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General anesthetics variably enhance inhibitory synaptic transmission that relies on (-aminobutyric acid (GABA) and GABAA receptor function with distinct differences across brain regions. Activation of "extra-synaptic" GABAA receptors produces a tonic current considered the most sensitive target for general anesthetics, particularly in forebrain neurons. To evaluate the contribution of poor drug access to neurons in slices, we tested the intravenous anesthetic propofol in mechanically isolated neurons from the solitary tract nucleus (NTS). Setting chloride concentrations to ECl=-29 mV made GABA currents inward at holding potentials of -60 mV. Propofol triggered pronounced but slowly-developing tonic currents that reversed with 5 min washing. Effective concentrations in isolated cells were lower than in slices and propofol enhanced phasic IPSCs more potently than tonic currents (1 microM increased phasic decay-time constant vs. >3 microM tonic currents). Propofol increased IPSC frequency (>3 microM), a presynaptic action. Bicuculline blocked all propofol actions. Gabazine blocked only phasic IPSCs. IPSCs persisted in TTX and/or cadmium but these agents prevented propofol-induced increases in IPSC frequency. Furosemide (>1 mM) reversibly blocked propofol-evoked IPSC frequency changes without altering waveforms. We conclude that presynaptic actions of propofol depend on a depolarizing chloride gradient across presynaptic inhibitory terminals. Our results in isolated neurons indicate that propofol pharmacokinetics intrinsically trigger the tonic currents slowly and the time course is not related to slow permeation or delivery. Unlike forebrain, phasic NTS GABAA receptors are more sensitive to propofol than tonic receptors but that presynaptic GABAA receptor mechanisms regulate GABA release.

Sighs, a well-known phenomenon in mammals, are substantially augmented by hypoxia and hypercapnia. Because (d-Ala2,N-Me-Phe4,Gly-ol)-enkephalin (DAMGO), a μ-receptor agonist, injected intravenously and locally in the caudal medullary raphe region (cMRR) decreased the ventilatory response to hypoxia and hypercapnia, we hypothesized that these treatments could inhibit sigh responses to these chemical stimuli. The number and amplitude of sighs were recorded during three levels of isocapnic hypoxia (15%, 10%, and 5% O2for 1.5 min) or hypercapnia (3%, 7%, and 10% CO2for 4 min) to test the dependence of sigh responses on the intensity of chemical drive in anesthetized and spontaneously breathing rats. The role of μ-receptors in modulating sigh responses to 10% O2or 7% CO2was subsequently evaluated by comparing the sighs before and after 1) intravenous administration of DAMGO (100 μg/kg), 2) microinjection of DAMGO (35 ng/100 nl) into the cMRR, and 3) intravenous administration of DAMGO after microinjection of d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2(CTAP, 100 ng/100 nl), a μ-receptor antagonist, into the cMRR. Hypoxia and hypercapnia increased the number, but not amplitude, of sighs in a concentration-dependent manner, and the responses to hypoxia were significantly greater than those to hypercapnia. Systemic and local injection of DAMGO into the cMRR predominantly decreased the number of sighs, while microinjection into the rostral and middle MRR had no or limited effects. Microinjecting CTAP into the cMRR significantly diminished the systemic DAMGO-induced reduction of the number of sighs in response to hypoxia, but not to hypercapnia. Thus we conclude that hypoxia and hypercapnia elevate the number of sighs in a concentration-dependent manner in anesthetized rats, and this response is significantly depressed by activating systemic μ-receptors, especially those within the cMRR.

Background : Opioids, extensively used as analgesics, markedly depress ventilation, particularly the ventilatory responsiveness to hypercapnia in humans and animals predominantly via acting on mu receptors. The medullary raphe region (MRR) contains abundant mu receptors responsible for analgesia and is also an important central area involving carbon dioxide chemoreception and contributing to the ventilatory responsiveness to hypercapnia. Therefore, the authors asked whether activation of mu receptors in the caudal, medial, or rostral MRR depressed ventilation and the response to hypercapnia, respectively. Methods : Experiments were conducted in 32 anesthetized and spontaneously breathing rats. Ventilation and it response to progressive hypercapnia were recorded. The slopes obtained from plotting minute ventilation, respiratory frequency, and tidal volume against the corresponding levels of end-tidal pressure of carbon dioxide were used as the indices of the respiratory responsiveness to carbon dioxide. DAMGO ([d-Ala2, N-Me-Phe4, Gly-ol]-enkephalin), a mu-receptor agonist, was systemically administered (100 mug/kg) before and/or after local injection of CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) (100 ng/100 nl), a mu-receptor antagonist, into the caudal MRR, or locally administered (35 ng/100 nl) into the MRR subnuclei. Results : The authors found that systemic DAMGO significantly inhibited ventilation and the response to carbon dioxide by 20% and 31%, respectively, and these responses were significantly diminished to 11% and 14% after pretreatment of the caudal MRR with CTAP. Local administration of DAMGO into the caudal MRR also reduced ventilation and the response to carbon dioxide by 22% and 28%, respectively. In sharp contrast, these responses were not observed when the DAMGO microinjection was made in the middle MRR or rostral MRR. Conclusions : These results lead to the conclusion that mu receptors in the caudal MRR rather than the middle MRR or rostral MRR are important but not exclusive for attenuating the hypercapnic ventilatory response.

