Cholecystokinin release triggered by NMDA receptors produces LTP and sound-sound associative memory.

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.

Engineered core-shell magnetic nanoparticle for MR dual-modal tracking and safe magnetic manipulation of ependymal cells in live rodents.

Tagging recognition group(s) on superparamagnetic iron oxide is known to aid localisation (imaging), stimulation and separation of biological entities using magnetic resonance imaging (MRI) and magnetic agitation/separation (MAS) techniques. Despite the wide applicability of iron oxide nanoparticles in T 2-weighted MRI and MAS, the quality of the images and safe manipulation of the exceptionally delicate neural cells in a live brain are currently the key challenges. Here, we demonstrate the engineered manganese oxide clusters-iron oxide core-shell nanoparticle as an MR dual-modal contrast agent for neural stem cells (NSCs) imaging and magnetic manipulation in live rodents. As a result, using this engineered nanoparticle and associated technologies, identification, stimulation and transportation of labelled potentially multipotent NSCs from a specific location of a live brain to another by magnetic means for self-healing therapy can therefore be made possible.

Small interfering RNA specific for N-methyl-D-aspartate receptor 2B offers neuroprotection to dopamine neurons through activation of MAP kinase.

In the present study, N-methyl-D-aspartate receptor 2B (NR2B)-specific siRNA was applied in parkinsonian models. Our previous results showed that reduction in expression of N-methyl-D-aspartate receptor 1 (NR1), the key subunit of N-methyl-D-aspartate receptors, by antisense oligos ameliorated the motor symptoms in the 6-hydroxydopamine (6-OHDA)-lesioned rat, an animal model of Parkinson's disease (PD).

20(S)-protopanaxadiol promotes the migration, proliferation, and differentiation of neural stem cells by targeting GSK-3ß in the Wnt/GSK-3ß/ß-catenin pathway.

BACKGROUND: Active natural ingredients, especially small molecules, have recently received wide attention as modifiers used to treat neurodegenerative disease by promoting neurogenic regeneration of neural stem cell (NSC) in situ. 20(S)-protopanaxadiol (PPD), one of the bioactive ingredients in ginseng, possesses neuroprotective properties. However, the effect of PPD on NSC proliferation and differentiation and its mechanism of action are incompletely understood. METHODS: In this study, we investigated the impact of PPD on NSC proliferation and neuronal lineage differentiation through activation of the Wnt/glycogen synthase kinase (GSK)-3ß/ß-catenin pathway. NSC migration and proliferation were investigated by neurosphere assay, Cell Counting Kit-8 assay, and EdU assay. NSC differentiation was analyzed by Western blot and immunofluorescence staining. Involvement of the Wnt/GSK3ß/ß-catenin pathway was examined by molecular simulation and Western blot and verified using gene transfection. RESULTS: PPD significantly promoted neural migration and induced a significant increase in NSC proliferation in a time- and dose-dependent manner. Furthermore, a remarkable increase in antimicrotubule-associated protein 2 expression and decrease in nestin protein expression were induced by PPD. During the differentiation process, PPD targeted and stimulated the phosphorylation of GSK-3ß at Ser9 and the active forms of ß-catenin, resulting in activation of the Wnt/GSK-3ß/ß-catenin pathway. Transfection of NSCs with a constitutively active GSK-3ß mutant at S9A significantly hampered the proliferation and neural differentiation mediated by PPD. CONCLUSION: PPD promotes NSC proliferation and neural differentiation in vitro via activation of the Wnt/GSK-3ß/ß-catenin pathway by targeting GSK-3ß, potentially having great significance for the treatment of neurodegenerative diseases.

The selective vulnerability of retinal ganglion cells in rat chronic ocular hypertension model at early phase.

