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Dextromethorphan (DEX) presynaptically decreases glutamatergic transmission in second-order neurons of the nucleus tractus solitarius (TS). To clarify the inhibitory mechanism of DEX, the present study examined the interaction of DEX with cAMP. The effects of DEX on miniature and TS-evoked excitatory postsynaptic currents (mEPSCs and eEPSCs) were recorded under activation of the cAMP-dependent pathway using the brainstem slices. An increase in cAMP by forskolin counteracted the inhibitory effect of DEX on mEPSCs. Eight-Bromo-cAMP and N-ethylmaleimide also attenuated the DEX effect. However, forskolin had negligible effects on the DEX-induced inhibition of eEPSCs. This suggests that DEX decreases spontaneous glutamate release by inhibiting the cAMP-dependent pathway and synchronous release by another unknown mechanism., (© 2018 S. Karger AG, Basel.)

The spinal trigeminal subnucleus caudalis (Vc) receives preferentially nociceptive afferent signals from the orofacial area. Nociceptive stimuli to the orofacial area induce cyclooxygenase both peripherally and centrally, which can synthesize a major prostanoid prostaglandin E2 (PGE2) that implicates in diverse physiological functions. To clarify the roles of centrally-synthesized PGE2 in nociception, effects of exogenous PGE2 on synaptic transmission in the Vc neurons were investigated in the rat brainstem slice. Spontaneously occurring excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) were recorded, respectively, under pharmacological blockade of inhibitory and excitatory transmission by whole-cell patch-clamp mode. Perfusion of PGE2 (1-5 μM) increased the frequency of sIPSCs in a concentration-dependent manner but had no significant effect on the amplitude. Similarly to the effects on sIPSCs, PGE2 increased the sEPSC frequency without any effect on the amplitude. These facilitatory effects of PGE2 on spontaneous synaptic transmissions were blocked by an EP1 antagonist SC19220 but not by an EP4 antagonist AH23848. Electrical stimulation of the trigeminal tract evoked short latency EPSCs (eEPSCs) in the Vc neurons. PGE2 (5 μM) was ineffective on the eEPSCs. The present study demonstrated that PGE2 facilitated spontaneous synaptic transmissions in the Vc neurons through activating the presynaptic EP1 receptors but had no effect on the trigeminal tract-mediated excitatory transmission. These results suggest that centrally-synthesized PGE2 modifies the synaptic transmission in the Vc region, thereby contributing to the processing of nociceptive signals originated from the orofacial area., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study investigated the physiological function of presynaptic N-methyl-d aspartate (NMDA) mechanisms in glutamatergic transmission in the rat nucleus tractus solitarius (NTS). Membrane currents were recorded from the NTS second-order neurons by using whole-cell patch pipettes including MK-801 to block postsynaptic NMDA receptors. All experiments were performed under blockade of inhibitory synaptic transmission. Co-application of NMDA and d-serine decreased the tractus solitarius (TS)-evoked excitatory postsynaptic currents (eEPSCs) in 7/12 (58%) of neurons, and increased the paired pulse ratio. The remaining neurons were insensitive to NMDA and d-serine. Application of an NMDA antagonist D-AP5 had no effect on eEPSCs in all 8 neurons tested. Action potential-independent EPSCs (miniature EPSCs; mEPSCs) were recorded in the presence of tetrodotoxin. Co-application of NMDA and d-serine increased the mEPSC frequency but had no significant effect on the amplitude in 5/28 (18%) of neurons. D-AP5 decreased the mEPSC frequency without effect on the amplitude in 6/18 (33%) of neurons. This study demonstrated that (1) NMDA receptors were presynaptically distributed in a subset of NTS second-order neurons and that (2) the presynaptic NMDA receptors played an inhibitory role in TS-mediated release of glutamate and a facilitatory role in spontaneous release of glutamate. The present results suggest that the activation of presynaptic NMDA receptors modulates glutamatergic transmissions in the rat NTS second-order neurons., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Activity of second-order relay neurons in the nucleus tractus solitarius (NTS) is regulated by peripheral and intrinsic synaptic inputs, and modulation of those inputs by metabotropic glutamate receptors (mGluRs) has been proposed. This study investigated effects of mGluR activation on glutamatergic transmission in the NTS second-order neurons of guinea pigs. Whole-cell patch-clamp recordings from the brainstem slices revealed that activation of mGluRs exerted its effects on the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but not on the amplitude. The sEPSC frequency was increased by an agonist of group I mGluRs, and it was decreased by an mGluR1 antagonist but not by an mGluR5 antagonist. The agonists of group II and III mGluRs decreased the sEPSC frequency, while their antagonists alone had no effect. Perfusion of cystine or TBOA, either of which elevates extracellular glutamate concentration, resulted in an increase in the sEPSC frequency, leaving the amplitude unchanged. The increased frequency of sEPSCs was returned to control by an mGluR1 antagonist. The tractus solitarius-evoked EPSCs were not altered by an agonist of group I mGluRs, whereas they were decreased along with an increase in paired-pulse ratio by agonists of group II and III mGluRs. These results suggest that mGluRs are present at the presynaptic sites in the NTS second-order neurons in guinea pigs. The mGluR1s function to facilitate the release of glutamate from axon terminals of intrinsic interneurons and the group II and III mGluRs play an inhibitory role in glutamatergic transmission., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Dextromethorphan (DEX) is a widely used non-opioid antitussive. However, the precise site of action and its mechanism were not fully understood. We examined the effects of DEX on AMPA receptor-mediated glutamatergic transmission in the nucleus tractus solitarius (NTS) of guinea pigs. Excitatory postsynaptic currents (evoked EPSCs: eEPSCs) were evoked in the second-order neurons by electrical stimulation of the tractus solitarius. DEX reversibly decreased the eEPSC amplitude in a concentration-dependent manner. The DEX-induced inhibition of eEPSC was accompanied by an increased paired-pulse ratio. Miniature EPSCs (mEPSCs) were also recorded in the presence of Cd(2+) or tetrodotoxin. DEX decreased the frequency of mEPSCs without affecting their amplitude. Topically applied AMPA provoked an inward current in the neurons, which was unchanged during the perfusion of DEX. BD1047, a σ-1-receptor antagonist, did not block the inhibitory effect of DEX on the eEPSCs, but antagonized the inhibition of eEPSCs induced by SKF-10047, a σ-1 agonist. Haloperidol, a σ-1 and -2 receptor ligand, had no influence on the inhibitory action of DEX. These results suggest that DEX inhibits glutamate release from the presynaptic terminals projecting to the second-order NTS neurons, but this effect of DEX is not mediated by the activation of σ receptors.

