Kazuhiko Narita

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs.

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn2+, verapamil (40 microM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg2+, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca2+ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca2+ (nominally Ca2(+)-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn2+ and Mg2+, as well as Ca2+, enter the nerve terminal through Ca2+ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca2+ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na+ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.

Tetanic stimulation increases the frequency of miniature end-plate potentials at the frog neuromuscular junction in Mn2+-, CO2+-, and Ni2+-saline solutions.

The effects of Mn2+, Co2+, and of Ni2+ on quantal acetylcholine (ACh) release have been studied with conventional microelectrode techniques. Increasing the [Co2+]0 or [Ni2+]0 (in the absence of extracellular Ca2+) caused an increase in miniature endplate potential (MEPP) frequency. [Mn2+]0 caused some increase in frequency at low levels, but then there was no rise as the concentration was increased further. In preparations depolarized with 20 mM K+, the MEPP frequency was a monotonically increasing function of [Co2+]0 or [Ni2+]0. In increasing concentrations of [Mn2+]0 there was an increase followed by a levelling off or a depression at higher concentrations. Tetanic stimulation of the motor nerve in solutions containing no added divalent cations or containing MgEGTA produced slight or no increases in MEPP frequency. In Mn2+-, Co2+- or Ni2+- saline solution stimulation of the motor nerve led to substantial increases in MEPP frequencies. The maximum frequency attained in Mn2+, Co2+, or Ni2+ was a power function of: (a) the duration of the tetanus; (b) the frequency of stimulation during the tetanus; or (c) the extracellular concentration of the divalent cation. During stimulation in Mn2+-saline solution the MEPP frequency reached a maximum; further stimulation led to a fall in frequency. We conclude that Mn2+, Co2+, and Ni2+ can enter the nerve terminal through a voltage-gated channel. Once within the terminal, they can stimulate quantal release by releasing Ca2+ or by causing the liberation of an activator, like H+, within the terminal.

Neural network remodeling underlying motor map reorganization induced by rehabilitative training after ischemic stroke

Motor map reorganization is believed to be one mechanism underlying rehabilitation-induced functional recovery. Although the ipsilesional secondary motor area has been known to reorganize motor maps and contribute to rehabilitation-induced functional recovery, it is unknown how the secondary motor area is reorganized by rehabilitative training. In the present study, using skilled forelimb reaching tasks, we investigated neural network remodeling in the rat rostral forelimb area (RFA) of the secondary motor area during 4weeks of rehabilitative training. Following photothrombotic stroke in the caudal forelimb area (CFA), rehabilitative training led to task-specific recovery and motor map reorganization in the RFA. A second injury to the RFA resulted in reappearance of motor deficits. Further, when both the CFA and RFA were destroyed simultaneously, rehabilitative training no longer improved task-specific recovery. In neural tracer studies, although rehabilitative training did not alter neural projection to the RFA from other brain areas, rehabilitative training increased neural projection from the RFA to the lower spinal cord, which innervates the muscles in the forelimb. Double retrograde tracer studies revealed that rehabilitative training increased the neurons projecting from the RFA to both the upper cervical cord, which innervates the muscles in the neck, trunk, and part of the proximal forelimb, and the lower cervical cord. These results suggest that neurons projecting to the upper cervical cord provide new connections to the denervated forelimb area of the spinal cord, and these new connections may contribute to rehabilitation-induced task-specific recovery and motor map reorganization in the secondary motor area.

Exercise in the Early Stage after Stroke Enhances Hippocampal Brain-Derived Neurotrophic Factor Expression and Memory Function Recovery

BACKGROUND Exercise in the early stage after stroke onset has been shown to facilitate the recovery from physical dysfunction. However, the mechanism of recovery has not been clarified. In this study, the effect of exercise on spatial memory function recovery in the early stage was shown, and the mechanism of recovery was discussed using a rat model of brain embolism. METHODS Intra-arterial microsphere (MS) injection induced small emboli in the rat brain. Treadmill exercise was started at 24 hours (early group) or 8 days (late group) after MS injection. The non-exercise (NE) and sham-operated groups were included as controls. Memory function was evaluated by the Morris water maze test, and hippocampal levels of brain-derived neurotrophic factor (BDNF) were measured by enzyme-linked immunosorbent assays. To further investigate the effect of BDNF on memory function, BDNF was continuously infused into the hippocampus via implantable osmotic pumps in the early or late stage after stroke. RESULTS Memory function significantly improved only in the early group compared with the late and the NE groups, although hippocampal BDNF concentrations were temporarily elevated after exercise in both the early and the late groups. Rats infused with BDNF in the early stage exhibited significant memory function recovery; however, rats that received BDNF infusion in the late stage showed no improvement. CONCLUSION Exercise elevates hippocampal BDNF levels in the early stage after cerebral embolism, and this event facilitates memory function recovery.

