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. 2012 Nov;108(9):2430-41.
doi: 10.1152/jn.00185.2012. Epub 2012 Aug 8.

Pontine μ-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs

Ivana Prkic  1 , Sanda Mustapic, Tomislav Radocaj, Astrid G Stucke, Eckehard A E Stuth, Francis A Hopp, Caron Dean, Edward J Zuperku
Affiliations
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Pontine μ-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs

Ivana Prkic et al. J Neurophysiol. 2012 Nov.
Free PMC article

Abstract

Life-threatening side effects such as profound bradypnea or apnea and variable upper airway obstruction limit the use of opioids for analgesia. It is yet unclear which sites containing μ-opioid receptors (μORs) within the intact in vivo mammalian respiratory control network are responsible. The purpose of this study was 1) to define the pontine region in which μOR agonists produce bradypnea and 2) to determine whether antagonism of those μORs reverses bradypnea produced by intravenous remifentanil (remi; 0.1-1.0 μg·kg(-1)·min(-1)). The effects of microinjections of agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO; 100 μM) and antagonist naloxone (NAL; 100 μM) into the dorsal rostral pons on the phrenic neurogram were studied in a decerebrate, vagotomized, ventilated, paralyzed canine preparation during hyperoxia. A 1-mm grid pattern of microinjections was used. The DAMGO-sensitive region extended from 5 to 7 mm lateral of midline and from 0 to 2 mm caudal of the inferior colliculus at a depth of 3-4 mm. During remi-induced bradypnea (~72% reduction in fictive breathing rate) NAL microinjections (~500 nl each) within the region defined by the DAMGO protocol were able to reverse bradypnea by 47% (SD 48.0%) per microinjection, with 13 of 84 microinjections producing complete reversal. Histological examination of fluorescent microsphere injections shows that the sensitive region corresponds to the parabrachial/Kölliker-Fuse complex.

