Hiroshi Washio

Early postnatal development of substantia nigra neurons in rat midbrain slices: hyperpolarization-activated inward current and dopamine-activated current

Whole-cell voltage-clamp recordings in rat midbrain slices were used to determine the characteristics and postnatal development of a hyperpolarization-activated inward current (Ih) and a dopamine receptor-activated current of neurons in the substantia nigra pars compacta (SNC). Ih was activated by membrane hyperpolarization beyond the resting membrane potentials, and displayed strong inward rectification. No sign of time- and voltage-dependent inactivation was apparent. The reversal potential of Ih in mature (P12-P13) SNC neurons was -39.2 +/- 4.3 mV, and the activation curve of Ih was well fitted by a sigmoidal function with half-maximal activation at -114.0 +/- 5.6 mV. Those values are essentially the same in immature (P3-P5) neurons. The rate of Ih activation was best approximated by a single exponential function. The time constants were voltage dependent, ranging from 692.3 ms at -110 mV to 342.8 ms at -150 mV at P12 neurons, and 1142.5 ms at -110 mV to 593.2 ms at -150 mV at P3 neurons. There was a marked increase in the amplitude of Ih during postnatal development of SNC neurons, while the membrane capacitance did not alter after P5. The increase in Ih after P5 thus appears to result from an increase in Ih current density. Application of dopamine produced a hyperpolarization of mature SNC neurons, leading to suppression of action potentials. Dopamine-activated outward current was negligible during the first postnatal week, and increased rapidly after the second week. Our results indicate that the SNC neuron of the rat midbrain is not fully mature at the time of birth in terms of voltage-gated ion channels and neurotransmitter receptors, but develops over the first two postnatal weeks.

Potent excitatory effect of maitotoxin on Ca channels in the insect skeletal muscle

The effect of maitotoxin (MTX), the most potent marine toxin as yet known, was studied using the skeletal muscle of the larval meal worm,Tenebrio molitor. In normal saline,Tenebrio muscles responded with the spike to direct stimulation. In the saline containing tetraethylammonium (TEA) the all-or-none action potential which had characteristic plateau was elicited by membrane depolarization. When MTX (5×10−9 to 10−8 g/ml) in the TEA saline was added, the plateau of action potential was prolonged more than in the saline containing TEA alone. Furthermore, MTX lowered the threshold, so that action potentials were readily evoked in the saline containing MTX. In either case, effects, of MTX were antagonized by Co2+. These results suggest that MTX activates the voltage-dependent Ca2+ channels in the insect muscle.

Some effects of octopamine, proctolin and serotonin on dorsal unpaired median neurones of cockroach (Periplaneta americana) thoracic ganglia

Abstract Effects of neuromodulators, two monoamines, octopamine and serotonin, and the pentapeptide, proctolin, known to be present in the cockroach central nervous system, on the excitability of dorsal unpaired median neurones in the thoracic ganglia of the cockroach, Periplaneta americana, were investigated using intracellular recording technique. Octopamine had a marked excitatory effect on these neurones. Bath application of octopamine increased the frequency of spontaneous spikes in a dose-dependent manner associated with an increase in membrane input resistance. Serotonin increased the frequency and the resistance, but much less than octopamine. Proctolin also had an excitatory effect on the dorsal unpaired median somata, but the effect was somewhat equivocal. No appreciable change in the membrane input resistance occurred in the presence of proctolin. Among these neuromodulators tested, octopamine may alter the intrinsic properties of the above somata, resulting in the modulation of neural networks.

Effects of putative neurotransmitters on dorsal unpaired median neurons of cockroach (Periplaneta americana) thoracic ganglia

Abstract Effects of bath applications of glutamate and gamma-aminobutyric acid (GABA) were studied to characterize the receptors for putative neurotransmitters present on soma membranes of the dorsal unpaired median (DUM) neurons of the thoracic ganglia in the cockroach, Periplaneta americana , using the intracellular recording technique. Both l -glutamate and GABA at concentrations of 10 −4 M produced a depolarization or a hyperpolarization at resting membrane potentials accompanied by an increase in membrane conductance, and inhibited spontaneous spikes. The reversal potential (about −40 mV) was almost the same for l -glutamate and GABA. Responses to the amino acids were completely blocked by picrotoxin, and were sensitive to a low chloride saline. The responses were reversed when chloride was injected into the cell. These results provide evidence for l -glutamate and GABA inhibitory receptors on the soma membrane of the DUM neurons linked to a chloride channel. Possible functional significance of these receptors are discussed.

