Yoshifumi Katayama

Characteristics of rabbit ClC-2 current expressed in Xenopus oocytes and its contribution to volume regulation

In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of ×40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- ≥ Br- ≫ I- ≥ F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2ΔNT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2ΔNT-injected oocytes (-5.9 ± 0.4 μA), followed by ClC-2-injected oocytes (-4.3 ± 0.6 μA), and smallest in water-injected oocytes (-0.2 ± 0.2 μA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 ± 0.2%) > ClC-2-injected oocytes (5.3 ± 0.5%) > ClC-2ΔNT-injected oocytes (1.1 ± 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopusoocytes appear to act for volume regulation when exposed to a hyposmolar environment.In the Xenopus oocyte heterologous expression system, the electrophysiological characteristics of rabbit ClC-2 current and its contribution to volume regulation were examined. Expressed currents on oocytes were recorded with a two-electrode voltage-clamp technique. Oocyte volume was assessed by taking pictures of oocytes with a magnification of x 40. Rabbit ClC-2 currents exhibited inward rectification and had a halide anion permeability sequence of Cl- > or = Br- >> I- > or = F-. ClC-2 currents were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), diphenylamine-2-carboxylic acid (DPC), and anthracene-9-carboxylic acid (9-AC), with a potency order of NPPB > DPC = 9-AC, but were resistant to stilbene disulfonates. These characteristics are similar to those of rat ClC-2, suggesting rabbit ClC-2 as a counterpart of rat ClC-2. During a 30-min perfusion with hyposmolar solution, current amplitude at -160 mV and oocyte diameter were compared among three groups: oocytes injected with distilled water, oocytes injected with ClC-2 cRNA, and oocytes injected with ClC-2 delta NT cRNA (an open channel mutant with NH2-terminal truncation). Maximum inward current was largest in ClC-2 delta NT-injected oocytes (-5.9 +/- 0.4 microA), followed by ClC-2-injected oocytes (-4.3 +/- 0.6 microA), and smallest in water-injected oocytes (-0.2 +/- 0.2 microA), whereas the order of increase in oocyte diameter was as follows: water-injected oocytes (9.0 +/- 0.2%) > ClC-2-injected oocytes (5.3 +/- 0.5%) > ClC-2 delta NT-injected oocytes (1.1 +/- 0.2%). The findings that oocyte swelling was smallest in oocytes with the largest expressed currents suggest that ClC-2 currents expressed in Xenopus oocytes appear to act for volume regulation when exposed to a hyposmolar environment.

Regulation of the intracellular free calcium concentration in acutely dissociated neurones from rat nucleus basalis.

1. Neurones were acutely dissociated from the rat nucleus basalis. Whole‐cell patch clamp recordings of calcium currents (ICa) and fura‐2 microfluorimetric recordings of intracellular free Ca2+ concentration ([Ca2+]i) were made simultaneously. 2. Depolarization from ‐60 to 0 mV elicited ICa and a gradual increase in [Ca2+]i. After repolarization, ICa terminated in 0.7 ms, and [Ca2+]i recovered to control exponentially (1‐5 s). 3. Both ICa and the transient [Ca2+]i increase in response to step depolarizations, were abolished in Ca2+ free extracellular solution and in Cd(2+)‐containing solution. 4. Depolarizations from ‐90 mV to membrane potentials less negative than ‐40 mV induced ICa and an increase in [Ca2+]i. Depolarization to 0 mV elicited the maximum ICa, and produced the largest increase in [Ca2+]i. There was a parallel relationship between the [Ca2+]i increase and the magnitude of the ICa. 5. The [Ca2+]i increase was associated with an increase in total Ca2+ influx when the duration of the step depolarization was varied. The relationship between the total Ca2+ influx and the peak of [Ca2+]i transient reached an asymptote as total Ca2+ influx exceeded 200 pC. A similar finding was made when more than thirty action potentials were used in increasing [Ca2+]i. 6. The process of the [Ca2+]i recovery was slowed down by lowering the temperature, by an intracellular dialysis with vanadate, by extracellular application of a mitochondrial inhibitor, carbonyl cyanide m‐chlorophenyl‐hydrazone (CCCP), and by Na(+)‐free external solution. It was unaffected by membrane potential (‐50 to ‐130 mV). 7. When pipette solution contained a high concentration of fura‐2 (200 microM), the [Ca2+]i increase per 1 pC of Ca2+ influx decreased, and the [Ca2+]i recovery was slowed. 8. The results indicate that the ICa through voltage‐dependent Ca2+ channels elevates [Ca2+]i. The neurones possess a large capacity for Ca2+ buffering, and the recovery of [Ca2+]i requires both the Ca2+ pump and membrane Na(+)‐Ca2+ exchange.

