Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin.

Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined fluorescence of ZnAF-2 DA staining as a marker of intact mossy fibers and increased fluorescence of Fluoro-Jade B staining as a marker of degenerated neurons during the initial 2 to 5 days after the administration with later ameliorations within 30 days in the hippocampal dentate gyrus (DG) and CA3 region in mice. On immunoblotting analysis, both GABABR1 and GABABR2 subunit levels increased during 15 to 30 days after TMT along with significant decreases in glutamatergic GluA1 and GluA2/3 receptor subunit levels during 2 to 7 days in the DG, but not in other hippocampal regions such as CA1 and CA3 regions. Immunohistochemical analysis revealed the constitutive and inducible expression of GABABR2 subunit in cells immunoreactive to an astrocytic marker as well as neuronal markers in the DG with the absence of neither GABABR1a nor GABABR1b subunit from cells positive to an astrocytic marker. These results suggest that both GABABR1 and GABABR2 subunits may be up-regulated in cells other than neurons and astroglia in the DG at a late stage of TMT intoxication in mice.

Bacopa monnieri (L.) Wettst. Extract Improves Memory Performance via Promotion of Neurogenesis in the Hippocampal Dentate Gyrus of Adolescent Mice.

Bacopa monnieri L. Wettst. (BM) is a botanical component of Ayurvedic medicines and of dietary supplements used worldwide for cognitive health and function. We previously reported that administration of BM alcoholic extract (BME) prevents trimethyltin (TMT)-induced cognitive deficits and hippocampal cell damage and promotes TMT-induced hippocampal neurogenesis. In this study, we demonstrate that administration of BME improves spatial working memory in adolescent (5-week- old) healthy mice but not adult (8-week-old) mice. Moreover, improved spatial working memory was retained even at 4 weeks after terminating 1-week treatment of adolescent mice. One-week BME treatment of adolescent mice significantly enhanced hippocampal BrdU incorporation and expression of genes involved in neurogenesis determined by RNAseq analysis. Cell death, as detected by histochemistry, appeared not to be significant. A significant increase in neurogenesis was observed in the dentate gyrus region 4 weeks after terminating 1-week treatment of adolescent mice with BME. Bacopaside I, an active component of BME, promoted the proliferation of neural progenitor cells in vitro in a concentration-dependent manner via the facilitation of the Akt and ERK1/2 signaling. These results suggest that BME enhances spatial working memory in healthy adolescent mice by promoting hippocampal neurogenesis and that the effects of BME are due, in significant amounts, to bacopaside I.

Disruption of Gap Junction-Mediated Intercellular Communication in the Spiral Ligament Causes Hearing and Outer Hair Cell Loss in the Cochlea of Mice.

It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

Bacopa monnieri (L.) Ameliorates Cognitive Deficits Caused in a Trimethyltin-Induced Neurotoxicity Model Mice.

We previously demonstrated that Bacopa monnier (L.) WETTST. extract (BME) ameliorated cognitive dysfunction in animal models of dementia by enhancing synaptic plasticity-related signaling in the hippocampus and protecting cholinergic neurons in the medial septum. To further clarify the pharmacological features and availability of BME as a novel anti-dementia agent, we investigated whether BME affects neuronal repair using a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampus. Mice pretreated with TMT (2.8 mg/kg, intraperitoneally (i.p.)) on day 0 were given BME (50 mg/kg, per os (p.o.)) once daily for 15-30 d. Cognitive performance of the animals was elucidated twice by the object location test and modified Y maze test on days 17-20 (Phase I) and days 32-35 (Phase II) or by the passive avoidance test on Phase II. TMT impaired hippocampus-dependent spatial working memory and amygdala-dependent fear-motivated memory. The administration of BME significantly prevented TMT-induced cognitive deficits. The protective effects of BME on the spatial memory deficits were confirmed by Nissl staining of hippocampal tissues and propidium iodide staining of organotypic hippocampal slice cultures. Immunohistochemical studies conducted on days 17 and 32 revealed that thirty days of treatment with BME increased the number of 5-bromo-2'-deoxyuridine (BrdU)-immunopositive cells in the dentate gyrus region of TMT-treated mice, whereas fifteen days of treatment with BME had no effect. These results suggest that BME ameliorates TMT-induced cognition dysfunction mainly via protecting the hippocampal neurons from TMT-induced hippocampal lesions and partly via promoting neuroregeneration in the dentate gyrus regions.

