Opioid-resistant respiratory pathway from the preinspiratory neurones to abdominal muscles: in vivo and in vitro study in the newborn rat

Abstract

We report that after spontaneous breathing movements are stopped by administration of opioids (opioid-induced apnoea) in neonatal rats, abdominal muscles continue to contract at a rate similar to that observed during periods of ventilation. Correspondingly, in vitro bath application of a mu opioid receptor agonist suppresses the activity of the fourth cervical root (C4) supplying the diaphragm, but not the rhythmic activity of the first lumbar root (L1) innervating the abdominal muscles. This indicates the existence of opioid-resistant rhythmogenic neurones and a neuronal pathway transmitting their activity to the abdominal motoneurones. We have investigated this pathway by using a brainstem-spinal cord preparation of the neonatal rat. We identified bulbospinal neurones with a firing pattern identical to that of the L1 root. These neurones were located caudal to the obex in the vicinity of the nucleus retroambiguus. Resting potentials ranged from -49 to -40 mV (mean +/- S.D. -44.0 +/- 4.3 mV). The mean input resistance was 315.5 +/- 54.8 MOmega. The mean antidromic latency from the L1 level was 42.8 +/- 4.4 ms. Axons crossed the midline at the level of the cell body. The activity pattern of the bulbospinal neurones and the L1 root consisted of two bursts per respiratory cycle with a silent period during inspiration. This pattern is characteristic of preinspiratory neurones. We found that 11 % of the preinspiratory neurones projected to the area where the bulbospinal neurones were located. These preinspiratory neurones were found in the rostral ventrolateral medulla close (200-350 microm) to the ventral surface at the level of the rostral half of the nucleus retrofacialis. Our data suggest the operation of a disynaptic pathway from the preinspiratory neurones to the L1 motoneurones in the in vitro preparation. We propose that the same pathway is responsible for rhythmic activation of the abdominal muscles during opioid-induced apnoea in the newborn rat.

Figure 1. Abdominal muscle activity and inspiratory activity in a 5-day-old rat after administration of fentanyl

An absence of respiratory flow during apnoeic cycles indicates that neither the diaphragm nor the inspiratory intercostal muscles contract during these cycles. Due to an integration process, the S_p,O2 signal lags behind other signals. Each inspiration has an effect on haemoglobin oxygen saturation. This is particularly clear after three consecutive apnoeic cycles. Traces, from top to bottom: raw and integrated abdominal EMG; tidal volume; tracheal flow; percentage haemoglobin saturation.

Figure 2. Activity of the C4 root and the L1 root in the brainstem-spinal cord preparation

A, a control recording. B, a response to the μ opioid receptor agonist. Note that in the absence of C4 activity, the L1 activity is transformed into a single burst. A model for the mechanism of this transformation is shown in Fig. 6.

Figure 6. Model for the generation of the L1 activity after administration of opioids

A, the diagram of the ventral aspect of the brainstem-spinal cord preparation showing a disynaptic pathway from the preinspiratory neurones to the L1 motoneurones. B, the activity of the preinspiratory neurones driving the twice-bursting bulbospinal neurones in the cVRG. Inside the dotted rectangular there are activities of the inspiratory neurones shaping the twice bursting pattern of the preinspiratory neurones (Onimaru et al. 1990; Ballanyi et al. 1999) and bulbospinal inspiratory neurones driving the C4 root. The plus and minus signs indicate synaptic excitation and inhibition, respectively. Arrows indicate the direction in which excitation/inhibition is transmitted.

Figure 3. Activity of a bulbospinal neurone supplying L1 root motoneurones

The left panel shows activity of the twice-bursting bulbospinal neurone, together with activity of the L1 and C4 root. The right panel shows a response to the antidromic stimulation with sub- and suprathreshold current. The arrow indicates the time of stimulation. The antidromic action potential was not preceded by an EPSP. The threshold current was constant and the antidromic latency (37 ms) was constant at various stimulus strengths.

Figure 4. Transverse section through the rat medulla caudal to the obex, showing reconstruction of an electrophysiologically identified, intracellularly filled, twice-bursting bulbospinal neurone projecting to the L1 level

Twice-bursting neurones that could be antidromically activated at the L1 level had a soma diameter of 10-30 μm, and their locations coexisted with, or were just ventral to, the nucleus retroambiguus. Axons crossed the midline at the same rostrocaudal level as the cell bodies. The primary dendrites projected ventromedially and dorsolaterally.

Figure 5. Activity of the preinspiratory neurone projecting to the nucleus retroambiguus and traces of the C4 and L1 root activity

Five out of 44 preinspiratory neurones projected to the nucleus retroambiguus and none projected directly to the L1 level. These five neurones were found close (200-350 μm) to the ventral surface at the level of the rostral half of the nucleus retrofacialis.