Yoshiichiro Kitamura

Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels

As a neurotransmitter and neuromodulator, serotonin (5‐HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5‐HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5‐HT‐induced increase in intracellular Ca2+ concentration ([Ca2+]i) could be involved in mediating the effects of 5‐HT. Application of 5‐HT to PC12 cells enhanced nerve growth factor (NGF)‐induced neurite outgrowth in a dose‐dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca2+]i was observed following application of 5‐HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5‐HT‐induced increases in [Ca2+]i were inhibited by MDL 72222, a selective 5‐HT3 receptor antagonist, and nifedipine, an L‐type calcium channel blocker, but not by ketanserin, a 5‐HT2 receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+‐ATPase. These pharmacological tests indicated that 5‐HT‐induced increases in [Ca2+]i are mediated by activation of voltage‐gated calcium channels via 5‐HT3 receptors and that 5‐HT‐induced increases in [Ca2+]i are likely to be independent of activation of 5‐HT2 receptors in PC12 cells. Furthermore, the neuritogenic effect of 5‐HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5‐HT‐induced increases in [Ca2+]i, which are mediated via 5‐HT3 receptors and L‐type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF‐induced neurite outgrowth.

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.

Identification of two types of synaptic activity in the earthworm nervous system during locomotion

In the ventral nervous system of the earthworm, a central pattern generator and motor neurons are activated during locomotion. We have previously reported that bath application of octopamine (OA) induces fictive locomotion in the earthworm, and the burst frequency of electrical activity from the first lateral nerves increases with OA concentration. However, there are no reports concerning locomotor neural networks in the earthworm. To identify neural networks involved in fictive locomotion, we optically monitored activity-dependent fluorescent staining in the earthworm ventral nerve cord (VNC) with a styryl dye, N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43), and a confocal laser scanning microscope. OA induces FM1-43 fluorescence in a dose-dependent manner, with bright fluorescent spots of 3-10 microm in diameter observed to be localized around specified neurons in the segmental ganglion of the VNC. We compared OA dose-response curves for FM1-43 fluorescence with the bursting frequency for fictive locomotion, and found that two types of curves could be identified: one fluorescence response shows a similar dose-dependency to that of the burst frequency, while another response has a higher sensitivity to OA. From these results, we suggest that OA acts as one of the neuromodulators for the earthworm locomotion. This is the first attempt to record motor and inter-neuronal activities simultaneously in a locomotor network in the earthworm.

Serotonin-induced nitric oxide production in the ventral nerve cord of the earthworm, Eisenia fetida

Effect of serotonin on nitric oxide (NO) production in the ventral nerve cord (VNC) of the earthworm Eisenia fetida was investigated by a bio-imaging and an electrochemical technique. In the bio-imaging, the spatial pattern of NO production in VNC was visualized using an NO-specific fluorescent dye, diaminofluorescein-2 diacethyl (DAF-2 DA). Application of serotonin (100 microM) increased NO production in VNC by about 65% (P<0.05), compared with basal NO production. The increase was mainly from the nitergic neurons in the ventral side of VNC. In the electrochemical technique, real-time basal and serotonin-induced NO production was estimated with an NO-specific electrode. On the ventral surface of VNC, the estimated basal NO production was stable at 200+/-52 nM, and was transiently augmented to 840+/-193 nM by the addition of 10 microM serotonin. In conclusion, the estimated basal NO production in the earthworm VNC is relatively high compared with other nervous systems earlier reported, and transiently augmented by serotonin. Our results suggest that NO signaling in VNC is involved in neuromodulation by serotonin.

Modulation of motor patterns by sensory feedback during earthworm locomotion

Role of sensory feedback to motor pattern activity concerning locomotion in the earthworm, Eisenia fetida, was investigated. We have previously reported that bath application of octopamine induces fictive locomotion in the earthworm. In this study, we have examined the role of sensory feedback during fictive locomotion by analyzing electrical activities from the cut end and intact first lateral nerves of the ventral nerve cord (VNC). From the cut end recordings, motor activity associated with fictive locomotion was measured. A mixture of sensory and motor activities was measured from the intact first lateral nerve using en passant recordings, and sensory activity was separated by subtraction of the cut end recording (mainly motor activities) from the intact first lateral nerve recording. We estimated the effect of sensory feedback from the earthworm body wall by comparing recordings that made when the preparation was in-contact with a substrate or suspended above it. Motor pattern activities and the coefficient of variation for inter-spike-interval of motor outputs were increased under suspended conditions during circular muscle contraction. These results indicate that sensory feedback modulates the pattern of motor activity in the earthworm during locomotion.

Visualization of nitric oxide production in the earthworm ventral nerve cord

Distribution of nitric oxide (NO)-producible neurons in the ventral nerve cord (VNC) of the earthworm was investigated by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry. Some neurons (20-30 microm in diameter) were intensely stained and were localized in areas between the 1st and 2nd lateral nerves in the ventral side of VNC. In contrast, no neurons including giant fibers were stained in the dorsal side. Endogenous NO production from VNC was visualized using a fluorescent dye, diaminofluorescein-2 diacethyl (DAF-2 DA). When VNC was incubated in a saline, a relative high level of NO was produced from the ventral side, especially from NADPH-d-positive neurons. Under high-K+ stimulation, NO was also detected in the giant fibers in the dorsal side of VNC. Our results suggest that the earthworm VNC constantly and relative highly produces NO as a neuromodulator, and that NO produced from the ventral side sometimes reaches and affects the giant fibers. In conclusion, we successfully visualized NO in the earthworm VNC by clarifying both the distribution of NO-producible neurons and the endogenous NO production.