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.

Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

Alleviation by GABAB Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to N-Methyl-D-aspartate.

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.

Preventive effect of curcumin and its highly bioavailable preparation on hearing loss induced by single or repeated exposure to noise: A comparative and mechanistic study.

We sought to determine the preventive effects of curcumin and its highly bioavailable preparation on noise-induced hearing loss in a novel murine model of permanent hearing loss developed by repeated exposure to noise. Upon exposure to noise (8-kHz octave band noise, 90 dB sound pressure level, 1 h), hearing ability was impaired in a temporary and reversible manner. During repeated noise exposure (1-h exposure per day, 5 days), there was a progressive increase in the auditory threshold shift at 12 and 20 kHz. The threshold shift persisted for at least 6 days after noise exposure. Oral administration of curcumin for 3 days before and each day during noise exposure significantly alleviated the hearing loss induced by repeated noise exposure. Curcumin abolished intranuclear translocation of nuclear factor-κB-p65 and generation of 4-hydroxynonenal-adducted proteins found in the cochlea after noise exposure. Theracurmin®, a highly absorbable and bioavailable preparation of curcumin, had strong preventive effects on hearing loss induced by repeated noise exposure. Together, these data suggest that curcumin exerts a preventive effect on noise-induced hearing loss and is therefore a good therapeutic candidate for preventing sensorineural hearing loss.

Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse.

Thrombin-activated protease-activated receptor (PAR)-1 regulates the proliferation of neural cells following brain injury. To elucidate the involvement of PAR-1 in the neurogenesis that occurs in the adult hippocampus, we examined whether PAR-1 regulated the proliferation of neural stem/progenitor cells (NPCs) derived from the murine hippocampal dentate gyrus. NPC cultures expressed PAR-1 protein and mRNA encoding all subtypes of PAR. Direct exposure of the cells to thrombin dramatically attenuated the cell proliferation without causing cell damage. This thrombin-induced attenuation was almost completely abolished by the PAR antagonist RWJ 56110, as well as by dabigatran and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), which are selective and non-selective thrombin inhibitors, respectively. Expectedly, the PAR-1 agonist peptide (AP) SFLLR-NH2 also attenuated the cell proliferation. The cell proliferation was not affected by the PAR-1 negative control peptide RLLFT-NH2, which is an inactive peptide for PAR-1. Independently, we determined the effect of in vivo treatment with AEBSF or AP on hippocampal neurogenesis in the adult mouse. The administration of AEBSF, but not that of AP, significantly increased the number of newly-generated cells in the hippocampal subgranular zone. These data suggest that PAR-1 negatively regulated adult neurogenesis in the hippocampus by inhibiting the proliferative activity of the NPCs.

Beneficial effect of cilostazol-mediated neuronal repair following trimethyltin-induced neuronal loss in the dentate gyrus.

Cilostazol acts as an antiplatelet agent and has other pleiotropic effects based on phosphodiesterase-3-dependent mechanisms. We evaluated whether cilostazol would have a beneficial effect on neuronal repair following hippocampal neuronal damage by using a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampal dentate gyrus [Ogita et al. (2005) J Neurosci Res 82:609-621]; these mice will hereafter be referred to as impaired animals. A single treatment with cilostazol (10 mg/kg, i.p.) produced no significant change in the number of 5-bromo-2'-deoxyuridine (BrdU)-incorporating cells in the dentate granule cell layer (GCL) or subgranular zone on day 3 after TMT treatment. However, chronic treatment with cilostazol on days 3-15 posttreatment resulted in an increase in the number of BrdU-incorporating cells in the dentate GCL of the impaired animals, and these cells were positive for neuronal nuclear antigen or doublecortin. Cilostazol was effective in elevating the level of phosphorylated cyclic adrenosine monophosphate response element-binding protein (pCREB) in the dentate gyrus of impaired animals. The results of a forced swimming test revealed that the chronic treatment with cilostazol improved the depression-like behavior seen in the impaired animals. In the cultures of hippocampal neural stem/progenitor cells, exposure to cilostazol produced not only enhancement of proliferation activity but also elevation of pCREB levels. Taken together, our data suggest that cilostazol has a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promotion of proliferation and/or neuronal differentiation of neural progenitor cells in the subgranular zone.

Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament.

The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear spiral ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, ß-catenin, and Cx43/ß-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and ß-catenin in primary cultures of fibrocytes derived from the cochlear spiral ligament.

A new VGLUT-specific potent inhibitor: pharmacophore of Brilliant Yellow.

The increased concentration of glutamate in synaptic vesicles, mediated by the vesicular glutamate transporter (VGLUT), is an initial vital step in glutamate synaptic transmission. Evidence indicates that aberrant overexpression of VGLUT is involved in certain pathophysiologies of the central nervous system. VGLUT is subject to inhibition by various types of agents. The most potent VGLUT-specific inhibitor currently known is Trypan Blue, which is highly charged, hence membrane-impermeable. We have sought a potent, VGLUT-specific agent amenable to easy modification to a membrane-permeable analog. We provide evidence that Brilliant Yellow exhibits potent, VGLUT-specific inhibition, with a Ki value of 12 nM. Based upon structure-activity relationship studies and molecular modeling, we have defined the potent inhibitory pharmacophore of Brilliant Yellow. This study provides new insight into development of a membrane-permeable agent to lead to specific blockade, with high potency, of accumulation of glutamate into synaptic vesicles in neurons.

Beneficial in vivo effect of aripiprazole on neuronal regeneration following neuronal loss in the dentate gyrus: evaluation using a mouse model of trimethyltin-induced neuronal loss/self-repair in the dentate gyrus.

Aripiprazole is used clinically as an atypical antipsychotic. We evaluated the effect of in vivo treatment with aripiprazole on the proliferation and differentiation of neural stem/progenitor cells in a mouse model, trimethyltin-induced neuronal loss/self-repair in the hippocampal dentate gyrus (referred as "impaired animals") [Ogita et al., J Neurosci Res. 82, 609 - 621 (2005)]. In the impaired animals, an increased number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells was seen in the dentate gyrus at the initial time window of the self-repair stage. At the same time window, a single treatment with aripiprazole significantly increased the number of cells positive for both BrdU and nestin in the dentate gyrus of the impaired animals. Chronic treatment with aripiprazole promoted the proliferation/survival and neuronal differentiation of the cells newly-generated following the neuronal loss in the dentate gyrus of the impaired animals. The chronic treatment with aripiprazole improved depression-like behavior seen in the impaired animals. Taken together, our data suggest that aripiprazole had a beneficial effect on neuronal regeneration following neuronal loss in the dentate gyrus through indirectly promoted proliferation/survival and neuronal differentiation of neural stem/progenitor cells in the subgranular zone of the dentate gyrus.

Possible involvement of caspases in proliferation of neocortical neural stem/progenitor cells in the developing mouse brain.

Caspases are well-known enzymes that work as initiators and effectors of apoptosis. To elucidate the role of caspases in neurodevelopment, we sought to determine if caspases are involved in the proliferation of neural stem/progenitor cells (NPCs) in the developing mouse brain. Labeling with 5-bromo-2'-deoxyuridine (BrdU) from days 14 to 18 of pregnant mice revealed that the 18-d old fetus had many BudU-positive cells in its brain. Double-labeling revealed that active caspase-3 was co-localized with these BrdU-positive cells in the neocortex, hippocampus, and subventricular zone of the fetal brain. Active caspase-3 was detected in cultures of NPCs derived from the neocortex of 15-d old fetuses during culture periods. Importantly, the pan-caspase inhibitor z-VAD-FMK was effective at completely inhibiting neurosphere formation by the NPCs. These results suggest the possibility that the caspase cascade is essential for the proliferation of neocortical NPCs in the developing mouse brain.

A novel model of sensorineural hearing loss induced by repeated exposure to moderate noise in mice: the preventive effect of resveratrol.