Glutamate neurotoxicity has been postulated to play a prominent role in glaucoma. In this study the possible roles of two subunits of glutamate receptors during the early phase of retinal ganglion cell (RGC) loss in a rat chronic ocular hypertension (COH) model were investigated. COH was induced by applying argon laser to the episcleral and limbal veins of the right eye of rats, the observation times were at 4, 14 and 28 days after the first laser. RGCs were retrogradely labeled by putting Fluoro-Gold (FG) on the surface of both side superior colliculus. Immunohistochemical staining using specific antibodies against N-methyl-D-aspartate receptor 1 (NR1) or glutamate receptor 2/3 (GluR2/3) was performed on the retinal sections of normal and COH eyes. Fluorescent images were captured using confocal laser scanning microscope and the number of NR1 and GluR2/3 labeled cells were counted and cell size was measured using Stereo Investigator. During the observation period, the numbers of NR1 and GluR2/3 positive RGCs in the RGC layer were reduced parallel to the loss of RGC. The dramatic loss of GluR2/3 immunoreactive neurons occurred starting immediately after the first laser to 4 days while the dramatic loss of NR1 immunoreactive neurons occurred from 14 to 28 days after the first laser. Size difference was detected in NR1 immunoreactive RGCs, large ones were more sensitive to the high ocular pressure. These results suggest that both NR1 and GluR2/3 are involved in the mediation of RGC death in the early stage of COH.

Dynamic effect of beta-amyloid 42 on cell mechanics.

Aß1-42, which is highly toxic to neural cells, is commonly present in the brains of people with Alzheimer's disease. In this study, dynamic changes in cell mechanics were monitored under Aß-induced toxicity. To investigate the changes in cellular mechanical properties, we used Aß1-42 oligomer at different concentrations to treat human neuroblastoma SH-SY5H cells. Results demonstrated a two-stage dynamic change in cell mechanics during neurodegeneration. Additionally, Young's modulus (YM) of the treated cells increased in a short period. The reasons include alteration in surface tension, osmotic pressure, and actin polymerization. Rough cellular membranes were observed from atomic force microscope (AFM) measurement. However, the cellular YM gradually decreased when the cells were continuously exposed to Aß1-42 or to a high concentration of Aß1-42. The major reason for the decreased YM was microtubule disassembly. Dynamic change in YM reflects different activities in cytoplasm in response to Aß1-42. The characteristic changes in cell mechanics provided insights into the dynamic neurodegeneration process of cells induced by Aß1-42 oligomer.

Developmental maturation of ionotropic glutamate receptor subunits in rat vestibular nuclear neurons responsive to vertical linear acceleration.

We investigated the maturation profile of subunits of ionotropic glutamate receptors in vestibular nuclear neurons that were activated by sinusoidal linear acceleration along the vertical plane. The otolithic origin of Fos expression in these neurons was confirmed as a marker of functional activation when labyrinthectomized and/or stationary control rats contrasted by showing sporadically scattered Fos-labeled neurons in the vestibular nuclei. By double immunohistochemistry for Fos and one of the receptor subunits, otolith-related neurons that expressed either alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate or N-methyl-d-aspartate subunits were first identified in the medial vestibular nucleus, spinal vestibular nucleus and Group x by postnatal day (P)7, and in the lateral vestibular nucleus and Group y by P9. No double-labeled neurons were found in the superior vestibular nucleus. Within each vestibular subnucleus, these double-labeled neurons constituted approximately 90% of the total Fos-labeled neurons. The percentage of Fos-labeled neurons expressing the GluR1 or NR2A subunit showed developmental invariance in all subnuclei. For Fos-labeled neurons expressing the NR1 subunit, similar invariance was observed except that, in Group y, these neurons decreased from P14 onwards. For Fos-labeled neurons expressing the GluR2, GluR2/3, GluR4 or NR2B subunit, a significant decrease was found by the adult stage. In particular, those expressing the GluR4 subunit showed a two- to threefold decrease in the medial vestibular nucleus, spinal vestibular nucleus and Group y. Also, those expressing the NR2B subunit showed a twofold decrease in Group y. Taken together, the postsynaptic expression of ionotropic glutamate receptor subunits in different vestibular subnuclei suggests that glutamatergic transmission within subregions plays differential developmental roles in the coding of gravity-related vertical spatial information.

