Yoshiaki Ohi

Functional and morphological organization of the nucleus tractus solitarius in the fictive cough reflex of guinea pigs

Projection of the superior laryngeal nerve (SLN) afferent fibers into the nucleus tractus solitarius (NTS) was investigated using a fluorescent tracer in guinea pigs. High density of fluorescence was detected in the ipsilateral NTS extending from 0.5 mm caudal to 1.2 mm rostral to the obex. At coronal slices, the fluorescent granules, lines and patches were located in the interstitial, medial and dorsal regions of NTS. Fluorescence was also found in the dorsal region of contralateral commissural NTS. Microstimulation of the rostral NTS, which corresponded to the region showing the strong fluorescence, induced an increase in the inspiratory discharge of phrenic nerve that was immediately followed by a large burst discharge of the iliohypogastric nerve in decerebrate, paralyzed and artificially ventilated guinea pigs. This serial response of the two nerves was identical to that induced by electrical stimulation of the SLN. Intravenous injection of codeine suppressed both NTS and SLN-induced responses. The SLN-induced response was inhibited by microinjection of codeine into the ipsilateral NTS and abolished by lesion of the ipsilateral NTS. These results suggest that the NTS has an integrative function in production of cough reflex and is possible sites of action of central antitussive agents.

Biphasic effects of morphine on bulbar respiratory neuronal activities in decerebrate cats

To understand neuronal mechanisms underlying respiratory depression induced by morphine, membrane potential, input resistance and burst discharge in different types of respiratory neurons were recorded in decerebrate and vagotomized cats. Intravenous morphine (0.3-3.0 mg/kg) dose-dependently decreased the respiratory discharge in the phrenic and iliohypogastric nerves. The drug changed the respiratory frequency in a biphasic fashion, a transient increase (early phase) followed by a long-lasting decrease (late phase). During the early phase, the membrane was hyperpolarized throughout the respiratory cycle and the burst discharge was decreased in all types of respiratory neurons. During the late phase, the active phase depolarization and the inactive phase hyperpolarization were decreased, resulting in a decline of membrane potential fluctuations. Input resistance was decreased during the early phase and increased during the late phase. Iontophoresed (50-100 nA) morphine produced hyperpolarization of the membrane and a decrease in input resistance in respiratory neurons. This hyperpolarization remained unaltered after iontophoresed tetrodotoxin depressed the synaptic transmission. These effects of morphine were completely blocked by naloxone and beta-funaltrexamine, but not by naltrindole. The present results suggest that morphine depresses the respiratory neuronal activity through two different mechanisms, both of which are mediated by mu receptors.

Distribution of μ receptors in the ventral respiratory group neurons; immunohistochemical and pharmacological studies in decerebrate cats

Immunoreactivity for mu receptors was investigated in 21 bulbar respiratory neurons, individually identified by intracellular recording and labeling with neurobiotin. In 14 of these neurons, effects of iontophoresed morphine were examined. Morphine hyperpolarized the membrane and decreased spike discharges in 4/6 augmenting inspiratory (aug-I), 4/5 postinspiratory (post-I) and 3/3 augmenting expiratory (aug-E) neurons. It had no effect on two aug-I and one post-I neurons. Strong immunoreactivity for mu receptor was detected in the soma and dendrites of 5/8 aug-I, 5/7 post-I and 6/6 aug-E neurons. In the remaining three aug-I and two post-I neurons that included cells unresponsive to morphine, weak immunoreactivity was detected only in the dendrites. These results demonstrated wide, but uneven, distribution of mu receptors in bulbar respiratory neurons and suggest their contribution to respiratory depression by opioids.

Phrenic and iliohypogastric nerve discharges during tussigenic stimulation in paralyzed and decerebrate guinea pigs and rats

Although effects of antitussive drugs have been examined in inbred small animals using a whole body plethysmography, neuronal mechanisms underlying the cough reflex are not fully understood. The present study analyzed the reflex discharge patterns of the phrenic (PN) and iliohypogastric nerves (IHN) evoked in decerebrate and paralyzed guinea pigs and rats. In guinea pigs, electrical stimulation of the superior laryngeal nerve, chemical stimulation with capsaicin and mechanical stimulation to the intratracheal mucosa equally produced a serial PN-IHN response. This response was characterized by an increased PN discharge and following spindle-shaped burst of the IHN. The evoked discharges overlapped for 20 ms. In rats, by contrast, mechanical stimulation was without effect while capsaicin and electrical stimulation produced two types of responses, both of which differed from that observed in guinea pigs. The first type consisted of an augmented burst of the IHN that was immediately followed by an increased PN discharge. The second type was a large spindle-shaped burst of the IHN that occurred 80 ms after the end of the preceding PN discharge. Codeine (3 mg/kg i.v.) depressed all types of responses evoked in guinea pigs and rats. The present study demonstrated that the fictive cough comparable with those induced in other experimental animals was produced consistently in guinea pigs, but not in rats. Therefore, guinea pigs are suitable for investigation of the neuronal mechanisms underlying the cough reflex and assessment of antitussive drugs.