Neurotransmitter release from growth cones of rat dorsal root ganglion neurons in culture

Growing neurites of rat dorsal root ganglion neurons in culture formed growth cones at the tips. Possible release of glutamate from these growth cones was investigated by using a whole-cell patch-clamp recording from an acutely dissociated hippocampal neuron containing glutamate receptors. The hippocampal neuron was placed in contact to various regions of the dorsal root ganglion neurons. Inward currents were recorded from the hippocampal neuron positioned on the growth cones of the dorsal root ganglion neurons (diameter, 12-16 microm) in response to the dorsal root ganglion cell body stimulation. The inward currents were associated with an increase in membrane conductance, and the reversal potential was estimated at -6.5 mV (n=8). The inward currents were blocked by 6-cyano-7-nitroquinoxaline (10 microM), but not blocked by 2-amino-5-phosphonovaleric acid (50 microM) and bicuculline (10 microM). The inward currents were abolished by tetrodotoxin (1 microM), EGTA-buffered Ca2+-free external solution or omega-agatoxin IVA (300 nM), and were inhibited by omega-conotoxin GVIA (3 microM), but were not affected by nicardipine (10 microM). Intracellular calcium ion concentration ([Ca2+]i) in growth cones of the dorsal root ganglion neurons increased in response to dorsal root ganglion cell body stimulation, whereas the elevation of [Ca2+]i was not observed either in the presence of tetrodotoxin (1 microM) or in a Ca2+-free external solution. These results indicate that the inward currents were evoked by glutamate released from the growth cones via a Ca2+-dependent process, and suggest that the growth cones are already endowed with much of the machinery for neurotransmitter release, even before making a structure for synaptic transmission.

Substance P inhibits activation of calcium-dependent potassium conductances in guinea-pig myenteric neurones.

1. Intracellular recordings were made from myenteric AH neurones of the guinea‐pig ileum in vitro. Some experiments were done with a single‐electrode voltage clamp to measure membrane currents. 2. Substance P (SP) applied by superfusion (10 nM‐300 nM), pressure ejection (100 nM‐10 microM, 760 mmHg, for 10‐20 ms) or ionophoresis (1 mM, 100 nA, for 0.2 s) caused a membrane depolarization and an inward current, associated with a decrease in potassium conductance. 3. The SP‐induced depolarization was abolished within 15 min by superfusion with calcium‐free/high‐magnesium (10 mM) solution or solutions containing cobalt, manganese or nickel at 1‐3 mM. The response persisted even after 40‐60 min of superfusion with calcium‐free/normal‐magnesium (1.2 mM) solution. In all these solutions, synaptic potentials were abolished within 5 min. 4. SP inhibited a slowly developing outward current and an outward tail current during and after a long depolarizing command pulse (2‐10 s), and an outward after‐current following single or multiple brief depolarizing command pulses (10‐50 ms). These outward currents were suppressed in calcium‐free/high‐magnesium solution. 5. SP depressed both a calcium‐dependent slow after‐hyperpolarization following the action potential and an outward after‐current preceded by a brief depolarizing command. Both the SP‐induced depolarization and the SP‐induced inward current were augmented when the peptide was pressure‐ejected during the recovery phase of the slow after‐hyperpolarization and during that of the slow outward after‐current, but both of them were inhibited or almost abolished when SP was applied immediately after spike initiation or a brief depolarizing command. 6. The SP‐induced response was depressed by barium (1‐2 mM). The SP response was not inhibited by tetraethylammonium at low concentrations (5‐10 mM), but was depressed at high concentration (20 mM). 7. Superfusion (1‐10 nM) or pressure application of a calcium ionophore, A23187, inhibited or even reversed the SP depolarization and the SP‐induced inward current. 8. These results indicate that SP inhibits activation of a calcium‐dependent potassium conductance which contributes to both the slow after‐hyperpolarization and the resting membrane potential. SP may affect the process by which calcium activates this potassium conductance.

