Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control

Abstract

Acute ethanol depresses respiration, but little is known about chronic ethanol exposure during gestation and breathing, while the deleterious effects of ethanol on CNS development have been clearly described. In a recent study we demonstrated that pre- and postnatal ethanol exposure induced low minute ventilation in juvenile rats. The present study analysed in juvenile rats the respiratory response to hypoxia in vivo by plethysmography and the phrenic (Phr) nerve response to ischaemia in situ. Glycinergic neurotransmission was assessed in situ with strychnine application and [(3)H]strychnine binding experiments performed in the medulla. After chronic ethanol exposure, hyperventilation during hypoxia was blunted in vivo. In situ Phr nerve response to ischaemia was also impaired, while gasping activity occurred earlier and recovery was delayed. Strychnine applications in situ (0.05-0.5 microM) demonstrated a higher sensitivity of expiratory duration in ethanol-exposed animals compared to control animals. Moreover, [(3)H]strychnine binding density was increased after ethanol and was associated with higher affinity. Furthermore, 0.2 microM strychnine in ethanol-exposed animals restored the low basal Phr nerve frequency, but also the Phr nerve response to ischaemia and the time to recovery, while gasping activity appeared even earlier with a higher frequency. Polycythaemia was present after ethanol exposure whereas lung and heart weights were not altered. We conclude that chronic ethanol exposure during rat brain development (i) induced polycythaemia to compensate for low minute ventilation at rest; (ii) impaired the respiratory network adaptive response to low oxygen because of an increase in central glycinergic tonic inhibitions, and (iii) did not affect gasping mechanisms. We suggest that ethanol exposure during early life can be a risk factor for the newborn respiratory adaptive mechanisms to a low oxygen environment.

Figure 1. Definition of measuring times during phrenic nerve response to ischaemia in situ

A, definition of the three phases of the respiratory cycle as measured on phrenic nerve discharge in situ during baseline activity. T_i: inspiration; T_e: expiration; PI: post-inspiration; T_e2: second phase of expiration. B, top trace is the time course of incrementing burst phrenic (Phr) nerve frequency before, during and after ischaemia. Bottom trace is the corresponding integrated Phr nerve activity. The measuring times are defined as follows: (1) hyperventilation with increased amplitude and frequency of incrementing Phr bursts; (2) apnoea duration; (3) time to first gasping burst; (4) duration of gasping activity; (5) recovery time to first ramping Phr burst after reoxygenation. C, Phr burst pattern showing the difference in ramp slope according to the type of bursting activity. Incrementing ramp discharges defined eupnoea. Gasping was characterized by decrementing ramp discharge. Bell-shaped discharges are observed at the transition between eupnoeic and gasping rhythms or at the beginning of recovery. R_f: respiratory frequency (cycles min⁻¹); ∫Phr: integrated phrenic nerve activity.

Figure 2. Response to hypoxia in vivo

A, recordings of spontaneous breathing activity in the two animal populations under normoxic conditions and at the end of hypoxia. Inspiration is upwards. B, analysis of ventilatory parameters shows that respiratory frequency was significantly increased in both populations (upper left panel), while tidal volume increased only in control animals (upper right panel). Minute ventilation and inspiratory inflow index were both increased only in the control population (lower left and right panels, respectively). **P < 0.01, ***P < 0.001, compared to baseline values; ##P < 0.01, ###P < 0.001, comparison between populations.

Figure 3. Phrenic nerve response to ischaemia in control and ethanol-exposed populations in situ

A, recordings of integrated Phr nerve activity illustrating the absence of hyperventilation in ethanol-exposed animal and the earlier onset of gasping activity (single vertical arrows) due to shorter apnoea duration (horizontal arrows). During reoxygenation, recovery of eupnoeic activity was significantly delayed in the ethanol-exposed group (double arrows indicate first incrementing Phr burst). B, ethanol-exposed animals did not present a significant increase in Phr burst frequency and amplitude. ∫Phr: integrated phrenic nerve activity. Same statistical symbols as in Fig. 2. The simple arrow points to the first gasping burst and the double arrow to the first incrementing burst. Horizontal arrow delineates apnoea.

Figure 4. Strychnine and phrenic nerve activity in situ

A, recording of Phr nerve activity in the presence of 0.2 μm strychnine. At this concentration, phase durations and frequency were similar in the two populations. B, strychnine decreased the duration of inspiration (left) to a similar degree in the two populations, but decreased expiratory duration only in ethanol-exposed animals (right). C, post-inspiratory time was reduced to a similar degree in the two populations (left), whereas the second expiratory phase was reduced only in ethanol-exposed animals (right). D, Phr frequency was increased in both populations, but at a lower dose for the control group (left) and burst amplitude was not affected up to a concentration of 0.5 μm strychnine in both populations. Data for B–D are expressed as means ±s.e.m.ⁱP = 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 as compared to baseline values before strychnine. #P < 0.05 and ##P < 0.01, between populations.

Figure 5. Phrenic nerve response to ischaemia in situ in the presence of 0.2 μm strychnine or 8 mm[K⁺]_o

A, recordings of Phr nerve response. Tonic activity was observed during the first minute of ischaemia in most cases. Note the same gasping latency and recovery time to the first incrementing burst (single and double arrows, respectively). B, Phr response to ischaemia of an ethanol-exposed animal in the presence of 8 mm potassium instead of 6.25 mm. Note the absence of increase in frequency and amplitude, the time to first gasp and the very slow recovery. C, increase in Phr nerve frequency (left) was restored in ethanol-exposed animals and the difference between populations was abolished. The increase in burst amplitude (right) was similar between the two populations.