Cough-related neurons in the nucleus tractus solitarius of decerebrate cats

This study was carried out on decerebrate, paralyzed and artificially ventilated cats to investigate the central regulatory mechanism for cough reflex. Fictive cough was induced by repetitive stimulation of the superior laryngeal nerve (SLN) or the nucleus tractus solitarius (NTS), and characterized by an increased inspiratory discharge in the phrenic nerve (stage 1 of cough; S1C) and large burst discharge in the iliohypogastric nerve (stage 2 of cough; S2C). Membrane potential was recorded from the neurons located in the cough-inducible sites of the NTS. Seven augmenting inspiratory (aug-I), 25 inspiratory-modulated (I-mod) and 16 non-respiratory (non-R) neurons were encountered, all of which showed short-latency (7.5 ± 1.6 ms, n=48) waves of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) in response to single pulse stimulation of the SLN. Out of these, all 7 aug-I and 12 I-mod neurons depolarized during the S1C and hyperpolarized during the S2C (DH-type response). Three I-mod and five non-R neurons showed membrane hyperpolarization during both stages (HH-type response). Ten I-mod and three non-R neurons displayed membrane depolarization during the S1C and S2C (DD-type response). The remaining eight non-R neurons showed no response during the fictive cough (NN-type response) but a long-lasting EPSP wave to single SLN stimulation. The NTS neurons recorded here were divided into three groups. Group I neurons with the NN-type response may be the second-order relay neurons. Group II neurons with the DD-type response may integrate the tussigenic afferent information and send a gate signal to the cough pattern generator. Group III neurons with either DH-type or HH-type response may constitute the network of cough pattern generation or modulatory circuits recruited during the cough reflex. The present study suggests that Group II neurons may play a gating role in generating the cough reflex.

The NADPH oxidase inhibitor diphenyleneiodonium activates the human TRPA1 nociceptor

Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX.

Distinct modulatory effects of 5-HT on excitatory synaptic transmissions in the nucleus tractus solitarius of the rat

The second-order relay neurons in the nucleus tractus solitarius (NTS) receive numerous peripheral afferent inputs mainly from the vagus nerve. Their activity is modified by several neuromodulators and hence autonomic responses are properly regulated. Serotonin (5-HT) is an important candidate for such neuromodulators, since serotonergic inputs and distribution of 5-HT receptors are provided in the NTS. However, its mechanism of action remains unclear. To evaluate the serotonergic modulation of synaptic transmission, we examined the effects of 5-HT (1.0-10.0 μM) on the solitary tract-evoked excitatory postsynaptic currents (eEPSCs) and spontaneously occurring EPSCs (sEPSCs) in the preselected second-order neurons of the rat NTS. 5-HT concentration-dependently decreased the amplitude of eEPSCs, which was accompanied by an increase in paired-pulse ratio. The inhibitory effect of 5-HT was mimicked by α-methylserotonin and blocked by ketanserin. 5-HT had no effect on the inward current induced in the NTS neurons by topically applied α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). On the other hand, 5-HT increased the frequency of sEPCSs without effect on their amplitude. This excitatory effect of 5-HT was mimicked by 2-methylserotonin and antagonized by ondansetron. The results suggest a dual modulation of the excitatory synaptic transmission by 5-HT in the NTS; presynaptic inhibition of the peripheral inputs synapsing to the relay neurons via 5-HT(2) receptors and presynaptic excitation of inputs from the intrinsic local network via 5-HT(3) receptors. These effects of 5-HT may provide important means of optimizing the autonomic responses mediated by the NTS network.

