Mu-opioid receptor-dependent transformation of respiratory motor pattern in neonates in vitro

Abstract

Endogenous opioid peptides activating mu-opioid receptors (MORs) are part of an intricate neuromodulatory system that coordinates and optimizes respiratory motor output to maintain blood-gas homeostasis. MOR activation is typically associated with respiratory depression but also has excitatory effects on breathing and respiratory neurons. We hypothesized that low level MOR activation induces excitatory effects on the respiratory motor pattern. Thus, low concentrations of an MOR agonist drug (DAMGO, 10-200 nM) were bath-applied to neonatal rat brainstem-spinal cord preparations while recording inspiratory-related motor output on cervical spinal roots (C4-C5). Bath-applied DAMGO (50-200 nM) increased inspiratory motor burst amplitude by 40-60% during (and shortly following) drug application with decreased burst frequency and minute activity. Reciprocal changes in inspiratory burst amplitude and frequency were balanced such that 20 min after DAMGO (50-200 nM) application, minute activity was unaltered compared to pre-DAMGO levels. The DAMGO-induced inspiratory burst amplitude increase did not require crossed cervical spinal pathways, was expressed on thoracic ventral spinal roots (T4-T8) and remained unaltered by riluzole pretreatment (blocks persistent sodium currents associated with gasping). Split-bath experiments showed that the inspiratory burst amplitude increase was induced only when DAMGO was bath-applied to the brainstem and not the spinal cord. Thus, MOR activation in neonates induces a respiratory burst amplitude increase via brainstem-specific mechanisms. The burst amplitude increase counteracts the expected MOR-dependent frequency depression and may represent a new mechanism by which MOR activation influences respiratory motor output.

Keywords: in vitro; mu-opiod receptor; neonatal rat; neuromodulation; respiratory motor control.

FIGURE 1

DAMGO produced changes in inspiratory-related motor output in vitro. (A) Drawing of a brainstem-spinal cord preparation with suction electrode attached at ventral cervical spinal root C4. At right is a sample voltage trace showing inspiratory-related motor bursts. (B) Effects of bath-applied DAMGO on inspiratory-related motor output are shown. DAMGO typically increased burst amplitude and decreased burst frequency.

FIGURE 2

DAMGO increased inspiratory-related motor burst amplitude and decreased burst frequency. (A) Percent change in burst amplitude relative to baseline for untreated time controls (white squares) and preparations exposed to DAMGO at 10 nM (white circles), 20 nM (gray circles), 50 nM (blue circles), 100 nM (brown circles), and 200 nM (black circles). The 15-min time point represents the steady-state response after a 15-min DAMGO application, and the 35-min time point represents the time after 20 min of washout. The 75-min time point represents the time after 60 min of washout. (B) The maximum percent change in burst amplitude is shown for the time controls and each DAMGO concentration. The maximum percent change in burst amplitude occurred during DAMGO application or the washout period. The circles represent individual experiments and the triangles (following the same color scheme as the circles for the DAMGO concentrations) represent the mean maximum percent change in burst amplitude. (C) Burst frequency was decreased at nearly all DAMGO concentrations and returned to near baseline levels after 60 min of washout. (D) Minute activity decreased during DAMGO application but returned to near time control levels at the 35-min time point. The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD.

FIGURE 3

DAMGO-induced inspiratory burst amplitude increase was dependent on baseline properties of inspiratory motor burst output. (A) Voltage traces of inspiratory motor output from a brainstem-spinal cord preparation with a relatively high baseline burst frequency (left), and the large burst amplitude increase at 10-min post-DAMGO administration (right). Linear regression analysis of maximum change in burst amplitude were compared to: (B) baseline burst frequency, (C) baseline burst duration, (D) baseline burst peak percent, and (E) maximum percent decrease caused by DAMGO applications. Preparations with relatively higher baseline burst frequencies (B) and longer burst durations (C) exhibited larger DAMGO-induced increases in inspiratory burst amplitude. DAMGO-induced increases in inspiratory burst amplitude were inversely proportional to the magnitude of burst frequency decreases during DAMGO application (E).

