Hiroto Ogawa

Nitric oxide suppresses fictive feeding response in Lymnaea stagnalis

Fictive feeding activity was monitored in the buccal ganglia of semi-intact preparations of the pond snail, Lymnaea stagnalis, to examine the effects of nitric oxide (NO) released from motoneurons innervating the esophagus on the feeding response. The present results suggest that first; even the low concentration of constitutive NO precisely regulates the feeding rhythm by suppressing high frequency feeding responses; second, that the high concentration of NO released after activation of the feeding central pattern generator following appetitive stimulation of the lips suppresses the feeding rate, resulting in recurrent inhibition. This is the first direct evidence that NO can function to suppress rhythmic activity in the brain.

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.

Dendritic Design Implements Algorithm for Synaptic Extraction of Sensory Information

While sensory information is encoded by firing patterns of individual sensory neurons, it is also represented by spatiotemporal patterns of activity in populations of the neurons. Postsynaptic interneurons decode the population response and extract specific sensory information. This extraction of information represented by presynaptic activities is a process critical to defining the input–output function of postsynaptic neuron. To understand the “algorithm” for the extraction, we examined directional sensitivities of presynaptic and postsynaptic Ca2+ responses in dendrites of two types of wind-sensitive interneurons (INs) with different dendritic geometries in the cricket cercal sensory system. In IN 10-3, whose dendrites arborize with various electrotonic distances to the spike-initiating zone (SIZ), the directional sensitivity of dendritic Ca2+ responses corresponded to those indicated by Ca2+ signals in presynaptic afferents arborizing on that dendrite. The directional tuning properties of individual dendrites varied from each other, and the directional sensitivity of the nearest dendrite to the SIZ dominates the tuning properties of the spiking response. In IN 10-2 with dendrites isometric to the SIZ, directional tuning properties of different dendrites were similar to each other, and each response property could be explained by the directional profile of the spatial overlap between that dendrite and Ca2+-elevated presynaptic terminals. For IN 10-2, the directional sensitivities extracted by the different dendritic-branches would contribute equally to the overall tuning. It is possible that the differences in the distribution of synaptic weights because of the dendritic geometry are related to the algorithm for extraction of sensory information in the postsynaptic interneurons.

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.

Dendritic calcium accumulation regulates wind sensitivity via short-term depression at cercal sensory-to-giant interneuron synapses in the cricket

An in vivo Ca2+ imaging technique was applied to examine the cellular mechanisms for attenuation of wind sensitivity in the identified primary sensory interneurons in the cricket cercal system. Simultaneous measurement of the cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential of a wind-sensitive giant interneuron (GI) revealed that successive air puffs caused the Ca2+ accumulation in dendrites and diminished the wind-evoked bursting response in the GI. After tetanic stimulation of the presynaptic cercal sensory nerves induced a larger Ca2+ accumulation in the GI, the wind-evoked bursting response was reversibly decreased in its spike number. When hyperpolarizing current injection suppressed the [Ca2+]i elevation during tetanic stimulation, the wind-evoked EPSPs were not changed. Moreover, after suprathreshold tetanic stimulation to one side of the cercal nerve resulted in Ca2+ accumulation in the GIs dendrites, the slope of EPSP evoked by presynaptic stimulation of the other side of the cercal nerve was also attenuated for a few minutes after the [Ca2+]i had returned to the prestimulation level. This short-term depression at synapses between the cercal sensory neurons and the GI (cercal-to-giant synapses) was also induced by a depolarizing current injection, which increased the [Ca2+]i, and buffering of the Ca2+ rise with a high concentration of a Ca2+ chelator blocked the induction of short-term depression. These results indicate that the postsynaptic Ca2+ accumulation causes short-term synaptic depression at the cercal-to-giant synapses. The dendritic excitability of the GI may contribute to postsynaptic regulation of the wind-sensitivity via Ca2+-dependent depression.