Figures

Fig. 1.
Fig. 1.
Example of data illustrating the methods used to quantify the effects of [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) microinjections during the pontine mapping protocol (protocol 1). Microinjections of DAMGO (100 μM, 500 nl) were sequentially injected beginning at 0 rostral-caudal (R-C) and 4 lateral and progressed laterally until 8 mm lateral. The medial to lateral sequence of microinjections was subsequently repeated for −1, −2, −3, +1 R-C. Typical depth of injections was 3 mm from dorsal surface (see 3rd coordinate). A, top: effects of a series of DAMGO microinjections [vertical arrows with coordinates as indicated: R-C/medial-lateral (M-L)/depth (D)] on the phrenic neurogram (PNG) and breath-by breath values of TI (blue symbols), TE (red symbols) and fictive breathing rate [fB, breaths/min (BPM), black symbols]. The corresponding locations are shown by the 3 black circles at 0 R-C on the contour plot (A, bottom). Thick red line in rate data is the local regression curve (LOWESS function) indicating the best-fit trend. Note that there is a progressive increase in TI and TE, a decrease in rate, and no change in peak PNG. B: time-expanded plots of fB for the corresponding microinjections indicated on the contour plot (black circles) with time of injection set to 0 (vertical dashed lines). Red lines: best-fit regression curves. Because of cumulative effects of DAMGO, incremental % changes relative to the preinjection values (dashed horizontal lines) were calculated 6–10 min after injection (arrows with values). Arrowheads: time of microinjections. R-C, lateral, and depth coordinates are given at right of each plot. Contour plot (A, bottom) shows % reduction in breathing rate caused with DAMGO microinjections at the various R-C and M-L locations. In this example, the maximum change in fB of ∼25% occurred at −2 R-C and 6 lateral. Corresponding data are shown in B, bottom, with changed scale.
Fig. 2.
Fig. 2.
Preliminary data suggesting that μ-opioid receptors (μORs) in the dorsolateral pons may be the targets of systemically administered opioids that produce bradypnea at clinically relevant concentrations. A: example of the effects of DAMGO-saturated pledgets applied to the dorsal surface of the pons between the inferior colliculus (IC) and the superior cerebellar peduncle (CP) of a dog on PNG and fB. Note marked bradypnea and increases in TI and TE, with a small increase in peak PNG. Naloxone (NAL)-saturated pledgets gradually reversed the DAMGO-induced bradypnea. B: distribution of μOR immunoreactivity (μOR-ir) in the rostral dorsal lateral pons showing the parabrachial/Kölliker-Fuse (PB-KF) complex. Center: fluorescence photomicrograph of a coronal section that shows a relatively high level of μOR-ir lateral to the superior cerebellar peduncle (arrowheads a–c) corresponding to the lateral PB complex. a and c: Higher-magnification images near arrowheads a and c show discrete points of high-density μOR-ir on cell somas. b: High level of μOR-ir of a filamentous network, possibly dendritic, near arrowheads b.
Fig. 3.
Fig. 3.
Schematic of the dorsal view of the canine pons/medulla with cerebellum removed showing the coordinate system used in this study. Shaded areas indicate the pontine region explored for opioid-induced bradypnea. The caudal pole of IC and the midline were used as the R-C and M-L initial (0/0) reference points, respectively.
Fig. 4.
Fig. 4.
Example of the effects of a series of unilateral DAMGO microinjections (diamond symbols with pontine R-C/lateral coordinates). Note the progressive increases in TI and TE and decrease in fB and subsequent apnea. An IV bolus of NAL (10 μg/kg) partially antagonized the apnea. Larger subsequent doses were required to fully antagonize the DAMGO effects. A moderate increase in peak PNG is entirely due to the increase in TI without changes in drive (see Fig. 5 and text for further explanation).
Fig. 5.
Fig. 5.
Pontine DAMGO microinjections produce changes in timing but not drive. A: time-expanded segments of PNG record in Fig. 4, top, at the times indicated (a–e) below the PNG trace. The 1st PNG cycle in each segment has been time aligned at 0 s. B: superimposed traces of the 1st breath cycle in each record. Note that the slope of the PNG ramp is not altered and the peak height is dependent on magnitude of TI, suggesting that respiratory drive has not changed. C: plot of TI vs. the subsequent TE of each cycle during vagotomy appears to be nonlinear. While DAMGO caused increases in both TI and TE, the large decreases in fB are mainly due to the large increases in TE.
Fig. 6.
Fig. 6.
Top: contour plot for DAMGO-induced decreases in breathing frequency based on mean % change in fB values obtained in 10 dogs. The most opioid-responsive region was confined to an area of <2 × 2 mm. The largest decreases in fB at any injection site were ∼20% and were located at −1 R-C and 5 lateral. Superimposed open circles indicate sites from which data were tested for statistical significance (see text for details) (Fig. 7A). Bottom: contour plot for NAL microinjection reversal of IV remifentanil (remi)-induced bradypnea. Values are the average % reversal of the maximum depression of fB caused by remi from 21 dogs (see methods for more details regarding calculations). The largest mean values were ∼60% (red). Open circles indicate sites where data were statistically analyzed as shown in Fig. 7C.
Fig. 7.
Fig. 7.
A: % change in fB for each microinjection of 300–500 nl of 100 μM DAMGO at the site indicated above each bar (also in Fig. 6A, open circles; n = 10 dogs). #P < 0.05 difference from zero change (1-way ANOVA). B: types of responses to DAMGO as % of the number (N) of DAMGO microinjections per lateral coordinate (all R-C locations pooled). Tachypnea was rarely observed throughout the mapped region (darkest bars). C: % reversal of the remi-induced depression of fB per NAL microinjection (540 nl of 100 μM NAL). *P < 0.05, **P < 0.01 changes relative to no effect (1-way ANOVA).
Fig. 8.
Fig. 8.
Example of the reversal of IV remi-induced bradypnea/apnea by NAL (100 μM) microinjections in the dorsolateral pons (−1 mm R-C, 7 mm lateral to midline, 3 mm ventral to dorsal surface). The IV remi infusion at a rate of 0.8 μg·kg−1·min−1 decreased peak PNG, PNG slope, and breathing rate fB until transient apneas ensued (PNG record, arrows). Intermittently, large sighlike respiratory cycles were also observed. A transient increase in fB above control baseline (RATE record, horizontal dashed line) was observed after initiation of remi infusion prior to bradypnea. Throughout the infusion, peak PNG gradually decreased until a steady-state plateau at ∼20% peak PNG was reached. Peak PNG remained depressed after the two bilaterally symmetrical 1-μl NAL microinjections (compare records at times c, d, e, and f), while respiratory rate transiently recovered despite ongoing remi infusion. During the initial apneic period, short periods of phrenic activity reappeared (between times c and e), suggesting that the opioid plasma concentration was close to the phrenic apneic threshold. After 10 min of apnea, the first unilateral NAL microinjection reversed the apnea (the 2nd contralateral microinjection given as part of the protocol), and a transient tachypnea similar to the initial one resulted without any notable change in peak PNG. As the effect of the bilaterally microinjected NAL gradually declined during sustained IV remi infusion, bradypnea and apnea eventually reoccurred. After termination of the remi infusion, both peak PNG and fB gradually returned to pre-remi levels. The entire protocol lasted almost 2 h. Time-expanded records of PNG are shown for times indicated by the labeled arrows (a–g). e: Onset of reversal of the apnea by the 1st NAL microinjection, where the 1st PNG cycle shows a sigh in this case.
Fig. 9.
Fig. 9.
Left: section of rostral pons at the level of the caudal pole of the inferior colliculus (0 R-C) indicating the location of a microinjection in the most sensitive response area in this dog. The open circle indicates the location of the tip of the electrode at the bottom of the track (0 mm R-C, 6 mm lateral, 4.2 mm deep) that has been filled by the fluorescent beads (arrow) during injection and is just medial to KF. Right: diagram shows the most effective sites for NAL reversal of IV remi-induced bradypnea, where asterisks indicate sites based on the fluorescent bead microinjections and open circles indicate sites based on stereotaxic coordinates relative to the midline and depth for R-C coordinates from 0 to −2 mm relative to the caudal pole of the IC. NAL microinjections in and near the brachium pontis (lateral-most asterisks) resulted in nearly complete (78–100%) reversal of the remi-induced bradypnea, suggesting that μORs in the lateral parabrachial-KF complex mediate the remi-induced bradypnea. DTN, dorsal tegmental nucleus; KF, Kölliker-Fuse nucleus; LC, locus coeruleus; LLV, lateral lemniscus ventral nucleus; LPB, lateral parabrachial nucleus; MLB, medial longitudinal bundle; MPB, medial parabrachial nucleus; PG, pontine gray; SCN, superior central nucleus; SCP, superior cerebellar peduncle; TF, tegmental field.
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