A previous study by the authors has shown that isoflurane (ISO), a commonly used volatile anesthetic, has an excitatory effect on bronchopulmonary C-fibers (PCFs). Since selective stimulation of PCFs by action on local 5-HT 3 receptors could evoke an apnea, this current study addresses whether inhalation of ISO would facilitate the PCF 5-HT 3 receptor-mediated apneic response and, if so, how. In anesthetized and spontaneously breathing rats, inhalation of 5% ISO markedly inhibited the apneic response to intra-atrium injection of phenylbiguanide (PBG, 25 μg/kg), a 5-HT 3 receptor agonist, which was contrary to the hypothesis. Extracellular recording of the nodose ganglion neurons in anesthetized, paralyzed and ventilated rats revealed that ISO attenuated the PBG-elicited excitation of pulmonary C neurons. Furthermore, using the patch clamp technique, it was found that ISO depressed the PBG-induced inward current of the pulmonary C neurons labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) instilled previously into the lungs. These results suggest that ISO inhibits PCF 5-HT 3 channel functions, and thereby attenuates PCF excitatory response to PBG, likely contributing to the diminution of the PBG-induced apnea by ISO in rats.

Inhalation of isoflurane (ISO), a widely used volatile anesthetic, can produce clinical tachypnea. In dogs, this response is reportedly mediated by bronchopulmonary C-fibers (PCFs), but the relevant mechanisms remain unclear. Activation of transient A-type potassium current ( I A ) channels and delayed rectifier potassium current ( I K ) channels hyperpolarizes neurons, and inhibition of both channels by ISO increases neural firing. Due to the presence of these channels in the cell bodies of rat PCFs, we determined whether ISO could stimulate PCFs to produce tachypnea in anesthetized rats, and, if so, whether this response resulted from ISO-induced depolarization of the pulmonary C neurons via the inhibition of I A and I K . We recorded ventilatory responses to 5% ISO exposure in anesthetized rats before and after blocking PCF conduction and the responses of pulmonary C neurons (extracellularly recorded) to ISO exposure. ISO-induced (1 mM) changes in pulmonary C neuron membrane potential and I A / I K were tested using the perforated patch clamp technique. We found that: (1) ISO inhalation evoked a brief tachypnea (∼7 s) and that this response disappeared after blocking PCF conduction; (2) the ISO significantly elevated (by 138%) the firing rate of most pulmonary C neurons (17 out of 21) in the nodose ganglion; and (3) ISO perfusion depolarized the pulmonary C neurons in the vitro and inhibited both I A and I K , and this evoked-depolarization was largely diminished after blocking both I A and I K . Our results suggest that ISO is able to stimulate PCFs to elicit tachypnea in rats, at least partly, via inhibiting I A and I K , thereby depolarizing the pulmonary C neurons.

Rapid shallow breathing (RSB) is mainly mediated by bronchopulmonary C-fibers (PCFs). We asked whether this RSB could be modulated by opioids. In anesthetized rats right atrial bolus injection of phenylbiguanide (PBG) to evoke RSB was repeated after: (1) intravenously giving fentanyl (μ-receptor agonist), DPDPE (δ-receptor agonist), or U-50488H (κ-receptor agonist); (2) fentanyl (iv) following naloxone methiodide, a peripheral opioid receptor antagonist; (3) bilateral microinjection of fentanyl into the nodose ganglia; (4) fentanyl (iv) with pre-blocking histamine H 1 and H 2 receptors by diphenhydramine and ranitidine. Systemic fentanyl challenge, but not DPDPE or U-50488H, switched the PBG-induced RSB to a long lasting apnea. This switch was blocked by naloxone methiodide rather than diphenhydramine and ranitidine. After microinjecting fentanyl into the nodose ganglia, PBG also produced an apnea. Our results suggest that activating μ-receptors is capable of turning the PCF-mediated RSB into an apnea, at least partly, via facilitating PCFs’ activity and this switching effect appears independent of the released histamine.