Biophysical Characteristics of Human Neuroblastoma Cell in Oligomeric $\beta $ -Amyloid (1-40) Cytotoxicity.

Beta amyloid ( ) peptide, which is a common neuropathological hallmark deposit in the brain of patients with Alzheimer's disease, typically comprises 39-43 amino acid residues. peptides exist as isoforms of and with various lengths. In this research, atomic force microscopy (AFM) was applied to investigate aggregations in Hank's Balanced Salt Solution. Toxic effect of oligomer was investigated in live SH-SY5Y neuroblastoma cells by characterizing cell morphology and cell mechanics using high-resolution AFM scanning. oligomer-induced cytoskeleton reorganization was also observed under confocal microscopy, and it can account for reduction in Young's modulus of cells. Meanwhile, phosphorylation of tau increased after oligomer treatment, possibly resulting in microtubule disassembly. This paper demonstrates the linkage between cellular mechanical changes and neurodegeneration mediated by . The method used implies promising applications of real-time monitoring of cellular mechanical properties given the toxic effects of on living neuronal cells.

Fluoro-substituted cyanine for reliable in vivo labelling of amyloid-ß oligomers and neuroprotection against amyloid-ß induced toxicity.

Alzheimer's disease (AD) is the most prevalent but still incurable neurodegenerative form of dementia. Early diagnosis and intervention are crucial for delaying the onset and progression of the disease. We herein report a novel fluoro-substituted cyanine, F-SLOH, which exhibits good Aß oligomer selectivity with a high binding affinity, attributed to the synergistic effect of strong π-π stacking and intermolecular CH···O and CH···F interactions. The selectivity towards the Aß oligomers in the brain was ascertained by in vitro labelling on tissue sections and in vivo labelling through the systemic administration of F-SLOH in 7 month APP/PS1 double transgenic (Tg) and APP/PS1/Tau triple Tg mouse models. F-SLOH also shows remarkably effective inhibition on Aß aggregation and highly desirable neuroprotective effects against Aß-induced toxicities, including the inhibition of ROS production and Ca2+ influx. Its excellent blood-brain barrier (BBB) penetrability and low bio-toxicity further support its tremendous potential as a novel theranostic agent for both early diagnosis and therapy of AD.

Quantitative study of the coexpression of Fos and N-methyl-D aspartate (NMDA) receptor subunits in otolith-related vestibular nuclear neurons of rats.

The expression of NMDA receptor subunits (NR1 and NR2A/B) was demonstrated immunocytochemically in otolith-related neurons within the vestibular nuclear complex and its subnuclei of conscious Sprague-Dawley adult rats. All experimental animals were subjected to constant velocity off-vertical axis rotation (OVAR). The rotating gravity vector during OVAR sequentially activates hair cells on all sectors of the utricular maculae; neurons so activated within the vestibular nuclei were denoted by the expression of Fos protein. Control animals, i.e., labyrinthectomized rats subjected to OVAR and normal rats that remained stationary, showed only a few sporadically scattered labeled neurons. In the brainstem of normal rats subjected to OVAR, a high density of Fos-immunoreactive (Fos-ir) neurons was found in the vestibular nuclear complex (namely, spinal vestibular nucleus, SpVe; medial vestibular nucleus, Mve; superior vestibular nucleus, SuVe) and subnuclei (namely, group x and group y), whereas a lower density was found in the lateral vestibular nucleus (LVe). A double-immunofluorescence study indicated that both NR1 and NR2A/B subunits were highly expressed in Fos-ir neurons within the vestibular nuclei. Fos/NR1 or Fos/NR2A/B double-labeled neurons constitute over three-quarters of the total number of Fos-ir neurons in SpVe, MVe, LVe, SuVe, and groups x and y. Our findings suggest that NMDA-type ionotropic glutamate receptors play a key role in the OVAR-induced neuronal activation of the vestibular nuclei, thus providing a morphological basis for further study of glutamatergic central otolith neurons and their involvement in sensorimotor regulation and autonomic functions of rats.