Sensorineural hearing loss (SNHL) induced by noise has increased in recent years due to personal headphone use and noisy urban environments. The study shows a novel model of gradually progressive SNHL induced by repeated exposure to moderate noise (8-kHz octave band noise, 90-dB sound pressure level) for 1 hr exposure per day in BALB/cCr mice. The results showed that the repeated exposure led to gradually progressive SNHL, which was dependent on the number of exposures, and resulted in permanent hearing loss after 5 exposures. Repeated exposure to noise causes a loss of synapses between the inner hair cells and the peripheral terminals of the auditory nerve fibers. Additionally, there is a reduction in the expression levels of c-fos and Arc, both of which are indicators of cochlear nerve responses to noise exposure. Oral administration of resveratrol (RSV, 50 mg/kg/day) during the noise exposure period significantly prevented the noise exposure-induced synapse loss and SNHL. Furthermore, the study found that RSV treatment prevented the noise-induced increase in the gene expression levels of the proinflammatory cytokine interleukin-1ß in the cochlea. These results demonstrated the potential usefulness of RSV in preventing noise-induced SNHL in the animal model established as gradually progressive SNHL.

Long-lasting increases in GABAB receptor subunit levels in hippocampal dentate gyrus of mice with a single systemic injection of trimethyltin.

We have recently shown delayed increases in GABAB receptor (GABABR) subunit protein levels in the hippocampal dentate gyrus (DG), but not in the pyramidal CA1 and CA3 regions, at 15-30 days after the systemic single administration of trimethyltin (TMT) in mice. An attempt was thus made to determine whether the delayed increases return to the control levels found in naive mice afterward. In the DG on hippocampal slices obtained at 90 days after the administration, however, marked increases were still seen in protein levels of both GABABR1 and GABABR2 subunits without significant changes in calbindin and glial fibrillary acidic protein (GFAP) levels on immunoblotting analysis. Fluoro-Jade B staining clearly revealed the absence of degenerated neurons from the DG at 90 days after the administration. Although co-localization was invariably detected between GABABR2 subunit and GFAP in the DG at 30 days on immunohistochemical analysis, GABABR2-positive cells did not merge well with GFAP-positive cells in the DG at 90 days. These results suggest that both GABABR1 and GABABR2 subunits would be tardily and sustainably up-regulated by cells other than neurons and astrocytes in the murine DG at 90 days after a systemic single injection of TMT.

In vivo and in vitro treatment with edaravone promotes proliferation of neural progenitor cells generated following neuronal loss in the mouse dentate gyrus.

Edaravone is clinically used in Japan for treatment of patients with acute cerebral infarction. To clarify the effect of edaravone on neurogenesis in the hippocampus following neuronal injury in the hippocampal dentate gyrus, we investigated the effect of in vitro and in vivo treatment with edaravone on the proliferation of neural stem/progenitor cells prepared from the mouse dentate gyrus damaged by trimethyltin (TMT). Histological assessment revealed the presence of large number of nestin(+) cells in the dentate gyrus on days 3 - 5 post-TMT treatment. We prepared cells from the dentate gyrus of naïve, TMT-treated mice or TMT/edaravone-treated mice. The cells obtained from the dentate gyrus of TMT-treated animals were capable of BrdU incorporation and neurosphere formation when cultured in the presence of growth factors. The TMT-treated group had a larger number of nestin(+) cells and nestin(+)GFAP(+) cells than the naïve one. Under the culture condition used, sustained exposure of the cells from the damaged dentate gyrus to edaravone at 10(-11) and 10(-8) M promoted the proliferation of nestin(+) cells. The systemic in vivo treatment with edaravone for 2 days produced a significant increase in the number of nestin(+) cells among the cells prepared from the dentate gyrus on day 4 post-TMT treatment, and as well as one in the number of neurospheres formed from these cells in the culture. Taken together, our data indicated that edaravone had the ability to promote the proliferation of neural stem/progenitor cells generated following neuronal damage in the dentate gyrus.

[Activated microglial cells trigger neurogenesis following neuronal loss in the dentate gyrus of adult mice].

