Early chronic ethanol exposure in rats disturbs respiratory network activity and increases sensitivity to ethanol

Abstract

Chronic ethanol exposure during the fetal period alters spontaneous neuronal discharge, excitatory and inhibitory amino acid neurotransmission and neuronal sensitivity to ethanol in the adult brain. However, nothing is known about the effects of such exposure on the central respiratory rhythmic network, which is highly dependent on ethanol-sensitive amino acid neurotransmission. In 3- to 4-week-old rats, we investigated (1) the effects of chronic ethanol exposure (10% v/v as only source of fluid) during gestation and lactation on phrenic (Phr) and hypoglossal (XII) nerve activity using an in situ preparation and on spontaneous breathing at rest in unanaesthetized animals using plethysmography; (2) the sensitivity of the respiratory system to ethanol re-exposure in situ; and (3) the phrenic nerve response to muscimol, a GABA(A) receptor agonist, applied systemically in an in situ preparation. In control rats, ethanol (10-80 mm) induced a concentration-dependent decrease in the amplitude of both XII and Phr motor outflows. At 80 mm ethanol, the amplitude of the activity of the two nerves displayed a difference in sensitivity to ethanol and respiratory frequency increased as a result of shortening of postinspiratory duration period. After chronic ethanol exposure, respiratory frequency was significantly reduced by 43% in situ and by 23% in unanaesthetized animals, as a result of a selective increase in expiratory duration. During Phr burst, the ramp was steeper, revealing modification of inspiratory patterning. Interestingly that re-exposure to ethanol in situ elicited a dramatic inhibitory effect. At 80 mm, ethanol abolished rhythmic XII nerve outflow in all cases and Phr nerve outflow in only 50% of cases. Furthermore, administration of 50 microm muscimol abolished Phr nerve activity in all control rats, but only in 50% of ethanol-exposed animals. Our results demonstrate that chronic ethanol exposure at an early stage of brain development depresses breathing in juvenile rats, and sensitizes the respiratory network to re-exposure to ethanol, which does not seem to involve GABAergic neurotransmission.

Figure 1. Acute ethanol exposure in control animals

A, illustration of respiratory phase duration measurements performed in situ. Pre-inspiration (Pre-I) phase, only present in hypoglossal (XII) nerve recording, corresponds to burst activity prior to onset of phrenic nerve (Phr) activity. Inspiration (Ti) is burst activity recorded on the phrenic nerve. Postinspiration (PI) duration was defined on the phrenic nerve, and corresponds to decreasing nerve activity after the end of Ti until silence is observed. Note that hypoglossal nerve also displayed a postinspiration discharge that was usually longer than the PI phase. The second part of expiration (TeII) is the period between the end of PI and the next inspiratory burst (not illustrated here). Expiration was the sum of phrenic PI and TeII durations. The equivalent period to phrenic expiration on hypoglossal nerve corresponds to the sum of hypoglossal postinspiratory discharge, the silence period and the Pre-I phase. B, ethanol effects on amplitude were significant from 10 mm for hypoglossal and 20 mm for the phrenic nerve. At 80 mm, a difference in sensitivity between the two nerves was observed (#P < 0.05). C, paired recordings of integrated phrenic (Int Phr) and hypoglossal (Int XII) nerves in the presence of 40 and 80 mm ethanol. The most intense effect was observed for 80 mm ethanol on hypoglossal nerve. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Acute ethanol exposure and respiratory phase durations in control animals studied in situ

A, phrenic postinspiration duration was reduced from 10 mm ethanol. B, the second expiratory phase on the phrenic nerve was not significantly reduced. C, on hypoglossal nerve, preinspiration burst duration (pre-I) increased but changes were significant only at 40 mm ethanol. D, respiratory frequency (R_f), as measured on phrenic nerve recording, increased with high doses of ethanol. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Early and chronic ethanol exposure alters spontaneous phrenic nerve activity in situ

A, no difference was observed in inspiratory duration. Expiration was increased (***P < 0001). During expiration, postinspiration was not significantly affected whereas the second expiratory phase was increased (***P < 0001). B, superimposed average of 15 consecutive phrenic nerve bursts from one control and one ethanol-exposed animal showing that the ramp became steeper in the ethanol-exposed animal whereas no difference was measured in inspiratory duration.

Figure 4. Respiratory activity under resting conditions recorded by whole-body plethysmography in intact and awake animals

A, respiratory phase durations in control animal (filled bars) and ethanol-exposed animals (open bars) showing that only expiratory duration was significantly increased after chronic ethanol exposure. B, examples of raw plethysmographic recordings from a control and an ethanol (EtOH)-exposed animal. Inspiration corresponds to the upward deflection of the trace and expiration to the downward deflection until the next upward deflection. Note that frequency was markedly reduced (−25%) whereas V_T was not significantly affected.

Figure 5. Ethanol concentration–response curve in ethanol-exposed animals studied in situ

A, paired recordings of phrenic (Phr) and hypoglossal (XII) nerve activity illustrated as integrated form (Int) showing effects of ethanol (40 and 80 mm) on control and ethanol-exposed animals. Rhythmic activity in ethanol (EtOH)-exposed animals is slower prior to ethanol application and is more sensitive to acute ethanol exposure. Arrows in the lower set of traces indicate the remaining phrenic nerve activity after application of 40 mm ethanol. No activity was observed on the hypoglossal nerve. B, effects of acute ethanol on amplitude in ethanol-exposed animals. Compare this with Fig. 1B. (***P < 0.01, dose-effect; #P < 0.05, response between nerves).

Figure 6. Effects of muscimol in control and ethanol-exposed animals

A, phrenic nerve activity in control animals in situ was rapidly abolished by application of 50 µm muscimol into the perfusate. B, in contrast to control animals, muscimol (50 µm) did not abolish phrenic nerve activity in two of four ethanol (EtOH)-exposed animals tested, whereas this activity disappeared in the two other animals after an interval three times longer than that observed in control animals.