Electroantennogram responses to the sex pheromone and other odours in the American cockroach

Abstract Electroantennogram responses of adult American cockroaches were recorded for the sex pheromone and 30 kinds of monoterpenoids. Strong responses to the sex pheromone were obtained from the antennae of male adults, but not from females. The compounds possessing a hydroxyl or carbonyl group produced significant EAG responses in the antennae. Bornyl acetate, however, was an exception and gave strong responses in both male and female antennae. There was basically no difference between the antennal responsiveness to monoterpenoids tested in both sexes.

L-glutamate as an excitatory transmitter at the neuromuscular junction of a beetle larva

Abstract The effects of L-glutamate and acetylcholine on the ventral muscle fibres of the larval mealworm Tenebrio molitor were studied by means of microelectrodes. Bath application of L-glutamate at concentrations higher than 1 × 10 4M suppressed excitatory postsynaptic potentials (EPSPs) and evoked both a depolarisation and a reduction in the input resistance of the muscle fibre. In contrast, acetylcholine chloride (up to 1 mM) had no effect at all. Circumscribed spots could be detected on the fibre surface where iontophoretic applications of L-glutamate caused transient depolarizations (glutamate potentials). Focal extracellular recordings revealed that the glutamate sensitive spots were identical with synaptic sites. The reversal potentials of the EPSP and the L-glutamate potential were identical. These results are compatible with the hypothesis that L-glutamate is an excitatory transmitter at the neuromuscular junction.

Proctolin and octopamine actions on the contractile systems of insect leg muscles.

1. The pentapeptide proctolin produced contractions of the coxal depressor muscle of the cockroach, Periplaneta americana. 2. The contraction was dependent upon extracellular calcium and the contraction was completely blocked by a Ca-free EGTA saline. 3. Caffeine elicited transient contractions which were unaffected by manganese treatment. 4. When the muscle was pre-treated with the conditioning solution with different K+ concentrations (1–100 mM), the amplitude of proctolin-induced contractions was reduced in the low K+ saline as well as in the high K+ saline. 5. The results suggest that voltage sensitive calcium channels account for the proctolin-induced contractions. 6. Octopamine (OA) reduced the contractions resulting from brief applications of elevated K+ concentration and of caffeine. 7. The effect of OA on the response to elevated K+ concentrations was blocked by the α-adrenergic blocker, phentolamine.

Antennal receptor response to sex pheromone mimics in the American cockroach

THE essential oils D-bornyl acetate, α- and β-santalol and several plant sesquiterpene hydrocarbons have been shown to induce sexual excitement in male American cockroaches1, and thus seem to mimic the cockroach sex pheromone. Tahara et al.2 have identified one of the active sesquiterpene hydrocarbon species obtained from Compositae plants as germacrene D. The contrast in structures of these various active compounds poses a serious problem as to the specificity of the sex pheromone receptor. To resolve this we have undertaken an electrophysiological study of the antennal receptors of both male and female cockroaches. The electroantennogram (EAG) responses of both sexes were examined during exposure to the compounds in question. Theoretically the sex specific receptors of the male antennae should respond singularly to natural sex-pheromone (and stereochemical mimics) whereas the female antennae should be unresponsive other than to general irritants and/or to food odours. In fact, the EAG response to the female sex attractant has been obtained in males but not in females3,4.

Choline acetyltransferase and acetylcholinesterase activities in muscles of aged mice

The activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were assayed in intact diaphragm, extensor digitorum longus (EDL), and soleus muscles or their homogenates of young (2-6 months) and aged (24-34 months) mice. CAT activity (per mg of protein) was significantly higher in diaphragm and soleus of old mice in comparison with the young but the age change in EDL was negligible. On the other hand, AChE activity (per mg of protein) was significantly higher in EDL of old mice but in diaphragm and soleus muscles the enzyme activity did not show any significant change statistically. The diaphragm muscle was divided into two fractions, one being neuromuscular (NM) fraction and the other the remainder of the muscle (M fraction). No appreciable change in the ratio of the enzyme activities of NM fraction to the one of M fraction was obtained between the young and aged preparations. Thus, it seems likely that there is an age-related change in CAT and AChE activities which might be affected by the degree to which muscle activity is maintained.

Effect of uncoupling agents of oxidative phosphorylation on the spontaneous release of transmitter from insect motor nerve terminals

1. The effect of uncoupling agents of oxidative phosphorylation, potassium warfarin and carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (p-CCP), on the spontaneous release of transmitter was studied at the neuromuscular junction of cockroach muscles. 2. The agents produced a large increase in the frequency of occurrence of miniature excitatory postsynaptic potentials (MEPSPs). This increase also was observed in calcium-free saline. 3. The results may be explained on the hypothesis that the increase in the spontaneous release is due to the increase in free calcium concentration derived from an intracellular origin in the terminal. The mitochondria may play an important role in regulating the intracellular calcium concentration in the nerve terminals of insect muscles.