Motoy Kuno

Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle

The nicotinic acetylcholine receptor (AChR) from fish electric organ has a subunit structure of α2βγδ, and this is thought to be also the case for the mammalian skeletal muscle AChR1–3. By cloning and sequencing the complementary or genomic DNAs, we have previously elucidated the primary structures of all four sub-units of the Torpedo californica electroplax4–6 and calf muscle AChR7–10 and of the α- and γ-subunits of the human muscle AChR7,11; the primary structures of the γ-subunit of the T. californien AChR12 and the α-subunit of the Torpedo marmorata AChR13,14 have also been deduced elsewhere. We have now cloned DNA complementary to the calf muscle messenger RNA encoding a novel polypeptide (the ε-subunit) whose deduced amino-acid sequence has features characteristic of the AChR subunits and which shows higher sequence homology with the γ-subunit than with the other subunits. cDNA expression studies indicate that the calf ε-subunit, as well as the calf γ-subunit, can replace the Torpedo γ-subunit to form the functional receptor in combination with the Torpedo α-, β- and δ-subunits.

Effects of nerve growth factor on the survival and synaptic function of Ia sensory neurons axotomized in neonatal rats

Sensory neurons with small diameters (A delta and C cells) are known to be responsive to exogenous NGF even at postnatal stages. We have examined whether large Group Ia sensory neurons (A alpha cells) arising from muscle spindles are also responsive to NGF in neonatal rats. For this purpose, monosynaptic excitatory postsynaptic potentials (EPSPs) were evoked in spinal motoneurons by Group Ia muscle afferent volleys. When a muscle nerve was crushed on the day after birth, the monosynaptic EPSPs elicited by afferent volleys from the muscle were depressed within several weeks. This synaptic depression was partially reversed by daily treatment with NGF. NGF treatment also enhanced the EPSPs evoked by stimulation of intact muscle nerves, but this effect was less marked than that on the EPSPs produced by stimulation of the previously crushed muscle nerve. Exogenous NGF was effective for the EPSPs when the treatment began on the day after birth but not when the treatment began 4 d after birth. Following crush of a muscle nerve on the day after birth, about 45% of the sensory neurons derived from the muscle were lost. The cell death of small sensory neurons was prevented by daily treatment with NGF, whereas the NGF treatment was ineffective in preventing the cell death of large sensory neurons. The results indicate that Group Ia sensory neurons are responsive to NGF during early postnatal life.

Terminal sprouting is not responsible for enhanced transmitter release at disused neuromuscular junctions of the rat

Chronic block of nerve-muscle activity is known to induce sprouting of motor nerve terminals and to enhance transmitter release at the neuromuscular junction. Increased transmitter release has been assumed to be a physiological correlate of disuse-induced sprouting of nerve terminals. We examined this assumption in the rat extensor digitorum longus muscle following chronic conduction block of the sciatic nerve with TTX. The minimal period of nerve block required for the expression of terminal sprouting was 3 d, whereas transmitter release, measured by the quantal analysis of end-plate potentials, was already enhanced within 24 hr of nerve block. Following 6 d of nerve block, sprouting was observed in about 35% of the motor nerve terminals examined. Under this condition, the total length of individual terminals was significantly greater in the terminals with sprouts than those without sprouts. However, enhancement of transmitter release occurred uniformly at these junctions regardless of the presence or absence of terminal sprouts. Also, transmitter release enhanced by nerve block for 2 d remained elevated for at least 4 d even after resumption of nerve activity without the formation of terminal sprouts. It is concluded that terminal sprouting and increased transmitter release induced in disused neuromuscular junctions are not causally related and that the signals for inducing these 2 events are at least quantitatively different.

Central chemosensitivity to H+ and CO2 in the rat respiratory center in vitro

The brainstem, cervical cord and attached phrenic nerve were excised from newborn rats and superfused in vitro. Respiratory output was measured by integration of phrenic nerve discharges. Respiratory output was enhanced by an increase in pCO2 at constant pH as well as by decreased pH with constant pCO2. It is concluded that the adequate stimulus to central chemoreceptors is not restricted only to H+.