Fos expression in otolith-related brainstem neurons of postnatal rats following off-vertical axis rotation.

To determine the critical time of responsiveness of developing otolith organ-related brainstem neurons and their distribution, Fos protein expression in response to off-vertical axis rotations (OVAR) was mapped in conscious Sprague Dawley rats from P5 to adulthood. OVAR was used to activate sequentially all utricular hair cells per 360 degrees revolution. We detected the coding of horizontal head positions in otolith organ-related neurons within the vestibular nucleus as early as P7. In the vestibular nuclear complex and its subgroups, the density of Fos-immunoreactive (Fos-ir) neurons increased steadily with age and reached the adult level by P21. In both labyrinthectomized rats subjected to OVAR and normal rats kept stationary, labeled neurons were found sporadically in the aforementioned brain regions in each age group, confirming that Fos labeling observed in neurons of normal experimental rats subjected to OVAR was due to otolith organ stimulation. Whereas OVAR-induced Fos-ir neurons were also first observed in vestibular-related brain areas, such as the prepositus hypoglossal nucleus, gigantocellular reticular nucleus, and locus coeruleus, of normal experimental rats at P7, those in the inferior olive were observed only from P14 onward. This indicates the unique maturation time of inferior olivary neurons in gravity-related spatial coding. In general, age-dependent increase in OVAR-induced Fos-ir neurons was observed in brain areas that received otolith inputs. The locus coeruleus was exceptional in that prominent OVAR-induced Fos-ir neuronal number did not change with maturation, and this was well above the low but significant number of Fos-ir neurons in control preparations. Taken together, our results suggest that neuronal subpopulations within the developing network of the horizontal otolith system provide an anatomical basis for the postnatal development of otolith organ-related sensorimotor functions. J. Comp. Neurol. 470:282-296, 2004.

Non-opioid-dependent anti-inflammatory effects of low frequency electroacupuncture.

Low frequency electroacupuncture, which is commonly used in pain relief, is known to induce opioid-mediated analgesia. This study examined the contribution of the opioid system in mediating the anti-inflammatory effects of low frequency EA in a standard model of acute inflammation, the carrageenan-induced edema model. Carrageenan was injected in the hind paw of anesthetized rats and low frequency electroacupuncture was applied to acupoints equivalent to Zusanli (St 36) and Sanyinjiao (Sp 6) in humans just prior to the induction of inflammation in the ipsilateral leg. Induction of Fos protein, reflecting neuronal activation, was investigated in the spinal cord with immunohistochemistry. It was found that electroacupuncture strongly inhibited the carrageenan-induced edema by over 60%, and suppressed the associated Fos expression in the superficial laminae (I-II) of the ipsilateral dorsal horn by 50%. Neither the anti-edematous effect nor the suppression of Fos expression in the superficial spinal laminae was affected by intraperitoneal injection of the opioid antagonist naloxone. These results demonstrate that low frequency electroacupuncture is capable of inhibiting peripheral inflammation and the associated central neuronal activity via a non-opioid-dependent mechanism.

Multifunctional silica-coated iron oxide nanoparticles: a facile four-in-one system for in situ study of neural stem cell harvesting.

