Tadashi Kawakami

Coexistence of nitric oxide synthase and neuropeptides in the mouse vomeronasal organ demonstrated by a combination of double immunofluorescence labeling and a multiple dye filter

Nitric oxide synthase (NOS)-immunofluorescence techniques were applied to the mouse vomeronasal organ. Immunoreactivity for NOS was found in the nerve fibers distributed in the receptor-free epithelium, and around the blood vessels and glands in the cavernous tissue. No NOS fibers were seen in the receptor area. A combination of double immunofluorescence labeling and multiple dye filter revealed that a part of the substance P (SP)-immunoreactive nerve fibers in the cavernous tissue contained NOS and that all the vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers around the blood vessels and glands in the cavernous tissue contained NOS. A few SP-immunoreactive cell bodies in the trigeminal ganglion showed coexistence with NOS, and almost all VIP-immunoreactive cell bodies in the sphenopalatine ganglion showed coexistence with NOS. Immunoreactivity for NOS without VIP in the cell bodies in the sphenopalatine ganglion was also found. These results suggest that NOS-immunoreactive nerve fibers in the mouse vomeronasal organ originate from the trigeminal and the sphenopalatine ganglia, and may modulate the vascular tone and the glandular secretion. In addition, these functions may be controlled in part by the interaction of nitric oxide and neuropeptides.

Axoplasmic transport of mitochondria in cultured dorsal root ganglion cells

The movements of individual mitochondria in cultured mouse dorsal root ganglion cells were directly observed by using fluorescent staining with rhodamine 123 in combination with video microscopic techniques. This gives greater spatial and temporal resolution and much higher specificity than possible by conventional methods. The instantaneous velocities were 0.55 +/- 0.11 microns/s anterograde and 0.60 +/- 0.10 microns/s retrograde. Movement of the mitochondria was in fits and starts, and some reversed direction. The number of mitochondria moving retrogradely was 1.5-1.9 times greater than the number moving anterogradely. The average length of mitochondria moving retrogradely was 2.8 microns and of mitochondria moving anterogradely was 4.1 microns. These results suggest that mitochondria increase their numbers by division in the nerve fiber terminal.

Effects of Arachidonic Acid and the Other Long-Chain Fatty Acids on the Membrane Currents in the Squid Giant Axon

SummaryThe effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using α-cyclodextrin as a solvent. α-cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED50) was 0.18mm, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952)J. Physiol (London)117:500–544) and the change in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m∞) for the Na current against membrane potential and the time constant of activation (τm) were shifted 10 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation (τh) has almost unaffected by application of these fatty acids.

Coexistence of substance P, neuropeptide Y, VIP, and CGRP in the nerve fibers of the carotid labyrinth of the bullfrog, Rana catesbeiana: a double-labelling immunofluorescence study in combination with alternate consecutive sections.

Double immunohistochemical staining with rhodamine- and fluorescein isothiocyanate (FITC)-conjugated antisera revealed the coexistence of substance P (SP) and neuropeptide Y (NPY), and SP and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bull-frog, Rana catesbeiana, although there were a few fibers which showed only SP- or NPY-immunoreactivity. Approximately one third of SP-immunoreactive fibers also showed coexistence with vasoactive intestinal polypeptide (VIP)-immunoreactivity, and a few fibers contained VIP without SP. The combination of the double immunofluorescence technique and alternate consecutive sections further demonstrated the possible coexistence of SP, VIP, NPY, and CGRP. This coexistence of four different peptides in the same nerve fibers was proved by the following two evident facts: 1) some SP fibers which demonstrated coexistence with NPY-immunoreactivity were assumed to be continuous with those showing VIP-immunoreactivity, and 2) almost all of the SP fibers showed coexistence with CGRP-immunoreactivity. By this reasoning, nearly one third of SP fibers may demonstrate coexistence with NPY-, VIP-, and CGRP-immunoreactivities. These multiple peptides might be involved in vascular regulatory function, which is a possible function of the amphibian carotid labyrinth.