Delayed administration of the nucleic acid analog 2Cl-C.OXT-A attenuates brain damage and enhances functional recovery after ischemic stroke

2Cl-C.OXT-A (COA-Cl) is a novel nucleic acid analog that enhances angiogenesis through extracellular signal-regulated kinase 1 or 2 (ERK1/2) activation. ERK1/2 is a well-known kinase that regulates cell survival, proliferation and differentiation in the central nervous system. We performed in vitro and in vivo experiments to investigate whether COA-Cl can attenuate neuronal damage and enhance recovery after brain ischemia. In primary cortical neuron cultures, COA-Cl prevented neuronal injury after 2h of oxygen-glucose deprivation. COA-Cl increased phospho-ERK levels in a dose-dependent manner and COA-Cl-induced neuroprotection and ERK1/2 activation was inhibited by suramin or PD98059. The effect of COA-Cl was evaluated in vivo with 60min of middle cerebral artery occlusion combined with bilateral common carotid artery occlusion. COA-Cl or saline was injected intracerebroventricularly 5min after reperfusion. COA-Cl significantly reduced infarct volume and improved neurological deficits upon injection of 15 or 30μg/kg COA-Cl. Moreover, COA-Cl reduced the number of TUNEL positive cells in ischemic boundary, while rCBF was not significantly changed by COA-Cl administration. We also evaluated the effect of delayed COA-Cl administration on recovery from brain ischemia by continuous administration of COA-Cl from 1 to 8 days after reperfusion. Delayed continuous COA-Cl administration also reduced infarct volume. Furthermore, COA-Cl enhanced peri-infarct angiogenesis and synaptogenesis, resulting in improved motor function recovery. Our findings demonstrate that COA-Cl exerts both neuroprotective and neurorestorative effects over a broad therapeutic time window, suggesting COA-Cl might be a novel and potent therapeutic agent for ischemic stroke.

The Role of Endogenous Neurogenesis in Functional Recovery and Motor Map Reorganization Induced by Rehabilitative Therapy after Stroke in Rats

BACKGROUND AND OBJECTIVE Endogenous neurogenesis is associated with functional recovery after stroke, but the roles it plays in such recovery processes are unknown. This study aims to clarify the roles of endogenous neurogenesis in functional recovery and motor map reorganization induced by rehabilitative therapy after stroke by using a rat model of cerebral ischemia (CI). METHODS Ischemia was induced via photothrombosis in the caudal forelimb area of the rat cortex. First, we examined the effect of rehabilitative therapy on functional recovery and motor map reorganization, using the skilled forelimb reaching test and intracortical microstimulation. Next, using the same approaches, we examined how motor map reorganization changed when endogenous neurogenesis after stroke was inhibited by cytosine-β-d-arabinofuranoside (Ara-C). RESULTS Rehabilitative therapy for 4 weeks after the induction of stroke significantly improved functional recovery and expanded the rostral forelimb area (RFA). Intraventricular Ara-C administration for 4-10 days after stroke significantly suppressed endogenous neurogenesis compared to vehicle, but did not appear to influence non-neural cells (e.g., microglia, astrocytes, and vascular endothelial cells). Suppressing endogenous neurogenesis via Ara-C administration significantly inhibited (~50% less than vehicle) functional recovery and RFA expansion (~33% of vehicle) induced by rehabilitative therapy after CI. CONCLUSIONS After CI, inhibition of endogenous neurogenesis suppressed both the functional and anatomical markers of rehabilitative therapy. These results suggest that endogenous neurogenesis contributes to functional recovery after CI related to rehabilitative therapy, possibly through its promotion of motor map reorganization, although other additional roles cannot be ruled out.

Calcium influx through the TRPV1 channel of endothelial cells (ECs) correlates with a stronger adhesion between monocytes and ECs

PURPOSE Atherosclerosis is thought to be initiated by the transendothelial migration of monocytes. In the early stage of this process, the adhesion of monocytes to endothelial cells is supported by an increase in the intracellular concentration of calcium ion ([Ca(2+)]i) in endothelial cells. However, the main source of Ca(2+) has been unclear. In this study, the changes in ionic transmittance and [Ca(2+)]i due to the adhesion of monocytes were continuously measured by an electrophysiological technique and fluorescent imaging. Especially, we focused on transient receptor potential vanilloid channel 1 (TRPV1) as a Ca(2+) channel that could influence the adhesion of monocytes. MATERIAL AND METHODS Whole-cell current was continuously recorded in human umbilical vein endothelial cells (HUVECs) by a patch electrode. RESULTS The adhesion of monocytes (THP-1) induced a transient inward current in HUVECs, as well as an elevation of [Ca(2+)]i. This inward element was abolished by the application of 100 nM SB366,791, a selective antagonist of TRPV1 channel. Furthermore, SB366,791 significantly decreased the number of THP-1 cells that adhered to HUVECs (control: 231 ± 38, SB366,791: 96 ± 16 cells/mm2). CONCLUSION These results suggest that an inward calcium current via the TRPV1 channels of endothelial cells correlates with a stronger adhesion between monocytes and endothelial cells.

