Takashi Katakura

Low-concentration lidocaine rapidly inhibits axonal transport in cultured mouse dorsal root ganglion neurons.

BackgroundAxonal transport plays a critical role in supplying materials for a variety of neuronal functions such as morphogenetic plasticity, synaptic transmission, and cell survival. In the current study, the authors investigated the effects of the analgesic agent lidocaine on axonal transport in neurites of cultured mouse dorsal root ganglion neurons. In relation to their effects, the effects of lidocaine on the growth rate of the neurite were also examined. MethodsIsolated mouse dorsal root ganglion cells were cultured for 48 h until full growth of neurites. Video-enhanced microscopy was used to observe particles transported within neurites and to measure the neurite growth during control conditions and in the presence of lidocaine. ResultsApplication of 30 &mgr;m lidocaine immediately reduced the number of particles transported in anterograde and retrograde axonal directions. These effects were persistently observed during the application (26 min) and were reversed by lidocaine washout. The inhibitory effect was dose-dependent at concentrations from 0.1 to 1,000 &mgr;m (IC50= 10 &mgr;m). In Ca2+-free extracellular medium, lidocaine failed to inhibit axonal transport. Calcium ionophore A23187 (0.1 &mgr;m) reduced axonal transport in both directions. The inhibitory effects of lidocaine and A23187 were abrogated by 10 &mgr;m KN-62, a Ca2+–calmodulin-dependent protein kinase II inhibitor. Application of such low-concentration lidocaine (30 &mgr;m) for 30 min reduced the growth rate of neurites, and this effect was also blocked by KN-62. ConclusionsLow-concentration lidocaine rapidly inhibits axonal transport and neurite growth via activation of calmodulin-dependent protein kinase II.

Circadian rhythm in the locomotor behavior in a population of Paramecium multimicronucleatum

Abstract The circadian behavioral rhythm in a population of Paramecium multimicronucleatum was examined by means of a computer/video system. A parameter, “traverse frequency”;, was introduced to index a locomotor activity of the specimens. The frequency is the average number of paramecia images per hour which individually traversed underneath photo‐cells placed on the CRT‐screen of the TV monitor. The traverse frequency was entrained and phase‐shifted by light‐dark (LD) cycles. It was highest at about 4 h after the beginning of the dark period and lowest at about 4 h after the beginning of the light period in LD 12:12 (20°C). Its rhythm free‐ran in DD and LL with about a 24 h period, the LL rhythm being relatively less stable than the DD rhythm. The free‐running period of DD rhythm was temperature‐compensated for 15–25°C. The circadian rhythmicity in the traverse frequency implies that the daily locomotor behavior of Paramecium is controlled by its circadian pacemaker.

Axonal transport is inhibited by a protein kinase C inhibitor in cultured isolated mouse dorsal root ganglion cells

We investigated roles of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein II (CAM II) kinase activities in the maintenance of axonal transport in cultured isolated mouse dorsal root ganglion (DRG) cells. Video-enhanced microscopic recordings revealed that the PKC inhibitor chelerythrine (1 microM) reduced anterograde and retrograde axonal transport, while the CAM II kinase inhibitor KN-62 (10 microM) had no effect. Morphological observation showed that neurite growth was prevented by the presence of chelerythrine (1 microM). From these results, we conclude that PKC activity is required to maintain axonal transport and thereby neurite growth.

Neurotropin inhibits axonal transport in cultured mouse dorsal root ganglion neurons.

Axonal transport is a basic neuronal cell function and important for the supply of materials that maintain neuronal cells, and any increase or decrease in axonal transport expresses the state of neurons. Neurotropin is an analgesic agent commonly used for the treatment of chronic pain, but its mechanism of action remains not fully understood. The effects of neurotropin have been investigated in various animal models of nerve injury and chronic pain. In the present study, we dissected the effects of neurotropin on sensory neurons with a special focus on axonal transport using cultured mouse dorsal root ganglion (DRG) neurons. Movement of organelles in neurites was recorded by real-time video-enhanced microscopy. Neurotropin significantly reduced bidirectional axonal transport in time- and concentration-dependent manners without affecting the diameter of these neurites. This is the first report to show the inhibitory effect of neurotropin on axonal transport, and suggest that this action may mediate, at least in part, the analgesic effects of this agent.