Effect of neurotransmitters on axoplasmic transport: how adrenaline affects superior cervical ganglion cells

The effect of adrenaline on the axoplasmic transport of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Adrenaline increased the axoplasmic transport reversibly in both anterograde and retrograde directions. A beta 2-antagonist, butoxamine, antagonized the increasing effects of adrenaline, but alpha-antagonists and beta 1-antagonists did not. A beta 2-agonist, albuterol, mimicked the adrenaline effect, but beta 1-, alpha 1-, alpha 2-agonists did not. The adrenaline receptor may be a beta 2-receptor. Dibutyryl cyclic AMP and forskolin increased the axoplasmic transport. Therefore, adrenaline increases the axoplasmic transport by raising the cyclic AMP level. In light of our former report that acetylcholine suppresses the axoplasmic transport, neurotransmitters control axoplasmic transport and this neurotransmitter control reflects the activity of the nerve cell.

Bradykinin-responsive cells of dorsal root ganglia in culture: Cell size, firing, cytosolic calcium, and substance P

Summary1. We analyze bradykinin-sensitive cells of the mouse dorsal root ganglion in culture from the viewpoints of cell size, electrical responses, and Ca2+ concentration change due to bradykinin and immunocytochemistry of substance P.2. Sixteen percent of cells in the cell group 26–30 µm in diameter fired in response to 10 µM bradykinin. None of other cell groups showed a firing response to bradykinin.3. We measured a cytosolic Ca2+ change due to bradykinin using a Ca2+-sensitive fluorescent dye, Fura 2. The rapid rise (peak time, 20 sec) in the Ca2+ concentration was ascribed to Ca2+ release from intracellular Ca2+ stores. The profound change in the Ca2+ concentration was observed again in the cell group 26–30 µm in diameter. Seventeen percent of cells in this group increased the Ca2+ concentration by approximately seven times that at resting level.4. Among cells which increase Ca2+ concentration responding to bradykinin, 83% of them contain substance P (an immunocytochemical study).5. We conclude that 16–17% of the cell group 26–30 µm in diameter of the dorsal root ganglia in culture are polymodal nociceptors and respond to bradykinin.

Coexistence of substance P and calcitonin gene-related peptide in the nerve fibers of the carotid labyrinth of the bullfrog,Rana catesbeiana

Double immunohistochemical staining with fluorescein isothiocyanate (FITC)- and rhodamine-conjugated antisera revealed the coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in most nerve fibers in the intervascular stroma of the carotid labyrinth of the bullfrog, Rana catesbeiana. A few fibers contained SP without CGRP. The results suggest that the vascular regulatory function, which is one of the possible functions of the carotid labyrinth, may be controlled in part by the interaction of SP and CGRP.

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca(2+)-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca2+ concentration. ACh also suppressed the axoplasmic transport in Ca(2+)-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca2+ ions.

Heart Stimulation by Time-Varying Magnetic Fields

A strong magnetic stimulator adopted for cardiac muscle was constructed with the stored energy of 50 kJ. Pulsed magnetic fields were applied to dog hearts with normal activity from outside of the body. The magnetic stimulus triggered on the T wave of the electrocardiograph caused arrhythmias in the first and second beats after the stimulus. It has been confirmed that this magnetic effect is due to a direct stimulation of cardiac muscle, not to an indirect stimulation on the vagus nerve. The threshold strength was determined for different pulse durations. The obtained strength-duration relationship is comparable to that for the electric stimulation of the dog heart.

Electromagnetic mechanism of magnetic nerve stimulation

Rabbit sciatic nerves were stimulated by pulsed magnetic fields and nerve responses were analyzed on the basis of electromagnetic theory to understand the dominant factors in magnetic stimulation. Some conducting substance surrounding the nerve is required to induce the currents exciting it. The strength of a magnetic stimulus is evaluated by the rate of change in the vector potential at the nerve, dA/dt, which equals the magnitude of the induced electric field E. The minimum strength of dA/dt for exciting the nerve is 18 Wb/ms (18 V/m) in the agar with a conductivity of 0.6 S/m. The induced current density of 10 A/m2 is comparable to that used in the electric stimulation of peripheral nerves. The component of the vector potential parallel to the nerve is more effective in stimulating the nerve than the component perpendicular to it.