Adenosine depresses a Ca2+-independent step in transmitter exocytosis at frog motor nerve terminals

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca2+‐imaging techniques. Adenosine (200 µm) quickly and reversibly decreased the amplitude and quantal content of end‐plate potentials (EPPs) with no change in quantal size in a low‐Ca2+, high‐Mg2+ solution, and EPP amplitude in normal Ringer containing d‐tubocurarine. Likewise, adenosine (200 µm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high‐K+ (6 mm) solution. Adenosine (40–200 µm), however, did not affect single or repetitive impulse(s)‐induced rises in Ca2+ in the nerve terminals or its basal level. Adenosine (100–200 µm) reduced the Ca2+‐independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d‐tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 µm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca2+‐independent step of transmitter exocytosis at frog motor nerve terminals.

Elevated tonicity increases miniature end-plate potential frequency during tetanic stimulation at frog neuromuscular junction in low calcium and in manganese saline solutions

We have followed the increase in miniature end-plate potential (MEPP) frequency that occurs during tetanic stimulation of the motor nerve in low Ca2+ and in Mn2+ saline solutions. During stimulation in either solution the frequency rises over the first minutes to reach an asymptote. In low Ca2+ solutions, hypertonicity increases the rate at which the MEPP frequency rises. Raising the [Mn2+] also increases the rate at which the MEPP frequency rises and elevates the level of the asymptote. The data are interpreted by using a model in which quantal release frequency is proportional to the [( Ca2+])n in the nerve terminal; the model specifically includes the [Ca2+] in the terminal before stimulation. There is evidence that hypertonic solutions elevate the [Ca2+]i. We suggest that the effects of tonicity increases can be accounted for solely by the rise in [Ca2+]i before the beginning of the tetanus. If the number of Ca2+ cooperating in release, n, is 4, then with each stimulus there must be an increase in Ca2+ influx. If the value of n is higher, for example 50, the amount of Ca2+ entering with each stimulus is roughly constant. The data from the Mn2+ experiments also suggest than n may be greater than 4. It is pointed out that for exocytotic transmitter release, many Ca2+ ions may cooperate in the release process, so high values of n are not unreasonable.

Calcium dependence of the priming, activation and inactivation of ryanodine receptors in frog motor nerve terminals.

We studied the effects of varying extracellular Ca2+ ([Ca2+]o) and Ca2+ channel density and intracellular loading of Ca2+ chelators on stimulation‐induced rises in intracellular Ca2+ ([Ca2+]i) in frog motor nerve terminals with Ca2+ imaging. The slowly waxing and waning components of rises in [Ca2+]i induced by repetitive tetani were suppressed by blockers of Ca2+ pumps of the endoplasmic reticulum (thapsigargin and cyclopiazonic acid) and a blocker of ryanodine receptors [8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxybenzoate hydrochloride] without affecting the initial quickly‐rising component, thus reflecting the priming (and then subsequent rapid activation) and inactivation phases of Ca2+‐induced Ca2+ release (CICR) from the endoplasmic reticulum. A short tetanus‐induced rise in [Ca2+]i was proportional to [Ca2+]o, whereas the component of CICR was non‐linearly related to [Ca2+]o with saturation at 0.9 mm. The progressive blockade of Ca2+ channels by ω‐conotoxin GVIA caused proportional decreases in CICR and short tetanus‐induced [Ca2+]i rises. Intracellular loading of BAPTA and EGTA reduced the magnitude of CICR as well as short tetanus‐induced rises in [Ca2+]i with a greater effect of BAPTA than EGTA on CICR. The time to peak and the half decay time of CICR were prolonged by a low [Ca2+]o or Ca2+ channel blocker or [Ca2+]i chelators. These results suggest that ryanodine receptors sense the high [Ca2+]i transient following single action potentials for triggering CICR, whereas the priming and inactivation processes of CICR sense a slower, persisting rise in [Ca2+]i during and after action potential trains. A model is presented that includes CICR activation in elementary units.