G proteins mediate signal transductions generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain, but its precise role in the nervous tissue is not fully understood. In addition, Go is one of the major proteins in growth cone membranes, which implies an important role of Go in the regulation of axon outgrowth. In this study, we attempted to determine the role of Go in axon outgrowth. We overexpressed the a subunit of Go (ao) in F11 neuroblatoma cells and examined the effect of ao on the neurite outgrowth. In F11 cells, dibutyryl cAMP increased neurite outgrowth remarkably upto 0.1 mM in a concentration dependent manner, but in a less degree at higher concentration. In the presence of 0.5 mM dibutyryl cAMP, the differentiation of F11 cells was almost saturated and the cells exhibited a typical neuronal morphology. Overexpression of ao caused a reduction of neurite outgrowth by 77.4% in length while increasing the number of neurites by 2.2 fold. The average neurite length was 38.9+/-12.5 mm in the ao-overexpressing F11 cells but 172.3+/-25.9 mm in the untransfected cells The total number of nurites per cell was 5.6+/-0.4 in the ao-overexpressing cells but 2.5 0.2 in the untransfected cells. This result suggests that Go may play an important role in growth cone collapse during neuronal cell differentiation.
Axons ; Brain ; Cell Differentiation ; Growth Cones ; GTP-Binding Proteins ; Membranes ; Neurites* ; Neurons ; Neurotransmitter Agents ; Signal Transduction

Heterotrimeric G proteins mediate signals generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain and growth cone membranes of neurons. In spite of its abundance in neurons, the role of Go is not fully understood. In the previous study, we showed that transient expression of the alpha subunit of Go (alpha o) modulated neurite outgrowth in F11 cells. It is possible that transient transfection may cause transient accumulation of the protein, which itself may alter differentiation process in non-specific manner. In this study, we determined that modulation of neurite outgrowth by alpha o was specific by evaluating the effect of alpha o in stably transformed F11 cells. F11 cells stably expressing the wild type alpha o (alphao(wt)) and a constitutively active form of alpha o (alpha oQ205L) were established. In normal F11 cells and alpha o-stable cell lines, the neurite length was measured in the presence of dibutyryl cAMP. In normal F11 cells, the average length of neurites was 57.9+/-7.0 microgram. In alpha o(wt)- and alpha o(Q205L)-expressing cells, the average length were 34.4+/-5.1 microgram 30.5+/-3.6 microgram, respectively. Thus, stable expression of alpha o(wt) and alpha o(Q205L) caused a decrease in neurite outgrowth by 40.6%, 47.3% respectively. This result indicates that modulation of neurite by alpha o was specific to the function of alpha o but not due to accumulation of exogenous proteins.
Brain ; Cell Line ; Growth Cones ; GTP-Binding Proteins ; Heterotrimeric GTP-Binding Proteins ; Membranes ; Neurites* ; Neurons ; Neurotransmitter Agents ; Transfection

Neuronal differentiation is a complex biological process accompanying cytoskeletal reorganization, including neurite outgrowth and growth cone formation. Therefore, neuronal differentiation is critically regulated by actin-related signaling proteins, such as small Rho GTPases, guanine nucleotide exchange factors (GEFs), and myosins. This study will demonstrate the change in activity of three small Rho GTPases, Rac, Cdc42, and Rho A, by treatment with blebbistatin (BBS), a specific inhibitor for myosin, during bFGF-induced neurite outgrowth in PC12 cells. Treatment with BBS induced morphological changes in growth cones and neurites during differentiation. A marked increase in protrusion and filopodia structures in growth cones, the shaft of neuritis, and cell membranes was observed in the cells treated with BBS. Activity of Rho GTPases showed the alterations in response to BBS. Activities of both Rac and Rho A were inhibited by BBS in a time-dependent manner. By contrast, Cdc42 activity was not changed by BBS. These results suggest that inactivation of myosin II by BBS induced morphological changes in neurites and growth cones and distinct regulation of three Rho GTPases during differentiation of PC12 cells.
Animals ; Biological Processes ; Cell Membrane ; Growth Cones ; Guanine Nucleotide Exchange Factors ; Heterocyclic Compounds with 4 or More Rings ; Myosin Type II ; Myosins ; Neurites ; Neuritis ; Neurons ; PC12 Cells* ; Proteins ; Pseudopodia ; rho GTP-Binding Proteins*

The perception of motion is an important function of vision. Neural wiring diagrams for extracting directional information have been obtained by connectome reconstruction. Direction selectivity in Drosophila is thought to originate in T4/T5 neurons through integrating inputs with different temporal filtering properties. Through genetic screening based on synaptic distribution, we isolated a new type of TmY neuron, termed TmY-ds, that form reciprocal synaptic connections with T4/T5 neurons. Its neurites responded to grating motion along the four cardinal directions and showed a variety of direction selectivity. Intriguingly, its direction selectivity originated from temporal filtering neurons rather than T4/T5. Genetic silencing and activation experiments showed that TmY-ds neurons are functionally upstream of T4/T5. Our results suggest that direction selectivity is generated in a tripartite circuit formed among these three neurons-temporal filtering, TmY-ds, and T4/T5 neurons, in which TmY-ds plays a role in the enhancement of direction selectivity in T4/T5 neurons.
Animals ; Neurites ; Drosophila ; Neurons ; Connectome

