Jun Fukuda

Presynaptic Ca-antagonist ω-conotoxin irreversibly blocks N-type Ca-channels in chick sensory neurons

Abstract The present studies on electrophysiological and pharmacological differences of the three types of Ca-currents (N-, L- and T-types) in whole-cell clamped, cultured embryonic chick sensory neurons revealed that the majority (94%) of the Ca-currents in the nerve cells were the N-type. ω-Conotoxin (ωCTX, 5 μM), a blocker of transmitter release at the presynaptic terminals, induced a complete and irreversible blockage of Ca-currents elicited from the resting membrane potential (− 60 mV) in 29 cells among 58. The Ca-currents thus irreversibly blocked by the ωCTX were determined as the N-type (neuronal), as they were insensitive to nifedipine (5 μM) or were reduced in amplitude by Bay K 8644 (5 μM). A small fraction (12%) of the total Ca-currents, which were still present after the ωCTX treatment (in the rest of 29 cells), were pure L-type (long-lasting) Ca-currents, as they were enhanced by the Bay K and were blocked by the nifedipine. ωCTX was a partial and reversible blocker of the L-type Ca-currents. Furthermore, T-type (transient) Ca-currents elicited in the hyperpolarized membrane (at − 100 mV) were blocked by ωCTX in an incomplete and reversible manner. The N-type Ca-currents thus separated in the nerve cells exhibited various differences in features of the voltage-dependence and ionic selectivity from the L- and T-type Ca-currents.

Cerebellar inhibitory control of the vestibulo-ocular reflex investigated in rabbit IIIrd nucleus

SummaryIn anaesthetized rabbits, the vestibulo-ocular reflex was evoked by electric stimulation of VIIIth nerve and was observed by recording postsynaptic potentials and relevant field potentials in Illrd nucleus. The electric stimulation of flocculus produced a prominent inhibition of the vestibulo-ocular reflex in both the inhibitory component relayed by the superior vestibular nucleus and the excitatory component mediated by the brachium conjunctivum. The excitatory component mediated by the medial vestibular nucleus appeared to be free of the flocculus inhibition. The flocculus inhibition was blocked very effectively by systemic injection of picrotoxin. That the flocculus inhibitory action is due to monosynaptic postsynaptic inhibition of secondary vestibular neurones was demonstrated by direct stimulation of, and also by recording from, the superior nucleus. Recording from the superior nucleus was also performed in anaesthetized cats. All of these above results indicate that Purkinje cells in flocculus projecting to vestibular and cerebellar nuclei cells have inhibitory synaptic action. Flocculus stimulation produced also an excitatory effect upon vestibular nuclei neurones. However, this effect could be attributed to intracerebellar activation of the primary vestibular fibers which pass into the flocculus.

Recognition of artificial microstructures by sensory nerve fibers in culture

Dissociated culture of adult mouse dorsal root ganglion cells on glass plates, on which grating-associated microstructures (a repetition of microgrooves [mGRV] and microsteps [mSTP] of 0.1-10 micron) are fabricated by the conventional lithographic techniques, represents a remarkable bi-directional growth of their nerve fibers in the axial direction of the grating. Microscopical observation shows that the nerve fibers prefer to grow in the mGRV (70%), while their growth cones exhibit an even distribution onto the mGRV and mSTP. The efficiency of the nerve fibers to grow along the grating-axis are highly sensitive to a fine alteration of the width and depth of the mGRV. The preferential growth of the nerve fibers is thus due to a mechanical recognition of the microstructures by the growth cones and neurites.

Single transient K channels in mammalian sensory neurons.

A single-channel recording of the transient outward current (A-current) was obtained from dorsal root ganglion cells in culture using patch-clamp techniques. Depolarization of the membrane patch elicited pulse like current of a uniform amplitude in an outward direction, of which the unitary conductance was 20 pS. Alteration of extracellular ionic compositions indicated that the charge carriers were K ions. A systematic study was made on the voltage-dependence of the ensemble average current; (a) the activation started at a potential around -60 mV; (b) the time course of the activation was relatively rapid; (c) the channel was completely inactivated at a potential positive to -40 mV. Two time constants (tau f = 100 ms and tau s = 4,000 ms) were detected in the decay of the current indicating that the channels had two different states of inactivation. A convulsant, 4-aminopyridine (4-AP), acted on the channel only from the intracellular side of the membrane. 4-AP (5 mM) reduced not only mean open time (by 50%) but also the single-channel conductance (by 20%). The properties of the channel were independent of Ca ions in the intracellular space.

Inhibition by K-252a, a new inhibitor of protein kinase, of nerve growth factor-induced neurite outgrowth of chick embryo dorsal root ganglion cells.

The effect of K-252a, a new alkaloid-like kinase inhibitor from the culture broth of Nocardiopsis sp., on the action of nerve growth factor on normal nerve cells was examined. Nerve cells were isolated from the dorsal root ganglia of chick embryo by trituration without using digesting enzymes, and were seeded on collagen- or poly-L-lysine-coated plastic dishes containing serum-free medium. K-252a at 50-100 nM reversibly inhibited the formation of neurites by dorsal root ganglion cells elicited by the treatment with nerve growth factor.

