurotrophic factors, we performed reverse transcription polymerase chain reaction to investigate rat bone marrow stromal cells' effect on astrocyte growth factor gene expression post-ischemia. We observed that brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor gene expression was enhanced by rat bone marrow stromal cell coculture. These data suggest that bone marrow stromal cells increase astrocytic survival post-ischemic injury. This protective function might involve the activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/threonine protein kinase pathways. Upregulation of brain-derived neurotrophic factor, vascular endothelial growth factor and basic fibroblast growth factor may also contribute to astrocyte survival.

Studies have shown a few cerebral metabolites modified by cocaine in brain regions; however, endogenous metabolic profiling has been lacking. Ex vivo (1)H NMR (hydrogen-1 nuclear magnetic resonance) spectroscopy-based metabonomic approach coupled with partial least squares was applied to investigate the changes of cerebral metabolites in nucleus accumbens (NAc) and striatum of rats subjected to cocaine treatment. Our results showed that both single and repeated cocaine treatment can induce significant changes in a couple of cerebral metabolites. The increase of neurotransmitters glutamate and gamma-amino butyric acid (GABA) were observed in NAc and striatum from the rats repeatedly treated with cocaine. Creatine and taurine increased in NAc whereas taurine increased and creatine decreased in striatum after repeated cocaine treatment. Elevation of N-acetylaspartate in NAc and striatum and decrease of lactate in striatum were observed, which may reflect the mitochondria dysregulation caused by cocaine; moreover, alterations of choline, phosphocholine and glycerol in NAc and striatum could be related to membrane disruption. Moreover, groups of rats with and without conditioned place preference (CPP) apparatus are presenting difference in metabolites. Collectively, our results provide the first evidence of metabonomic profiling of NAc and striatum in response to cocaine, exhibiting a regionally-specific alteration patterns. We find that repeated cocaine administration leads to significant metabolite alterations, which are involved in neurotransmitter disturbance, oxidative stress, mitochondria dysregulation and membrane disruption in brain.

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.

In the present study, we hypothesized that thymosin beta 4 (Tbeta4) is a potential therapy of multiple sclerosis (MS). To test this hypothesis, SJL/J mice (n=21) were subjected to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated with saline or Tbeta4 (6 mg/kg, n=10) every 3 days starting on the day of myelin proteolipid protein (PLP) immunization for total five doses. Neurological function, inflammatory infiltration, oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes were measured in the brain of EAE mice. Double immunohistochemical staining was used to detect proliferation and differentiation of OPCs. Tbeta4 was used to treat N20.1 cells (premature oligodendrocyte cell line) in vitro, and proliferation of N20.1 cells was measured by bromodeoxyuridine (BrdU) immunostaining. Tbeta4 treatment improved functional recovery after EAE. Inflammatory infiltrates were significantly reduced in the Tbeta4 treatment group compared to the saline groups (3.6+/-0.3/slide vs 5+/-0.5/slide, P<0.05). NG2(+) OPCs (447.7+/-41.9 vs 195.2+/-31/mm(2) in subventricular zone (SVZ), 75.1+/-4.7 vs 41.7+/-3.2/mm(2) in white matter), CNPase(+) mature oligodendrocytes (267.5+/-10.3 vs 141.4+/-22.9/mm(2)), BrdU(+) with NG2(+) OPCs (32.9+/-3.7 vs 17.9+/-3.6/mm(2)), BrdU(+) with CNPase(+) mature oligodendrocytes (18.2+/-1.7 vs 10.7+/-2.2/mm(2)) were significantly increased in the Tbeta4 treated mice compared to those of saline controls (P<0.05). These data indicate that Tbeta4 treatment improved functional recovery after EAE, possibly, via reducing inflammatory infiltrates, and stimulating oligodendrogenesis.

Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway.

Silent mating type information regulation 2 homolog 1 (SIRT1) is a class III histone deacetylase and activates peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) which attenuates oxidative damage. Alpha-lipoic acid (ALA) has been proven to protect the rat brain against cerebral ischemia injury by reducing oxidative stress. However, the underlying mechanisms are poorly understood. In this study, we investigated the potential neuroprotection and the possible role of ALA in SIRT1 pathway.

Reactivation of consolidated memory initiates a memory reconsolidation process, during which the reactivated memory is susceptible to strengthening, weakening or updating. Therefore, effective interference with the memory reconsolidation process is expected to be an important treatment for drug addiction. The nucleus accumbens (NAc) has been well recognized as a pathway component that can prevent drug relapse, although the mechanism underlying this function is poorly understood. We aimed to clarify the regulatory role of the NAc in the cocaine memory reconsolidation process, by examining the effect of applying different pharmacological interventions to the NAc on Zif 268 and Fos B expression in the entire reward circuit after cocaine memory reactivation. Through the cocaine-induced conditioned place preference (CPP) model, immunohistochemical and immunofluorescence staining for Zif 268 and Fos B were used to explore the functional activated brain nuclei after cocaine memory reactivation. Our results showed that the expression of Zif 268 and Fos B was commonly increased in the medial prefrontal cortex (mPFC), the infralimbic cortex (IL), the NAc-core, the NAc-shell, the hippocampus (CA1, CA2, and CA3 subregions), the amygdala, the ventral tegmental area (VTA), and the supramammillary nucleus (SuM) following memory reconsolidation, and Zif 268/Fos B co-expression was commonly observed (for Zif 268: 51-68%; for Fos B: 52-66%). Further, bilateral NAc-shell infusion of MK 801 and SCH 23390, but not raclopride or propranolol, prior to addictive memory reconsolidation, decreased Zif 268 and Fos B expression in the entire reward circuit, except for the amygdala, and effectively disturbed subsequent CPP-related behavior. In summary, N-methyl-d-aspartate (NMDA) and dopamine D1 receptors, but not dopamine D2 or β adrenergic receptors, within the NAc-shell, may regulate Zif 268 and Fos B expression in most brain nuclei of the reward circuit after cocaine memory reactivation. These findings indicated that the NAc played a key role in regulating addictive memory reconsolidation by influencing the function of the entire addictive memory network.