Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats

Abstract

Exposure to episodic hypoxia induces a persistent augmentation of respiratory activity, known as long-term facilitation (LTF). LTF of phrenic nerve activity has been reported to require serotonin receptor activation and protein syntheses. However, the underlying cellular mechanism still remains poorly understood. NMDA receptors play key roles in synaptic plasticity (e.g. some forms of hippocampal long-term potentiation). The present study was designed to examine the role of NMDA receptors in phrenic LTF and test if the relevant receptors are located in the phrenic motonucleus. Integrated phrenic nerve activity was measured in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats before, during and after three episodes of 5 min isocapnic hypoxia (P(a,O2) = 30-45 mmHg), separated by 5 min hyperoxia (50% O2). Either saline (as control) or the NMDA receptor antagonist MK-801 (0.2 mg kg(-1), i.p.) was systemically injected approximately 1 h before hypoxia. Phrenic LTF was eliminated by the MK-801 injection (vehicle, 32.8 +/- 3.7% above baseline in phrenic amplitude at 60 min post-hypoxia; MK-801, -0.5 +/- 4.1%, means +/- S.E.M.), with little change in both the CO2-apnoeic threshold and the hypoxic phrenic response (HPR). Vehicle (saline, 5 x 100 nl) or MK-801 (10 microM; 5 x 100 nl) was also microinjected into the phrenic motonucleus region in other groups. Phrenic LTF was eliminated by the MK-801 microinjection (vehicle, 34.2 +/- 3.4%; MK-801, -2.5 +/- 2.8%), with minimal change in HPR. Collectively, these results suggest that the activation of NMDA receptors in the phrenic motonucleus is required for the episodic hypoxia-induced phrenic LTF.

Figure 1. Superimposed schematic drawing of the spinal microinjection sites in one vehicle and one MK 801 rat

This diagram depicts a cross-section of the spinal cord at C3–C5 segments. Eight black dots in the lower right portion of the section represent the tip sites of the micropipettes as seen under a light microscope. Note that actual volumes and diffusion areas of the injected fluid are much larger than those spots. The dimensions shown here are similar to those reported by other laboratories (Furicchia & Goshgarian, 1987; Chitravanshi & Sapru, 1996).

Figure 2. The effect of NMDA receptor antagonism on hypoxic phrenic response (HPR) in the peak amplitude of integrated phrenic nerve activity

This HPR is the average of 3 episodes of hypoxia and expressed as a percentage increase above baseline. Saline or MK-801 was systemically injected and microinjected (into the phrenic motonucleus region). Data are presented as means ± s.e.m. There is no significant difference between these groups (all P > 0.7).

Figure 3. The effect of MK-801 (systemic injection) on phrenic long-term facilitation (LTF)

A, average changes from baseline in peak amplitude of integrated phrenic nerve activity, normalized as a percentage of the baseline (%baseline). B, changes from baseline in phrenic burst frequency (bursts min⁻¹). C, changes from baseline in minute phrenic nerve activity, normalized as a percentage of the baseline (%baseline). These were obtained following 3 episodes of 5 min isocapnic hypoxia in vehicle (n = 14, □) and MK-801 (n = 7, ▪) rats. Data are expressed as means ± s.e.m. * Significant difference from baseline; † significant difference from vehicle group (P < 0.05).

Figure 4. Representative tracings of the integrated phrenic neurogram (Phr) and mean arterial blood pressure (BP)

These were recorded before (baseline), during (hypoxia) and after 3 episodes of isocapnic hypoxia in one control (vehicle) and one MK-801 rat in the microinjection (into the phrenic motonucleus region) study. Note that the phrenic responses to hypercapnia (CO₂) are also presented but the data for only one hypoxic episode are presented.

Figure 5. The effect of MK-801 microinjection (into the phrenic motonucleus region) on phrenic long-term facilitation (LTF)

A, average changes from baseline in peak amplitude of integrated phrenic nerve activity, normalized as a percentage of the baseline (%baseline). B, changes from baseline in phrenic burst frequency (bursts min⁻¹). C, changes from baseline in minute phrenic nerve activity, normalized as a percentage of the baseline (%baseline). These were obtained following 3 episodes of 5 min isocapnic hypoxia in vehicle (n = 8, □) and MK-801 (n = 6, ▪) rats. Data are expressed as means ± s.e.m. * Significant difference from baseline; † significant difference from vehicle group (P < 0.05).