EXTRACELLULAR POTASSIUM RAPIDLY INHIBITS AXONAL TRANSPORT OF PARTICLES IN CULTURED MOUSE DORSAL ROOT GANGLION NEURITES

Changes in extracellular potassium concentration ([K+]o) modulate a variety of neuronal functions. However, whether axonal transport, which conveys materials to the appropriate destination for morphogenesis and other neuronal functions, depends on the extracellular K+ environment remains unclear. We therefore examined the effects of changes in [K+]o on axonal transport of particles visualized by video-enhanced microscopy in cultured mouse dorsal root gan-glion neurites. Increases in [K+]o (delta[K+]o > or = 2.5 mM) from control concentration (5 mM) inhibited both anterograde and retrograde axonal transport within a few minutes in a concentration-dependent manner. Conversely, removal of extracellular K+ induced the rapid facilitation of transport in both directions. These inhibitory and facilitatory responses were completely blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting that the effect of changes in [K+]o involves the TEA-sensitive K+ channels. Increases in [K+]o provoked membrane depolarization in the absence and presence of TEA. Another depolarizing agent, veratridine, did not produce an effect on axonal transport. These results suggest that the extracellular K+-mediated inhibition of axonal transport does not depend on membrane depolarization. The inhibitory effect of increasing [K+]o on axonal transport was retained in calcium (Ca2+)-free extracellular medium, indicating that the inhibitory effect of extracellular K+ does not result from Ca2+ influx through voltage-dependent Ca2+ channels. In chloride (CI-)-free medium, increasing [K+]o failed to inhibit axonal transport, implying that the extracellular K+-mediated inhibition of axonal transport may be due to an increase in intracellular Cl- concentration associated with increases in the net inward movement of K+ and CI- across the membrane. Our results suggest that the extracellular K+ environment is involved in the rapid modulation of axonal transport of particles in dorsal root ganglion neurites.

Effect of inhibition of superoxide dismutase on motor neurons during growth: Comparison of phosphorylated and non-phosphorylated neurofilament-containing spinal neurons by histogram distribution

We reported recently that non-phosphorylated neurofilaments (NF)-positive neurons were more sensitive to the growth inhibitory effects of Cu/Zn superoxide dismutase (SOD1) than phosphorylated NF-positive neurons. The findings suggested that non-phosphorylated NF-positive neurons, presumed to represent spinal motor neurons, are more vulnerable to oxidative stress than other neurons, and thus explain in part the selective degeneration of motor neurons in amyotrophic lateral sclerosis. The present investigation is an extension to our previous study and examined the neurite growth process in the presence of diethyldithiocarbamate (DDC), an SOD1 inhibitor. Non-phosphorylated NF, representing spinal motor neurons, and phosphorylated NF, representing other spinal neurons, were stained with SMI-32 and SMI-31 antibodies, respectively. The distribution histogram of neurite length after treatment with 0 nM DDC (control) for 72 h appeared flatter compared with that of 24h. Although the addition of DDC (1 nM, 10 nM, 100 nM, or 1000 nM) to the culture medium for 72 h shifted the histogram of neurite length to a shorter range in a concentration-dependent manner, the neurite of SMI-31-immunoreactive neurons grew under DDC. On the other hand, DDC-treatment for 72 h altered the neurite growth of SMI-32-immunoreactive neurons compared with that for 24-h. The results suggest that SOD1 inhibition, representing accumulation of endogenous oxidative stress, suppresses neurite growth of spinal motor neurons, and that the growth of spinal motor neurons is more sensitive to oxidative stress than other types of neurons.

Inhibition of superoxide dismutase selectively suppresses growth of rat spinal motor neurons: comparison with phosphorylated neurofilament-containing spinal neurons.

Amyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor neurons. The reason why only motor neurons are targeted is unknown. Since ALS has been linked to mutations in Cu/Zn superoxide dismutase (SOD1), oxidative stress is regarded as a major cause of ALS. We hypothesized that motor neurons are more susceptible to oxidative stress than other neurons. To test our hypothesis, we investigated differences in neurite growth between motor and non-motor neurons under SOD1 inhibition. Spinal motor neurons were identified by immunocytochemistry using anti-non-phosphorylated neurofilament (NF) antibody (SMI-32). Other neurons immunoreactive to an antibody against phosphorylated NF (SMI-31) were used as a control. Cultured rat spinal neurons were treated with the SOD1 inhibitor diethyldithiocarbamate (DDC). SMI-32-immunoreactive neurons were more sensitive to the growth inhibitory effects of DDC than SMI-31-immunoreactive neurons. Such inhibition was blocked by the antioxidants, L-ascorbic acid, L-histidine, astaxanthin, α-tocopherol, and β-carotene. The results suggested that spinal motor neurons are more vulnerable to oxidative stress than other neurons, which may explain in part the selective degeneration of motor neurons in ALS.

Modulatory effect of prostaglandin E2 on axonal transport in cultured mouse dorsal root ganglion cells: Involvement of cyclic AMP/protein kinase a cascade

‘Dept. of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183-8526, ‘Dept. of Nemo-Cell Biology, Tokyo Metropolitan Institute for Gerontology, Sakaecho, Itabashi-ku, Tokyo 173-0015 We have found that three-dimensional (3-D) fibrin and collagen gels containing laminin (with entactin. LM) promote the migration of cerebellar granule cells”. To determine whether or not other CNS neurons migrate in such gels, smaII tissues were dissected from mouse cerebral cortices (E12), rat hippocampus (E18) or mouse cerebellar nuclei with white matter (P4), embedded in gel matrices formed of plasminogen-free fibrin (FIB) and collagen-type I (COL) with (FIB-LM, COL-LM) or without LM, cultured in a serum-free synthetic medium and observed by time-lapse video microscopy. Single neuroblasts migrated out from these explants and moved freely in both FIB-LM and COL-L.M, but not FIB and COL alone. The migratory dynamics of neuroblasts in FIB-LM was as usual but that in COL-LM was different. Neuroblasts in COL-LM had unique cytoplasma segmented with some constrictions, and they appeared to move Iike toothpaste squeezed in a tube. These results suggest that LM is essential for the migration of CNS neuroblasts in 3-D gels and may serve to elucidate the migratory mechanism of neuroblasts in vivo. 1) Kimura-Kuroda, J. et al. in Neural Development (Uyemura, K. et al. eds.) Springer-Verlag Tokyo (in press)

Regulation by an extract of embryonic chick brain of the densities of voltage-dependent Na+ and Ca2+ channels in embryonic chick skeletal muscle cells during their development in culture

We studied the chronic effects of a brain extract (BE) prepared from chick embryonic brains on voltage-dependent Na+ channels (VDNCs) and Ca2+ channels (VDCCs) during the development of chick skeletal muscle cells in culture. The maximum rates of rise of Na+ and Ca2+ action potentials were measured electrophysiologically in an attempt to determine the effects of BE on the densities of these channels. The basic culture medium was supplemented with chick transferrin instead of whole-embryo extract and skeletal muscle cells were grown in the absence or in the presence of crude BE or fractionated BE. Long-term inclusion of BE to the culture medium increased the densities of both VDNCs and L-type VDCCs. By contrast, BE apparently decreased the density of T-type VDCCs. Our results indicate that BE contains some protein(s) that has a negative effect on the density of T-type VDCCs of skeletal muscle cells at a less differentiated stage and that this effect of BE is closely associated with subsequent regulation of the densities of VDNCs and L-type VDCCs. Possible roles of the influx of Ca2+ ions through T-type and L-type VDCCs in the control of the densities of VDNCs and L-type VDCCs are discussed.