A tissue-culture of nerve cells from adult mammalian ganglia and some electrophysiological properties of the nerve cells in vitro *

A new technique for primary culture of nerve cells isolated from peripheral ganglia of adult mammals has been developed. Nerve cells were isolated from trigeminal ganglia, nodose ganglia, sympathetic ganglia or dorsal root ganglia of adult guinea pigs by collagenase or dispase, and then were grown on collagen-coated plastic dishes for more than 4 weeks. Intracellular recording with a glass microelectrode reveals that the nerve cells in vitro generate not only Na spikes but also tetrodotoxin-resistant Na and Ca spikes. An exception was that the sympathetic ganglion cells in vitro failed to generate tetrodotoxin-resistant Na spikes. Difference in values of the Vmax of these spike components is observed among nerve cells (3-8 days in vitro) of different origin, suggesting that distribution of ionic channels on the plasma membrane may be different among species of nerve cells.

Loss of membrane excitability after herpes simplex virus infection in tissue-cultured nerve cells from adult mammals

Dorsal root ganglion cells of adult guinea-pigs in vitro were inoculated with herpes simplex virus (HSV). Intracellular recording with a glass microelectrode revealed that HSV-infection caused a marked decrease in membrane excitability of the nerve cells within 24 h, which could be explained as a decrease in Na+ channel activity in the plasma membrane; while changes in other physiological properties (resting membrane potential, membrane resistance and capacitance) remain small. Viral antigens were detected in only 10-15% of the HSV-infected nerve cells. These indicate that the loss of the membrane excitability occurs much earlier than any other change in the HSV-infected nerve cells.

Primary culture of postnatal rat hypothalamic neurons in astrocyte-conditioned medium.

Mature functional hypothalamic neurons of male and female rats (21-day postnatal) were successfully cultured without attachment to non-neuronal cells in serum-free astrocyte-conditioned medium (ACM). A novel cell-collecting method was designed for these vulnerable cells by allowing the dissociated cell suspension to stand in a vertically held, wide-tipped syringe so that the cells were concentrated near the lower liquid surface, from which position they could be easily dropped into the medium, leaving most of the small debris in the syringe. This method made it possible to study statistically the survival of cultured neurons. It was impossible to collect many viable cells by the commonly used dissociating technique for fetal rat brain. However, neuron-like cells with a few processes could be isolated from sliced hypothalamic tissues by means of enzymatic and mechanical treatments. The original processes disappeared within 1-2 days and some new processes were generated after 2-3 days in vitro; the cells survived for 28 days in vitro. The cells were identified as neurons by the immunostaining method for microtubule-associated protein 2 (MAP2) and neurofilament (NF). Such neurons were obtained from every site of hypothalamic tissue sampled. These phenomena were not observed in chemically-defined medium (CDM), CDM supplemented with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) or nerve growth factor (NGF).

Nerve Cells of Adult and Aged Mice Grown in a Monolayer Culture: Age-Associated Changes in Morphological and Physiological Properties of Dorsal Root Ganglion Cells in vitro

In order to differentiate age-associated changes in morphological and physiological properties of mammalian nerve cells, dorsal root ganglion (DRG) cells of aged mice (C57BL/6; 98-99 weeks old) were grown in a monolayer culture. Neurite outgrowth, changes in shape and size of their soma and functional properties of their plasma membranes were compared to those of tissue-cultured DRG cells from young adult mice (4-8 weeks old). Trigeminal root ganglion (TRG) cells of aged mice were also grown in a monolayer culture, and their in vitro growth was compared to that of the aged DRG cells. Nerve cells were dissociated from DRG (or TRG) by digestion with collagenase and by trituration and were grown on collagen-coated plastic dishes for more than 14 days. Growth of neurites and changes in the size and shape of the nerve cell soma were viewed under a phase-contrast microscope, and physiological properties of the plasma membrane were studied by conventional intracellular recordings with a glass microelectrode. Both adult and aged DRG cells grew neurites of various length and underwent changes in shape and size of their soma, which could be divided into 2 stages; early and late. In the early stage of tissue culture (0-60 h in vitro), nerve cells altered their shape from a spherical to a spindle-like form. This change was not associated with the reduction in cell size. In the late stage of the tissue culture (3-14 days and thereafter), the DRG reduced their cell size, while changes in shape remained small. Quantitative comparison of the adult and aged DRG nerve cells revealed the following 3 major differences between 2 cultures: the survival fraction of the aged DRG cells counted at 36-48 h in vitro was 1/4 to 1/10 of that of the adult DRG cells in 3 different tissue culture trials; reduction in the cell size occurred much earlier in the aged than in the adult nerve cells; the rate of reduction in size of the aged DRG cells was large in comparison with that of the adult DRG cells. No difference in neurite growth or in physiological properties (resting membrane potential, input resistance, input capacitance or capability of generating both Na and Ca spikes) was detected between the aged and adult nerve cells in tissue culture.

ATP content in isolated mammalian nerve cells assayed by a modified luciferin-luciferase method.

ATP content in a nerve cell isolated from dorsal root ganglia of adult guinea-pigs by collagenase was measured by a newly developed technique modified from the conventional luciferin-luciferase methods. A small volume (4 microliters) of the nerve cell suspension, which contained 10-300 nerve cells (3-100 X 10(-4) microliters of cellular volume) under view of an inverted, phase-contrast microscope, was heat-treated for about 1 s by flame of an alcohol lamp. This heat-treated cell suspension was then reacted with a luciferin-luciferase solution. Light flux from the bioluminescence thus elicited gave an ATP content in single nerve cell, 27 pg (mean) +/- 10 pg (S.D.). ATP concentration in a nerve cell was calculated as 1.7 mM (mean) +/- 0.6 mM. The ATP content in a nerve cell was reduced when the nerve cells were exposed to KCN (5 microM) or dinitrophenol (20 microM), respectively.