Neural stem cells (NSCs), which generate the main phenotypes of the nervous system, are multipotent cells and are able to differentiate into multiple cell types via external stimuli from the environment. The extraction, modification and re-application of NSCs have thus attracted much attention and raised hopes for novel neural stem cell therapies and regenerative medicine. However, few studies have successfully identified the distribution of NSCs in a live brain and monitored the corresponding extraction processes both in vitro and in vivo. To address those difficulties, in this study multi-functional uniform nanoparticles comprising an iron oxide core and a functionalized silica shell (Fe(3)O(4)@SiO(2)(FITC)-CD133, FITC: a green emissive dye, CD133: anti-CD133 antibody) have been strategically designed and synthesized for use as probe nanocomposites that provide four-in-one functionality, i.e., magnetic agitation, dual imaging (both magnetic resonance and optical) and specific targeting. It is shown that these newly synthesized Fe(3)O(4)@SiO(2)(FITC)-CD133 particles have clearly demonstrated their versatility in various applications. (1) The magnetic core enables magnetic cell collection and T(2) magnetic resonance imaging. (2) The fluorescent FITC embedded in the silica framework enables optical imaging. (3) CD133 anchored on the outermost surface is demonstrated to be capable of targeting neural stem cells for cell collection and bimodal imaging.

Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration.

Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.

Ginsenosides attenuate methylglyoxal-induced impairment of insulin signaling and subsequent apoptosis in primary astrocytes.

Diabetes mellitus (DM), which is characterized by chronic hyperglycemia, is known to increase the risk of neurodegeneration. In type 2 diabetes, hyperglycemia could cause insulin resistance and neurodegeneration in various cells including neurons and astrocytes. Hyperglycemia is also known to result in the formation of advanced glycation end-products (AGE) Methylglyoxal (MG) is one of the most reactive AGE precursors in which its abnormal accumulation is usually found in diabetic patients and induces neuronal cell death in central nervous system. Ginseng is a herb that has been widely used to treat various diseases in traditional Chinese medicine. Ginsenosides, the pharmacologically active component isolated from ginseng, have been shown to have cryoprotective effects in different neural cells. In the present study we investigated the effects of MG in disturbing insulin signaling and leading to further cellular apoptosis in rat primary astrocytes. Furthermore, the protective effects of different subtypes of ginsenosides were studied. From the results, impairment of insulin signaling was found in astrocytes under MG treatment. Moreover, cleavage of caspase and Poly ADP ribose polymerase (PARP) was observed in line with insulin signaling disruption, showing the neurotoxic effects of MG towards astrocytes. The effects of ginsenosides in MG treated astrocytes were also investigated. After treatment, ginsenosides Rd and R-Rh2 were shown to ameliorate the cell viability of MG-treated astrocytes. In addition, Rd and R-Rh2 could improve insulin signaling and inhibit apoptosis, indicating that Rd, R-Rh2 and related compounds may have therapeutic potential in treating diabetes-induced neurodegeneration.

Assessments of the effects of nicotine and ketamine using tyrosine hydroxylase-green fluorescent protein transgenic zebrafish as biosensors.

Transgenic zebrafish are a common vertebrate model system for the study of addictive behavior. In the present study, plasmid constructs containing green fluorescent protein (GFP) and the promoter of tyrosine hydroxylase (TH), a key synthetic enzyme for catecholamines, were produced. The TH-GFP constructs were microinjected into zebrafish embryonic cells. Three days post-fertilization, GFP began expressing in distinct catecholaminergic areas. The TH-GFP transgenic zebrafish were employed as live biosensors to test the effects of the commonly abused drugs nicotine and ketamine. First, locomotion assays were used to study the general excitatory effects of the drugs. Maximal locomotor activity was obtained after treatment with a high concentration of nicotine (10 µM), but with a much lower concentration of ketamine (0.1 µM). Second, TH protein levels in zebrafish brains were assessed by Western blot. TH protein levels were significantly increased, with maximal protein levels found after treatment with the same drug concentrations that gave maximal locomotor activity. Importantly, analysis of GFP in the zebrafish catecholaminergic areas revealed the same expression patterns as was obtained by Western blot. The present results indicate that increased locomotor activity can be correlated to TH protein expression, as indicated by Western blot and expression of TH-GFP. We have shown that TH-GFP expression is a reliable method to show the effects of drugs on TH expression that may be employed as a novel high-throughput live biosensor for screening drugs of abuse.