ATP regulates synaptic transmission by pre- and postsynaptic mechanisms in guinea-pig myenteric neurons

Intracellular recordings were made from myenteric neurons of the guinea-pig ileum in vitro; they were classified into S and AH neurons according to electrophysiological criteria. ATP (10 nM-100 microM) inhibited excitatory synaptic potentials in the myenteric plexus; fast excitatory postsynaptic potentials and slow excitatory postsynaptic potentials of S neurons and slow excitatory postsynaptic potentials in AH neurons. This inhibitory action was reversible and dose-dependent, and was usually followed by a transient augmentation of the synaptic potentials after washing of ATP. The actions of ATP on the synaptic potentials were prevented by pretreatment with theophylline, caffeine, quinidine and 8-phenyl theophylline. The ATP analogues, ATP-gamma-s (100 nM-100 microM) and alpha-beta-methylene ATP (100 nM-100 microM) also depressed the synaptic potentials recorded from both types of neurons. The inhibitory effect of adenosine on the synaptic potentials was 10 times weaker than that of ATP. Thus, it seems clear that the presynaptic inhibition is not occurring through adenosine A1 or A2 receptors. Furthermore, ATP at high concentrations ( > or = 1 microM) augmented nicotinic fast depolarizations of S neurons produced by extracellular acetylcholine. However, ATP at the same concentrations inhibited the slow depolarizations of S and AH neurons caused by exogenous acetylcholine (muscarinic) and substance P. It is concluded that ATP regulates synaptic transmission in the myenteric plexus of the guinea-pig ileum and the sites of ATP actions are pre- and postsynaptic.

Observation of subcellular nanostructure of single neurons with an illumination mode photon scanning tunneling microscope

We report about the observation of microtubules lying underneath the cell membrane of neural process in neurons with a resolution as high as that of an electron microscope by an illumination mode photon scanning tunneling microscope. Nanoapertures used in our observations were fabricated by means of selective chemical etching and metal coating of an optical fiber. The narrowest observed tube has got an average diameter of 26 nm. Comparing this with its nominal value of 25 nm, the difference which is considered as a measure of resolution (δ) is 1 nm implying a resolution comparable to that of an electron microscope in imaging dielectric specimens. This was possible due to the presence of a boundary between the glass and the metal coating and also due to the use of an aperture of almost the same size as that of the microtubule that enhances the detection.

5-Hydroxytryptamine effects on the somata of bullfrog primary afferent neurons

Intracellular recordings were made from neurons in the isolated dorsal root ganglia of bullfrogs. 5-Hydroxytryptamine was applied by superfusion and by ionophoresis. The most common response to 5-hydroxytryptamine in C neurons was a membrane hyperpolarization and this was observed in 80% of cells. This was due to an increase in membrane potassium conductance because it reversed its polarity at about -90 mV. It was blocked by removal of calcium or addition of calcium blockers. (+)-Tubocurarine, methysergide, ketanserin, quipazine, picrotoxin, caffeine and ouabain blocked this response. The next most common response in C neurons was a fast depolarization, particularly readily observed when 5-hydroxytryptamine was applied by ionophoresis. Since this response reversed its polarity at about -10 mV and was blocked by removal of sodium, this was due to an increase in membrane conductance to both sodium and potassium ions. This response was reduced by superfusion of acetylcholine and gamma-aminobutyric acid. (+)-Tubocurarine, quipazine, picrotoxin and caffeine blocked the response. A small proportion of C neurons (16%) responded to superfusion of 5-hydroxytryptamine with a slow depolarization accompanied by an increase in input resistance. This response reversed its polarity at about -90 mV and, therefore, is presumed to result from potassium inactivation. It was blocked by methysergide and ketanserin but not by (+)-tubocurarine or quipazine. A few type A neurons (8%) caused a fast and transient depolarization like the fast depolarization of C neurons. About half of the A neurons showed a slow depolarization associated with a fall in input resistance. This slow response was assumed to be due to an increase in membrane conductance to both potassium and calcium ions because the response reversed its polarity at about -65 mV and was sensitive to change in external concentrations of those ions. This slow response was blocked by (+)-tubocurarine, methysergide, ketanserin, picrotoxin, caffeine and ouabain but not by quipazine. The effects of 5-hydroxytryptamine are discussed in relation to the similar actions described on a variety of other vertebrate and invertebrate nerve cells. The findings imply that dorsal root ganglion cells of bullfrogs are sensitive to 5-hydroxytryptamine and causes multiple types of 5-hydroxytryptamine responses.

