Hitoshi Tatsumi

Measurement of the intracellular calcium concentration in guinea-pig myenteric neurons by using fura-2

Intracellular free calcium concentration [( Ca2+]i) was measured in guinea-pig myenteric neurons by using the fluorescent calcium indicator, fura-2; at the same time, intracellular recordings were made from the myenteric neurons. The [Ca2+]i of the myenteric neurons was about 100 nM at the resting state. The slow after-hyperpolarization that followed an action potential was associated with an increase in [Ca2+]i [Ca2+]i decreased during superfusion with calcium-free/high (6 mM) magnesium solution, and increased during superfusion with high (20 mM) potassium solution. However, [Ca2+]i did not change when the depolarizations caused in the high potassium solution were prevented by passing inward current through the recording electrode. The present experiments provide direct evidence that depolarization of the myenteric neurons allows calcium to enter the cell, and that rises in intracellular calcium concentration hyperpolarize the neuron.

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.

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.

Attachment of growth cones on substrate observed by multi-mode light microscopy.

Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rhodamine B). Dynamic changes of the cell substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed.

The actions of 5-hydroxytryptamine in the rabbit ciliary ganglion

Intracellular recordings were made from ciliary ganglion neurones of the rabbit in vitro. 5-Hydroxytryptamine (5-HT) produced a rapid depolarization in 67% of the neurones examined. The 5-HT-induced depolarization persisted in solutions containing zero (1-100 microM), or on the amplitude and the duration of the current used for ionophoresis. The 5-HT-induced depolarization was associated with a marked fall of input resistance and reversed in polarity at about -10 mV. The 5-HT-induced response was reversibly abolished in sodium-free solution. A marked desensitization to this action of 5-HT was observed. After the 5-HT depolarization passed off due to desensitization, 5-HT (1 microM-1 mM) reversibly depressed the amplitude of cholinergic excitatory postsynaptic potentials (EPSPs) in 91% of the neurones tested in a dose-dependent manner, whereas 5-HT did not detectably affect the depolarization induced by ionophoresis of acetylcholine (ACh). The frequency of spontaneous miniature EPSPs (in 20 mM potassium solution) was decreased by 5-HT (30-100 microM). These results demonstrate that 5-HT exerts two separate actions in the rabbit parasympathetic ciliary ganglion. One is a depolarization of ganglion cells due to an increase in membrane permeability to sodium and presumably potassium ions; the other is a depression of ACh release from presynaptic fibres. Further pharmacological characteristics of these actions of 5-HT were investigated and discussed.

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.

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.

Growth cones exhibit enhanced cell-cell adhesion after neurotransmitter release

Evoked release of acetylcholine and subsequent cell-cell adhesive contacts between growth cones and acetylcholine sensing neurons were observed using cultured neurons dissociated from the diagonal band of Broca of the rat. Stimulation to the soma of the diagonal band of Broca neurons evoked release of acetylcholine from the growth cones. The release of acetylcholine was monitored using whole-cell patch-clamp recording from acetylcholine receptor-rich superior cervical ganglion neuron positioned on the growth cone as a sensor of acetylcholine release. By measuring changes in fluorescence from the growth cone using Ca2(+)-sensitive dye while voltage-clamping the superior cervical ganglion neuron, transient intracellular Ca2+ concentration increase and acetylcholine release from growth cone were recorded simultaneously. Video-enhanced differential interference contrast imaging of the growth cones demonstrated tether formation between the growth cone and superior cervical ganglion cell soma when the superior cervical ganglion cell soma was moved away from the growth cone after acetylcholine release, suggesting formation of adhesive contacts between the growth cone and the sensor neuron. Adhesive contacts between growth cones and sensor neurons were also detected when a high K+ solution or alpha-latrotoxin was applied to the growth cone. Adhesions were also observed between growth cones and latex beads, when growth cones were exposed to high K+ solution. The properties of the adhesive contacts at the growth cone were studied by optically manipulating a latex bead attached to the growth cone surface. These results suggest that growth cones exhibit cell-cell adhesion after neurotransmitter release.

Calcium homeostasis in the presence of fura-2 in neurons dissociated from rat nucleus basalis: theoretical and experimental analysis of chelating action of fura-2

Intracellular calcium ions (Ca2+) play important roles in cell functions. Measurements of intracellular calcium ion concentration ([Ca2+]i) are often made with the fura-2 fluorescence recording technique in various preparations including neurons. Fura-2 has, however, a Ca(2+)-chelating action which complicates the interpretation of experimental results. In this report the chelating action of intracellular fura-2 was studied by means of computer simulations. The chelating action of an endogenous Ca(2+)-binding protein, calmodulin, was also estimated. Furthermore, whole-cell patch-clamp recordings of calcium currents (ICa) and fura-2 microfluorimetric recordings of [Ca2+]i were simultaneously made from neurons which were acutely dissociated from the rat nucleus basalis. Since Ca2+ influx can be initiated and terminated by using the voltage-clamp technique, the relationship between Ca2+ influx and rapid [Ca2+]i increase was examined. The present theoretical evaluations and experimental results disclosed the relationship between fura-2 and endogenous Ca(2+)-binding proteins; fura-2 at low concentration (10 microM) did not substantially affect the endogenous Ca2+ buffering mechanisms, but at high concentration (200 microM) effectively buffered cytosolic Ca2+ instead of endogenous Ca2+ buffers. Calcium homeostasis in neurons is furthermore discussed.