N-Acetyl-l-cysteine capped quantum dots offer neuronal cell protection by inhibiting beta (1-40) amyloid fibrillation.

This is the first work that revealed the neuro-protective effect of functionalized quantum dots against the cytotoxicity induced by beta-amyloid peptides. This study gives insight into the future treatment of Alzheimer's disease. It opens many avenues for the development of the next generation nanotechnology for biomedical and therapeutic applications.

Modulation of endoplasmic reticulum chaperone GRP78 by high glucose in hippocampus of streptozotocin-induced diabetic mice and C6 astrocytic cells.

Diabetes mellitus is known to increase the risk of neurodegeneration, and both diseases are reported to be linked to dysfunction of endoplasmic reticulum (ER). Astrocytes are important in the defense mechanism of central nervous system (CNS), with great ability of tolerating accumulation of toxic substances and sensitivity in Ca(2+) homeostasis which are two key functions of ER. Here, we investigated the modulation of the glucose-regulated protein 78 (GRP78) in streptozotocin (STZ)-induced diabetic mice and C6 cells cultured in high glucose condition. Our results showed that more reactive astrocytes were presented in the hippocampus of STZ-induced diabetic mice. Simultaneously, decrease of GRP78 expression was found in the astrocytes of diabetic mice hippocampus. In in vitro study, C6 cells were treated with high glucose to investigate the role of high glucose in GRP78 modulation in astrocytic cells. GRP78 as well as other chaperones like GRP94, calreticulin and calnexin, transcription levels were down-regulated after high glucose treatment. Also C6 cells challenged with 48h high glucose were activated, as indicated by increased level of glial fibrillary acidic protein (GFAP). Activated C6 cells simultaneously exhibited significant decrease of GRP78 level and was followed by reduced phosphorylation of Akt. Moreover, unfolded protein response was induced as an early event, which was marked by the induction of CHOP with high glucose treatment, followed by the reduction of GRP78 after 48h. Finally, the upsurge of ROS production was found in high glucose treated C6 cells and chelation of ROS could partially restore the GRP78 expression. Taken together, these data provide evidences that high glucose induced astrocytic activation in both in vivo and in vitro diabetic models, in which modulation of GRP78 would be an important event in this activation.

Detection of water toxicity using cytochrome P450 transgenic zebrafish as live biosensor: for polychlorinated biphenyls toxicity.

Cytochrome P450 (CYPs) is significant in degradation of endogenous substrates and detoxification of carcinogens, therefore it is a biomarker for assessment of polycyclic aromatic hydrocarbons (PAHs) level in aquatic environment. In the present study, a transgenic line of zebrafish had been generated using a CYP-green fluorescence protein (CYP-GFP) construct, driven by CYP1A1 promoter. Polychlorinated biphenyls (PCBs) were used as toxicant, in concentrations of 0.02 µg/ml, 0.04 µg/ml, 0.08 µg/ml, 0.4 µg/ml, and 0.8 µg/ml. The transgenic control fish showed low intensity of fluorescence in the liver. After exposed to PCBs, zebrafish had morphological changes such as expansion of yolk, contortion of tails and inflation of pericardial area. Green fluorescence signals were found to express according to concentrations and time. The green fluorescence signal was most intense after treatment with 0.08 µg/ml PCBs. However, the maximum area of green fluorescent signal was found at 0.04 µg/ml PCBs. GFP started to express at 3h exposure to PCBs, increasing its intensity until 6 h exposure, and then level off. Since the GFP expression is fast responding and is sensitive to low PAHs concentrations, transgenic fish is a good tool for live imaging and monitoring of aquatic contamination.