FIGURE 4

Naloxone reversed persistent DAMGO-induced effects on inspiratory burst amplitude and frequency. Six/10 brainstem spinal cord preparations exposed to 200 nM DAMGO (15 min) had a persistent increase in burst amplitude after 60 min of washout. Naloxone (1.0 µM) was bath-applied to test whether DAMGO effects could be reversed. (A) Compressed voltage trace showing baseline (left) and DAMGO-induced changes in burst amplitude and frequency that rapidly returned to baseline with naloxone application. The blue box highlights the part of voltage traces that were analyzed for the graphs below. (B) Naloxone application rapidly returned increased burst amplitude to time control levels. (C) Naloxone application rapidly increased burst frequency above time control levels. (D) Minute activity was not altered by naloxone application. The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD. These data indicate that the persistent DAMGO-induced inspiratory motor burst increase was not likely due to neuroplasticity.

FIGURE 5

Cervical spinal midline-lesion did not alter the DAMGO-induced inspiratory burst amplitude increase. (A) Drawing of a brainstem-spinal cord preparation with the cervical spinal midline-lesion from C3 to C7 with a suction electrode attached at ventral cervical spinal root C4. At right is a sample voltage trace showing DAMGO-induced changes in burst amplitude and frequency. DAMGO (50–200 nM; green diamond symbols) increased burst amplitude (B) and decreased burst frequency (C) compared to time controls. (D) Minute activity was also decreased by DAMGO. The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD. These data indicate that crossed bulbospinal pathways in the cervical spinal cord at C3-C7 were not necessary for the DAMGO-induced inspiratory burst amplitude increase.

FIGURE 6

The DAMGO-induced inspiratory burst amplitude increase was expressed in motor output from the thoracic spinal cord. (A) Drawing of a brainstem-spinal cord preparation extended to thoracic spinal segment T8 with suction electrodes attached to ventral roots of spinal segments C4 and T7. At right are sample voltage traces showing inspiratory-related motor bursts in cervical (upper trace) and thoracic (lower trace) during a 50 nM DAMGO application. (B) Burst amplitude increased in both cervical (blue circles) and thoracic (blue triangles) spinal cord motor output. (C) Burst frequency was decreased by DAMGO. The decrease was identical for brainstem and spinal cord recordings (blue circles), while (D) minute activity was relatively unaltered by the DAMGO application except for the cervical motor output at the 15-min time point. The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD. These data indicate that the inspiratory burst amplitude increase was not restricted to phrenic motoneurons in the cervical spinal cord.

FIGURE 7

The DAMGO-induced burst amplitude increase was not altered by riluzole. (A) Voltage trace showing that DAMGO increased burst amplitude and decreased burst frequency during a background riluzole application. (B) Burst amplitude increased and (C) burst frequency decreased, while (D) minute activity was not altered (blue circles) compared to riluzole time controls (white squares). The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD. These data suggest that the DAMGO-induced burst amplitude increase was not related to gasping since riluzole blocks persistent sodium currents associated with gasping.

FIGURE 8

The DAMGO-induced burst amplitude increase was due to brainstem MOR activation. (A) Drawing of brainstem-spinal cord preparation with a plastic and petroleum jelly barrier that separated the recording chamber into separately perfused brainstem and spinal compartments. At right is a voltage trace showing the effects of bath-applied DAMGO (50 nM) in the spinal compartment first, followed by a similar bath-application in the brainstem compartment. (B) The burst amplitude increase occurred only when DAMGO was bath-applied in the brainstem compartment. Data for time controls and DAMGO application experiments are indicated by the white and blue symbols, respectively (spinal application = circles; brainstem application = triangles). (C) Burst frequency depression was only observed when DAMGO was bath-applied to the brainstem compartment. (D) Minute activity decreased at the 15-min time point when DAMGO was applied to the brainstem and not the spinal cord. Otherwise, there were no overall DAMGO concentration effect of DAMGO on minute activity. The pound sign (#) indicates a significant pairwise comparison difference from time controls at that time point, and the dagger (†) indicates a significant overall DAMGO concentration effect. Data shown as mean ± SD.