Neurological injuries are widely known to promote neurogenesis in the adult hippocampal dentate gyrus. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and eradicated by treatment with trimethyltin (TMT), with being regenerated in the dentate granule cell layer (GCL) after neuronal loss. Recent collective reports indicate that during brain injury and in neurodegenerative disorders, neurogenesis is controlled by cytokines, chemokines, neurotransmitters, and reactive oxygen/nitrogen species, which are released by dying neurons as well as by activated macrophages, micro-glia, and astrocytes. To elucidate the role of activated microglia in the neuroregeneration following the dentate granule cell loss, in this study, we evaluated the involvement of activated microglial cells and a related factor in the generation of newly-generated cells of the hippocampal dentate gyrus following neuronal loss induced by TMT. Our results support the possibility that pro-inflammatory cytokines released from activated microglial cells may be involved in promotion of the neurogenesis mechanism through activation of the NF-kappaB signaling pathway following the dentate neuronal loss induced by TMT treatment.

Trimethyltin initially activates the caspase 8/caspase 3 pathway for damaging the primary cultured cortical neurons derived from embryonic mice.

The organotin trimethyltin (TMT) is well known to cause neuronal damage in the central nervous system. To elucidate the mechanisms underlying the toxicity of TMT toward neurons, we prepared primary cultures of neurons from the neocortex of mouse embryos. A continuous exposure to TMT produced a decrease in cell viability as well as an increase in the number of cells with nuclear condensation/shrinkage at the exposure time window up to 24 hr. In addition to the events at the early time window, lactate dehydrogenase released was significantly elevated at the later exposure time from 36 to 48 hr. With a 3-hr exposure to TMT, a significant increase was observed in the activity of caspase 8, but not in that of caspase 9. TMT exposure produced no elevation in the level of cytochrome c released from mitochondria until 12 hr of exposure, with a significant facilitation of cytochrome c release at the exposure times of 16 and 24 hr. After the activation of caspase 8 by TMT exposure, caspase 3 activation and nuclear translocation of caspase-activated DNase were caused by exposure for 6 hr or longer. However, nuclear DNase II was elevated at the later time window of exposure. A caspase inhibitor completely prevented TMT from damaging the cells in any time window. Taken together, our data are the first demonstration that TMT toxicity is initially caused by activation of the caspase 8/caspase 3 pathway for nuclear translocation of DNases in cortical neurons in primary culture.

Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP / protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus.

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO(2), and intracellular cGMP. Like L-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the L-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca(2+)-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO(2) level was decreased by dantrolene, but not by the other 3 blockers. L-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.

Endogenous Nitric Oxide Generation Linked to Ryanodine Receptors Activates Cyclic GMP / Protein Kinase G Pathway for Cell Proliferation of Neural Stem/Progenitor Cells Derived From Embryonic Hippocampus.

Nitric oxide (NO) activates the cyclic GMP (cGMP) / protein kinase G (PKG) pathway during physiological processes in numerous types of cells. Here, we evaluated whether this NO/cGMP/PKG pathway is involved in the proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampus of embryonic mice. In culture, the exposure to the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) significantly decreased the number of viable cells and 5-bromo-2'-deoxyuridine (BrdU) incorporation into the cells, as well as the levels of intracellular reactive oxygen species, extracellular NO2, and intracellular cGMP. Like l-NAME, the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and PKG inhibitor KT5823 also decreased cell viability and BrdU incorporation. The membrane-permeable cGMP analogue 8-bromo-cGMP partially abolished the l-NAME-induced decrease in the BrdU incorporation. BrdU incorporation was decreased by Ca2+-channel blockers, including dantrolene, MK-801, ifenprodil, and nifedipine. Interestingly, the NO2 level was decreased by dantrolene, but not by the other 3 blockers. l-NAME and ODQ attenuated phosphorylation of Akt, but not that of extracellular signal-regulated kinases or epidermal growth factor receptors. Our data suggest that endogenous NO generation linked to dantrolene-sensitive ryanodine receptors activates the cGMP/PKG signaling pathway for positive regulation of proliferation of hippocampal NPCs derived from embryonic mice.