N-methyl-d-aspartate mechanisms in depolarization of augmenting expiratory neurons during the expulsive phase of fictive cough in decerebrate cats

Cough reflex is characterized by a large expulsive phase for expelling the mucus or particles from the airway. The present study investigated the involvement of N-methyl-D-aspartate (NMDA) mechanisms in the expulsive phase of cough reflex using decerebrate and paralyzed cats. A fictive cough was induced by repetitive stimulation of the superior laryngeal nerve, which was characterized by an increased inspiratory discharge in the phrenic nerve (the stage 1 of fictive cough; SC1) and large spindle-shaped discharge in the iliohypogastric nerve (the stage 2 of fictive cough; SC2). Intravenous injection of an antagonist of NMDA receptors, dizocilpine (0.1mg/kg), increased the threshold intensity of stimulation for inducing a fictive cough. The SC2 iliohypogastric response was more vulnerable to dizocilpine than the SC1 phrenic response. Membrane potential of augmenting expiratory (aug-E) neurons was recorded from the caudal ventral respiratory group. Aug-E neurons showed a large depolarization with a high frequency discharge during the SC2 in major cases (n=35) and hyperpolarization in minor cases (n=6). Dizocilpine inhibited the occurrence of these SC2 responses of aug-E neurons without any effect on the basal respiratory fluctuations of membrane potential. This drug had no significant effect on waves of excitatory and inhibitory postsynaptic potentials evoked in aug-E neurons by single pulse stimulation of the SLN. The present results demonstrated that NMDA mechanisms contribute preferentially to the expulsive phase response in aug-E neurons during fictive cough reflex.

Effects of cholinesterase inhibitors and serotonin-1A receptor agonists on morphine-induced ventilatory depression and antinociception in rats

Ventilatory depression is a serious side-effect of opioid analgesics. Naloxone, an antagonist of opioid receptors, eliminates not only ventilatory depression but also analgesic effect of opioids. Pharmacological dissociation of adverse reactions from the main action is important clinically and basically. Cholinergic and serotonergic mechanisms are suggested to counteract the opioid-induced ventilatory disturbances, but their influence on analgesia is still controversial. The present study evaluated the effects of cholinesterase inhibitors and serotonin-1A (5-HT1A) receptor agonists on morphine (1.0mg/kg, i.v.)-induced ventilatory depression and analgesia in rats. In anesthetized animals, spontaneous ventilation and hind leg withdrawal reflexes against nociceptive thermal stimuli were measured simultaneously. Physostigmine (0.1 and 0.2mg/kg, i.v.) and donepezil (0.5 and 1.0mg/kg, i.v.) relieved the morphine-induced ventilatory depression and enhanced its antinociception. On the other hand, (±)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.03 and 0.1mg/kg, i.v.) and buspirone (0.1 and 0.3mg/kg, i.v.) did not influence antinociception of morphine while they restored the decreased ventilation. In unanesthetized animals, hypercapnic ventilatory response was measured by using whole-body plethysmography. Physostigmine (0.3mg/kg, i.p.), donepezil (1.0mg/kg, i.p.), 8-OH-DPAT (0.3mg/kg, i.p.) and buspirone (3.0mg/kg, i.p.) all recovered the morphine (10mg/kg, i.p.)-induced depression of hypercapnic ventilatory response. The present study suggests that activation of cholinergic or serotonergic (5-HT1A) mechanisms may be a useful therapeutic approach for morphine-induced ventilatory depression without loss of its analgesic action.

High Doses of Oseltamivir Phosphate Induce Acute Respiratory Arrest in Anaesthetized Rats

It has been reported that one of the serious adverse events after the treatment of oseltamivir phosphate (OP) for influenza patients is sudden death resulting from cardiorespiratory arrest. To investigate the aetiology of such an adverse consequence, we examined effects of OP (expressed as free base) on blood pressure and ventilation in anaesthetized rats with vagotomy. Intravenous OP (30–200 mg/kg) caused dose‐dependent hypotension and bradycardia in spontaneously breathing animals. Concomitantly with changes in blood pressure, the tracheal airflow increased. The ventilatory rate hastened during the injection and then transiently slowed around 1 min. after the administration (transient hypopnea). Thereafter, it gradually returned to control. The hypopnea increased with increasing dose and ventilatory arrest occurred at 200 mg/kg. Intraduodenal OP (500–1000 mg/kg) provoked cardioventilatory arrest 72–218 min. after the injection. Oseltamivir carboxylate (100–200 mg/kg, i.v.), an active metabolite of OP, had no significant effect on ventilation and blood pressure. In artificially ventilated animals, intravenous OP caused slowing of the respiratory rate around 1 min. after the injection in a dose‐dependent manner. This effect of OP waned in 5 min. after the administration. The amplitude of phrenic nerve discharge was not changed at lower doses (30–100 mg/kg). The phrenic nerve stopped to discharge immediately after higher doses (150–200 mg/kg). We demonstrated that OP causes central suppression of the respiratory function in rats and suggest a relationship between the OP‐induced cardiorespiratory arrest and sudden death observed in influenza patients after taking OP.