Activation of opioid micro-receptors in medullary raphe depresses sighs

Abstract

Sighs, a well-known phenomenon in mammals, are substantially augmented by hypoxia and hypercapnia. Because (d-Ala(2),N-Me-Phe(4),Gly-ol)-enkephalin (DAMGO), a mu-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% O(2) for 1.5 min) or hypercapnia (3%, 7%, and 10% CO(2) for 4 min) to test the dependence of sigh responses on the intensity of chemical drive in anesthetized and spontaneously breathing rats. The role of mu-receptors in modulating sigh responses to 10% O(2) or 7% CO(2) was subsequently evaluated by comparing the sighs before and after 1) intravenous administration of DAMGO (100 microg/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-NH(2) (CTAP, 100 ng/100 nl), a micro-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 mu-receptors, especially those within the cMRR.

Fig. 1.

Effects of different hypoxic and hypercapnic degrees on sighs. A: representative recordings of sighs induced by 3 different hypoxic (1.5 min) and hypercapnic (4 min) exposures. To emphasize the sigh responses, the moving average of the tidal volume (Vt) is presented and the spikes reflect sighs. B and C: grouped responses of sigh number (left) and amplitude (right) to hypoxia (B) or hypercapnia (C). Data are presented as means ± SE; n = 6.

Fig. 2.

Representative recordings exhibiting the impact of systemic (d-Ala²,N-Me-Phe⁴,Gly-ol)-enkephalin (DAMGO; 100 μg/kg) on hypoxia (A)- or hypercapnia (B)-induced changes in sigh number and amplitude. In each panel, the chemical challenges (10% O₂ for 1.5 min and 7% CO₂ for 4 min) were applied before (left) and 5 min (center) and 30 min (right) after systemic DAMGO. Traces from top to bottom are arterial blood pressure (BP), Vt, and end-tidal pressure of carbon dioxide (Pet_CO2), with stimulating durations of chemical challenges indicated.

Fig. 3.

Effect of systemic DAMGO (100 μg/kg) on sigh number (left) and amplitude (right) induced by 10% O₂ (A) or 7% CO₂ (B). Data are presented as means ± SE; n = 6. *P < 0.05, **P < 0.01 compared with before DAMGO. “0” on x-axis indicates onset of intravenous administration of DAMGO.

Fig. 4.

Effect of microinjection of DAMGO into the caudal medullary raphe region (cMRR) on the number (left) and amplitude (right) of sighs induced by 10% O₂ (A) or 7% CO₂ (B). Data are presented as means ± SE; n = 6. *P < 0.05, **P < 0.01 compared with before DAMGO. “0” on x-axis indicates onset of intravenous DAMGO administration. Note that there was no amplitude response to 7% CO₂ at the 30-min point in B because of the absence of a sigh at that time point.

Fig. 5.

Diagram showing the sites where the microinjections occurred. A, left: cartoon of a coronal slice. ▪ and ▴, Locations of the microinjections in the cMRR of 6 rats;×, 4 microinjections outside the cMRR in 2 rats. A, right: representative slice containing the cMRR. Areas stained by Chicago Sky Blue are circled. B and C: ▪, locations of the microinjections in the middle MRR (mMRR; B) and rostral MRR (rMRR; C), respectively. Amb, nucleus ambiguus; IRt, intermediate reticular nucleus; LPB, lateral parabrachial nucleus; ml, medial lemniscus; Mo5, motor 5 nucleus; NTS, nucleus of solitary tract; PCRtA, parvicellular reticular nucleus alpha; Pr5VL, ventrolateral part of principal sensory 5 nucleus; ROb, raphe obscurus nucleus; RMg, raphe magnus nucleus; RPa, raphe pallidus nucleus; RVL, rostroventrolateral reticular nucleus; 7, facial nucleus; Sp5I, interpolar part of spinal 5 nucleus; Sp5O, oral part of spinal 5 nucleus.

Fig. 6.

Influence of DAMGO microinjection into the mMRR on the responses of sigh number (left) and amplitude (right) to 10% O₂ (A) and 7% CO₂ (B). Data are presented as means ± SE; n = 6. *P < 0.05, **P < 0.01 compared with before DAMGO. “0” on x-axis indicates onset of intravenous DAMGO administration.

Fig. 7.

Effect of DAMGO microinjection into the rMRR on the responses of sigh number (left) and amplitude (right) to 10% O₂ (A) and 7% CO₂ (B). Data are presented as means ± SE; n = 6. “0” on x-axis indicates onset of intravenous DAMGO administration.

Fig. 8.

Comparison of the systemic DAMGO-induced attenuation of the responses of sigh number (left) and amplitude (right) to 10% O₂ (A) or 7% CO₂ (B) before and after microinjection of d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP) into the cMRR. All variables are presented as % change (Δ%) from control (indicated as “0” on y-axis). Data are means ± SE; n = 6. *P < 0.05, **P < 0.01 compared with control; †P < 0.05 between DAMGO-induced changes before and after CTAP microinjection.