Nitric oxide-cyclic guanosine monophosphate signaling in the local circuit of the cricket abdominal nervous system

The distribution of potential nitric oxide (NO) donor neurons and NO-responsive target neurons was revealed in the terminal abdominal ganglion (TAG) of the cricket. The expression of nitric oxide synthase (NOS) in the nervous system was examined by Western blotting using universal nitric oxide synthase (uNOS) antibody that gave about a 130 kDa protein band. Immunohistochemistry using the uNOS antibody detected neurons whose cell bodies are located at the lateral region of the TAG. These neurons expanded their neuronal branches into the dorsal-median region or the dorsal-lateral region of the TAG. NADPH-diaphorase histochemistry was performed to confirm the distribution of NOS-containing neurons. The distributions of cell bodies and stained neuronal branches were similar to those revealed by uNOS immunohistochemistry. NO-induced cGMP immunohistochemistry was performed to reveal NO-responsive target neurons. Most of the cell bodies stained by immunohistochemistry appeared at the dorsal side of the TAG. At the dorsal-median region, some unpaired neuronal cell bodies were strongly stained. Some efferent neurons whose axon innervate into each nerve root were strongly stained. The generation of NO in the TAG was detected by NO electrode. We found that NO is generally produced to maintain a basal concentration of 70 nM. Hemoglobin scavenged released NO from the ganglion. The concentration of NO was partly recovered when hemoglobin was replaced by normal saline. Application of 10 microM L-arginine that is a substrate of NOS increased NO release by approximately 10 nM. Furthermore, an excitatory neurotransmitter acetylcholine (ACh) also increased NO generation by approximately 40-50 nM in concentration in addition to the basal level of 70 nM. Optical imaging with fluorescent NO-indicator demonstrated that ACh-induced enhancement of NO release was transiently observed in the outer-edge region of TAG, where cell bodies of NOS-immunoreactive neurons were located. These results suggest that ACh accelerates NO production via neuronal events activated by ACh in the TAG.

In Vivo Nitric Oxide Measurements Using a Microcoaxial Electrode

Nitric oxide (NO) is a vital molecule contributing to numerous physiological phenomena in various biological systems. To investigate the physiological role of NO, a range of NO-specific electrodes allowing direct and continuous NO measurement have been developed for in vitro and in vivo NO detection. A microcoaxial electrode has also been developed for the measurement of real-time NO levels. Because the working and reference electrodes in this device are situated close together, the microcoaxial electrode is considered to be ideal for the measurement of local NO concentrations with high spatial resolution. The microcoaxial electrode has been successfully applied to the real-time measurement of NO in endothelial cells, thereby demonstrating its effectiveness as an NO-specific electrode. In this chapter, we describe our experimental protocol for performing real-time NO measurements that was developed during physiological experiments using the microcoaxial electrode.

Investigation of intracellular magnesium mobilization pathways I PC12 cells B simultaneous Mg-Ca fluorescent imaging

Objective and Methods: PC12 cells were loaded with a novel Mg indicator KMG-104 and Ca indicator fura-2, and intracellular Mg was studied in the endoplasmic reticulums (ERs), mitochondria, and Mg-ATP. Under coexistence of the two indicators, fluorescent signals of Mg and Ca can be measured separately. Mg release from the ER was investigated by photolysis of caged compounds. Results: Transient [Ca] i increase by uncaging of caged Ca or caged IP 3 or bath-application of caffeine (10 mM) induced no [Mg] i increase. These results suggest that there is no mechanism for Mg release from the ER through ryanodine receptors or IP 3 receptors. In order to investigate the possibility of Mg release from Mg-ATP by energy consumption, we depleted ATP by oligomycin, an inhibitor of mitochondrial ATP synthase. Treating with oligomycin (4 μM) for several minutes showed no change of [Mg] i and [Ca] i. Conclusions: This result shows that Mg-ATP is not a Mg store. Since, when cells were treated by an uncoupler FCCP (3 μM), [Mg] i and [Ca] i increased, we concluded that mitochondria participate in maintenance of intracellular Mg stores.

Real-time measurement of nitric oxide using a bio-imaging and an electrochemical technique

Nitric oxide (NO) is an important mediator responsible for numerous physiological phenomena. Transient levels of NO in biological systems usually range from nanomolar to micromolar concentrations, with a rapid return to basal levels normally seen following these increases. Because NO can diffuse only over a local area in limited time due to such low levels of production and due to its short life-time prior to degradation, high spatial and temporal resolutions are required for direct and continuous NO measurement if the physiological role of NO is to be investigated in any system. For such purposes, analytical methods based on bio-imaging and electrochemical techniques for the measurement of NO are useful. In this paper, we describe the successful application of these methods to a number of biological systems. Specifically, complementary application of these methods demonstrate that it is possible to detect real-time NO production from nervous tissue with high spatial and temporal resolutions.