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.

The cytosolic Ca(2+) released from internal stores is important for distinctive cell functions. To assess the role of ryanodine/Ca(2+) releasing mechanisms in the rhythmic activity of respiratory neurons, effects of intracellular injection of ryanodine on the membrane potential trajectory of postinspiratory and augmenting inspiratory neurons were investigated in unanesthetized, decerebrate, paralyzed and artificially ventilated cats. Ryanodine injection hyperpolarized the membrane and decreased input resistance throughout the respiratory cycle in both types of respiratory neurons. Specifically, membrane repolarization during postinspiration was accelerated in postinspiratory neurons, and the large hyperpolarization at the onset of postinspiration was increased in augmenting inspiratory neurons. Spike-afterhyperpolarization consisting of a fast, early component and slow, late component increased in size after ryanodine, resulting in prolongation of inter-spike intervals and decrease of burst discharge. Intracellular injection of caffeine produced similar effects on these respiratory neurons, and Ruthenium Red, an antagonist of ryanodine receptors, had opposite effects. Immunoreactivity for ryanodine receptors was detected in all respiratory neurons labeled intracellularly with neurobiotin. These results demonstrate that ryanodine-sensitive Ca(2+) stores modulate the periodic membrane potential fluctuations and spike activity in respiratory neurons.

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.

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.

Abstract: Although codeine is the most prominent and centrally acting antitussive agent, the precise sites and mode of its action have not been fully understood yet. In the present study, we examined the effects of codeine on synaptic transmission in second-order neurons of the nucleus tractus solitarius (NTS), which is the first central relay site receiving tussigenic afferent fibers, by using whole-cell patch-clamp recordings in guinea-pig brainstem slices. Codeine (0.3–3 mM) significantly decreased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of the tractus solitarius in a naloxone-reversible and concentration-dependent manner, but it had no effect on the decay time of evoked EPSCs (eEPSCs). The inhibition of eEPSCs was accompanied by an increased paired-pulse ratio of two consecutive eEPSCs. The inward current induced by application of AMPA remained unchanged after codeine application. A voltage-sensitive K+ channel blocker, 4-aminopyridine (4-AP) attenuated the inhibitory effect of codeine on eEPSCs. These results suggest that codeine inhibits excitatory transmission from the primary afferent fibers to the second-order NTS neurons through the opioid receptors that activate the 4-AP sensitive K+ channels located at presynaptic terminals. [Copyright &y& Elsevier]