Dendritic Ca2+ transient increase evoked by wind stimulus in the cricket giant interneuron

In vivo Ca2+ imaging was applied to the cricket median giant interneuron (MGI), to visualize the dendritic processing of mechanosensory signals. Wind stimulation (air-puff) to the cerci induced a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in the MGI with a latency of a few seconds, suggesting the release of Ca2+ from the intracellular store site in the MGI. The amplitude of the transient increase in [Ca2+]i in the dendrites depended on the direction of the air-puff, and the increase in [Ca2+]i evoked by the air-puff was suppressed by the hyperpolarizing current injection which blocked the generation of action potentials. These results indicate that the action potential is necessary to the direction-sensitive increase in [Ca2+]i induced by wind stimulation.

Directional sensitivity of dendritic calcium responses to wind stimuli in the cricket giant interneuron

We examined directional sensitivities in the dendritic activity of the identified giant interneurons (GIs) in the cricket, using in vivo Ca(2+) imaging during different directional air-current stimuli. Air current stimulus evoked action potential burst and quick Ca(2+) increase in GI. The stimulus direction of the maximal Ca(2+) responses corresponded to that of the maximal voltage response. However, the shapes of the directional tuning curves based on the Ca(2+) responses for each dendritic branch were different from the overall tuning curve based on spike counts for the cell. Moreover, different dendritic branches displayed distinct directional sensitivity profiles to the air-current stimuli. We propose that postsynaptic activities will influence the local Ca(2+) signals in the distal dendrites, and produce the difference in directional sensitivity of the dendritic Ca(2+) response.

Neural Basis of Stimulus-Angle-Dependent Motor Control of Wind-Elicited Walking Behavior in the Cricket Gryllus bimaculatus

Crickets exhibit oriented walking behavior in response to air-current stimuli. Because crickets move in the opposite direction from the stimulus source, this behavior is considered to represent ‘escape behavior’ from an approaching predator. However, details of the stimulus-angle-dependent control of locomotion during the immediate phase, and the neural basis underlying the directional motor control of this behavior remain unclear. In this study, we used a spherical-treadmill system to measure locomotory parameters including trajectory, turn angle and velocity during the immediate phase of responses to air-puff stimuli applied from various angles. Both walking direction and turn angle were correlated with stimulus angle, but their relationships followed different rules. A shorter stimulus also induced directionally-controlled walking, but reduced the yaw rotation in stimulus-angle-dependent turning. These results suggest that neural control of the turn angle requires different sensory information than that required for oriented walking. Hemi-severance of the ventral nerve cords containing descending axons from the cephalic to the prothoracic ganglion abolished stimulus-angle-dependent control, indicating that this control required descending signals from the brain. Furthermore, we selectively ablated identified ascending giant interneurons (GIs) in vivo to examine their functional roles in wind-elicited walking. Ablation of GI8-1 diminished control of the turn angle and decreased walking distance in the initial response. Meanwhile, GI9-1b ablation had no discernible effect on stimulus-angle-dependent control or walking distance, but delayed the reaction time. These results suggest that the ascending signals conveyed by GI8-1 are required for turn-angle control and maintenance of walking behavior, and that GI9-1b is responsible for rapid initiation of walking. It is possible that individual types of GIs separately supply the sensory signals required to control wind-elicited walking.

Optical monitoring of the neural activity evoked by mechanical stimulation in the earthworm nervous system with a fluorescent dye, FM1-43

In the central nervous system of the earthworm, sensory and motor neurons have direct synapses on three giant fibers. To determine the locations of synapses and neural network activated by mechanical stimuli, we optically monitored the activity-dependent staining in the earthworm ventral nerve cord with a styryl dye, N-(3-triethylammoniumpropyl)-4- (4-(dibutylamino)styryl)pyridinium dibromide (FM1-43), and a confocal laser scanning microscope. When scratch stimulus was applied to the body wall of the earthworm, bright fluorescent spots with 3-10 microns in diameter localized only in the stimulated segmental ganglion of the ventral nerve cord. The fluorescent intensity of these spots decreased during dye-free high K+ saline incubation. These results suggest that FM1-43 is useful for activity-dependent staining of invertebrate neurons and their synaptic regions as well as vertebrate nervous system.

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.