Intravenous bolus injection of morphine causes a vagal-mediated brief apnea (∼3 s), while continuous injection, via action upon central μ-opioid receptor (MOR), arrests ventilation (>20 s) that is eliminated by stimulating central 5-hydroxytryptamine 1A receptors (5HT1ARs). Bronchopulmonary C-fibers (PCFs) are essential for triggering a brief apnea, and their afferents terminate at the caudomedial region of the nucleus tractus solitarius (mNTS) that densely expresses 5HT1ARs. Thus we asked whether the vagal-mediated apneic response to MOR agonists was PCF dependent, and if so, whether this apnea was abolished by systemic administration of 8-hydroxy-2-(di- n-propylamino)tetral (8-OH-DPAT) largely through action upon mNTS 5HT1ARs. Right atrial bolus injection of fentanyl (5.0 μg/kg, a MOR agonist) was performed in the anesthetized and spontaneously breathing rats before and after: 1) selective blockade of PCFs' conduction and subsequent bivagotomy; 2) intravenous administration of 5HT1AR agonist 8-OH-DPAT; 3) intra-mNTS injection of 8-OH-DPAT; and 4) intra-mNTS injection of 5HT1AR antagonist WAY-100635 followed by 8-OH-DPAT (iv). We found the following: First, fentanyl evoked an immediate apnea (2.5 ± 0.4 s, ∼6-fold longer than the baseline expiratory duration, TE), which was abolished by either blocking PCFs' conduction or bivagotomy. Second, this apnea was prevented by systemic 8-OH-DPAT challenge. Third, intra-mNTS injection of 8-OH-DPAT greatly attenuated the apnea by 64%. Finally, intra-mNTS microinjection of WAY-100635 significantly attenuated (58%) the apneic blockade by 8-OH-DPAT (iv). We conclude that the vagal-mediated apneic response to MOR activation depends on PCFs, which is fully antagonized by systemic 8-OH-DPAT challenge largely via acting on mNTS 5HT1ARs.

Background : The commissural subnucleus of the nucleus tractus solitarius (comNTS) is a key region in the brainstem responsible for the hypoxic ventilatory response (HVR) because it contains the input terminals of the carotid chemoreceptor. Because opioids inhibit the HVR via activating central μ-receptors that are expressed abundantly in the comNTS, the authors of the current study asked whether activating local μ-receptors attenuated the carotid body-mediated HVR. Methods : To primarily stimulate the carotid body, brief hypoxia (100% N2) and hypercapnia (15% CO2) for 10 s and/or intracarotid injection of NaCN (10 μg/100 μl) were performed in anesthetized and spontaneously breathing rats. These stimulations were repeated after: (1) microinjecting three doses of μ-receptor agonist [d-Ala2, N-Me-Phe4, Gly-ol]-Enkephalin (DAMGO) (approximately 3.5 nl) into the comNTS; (2) carotid body denervation; and (3) systemic administration of DAMGO (300 μg/kg) without and with previous intracomNTS injection of d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2, a μ-receptor antagonist. Results : Study results showed that DAMGO at 0.25 and 2.5, but not 0.025 mM, caused a similar decrease in baseline ventilation (approximately 12%). DAMGO at 0.25 mM largely reduced (64%) the HVR, whereas DAMGO at 2.5 mM abolished the HVR (and the VE response to NaCN) and moderately attenuated (31%) the hypercapnic ventilatory response. Interestingly, similar HVR abolition and depression of the hypercapnic ventilatory response were observed after carotid body denervation. Blocking comNTS μ-receptors by d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2 significantly attenuated the HVR depression by systemic DAMGO with little change in the DAMGO modulatory effects on baseline ventilation and the hypercapnic ventilatory response. Conclusion : The data suggest that opioids within the comNTS, via acting on μ-receptors, are able to abolish the HVR by affecting the afferent pathway of the carotid chemoreceptor.

Opioids can attenuate the peripheral chemoreceptor-mediated hypoxic ventilatory response (HVR) by acting on central mu-type opioid receptors. Since the medullary raphe region (MRR) expresses abundant mu-receptors and participates in modulating HVR, we tested the role of mu-receptors within the caudal, medial, and rostral MRR (cMRR, mMRR, and rMRR) in modulating the HVR. We recorded cardiorespiratory activities and their responses to isocapnic hypoxia in anesthetized rats before and after local microinjection of DAMGO into the MRR, and intravenous administration of DAMGO (100 microg/kg) alone or coupled with a previous local injection of CTAP. Microinjecting DAMGO into the cMRR or mMRR but not the rMRR significantly attenuated the HVR. However, systemic DAMGO-induced HVR attenuation was not significantly affected by pretreating the cMRR and mMRR with CTAP. Our data suggest that cMRR and mMRR mu-receptors are capable of depressing the HVR, while their contribution to the attenuated HVR by systemic DAMGO is limited.