Synthesis, storage and release of acetylcholine at and from growth cones of rat central cholinergic neurons in culture

Neurons from the nucleus diagonal band of Broca (DBB) from new born rats protrude neuronal processes and growth cones in culture. Cytochemical observations with the light and electron microscope indicate that growth cones of these neurons take up choline, synthesize acetylcholine (ACh) and store ACh in the vesicles. Electrical stimuli at the soma of DBB neurons evoked inward currents in ACh-sensitive neurons attached to DBB growth cones. These currents were suppressed by TTX, a Ca2+ channel blocker (Cd2+), and an ACh nicotinic antagonist (C6). These results suggest that ACh is synthesized, stored and released from the growth cones of DBB neurons prior to synapse formation.

Calcium-dependent slow outward current in visceral primary afferent neurones of the rabbit.

Slow outward currents were recorded from voltage-clamped neurones in nodose ganglia excised from rabbits. In the majority of Type C neurones, a short depolarizing command pulse evoked a slow outward tail current (ISAH) with a decay time constant ranging from 0.5 to 2 s. TheISAH was due to an increase in membrane conductance to K+ because its reversal potential was approximately equal to the Nernst potential for K+. TheISAH was reversibly blocked by removal of external Ca2+ or by Ca2+ antagonists. A Ca2+ ionophore, A23187, produced an outward current which was similar to theISAH. TheISAH was resistant to tetraethylammonium and depressed by Ba2+, whereas it was not affected by Cs+ and 4-aminopyridine. TheISAH was initially augmented and subsequently depressed by apamin (1–10 nM) and (+)-tubocurarine (100–600 μM). It is concluded that theISAH in visceral primary neurones may be due to a long-lasting increase in K+ conductance caused by an increase in the concentration of intracellular Ca2+, resulting from Ca2+ entry during the depolarizing command pulse.

Brief increases in intracellular Ca2+ activate K+ current and non-selective cation current in rat nucleus basalis neurons

Neurons were acutely dissociated from the rat nucleus basalis, and membrane currents (whole-cell patch-clamp) and intracellular free Ca2+ concentrations (Fura-2) were measured simultaneously from large neurons (approximately 25 microns in diameter). A brief depolarization from -60 to 0 mV for 200 ms evoked an increase in intracellular free calcium and a slow outward tail current (72 +/- 8 pA, n = 30). The outward current reversed polarity at -75.5 +/- 2.7 mV (n = 14). The tail current declined and the intracellular calcium recovered its resting level exponentially with time-constants of 1.0 +/- 0.1 s and 2.5 +/- 0.2 s, respectively (n = 17). In neurons loaded with Cs-gluconate, a similar depolarizing pulse evoked a similar increase in intracellular free calcium, but this was now followed by an inward tail current (118 +/- 8 pA, n = 44). The inward tail current reversed polarity at -27.8 +/- 3.8 mV (n = 7), and was suppressed by removal of external sodium ions. Neither outward nor inward tail currents were observed, when the external solution was calcium-free or when the pipette solution contained EGTA (10 mM). These results indicate that a depolarization causes a calcium entry and that this consequently increases both K+ conductance and non-selective cation conductance.