Neurons in the nucleus raphe magnus are involved in descending modulation of nociceptive transmission. In this study, we attempted to investigate electrophysiological properties of the NRM neurons dissociated from the postnatal rat medulla. The NRM neurons in the coronal slices of and the dissociated neurons from the postnatal rat medullae were immunohistochemically identified using antibody against serotonin. Relatively small number of neurons were positively stained in both preparations. The positively stained neurons displayed large cell body with double or multiple neurites. Using whole-cell patch clamp configuration ionic currents were recorded from the dissociated NRM-like neurons selected by criteria such as size and shape of cell body and cell population. Two types, high- and low-threshold, of voltage-dependent calcium currents were recorded from the dissociated NRM-like neurons. Some neurons displayed both types of calcium currents, whereas others displayed only high-threshold calcium current. Voltage-dependent potassium currents were also recorded from the dissociated NRM neurons. Some neurons displayed both transient outward and delayed rectifier currents but others showed only delayed rectifier current. These results suggest that there are at least two types of calcium currents and two types of potassium currents in the dissociated NRM neurons.
Animals ; Calcium ; Neurites ; Neurons* ; Potassium ; Rats* ; Serotonin

Inhibition of Rho-associated coiled coil-containing kinase (ROCK) has been reported to promote differentiation of neuronal cells. Here, we examined the effect of Y-27632, a ROCK inhibitor, on the outgrowth of neurites in PC12 cells. Y-27632 caused a rapid induction of neurite outgrowth in PC12 cells in a time-dependent manner. The neurite outgrowth, triggered by Y-27632, was accompanied by Rac1 activation, and was attenuated by Rac1 inhibitor NSC23766, in a concentration-dependent manner. Y-27632 also induced an increase in the production of reactive oxygen species (ROS). Pretreatment with N-acetylcysteine, an ROS scavenger, inhibited the ROS generation and neurite outgrowth in response to Y-27632. These results indicate that the activation of Rac1 and the generation of ROS contribute to the neurite outgrowth triggered by Y-27632 in PC12 cells.
Acetylcysteine ; Animals ; Neurites* ; Neurons ; PC12 Cells* ; Phosphotransferases ; Reactive Oxygen Species*

We examined the neuroprotective and neurotrophic effects of Tremella fuciformis. The neurotrophic effects of the hot water extract of T. fuciformis was evaluated by microscopically monitoring its potency to induce neurite outgrowth in PC12h cells. The hot water extract of T. fuciformis promoted neurite outgrowth in PC12h cells in this study, superior to other natural substances which was reported previously. When cells were treated with the hot water extract of T. fuciformis prior to beta-amyloid peptide treatment (active domain of A peptide 25~35 treated), toxicity was significantly diminished (p<0.01). These results suggest that T. fuciformis might potentially be used as a precautionary agent in neurodegenerative disease, such as Alzheimer's disease, etc.
Alzheimer Disease ; Neurites ; Neurodegenerative Diseases ; Neuroprotective Agents ; Water

Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.
Animals ; Embryonic Structures ; GAP-43 Protein ; Mice ; Neurites ; Neurons ; Peripheral Nervous System ; Plastics ; Synapses ; Synaptophysin

OBJECTIVE: The aim of this study was to identify diffrentially regulated genes after the treatment of fluoxetine in rat C6 glioma cells using cDNA microarray chip techniques and real-time RT-PCR. METHODS: Cells were incubated for 24 hours, and for 72 hours with or without 10 uM fluoxetine. Total RNAs extracted from cells were reversely transcribed to cDNA. These cDNA were used to carry out cDNA microarray chip. A part of the up-/down-regulated genes in cDNA microarray result were confirmed by real-time RT-PCR. RESULTS: 1) Genes in fluoxetinetreated cells for 72 hours (chronic treatment) were more regulated than that in fluoxetine-treated cells for 24 hours (acute treatment). 2) The expression level of Gs gene in fluoxetine-treated cells for 24 hours hardly altered, but that of Gs in fluoxetine-treated cells for 72 hours significantly increased. The expression of Gi2 also decreased in 72 hours in relation to 24 hours after the administration of fluoxetine. 3) The expression level of NCAM140 gene in fluoxetine-treated cells was higher than that in control cells. CONCLUSION: We identified genes (Gs, Gi2 and NCAM140) related to neural plasticity and intracellular signal transduction cascade from our result. This implies that fluoxetine may inhibit atrophy or death of impaired neural cells by promoting neurite outgrowth.
Animals ; Atrophy ; DNA, Complementary ; Fluoxetine* ; Glioma* ; Neurites ; Oligonucleotide Array Sequence Analysis ; Plastics ; Rats* ; RNA ; Signal Transduction

Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent CoCl2. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus CoCl2 conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus CoCl2. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus CoCl2 upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.
Anoxia ; Chondroitin Sulfate Proteoglycans* ; Chondroitin Sulfates* ; Chondroitin* ; Humans ; Neurites ; Neurons ; Protein Kinase C ; Stem Cells