An aromatic substance, eugenol induces distinct depressant effects on respiratory activity in different postnatal developmental stages of the rat.

Eugenol modulates neuronal activity through actions on voltage-gated ionic channels and/or transient receptor potential channels. We previously suggested that eugenol inhibited cellular (and/or network) mechanisms essential for the maintenance of the respiratory burst activity in a brainstem-spinal cord preparation from newborn rat (postnatal day 0-3). Study of the distinct effects of eugenol in neonatal and later developmental stage rats may offer new information about postnatal developmental changes of respiratory neuron networks. In the present study, therefore, we compared effects of eugenol in an in vitro newborn rat preparation with those in an arterially perfused in situ preparation from juvenile rat (postnatal day 12-15). In the former preparation, application of 1 mM eugenol decreased respiratory rate and inspiratory burst duration. In contrast, in the latter preparation, 1 mM eugenol induced a gradual decrease in the amplitude of integrated phrenic nerve activity. Phrenic nerve activity gradually recovered at 25-30 min after washout with a burst duration similar to control values. We hypothesized that the depressant effects of eugenol were caused by inhibition of cell excitability in the neonatal rat in vitro preparation but by a reduction of synaptic interactions in the juvenile rat in situ preparation.

Effects of Feeding-Related Peptides on Neuronal Oscillation in the Ventromedial Hypothalamus.

The ventromedial hypothalamus (VMH) plays an important role in feeding behavior, obesity, and thermoregulation. The VMH contains glucose-sensing neurons, the firing of which depends on the level of extracellular glucose and which are involved in maintaining the blood glucose level via the sympathetic nervous system. The VMH also expresses various receptors of the peptides related to feeding. However, it is not well-understood whether the action of feeding-related peptides mediates the activity of glucose-sensing neurons in the VMH. In the present study, we examined the effects of feeding-related peptides on the burst-generating property of the VMH. Superfusion with insulin, pituitary adenylate cyclase-activating polypeptide, corticotropin-releasing factor, and orexin increased the frequency of the VMH oscillation. In contrast, superfusion with leptin, cholecystokinin, cocaine- and amphetamine-regulated transcript, galanin, ghrelin, and neuropeptide Y decreased the frequency of the oscillation. Our findings indicated that the frequency changes of VMH oscillation in response to the application of feeding-related peptides showed a tendency similar to changes of sympathetic nerve activity in response to the application of these substances to the brain.

Effects of eugenol on respiratory burst generation in newborn rat brainstem-spinal cord preparations.

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABAA antagonist bicuculline and glycine antagonist strychnine or GABAB antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na+ current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 µM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 µM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.