A hypothesis for neural control of the speed of muscle contraction in the mammal

A hypothesis for neural control of the contractile properties of muscle is presented. The hypothesis is based on two assumptions: i) the motoneurons innervating both the fast- and slow-twitch muscles in the mammal possess an identical neurotrophic substance which accelerates the speed of muscle contraction, and ii) slow contractions of the slow-twitch muscle are induced by the low frequency discharges of the innervating motoneurons. The presence of such a trophic substance in the motoneurons is supported by the prolongation of contraction time in both the fast- and slow-twitch muscles following denervation. Furthermore, a given slow-twitch muscle dually reinnervated by the fast and slow muscle nerves contracts at the same speed regardless of which nerve is stimulated, if identical neural activity patterns are chronically imposed upon the two nerves. The presence of effective neural activity patterns in the motoneurons innervating the slow-twitch muscle is supported by an increase in the contraction speed of the slow-twitch muscle following virtual elimination of motoneuron activity by transection of the spinal cord. Under such conditions, contraction time of the fast-twitch muscle remains unchanged. Also, cord transection of newborn animals does not affect the postnatal acceleration in contraction time of the fast-twitch muscle, while the postnatal changes of contraction of the slow-twitch muscle mimic those of the fast-twitch muscle. The contraction time of slow-twitch muscle fibers is correlated with the duration of after-hyperpolarization (AHP) in the motoneurons, which regulates the discharge frequency. The duration of AHP increases with age in the motoneurons innervating the slow-twitch muscle but not in those innervating the fast-twitch muscle. It is proposed that the maintenance of slow contractions in the slow-twitch muscle during muscle differentiation is due to the appearance of low frequency discharges in the innervating motoneurons as a result of the postnatal prolongation of the AHP. The implications and limitations of the proposed hypothesis are discussed.

Disparity of cell swelling and rapid neuronal death by excitotoxic insults in rat hippocampal slice cultures

The rapidly (< 1 h) developing neuronal death induced by a 15-min-exposure to N-methyl-D-aspartate (NMDA) in rat hippocampal slice cultures is associated with cell swelling. We examined whether the swelling directly leads to neuronal death. The rapid neuronal death assayed by propidium iodide was Cl(-)-dependent, as reported for the cell swelling. However, the dose-dependence for NMDA-induced neuronal death differed from that for the cell swelling. Also, cell swelling alone induced by hypotonic insults led to neuronal death only when the cell size increased far more than the extent achieved by NMDA insults. Moreover, contrary to the previous notion, the rapid neuronal death was Ca2+-dependent. Thus, the primary cause of the rapid neuronal death induced by NMDA cannot be attributed to cell swelling.

1209 Rapid NMDA-induced neuronal death in hippocampal slice cultures: Disparity of cell swelling and cell death

Takuya Sakaguchi, Motoy Kuno, Kazuo Kawasaki Neuronal death induced by glutamate or other excitatory amino acids can be classified into the rapidly (<a few hrs) developing and slowly evolving forms. The rapid neuronal death has been postulated to result from osmotic uptake of water (swelling) and lysis. We applied NMDA to rat hippocampal slice cultures for 15 min, and the resultant neuronal death was quantified by propidium iodide 1 hr later. The magnitude of NMDA-induced neuronal death occurred in a dose-dependent manner, but the magnitude of cell swelling was independent of the NMDA dose applied in a range of 20-100pM. Cell swelling was also induced by application of hypotonic solutions for 15 min. The hypotonic insult failed to induce cell death even when the magnitude of cell swelling was much greater than that observed by NMDA application. It is concluded that cell swelling is no the primary

1322 NMDA-Induced early and delayed neuronal death in rat hippocampal slice cultures

An increase in glutamine synthetase expression after the hypoglossal nerve injury was demonstrated by the differential display PCR (DD-PCR) using single arbitrary primer coupled with in situ hybridization screening called in situ display. DD-PCR was carried out to compare differences in expression of mRNAs between axotomized (6 hours after the transection) and normal hypoglossal nuclei in the mice. One of several gene fragments which were increased after nerve injury, was identified as the glutamine synthetase (GS). Our previous finding that neuronal glutamate transporter EAACl expression was enhanced after the nerve injury (Kiryu et al, J. Neurosci.15:7872, 1995) and the present results strongly suggest that the glutamate uptake and metabolic systems in injured neurons appeared to be enhanced probably for the resistance of these injured motoneurons against neurotoxic glutamate accumulation.

Messenger RNAs from chick muscle encode a motoneuronal survival-promoting factor

The survival of motoneurons requires a trophic factor derived from the target muscles, but the nature of this trophic factor is not known. We examined whether skeletal muscle expresses mRNAs encoding survival-promoting factors for motoneurons. Messenger RNAs were purified from hindlimbs of chick embryos and injected into Xenopus oocytes for translation. The media conditioned by incubation of the injected oocytes were applied to chick embryos during the natural cell death period of lumbar motoneurons. This procedure significantly reduced the magnitude of natural motoneuronal death. The conditioned media also promoted the survival of motoneurons cultured from chick embryos.